Multi-array, multi-specific electrochemiluminescence testing.
Abstract
Materials and methods are provided for producing patterned multi-array, multi-specific surfaces which are electronically excited for use in electrochemiluminescence based tests. Materials and methods are provided for the chemical and/or physical control of conducting domains and reagent deposition for use in flat panel displays and multiply specific testing procedures.
Term
Term ended
Expired 25 August 2017, 9.1 years ago.
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- Today
11 claims: 10 independent, 1 dependent
- 1CLAIMS REIVINDICACIONES 1. Un aparato para su uso en la detección de un analito one. An apparatus for use in detecting an analyte either a predetermined amount of a reagent capable of binding an electrochemical-luminescent labeled species or a binding partner of a labeled species elec t roqu im i coluro i. niscent. mismo una cantidad predeterminada de un reactivo capaz de enlazar una especie marcada electroquimicoluíniniscente o bien un socio de enlace de una especie marcada elec t roqu im i coluro i. niscente. 10 2. An apparatus for use in the detection of an analyte by means of an electroluminescence electrode, comprising an electrode and a counter electrode, said electrode or counter electrode having immobilized on a surface thereof a predetermined amount of a reagent capable of directly or indirectly binding an electrochemical luminescent labeled species. 10 2. Un aparato para su uso en 1¿l detección de un analito med i ante elec troqu imicoluminiscenci a, que comprende un electrodo y un contraeleetrodo, dicho electrodo o bien contraelectrodo tiene inmovi1 izado en una superficie del mismo una cantidad predeterminada de un reactivo capaz de enlazar directa o indirectamente una especie marcada electroquimicoluminiscente. 3. An apparatus for use in detecting an elect rodo analyte, said e 1 ec t rodo t i. a child who surpasses a quantity that corrods i nroovi 1 i zed in redetermined component of an essay one one of 3. Un aparato para su uso en la detección de un analito elect rodo, d icho e 1 ec t rodo t i. e ne supe r f ic ie del ni i smo una cantidad un que coroprende i nroovi 1 i zado en p redetermi nada de componente de un ensayo un una un de de 4. Un aparato de conformidad con la reivindicación 3 donde dicho electrodo es el electrodo de trabajo. Four. An apparatus according to claim 3 wherein said electrode is the working electrode. 25 5. An apparatus according to claim 3 wherein 25 5. Un aparato de conformidad con la reivindicación 3 donde -216ι -216ι 216 Said electrode is the counter electrode. 216 dicho electrodo es el contraelectrodo. 6 «An apparatus in accordance with re vi nd icae i on 3 where the immobilized reagent and the electrode complex substantially allows the transfer of electrons between said electrode and an electrochemiluminescent marker. 6« Un aparato de conformidad con la reí v i nd icae i ón 3 donde el reactivo inmovilizado y el complejo de electrodos permite substancia 1mente la transferencia de electrones entre dicho electrodo y un marcador eleetroquimicoluminiscente. 7 - An apparatus in accordance with claim 3 wherein said electrode consists of a porous material. 7 - Un aparato de conformidad con la re iv i ndicación 3 donde dicho electrodo consiste de un material poroso. 8. An apparatus for the detection of an analyte by means of the ec t roqu im i colum ini scenci a comprising a porous ro ect ect and a electrode control. 8. Un aparato para la detección de un analito mediante e1 ec t roqu im i colum i n i scenci a que comprende un e1ect rodo poroso y un cont raelec trodo. 9. An apparatus for use in the detection of an analyte by means of the troqu imi co1um ini scenc ia, comprising a working electrode and a counter electrode, said working electrode having immobilized on a surface thereof and in electrochemical contact with it. , a predetermined amount of a reagent capable of binding a component of a binding electrochemiluminescence assay. 9. Un aparato para su uso en la detección de un analito med i a n te e1ec troqu i m i co1um i n i scenc i a, que comprende un electrodo de trabaja y un contraelectrado, dicho electrodo de trabajo tiene inmovilizado en una superficie del mismo y en contacto electroquímico con el mismo, una cantidad predeterminada de un reactiva capaz de enlazar un componente de un ensayo de electroquimicoluminiscencia de enlace. 10. A device for use in the detection of an ana 1 i to by means of an elec trochicolis niscence, which comprises a working electrode and a center electroelectrode, said counter electrode has i nmov i 1 ized in a surface of the same and in electrochemical contact therewith, a predetermined amount of a reagent capable of binding a component of a binding electrochemical luminescence assay. 10. Un aparata para su uso en la detecc ión de un ana 1 i to mediante elec t roqu imicolumi niscencia, que comprende un electrodo de traba jo y un cent raeleetrodo, dicho contraelectrodo tiene i nmov i 1 i zado en una superf ic ie del mismo y en contacto electroquímico con el mismo, una cantidad predeterminada de un reactivo capaz de enlazar un componente de un ensayo de electroquimicoluminiscencia de enlace. -217I -217I 11. Un aparato para su uso en la detección de un analito med iante elect roqu. imicoluminiscencia, que comprende un electrodo, un contraelectrodo y una matriz porosa en contacto electroquímico con dicho electrodo, dicha matriz eleven. An apparatus for use in detecting an analyte by elect roqu. photoluminescence, comprising an electrode, a counter electrode and a porous matrix in electrochemical contact with said electrode, said matrix 5 Porous contains on a surface thereof a predetermined amount of a reagent capable of binding a component of a binding electrochemical luminescence assay. 5 porosa contlene en una superficie de la misma una canti dad predeterminada de un reactivo capaz de enlazar un componente de un ensayo de electroquimicoluminiscencia de enlace. 12. An apparatus for use in the detection of an analyte by electrochemiluminescence, comprising a 12. Un aparato para su uso en la detección de un analito mediante electroquimicoluminiscencía, que comprende un 10 electrode and a plurality of discrete link domains, such link domains can link a component of an e 1 ec t roch imic 1 um i assay. no link scenc ia. 10 electrodo y una pluralidad de dominios de enlace discretos, dichos dominios de enlace pueden enlazar un componente de un ensayo de e 1 ec t roqu i m i c o 1 um i. n i scenc i a de enlace. 13. An apparatus for use in detecting an analyte by electrochemiluminescence, comprising a discrete electrode each containing a predetermined amount of a reagent capable of binding a component of an assay 13. Un aparato para su uso en la detección de un analito med i ante electroquimicoluminiscencia, que comprende un electrodo discretos cada uno conteniendo una cantidad predeterminada de un reactivo capaz de enlazar un componente de un ensayo 14. An apparatus in accordance with which further comprises a built-in counter. 14. Un aparato de conformidad con la que comprende además un con trae1ec t rodo. 15. Un aparato de conformidad con la reivindicación que comprende además un contrae1ec trodo, un d isposit i vo de suministro de muestras y un dispositivo de detección de luz fifteen. An apparatus according to claim further comprising a counter trod, a specimen supply device and a light detection device 16. An apparatus for use in detecting an analyte by elect roquimicolumi niscenc ia, comprising 16. Un aparato para su uso en la detección de un analito med iante elect roquimicolumi niscenc i a, que comprende -2181 -2181 Ca ) u.n elec t reído , (b) una pluralibad de dominios de enlace discretos cada uno conteniendo una cantidad predeterminada de un reactivo capaz de enlazar un componente de un ensayo de electroquimicoluminiscencia de enlace, Ca) a reacted elec t, (b) a plurality of discrete binding domains each containing a predetermined amount of a reagent capable of binding a component of a binding electrochemical luminescence assay, Cc) a counter electrode, Cc) un contraelectrodo, Cd) a device for supplying samples of said link domains, Cd) un dispositivo para suministro de muestras di chos dominios de enlace, Ce > Ce> It is available to trigger the electroluminescence elect, and <f) it is available to detect the electrochemistry. dispos i t i vo pa ra desencadenar la elect roquimicoluminiscencia, y <f) d ispos i t i vo pa ra detectar la electroquimicolumi niscenc ia. 17. An apparatus for use in detecting an analyte by electrochemistry. niscence, comprising 17. Un aparato para su uso en la detección de un analitci mediante electroquimicolumi. niscencia, que comprende 15 Ca) un electrodo, (b) un soporte que tiene inmovilizado ahí una pluralidad de dominios de enlace discretos cada uno conteniendo una cantidad predetermi nada de un reactivo capaz de enlazar un componente de un ensayo de eleetroquimicoluminiscencia de 20 enlace, (c) un contraelectrodo, <d) un dispositivo para suministrar muestras a dichos dominios de enlace, fifteen Ca) an electrode, (b) a support having there immobilized a plurality of discrete binding domains each containing a predetermined amount of a reagent capable of binding a component of a 20-bond electrochemiluminescence assay, (c) a counter electrode , <d) a device for supplying samples to said linking domains, Ce) dispos i t i vo pa ra desencadenar la Ce) dispos iti vo to trigger the 25 elec t roqu.imicolumi niscenc ia, and 25 elec t roqu.imicolumi niscenc ia , y -219t (f ) pa ra la e 1 ec t roqu i m i c □ 1. um iniscericia -219t (f) for e 1 ec t roqu imic □ 1. um iniscericia 18. An apparatus in accordance with claim 17, is in electrochemical contact with said electrode claim 18. Un aparato de conformidad con la reí vindicación 17, están en contacto electroquímico con dicho electrodo la reivindicación 17, donde dicho electrodo es poroso» 17, where said electrode is porous » lee e lee t roqu imicolum ini scenc ia detects one or more of these ΐ e lee t roqu imicolum i n i scenc i a detecta la uno o varios de dichos 2. 3. An apparatus in accordance with claim where said support is a porous matrix 23. Un apa rato de conformidad con la reivindicaeión donde dicho soporte es una matriz porosa 24. An appliance in accordance with the re ivi nd ication 24. Un aparato de conformidad con la re i v i nd icac ión 17, donde dicho soporte es una matriz es poroso» la re i v i nd i cae i ón 17, donde dicho soporte es una ma t r iz porosa, dicha ma t r i z se posiciona entre dicho electrodo y contraelectrodo 17, where said support is a matrix is porous »the re ivi nd i falls i on 17, where said support is a porous matrix, said matrix is positioned between said electrode and counter electrode -220I by elect roquimicolum i riiscenc ia, comprising (a) one electrode, (b? A plurality of discrete binding domains, each containing a predetermined amount of a reagent capable -220I mediante elect roquimicolum i riiscenc i a , que comp rende (a) un electrodo, (b? una pluralidad de dominios de enlace discretos, cada uno conteniendo una cantidad predeterffii nada de un reactivo capaz 5 of linking a component of a test 5 de enlazar un componente de un ensayo de <f) a plurality of devices for electrochemical illumination detection. <f) una pluralidad de dispositivos para la detección la electroquimicoluminiscenc ia. 27 »An apparatus according to claim 26, 27» Un aparato de conformidad con la reivindicación 26, 15 donde dicha pluralidad de dispositivos para la detección de la elect roqu i m i co 1 u.m i n i scenc i a se emp lea pa ra forma r una imagen de uno o varios de dichos dominios de enlace. fifteen where said plurality of devices for the detection of the elect roqu imi co 1 um ini scenc ia is used to form an image of one or more of said linking domains. 28. An apparatus in accordance with re ivi nd icae i on 27, where at least one of said plurality of devices 28. Un apa ra to de conformidad con la re i v i nd icae i ón 27, donde al menos uno de dicha pluralidad de dispositivos de 20 detección detecta la electroquimicoluminiscencia proveniente de un dominio de enlace. twenty Detection detects electrochemiluminescence from a binding domain.
- 22? An apparatus in accordance with claim. ion 27, where said plurality of devices for the detection of electrochemiluminescence is a CCD set or a set of diodes. 2? Un aparato de conformidad con la reivindicar. i ón 27, donde dicha pluralidad de dispositivos para la detección de la electroquimicoluminiscencia es un conjunto CCD o bien un 25 conjunto de diodos. -221i -221i 30. An apparatus for use in detecting an analyte by elect rochimicolumiscence, comprising an electrode having a plurality of discrete link domains immobilized on one surface thereof, such link domains may link a component of a link electrochemiluminescence assay. 30. Un apa rato para su uso en la detecc ión de u.n anal i to mediante elect roquimicolumi niscencia, que comprende un electrodo que tiene inmovilizado en una superficie del mismo una pluralidad de dominios de enlace discretos, dichos dominios de enlace pueden enlazar un componente de un ensayo de electroquimicoluminiscencia de enlace. 31. An apparatus for use in detecting an analyte through 1 ec t roqu imi co 1 um ini scenc ia, comprising a surface thereof with a plurality of d erates, each containing one of a reactive layer z to link a component of an electrochemical link luminescence assay. 31. Un aparato para su uso en la detección de un analito med i a n te e 1 ec t roqu i m i co 1 u.m i n i scenc i a , que comprende un superficie del mismo una plura1 idad d iseratos, cada uno conteniendo una de un react i vo capa z de enlazar un componente de un ensayo elec t roquimicoluminiscenc ia de enlace. 32. An apparatus for use in the detection of an analyte by means of electrochemistry and my iscency column, comprising an electrode that has immobilized on a surface thereof a plurality of domains binding domains a rooter in the detection of an arialite by electrochemiluminescence, which comprises a 32. Un aparato para su uso en la detección de un analito med iante elec troqu i mi columi n iscenc i a , que comprende un electrodo que tiene inmovilizado en una superficie del mismo una pluralidad de dominios dominios de enlace un roa rcador en la detección de un arialito mediante electroquimicoluminiscencia, que comprende un de electroquimicoluminiscencia? y un contraelectrodo. electrochemiluminescence? and a counter electrode. -222i -222i ITS use in the detection of an analyte using electroqu i mi colum i. n iscenc ia, which includes a support that has i nmov i 1 i zado ah i uria plurality of domains each containing a predetermined quantity of a reagent capable of binding one or the other link SU uso en la detección de un analito med ¿ante electroqu i mi colum i. n iscenc i a , que comprende un soporte que t i ene i nmov i 1 i zado ah i uria plural idad de dominios cada uno conteniendo una cantidad predeterminada de un reactivo capaz de enlazar u n c omp o ríe nte enlace 35. An apparatus for use in the detection of a year by means of electrochemical immersion in the electrode and a support that has immobilized in one of the mystnci a plurality each containing a predetermined quantity capable of ele bind a component of a bonding assay, that bonding electrode and bond are in contact 35. Un aparato para su uso en la detección de un a na 1 i to med iante electroqu imicolumi niscencia un el ec t rodo y un soporte que t i ene inmovilizado en una del m i stnci una plural i.dad cada uno conteniendo una cantidad predeterminada capaz ele enlazar un componente de un ensayo de enlace, dicho electrodo y de enlace se encuentran en contacto 36. An apparatus in accordance with reí vi nd icae i óπ 36. Un aparato de conformidad con la reí v i nd icae i óπ 35, donde el electrodo es el electrodo de trabajo conformidad con la reí vindicación 35, where the electrode is the working electrode in accordance with the above vindication 35, donde el electrodo es el cent raelee t rodo 35, where the electrode is the cent raelee t rodo 38. An apparatus for use in the detection of an analyte by electrochemistry and luminescence, which would comprise a ce Ida e 1 ec t roqu í.mi. ca, (b) a first surface containing an electrode, (c) a second surface having immobilized there i a plurality of binding domains, each containing a 38. Un aparato para su uso en la detección de un analito mediante electroquimicolumiπiscencia, que comprendeí ía) una ce Ida e 1 ec t roqu í.mi. ca , (b) una primera superficie que contiene un electrodo, (c) una segunda superficie que tiene inmovilizado ah i una pluralidad de dominios de enlace, cada uno conteniendo un -223i component of a bond, said surface are spatially aligned to allow the transfer of a marker axis electroquimicolumi niscenc¿adi rec tai nd i straight link. -223i componente de un de enlace, d i cha superficie están espacialmente alineadas para permitir la transferencia de un marcador eje electroquimicolumi niscenc¿a d i rec t a i nd i rectamente enlace. 39. An apparatus for use in the detection of an analyte, comprising an electrochemical cell and an electrode having imovi 1 located therein a plurality of binding domains, each containing no capia z to link a component of from elec traquimi cα1um ini scenci to link 39. Un aparato para su uso en la detección de un ana lito , que comprende una celda electroquímica y u.n electrodo que t i ene i nmovi 1 i zado ahí u na pluralidad de dominios de enlace, cada u no conteniendo un capia z de enlazar un componente de de elec traquimi cα1um i n i scenci a de enlace
- 34D. A method of carrying out a plurality of electrochemiluminescence assays, comprising the steps of (a) contacting a number of discrete binding domains with a sample containing one or more analytes of interest under conditions of test; 4D. Un método para llevar a cabo una pluralidad de ensayos de electroquimicoluminiscencia, que comprende los pasos des (a) poner en contacto una piara 1 idad de dominios de enlace discretos con una muestra que contiene uno o varios ana 1 i tos de interés bajo condiciones de ensayo; (b) usar electroquimicoluminiscencia en uno o varios de e 1 ec t r oqu i m i co 3. um i. n i scenc i a en una plural idad de d ichos dominios de enlace. (b) use electrochemiluminescence in one or more of the e 1 ec tr oqu imi co 3. um i. nor scenc ia in a plurality of said link domains. 41. A method for carrying out a plurality of elec troqu imicolum i tests, niscenc ia for a plural i ty of 41. Un método para llevar a cabo una pluralidad de ensayos de elec troqu imicolum i, niscenc i a para una plural i dad de -224ι analitos de interés diferentes, que comprende los pasos de:-224ι analytes of different interest, comprising the steps of: (a) contacting a plurality of discrete binding domains with a sample containing a plurality of analytes of interest under assay conditions;(a) poner en contacto una pluralidad de dominios de enlace discretos con una muestra que contiene una pluralidad de analitos de interés bajo condiciones de ensayo;ib) use elec tr oqu imi co 1 um ini se e nc ia si mu 11 online in said plurality of discrete link domains;and (c) detecting whether the electrochromy is simultaneously my incisence of each of said discrete binding domains. íb) usar elec t r oqu i m i co 1 u.m i n i se e nc i a si mu 11 á nea m ente e n dicha pluralidad de dominios de enlace discretos;y (c) detec ta r si mu1táneamente la electroqui mi co1um iniscenci a de cada uno de dichos dominios de enlace discretos. 42. An article comprising a plurality of discrete binding domains on a support, said binding domains have a relative spatial organization relative to each other and have different binding specificities for binding a plurality of different analytes of interest in an elec t roqu assay .imicolumi ni. scenc ia. 42. Un articulo que comprende una pluralidad de dominios de enlace discretos en un soporte, dichos dominios de enlace tienen una organización espacial relativa en relación entre ellos y tienen especificidades de enlace diferentes para enlazar una pluralidad de analitos de interés diferentes en un ensayo de elec t roqu.imicolumi ni. scenc i a . 43. A linkage has article comprising a plurality of discrete domains on a support, each of these domains a relative spatial organization in relation to the 11ths and each have a different linkage specificity for an analyte of interest in an assay of e1ec t roqui mi co1um i π i scenc ia, each domain contains a different link component linked to an analyte of different interest 43. Un enlace tienen artículo que comprende una pluralidad de dominios de discretos en un soporte, cada uno de dichos dominios una organización espacial relativa en relación entre e 11 os y cada uno t ienen una especificidad de enlace diferente para un analitos de interés en un ensayo de e1ec t roqui m i co1um i π i scenc i a, cada dominio cont¡ene componente de enlace diferente enlazado a un analito de interés d i ferente 44. An article comprising a plurality of domains of relative spatial organization in relation to each other and 44. Un artículo que comprende una pluralidad de dominios de organización espacial relativa en relación entre ellos y -225i cada u no t i e ne u na especificidad de enlace di ferente para un ana 1 i. tos de i nterés en un ensayo de elec t roq u i m icol uro i n i se e nc i a , cada i nd i rectamente con un ana 1 i to de interés diferente y a una porción capaz de electroquimicolumi ni seene ia. -225i each u does not have a different link specificity for an ana 1 i. coughs of interest in an elec t roq uim icol uro ini se e nc ia trial, each directly with an ana1 i of different interest and a portion capable of electrochemicolumi or sene ia. comprising a plurality of discrete binding domains on a support comprising a porous material, each having an organization relative to each other and each having an aspee ifity of interest in an electrochemiluminescence assay, each domain contains a different binding component linked to a different analyte of interest. que comprenda una plural idad de dominios de enlace discretos en un soporte que comprende un material poroso, cada uno da t iene una organizac i ón en relación entre ellos y cada uno tiene una aspee i f ¿cidad de interés en un ensayo de electroquimicoluminiscencia, cada dominio contiene un componente de enlace diferente enlazado a un analito de interés diferente. 46. An article where said material 46. Un articulo de donde dicho material 47. An article in accordance with porous comprises according to claim 45, fibrils. 47. Un articulo de conformidad con poroso comprende conformidad con la reivindicación 45, f ibrilas. la rei v ind icac i ón 45 donde dicho material poroso comprende carbono. the rei v ind ica tion 45 where said porous material comprises carbon. 48. An article comprising a plurality of discrete binding domains on a support comprising a functionalized fibril, these domains have a relative spatial organization relative to each other and each has a different binding specificity for an analyte of interest in an electrochemiluminescence assay, each domain is directly or indirectly linked to a different analyte of interest and to a portion capable of 48. Un artículo que comprende una pluralidad de dominios de enlace discretos en un soporte que comprende una fibrila funcional izada, dichos dominios tienen una organización espacial relativa en relación entre ellos y cada uno tiene una espec i f ic idad de enlace diferente para un analito de interés en un ensayo de electroquimicoluminiscencia, cada dominio se encuentra enlazado directa o indirectamente a un analito de interés diferente y a una porción capaz de -226- -226- 49. An article that includes a plurality of polymer domains, said domains do not have a relative relationship to each other, each having a different binding specificity for an analyte, each domain is directly or directly linked to interest. to an analysis of the z-layer of an electrochemical luminescence sample, comprising (a) a plurality of supports;and <b) one or more pairs of electrodes and counter electrodes 49. Un artículo que comprende una plura1 idad da domi π ios de pol1mero, dichos domi ni os t ienen una relativa en relación entre ellos cada uno tiene una especificidad de enlace diferente para un analito cada dominio se encuentra enlazado di recta o i ndi rectamente interés u ría a un anal i to capa z de de en una muestra electroquimicoluminiscencia, que comprende (a) una pluralidad de soporte;y < b) uno o varios pares de electrodos y contraelectrodos 51. A cassette to use the detection of an analyte in a sample supporting (b) one or more pairs of electrodes with said di: challenges;and (c) a device to supply samples on said 51. Un cassette para usar la detección de un analito en una muestra soporte (b) uno o varios pares de electrodos con dichos d i se: retos;y (c ) un disposi t ivo para suministrar muestras en d i cha -227i -227i 52. A cassette in accordance with what is claimed in the i da d of discrete link domains form at least one surface layer a component of a test of re iv i nd icaeión 50, where said number of link domains includes domains linkages having different linkage speci fi cies to provide the simultaneous linkage of a plurality of different present in a sample. 52. Un cassette de conformidad con lo reivindicado en la i da d de domi n i os enlace discretos forman al menos una superf i c i e capa un componente de un ensayo de en re iv i nd icaeión 50, donde dicha piuca 1 idad de dominios de enlace incluye dominios de enlace que tienen especi fic idades de enlace di ferentes pa ra proporcionar el enlace simuítáneo de una pluralidad de di ferentes presentes en una muestra. A para bi in med go the e1ectraquÍmicolumi niscenc ia (a) that of discrete link domains in (b> Un para b i en med i r la e1ectraquÍmicolumi niscenc ia (a ) que de dominios enlace discretos en (b > pairs of electrode and counter electrode, each of said plurality of is in line with one of electrode and said gold electrode bonding and counter electrode forms form a plurality of cells to carry out elect rochimicolumi measurements, di oh pairs of airships by a source of a voltage waveform pares de electrodo y contraelectrodo, cada uno de dicha pluralidad de se encuentra a lineado con uno de electrodo y d i c h o s d oro i n i o s de enlace d de electrodo y contraeleet rodo forman una p1ura1 idad de celdas pa ra cabo mediciones de elect roquimicolumi n iscenci a, di oh os pares de dirigibles por una fuente de una forma de onda de voltaje -228i ef ec t i va para desencadena r la elec t roqu imicolumi ni scenc i a ;y (c) dispositivo para suministrar muestras en dicha pluralidad de dominios de enlace discretos. -228i ef ec ti va to trigger the elec t roqu imicolumi ni scenc ia;and (c) device for supplying samples in said plurality of discrete link domains. 55, A cassette as claimed in claim v 53, wherein said plurality of link domains includes link domains having different link specificities to provide simultaneous linking of a plurality of years of interest different presents in a sample. 55, Un cassette de conformidad con lo reivindicado en la reí vindicación 53, donde dicha pluralidad de dominios de enlace incluye dominios de enlace que tienen especificidades de enlace diferentes p¿ira proveer el enlace simultáneo de una plura1 idad de ana 1 i tos de interés d i ferentes presentes en una mués t ra. 56. A cassette to detect or measure an analyte of interest in a sample, comprising: 56. Un cassette para detectar o medir un analito de interés en una muestra, que comprende: (a) a first support having a plurality of discrete link domains on a surface thereof, at least one of said discrete link domains is of different link specificity from the other link domains, each of said plurality of domains discrete binding is hydrophilic and surrounded by hydrophobic regions;(a) un primer soporte que tiene una pluralidad de dominios de enlace discretos en una superficie del misma, al menos uno de dichos dominios de enlace discretos es de especificidad de enlace diferente de los demás dominios de enlace, cada uno de dicha pluralidad de dominios de enlace discretos es hidrofílico y rodeado por regiones h idrofób icas;(b) a second support having a plurality of domains (b) un segunda soporte que tiene una pluralidad de dominios discrete link domains and said plurality of reaction means such that a sample to be analyzed that is present in each link domain is found in dominios de enlace discretas y dicha pluralidad de medios de reacción de tal manera que una muestra a analizar que se encuentra presente en cada dominio de enlace se encuentra en -229ι contact with a reaction medium. -229ι contacto con un medi o de reacci ón. 57. A cassette according to claim 50 wherein said discrete link domains further comprise an internal control. 57. Un cassette de conformidad con la reivindicación 50 en donde dichos dominios de enlace discretos comprenden además un control interno. 58. A method of preparing a plurality of discrete binding domains on a support containing binding reagents capable of binding the ana 1 ites of interest, comprising the steps of (a) forming a self-assembled monolayer on a support , said monolayer includes a group of support monolayer, said first linking group A specifically with linking group B;58. Un método para preparar una p l.ura 1 idad de dominios de enlace discretos en u.n soporte que contiene reactivos de enlace capaces de enlazar los ana 1 i tos de interés, que comprende las pasos de (a) formar una monocapa autoensamblada en un soporte, dicha monocapa inc1uye un g rupo de monocapa soporte, dicho primer grupo de enlace A específicamente con un grupo de enlace B;(b) contacting said first linking group A with one with an analyte of interest, said binding reagent is linked on said linking group B, such that said linked to said monolayer by an AIE bond to form link is a link surface, it organizes in the form of a plurality of domains of (b) poner en contacto dicho primer grupo de enlace A con un con un analito de interés, dicho reactivo de enlace se encuentra enlazado sobre dicho grupo de enlace B, de ta 1 manera que dicho unido con dicha monocapa por una unión AíE para formar enlace se una superficie de enlace, dicha organiza en forma de una pluralidad de dominios de 59. A process according to claim 57 where a plurality of different binding reagents are bound over the plurality of discrete binding domains and wherein said contacting step is carried out by 59. Un proceso de conformidad con la reivindicación 57 donde una pluralidad de reactivos de enlace diferentes se enlaza sobre la pluralidad de dominios de enlace discretos y donde dicho paso de la puesta en contacto se lleva a cabo mediante -2301 el suministro de una plural idad de muestras de fluidos, cada muestra de fluido incluye un reactivo de enlace diferente, sobre dicha monocapa a partir de una pluralidad de guías de f1u i do. -2301 supplying a plurality of fluid samples, each fluid sample includes a different binding reagent, on said monolayer from a plurality of fluid guides.
- 45 60- A method for detecting or measuring an analyte in an electrochemical luminescence binding assay, comprising the steps of:5 60- Un método para detectar o medir un analito en un ensayo de enlace de electroquimicoluminiscencia, que comprende los pasos de: (a) contacting a plurality of discrete binding domains immobilized on a surface of one or 10 multiple supports with a sample containing a plurality of analytes and a component of said assay linked to an electrochemical-luminescence primer;(a) la puesta en contacto de una pluralidad de dominios de enlace discretos inmovilizados en una superficie de uno o 1O varios soportes con una muestra que contiene una pluralidad de analitos y un componente de dicho ensayo enlazado a un ma reador de electroquímicoluminiscenci a;<b) la aplicación de una forma de onda de volta je efectiva pa ría desencadena r vari os d oroi ni os adecuado para la en electroquimicoluminiscencia en uno o de un roed i o de reacción <b) the application of an effective voltage waveform for the triggering of suitable gold vari os for electrochemiluminescence in one or one reaction roed io 1 levar cabo un ensayo de elec t roqu i mi coluroi n i scenc i a <c) la med i c i ó n de la e1ec t roqu i m i co1um ΐ n i scenc i a plural idad de one carry out a test of elec t roqu i mi coluroi ni scenc ia <c) the measurement of the e1ec t roqu imi co1um ΐ ni scenc ia plural idad 61. A set of elements for their use in the performance of a pluira 1 iity for a p1ure testing of the tests on analyte scenarios of interest, comprising: 61. Un conjunto de elementas pa ra su uso en e1 desempeña de una pluirá 1 idad de pa ra una p1ura1 i dad ensayos de e1ec t roqu i m i colum i n i scenci a de analitos de interés, que comprende: (a) an article comprising a plurality of domains of (a) un artículo que comprende una pluralidad de dominios de -231i have a relative spatial organization in relation to each other and have spec i. different liaison features for a length of anality i. different coughs of interest in a trochimicolumi n iscency test;and carry out said tests. -231i tienen una organización espacial relativa en relación entre ellos y tienen espec i. f ic idades de enlace diferentes para una plora 1 i dad de anal i. tos de interés diferentes en un ensayo de e1ec troquimicolumi n iscenci a;y llevar a cabo dichos ensayos. 62. A set of elements complies with claim 60, which further includes a display unit for carrying out said chases and tests. 62. Un conjunto de elementos da conformidad con la reivindicación 60 que incluye además un aparata para llevar a cabo di chas e nsay os. 63. A set of elements for use in performing a plurality of electrocyclolylaminescence assays for a plurality of analytes of interest comprising a container containing specific binding components for a plurality of different analytes for use in a plurality of electrochemiluminescence assays, 63. Un conjunto de elementos para su uso en el desempeño de una pluralidad de ensayos de electroquifliicolaminiscencia pa ra una plural idad de analitos de interés que comp rende i un recipiente que contiene componentes de enlace específicos pa ra una plura1 idad de analitos di ferentes para su uso en una plural idad de ensayos de electroquimicoluminiscencia, 64. An assembly of elements according to the claimed in claim 62, which also contains one or more containers that contain non-binding components for a plurality of electrochemical assays. niscenc i¿i. 64. Un conjunto de elementos de conformidad con lo reivindicado en la reivindicación 62 que contiene además uno o varios recipientes que contienen componentes que no son de enlace para una plura1 idad de ensayos de elec t roquimicolumi. niscenc i¿i. 65. A set of elements in accordance with claim 62 further containing an article comprising a plurality of discrete link domains on a carrier, said link domains have a relative spatial organization relative to each other and 65. Un conjunto de elementos de conformidad con la reí vindicación 62 que contiene además un artículo que comprende una pluralidad de dominios de enlace discretas en un soporte, dichos dominios de enlace tienen una organización espacial relativa en relación entre ellos y -2321 t i. Jan? n different speci fi cations of different links for a plurality of analysts of different interest in an essay on e 1 ec t roqu.i. ai icolum i niscenc ia. -2321 t i. ene? n unas espec i fieldades de enlace d i fe rentes para una plural idad de anal i tos de interés di ferentes en un ensaya de e 1 ec t roqu.i. ai icolum i niscenc i a . 66. A cassette comprising: 66. Un cassette que comprende: 5 (a) a plurality of discrete link domains on a support, which form at least one link surface;5 (a) una plural idad de dominios de enlace discretos en un soporte, que forman al menos una superficie de enlace;(b) a plurality of pairs of electrodes and counter electrodes, wherein said discrete link domains are spatially aligned with said plurality (b) una plura1 idad de pares de electrodos y contraelectrodos, en donde dichas dominios de enlace discretos están espacialmente alineados con dicha pluralidad 1O de pares de electrodos y contraelectrodos;y (c) un dispositivo para suministrar muestras en dicha pluralidad de dominios de enlace discretos. 1O pairs of electrodes and counter electrodes;and (c) a device for supplying samples in said plurality of discrete link domains. 67. A cassette comprising: 67. Un cassette que comprende: (a) a plurality of discrete link domains in one (a) una pluralidad de dominios de enlace discretos en un 15 soporte, que forman al menos una superficie de enlace;y <b) una pluralidad de pares de electrodos y contraelectrodos, en donde dichos dominios de enlace discretas están espacialmente alineados con dicha pluralidad de pares de electrodos y contraelectrodos y pueden acercarse fifteen support, which form at least one bonding surface;and <b) a plurality of pairs of electrodes and counter electrodes, wherein said discrete bonding domains are spatially aligned with said plurality of pairs of electrodes and counter electrodes and can be brought closer together. 20 a dicha pluralidad de pares de electrodos y contraelectrodos, donde dichos dominios de enlace son hidrofílicos o bien hidrofóbicos en relación a la superficie del soporte. twenty to said plurality of pairs of electrodes and counter electrodes, where said binding domains are hydrophilic or hydrophobic in relation to the surface of the support. 68. A cassette that I understood 68. Un cassette que comprendeí 25 (a) a plurality of discrete link domains in a 25 (a) una plural idad de dominios de enlace discretos en un -233ι -233ι de pares de electrodos y contraelectrodos, la pluralidad de electrodos se encuentra en di.cha superf ic te de enlace, cada electrodo adyacente a un dominio de enlace, y los contraelectrodos se encuentran en un segundo soparte;y (c) un dispos i ti vo para sumí nist rar muestras a di cha pluralidad de dominios de enlace discretos, of pairs of electrodes and counter electrodes, the plurality of electrodes is on the right bonding surface, each electrode adjacent to a bonding domain, and the counter electrodes are on a second part;and (c) a device for supplying samples to said plurality of discrete link domains, 69. The cassette according to claim 68 wherein said binding domains are hydrophilic or hydrophobic relative to the surface of the support. 69. El cassette de conformidad con la reívindicae ión 68 en donde dichos dominios de enlace son hidrofílicos o bien hidrofóbicos en relación a la superficie del soporte. 70. A cassette in accordance with claim 66, wherein said part contains from 2 to 70. Un cassette de conformidad con la reivindicaeión 66, donde dicho soparte contiene de 2 a 500 link domains, 500 dominios de enlace, 71. A cassette in accordance with re i. vi nd i falls ión 66, where said plurality of link domains comprises link domains that have different link speci fi icities to provide 'the simultaneous link of several years with different interests present in a sample' 71. Un cassette de conformidad con la re i. v i nd i cae ión 66, donde dicha pluralidad de dominios de enlace comprende dominios de enlace que t ienen especi f ic idades de enlace diferentes para proporcionar' el enlace simultáneo de varios ana 1 i tos de i nterés di ferentes presentes en una muestra» 72. A cassette for detecting or measuring electrochemiluminescence, comprising (a) a first support having a plurality of discrete binding domains therein;72. Un cassette para detectar o medir la electroquimicoluminiscencia, que comprendes (a) un primer soporte que tiene una pluralidad de dominios de enlace discretos ahí;-234i -234i 2. 3. 4 (b) a plurality of pairs of electrodes and counter electrodes, said plurality of? Discrete link domains are aligned with said plurality of pairs of electrodes and counter electrodes and in the vicinity of said plurality of pairs of electrodes and counter electrodes, said domains with all types of electrodes form a plurality of electrode measurements: t roquim i. Columns and scenarios for these elec tor rods / w hich brings 1 ec trods can be rigid by a source of electric energy: in the form of a voltage waveform. you are going to unleash the e1ec t roqui mi colum ini scenc ia < 234 (b) una pluralidad de pares de electrodos y contraelectrodos, dicha pluralidad de? dominios de enlace discretos se encuentra alineada con dicha pluralidad de pares de electrodos y contraelectrodos y en la cercanía de dicha pluralidad de pares de electrodos y contraelectrodos, dichos domi nios con t rae1ect rodos forman una pluna 1 idad p>ara medic iones de elec: t roquim i. columi ni scenc i a dichos pa res de elec t rod o s / c on t r a e 1 ec t rod os pueden ser d i rígidos por u na f u e π t e de energía eléc t ríe: a en forma de una forma de onda de vol ta je afee t i va pa ra desencadenar la e1ec t roqui m i colum i n i scenc i a < (c) a device for supplying samples over a plurality of discrete link domains. (c) un dispositivo para suministrar muestras sobre pluralidad de dominios de enlace discretos. 73. The cassette in accordance with claim 72 further comprising a second bracket capable of being placed adjacent to said first bracket to provide a device containing samples therebetween, where said p 1 ti ra 1 i dad of pairs of electrodes and counter electrodes is fixed on said second support. 73. El cassette de conformidad con la re i v i nd icac ión 72 que comprende además un segundo soporte capaz de colocarse de manera adyacente a dicho primer soporte para proporcionar un dispositivo que contiene muestras entre ellos, donde dicha p 1 tí ra 1 i dad de pa res de electrodos y contraelectrodos se fija sobre dicho segunda soporte. 74. The cassette according to claim wherein said plurality of pairs of electrodes and counter electrodes is fixed on said first support. 74. El cassette de conformidad con la reivindicación donde dicha pluralidad de pares de electrodos y contraelectrodos se f i ja sobre dicho primer sopor te. 75. The cassette according to claim 72 which 75. El cassette de conformidad con la reivindicación 72 que -235L -235L 235 it further comprises a second support capable of being placed adjacent to said first support to provide a device containing samples between them, where a plurality of electrodes are weighed on said first support and a plurality of counter electrodes are positioned in said second support, in such a way that said plurality of electrodes and counter electrodes can be positioned close to each other. 235 comprende además un segunda soporte capaz de colocarse de manera adyacente a dicho primer soporte para proporcionar un dispositivo que contiene muestras entre ellos, donde una plura1 idad de electrodos se pesie lona en dicho primer soporte y una plora 1 idad de contraelectrodos se pos ic iona en dicho segundo soporte, de tal manera que dicha plura1 idad de electrodos y contraelectrodos puede posicionarse cerca entre ellos. 76. The cassette in accordance with which said plurality of domains contains from 5 to 1000 linking domains 76. El cassette de conformidad con la donde dicha pluralidad de dominios de contiene de 5 a 1000 dominios de enlace El cassette de conformidad con la de dominio de comprende a 1 menos un dominio de enlace que contiene react i vos de enlace que son idénticos entre ellos y que difieren en cuanto a especificidad de los reactivos de enlace contenidas dentro de otros dominios de enlace, para proporcionar el enlace de múltiples ana litas de interés di ferentes la re ivindicao ión 72, donde dicha pluralidad de dominios comp rende a 1 menos un dominio de enlace que cont iene d i f ieren en cuanto a especificidad de enlace la rei v i ndicae i ón 78, que The domain domain cassette comprises 1 minus one binding domain containing binding reagents that are identical to each other and differ in specificity of the binding reagents contained within other binding domains, for provide the link of multiple analytes of interest other than claim 72, where said plurality of domains comprises 1 minus one link domain that contains, they differ in terms of link specificity, the rei vi ndicae ion 78, which -236i also includes a reflective surface adjacent to said first support or to said second support, -236i cciffiprende además una superf icié reflee tora adyacente a dicho primer soporte o bien a d icho segundo soporte, 80. The cassette in accordance with claim 78 wherein said first support and said second part, the plurality of discrete link domains and the plurality of counter-electrode electrodes are substantially transparent to each other where said pairs of electrodes and contain electrodes of diameter to width. 80. El cassette de conformidad con la reívindicación 78 donde dicho primer soporte y dicho segunda soparte, la 5 pluralidad de dominios de enlace discretos y la pluralidad de electrodos contraeleetrodos son substancia 1mente transparentes confo rm i dad coπ donde dichos pares de electrodos v cont raelee trodos de d i ámetro a a nchura. 82. The cassette according to claim 80, wherein said binding domains are within a size range of 0.001 to 10 mm in diameter or width, 82. El cassette de conformidad con la reivindicación 80, donde dichos dominios de enlace se encuentran dentro de un rango de tamaños de 0.001 a 10 mm de diámetro o anchura, 15 83. El cassette de conformidad con la reívindicación 72, donde dichos pares de electrodos y contraelectrodos pueden recibir energía de al menos una fuente de energía eléctrica en forma sepa rada. fifteen 83. The cassette according to claim 72, wherein said pairs of electrodes and counter electrodes can receive power from at least one source of electrical energy separately. 84. The cassette according to claim 72. 84. El cassette de conformidad can la reivindicación 72. 20 donde dichos dominios de enlace san hidrof11 icos y están rodeadas por una superficie hidrofóbica. twenty where said binding domains are hydrophobic and surrounded by a hydrophobic surface. 85. The cassette according to claim 72, wherein said binding domains are hydrophobic and surrounded by an hydrophilic surface. 85. El cassette de conformidad con la reivindicación 72, donde dichos dominios de enlace son h idrofób icos y están rodeados por una superf ic ie h idrof11ica. 25 66, The cassette in accordance with claim vindication 72, 25 66, El cassette de conformidad con la rei vindicaeión 72, -237I comprenden una monoc:apa autoensamblada, conf igurada -237I comprise a monoc: apa self-assembled, configured 87. The cassette in accordance with which said configured self-assembled monolayer comprises the candiols with which said binding domains can be linked to anates of the group consisting of nucleic acids, carbohydrate ions, antigens, cells and compounds, and organic organisms in accordance with the rei vi nd icae i ón 7G, where said link children contain functional reagents within consists of the initials, tests of which are my scenarios, Immunoassays and Nuclaic Acid Probe Assays 87. El cassette de conformidad con donde dicha monocapa autoensamblada conf igurada comprende al candióles con donde dichos dominios de enlace pueden enlazarse a ana Utos del grupo que consiste ácidos nucleicos, pare iones carbohidra to, ant1genos, células y compuestos o r g anometá1 i c os conformidad con la rei v i nd icae i ón 7G, donde dichos doro i ni os de enlace contienen react i vos fuñe ionales en dentro consiste de c1 Inicos, ensayos de qu i mi o1um i n i scenc i a, i nmunaensayos y ensayos con sondas de ác idos nuclaicos 90> El cassette de conformidad con la reí vindicarión 73, que comprende además una barrera de protección de electrodos remov ible i. nterpuesta entre d icho segundo soporte y dichos removerse antes de desencadenar d icha e1ec t roqui mi co1um i π i scenci a 90> The cassette in accordance with vindicarión 73, which also includes a removable electrode protection barrier i. Interposition between said second support and said removals before triggering said e1ec t roqui mi co1um i π i scenci a 91. The cassette according to claim 72, further comprising an electrically conductive material on 91. El cassette de conformidad con la reivindicación 72, que comprende además u.n material eléctricamente conductor sobre -238i -238i 238 a plurality of electrodes of said pairs of electrodes or adjacent to a plurality of electrodes of said pairs of electrodes, positioned to remove an electrical potential from the electrode surface in a test medium, 238 una pluralidad de electrodos de dichos pares de electrodos o bien adyacente a una pluralidad de electrodos de dichos pares de electrodos, posiclonado para alejar un potencial eléctrico de la superficie de electrodo en un medio de 5 ensayo, 92. An apparatus for measuring the electrochemistry of a sample, comprising: 92. Un aparato para medir la electroquimicoluaiiniscencia de una muestra, que comprende: (a) a plurality of cells to contain at least one sample, said plurality of cells is formed from a plurality of pairs of electrodes and counter electrodes and a first support comprising a plurality of discrete link domains, said plurality of domains discrete link lines align with the plurality of pairs of electrodes and counter electrodes and close to said plurality of pairs of 15 electrodes and counter electrodes, Said pairs of electrodes and counter electrodes can receive energy separately, these cells are suitable for carrying out measurements of the electro-imiscence, <b) a voltage control device adapted to (a) una pluralidad de celdas para contener al menos una muestra, dicha pluralidad de celdas se forma a partir de una 10 pluralidad de piares de electrodos y contraelectrodos y un primer soporte que comprende una pluralidad de dominios de enlace discretos, dicha pluralidad de dominios de enlace discretos se alinea con la pluralidad de pares de electrodos y contraelectrodos y cerca de dicha pluralidad de pares de 15 electrodos y contraelectrodos, dichos pares de electrodos y contraelectrodos pueden recib i r energ ía de manera sepa rada, dichas celdas son adecuadas para llevar a cabo mediciones de elec t roqu i m i co1um iniscencia, <b) un dispositivo de control de voltaje adaptado para 20 aplicar una forma de onda de voltaje controlada a dicha pluralidad de pares de electrodos y contraelectrodos, dicha forma de onda de voltaje es efectiva para desencadenar electroquimicoluminiscencia en dicha pluralidad de celdas, y Ce) un dispositivo detector de fotones para detectar la twenty applying a controlled voltage waveform to said plurality of pairs of electrodes and counter electrodes, said voltage waveform is effective to trigger electrochemiluminescence in said plurality of cells, and Ce) a photon detecting device to detect the 25 elec t roqu imi co1um i ni scenci a de dic ha mu t. 25 elec t roqu i m i co1um i ni scenci a de d i c ha mués t ra. -239ι -239ι 93 »A set of ele? mentosa de cuado to measure the e 1 ec t roqu imi co 1 um ini se ene i. a, comprising in one or more containers (a) a support, forming at least one connecting surface;and (b) a plurality of pairs of electrodes and counter electrodes spatially aligned with said plurality of discrete link domains and capable of approaching said plurality of discrete link domains. 93» Un c o n j u n t o de ele? m e n t o s a dec u a d o paira medir la e 1 ec t roqu i m i co 1 um i n i se ene i. a , que comp rende en uno o varios recip ientes (a ) una soporte, formando al menos una superficie de enlace;y (b) una plural idad de pares de electrodos y contraelectrodas espacia1mente a1 ineado con dicha pluralidad de dominios de enlace discretos y que pueden acercarse a dicha pluralidad de dominios de enlace discretos. 94. The assembly of elements in accordance with rei vi nd icae i ón 93, further comprising at least a second support capable of being able to be placed adjacent to the connection surface support to provide a sample. 94. El conjunto de elementos de conformidad con la rei v i nd icae i ón 93, que comprende además al menos un segundo soporte capaz de poder colocarse de manera adyacente soporte de superficie de enlace para proporcionar un muestras. 95. The set of elements in accordance with re iv indication 94, plurality of pairs of electrodes and cantraelectrodes capable of aligning discrete links and 95. El conjunto de elementos de conformidad con la re i v indicaeión 94, plura1 idad de pares de electrodos y cantraelectrodos capaces de poder alinearse enlace discretos y 96. The set of elements according to the iv ind i falls ion 93, to carry out an electrochemiluminescence assay on a predetermined amount of a purified analyte of interest. 96. El conjunto de elementos de conformidad con la re i v ind i cae ión 93, para 1 levar a cabo un ensayo de electroquimicoluminiscencia en una cantidad predeterminada de un analito de interés puri ficado. -240i -240i 240 240 97. A cassette for detecting or measuring an analyte of interest in a sample, comprising <a) a first support having a plurality of binding domains cl i to the surface thereof to form at least one surface of link, at least some of said discrete link domains have different link specificities than other link domains, each of said plurality of discrete link domains is f 97. Un cassette para detectar o medir un analito de interés en una muestra, que comprende <a) un primer soporte que tiene una pluralidad de dominios de enlace cl i se re tos en la superf ic ie del mismo para forma r al menos una superficie de enlace, al menos algunos de dichos dominios de enlace discretos tienen especificidades de enlace diferentes que otros dominios de enlace, cada uno de dicha pluralidad de dominios de enlace discretos es f h i d r o f í 1 ico y se e nc u entra r ocl ea d o por r e g i o ríe s hidrofóbicas, y (b) un segundo soporte que tiene una pluralidad de dominios hidrofílicos que comprende medios de reacción adecuados para llevar a cabo un ensaya químico ahí para formar una superficie de ensayo, dicha pluralidad de dominios de enlace discretos y dicha pluralidad de dichos medios de reacción pueden entrar en contacto de tal manera que una muestra a analizar que se encuentra presente en cada dominio de enlace está en contacta con un med io de reacc ión pa ra detecta r o med ir un analito de interés. hydrophilic and included by hydrophobic regions, and (b) a second support having a plurality of hydrophilic domains comprising suitable reaction means to carry out a chemical assay there to form a test surface, said plurality of discrete link domains and said plurality of said reaction means can come into contact in such a way that a sample to be analyzed that is present in each link domain is in contact with a reaction medium to detect ro med go an analyte of interest. 98- El cassette de conformidad con la reivindicación 72, que comp rende además un d ispos i ti vo de coπt ro1 de la tempera tura. 98- The cassette according to claim 72, which further comprises a temperature coπt ro1 device. 99. A cassette to carry out a reaction of interest, comprising: 99. Un cassette para llevar a cabo una reacción de interés, que comprende: -241¡ (a) a first support having a plurality of discrete domains on the surface of the support, each of said domains being hydrophilic and surrounded by a hydrophobic region on said first support surface;and -241¡ (a) un primer soporte que tiene una pluralidad de dominios discretos en la superficie del soporte, cada uno de dichos dominios es hídrofílico y se encuentra rodeado por una región hidrofóbica en dicha superficie de primer soporte;y 5 (b) a second support having a plurality of discrete domains on the surface of the second support, each of said domains (i) is hydrophilic and is surrounded by a hydrophobic region on said second support surface, (ii) comprises a medium reaction medium suitable for carrying out a reaction of interest, and ii) is spatially aligned with the domains on said first support surface such that said second support is located to bring each of said domains on said second support surface into contact with a domain aligned on said surface of first support. 5 (b) un segundo soporte que tiene una pluralidad de dominios discretos en la superficie del segundo soporte, cada uno de dichos dominios (i) es hídrofílico y está rodeado por una región hidrafóbica en dicha segunda superficie de soporte, (i i) comprende un medio de reacción adecuado para llevar a ID cabo una reacción de interés, y íi i i) se encuentra espacialmente alineado con los dominios en dicha superficie de primer soporte de tal manera que dicho segundo soporte esté ubicada para poner en contacto cada uno de dichos dominios en dicha superficie de segundo soporte con un 15 dominio alineado en dicha superficie de primer soporte. 100. The cassette according to claim 66 wherein said discrete link domains further comprise an internal control, 100. El cassette de conformidad con la reivindicación 66 en donde dichos dominios de enlace discretos comprenden además un control interno, 101. The apparatus according to claim 92 in 101. El aparato de conformidad con la re ivindicación 92 en 20 donde dicha pluralidad de dominios de enlace discretos incluye al menos dos dominios de enlace idéntico, twenty wherein said plurality of discrete link domains includes at least two identical link domains, 102. A bonding surface that is the product of the following process: 102. Una superficie de enlace que es el producto del siguiente proceso: (a) la formación de una monocapa autoensamblada en un (a) the formation of a self-assembled monolayer in a 25 support, said monolayer comprises a first linking group 25 soporte, dicha monocapa comprende un primer grupo de enlace -242ι -242ι A en la superficie de la monocapa no adyacente al soporte, dicho primer grupo de enlace A puede enlazarse específicamente sobre un segundo grupo de enlace 8, dicha monocapa se aplica al menos a un dominio en dicho soparte;y (b) poner en contacto dicho primer grupo de enlace A con un react i vo de enlace dicho reactivo de enlace puede en1a zarse con un analito de i nterés, d i cho reacti vo de enlace se enlaza con dicho grupo de enlace 8, de tal manera que dicho reactivo de enlace está enlazada con dicha monocapa por medio de un enlace A:B para formar una superficie de enlace, dicha superficie de enlace se organiza en forma de una pluralidad de dominios de enlace discretos que contienen dichos reactivos de enlace capaces de enlazarse con un analito de interés. A on the surface of the monolayer not adjacent to the support, said first linking group A can specifically bind on a second linking group 8, said monolayer applies to at least one domain in said support;and (b) contacting said first linking group A with a binding reagent said binding reagent can be linked with an analyte of interest, said binding reagent is linked to said linking group 8, of such that said binding reagent is linked to said monolayer via an A: B bond to form a binding surface, said binding surface is organized in the form of a plurality of discrete binding domains containing said binding reagents capable of binding to an analyte of interest. 103. An apparatus comprising the bonding surface of claim 102, 103. Un aparato que comprende la superficie de enlace de la reivindicación 102, 104. The cassette according to claim 106 wherein said support comprises an elastomeric material, 104. El cassette de conformidad con la reivindicación 106 en donde dicho soporte comprende un material elastomárico, 105. A process for the preparation of a plurality of eη 1 ac edi s domain s in a support, which comprises the steps of: 105. Un proceso para la preparación de una pluralidad de dom i n i os de eη 1 ac e d i sc re t os en u.n sopo r te , que comp r ende los pasos de: (a) formar una monocapa autoensamblada en un soporte, dicha monocapa comprende un primer grupo de enlace A en la superficie de la monocapa no adyacente al soporte, dicho primer grupo de enlace A puede enlazarse específicamente con (a) forming a self-assembled monolayer on a support, said monolayer comprises a first bonding group A on the surface of the monolayer not adjacent to the support, said first bonding group A can specifically bind to -243i ί1 a second linking group B, said monolayer is applied to at least one domain on said support;and (b) contacting said first linking group ñ with a binding reagent, said binding reagent can be linked to an analyte of interest, said binding reagent is linked to said binding group B, such that that said binding reagent is bound to said monolayer by means of an A: B bond to form a binding surface, said binding surface is organized as a plurality of discrete binding domains that it contains, ennea said binding reagents capable of binding on an analyte of interest -243i ί1 un segundo grupo de enlace B, dicha monocapa se aplica al menos a un dominio en dicho soporte;y (b) poner en contacto dicho primer grupo de enlace ñ con un reactivo de enlace, dicho reactivo de enlace puede enlazarse con un ana.lito de interés, dicho reactivo de enlace se encuentra enlazado con dicho gcupo de enlace B, de tal manera que dicho reactivo de enlace esté enlazado a dicha monocapa por medio de un enlace A:B para formar una superficie de enlace, d icha superf ic ie de enlace se organiza como una plura1 idad de dominios de enlace discretos que conti, enea d ichos reactivos de enlace capaces de enlazarse sobre un analito de interés 106. The process in accordance with rei vi nd icae i ón 105 where in step (a) said monolayer is produced in the form of a pattern on said support by a method selected from the group consisting of micro-attack and micro-coating 106. El proceso de conformidad con la rei v i nd icae i ón 105 donde en el paso (a) dicha monocapa se produce en forma de un patrón sob re dicho soporte mediante un método seleccionado dentro del grupo que consiste de microataque y microesLampado 1G7 1G7 El proceso de conformidad con la rei v ind icae i ón The process in accordance with rei v ind icae i ón 105 wherein said binding reagent is contained within 1 fragments derived therefrom, and nucleic acids and fragments and the same. 105 donde dicho reactivo de enlace se dentro de 1 fragmentos derivados de las mismas, y ácidos nucleicos y fragmentos y los mismos. IOS »The process of conforming to claim 105, wherein said binding reagent is selected from the IOS» El p roceso de con formi dad con la reivindicación 105, donde dicho reactivo de enlace se selecc iona dentro del -244ι group consisting of antibodies and binding fragments thereof, antigens and epitopes, cells and cellular components, enzymes, enzyme substrates, leetins, protein A, protein C3, organic compounds, organometallic compounds and carbohydrate portions. -244ι grupo que consiste de anticuerpos y fragmentos de enlace de los mismos, antígenos y epítopos, células y componentes celulares, enzimas, sustratos de enzimas, leetinas, proteína A, proteína C3, compuestos orgánicos, compuestos organometálicos y porciones de carbohidrato. 109. The process according to claim 105, wherein said binding reagent comprises a plurality of different binding reagents, and said contacting step is carried out by * supplying a plurality of fluid samples, each sample of fluid comprises a different binding reagent, on said monolayer to go from a multiple set of fluid guides, such that discrete binding domains on the binding surface in said monolayer have different binding reagents bound there. 109. El proceso de conformidad con la reivindicación 105, donde dicho reactivo de enlace comprende una pluralidad de reacti vos de enlace d i ferentes, y dicho paso de puesta en contacto se lleva a cabo mediante* el suministro de una pluralidad de muestras de fluido, cada muestra de fluido comprende un reactivo de enlace diferente, sobre dicha monocapa a pa rt i r de un con junto múltiple de guías de fluido, de tal manera que dominios de enlace discretos en la superficie de enlace en dicha monocapa tengan reactivos de enlace diferentes unidos ahí. 110. A method of detecting or measuring electrochemlin iscence, which comprises (a) contacting a plurality of discrete binding domains located on the surface of one or more supports with a sample comprising molecules bound to a luminescent electrochemical marker ;110. Un método detec tar o med ir la electroquimicolamín iscencia, que comprende (a) la puesta en contacto de una pluralidad de dominios de enlace discretos ubicados en una superficie de uno o varios soportes con una muestra que comprende moléculas unidas a un m a r c a d o r ele c t r o q u i m i c oluminiscente;<b) aplicar una forma de onda de voltaje efectiva para desencadenar la electroquimicoluminiscencia en cada uno de una pluralidad de pares de electrodos y contraelectrodos espacialmente alineados con dicha pluralidad de dominios de <b) applying an effective voltage waveform to trigger electrochemiluminescence on each of a plurality of pairs of electrodes and counter electrodes spatially aligned with said plurality of domains of -245ι discrete link and in the vicinity of said plurality of discrete link domains, and (c) detect or measure said elec t roqu imicolumi niscenc ia. -245ι enlace discretos y en la cercanía de dicha plural idad de dominios de enlace discretos, y (c) detectar o medir dicha elec t roqu imicolumi niscenc i.a . 111. The method according to claim 110, 111. El método de conformidad con la reivindicación 110, 5 where said plurality of discrete link domains is within a range of 5 to 1000 link domains. 5 donde dicha pluralidad de dominios de enlace discretos se encuentra dentro de un rango de 5 a 1000 dominios de enlace. 112. The method according to claim 110, wherein said plurality of discrete link domains comprises at least one link domain containing 112. El método de conformidad con la reivindicación 110, donde dicha pluralidad de dominios de enlace discretos comprende la menos un dominio de enlace que contiene
- 510 Binding reagents are identical to each other and differ in terms of specificity of the binding reagents contained within other binding domains, to provide binding of multiple different analytes of interest. 10 reactivos de enlace idénticos entre ellos y que difieren en cuanto a especificidad de los reactivos de enlace contenidos dentro de otros dominios de enlace, para proveer el enlace de múltiples analitos de interés diferentes. 113. The method in accordance with rei vi nd i falls:i ón 110, where said support comprises a reflecting surface. 113. El método de conformidad con la rei v i nd i cae: i ón 110, donde d icho soporte comprende una superficie refleetora. 114. The method according to claim 110, wherein said support, the bonding surface and the;Electrode pairs and counter electrodes are substantially transparent. 114. El método de conformidad con la reivindicación 110, donde d icho soporte, la superfic ie de enlace y los;pa res de electrodos y contraelect rodos son substañe iaImente transparentes. 20 115. El método de conformidad con la reivindicación 110, donde dichos dominios de enlace se encuentran dentro de un rango de tamaños de 0.001 a 10 mm de diámetro o anchura. twenty 115. The method according to claim 110, wherein said binding domains are within a size range of 0.001 to 10 mm in diameter or width. 116. The method according to claim 110, wherein said pairs of electrodes and counter electrodes can receive energy separately from at least one source of 116. El método de conformidad con la reivindicación 110, donde dichas pares de electrodos y contraelectrodos pueden 25 recibir energía de manera separada de al menos una fuente de -2461 electric power. -2461 energía eléctrica. 117. The method according to claim 110, wherein said binding domains are hydrophilic and surrounded by a hydrophobic surface. 117. El método de conformidad con la reivindicarión 110, donde dichos dominios de enlace son hidrofí1 icos y están rodeados por una superficie hidrof¿bica. 5 118. The method according to claim 110, wherein said binding domains are hydrophobic and surrounded by a hydrophilic surface » 5 118. El método de conformidad con la reivindicarión 110, donde dichos dominios de enlace son hidrofobicos y están rodeados por una superficie hidrofílica» 119. The method according to claim 110, wherein said binding domains are single-layered 119. El método de conformidad con 1.a reivindicación 110, donde dichos dominios de enlace son monorapas 10 self-assembled, configured, 10 autoensambladas, configuradas, 120. The method according to claim 119, wherein said binding domains can be specifically linked to an analyte selected from the group consisting of carbohydrate, cellular, protein, antibody, nucleic acid compounds, antigen moieties, cells and organic components, and compounds organometallic. 120. El método de conformidad con la reivindicaríón 119, donde dichos dominios de enlace pueden enlazarse espec1ficamente con un analito seleccionado dentro del grupo que consiste carbohidrato, celulares, de proteínas, anticuerpos, compuestos ácidos nucleicos, porciones de antígenos, células y componentes orgánicos, y compuestos organometálicos. 121. The method according to claim 110, wherein said method further comprises contacting 121. El método de conformidad con la reivindicarión 110, donde dichos método comprende además la puesta en contacto 20 de dichos dominios de enlace con un medio de reacción adecuado para llevar a cabo un ensayo seleccionado dentro del grupo que consiste de ensayos químico clínicos, inoiunaensayas así como ensayos de sondas de ácidos nucleicos, twenty of said binding domains with a suitable reaction medium to carry out an assay selected from the group consisting of clinical chemical assays, stainless steel assays as well as nucleic acid probe assays, 25 122. The method according to claim 110, 25 122. El método de conformidad con la reivindicarión 110, -247ι -247ι 247 where said support or rivers supports is a plurality of supports located in a cell and at least a part of said supports are substantially transparent to the light generated by electrochemiluminescence, each of said 247 donde d icho soporte o va ríos soportes es una plura1 idad de soportes ubicados en una pila y al menos una parte de dichos soportes son substanda 1 mente t ranspa rentes a la luz generada por electroquimicoluminiscencia, cada uno de dicha 5 plurality of supports is positioned adjacent to another of said supports. 5 plura1ldad de soportes se posiciona adyacente a otro de dichos soportes. 123. A method for detecting or measuring the e1ec t roqu im ico1umin i scenc ia in a sample, which involves) bringing one or more of a plurality into contact 123. Un método pa ra detoe tar o med ir la e1ec t roqu i m ico1umin i scenc i a en una mués t ra, que comp rende ía) la puesta en contacto de uno o varias de una pluralidad 10 of link domain ^ challenges, said plurality of link domains is located on a surface of one or more supports, with a sample comprising molecules linked to an electrochemical micro-luminescent marker, where a plurality of pairs of electrodes and counter electrodes 10 de dominio^ de enlace dise retos, dicha plural idad de dominios de enlace se ub ica en una superf ic ie de uno o varios soportes, con una muestra que comprende moléculas enlazadas a un marcador electroqu¿micoluminiscente, donde una pluralidad de pares de electrodos y contraelectrodos 15 están espaci a 1mente al i neados con dicha plura1 idad de dominios de enlace discretos, donde dicha pluralidad de pares de electrodos y contraelectrodos se encuentra presente en una superf ic ie de segundo soporte, (b) colocar dicha superficie de segunda soparte cerca de fifteen they are spatially aligned with said plurality of discrete link domains, where said plurality of pairs of electrodes and counter electrodes is present on a second support surface, (b) placing said second part surface near 20 dicha superficie de enlace de tal manera que cada uno de dichos pares de electrodos y contraelectrodos esté cerca de un dominio de enlace diferente, (c) aplicar una forma de onda de voltaje efectiva para desencadenar la electroquimicolumíniscencia en cada uno de twenty said bonding surface such that each of said pairs of electrodes and counter electrodes is close to a different bonding domain, (c) applying an effective voltage waveform to trigger electrochemiluminescence at each of 25 said plurality of pairs of electrodes and counter electrodes, 25 dicha pluralidad de pares de electrodos y contraelectrodos, -248248 <d ) detecta r □ med ir dicha electroquimicolumin iscencia. -248248 <d) detects r □ med ir said electrochemistry. 124. The method in accordance with rei vi nd icae i ón 110 where during step (a) said p l.ura 1 i dad of pairs of e1ect rodos and contraelect rodos is found by a removable protection barrier, and said The method further comprises reroating said barrier before applying said voltage waveform. 124. El método de conformidad con la rei vi nd icae i ón 110 donde durante el paso (a) dicha p l.ura 1 i dad de pa res de e1ect rodos y contraelect rodos se encuentra muestra por una barrera de protecc ión removible, y dicho método comprende además la rerooción de dicha barrera antes de aplicar dicha forma de onda de volta je. 125 n A method of detecting or measuring analytes of interest in a sample that involves (a) dropping of a sample containing an analyte to be detected or measured in a plurality of discrete binding domains on a surface of support, said plurality of discrete binding domains comprises at least one binding domain containing binding reagents that are identical to each other and differ in terms of the specificity of the binding reagents contained within other binding domains, each of said discrete binding domains is characterized by being either hydrophobic or hydrophilic, provided that the region of said support surface surrounding said binding domain is (i) hydrophobic if said binding domain is hydrophilic, and <ii) hydrophilic if said binding domain is hydrophobic, to allow said analyte or said anal i cough of interest 125 n Un método para detec tar o medir ana 1 i tos de interés en una muest ra que comp rende (a) la colocación de gotas de una muestra que contiene un analito a detectar o medir en una pluralidad de dominios de enlace discretos en una superficie de soporte, dicha plural idad de dominios de enlace discretos comprende al menos un dominio de enlace que contiene reactivos de enlace que son idénticos entre ellos y que difieren en cuanto a espec i fic idad de los react i vos de enlace contenidos dentro de otros dominios de enlace, cada uno de dichos dominios de enlace discretos se caracteriza por ser ya sea hidrofóbica o bien hidrofílico, a condición que la región de dicha superficie de soporte que rodea dicho dominio de enlace sea (i) hidrofóbica si dicho dominio de enlace es hidrofílico, y <i i) hidrofílica si dicho dominio de enlace es hidrofóbico, para permitir que dicho analito o dichos anal i tos de interés -249ι en la muestra se enlace <n) con dichos dominios de enlace, y -249ι in the sample link <n) with said link domains, and Cb) contacting a surface of a second discrete hydrophilic comprising suitable reaction means to carry out a chemical test there, and (c) determine the presence of said analytes of interest Cb) poner en contacto con una superficie de un segundo h idrofí1 icos discretos que comprenden medios de reacción adecuados para llevar a cabo un ensayo químico ahí, y (c) determinar la presencia de dichos analitos de interés 126 »A method of detecting or measuring analytes of interest 'in a sample, comprising ia) placing drops of a sample containing an analyte to be measured in a plurality of binding domains d hzha plura1ity of discrete binding domains comprises a1 me we are a binding domain that contains binding reagents that are identical to each other binding reagents contained within other binding domains, each one of said discrete binding domains is characterized by being either hydrophobic or hydrophilic, it surrounds each one of said binding domains, whether it is for said analyte or said analytes of interest 126» Un método para detectar o medir analitos de interés 'en una muestra, que comprende ía) colocar gotas de una muestra que contiene un analito a medir en una pluralidad de dominios de enlace d hzha plura1 idad de dominios de enlace discretos comprende a1 me nos un dominio de enlace que contiene reactivos de enlace que son idénticos entre ellos los react i vos de enlace contenidos dentro de otros dominios de enlace, cada uno de dichos dominios de enlace discretos caracteriza por ser ya sea hidrofóbico o bien hidrofílico, rodea cada uno de dichos dominios de enlace sea para perroit i r que d icho analito o dichos ana 1 i tos de interés -2501 -2501 250 (b) placing drops of a reaction medium in said sample drops;250 (b) colocar gotas de un medio de reacción en dichas gotas de muestra;(c) determinar la presencia de dichos analitos de interés unidos a dicho dominio de enlace. (c) determining the presence of said analytes of interest bound to said binding domain. 5 127. A method of detecting or measuring electrochemiluminescence in a sample that comprises the following steps in order is listed: 5 127. Un método para detectar o medir la electroquimicoluminiscencia en una muestra que comprende los siguientes pasos en el orden es tablee ido: (a) contacting a sample with a support surface, said surface contains a plurality of (a) poner en contacto una muestra con una superficie de r soporte, dicha superficie contiene una pluralidad de 10 discrete link domains, said link domains are spatially aligned with a plurality of pairs of electrodes and counter electrodes and may approach said plurality of pairs of electrodes and counter electrodes;10 dominios de enlace discretos, dichos dominios de enlace están espacialmente alineados con una pluralidad de pares de electrodos y contraelectrodos y pueden acercarse a dicha pluralidad de pares de electrodos y contraelectrodos;tb) acercar dichos dominios de enlace con dicha pluralidad tb) bringing said linking domains closer to said plurality 15 de pares de electrodos y contraelectrodos;fifteen of pairs of electrodes and counter electrodes;te) aplicar una forma de onda de voltaje efectiva para desencadenar electroquimicoluminiscencia;te) applying an effective voltage waveform to trigger electrochemiluminescence;d) detect or measure electrochemiluminescence. d) detectar o medir la electroquimicoluminiscencia. 128. A method of detecting or measuring 128. Un método para detectar o medir la 20 electroquimicoluminiscencia en una muestra que comprende;twenty electrochemiluminescence in a sample comprising;(a) contacting a sample with a surface of a support, said surface contains a plurality of discrete link domains;(a) poner en contacto una muestra con una superficie de un soporte, dicha superf icie contiene una plural idad de dominios de enlace discretos;(b) explorar un par de electrodos y contraelectrodos sobre (b) explore a pair of electrodes and counter electrodes on 25 the surface of said soparte in the vicinity of said 25 la superficie de dicho soparte en la cercanía de dichos -251ι dominios de enlace, mientras se api ica una forma de onda de voltaje efectiva para desencadenar la el ec t roquimicoluminiscencia;y (c) detectar o medir la elec:t roquimicoluminiscenc ia. -251ι link domains, while an effective voltage waveform is applied to trigger the electrochemical illumination;and (c) detect or measure the electrochemical luminescence. 5 129. The process in accordance with claim 109 in which each of said domains is prepared in such a way that it is spatially aligned with and close to said plurality of pairs of electrodes and counter electrodes t 5 129. El procesa de conformidad con la rei v i ndicac i ón 109 en el cual cada uno de dichos dominios se prepara de tal manera que esté espacialmente alineado con una pluralidad de pares de electrodos y contraelectrodos y cerca de dicha pluralidad t de fiares de electrodos y contraelectrodos, of electrode and counter electrode trusses, 10 130. A method of detecting or measuring an analyte of interest in a sample, comprising. * (A) contacting a sample with a surface of a support, said surface containing a plurality of discrete binding domains, said link domains 10 130. Un método para detectar o medir un ana 1 i to de interés en una muestra, que comprende.* (a) poner en contacto una muestra con una superficie de un soporte, dicha superficie contiene una pluralidad de dominios de enlace discretos, dichos dominios de enlace 15 están espaciaImente alineados con una pluralidad de pares de electrodos y contraelectrodos y pueden acercarse a dicha pluralidad de pares de electrodos y contraelectrodos, dichos dominios de enlace (i) contienen un marcador e 1 ectroquimic.oluminiscente, y (i. i) pueden enlazarse con un ana 1 i to de i nterés en donde dicho contacto se realiza en cond ic iones tales que puede ocurrir el enlace de cualquier analito en la muestra con dichos dominios de enlace;fifteen They are spatially aligned with a plurality of pairs of electrodes and counter electrodes and can approach said plurality of pairs of electrodes and counter electrodes, said binding domains (i) contain an e- electrochemical luminescent marker , and (i. i) can be linked to one year of interest where such contact is made under conditions such that the binding of any analyte in the sample with said binding domains may occur;(b) acercar dichos dominios de enlace a dicha pluralidad de pares de electrodos y contraelectrodos;(b) bringing said link domains closer to said plurality of pairs of electrodes and counter electrodes;25 (c) apply an effective voltage waveform to 25 (c) aplicar una forma de onda de voltaje efectiva para -252 triggers the elec t roqu im icol um in i. scenc ia;-252desencadena r la elec t roqu i m icol um i n i. scenc i a ;<d) detect to measure electrochemiluminescence, in <d) detectar a medir la electroquimicoluminiscencia, en it is carried out in the absence of any analyte of interest in the sample, indicates the review or the quantity of said analyte in the sample, se lleva a cabo en ausencia de cualquier analito de interés en la muestra, indica la p reseñe ia o la cantídad de d icho analito en la muestra, 131 »A method of detecting or measuring an analyte of interest in a sample, comprising;131» Un método para detectar o medir un analito de interés en una muestra, que comprenden;Ca) la puesta en contacto de una muestra con una superficie de un soporte, dicha superficie contiene una pluralidad de dominios de enlace discretos, dichos dominios de enlace se encuentran espacialmente alineados con una pluralidad de pares de electrodos y contraelectrodos y pueden acercarse a dicha pluralidad de pares de electrodos y contraelectrodos, dichos dominios de enlace pueden enlazar un analito de Ca) the contacting of a sample with a surface of a support, said surface contains a plurality of discrete link domains, said link domains are spatially aligned with a plurality of pairs of electrodes and counter electrodes and can approach said plurality of pairs of electrodes and counter electrodes, such binding domains can bind an analyte of enlazadas con un marcador electroqui.micolumi niscente;linked with a niscent electroqui.micolumi marker;pairs of electrodes and counter electrodes;pares de electrodos y contraelectrodos;<c) aplicar una forma de onda de voltaje efectiva para <c) apply an effective voltage waveform to -253ι desencadenar la electroquim icoluminiscencia,' y (d) detectar a medir la electroquimicoluminiscencia, en donde un incremento de la electroquimícoluminiscencia en campa ración con niveles de fondo i nd i.ca la preseñe i. a □ la 5 cantidad del ana lito en la muestra. -253ι trigger the electrochemiluminescence, 'and (d) detect to measure the electrochemiluminescence, where an increase in the electrochemiluminescence in field with background levels i nd i. a □ the 5th quantity of the analyte in the sample. 132. A method of detecting or measuring an analyte of interest in a sample, comprising: 132. Un método para detectar o medir un analito de interés en una muést ra, que comprende: (a) la puesta en coritac to una muestra con una superf ic ie de un soporte, dicha superficie contiene una pluralidad de (a) the coritac to a sample with a surface of a support, said surface contains a plurality of 10 Discrete link domains, said link domains are spatially 1 to 1 lines with a plurality of pairs of electrodes and counter electrodes and can approach said plurality of pairs of electrodes and counter electrodes, said domi. Link children cannot link a year 1 to 10 dominios de enlace discretos, dichos dominios de enlace se encuentran espacia1 mente a 1 ineados con una plura1 idad de pares de electrodos y contraelectrodos y pueden acercarse a dicha pluralidad de pares de electrodos y contraelectrodos, dichos domi. ni os de enlace pueden enlazar un ana 1 i to de 15 interés, en donde dicha puesta en contacto se real iza en condiciones tales que puede ocurrir el enlace de cualquier analito en la muestra con dichos dominios de enlace;fifteen interest, where said contacting is carried out under conditions such that the binding of any analyte in the sample with said binding domains can occur;(b) contacting said link domains with a link partner of the analyte of interest, where said link partner (b) poner en contacto dichos dominios de enlace con un socio de enlace del analito de interés, en donde dicho socio de 20 enlace se encuentra unido a un marcador elect roqu. im i co1um i n i scente;twenty link is attached to an elect roqu marker. im i co1um ini scente;(c) acercar dichos dominios de enlace a dicha pluralidad de pares de electrodos y contrael.eetrodos;(c) bringing said link domains closer to said plurality of pairs of electrodes and counter electrodes;(d) aplicar una forma de onda de voltaje efectiva para (d) apply an effective voltage waveform to 25 trigger electrochemiluminescence;and 25 desencadenar la electroquimicoluminiscencia;y -254i (e) detect or measure electrochemiluminescence, where an inc remen t of the ele ct roqu. im icol um ini so e nc ia in compared to background levels indicates the presence or analyte in the sample -254i (e) detectar o medir la electroquimicoluminiscencia, d o nd e u n inc remen t o de la ele c t roqu. i m icol um i n i so e nc i a en en comparae ión con las niveles de fondo indica la presencia o analito en la muestra 133. The method where steps (a) and (b) are carried out 133. El método donde los pasos (a) y (b) se llevan a cabo de 131, en manera ocurrente. 131, in a witty way. 131, en donde el paso (a) se lleva a cabo antes que el paso (b);y el lavado de los;dominios de enlace para remover el analito no enlazado se 1 leva a cabo después del paso (a) y antes del paso <b). 131, wherein step (a) is carried out before step (b);and the washing of the;Binding domains to remove the unbound analyte were carried out after step (a) and before step <b). 135. A method of detecting or measuring an analyte of interest in a sample, comprising: 135. Un método para detectar o medir un analito de interés en una muestra, que comprende: (a) contacting a first sample with a surface of a support, said surface contains a plurality of discrete bonding domains, said bonding domains are spatially 1 to 1 lined with a plurality of pairs of electrodes and counter electrodes and they can approach said p 1 urality of pa res cle e 1 ec: t rodos y cont rae 1 ec i rodos, where said sample contains an analyte of interest linked to an electrochemiluminescent marker, where said contacting is carried out under conditions such that said analyte in the sample can bind to said binding domains: (a) poner en contacto una primera muestra con una superficie de un soporte, dicha superficie contiene una pluralidad de dominios de enlace discretos, dichos dominios de enlace están espac ia 1mente a 1 i neados can una plura1 idad de pa res de electrodos y contraelectrodos y pueden acercarse a dicha p 1 urali dad de pa res cle e 1 ec: t rodos y cont rae 1 ec i rodos , en donde dicha muestra contiene un analito de interés enlazado a un marcador electraquimicoluminiscente, en donde dicha puesta en contacto se realiza en condiciones tales que dicho analito en la muestra puede enlazarse con dichos dominios de enlace: -255i <b) contacting a second sample with said binding domains, under conditions such that the binding of any analyte in said second sample with said binding domains may occur, in which an electrochemical-fluorescent marker is not linked to any analyte in said second sample;-255i <b) poner en contacto una segunda muestra con dichos dominios de enlace, bajo condiciones tales que puede ocurri r el enlace de cualquier analito en dicha segunda muestra con dichos dominios de enlace, en donde un marcador electroquimicoluaiiniscente no se encuentra enlazado a ningún analito en dicha segunda muestra;(c) acercar d ichos dom i nios de enlace a dicha plura1 idad de pares de electrodos y contraelectrodos;(c) bringing said link domains closer to said plurality of pairs of electrodes and counter electrodes;td) aplicar una forma de onda de volta je efect i va para íe) detectar o mecí i r la electroquimicoluffiiniscenci a, en donde una disminución de la electroqui mi columi ni seene ia en comparación con a la electroquimicolumiπiscencia observada cuando se omite el paso (b) o bien cuando no hay analito en la segunda muestra indica la preseñe ia o cantidad del analito en la muestra. td) apply a waveform of effective voltage to i) detect or rock the electrochemical dissolution, where a decrease in the electrochromy my column does not appear compared to the electrochemistry observed when the step is omitted (b ) or when there is no analyte in the second sample, it indicates the presence or quantity of the analyte in the sample. 136. The method according to claim 131 wherein the sample is derived from a mammal, and wherein said method is employed to determine or confirm the identity of the mammal. 136. El método de conformidad con la reivindicación 131 en donde la muestra se deriva de un mamífero, y en donde dicho método se emplea piara determinar o confirmar la identidad del mamífero. 137. The one where the sample contains method cells is used to quantify the one mammal, and said number of cells in said sample. 137. El donde la muestra contiene células de método se emplea para cuantificar el un mamífero, y dicho número de cé1u1as en dicha muestra. 138. The method of carrying out a reaction of interest, 138. El método para 11evar a cabo una reacción de i nterés, -256ι comprising: -256ι que comprende: Ca) apiica r a sample to each of a plurality of domains of d iserets on the surface of a first support, each of these domains is hydrophilic, and is 5 surrounded by a hydrophobic region in said surface first support;and (b) place a second support that has a plurality of discrete domains on the surface of the second support, each of said domains (i) is hydrophobic and is surrounded by an hydrophobic region in said second support surface, (ii) comprises a reaction medium suitable for carrying out a reaction of interest, iii) is spatially 1 to 1 lined with the supported domains, to put each one of said domains in said second domain surface to 1 in line on ta 1 so that said sample is in contact with said reaction medium Ca) apiica r una muestra a cada una de una plura1 idad de dominios de d iseretos en la superficio de un primer soporte, cada uno de dichos dominios es hidrofílico, y se encuentra 5 rodeado por una reg ión hidroí óbica en dicha superf ic ie de primer soporte;y (b) colocar un segundo soporte que tiene una pluralidad de dominios discretos en la superf ic ie del segundo soporte, cada uno de d i chos dominios ( i ) es hidrof11 ico y se 10 encuentra rodeado por una región h idrof¿bica en dicha superficie de segundo soporte, (ii) comprende un medio de reacción adecuado pa ra llevar a cabo una reacción de interés, í i i i) se encuentra espac ia 1mente a 1 i neado con los domi nios en soporte, pa ra poner cada uno de dichos dominios en dicha superf ic ie de segundo dominio a 1 i neado en d i cha de ta 1 manera que dicha muestra esté en contacto con dicho medio de reacción 139. The method according to claim 109 in 139. El método de conformidad con la reivindicación 109 en 20 donde dicha pluralidad de muestras de fluido se suministra concurrentemente en d icha monocapa. twenty where said plurality of fluid samples are supplied concurrently in said monolayer. 140. The method according to claim 110 wherein said support comprises an elastomeric material 1. 140. El método de conformidad con la re ivindicación 110 donde dicho soporte comprende un materia 1 elastoméríco. 141 »The method pursuant to revision iv 119 141» El método de conformidad con la re i v i nd icaeión 119 25 where said self-assembled monolayer, configured comprises 25 donde dicha monocapa autoensamblada, configurada comprende -257ι alcantioles . -257ι alcantiols. 142. A method for detecting or measuring a year of interest that you understand (a) bringing into contact one or more linking domains 142. Un método pa ra detecta r o med i r un ana 1 i to de i nterés que comprendes (a) poner en contacto uno o varios dominios de enlace 5 Discretes of a plurality 1 dity of domains of? link of rights, said plurality of link domains is found on a surface of one or more supports, where said contacting is performed with a sample comprising molecules linked to a marker 5 discretos de una plura 1 i dad de dominios de? enlace d i se re tos, dicha plural idad de dominios de enlace se encuentra en una superficie de uno o varios soportes, en donde dicha puesta en contacto se realiza con una muestra que comprende moléculas enlazadas a un marcador 10 electro-chemiluminescent, where said sample is not in contact with π electrodes nor with π t rae1ec t rad os during said contacting step;10 electroquimicoluminiscente, donde dicha muestra no está en canta cto co π electrodos ni c o π t rae1ec t rad os du r a n t e dicho paso de puesta en contacto;(b) acercar un electrodo a uno o varios dominios de enlace a dicha pluralidad de dominios de enlace. (b) bringing an electrode closer to one or more link domains to said plurality of link domains. (c ) apiicar una forma de onda de volta je efectiva para desencadenar e1ec t roqu i m i co1um i n i scenc i a en uno o varios dominios de enlace de dicha pluralidad de dominios de enlace;y <d) detectar o med i r la e1ectroqui m i c o1umi nise ene i a que comprende ía) poner en contacto uno o varios dominios de enlace discretos, de una pluralidad de dominios de enlace, dicha pluralidad de dominios de enlace (i) se encuentra ubicada en (c) applying a voltage waveform effective to trigger the rocket imi co1um ini scenc ia in one or more link domains of said plurality of link domains;and <d) detecting or measuring the microelectronic microscope that comprises ia) contacting one or more discrete link domains, of a plurality of link domains, said plurality of link domains (i) is located in 25 a surface of one or more supports, and iii) is 25 una superficie de uno o varios soportes, y íii) está -258258 espacia1 mente a 1 i neada con una p lura1 idad depa res de electrodos y contraelectrodos y cerca de dicha p lura1 idad de pa res de electrodos y contraeleetrodos, en donde dicha puesta en realiza con una muestra que comprende moléculas enlazadas un ma rcado τun electrodo y contraelectrodo a uno o varios dominios de enlace de dicha plural idad de dora i n ios de enlace;-258258 spaced at 1 line with a plurality of electrodes and counter electrodes and close to said plurality of pairs of electrodes and counter electrodes, where said implementation is performed with a sample comprising linked molecules a τ an electrode and counter electrode to one or more link domains of said plurality of dora link ios;c) apply trigger link;and (d) detect a voltage wave e 1 ec t roqu im i.co 1 um i π iscenc ia in one or more links or med i. r íc ) aplicar desencadenar enlace;y (d) detectar de onda de voltaje e 1 ec t roqu i m i.co 1 um i π iscenc i a en uno o varios enlace o med i. r 144. A cassette that of the e1ect roqu imi colum ini scenc ia comprises í (a) an electrode containing on its surface a predetermined quantity of a material 1 comprising organized in a discrete link domain or in various link link domains These discrete reagents can be specifically linked to a molecule that 144. Un cassette que de la e1ect roqu i m i colum i n i scenc i a comprende í (a) un electrodo que contiene en su superficie una cantidad predeterminada de un mater ia 1 que comprende organizados en un dominio de enlace discreto o de en va ríos enlace dominios de enlace discretos, dichos react i vos de pueden enlazarse especi f icamente con una molécula que
- 611eva a marker elee troqu i πiscent binding micolumi of a molecule that e1ect roq uimi co1um i π i scen te, and said transfer of electrons between 11eva un marcador elee troqu i micolumi πiscente de enlace de una molécula que e1ect roq u i m i co1um i π i scen t e, y di cho transferencia de electrones entre 1 leva material o bien un socio un ma rcador ( i ) perra i te una dicho electrodo y un con one cam material or a partner a brand (i) bitch and I said one electrode and a con -259259 iscent electrochemical cyclic marker attached to said binding reagent or to said binding satia when said sacia is linked to said binding reagent or (ii) allowing said electrode to generate photons from said electrochemiluminescent marker; and (b) a counter electrode. -259259 marcador electroqciimicolumiπ iscente unida a dicho reactiva de enlace o bien a dicha sacia de enlace cuando dicho sacio se encuentra enlazada a dicho reactivo de enlace o bien (ii) permitir que dicho electrodo genere fotones a partir de dicho marcador electroquimicoluminiscente; y (b) un contraelectrodo. 145 145 A cassette comprising an electrode containing on its surface a predetermined amount of a material comprising binding reagents organized in one or more discrete binding domains, said binding reagents being electrically chemiluminescently labeled, and said material allows the transfer of electrons between said electrode and said electrochemiluminescent marker. Un cassette que comprende un electrodo que contiene en su superficie una cantidad predeterminada de un material que comprende reactivas de enlace organizados en uno o varios dominios de enlace discretos, dichos reactivos de enlace se encuentran marcadas de manera electraquimiculuminiscente, y dicho material permite la transferencia de electrones entre dicho electrodo y dicho marcador electroquimicoluminiscente. 146. The cassette according to claim 143 wherein said electrode is porous. 146. El cassette de conformidad con la reivindicación 143 en donde dicho electrodo es poroso. 147. The cassette in accordance with claim 144 wherein said electrode is porous. 147. El cassette de conformidad con la reívindicación 144 en donde dicho electrodo es poroso. 148. A cassette comprising:148. Un cassette que comprende: a) a support that has one or more discrete binding domains that contain binding reagents? ía) un suporte que tiene una o varios dominios de enlace discretos que contienen reactivos de enlace? (b) an ionically permeable porous matrix, said binding domain is linked to said porous matrix;(b) una matriz porosa iónicamente permeable, dicho dominio de enlace se encuentra enlazado con dicha matriz porosa;(c) an electrode in electrochemical contact with said binding domain;and <d) a counter electrode. (c) un electrodo en contacto electroquímico con dicho dominio de enlace;y <d) un contraelectrodo. -260i -260i 149. A cassette in accordance with claim 147 where the eπ 1ace reagents are marked for elect rochimicholuminescence. 149. Un cassette de conformidad con la reívindicación 147 en donde los reac ti vos de eπ 1ace están marcados para elect roquimicoluminiscencia. 150. A cassette comprising: 150. Un cassette que comprende: (a) a binding domain containing binding reagents;and (b) a porous support, said link domain is linked to said porous support, said porous support is an e1ect all in contact link, (a) un dominio de enlace que contiene reactivos de enlace;y (b) un soporte poroso, dicho dominio de enlace se encuentra enlazado a d i cho soporte poroso, d icho soporte poraso es un e1ect rodo en contac enlace, 151. The cassette of which further comprises 151. El cassette de que comprende además 152. The cassette of;□ elect roqufmi co conforming to the 152. El cassette de ;□ elect roqufmi co co conformidad con la (.. (n with t rae 1 ec t rodo. (..(n con t rae 1 ec t rodo. I agree η 1 to said re ivi nd ic ation domain 149 rei vi nd icat ion 150 where said binding reagents are covalently linked with said electrode, c o n f o r m i d a d c o η 1 a dicho domin io de re i v i nd i c ación 149 rei v i nd icac i ón 150 donde dichos reactivos de enlace están enlazados covalentemente con dicho electrodo, 153 .. A cassette comprising: 153.. Un cassette que comprende: (a) a first support having a first surface containing an electrode;and ib) a second support that has a second surface that contains a binding domain, said first surface and said second surface are spatially 1 to 1 i, negated for aatab 1 ec: er> .. > π elec troqj contact λ í mic o. (a) un primer soporte que tiene una primera superficie que contiene un electrodo;y íb) un segundo soporte que tiene una segunda superficie que contlene un dominio de enlace, dicha primera superf ic ie y d i cha segunda super f ic i e están esípac i.a 1 mente a 1 i, neadas pa ra a a t a b 1 e c: e r >.. > π c o n t a c t o elec t r o q j λ í m i c o.
- 715 4 . U n c o π ¿ u. n t o de elementos q ue c om prendas <a) un primer soporte que tiene una primera superficie que c o π t i e ne un el ec:t r od o;fifteen Four . U nco π ¿u. nt of elements that contain garments <a) a first support that has a first surface that connects to the ec: tr od o;(b) a second support having a second surface that (b) un segundo soporte que tiene una segunda superficie que -261i -261i 261 it contains a binding domain, said binding domain contains a predetermined amount of binding reagents, said first surface and said second surface are spatially aligned to establish an electronic contact;and (c) a bottle containing a species marked for elect roqu im ico 1 um i π i, seenc ia. 261 contiene un dominio ele enlace, dicho dominio de enlace contiene una cantidad predeterminada de reactivos de enlace, dicha primera superficie y dicha segunda superficie están espacialmente alineadas para establecer un contacto e 1 ec t roq u 1 m i c o;y (c) frasco que contiene una especie marcada para elec t roqu im ico 1 um i π i,seenc i a . 155. A casse11e comprising;155. Un casse11e que comprende;ia) a first support that has a first surface that co π has an elec: t reid;and (b) a second support having a second surface containing one or more discrete binding domains, said first surface and said second surface being spatially aligned to allow transfer of electrons between an electrochemical luminescent marker associated with the binding domain and the electrode. ía) un primer soporte que tiene una primera superficie que c o π t i e n e un elec: t reíd o;y (b) un segundo soporte que tiene una segunda superficie que contiene uno o varios dominios de enlace discretos, dicha primera superficie y dicha segunda superficie están alineadas espacialmente piara permitir la transferencia de electrones entre un marcador electroquimicoluminiscente asociado con el dominio de enlace y el electrodo. 156. The cassette according to claim 154, wherein the second surface is a porous material, and said cassette further comprises a counter electrode. 156. El cassette de conformidad con la reivindicación 154, donde la segunda superficie es un material poroso, y dicho cassette comprende ademés un contraelectrodo. 157. A c se11 e qu ec omp re nde: 157. Un c as se11 e qu e c omp re nde: (a) an electrode having a first surface;(a) un electrodo que tiene una primera'súperficie;(b) a counter electrode having a second surface;(b) un contraelectrodo que tiene una segunda superficie;(c) a porous material between said first surface and said second surface, said porous material contains binding reagents that can bind a labeled molecule (c) un material poroso entre dicha primera superficie y dicha segunda superficie, dicho material poroso contiene reactivos de enlace que pueden enlazar una molécula marcada -262i dicha primera superficie están espacialmente alineadas para electrones entre un marcador e1ect roqu i m i co1um i n i seeπ t e en una molécula varios de dichos reactivos de enlace y dicha primera sup erficie. -262i said first surface are spatially aligned for electrons between an e1ect roqu imi co1um ini seeπ te in a molecule several of said binding reagents and said first surface. (A) a porous carbon dioxide;(¿a) un a1eci rodo poroso;(b) a co πt ra e1ect rodo;(b) un c o πt ra e1ect rodo;(c) soporte que t iene una p luralidad d i se retos ahí, di chos domi n i os espacia 1mente a 1 i neados pa ra permit i r la transferenci a de entre una para electroquimicoluminiscencia, enlazada sobre el dominio de enlace y dicho electrodo poroso (c) support that has a plurality of challenges there, said domains spaced 1 to 1 lines to allow the transfer between one for electrochemiluminescence, linked over the link domain and said porous electrode 159. A cassette comprising (b) a counter electrode;Said porous electrode contains on its surface a material that includes binding reagents, such as the transfer of electrons between one or several days. 159. Un cassette que comprende (b) un contraelectrodo;dicho electrodo poroso contiene en su superficie un material que comprende reactivos de enlace, d i cho la transferencia de electrones entre un ido uno o va rios da 160. A cassette that (a) a support that? 160. Un cassette que (a) un soporte que? -2631 -2631 263 (b) a compartment for transporting fluid to said linking domains and out of said linking domains;and (c) an e]. ect red o y co π brings 1 ec tr odo esp a cia lmente to 1 line two to generate an electrochemiluminescent signal from 263 (b) un compartimiento para transporte de fluido hacia dichos dominios de enlace y fuera de dichos dominios de enlace;y (c ) un e ]. e c t red o y co π trae 1 ec t r odo esp ac i a lmente a 1 i nea dos para generar una señal electroquimicoluminiscente a partir 5 of an electrochemical-luminescent marker attached to said plurality of binding domains. 5 de un marcador electroquimicaluminiscente unido a dicha pluralidad de dominios de enlace. 161. A cassette comprising: 161. Un cassette que comprende: (a> an electrode »that contains on its surface a tp 1 ura 1 ity of my π i os dee η 1 aced i. scr * et os;and (a> un electrodo» que contiene en su. superficie una t p 1 u r a 1 i d a d d e d o m i π i os d e e η 1 a c e d i. s c r* e t os;y 10 (b) a counter 1 electrode. 10 (b) un co n t r a e 1ectrodo. 1 62 . U n c a s se 11 e que c omp rende: one 62. O ncas 11 e que c omp rende: (a) a plurality of discrete link domains on a support;(a) una pluralidad de dominios de enlace discretos en un soporte;<b) an electrode;and <b) un electrodo;y 15 (c) un contraelectrodo, dicho electrodo puede generar una pluralidad de señales electroqu.imicolu.miniscentes a partir de una pluralidad de marcadores electroquimicoluminiscentes enlazados con dicha pluralidad de dominios de enlace discretos. fifteen (c) a counter electrode, said electrode may generate a plurality of electrolysis miniscent signals from a plurality of electrochemiluminescence markers linked to said plurality of discrete binding domains. 20 163. El cassette de la reivindicación 161 en donde dicha p 1 u. r a 1 i d a d d e d o m i n i. o s de e η 1 a c e c α n t i e ne u n m a r c a d o r e 1 ec t r c»q u i m i c o 1 um i n i sce n t e, y do ocle e 1 ca ese 11 e co n t i e ne además un dispositivo para suministrar muestras en dicha pluralidad de dominios de enlace discretas. twenty 163. The cassette of claim 161 wherein said p 1 u. ra 1 ity of domain i. os of η 1 acec α ntie ne unmarkerre 1 ec trc »chemical 1 um ini sce nte, and do ocle e 1 ca that 11 e also contains a device to supply samples in said plurality of discrete link domains. 25 164. The cassette of claim 161 comprising 25 164. El cassette de la reivindicación 161 que comprende -264264 además un dispositivo para suministrar muestras en dicha pluralidad de dominios de enlace discretos. -264264 further a device for supplying samples in said plurality of discrete link domains. 165 . The re ivi nd cassette i. Ion 160 falls where said electrode can generate an electrochemical luminescent signal from an electrochemical luminescent marker associated with 1 minus one of said binding domains. 165 . El cassette de la re i v i nd i. cae ión 160 en donde d icho electrodo puede generar una señal electroquimicoluminiscente a partir de un marcador electroquimicoluminiscente asociado con a 1 menos uno de dichas domi nios de enlace. 166. A ca ss et te that includes: 166. Un ca ss et te que comprende: (a) a support having a plurality of discrete link domains there;and (b / one or more pairs of electrodes and counter electrodes. (a) un soporte que tiene una pluralidad de dominios de enlace discretos ahí;y (b/ uno o varios pares de electrodos y contraelectrodos. 167. The cassette of claim 165 further comprising a device for supplying samples in said plurality of discrete link domains. 167. El cassette de la reiv i ndicae i ón 165 que comprende además un dispositivo para suministrar muestras en dicha plura1 idad de dominios de enlace d iseretos.
- 816/8. Un con junto de elementos que comp rende:8/16. A set of elements that comprise: (a> an electrode containing on its surface ie discrete binding domains organized in a configuration;(a> un electrodo que contiene en su superfic ie dominios de enlace discretos organizados en una configuración;(b) a compartment for transporting fluid to said link domains and out of said link domains;(b) un compartimiento para transportar fluido hacia dichos dominios de enlace y fuera de dichos dominios de enlace;(c) a counter electrode;and (d) a bottle that contains mo 1 éc u the marks for the ctroqu i. me. co 1 uminisce nc ia „ (c) un contraelectrodo;y (d ) u n f r a s c o qu.e c o n t i e ne mo 1 éc u las ma r c a d a s para ele c t r o q u i. m i. c o 1 u m i n i s c e nc i a „ 169. A cassette comprising: 169. Un cassette que comprende: (a) a length of discrete link domains linked to an electrode, said electrode is close to an ionically permeable porous matrix;and (a) una plora 1 idad de dominios de enlace discretas enlazados a un electrodo, dicho electrodo se encuentra cercano a una matri z porosa i ónicamente permeable;y -265I -265I C ti) a c. ci ntrae 1. ec trod o. C ti) u n c. ci n t r a e 1. ec t r o d o. 170. A cassette comprising: 170. Un cassette que comprende: (a) a p luraIty of di serete link domains, these link domains are linked to a porous matrix (a) una p luraI idad de domiπ ios de enlace di seretes, d ichos dominios de enlace están enlazados a una matriz porosa 5 ionically permeable;5 iónicamente permeable;(or) an electrode close to said ionically permeable porous matrix: and (c) a counter electrode. (ó) un electrodo cercano a dicha matriz porosa iónicamente permeable: y (c) un contraelectrodo. 171. A cassette comprising: ionic mente perme a b1e;and (b) an electrode in electrochemical contact with a plurality of said binding domains. 171. Un cassette que comprende: iónica me nte perme a b1e;y (b) un electrodo en contacto electroquímico con una pluralidad de dichos dominios de enlace 172. The cassette plus a counter electrode 172. El cassette de además un contraelectrodo 173. A cassette comprising: 173. Un cassette que comprende: (a) a plurality plurality of dom i. Neither link is linked to a porous support;and {b) uπ e1ectr ode in c on tact elect roquim i. With a plethora of said items, it includes: (a) una p1u rali dad de pluralidad de dom i. n i os de enlace están enlazados con un soporte poroso;y {b) uπ e1ectr oda en c on t a c t o elect r o q u i m i. c o c o n una plura1 idad de d ichos comprende: (a) an electrochemical cell comprising an electrode, a (a) una celda electroquímica que comprende un electrodo, un -2661 -2661 266 electrode;and (b) a support having a plurality of discrete binding domains therein, said plurality of binding domains is in electrochemical contact with said electrode. 266 electrodo;y (b) un soporte que tiene una pluralidad de dominios de enlace discretos ahí, dicha pluralidad de dominios de enlace se encuentra en contacto electroquímico con dicho electrodo. 5 175 »The cassette of claims 143, 144, or 5 175» El cassette de las reivindicaciones 143, 144, o bien 160 wherein the electrode comprises carbon fibrils. 160 en donde el electrodo comprende fibrilas de carbono. 176. A method of detecting or measuring the electrochemistry uroi ni scenci a, comprising;176. Un método para detectar o medir la e1ectroquimico1 uroi ni scenci a, que comprende;A (a) contact the linkage reagents in cassette 10 of re i. vi nd i. cac i. 143 with a sample that complies with links linked to a marker and 1 ec t roqu i. mico 1 um i. ni scen te;Γ (a) poner en contacto los reactivos de enláce en el cassette 10 déla re i. v i nd i. cac i. ó n 143 con una mués t ra que ccump rende mo1écu1a s enlazadas c o n un ma rcador e 1 ec t roqu i. m i c o 1 um i. n i scen t e;(b) aplicar una forma de onda de voltaje efectiva para desencadenar la electroquimicoluminiscencia en dichos (b) applying an effective voltage waveform to trigger electrochemiluminescence in said 15 electrodo y contraeleetrodo;y (c) detectar o medir dicha electroquimicoluminiscencia. fifteen electrode and counter electrode;and (c) detect or measure said electrochemiluminescence. 177. A method for. detect or measure the elect roqu imicolumi niscenci a, comprising: 177. Un método para. detectar o medí r la elect roqu imicolumi niscenci a, que comprende: (a) contacting the binding reagents in cassette 20 of re i, vi nd i. acion ion 147 with a sample comprising molecules bound to a niscent electrochemical column marker;ía) poner en contacto los reactivos de enlace en el cassette 20 de la re i, v i nd i.ac ión 147 con una mués t ra que comprende moléculas enlazadas a un marcador elec t roquimicolumi niscente;<b) aplicar una forma de onda de voltaje efectiva para desencadenar la elec: troqu imi columi niscenc i a en dichos 25 electrodo y contraelectrodo;y <b) applying an effective voltage waveform to trigger the elec: troqu imi columi niscenc ia in said electrode and counter electrode;and -2671 -2671 178 The method in accordance with rei vind icae ion 175 where said electrode is porous 178 El método de conformidad con la rei vind icae ión 175 en donde dicho electrodo es poroso 179 179 A method to measure the e 1 ec t roqu imicolu.mi π iscenc ia, comprising: Un método para med i r la e 1 ec t roqu imicolu.mi π iscenc ia , que comprende: (a) contacting them with a sample comprising a linked marker ib) applying a waveform of voltage at the end and at the end;and (a) poner en contacto los con una muestra que comprende enlazadas un marcador íb ) aplicar una forma de onda de volta je e1ec t rodo y cont rae1ec trodo;y Ce) detect or measure said electrochemicolum iniscence Ce) detectar o med ir dicha electroquimicolum iniscencia 180 180 A method to measure the elec troqu im icolumi niseene ia, comprising: Un método para med i r la elec troqu im icolumi niseene ia, que comprende: (a) contacting the binding reagents of the claim vindication 150 with a sample of bound molecules an electro-reactive rna coli luminescent wavelet with the dena r 1 to o1umi niscence (a) poner en contacto los reac t i vos de enlace de la reí vindicación 150 con u na muestra moléculas enlazadas un rna rezador elec troqu i m i columiniscente onda valta je deseπc a dena r 1 a o1umi niscencia A method to detect measure the e 1 ec t roqu imi co 1 um ini sce nc la, qua c omp 1-1 ende Un método pa ra detectar med i r la e 1 ec t roqu i m i co 1 um i n i sce nc la, qua c omp 1-1 ende -268i (a) contacting a binding domain containing a first surface with a sample containing molecules linked to an electrochemiluminescent marker, said first surface is aligned with a second surface that c: ontie laugh u π e 1 ec All in all, said first surface and said second surface are spaced 1. in alignment to establish electrochemical contact;-268i (a) poner en contacto un dominio de enlace que contiene una primera superficie con una muestra que contiene moléculas enlazadas a un marcador electroquimicoluminiscente, dicha primera superficie se encuentra alineada can una segunda superficie q ue c: o n t i e ríe u π e 1 ec t rod o, dicha p r i me r a superficie y dicha segunda superficie están espac ía 1. mente alineadas para establecer un contacto electroquímico;<b) aplicar una forma de onda de voltaje efectiva para de s e nc aderar 1 a e 1 ec t roq u i m i c o 1 u m i n i scenc i a e n d i c: ti os electrodo y contraelectrodo;y (c) detectar o medir dicha electroquimicoluminiscencia. <b) apply an effective voltage waveform to identify 1 ae 1 ec t roq uimico 1 umini scenc iaendic: you electrode and counter electrode;and (c) detect or measure said electrochemiluminescence. 182. The method according to claim 180 wherein the first surface is a porous material 182. El método de conformidad con la reivindicación 180 en donde la pr imera superfic ie es un material poroso 183. 183. A method of detecting the reduction of electrochemiluminescence, which comprises (a) contacting the binding reagents in the re-ivication cassette Un método para detecta r raed i r la electroquimicoluminiscencia, que comprende (a) poner en contacto los reactivos de enlace en el cassette de la re i v i nd i c a c i ó n 156 with a sample that includes molecules bound to a (b) apply dec: ade na rdi cha <c.) detect 156 con una muestra que comprende moléculas unidas a un (b) aplicar d e s e n c: a d e na r d i cha <c.) detectar 184. 184. A method to roed the electrochemical 1 umi.niscencía that includes: Un método pa ra roed i r la electroquimico 1 umi.niscencía que comprende: (a) contact the linking domains on the cassette (a) poner en contacto los dominios de enlace en el cassette -269f de la reivindicación 157 con una muestra que comprende moléculas anla zadas ma rezador electrodos y una forma de onda de voltaje afee t i va para la alee troquimicoluminiscenc ia en dichos contraelectrodo;y o mecí i r dicha elect roquimicoluminiscencia -269f of claim 157 with a sample comprising molecules linked to the hardener electrodes and a voltage waveform at the same time for the trochemicoluminescent alee in said counter electrode;I rocked to go said electroluminescence elect 185. A method of detecting or measuring the elec troqu imic-olumi niscenc ia, which comprises 185. Un método para detectar o medir la elec troqu imic-olumi niscenc i a , que comp rende s IC (a) contacting the binding reagents in the cassette gives re i. vi nd i falls ion 158 with a sample comprising molecules attached to an electrochemiluminescent marker;IC (a) poner en contacto los reactivos de enlace en el cassette da la re i. v i nd i cae ión 158 con una mués t ra que comprende moléculas unidas a un marcador electroquimicoluminiscente;<b) aplicar una forma de onda de voltaje efectiva para desencadenar la elect roquimicolumi niscencia en dicha <b) apply an effective voltage waveform to trigger the elect roquimicolumi niscence in said 15 superficie de electrodo y superficie de contraelectrodo;y (c ) da tac ta r o med ir dicha elec t roquímicoluiRiniscenc i a . fifteen electrode surface and counter electrode surface;and (c) it gives tac ta ro to measure said elec ro ro micoluiRiniscenc ia.
- 918Ó ·>. A method of detecting or measuring e 1 ec t roqu imi co 1 um i. nor scenc ia, which comprises:18ό·>. Un método para detectar o medir la e 1 ec t roqu i m i co 1 um i. n i scenc i a , que comp rende: (a) contact the linking domains on the cassette (a) poner en contacto los dominios de enlace en el cassette 2G de 1a re i vi nd i cae ión 159 con mo1éculas enlazadas e 1 ec t roqu.imicolum i niscente ;2G of the 1st re i vi nd i falls ion 159 with linked molecules and 1 ec t roqu. (b) aplicar una forma de onda u, n a m u ostra q u e c o m p r ende a un marcador de voltaje efectiva para desencadena r la el ec t roqu. i m i co 1 um i n i scenc i a en dicha (b) apply a waveform u, namu oyster that compresses an effective voltage marker to trigger r the ec t roqu. imi co 1 um ini scenc ia in bliss 25 electrode surface and counter electrode surface;and 25 superficie de electrodo y superficie de contraelectrodo;y -270ι (c) detect or measure said electrochemiluminescence. -270ι (c) detectar o medir dicha electroquimicoluminiscencia. 187, A method for 1 ec t roqtí imico 1 um ini scenc ia, which (a) put in contact the domi of the rei v i. nd i falls i ón 160 with bound molecules e 1 ec t rocfu im ico 1 um ini scente;187, Un mét odo para d e 1 ec t roqtí i m i c o 1 um i n i scenc i a , qu (a) poner en contacto los domi de la rei v i. nd i cae i ón 160 con moléculas enlazadas e 1 ec t rocfu i m ico 1 um i n i scente;ib) aplicar una forma de onda desencadenar 1a e1ectroqu i m L electrodo y contraelectrodo;y e t e r m i na r o med ir la e comprende: ib) applying a waveform to trigger the 1st electrode im L electrode and counter electrode;yetermi na ro med ir la e comprises: In the cassette, a sample comprising an effective voltage marker for luminescence in said cassettes (c) detects or measures said electrochemiluminescence. iios de enlace en el cassette una muestra que comprende a un marcador de voltaje efecti va para oluminiscencia en dichos (c) detectar o medir dicha electroquimicoluminiscencia. 188. A method to detect or measure Electrochemiluminescence, which comprises: 188. Un método pa ra detectar o med ir La electroquimicoluminiscencia, que comprende: (a) contacting the binding domains in the cassette of claim 161 with a sample comprising molecules bound to an electrochemiluminescent marker;(a) poner en contacto los domi nios de enlace en el cassette de la re ivindicaci¿n 161 con una muestra que comprende moléculas unidas a un marcador electroquimicoluminiscente;(b) aplicar una forma de onda de voltaje efectiva para d es encadenar la e 1 ec troquimico 1 uminiscencia en d i.ch os electrodo y contraelectrodo;y <c ) detecta r o med i. r d i.cha elec troqui micol uminiscenc i a . (b) applying an effective voltage waveform for d is to chain the e 1 ec trochemic 1 luminescence on the d i.ch os electrode and counter electrode;and <c) detects ro med i. rd i.cha elec troqui micol uminiscenc ia. 189. 189. A method for detecting ro med ir el elec troqu i mi, colu.mi ni se ene ia, which comprises: Un método para detecta r o med i r la elec troqu i mi, colu.mi ni se ene i a , que comp rende: <a) contacting a plurality of discrete binding domains on a support with a sample comprising m or 1 molecules bound to an electrochemical marker: or 1 luminescent <a) poner en contacto una pluralidad de dominios de enlace discretos en un soporte con una muestra que comprende mo 1 éculas enlazadas a un marcador eleetroquimic:o 1 uminiscente -271271 mediante e 1 sum i n i. st ro de uf ia mues t ra que cuntí ene dichas moléculas en dichos dominios de enlace, dicha muestra puede modul ar señales elec t roqu i mi col um i. niscentes, en donde dicha modula c i ó n p u e d e ¢: o r r e 1 a c i o na r s e c o n u n a conce n t r a c i ó n de u n analito de interés en la muestra;-271271 through e 1 sum in i. st ro uf the sample that holds these molecules in these binding domains, said sample can modulate elec t roqu i mi col um i signals. niscents, where said modulation can: a re to 1 action with a concentration of an analyte of interest in the sample;(b) apply an effective voltage waveform to trigger the 1st e1eu t roqu imi co1uroi ni scenci a in a e1ect rodo and counter electrode from a marker í (b) ¿aplicar una forma de onda de voltaje efectiva para desencadena r 1a e1eu t roqu i m i co1uroi n i scenci a en un e1ect rodo y contraelectrodo a partir de un marcador í elec troquimicoluffliniscente enlazado a una molécula unida al me nos a u no d e d i. c hos doro i n i os de e η 1 a c e;trochemicoluffliniscent elec linked to a molecule at tached to the minus au no ded i. c hosdoro ini os de e η 1 ace;(c) roed i r dicha elec t roquimicoluminiscencia;y (d) determinar dicha concentración del analito de interés. (c) roed ir said electrochemiluminescence;and (d) determining said concentration of the analyte of interest. 190. A method of detecting or measuring electrochemiluminescence, comprising: (a) contacting the binding reagents in the cassette of claim 143 with a sample comprising a first molecule and a second molecule linked to an electrochemiluminescent marker, wherein the second molecule is a specific binding dirty of the first roolécu1a;190. Un método para detectar o medir la elec troquimicoluminiscenc ia, que comprendeí (a) poner en contacto los reactivos de enlace en el cassette de la reivindicación 143 con una muestra que comprende una primera molécula y una segunda molécula enlazadas a un marcador electroquimicoluminiscente, en donde la segunda molécula es un sucio de enlace específico de la primera roolécu1a ;(b) aplicar una forma de onda de voltaje efectiva para desencadena r la elec t roqui.mi co1. umi π iscencía en d i.cho electrodo y contraelectrodo;y (c) detectar o medir dicha electroquimicoluminiscencia. (b) apply an effective voltage waveform to trigger the elec t roqui.mi co1. umi π iscency in d i.cho electrode and counter electrode;and (c) detect or measure said electrochemiluminescence. 191 the 191 la -272I el ec t roquim i col uní i niscenc i a , que comprende: -272I the ec t roquim i col uní i niscenc ia, comprising: desencadenar la aleetroquimicolumi.niscencia en al menos uno de d iohos pares de electrodos y contraelectrodos;y (c:) de tea ta r o roed i. r di chai elec troqu imicolumi niscenc i a . trigger aleetrochemicolumi.niscence in at least one of two pairs of electrodes and counter electrodes;and (c :) from tea ta ro roed i. r di chai elec troqu imicolumi niscenc ia. 10 192. A method to detect or measure i. r 1 a elec tr oqu im i. co 1 uni in i. scenc ia, which · ::: omp rendes 10 192. Un método pa ra detectar o med i. r 1 a elec t r oqu i m i. co 1 uni i n i. scenc ia , que ·::: omp rendes desencadenar la eleetroquimicoluminiscenci a en dichos e1ec trodo y con t raelec trodo;y (c ) de tec t a r o roed i r d i. cha elect roqu i m i co1 uro i. n i scenc i a . triggering the electrochemiluminescence at said electrode and with the electrode;and (c) from tec taro roed ird i. cha elect roqu imi co1 uro i. nor scenc ia. 2Ci 1.93. A method for detecting ro mecí go electroquimicoluro i ni scencia, which includes: 2Ci 1.93. Un método pa ra detecta r o mecí i r la electroquimicoluro i ni scencia, que coroprende: (a) contact the 1o linker doroi children of the 1st rei v ind ication 169 with a sample comprising molecules linked to a marker (a) poner en cont ac to 1os doroi ni os de enlace eπ e1 casse11 e de 1.a rei v ind icac ión 169 con una muestra que comprende moléculas enlazadas a un marcador 25 the ec t roqu imicolumi π i «icen te;25 el ec t roqu imicolumi π i «icen te ;-273273 (b) aplicar una forma de onda de voltaje efectiva para desencade na r la elect roqui mi co1um i π i se ene i a en d i c hos electrodo y contraelectrodo;y (c) detec tar o med i r d i cha elec t roqu im icol um i ni. scenc i a . -273273 (b) apply an effective voltage waveform to trigger the elect roqui mi co1um i π i e ene ia dic dic electrode and contraelectrode;and (c) detect or measure ir ir cha cha t roqu im icol um i ni. scenc ia. 5 194. A method to detect or measure the e 1 ec t roqu i mico1um i ni scenci a, which comprises: 5 194. Un método para detectar o med ir la e 1 ec t roqu i mico1um i ni scenci a, que comp rende: desencadena r la elec t roqu imicolufli iniscenc i a en cl i ches electrodo y contraelectrodo;y (c) detectar o medir dicha electroquimicoluminiscencla. it triggers the ele roqu imicolufli iniscenc ia in the electrode and counter electrode;and (c) detect or measure said electrochemiluminescent. 15 195. Un método para detectar o medir la electroquimieoluminiscencia, que comprende: fifteen 195. A method of detecting or measuring electrochemiluminescence, comprising: (a) bringing the link domains into contact in the cassette of claim 173 with a sample that compares the link with the links to the mother (a) la puesta en contacto de los dominios de enlace en el cassette de la reívindicación 173 con una muestra que c om p r end e melé c u las enlaza d as a u n ma r c at d o r
- 1020 elec t roqu imi columiniscente;twenty elec t roqu imi luminescent;<b) aplicar una forma de onda de voltaje efectiva para desencadenar la electroquimicoluminiscencia en dichos electrodo y contraelectrodo;y <c) detectar o med ir dicha elect roquimicoluminiscencia. <b) applying an effective voltage waveform to trigger electrochemiluminescence at said electrode and counter electrode;and <c) detect or measure said rochimicoluminescence elect.
Independent claims10
1,116 paragraphs in 104 sections, as filed
PCT WORLDINTELLECTUAL PROPERTY ORGANIZATION * iMentatioiuü Burean
INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
<td>(51) Internatkmal Patent Ctaerificetton 6: C12M l / DO, 1Λ0, C12Q 1 / 00,1Λ58, G01N 21/76, 33/53, 33 / 543,33 / 567</td><td>To the</td><td colspan="2">(11) International Publication Number: WO 96/28538 (43) Intemational Publication Date: 19 September 1996 (19.09.96)</td>
<td colspan="3">(21) IntermtiMal Applcatioti Number: PCT / US96 / 03190 (22) Internatfonal FUtag Date: 6 March 1996 (06.03.96) (30) Priority Dele: 08 / 402,076 10 March 1995 (10.03.95) US 08 / 402,277 10 March 1995 (10.03.95) US (71) AppUcant: MESO SCALE TECHNOLOGIES, LLC [US / US]; Corporation Service Cocnpany, 1013 Cerner Road, Wilmington, DE 19805 (US). (72) Invent: WOHLSTADTER, Jacob; Apaitment 34, 19 Ev- Erette Street, Cambridge, MA 02138 (US). WILBUR, James; Apartment A, 9903 Gable Ridge Tenace, Rockville, MD 20850 (US). SIGAL, Gecrge; Apartment 733. 304 Sunny Brook Terrece, Gaithersburg, MD 20877 (US). MARTIN, Mari; 6516 Listen F'm Court, Rockville, MD 20852 (US). GUO, Liang-Hong; Apartment 202. 14611 Philip Court, Laurel, MD 20708 (US). FISCHER, Alan; 80 Antrim Street, Cambridge, MA 02139 (US). LELAND, Jon; 14236 Amberieigb Tenace, Silvcr Spring, MD 20905 (US). (74) Agent: EVANS, Barry et al .; Curtís, Monis & Safford, PC, 530 Fifth Avenue, New York. NY 10036 (US).</td><td>(81) Desfgnated Status: AL, AM. AT. AU, AZ, BB, BG, BR, BY, CA, CH, CN, CZ. OF. DK, EE, ES. FI, GB, GE, HU, IS, JP, KE. KG, KP. KR. KZ, LK. LR, LS, LT, LU, LV, MD, MG, MK. MN, MW. MX, NO, NZ. PL, PT, RO. RU, SD, SE, SO, SI, SK, TJ, TM, IR, TT, UA, UG, UZ. VN, ARIPO patent (KE, LS, MW, SD, SZ, UG), Euruian patent (AM, AZ, BY, KG, KZ. MD, RU, 17, TM), European patent (AT, BE. CH. DE , DK, ES, FI, FR, GB, GR, ΙΕ, ΓΓ, LU, MC. NL. PT, SE), OAPI patent (BF, BJ, CF. CG. Cl. CM. GA. GN, ML. MR . NE, SN, TD, TG). PubHshed H'ftA intemafionaJ rearch repart. Bifm the expiration o / the time limit for amending the clama and to be republished üi the event of the receipt oj amendments.</td>
<td colspan="4">(54) Title: MULT1-ARRAY, MULTI-SPECJF1C ELECTROCHEMILUMINESCENCE TESTING (57) Abstract! Meteríais and merhods are provided for producing pattemed multí-auay, multi-spccific surfaces whích are elcctronically eacited for use in electrochemiluminescence based tests. Meteríais and methods are provided for the chemical and / or physical control of cooducting domains and reagent depoeition for use in flat panel displays and multiply specific testmg procedures.</td>
-1ι
TEST OF MULTI-SPECIFIC ELECTROCHEMICAL COLUMINISCENCE, OF
MULTIPLE ARRANGEMENTS
This application is a cent inuation in part of the filing application Serial No. 08 / 402,076 filed on March 10, 1995, and the co-pending application Serial No. 08 / 402,277, filed on March 10, 1995, which are incorporated herein by reference in their entirety.
one. INTRODUCTION
The present invention offers a multi-array, multi-array, speci fi c, configured surface for electrochemical illumination-based tests, as well as methods for preparing and employing PMAMS.
2. BACKGROUND OF THE INVENTION
2.1. DIAGNOSTIC TESTS
There is a significant financial need for fast and sensitive diagnostic technologies. Diagnostic technologies are important in a broad field of economic markets including the health, research, agriculture, veterinary, and industrial sectors. Improved sensitivity, time required, ease of use, robustness, or cost can open up entirely new diagnostic markets where previously no technology could meet the needs of the market. Certain diagnostic technologies can have high sensitivity but are too expensive to meet market needs. Other techniques may be inexpensive but not sufficiently robust for various markets. A novel diagnostic technique that can combine these qualities is a significant advance 5 and an opportunity in the field of diagnostics.
There are numerous different analytical techniques employed in diagnostic applications. These techniques include radioactive dyeing, enzyme-linked immunoassays, 10 chemical calorimetric assays, fluorescent labeling, chemilum πiscent labeling, and electro-niscent labeling. Each of these techniques has a unique combination of levels of sensitivity, ease of use, robustness, speed and caste that define and limit its usefulness in different diagnostic markets. These differences are due in part to the physical limitations inherent in each technique. Radioactive labeling, for example, presents a lack of robustness, the labeling itself disintegrates and the removal of environmental radioactive waste for many applications.
Many of those that are used today are essentially specialized today because they lead to
1imi tac ions noces idad carry out the market technical tests. The
-3I electrochemical luminescent procedures in use today, for example, require not only specialized technicians but also repeated washing and preparation steps. This increases both costs and the need to dispose of waste. Novel diagnostics that simplify testing procedures as well as lower cost per test will be very important and very useful to open new markets as well as to improve the t
performance of existing markets.
2.2 ELECTROCHEMICAL COLUMINISCENCE TESTS
Electroquimicolumiscence (ECL) is the phenomenon by which an electrically excited species emits a photon (see, eg, Leland and Powell, 1990 J. Electrochem. Soc. 137 (10); 3127-3131). Such species are called ECL markers and are also known here as
MARKERS. Commonly used ECL markers include: organometallic compounds where meta 1 comes for example from the noble metals of the Mili group, including organometallic compounds containing Ru and Os 20 such as the Ru <2.2 '~ b ipi r portion id i na) (+ - »+) (also known as Rubpy), presented, for example, by Bard et al. (North American Patent No. 5,238,808). The light generated by · * ECL markers can be used as a reporter signal in diagnostic procedures (Bard et al, 25 North American Patent No. 5,221, 605). For example, an ECL marker can be covalently coupled with a binding agent such as a nucleic acid probe or antibody. The ECL / binding agent marker complex can be used to test various supports (Bard 5 et al., US Patent No. 5,238,808). The need for an electrical potential to excite the ECL marker to emit a photon is fundamental 1 for ECL-based detection systems. An electric potential waveform is applied to an ECL test solution through an electrode surface, typically a metal surface, and a counter electrode (see for example, US Patent Nos. 5,068,088, 5,093,268,
5,061,445, 5,238,808, 5,147,806, 5,247,243, 5,296,191,
5,310,687, 5,221,605).
Various devices well known in the art are available to conduct and detect ECL reactions. For example, Zhang et al. (US Patent No. 5,324,457) presents exemplary electrodes for use in electrochemical cells to conduct ECL. Levantis et al. (North American Patent No. 5,093,268) presents electrochemical cells for use in ECL reactions. Kamin et al. (US Patent No. 5,147,806) features apparatus for carrying out and detecting ECL reactions, including voltage control device. Zoski et al. (North American Patent No. 5,061,445) presents an apparatus for carrying out and detecting ECL reactions, including electrical power waveform diagrams 1 for obtaining ECL reactions, converters from digital to analogue elements, control apparatus, detection apparatus, and methods for detecting the current generated by an ECL reaction at the work electrode to provide feedback information to the electronic control apparatus.
The ECL technique is presented in detail, for example, in US Patent No. 5,093,268. In summary, the ECL technique is a method of detecting in a volume of a sample an analyte of interest present in the sample at relatively low concentrations.
The portion of ECL, called TAG (MARKER) in the aforementioned prior patents, may or may not bind to an analyte, but is anyway promoted in an excited state as a result of a series of chemical reactions triggered by the electrical energy received from of the working electrode. A molecule promoting the MARKER ECL 20 is advantageously provided in the form of oxalate or, more preferably, tripropylamine (see US Patent No. 5,310,687).
2.3. ECL BUSINESS TESTS
To date, all commercial ECL reactions are performed on scale electrode surfaces.
-6i centimeter.
Scale electrodes. centimetric establish a sign 1 of desirability balance between
ECL that results from decreasing the magician and the electrostatic volume of increased majors and the total sample required for each test. However, even centimeter scale electrodes do not achieve the required sensitivity for many tests. In an attempt to overcome this problem, all commercial ECL systems addi tionally increase the seos ib i 1 idad by mediating the use of reversed magnetic beads to capture either reactive or ECL data. The beads are then displaced adjacent to a working electrode to increase sensitivity.
However, the use of magnetic beads has numerous features. The beads themselves are coated with proteins that break down and degrade over time, causing signal variations. Due to the complexity of handling and formatting the account-based tests »the ECL diagnostic study 1 requires a complex pre-formed series of procedures for each test carried out with a given sample, which increases the time and cost to carry out the tests. The 5 micron face of the beads prevents most ECL MARKER attached to the beads from reaching the thin film adjacent to the working electrodes, resulting in a
-7i inefficiency at ECL MARKER excitation level.
Leventis et al- (US Patent No. 5,093,268) have proposed a method for assaying more than one different analyte imuiteneously by using different ECL markers for each analyte, each emitting protons at different wavelengths for each analyte di front in a single trial. However, this technique is limited, for example, by the lack of availability of effective ECL markers sufficient that a number r
radiate at different wavelengths need to optimize chemical conditions for each ECL marker,
These practical limitations have impeded the commercialization of such multiple analyte ECL detection systems, at multiple wavelengths. Another approach to increasing ECL sensitivity is to improve electrode technology. Zhang et al. (US Patent No. 5,324,457) have directly deposited films of ECL species on various metal or semiconductor surfaces. The Zhang and Bard technique employing bulk saturation of the electrode surface results (as described by the authors) in an uneven patchy deposit unsuitable for high sensitivity assays.
The above methods of conducting an ECL test also require that the test cell, including the i
<img file="MX9706471A_D0001.tif" />
Electrodes must be cleaned by any of several methods, including the use of dilute acids, dilute bases, detergent solutions, etc. , in accordance with what is presented, for example in US Patent No. 5,147,806.
Accordingly, it is an object of the present invention to offer a novel and inexpensive assay for carrying out various ECL reactions, either sequentially 1 or r
Simultaneously, and in a preferred mode, to display integrated control standards to improve accuracy.
It is a further object of the present invention to offer a cassette comprising one or more supports suitable for carrying out a plurality of ECL reactions either simultaneously or sequentially, said cassette is disposable.
It is a further and related object of this invention.
<td>reduce time</td><td>and the cost</td><td>to carry</td><td>cape</td><td>essays</td>
<td>i nd iv idua1es pa ra</td><td>ana 1 i cough</td><td>of interest</td><td>in</td><td>samples</td>
<td>biological.</td><td></td><td></td><td></td><td></td>
<td colspan="2">It is another object to add iona1 and</td><td>related to</td><td>the</td><td>Present</td>
The invention offers methods and apparatus for conducting the plurality of simultaneous assays for a plurality of analytes of interest in a single biological sample.
3. SUMMARY OF THE INVENTION
The present invention relates to a cassette for carrying out ECL reactions and ECL assays comprising a plurality of discrete binding domains immobilized on a support, the discrete binding domains being spatially aligned with one or more pairs of electrodes and one or more pairs of counter electrodes. The cassette preferably includes a first support having a plurality of discrete binding domains immobilized on the surface. It may have one or more pairs of electrode and r
one or more pairs of counter electrodes. The electrode and cantraelectrode pairs can be accessed separately by an electrical power source in the form of an effective voltage waveform to trigger the electrochemiluminescence effect. The cassette may also comprise a second holder capable of being placed adjacent to the first holder to provide a sample containing device therebetween, and / or to serve as an electrode. The binding domains are configured on a support surface and are prepared in such a way that they bind the analytes or reagents of interest. The invention further relates to an apparatus for measuring the electrochemical luminescence of a sample, which provides a cassette or holder handling device, a voltage control device adapted to apply an effective controlled voltage waveform to trigger the electrochemiluminescence, a device
-1010 photon detector to detect the electrochemiluminescence coming from the (ours and a device to manipulate the sample.
The i π veneion further refers to methods for employing cassettes to measure electrochemiluminescence in a sample by contacting the plurality of binding domains of a cassette with a sample that contains a plurality of ana 1 i cough of interest, under ECL test conditions, and then by applying a voltage waveform effective to trigger the electrochemical luminescence at each of the plurality of pairs of electrodes and counter electrodes and the detection or measurement of the triggered electrochemiluminescence. In a broad aspect, the invention provides ECL assay methods where the sample does not contact an electrode. Additionally, as an alternative to the use of electrode and counter electrode pairs, the invention provides scanning of an electrode and counter electrode in the binding domains.
The invention also provides set of elements comprising components including cassettes suitable for simultaneously measuring a plurality of electrochemiluminescence reactions, support surface and on which a plurality of domains are immobilized, assay, means for carrying out the ECL assay that
-1111 produces chemical reactions.
The invention also offers electrodes prepared from graphite nanotubes.
Four. DESCRIPTION OF THE FISSURES
Figure 1 illustrates two supports that form a cassette according to the invention where a plurality of binding domain 14 are present in a support 10 and a plurality of corresponding electrodes 16 is present in the support of ta 1 so that the Iprox imation of the supports places a pair of electrodes adjacent to each link domain,
Figure 1A illustrates two supports that form a cassette in accordance with the present invention, where a plurality of link domain 14 are present in the support 10 and a plurality of corresponding electrodes is present in the part 12 of such so that the approximation of the supports places a pair of electrodes adjacent to each bonding domain.
Figure 2 illustrates two supports that form a cassette in accordance with the present invention where a plurality of link domain 30 in the support 26 are adjacent to each of the individual electrodes 32 such that the approximation of the supports 26 and 28 place each of the counter electrodes 38 adjacent to each of the link domains 30.
-12 Figure 3 illustrates two supports that form a cassette according to the present invention where a plurality of link domains 48 have pairs 50 of electrodes with adjacent electrodes on a support 44. The support 46 can optionally be positioned adjacent to the support 44 in such a way that support 46 provides a sample containing device adjacent to link domains 48 and electrodes 50.
J
Figure 4 illustrates two supports that form a cassette according to the invention where a plurality of link domains 64 in support 60 are in contact with a sample suspected of containing an analyte.
Support 62 has regions 66 containing reaction medium to detect either measure an analyte of interest or to carry out a desired reaction such that approaching support 60 and support 62 cause binding domains 64 and regions 66 come into contact with each other.
Figure 5A illustrates a top view of binding domain configured for a multi-specific, multi-array binding surface. Geometric shapes, triangles, squares, and circles represent specific binding domains for different analytes. The binding domains can be either hydrophobic or hydrochemical. The surrounding surface can have the opposite property (hydrochemical or
-1313 minimize ana lyte from link domains.
Figure 5B illustrates a top view of a myofluidic guide for supplying binding reagents and / or analytes to discrete binding domains.
Each dot illustrates a cut of a microfluidic gluttony (by a capi lar).
Γ
Figure 5C illustrates a side view of a microfluidic guide showing the approximation of microfluidic guides to 1 lines or to supply binding reagents and / or analytes to a multitude of configured binding domains. Each microfluidic guide can supply a different binding reagent to a discrete binding domain
Figure 6A illustrates the approximation of a multiple set of electrodes in correspondence with a surface having multi-set, multi-specific binding domains. A removable electrode rate barrier occurs between the electrode assembly and the bonding surface assembly. The complete assembly forms a cassette to carry out a plurality of ECL reactions.
Figure 6B illustrates the approximation of a set of working electrodes and corresponding counter electrodes or
-14 aligned to which power can be supplied. The electrodes can have a complementary shape with the bonding domain or it can have other shapes (for example interd ig i ta1 iation).
Figure 7 illustrates the side view of a rough set of matching or aligned counter electrodes and counter electrodes to which power can be supplied and the complementary bonding surface where conductive polymers are grown from 3rd electrode surface. through the gap between the electrode array and the binding domains in such a way that the potential field around the sample's ECL marker is extended to increase the reaction efficiency of ECL.
Figure 8 illustrates the side view of an approximate arrangement of corresponding or aligned working electrodes and counter electrodes and the complementary bonding surface with conductive particles scattered between both components to extend the potential field. By extending the ia1 potency field around the sample's ECL marker, the efficiency of the ECL reaction is increased. The conductive particles can be magnetic to allow easy manipulation i 1,
Figure 9 illustrates the side view of an approximate set of working electrode and corresponding or aligned counter electrodes and the complementary bonding surface where the electrodes have fixed projections that extend between the space between the electrode surface and binding domains to extend potential field 5 around an ECL marker in the sample, to increase the efficiency of the ECL reaction »
Figure 10 illustrates the view in Lera 1 of an approximate set of working electrodes and corresponding counter electrodes or 3. i neaclos and the complementary bonding surface 10 where the surfaces are not parallel but rather conform to each other. in a complementary way.
Figure 11 illustrates the side view of a support having a metal layer thereon to provide a unique electrode and bonding surface assembly in the form of a cassette. A set of self-assembled monolayers <* 'SAMs) is configured in the metal layer »
Figure 12 illustrates the side view of a support having a metal layer thereon to provide a unique electrode and bonding surface assembly in the form of a cassette. A set of SAMs is configured in the metal layer and conductive microparticles are scattered among the configured SAMs to extend the potential field around the sample's ECL marker to increase the efficiency of the reaction of the sample.
-1616
ECL.
Figure 13 illustrates the side view of a support having a metal layer on it to provide a unique assembly of bonding surfaces and electrodes in the form of a cassette. A set of self-assembled SAMs monolayers is configured in the metal layer and the growth of a conductive polymer and / or fiber from the ECL marker is illustrated to extend the potential field around the sample's ECL marker to increase the ECL reaction efficiency.
<td></td><td>Fig. 14</td><td>is</td><td>a</td><td>diagram of</td><td>a</td><td>support</td><td>than</td><td>has</td><td>a</td>
<td></td><td>set of</td><td colspan="2">pairs</td><td colspan="2">of electrodes</td><td colspan="2">controlled</td><td>by</td><td>a</td>
<td></td><td>computer.</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td>Fig. 15</td><td>is</td><td>a</td><td>diagram of</td><td>a</td><td>support</td><td>than</td><td>has</td><td>a</td>
<td> 15</td><td colspan="2">set of pairs</td><td>of</td><td>electrodes.</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td>Figure 16</td><td>is</td><td>a</td><td>diagram of</td><td>a</td><td>support</td><td>than</td><td>has</td><td>a</td>
set of pairs of electrodes and a computer system to control the sum in ist ro of energy of each pair of electrodes.
Figure 17 is a diagram of a holder having a set of pairs of electrodes and a computing system with a plurality of voltage source and multiples to control the power supply of each pair of electrode.
Figure 18 is a diagram of a support that has a set of pairs of electrodes and a computer system with
-171 a plurality of switched power supplies to control the sumí st st of energy of each pa r of electrodes.
Figures 19 (a) - (e) are plan views of various combi nac i cine a 1 terna ti ve of pairs of elect rodoscontraelectrode.
Figure 20 illustrates a support with a sandwich finished test.
Figure 21 illustrates two opposite surfaces of PMAMS on 10 supports.
Figure 22A illustrates a set of microfluidic glues (2201) and a fibril mat (2200).
Figure 22B illustrates link domains (2202).
Figure 23A illustrates an apparatus for forming a fibril mat by vacuum filtration.
Figure 23B illustrates a fibril mat (2304) in a fi 1 tro membrane (2303).
Figure 24 illustrates the use of rollers to produce fibril mats.
Figure 25 shows a schematic of a multi-layer fibril mat where the top layer has binding domains used for testing.
Figure 26 shows a schematic of a derived fibril with portions that increase non-specific binding, and several 25 species, both biological and non-biological, are found
-1818 linked I will know the surface.
Figure 7.7 shows a schematic of a derived fibril with portions that increase the nonspecific link and several species linked to a derived fibril with some 5 species linked adi clone 1 to 1 igand.
Figure 28 illustrates several species covalently fixed on a fibril and some species are linked additionally to additional entities.
Figure 29 illustrates the use of a mat of fibrils of
1O multiple layers edge an optical filter that, depending on the position of a light source on the mat or on said mat can allow the passage of light and / or can absorb and / or scatter light.
Figure 30A illustrates cyclic voltamograms from electrochemical measurements of carbon fibril mat electrodes.
Figure 30B illustrates cyclic valtamograms from electrochemical measurements on low gold electrodes.
Figure 31 compares an electrochemical property of fibril mats as a function of mat thickness and scanning speed.
Figure 32 shows a graph illustrating that the nonspecific binding in fibrils generally rises as the concentration of fibrils in a protein solution rises.
-191?
Figure 33 demonstrates that the use of surfactants can reduce the non-specific binding between ECL MARCAD0R1-labeled protein and carbon fibrils.
Figure 34 shows a top view schematic of an experimental cell used to measure electrochemical and ECL properties on a fibril mat electrode.
Figure 35 shows an ECL signal obtained using a fibril mat and electrode and 1000 pM of MARCADCRl
<td>(11 continuous line)</td><td colspan="2">in solution</td><td>and a sign</td><td>r coming from a</td>
<td>regulator</td><td>test</td><td>(without</td><td>MARKER1)</td><td>(dashed line</td>
<td>interrupted going). Figure 36</td><td>shows</td><td>a</td><td>schedule of</td><td>two days after you</td>
Surface PMAMS, where two sets of supported electrodes are separated by a configured dielectric layer.
Figure 37 illustrates an apparatus with a plurality of link domain (3702) on one holder and one electrode and counter electrode on another holder.
Figure 38 shows a cassette where binding domains are present on the surfaces of different objects supported on the counter electrode.
Figure 39 shows a gel in contact with a working electrode and a counter electrode.
Figure 4O shows an ECL intensity graph and a cyclic voltamogram from an ECL-labeled gel.
<td>in</td><td>Contact</td><td>with a</td><td>electrode</td><td>of</td><td>job</td><td>and a</td>
<td colspan="2">counter electrode.</td><td></td><td></td><td></td><td></td><td></td>
<td>The</td><td colspan="3">Figure 41 shows a graph of</td><td>the</td><td>intensity</td><td>ECL and</td>
<td>a</td><td>voltamogram</td><td>cyclic</td><td>coming</td><td>of</td><td>not a gel</td><td>marked</td>
for ECL in contact with a working electrode and a counter electrode.
Figure 42 shows a schematic for a shallow cassette used for ECL.
r
Figure 43 demonstrates that fibrile mats can be used as methods for ECL of antibody MARKER1 adsorbed on the mats.
Figure 44A shows the ECL intensity of a MARKER-labeled protein! immobilized on an electrode.
<td>The figure</td><td>44B shows</td><td>the</td><td>voltamogram</td><td>cyclical</td><td>a</td>
<td>electrode</td><td>coated.</td><td></td><td></td><td></td><td></td>
<td>The figure</td><td>45A sample</td><td>a</td><td>generation</td><td>repeat it goes</td><td>almost</td>
<td>reversible</td><td colspan="2">ECL signal</td><td>coming></td><td colspan="2">of a protein</td>
<td colspan="2">marked with MARKER for</td><td>ECL</td><td></td><td></td><td></td>
<td>The figure</td><td>45B shows</td><td>the</td><td>valtamogram</td><td>cyclical</td><td>a</td>
coated electrode indicating partial preservation of the coating.
Figure 4¿> A shows the irreversible generation of ECL signal from a marker-labeled protein for immovi 1 iZed ECL.
Figure 46B shows the cyclic voltamogram of a coated electrode indicating substantial loss of the coating.
Figure 47 shows a multi-array ECL apparatus and a microprocessor containing a controller device for generating and analyzing ECL signals,
5. DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention broadly includes cassettes for performing a plurality of electrochemiluminescence assays. Cassettes are formed from supports having a plurality of binding domains capable of specifically binding to one or more analytes of interest. Binding domains are prepared as multi-array, multi-array, specific surfaces (PMAMS) on the support. PMAMS offers significant improvement compared to previously known ECL test method by, for example, a significant increase in the density of tests that can be carried out and allowing a plurality of different tests that can be carried out quickly or simultaneously . The cassette may include a plurality of electrodes capable of selectively triggering light emission for ECL from labeled ECL reagents bound on the binding domains. Figure 47 shows a multi-array ECL apparatus having 4700, 4704 electrodes, a 4702 array with 4706 link domains, and a
-22i microprocessor containing a 4720 controller device to generate and analyze an ECL signal connected by means of 4710-4716 leads.
In the embodiment of the invention presented in Figure 1, a cassette comprises two supports 10, 12 where a plurality of link domain 14 are present on a surface of a first support 10 and a plurality
<td>pairs</td><td>16 of I</td><td colspan="3">electrodes / counter electrodes are</td><td>find</td>
<td>Present</td><td>in a</td><td>surface</td><td>second</td><td>support</td><td>12. The</td>
<td>domains</td><td>of</td><td>link</td><td>and the</td><td colspan="2">pairs of</td>
electrodes / counter electrodes are aligned such that each pair of the various electrate / counter electrode pairs 16 is adjacent to a different link domain of the plurality of link domain 14 when the first support 10 and the second support 12 are brought together. The first support 10 below the bonding domains 14 is preferably a PMAMS with a gold film surface and transparent bonding domains. The second support 12 is preferably a flat transparent plastic sheet having pairs 16 of Transparent electrodes / counter electrodes there. Binding domains 14 are preferably prepared by microstamping a configuration of self-assembled organic monolayers (composed of individual monomers) on the support surface, where the monomer has a portion
<img file="MX9706471A_D0002.tif" />
binding or biotin. Avidin or steptavidin are then bound to the exposed biotin (see, eg, US Patent No. 5,093,2681- Binding Reagents are then applied by applying a discrete amount of a suitable biotin-labeled binding reagent as per example biotin-labeled antibody, which can be selectively bound on the analyte of interest at the locations on the support surface where the moriolayer has been stamped - Figure IA illustrates a system comprising a cassette (Figure 1) contained in a frame (11).
In certain embodiments of the invention, it is desirable to reproducibly immobilize a specific or predetermined amount of one or more reagents on a surface. The immov i 1 i zaci ón applies in general terms to any method by which a reagent is fixed on a surface, including without limitation covalent chemical bonds; non-specific adsorption; drying a reagent on a surface; electrostatic interactions; hydrophobic and / or hydrophobic interactions; combination or drag in liquids or gels; biospecific binding, (for example ligand / receptor interactions or oligonucleotide hybridization); goal links 1/1 igando; chelation, and / or entanglement in polymers -24 The amount of reagent immobilized on a surface can be predetermined in several ways. For example, the amount of reagent on a surface can be specified by one or more elements of volume and / or area in which the reagent is present. It can also be specified by the number of individual molecules of a reagent that was immobilized. 1 i zan on a surface. The amount of reagent can be specified in terms of the density of r a particular reagent in a given region. The amount of reagent is to be specified as a percentage of one carrying a particular reagent, either in relation to the total surface area, or in relation to the amounts of other reagents present on the surface. The amount of react i vo can also be defined as the amount of react i vo that must be present on a particular surface to provide sufficient intensity of ECL for the assay to achieve a desired specificity. In a specific example, an area of cm2 of ring surface may be coated with a monolayer of alcandiales.
Reagents can also be immobilized reproducibly on reversed surfaces. The coating can serve to implement immobilization of some reagents and / or to reduce or prevent immobilization of other reagents. The surface can be completely
-2525 coated or the surface may be partially coated (ie, a configured coating). The coating may be uniform in composition, or it may contain different composition elements. In a specific example, the coating may be a configured monolayer film that immobilizes immunoglobulin G by chemical bonding to cova lenses in some areas, and prevents immobilization in others »
The coating can also serve to predetermine the quantity (s) of one or more reagents imoved in the surface in subsequent steps or in subsequent processes. Alternatively, the amount of a particular reagent can be controlled by limiting the amount of reagent deposited.
Having a surface that has reagents (or a coating) i nmov i 1 i ized in a quant i tat i va way, reproducible provides reproducible capacity and quantitatively one of
From measuring to splitting a sample, preferably, electrode / counter electrode pairs are less than one centimeter in size and are manufactured in conjunction with electrode connections 20 (eg, from a transparent metal film) by well known methods of manufacturing of liquid crystal displays and electrochronous presentation panels. Drivers of
-2626 electrodes 20 are connected by means of electrical connections 19 to a waveform generator device 18 »Advantageously, the pairs of e reads trodos / cont rae reads: All are individually managed with computerized control such that the electrical potential can be selectively applied to discrete link domains. A light detector device 22 and a digital computer device 24 are provided to record and analyze results when emissions ECL have been stimulated from a suitable marker found in a binding domain.
Figure 1A illustrates a system comprising a cassette, electrical conductors, waveform generator, light detection device, and digitized computer as described in Figure 1, contained in frame 11. The cassette is inserted into the frame through an opening (15).
In another embodiment, a working electrode is employed to simultaneously generate an ECL signal in a plurality of link domain. In this mode, the ECL signal from each link domain is identified through the use of light imaging equipment.
Generally speaking, tests performed using cassettes in accordance with the present invention are tests that benefit from the use of various discrete binding domains. For example, the use of such cassettes
-2727 enables rapid and / or concurrent measurement detection of a wide variety of analytes of interest. In a preferred embodiment, the assays in accordance with the present invention are also assays that benefit from the use of a reagent, analyte, or labeled binding surface for ECL. An ECL assay in accordance with the present invention comprises contacting a plurality of binding domain with a sample suspected of containing an analyte of interest and triggering an ECL emission from a marker of Bound ECL, where the ECL marker is found on either the analyte or a competitor of the analyte, in a reagent that binds to the analyte, or in the plurality of binding domain.
The invention also offers test methods for ECL to detect or measure an analyte of interest, comprising (a) contacting one or more of a plurality of discrete binding domains, said plurality of binding domains being immobilized on a surface of one or more supports, where said contacting is carried out with a sample containing molecules linked to a luminescent electrochemical marker, where said sample does not come into contact with any electrode or counter electrodes during said contacting step; <b) the approach of an electrode to said link domain or said link domains of a plurality of link domains; <c) the
-281 application of an effective voltage waveform to trigger ECLs in said link domain or in said link domains of a plurality of link domains; and the detection or measurement of ECL.
In another embodiment, the present invention offers ECL assay methods for (a) contacting one or more link domains of a plurality of discrete link domains, said plurality of link domains (i) being immobilized on a surface of one or more supports, and (ii) is spatially aligned with and in close proximity to pairs of electrodes and counter electrodes, wherein said contacting is a sample containing molecules linked to a marker an electrode and a counter electrode to said binding domain said binding domains of a plurality of binding domains; (c) applying an effective voltage waveform for said link r triggering plurality domain or electrochemiluminescence domains in said link binding domains; and (d>
detection or measurement of electrochemiluminescence
The plurality of binding domain on the support can interact with samples to be tested. The coming into contact with solutions that
PMAMS may also contain reagents necessary to complete an assay. The bonding surface
-291 is then contacted (eg by pressing) with a surface of a complementary electrode (preferably a clean or virgin electrode) which is then used to apply an electrical potential to stimulate 5 ECL.
In a preferred method of carrying out an assay using the apparatus of Figure 1, a sample of which suspicion contains an analyte of interest is applied to several r binding domains 14 together with ECL-labeled reagents suitable for detection in analyte . The support 11 and the support 12 are then brought together in such a way that each of the link domains 14 is located between the electrode and the counter electrode in a different pair of several pairs 16 of electrodes / counter electrodes and the sample is between them. It should be noted that the electrode and counter electrode pair do not make mechanical contact with the binding domain to stimulate the ECL when an appropriate potential is applied between the electrode and counter electrode pair waveform. One with an electrical potential suitable for triggering an ECL emission is applied by means of an electrical connection generating device 19 to
<td>so</td><td>of</td><td>wave</td><td>18</td><td>various</td><td>pairs</td><td> 16</td><td>of</td>
<td colspan="3">electro / counter electrode.</td><td>Any</td><td>signal</td><td>emi t ida</td><td>by</td><td>a</td>
<td>marker</td><td>ECL</td><td>Present</td><td>in several</td><td>domains</td><td colspan="2">link 14</td><td>is</td>
and detected by a light detection device 22
-30t registered and analyzed by a digital computing device
The iπveneion provides a method of detecting in a volume of a liquid, multi-component sample, a p lity of analyte of interest that may be shown at various concentrations
Generally speaking, a plurality of molar caneentrac ions less than 1/1000 can be detected. Preferably, a plurality of analytes can be detected at molar concentrations less than 1 / 1,000,000,000,000 from a multi-component sample.
The invention offers detection from a multi-component sample that can be carried out as assays which separate several labeled reagents not one i two from several labeled labeled reagents prior to that of the united labeled reagents. to electrochemical energy homogeneous assays, i.e. assays in which a plurality of unlinked labeled reacts and linked labeled reacts are exposed to electrochemical energy together
In the tests of the present invention, the electromagnetic radiation used to detect a particular year may radiate the radiation.
-31elec i romagnét ica correspond i. Entity other ana 1 i tos by identifying your. position and / or location as one or more characteristics of a pattern, said pattern corresponds to the pattern of the binding domains in the PMAMS.
In the homogeneous assays of the present invention, the electromagnetic radiation emitted by the bound labeled reagents either as an increase α or a
T decrease the amount of electromagnetic radiation emitted by bound labeled reagents compared to unlabeled reagents, or by detecting electromagnetic radiation emitted from sources that correspond in space to one or more patterns corresponding to the pattern of characteristics of the linking domains in the PMAMS.
In a specific example of the method of the invention presented in Figure 20, a sandwich test is carried out on a support (5) with a plurality of binding domain (BD) on its surface that are specific for bind to a particular analyte (An). When a sample suspected of containing the analyte is applied to the binding domains, the analyte is bound on the binding domains. Antibodies (Ab), which are suitable for selectively binding to analyte (An) and which have been labeled with a portion for ECL (MARKER) to form AbMARKER, are then applied to the analyte in the domains of 1 ace. After removal of the Ab-MARKER. In excess, unbound of the binding domains, a potential potential waveform is applied to the MARKER by means of electrodes (not illustrated) to trigger the electrochemiluminescence to trigger an ECL emission from any MARKER in the binding domains. The ECL signal is detected by a device r
light detection and registered by digitized computing devices (for example, in accordance with what is illustrated in 22 and 24 in figure 1).
Further embodiments, features and variations of the invention will now be described.
5.1. PREPARATION OF A LINKING SURFACE
To better understand the invention, a detailed description of the preparation of the binding domains on a support is provided. A configured set of binding domains on a surface that are specific to a plurality of analytes is referred to herein as a multi-array, multi-array, or PMAMS configured surface. PMAMS are prepared on a support, for example, by means of the self-assembled monolayers (SAMs) configuration (Ferguson et al., 1993, Macromolecules 26 (225: 5870-5875; Prime et al., 1991,
It starts 252: 1164-1167 $
Laibinis et al
1989, Science
245: 845-647? Kumar et al-, 1984, Langmuir 10 (5): 1498-1.511;
Saín et al., 1989, Angeui. Chem. 101: 522-528). Surface shaping methods also include the use of physical chemical etching (eg micro machining) (Abbott et al ,, 1992, Science 257: 1380-1382, - Abbott, 1994, Chem. Maten. 6 (5): 596-602 ), microlithograph (Laibinis et al., 1989, Science 245: 845-847), fixation of chemical groups on the surface using photoactive chemicals, (Sundberg et al., 1995, J. Am. Chem. Soc. 117 (49): 1205012057), and microstamping techniques (Kumar et al », 1994, Langmuir 10 (5): 1498-1511? Kumar et al., 1993, Appl. Phys. Lett. 63 (14): 2002-2004 ).
Surface configurations include procedures for the spatially controlled delivery of fluid or particles (eg, micro-stylus deposition (eg, using a microfluidic guide to deliver onto a surface using XY translation)), microcapillary filler (Kim et al ", 1995, Natura 376: 581), inkjet technology, or syringe suppliers. Combinations of these techniques can also be used to provide complete surface patterns. In Figure 5A, a support 600 with independent binding domains is shown so that they are represented, simply for illustration purposes, as geometric shapes 602 to indicate that different binding specificities may be present
-34 on a single stand. The surface 604 between the binding domains can alternatively be hydrophobic or hydrophilic to limit the deposition of binding reagent to form binding domains. Binding domains and / or the surface (s) between the binding domains can either ternationally tend to be non-specific binding or be resistant to non-specific binding, and / or may tend to bind binding reagents or okay resist the t
binding of binding reagents by means of emboldened or non-covalent interactions. In the case where nonspecific binding by hydrophobic interactions is not the desired method for fixing binding chemicals on the surface, detergent can be added to avoid the occurrence of nonspecific binding in ident 1.
The binding domains are, in general terms, 0.1 µm to 1 mm in terms of width or diameter, or wider dimension depending on the geometry of the domain. Surfaces are selectively derived to have specific binding components exposed for example to the ECL test solution. Additionally, non-specific interactions in the binding domains are decreased while maintaining a specific binding portion by incorporating portions such as polyethylene glycols on the exposed surface of the discrete binding domains (Prime et al -, 1993,
J. Chem Soc.
115: 10714-10721; Prime et; al., 1991, Science 252: 1164-1167;
Pale-Grosdemange et al., 1991, J. Am. Chem. Soc. 113: 12-20).
PMAMS can contain approximately 2 to 200 thousand link domain ions. For reference, the number of link domains is 50 to 500. In other ways, the number of link domains is 25 to 100.
The backing can include elastomeric materials, metals, alloys, composite sheets, semiconductors, insulators, silicon and / or layered materials, etc. without polymeric materials. Derived elastomeric supports can be repaired, for example, as provided by Ferguson et al., 1993, Macromolecules (Mac romomolecules) 26: 5870-5875; Ferguson et al., 1991, Science 253: 776-778; Chaudhury et al., 1992, Science 255: 1230-1232.
The surface of the support on which the PMAMS are prepared can contain various materials, for example, meshes, filters, fiber materials, gels, solids (for example formed of metals), elastomers, etc. The supporting surface may have various structural, chemical and / or optical properties. For example, the surface may be rigid, or flexible, flat or deformed, transparent, translucent, partially or fully reflective, or opaque, and may have composite properties, regions with different properties, and may be a composite of
-36 various materials. The surface may have glare regions with patterned surface and / or patterned regions where catalysis according to the invention may occur on one or more surfaces, and / or a set of electrodes to which power can be supplied on one or more surfaces. The surfaces of the supports can be configured in any suitable way including a flat, spheroid, cuboid, and cylindrical surface. In a specific embodiment, the support carried by a PMAMS is a rod. In another embodiment, the support carrying a PMAMS contains carbon, for example graphite, glassy carbon or carbon black.
In one embodiment, a support carrying a PMAMS contains one or more carbon fibers. These fibers can be amorphous or graphite carbon. They can also be nanotubes, carbon buckey tubes or members of the fullerenes family.
In a preferred embodiment, a support bearing a PMAMS contains one or more carbon fibrils (Hyperion Fibrils (MR)) (US Patent No. 4,663,230). Individual carbon fibrils (as presented in US Patent Nos. 4,663,230, 5,165,909, and 5,171,560) can have diameters that lie within a range of approximately 3.5 nm to 70 nm, and a length greater than 100 times the diameter, an outer region of essentially continuous multiple layers of ordered carbon atoms, and a distinct central core region. Simply for illustrative purposes, a typical diameter for a carbon fibril can be between approximately 7 and 25 nm, and a typical length range can be from 1 pem to 10 pm.
Carbon materials can be made to form aggregates. As shown in US Patent No. 5,110,693 and references mentioned herein, two or more suitable carbon fibers can form microscopic aggregates of entangled fibrils. These aggregates can have dimensions that are within a range of 5 nm to several cm. Merely for illustrative purposes, a type of microscopic aggregate (cotton or CC) resembles a spindle or tangled fiber rod with a diameter that can be within a range of 5nm to 20pm with a length that can be located within from a range of 0.1 pm to 1000 pm. Again for illustrative purposes, another type of microscopic aggregate of fibrils (bird's nest, or BN ") may be approximately spherical with a diameter that can be within the range of 0.1 pm to 1000 pm. Larger aggregates of each type (CC and / or BN) or mixtures thereof can be formed (see below).
Fibrils that can be employed in a support include, but are not limited to, individual fibrils, aggregates of one or
-38 more fibrils, suspensions of one or more fibrils, dispersions of fibrils, mixtures of fibrils at fairgrounds (for example, oils, paraffins, waxes, polymers, gels, plastics, adhesives, epoxy materials, Teflon, metals,
1 organic liquids, 1 organic solids, inorganic solid, acids, bases, ceramics, glasses, rubbers, elastorners, molecules and biological media, etc. ) as well as combinations of the same
Fibrils can be magnetic in some cases and non-magnetic in others
The magnitude in which the fibrils can be magnetized or not magnetized is controlled by the amount of tasting that is found in the fibril as a result of the fibril production process, such a process
5,165,909, and 5,171,560. The PMAMS are located in, inside or in the
PMAMS can be generated from different types of surface bonding groups. Self-assembling monolayers that can be used to form a monolayer on a surface to which they bond, include without
Imitation to Icantiols (which bind gold to other metals), alk i 11 richlorosi (by which they bind silicone), alkanocarboxylic acids, (for example, which bind aluminum oxides) as well as combinations thereof. The monolayer can be formed first and then a chemical bond is used to bind the binding reagents. Bypass after auto-linking produces a more perfect bid imensiona1 crystalline packing of the monolayer on a support surface with fewer holes or defects. The monolayer can be derived with the binding reagents before or after self-assembly. Regular defects in the monolayer may be desirable and can be obtained by bypassing the monolayer on the support surface prior to self-assembly. If the derived group (eg, exposed linking group) in the binding reagent is statically large, you can create a tight packing surface at the exposed end, but with regular gaps in the metal surface. This is useful to allow charge to flow through these regular gaps into the ECL marked portions attached to the part that comes in contact with the sample solution.
The preparation of incomplete monolayers is known in the art. Other procedures for the preparation of incomplete monolayers include without limitation: formation of monolayers from dilute binding reagent solutions, termination of the reaction to the monolayer prior to completion, damage to more complete monolayers with irradiation ( for example ionic particles), chemical reagents or light. In one embodiment, repeated stamping without re-inking the stamp may
-4040 cause a range of defective monolayers (Milbur et al., 1995, Langtnuir, 11; 825).
PMAMS can be generated on the matrix surface. The matrices can be highly conductive, for example, metallic electrodes or conductive polymer films; or the matrices can be insulating; or the matrices can be semiconductor and / or medium conductivity. The matrix material can be an ionic conductor or a porous material. Such porous materials can be used as a support material and / or a conductive material and / or a filter material and / or a reed material 1 i zac i ¿n (for example allowing the passage of fluids, ionic species etc. ).
The porous material can be combined with additional materials. For example, composite structures can be made of porous materials with additional porous materials, conductive materials, semiconductor materials, conduit structures, and / or solutions (eg, ionic fluids). Such compounds may be sheet-like structures, sandwich-like structures, and / or scattered compounds. A solid matrix can be used which is a porous material supported on a metal electrode. Alternatively, a porous material is found between conductive materials, semiconductor materials, or a combination of materials.
-4141 semiconductors and conductors. One or more binding domains may be on a continuous plate of the porous material and / or may be located on a plurality of discrete objects in the holder each with one or more binding domains. The surface of porous material (gel) can be flat, hemispherical, or any regular to irregular shape and / or can have a variety of physical properties (for example, elastomeric, rigid, low-density, high-density, density gradient , dry, wet, etc.) and / or optical properties (for example transparent, translucent, opaque, reflective, refractive, etc.) and / or electrical properties (for example conductive, semiconductor, insulating, variable conductive, for example wet vs. dry, etc.)
A channel configuration can be formed in the matrix. The layers of porous material can be from 5 microns to 2000 microns thick. The layers of porous material can also have a thickness greater than 2 mm.
The pores can extend partially and / or totally through the material or it can be part of a pore network. These pores can be dimensional and range from approximately 50 angstroms to 10,000 pm. In a preferred embodiment, the material has some pores with dimensions ranging from 200 angstroms to 500 angstroms and some pores with dimensions ranging from 0.5 pm to 100
-42 The porosity of the material can be constant throughout the material or it can be increased or decreased depending on the position of the material. The material can have a wide range of stops of different sizes distributed in a disorganized and / or random manner.
The porous material can be a composite of more than one material.
T
For example, the material may have some pores large enough for objects as large as biological cells to pass through, some pores may be large enough for protein or antibody-sized biological media to pass through, some pores may be sized to pass through. only small organic molecules (<1000 molecular weight), and / or combinations thereof.
The porosity of the materials can be such that one or more molecules, liquids, solids, emulsions, suspensions, gases, gels and / or dispersions can be dispersed in, in and / or through the material. The porosity of the material is such that biological media can be dispersed (passively activated α) or forced by some media in, within and / or through the material. Examples of biological media include, but are not limited to, whole blood, fractionated blood, plasma, serum, urine, protein solutions, antibodies or fragments thereof, cells, subcellular particles, viruses, nucleic acids, antigens, glycoproteins, 1-butterfly carids, fluids. , glycoproteins, carbohydrates, peptides, hormones or pharmacological agents. The porous material may have one or more layers of different porosity such that the biological media can pass through one or more layers, but not through other layers.
The porous material may have the ability to withstand a current caused by the flow of ionic species. In further refinement, the porous material is a water swollen porous gel, for example polyacrylamide or agar. Various other gel compositions are available (eg see Soane, DS Polymer Applications for Biotechnology; Soane, DS, Ed.; Simon & Schuster: Englewood Cliffs, NJ, 1992 or Hydrogels in Medicine and Pharmacy, Vol. Ι-ΣΙΙ ; Peppas, NA Ed. ; CRC Prese: Boca Ratón, FL, 19Θ7). Binding domains can be fixed on matrices using covalent and non-covalent bonds. (Numerous reviews and books have been written on this subject; some examples are Tampion J «and Tampion MD Immobilized Cells: Principies and Applications Cambridge University Pressí NY, 19S7; Salid Phase Biochemistrys Analytical and Synthetic Aspects Secuten, WH Ed., John Wiley and Sons: NY, 19Θ3; Methods in Enzymology, Immobilized Enzymes and Cells, Pt. B Mosbach, K.
-44ί
Ed., Elsevier Applied Science: London, 1988; Methods in Enzymology, Immobilized Enzymes and Cell, Pt-C Mosbach, K. Ed., Elsevier Applied Science: London, 1987; Methods in Enzymology, Immobilized Enzymes and Cells, Pt. C Mosbach, K. Ed., Elsevier Applied Science: London, 1987; see also Hydrogens in Medicine and Pharmacy, supra). For example, a protein can be fixed on a crosslinked copolymer of polyacrylamide and N-acryloyl succinimide by treatment with a protein solution. The binding domains can also be integrated into a porous matrix in a step prior to polymerization or gelation. In one embodiment, the binding domains can be attached to non-crosslinked polymers through the use of various coupling chemistries. The polymers can then be crosslinked (eg using chemistries including amide linkages, disulfides, nucleophilic attack on epoxides, etc.) (See for example: Pollack et. Al., 1980, J. Am. Chem · Soc. 102 ¢ 20) : 6324-36). Binding domains can be fixed on monomeric species that are then incorporated into a polymer chain during polymerization (see AdaIsteinsson, 0-, 1979, J. Mol. Catal. 6 (3): 199-225). In another embodiment, binding domains can be incorporated into gels by trapping the binding domains in pores during polymerization / gelation or by permeating the binding domains in the porous matrix.
-45 and / or film, analogously, binding domains can be absorbed on the surface of porous matrices (for example gels and polymer films) by non-specific adsorption caused for example by hydrophobic and / or ionic interactions. Biotin can be used for sale as the binding or binding agent. Avidin, streptavidin, and other biotin-binding agents can be incorporated into the binding domains.
t
PMAMS can be generated in porous materials (eg gels) with various pore sizes and solvent content. For example, polyacrylamide gels with various pore sizes can be prepared by varying the acrylamide concentration and the degree of crosslinking. In such PMAMS with smaller pore sizes than analyte, bonding reactions will occur substantially at the gel surface. In this case, fi 1 traction and / or electrophoresis through the gel can be used to concentrate analytes in the gel surface and to modulate the kinetic aspects (eg increase the rate) of the binding reaction. Faster kinetics are helpful in rapid tests (eg short times to results) and can generate increased sensitivity in a shorter period of time.
In PMAMS with pore sizes larger than the analyte, bonding reactions can occur on the surface as well as in the medullary part of the gel. In this case, rfi 1 traction and / or electrophoresis can be used to increase the bonding synthesis and unbound species from the surface.
Gels PMAMS can be stored in a wet state and / or can be stored in a dry state and reconstituted during the test. The reagents required for ECL assays can be incorporated into the gel prior to storage (by permeation into the gel or by incorporation during gel formation) and / or added during the assay,
Configured binding domains of a PMAMS can be generated by applying droplets or microdroplets that contain each binding domain in the matrix in a liquid form on a substrate. Solidification and / or gel formation of the liquid can then be caused by various well-cognated techniques (polymarization, crosslinking, cooling below the gel transition point, heat). Agents that cause solidification or gelation can be included in the drops, such that at some point after delivery, the drops solidify and / or form gels. Subsequent treatment (eg, light exposure, irradiation, and / or reduction-oxidation potential) can be employed to cause sol ification and / or gelation. In other embodiments, such droplets or microdroplets may be in pastes, prepolymer mixtures, groups of particles, and / or substantially solid droplets. Additionally, vapor phase deposition can be used.
Configuration can also be accomplished by forming a layered structure of matrices each containing one or more binding domains. For example, bound pink aga (by standard chemical procedures) can be emptied with an antibody into a container and allowed
gel is formed by cooling. Subsequent layers containing other antibodies could then subsequently be emptied into the first layer and allow a gel to form. Cutting this layered structure provides a continuous surface featuring a plurality of different link domains. Such cuts can be sharpened and other cuts can be cut to create an even density PMAM5 surface.
Alternatively, major lines of linking domains.
of an array containing a given link element are placed adjacent to each other and / or stacked. Such structures can also be cut into sections and used as a PMAMS surface.
Configuration can also be accomplished by taking advantage of the ability of some matrices to achieve separation. For example, a mixture of nucleic acid probes can be separated by electrophoresis on a polyacrylamide plate, generating a surface with a
-48p lura1ity from different linking domains »
Microfluidic guides can also be used to prepare the ΡΜΑΜΞ binding domains on a support. A partial list of micro-fluid guides includes hollow capillaries, capillaries made from a matrix, and / or attached to a matrix (for example, a porous or solvent-swollen medium), solid supports that can support a thin film, or well drop of liquid. The capillary can be solid and the reagents can flow 'along the outer surface of the capillary, a reactive fluid reservoir can be exposed to the tip of a porous matrix that comes in contact with a PMAMS surface. For example, the reagent reservoir can be filled continuously or periodically such that a given porous matrix tip can reproducibly deposit reagents several times (for example alkantiols to form monolayers and / or binding reagents etc.). Additionally, variation in tip porosity can be used to control the flow of reagent to the surface. Different or identical binding reagents may be present in a plurality of capillaries and / or multiple distinct binding agents may be present in a given capillary. The capillaries contact the PMAMS surface (eg, configured SAM) in such a way that certain regions are exposed to the link reactants to create discrete link domains.
Different binding reagents are supplied concurrently from the fluid guide assembly on a metal surface, SAM, etc., as desired. Microfluidic bulges can also be used to ink a microstamp with a desired molecule prior to application to the supporting surface. For example, identical microfluidic guides.
can be used to apply di. There are binding reagents attached to a portion that promotes adsorption on the surface of the support (for example, a thiol 1 ib re in a hydrocarbon linker that promotes adsorption on gold), to form a PMAMS. Therefore, for example, an inked microstamp using microfluidic guides with antibodies of different specificities that have a built-in linker with a free thiol can be used to apply such antibodies to desired areas on a gold surface to form domains. discrete link of a PMAMS.
Another approach to supplying a configured fluid includes the use of microprinting devices that deliver microdrops of fluid by ejecting the droplet through a small hole (for example an ink jet printer). Droplet ejection in these devices can be caused by different mechanisms
-5050 including slow motion, electrostatic charge, and / or pressure from a part device. Configuration of more than one liquid can be accomplished through the use of multiple ports and / or an appropriate port and valves.
In a method for the preparation of a PMAMS, microfluidic guides are employed to deliver (preferably concurrently) directly onto discrete regions on a surface droplets containing the desired binding reagents to form discrete binding domains. All 10 binding reagents can contain a functional chemical group that forms a bond to a chemical group on the surface to which it is applied. In another variation, droplet binding reagents are nonspecifically absorbed or bound on the surface (eg, dried on the surface).
Alternatively, the drop (s) deposited on a surface contains reagents that can form a matrix. This matrix can be a solid, polymer or gel.
Matrix formation can be carried out by evaporation of the solvent. It can be by polymerization of monomeric species. It can be by crosslinking preformed polymers. It can be carried out by modulating the temperature (for example cooling and / or heating). It can be carried out by other methods. For example, a polymeric species can be cooled through
-5151 from transitional cooling or by reagent that causes gel formation. The solid matrix may be induced by measurement. before the addition of reactive species formation in an electrode <including by means of light reagents that initiate the generation of the addition induce solidification or the formation of cooling or heating. Additionally, it can be formed eg by gel formation or polymerization).
In a preferred technique, configured hydrophobic / hydrophobic hours can be used to avoid spreading of applied fluids or gels. Such a fluid may contain surface binding reagents or a support to form the PMAMS.
In this case, the to bind on hydrophobic / hydrophobic helps to produced a discrete surface area and to bind reagents ge 1 to a binding domain using such an edge to limit the domain of
Ternat i vately, forming a defined region contents when deposited in an example, the edge aid be employed to limit the
In addition, areas already fluid bond matrix on the inside of a surface
Due to hydrophilicity / hydrophilicity, it can drop to a defined region, and bihydrophobic bihydrophobes may present groups that may be
-52 incorporated (for example covalently or covalently bonded) in the matrix, but I have a more stable matrix adhesive on the substrate (Itaya and Eard, 1978, Anal. Chem. 50 (10: 1487-1489 In another technique, the applied fluid or gel is the sample containing the analyte of interest, and the sample is applied on a prepared PMAMS. In a preferred example, capillaries containing hydrophilic solutions can be used to deposit a solution in discrete areas, creating hydrophilic domains surrounded by hydrophobic regions. Alternatively, hydrophobic binding domains surrounded by hydrophilic regions can be employed with a hydrophobic fluid containing binding reagents or analyte (s)).
Hydrophobic eh idrof i 1 ico are real terms, in relation to each other and / or in relation to the sample to be applied, that is, in such a way that the spreading or wetting of a sample of fluid or gel applied to the domains link can be controlled. In addition, controlled deposition of a solution from a set of microfluidics can be accomplished using surface physical characteristics (eg, surface wells or channels). A microfluidic guide can be included in a cassette, or more preferably, be used to apply specific reagents to a support prior to use. More than one chemical bonding procedure can be applied to the same support surface and / or a surface with both hydrophilic and hydrophobic bonding domains can be created using multiple stamps. For example, an area where a 1-position hydrophilic bonding domain is desired and a 2-position hydrophobic bonding domain may be prepared as follows. A first hydrophobic stamp is made which has a disc in position 1 and a larger ring in position 2. A second hydrophobic stamp is carried out with a disc 10 in position 2 that fits just inside the ring monolayer left by stamp 1. Finally, the surface is washed with an hydrophobic solution of monolayer components.
In particular, a PMAMS is generated by microcontact printing, i.e. stamping. The monolayer applied in this way is composed of a surface bonding group, for example, in the case of a gold surface, a tial group with an alkane spacer (for example, <CH2) n) is preferred. A spacer group is linked (preferably covalently linked) with a linking group A. A can be, for example, avidin, streptavid i na or biotin or any other suitable binding reagent with a complementary binding element. <sup>M</sup>B<sup>OR </sup>available. The A: B bond may be covalent or non-covalent and some chemical bonding techniques known in
The technique can be used in accordance with that presented, for example, by Bard et al. (North American Patents Nos. 5,221,605 and 5,310,687).<sup>M</sup>B '* binds additionally with a binding reagent such as an antibody, antigen, nucleic acid, pharmaceutical substance or another suitable substance to form a binding domain that can bind one or more analytes of interest in a sample to be tested . B may also be attached to an ECL MARKER or marker. Link group B can be supplied to SAM by means of a set of microfluidic or capillary guides (Figure 5A-5C) that can place a plurality of reagents "B" with different bond surface specificities on link A "Monolayer. A and B may also be bonded prior to attachment to the monolayer. In Figure 5A, shape-independent link domains are depicted, simply for the purpose of illustrating as geometric shapes 602 to indicate that different link specificities may be present on a single support 600. Figure 5B provides a top view of an assembly 606 of microfluidic guides (eg capillary). Points 610 are the sectional guides. Figure 5C provides a side view of the assembly 60Θ of microfluidic guides. The 1 lines emerging from the top and bottom are the individual 610 microfluid glues. The forms
-5555 Geometric 612 in the lower aspect represent specific binding domains formed by supplying the binding reagent from each individual capillary.
By way of example, after the first stamping presented above, the bare surface regions (for example, gold) may react with a second alkantiol that does not have the chemical properties of A bond and is hydrophobic / hydrophilic as opposed to the first monolayer f above. In this way, specific binding domains are prepared on a surface.
A binding reagent that is specific for an analyte of interest can be used for each binding domain, or a binding reagent that specifically binds to several analytes of interest can be used.
In another variant, a support surface may be stamped multiple times by materials (eg, binding reagents, ECL-markers, SAMs> having different binding chemical properties and / α different binding portions as shown in Figure 5A above. .
The binding reagents that are configured can be stable and / or robust chemical groups (for example, that survive the conditions to which they are subjected) that are poster-linked to less stable or robust binding groups, Multiple bonds can be used to optimize the conditions of each step in the preparation and in the
<td>surface of</td><td>PMAMS and / or simplify the manufacture of the</td>
<td>surface of</td><td>PMAMS. For example, a first surface of</td>
<td>PMAMS can</td><td>f I opened, I got tired in a generic way and then</td>
be modified to create different PMAMS surfaces »
<td>another example,</td><td>a generic PMAMS surface can</td>
<td>react with</td><td>a mixture of solution or reagents of</td>
bind themselves containing binding domains that are directed to particular regions (eg, r binding domains) on the surface of PMAMS. For example, a binding domain pattern each representing a different oligo (nucleotide) sequence binds to the surface
This surface is then treated with a solution containing a mixture of secondary binding reagents, each bound to a sequence of oligo (nucleotide) coiip 1 ementary to the surface. In this way, the configuration of these secondary link elements can be achieved. Preferably, the oligo sequences (nucleotides) are 6-30mers of DNA.
Some 6-30mer sequence sets may contain substantially similar sequence complementarity such that the approximate binding constants for hybridization are similar within a given set and are discernibly different from less complementary sequences. In another embodiment, the secondary binding elements are proteins (eg, antibodies).
Methods described to inhibit wetting or dispersion of applied reagents or sample on a surface are described in section 5.13 below, and these methods can also be used in the preparation of PMAMS (and / or in sample application).
The applied potential (for example, from the electrode / counter electrode pair) can be used to further control the tank and / or the dispersion of reagents and / or samples (see, for example, Abbott et al. , 1994, Langmuir 10 (5): 1493-1497).
The PMAMS binding reagents can be located on carbon-containing materials. They can also be located in individual carbon fibrils or the PMAMS binding reagents can be located in aggregates of one or more fibrils. In these modalities, the PMAMS binding reagents can be located on single or multiple fibril suspensions, diper fibril ions, fibril mixtures with other materials (described by way of example above) as well as combinations thereof.
The PMAMS binding reagents can be located on a plurality of individual fibrils and / or aggregates of fibrils located on or within or in the vicinity of a support. In one example, the binding reagents are located on dispersed individual fibrils or aggregates of * fibrils. These fibrils or these aggregates of fibrils
-58 can be located 1 mind spaced in your home; i rites in a medium, and may be your linking domain as defined in this application. In another example, such individual link domains or a plurality of such link domains are located in spatially distinct regions of the support. By way of non-limiting example, such individual link domains or sets of link domains may be located in depressions, cavities i
and / or holes in the support. In a further example, individual binding domains or a plurality of domain can be located in water droplets, gels, elastomers, plastics, oils, etc., which are located on the surface of the support. In yet another example, individual binding domains can be located on the support by a coating (which can be configured) having different binding affinities for different binding reagents and / or binding / fibril reagent sets.
The binding domains are preferably located on a plurality of individual and / or aggregated fibrils of fibrils and may be prepared on a support by one to several microfluidic guides (eg, a capillary). Different or identical binding reagents may be present within or on a plurality of microfluidic guides and / or multiple distinct binding agents may be present within or on a given microfluidic guide.
Capillaries can come into contact with the support (matted) and / or can supply the surfaces being scanned or translated relative to each other (ie a pen-like writing method). The microfluidic guide can supply the binding reagents located in the fibrils on the support in such a way that certain support regions are exposed to the * fibril binding reagent complex (s) to create some) domain (s). ) discrete <s) link.
In a preferred aspect, different binding reagents, each present in a different microfluidic guide, are supplied concurrently from the set of glues in the holder. In one example, binding reagents and / or the fibrils in which they are located are derived with a chemical functional group that forms a bond (eg, covalent or non-covalent interaction) on the surface of the support. In some embodiments, the binding reagents and fibrils are either non-specifically linked or adsorbed on the surface. In yet another aspect, the binding reagents located in the fibrils can be supplied in depressions, cavities and / or holes in the surface of the support. In another example, the binding reagents are supplied to a surface coated with a material 1 that has a stronger or more binding affinity.
-60ι weak for certain binding reagents or binding reagent / fibril assemblies and thus creates domains of the reagents that are spatially and distinctly located from other binding reagents.
The binding reagents are located on one or more identical or aggregated iv fibrils or aggregates of magnetic fibrils. In such a case, a magnetic support can attract the binding reagents located in magnetic fibrils to the support.
<td>The support</td><td>may contain multiple regions</td>
<td>magnetic and</td><td>surrounded by regions that are not magnetic.</td>
<td>Reagents</td><td>link located on magnetic f ibri1 as can</td>
locate in magnetic support regions. In one aspect, the support may contain one or more distinct regions that are magnetic and surrounded by non-magnetic regions, and the strength of the magnetic field in the magnetic regions can be modulated or switched. In this aspect, the use of such a modulated or switchable magnetic field helps to fix or release the binding reagents located in fibrils from the surface of the support and in this way they can serve to adjust or mix said domains.
Generally speaking, there are between 2 and 100 million link domains and preferably between 25 and 500 domains.
The binding domains can be located on working electrodes and / or the counter electrode.
-61 The different modalities described here for different types of PMAMS, mounts, and electrodes and configurations thereof can also be practiced in combination with each other.
PMAMS supports can be preserved (for example, by protective surface coatings, surface drying 1, robust vacuum or inert atmosphere packaging, refrigeration and related methods) for use r
later.
5.2. LINKING REAGENTS
The binding domains of the invention are prepared to contain binding reagents that specifically bind to 1 minus one ana 1 i to <1 igando) of interest. Binding reactants in discrete binding domains are selected such that binding domains have the desired binding specificity. Binding reagents can be selected from any molecule known in the art that can, or can putatively, specifically bind to an analyte of interest. The analyte of interest can be selected from among the rites in section 5.10 below, ECL tests that can be carried out. Accordingly, binding reagents include receptors, receptor ligands, antibodies, or binding parts thereof (eg, Fab, <Fab> '2>, proteins or fragments thereof, nucleic acids, oligonucleotides, glycoproteins, polysaccharides , antigens, epitopes, cells and cell components, subpart cells, carbohydrate ions, enzymes, enzyme substrates, organometallic computations, viruses, prions, viral raids,
1 (peps, fatty acids, lipopolysaccharides, peptides, cellular metabolites, hormones, pharmaceutical agents, ranks i za rites, barb i burates, alkaloids, non-biological polymers, biotin, av id i na, st reptav idi na, organic bonding compounds such as palm tree resin, ipop rotate, cytoe inas, lymphocytes, hormones, synthetic polymers, organic and inorganic molecules, mimic
Nucleic acids and oligonucleotides can refer to
DNA, RNA and / or oligonucleotide analogues oligonucleotides including but not limited to those dots containing modified bases or modified sugars, oligonucleotides containing structural chemistries other than phosphodiester linkages (see, eg, Nielsen, PE (1995) Anriu Rev. Biophys. Biomcl. Street. 24 167-183), and / or oligonucleotides, which have been synthesized or modified to present chemical groups that can be used to form icovalent or non-emboliant fixations) with other molecules (where we define a nucleic acid or aligo (nucleotide) as containing two or more nucleic acid bases and / or nucleic acid base derivatives).
The PMAMS of the present invention may have a plurality of described binding domains comprising at least one binding domain containing binding reagents that are identical to each other and differ in terms of the specificity of the reac Link you contained within other link domains, to provide the link of different analysts of interest through different link domains. By way of example, such a PMAMS comprises a binding domain containing antibody to thyroid stimulating hormone (TSH), a binding domain containing an oligonucleotide that hybridizes to the hepatitis C virus (HCV), a binding domain containing an oligonucleotide that hybridizes to HIV, a binding domain containing an antibody against an HIV protein or glycoprotein, a binding domain containing an antibody against prostate-specific antigen (PSA), and a binding domain containing an antibody 20 against hepatitis B virus (HEV), or any subset thereof.
A PMAMS can have a plurality of discrete binding domains some comprising a binding domain containing within it binding reagents that differ in binding specificity, such that a
-6464 single link domain can link to various analytes of interest. By way of example, such a PMAMS comprises a binding domain containing both antibody to a T-cell antigen receptor and antibody to a T-cell surface antigen such as CD4.
A PMAMS may have a plurality of discrete binding domains comprising (i) at least one binding domain containing binding reagents that are identical to each other and differ in specificity from at least one of the binding reagents contained within from the other linking domains; and (ii) at least one binding domain containing within the binding reagents that differ in binding specificities. By way of example, a PMAMS is made which has (a) at least one binding domain containing single identity binding reagents, eg, antibody against a T-cell antigen receptor, eg, alpha, beta T cell or receptor for gamma, delta T cell antigen, thus allowing this at least one binding domain to bind to all cells expressing this T cell antigen receptor; and (b) at least one binding domain containing two different binding reagents, eg, antibodies to T cell antigen receptor and antibodies to CD4, thus allowing this at least one binding domain to bind to CD4 lymphocytes * What
-65 express this T-cell antigen receptor (ie, a subpopulation of T lymphocytes).
In another fashion, at least one binding domain contains binding reagents that are different molecules and have the same binding specificities (for example, binding reagents such as epidermal growth factor and epidermal growth factor).
A plurality of reagents the binding reagents are different and have different binding specificities, recognize the same analyte (in an alternative modality, different analytes are recognized)
For example, when the analyte is an analyte numerous binding portions (eg, a cell that has different binding surface antigens that bind to different binding portions will recognize the same analyte. As another example, antigen antibodies Different cell surfaces in a single cell will recognize the same cell.Additional example, antibodies from ep i different types of a single antigen can be used antigen.
In another embodiment that binds (n) as reagents of en ad ic iona1, only specifically with
Lace to recognize the binding reagent (s) a unique analyte of interest are present in one or more binding domains. Ternatively, binding reagents that specifically bind to more than one analyte of interest are present in one or more binding domains (eg, a cross-reactive antibody). In a particular design, binding reagents can be used that bind a class of analytes, for example, with similar characteristics. Binding domains can also be incorporated into a PMAMS r that contain binding reagents that are specific for a desired standard analyte and that are used as an internal standard (for example a binding domain that may be in contact with a sample containing a defined amount of an analyte to which the binding reagents bind). Multiple binding domains containing specific binding reagents for the same analyte (s) may also be incorporated into a PMAF1S to allow statistical averaging of the analytical results. The binding reagents do not have to be analyte but can be the same binding portion only specific for the same to be identical, thus recognizing it in the analysis. Accordingly, several link domains can be prepared (eg within a range of 100 million) that specifically link to the same link portion, so that 1 ECL readings can be statistically averaged to control for variation and improve precision. The plurality of binding domain in a PMAMS can be specific for either a control analyte or an analyte of interest, or both, on a single carrier. »As another example, one or more discrete binding domains can be prepared with a concentrae cognate initial ion of ECL markers. The integrated ECL layer serves as a control for monitoring, for example marker degradation and the effects of temperature.
A binding reagent can be used which is a specific enzyme for a substrate (said substrate is the analyte of interest), where a product of the enzymatic reaction on the substrate is a reporter agent (an agent that can be detected ), for example, a product that triggers an ECL reaction, a fluorescent molecule, a substrate that changes color upon contact with the appropriate enzyme (for example, a chromogenic substrate for horseradish peroxidase), etc. In an example of such an embodiment, the enzyme used as the binding reagent is glucose dehydrogenase (GDH) which can be used to detect or measure glucose in a sample. An ECL marker is located within or near the binding domain that contains the GDH. NADH is produced by the action of the enzyme on glucose, NADH can react with ECL markers to promote ECL (Martín et al., 1993, Anal. Chim. Acta 281: 475).
-6868
Binding domains containing binding reagents that i nc: down-link the background (i.e., bind to a binding moiety present in the analyte of interest as well as other analytes in the sample) can be used to increase the ratios between signal and noise during the detection or measurement of electrochemiluminescence. By way of example, when the analyte of interest is a specific cell subpopulation (eg CD4 + cells) and r
the sample is a sample of fluid (eg blood) containing cells from a patient, sialic acid antibody can be used as a binding reagent to increase background binding to virtually all cells in the sample (since that sialic acid is a component of virtually all cell surfaces), and an antibody to a cell surface antigen (eg, antibody to CD4) can then be used as the same binding domain or different from that containing a binding reagent (in the sialic acid antibody binding domain either).
5.3. VOLTAGE WAVE SHAPE
<td>The waveform</td><td>voltage (change in potential / time</td>
<td>electrical) printed</td><td>in the plurality of electrode pairs and</td>
<td>counter electrode</td><td>the ECL cell should be enough to</td>
trigger an ECL reaction. This waveform of
-69voltage is usually in the form of a uniform voltage sweep that begins at a first voltage, constantly moves to a second voltage, returns through the first voltage to a third voltage, and then returns to the first voltage again. For example, the waveform can first voltage within a range of -0.5 to up to a second voltage located within a range of at 2.5 volts, and return through the first voltage to a third voltage located within from a range of OO to -1 volt. In another example, in simpler waves, the voltage can be changed from 0.0 to +3.5 to
OO Voltage waveforms can incorporate step function ramps, voltage waveforms can incorporate a period of time when the voltage remains fixed at a potential. The applied potential can be controlled in relation to one or several electrodes, or they can be presented from the use of reference electrodes. Optionally, a negative power1 can be used. Consequently, the voltages used to induce the
ECL from the present invention will be readily selected to achieve a specific intensity of ECL marker and the test medium.
In some applications, the voltage varies preferably as the light emitted from the bonding domain is measured. This is especially important in determining the threshold value of the electric field necessary to cause the link domain to emit light. In this case, the electrical patent 1 5 applied to the bonding domain starts at a value believed to be less than the threshold required to emit light, and a first measurement of the emitted light is made. If light is not measured, or if the light is below af
uflibra1 predetermined, the electrical potential applied through the pair of electrodes is increased under computer control, such as by means of a computer-controlled voltage source, and another light measurement is performed. This process can be repeated until the appropriate predetermined amount of light is received. In this way, the applied voltage can be used as the test signal.
The ECL signal can be generated from a voltage of
AC applied to the pairs of electrodes.
the person with the correct knowledge in the matter familiar with the voltage and current environments in accordance with what is presented, for example, in the Patents
North American Nos. 5,324,475 and 5,068,088 can easily select optimal operating voltages and voltage sweep to trigger an ECL emission.
The potential required to generate ECL can be generated
-71i for illumination of the working electrode surface if the working electrode is a semiconductor or contains another portion that generates an electric current in response to light.
5.4. ADDRESSABLE ELECTRODES AND METHODS FOR USING THEM
Numerous methods can be employed to supply power to the plurality of electrode / counter electrode pairs. Various illustrative techniques are presented in Figures 1418. In these figures, 4 electrode / counter electrode pairs (101, 102, 103, 104 and a waveform generator that is typically a digital computer and which preferably it is the same computer used to process the ECL detected by the detection means.
In Figure 14, each pair of electrodes (counter electrodes 101-104 is individually driven by a pair of lines connected to the waveform generator. By way of example, lines 105, 106 have access to the pair of electrodes / counter electrodes 101 A suitable waveform can be supplied by the waveform generator at any given time to one or more of the pairs of lines connected to the various pairs of electrode / counter electrode. To reduce the number of connections required to supply power to the electrode pairs, alternatives to the direct connection scheme are provided.
-7272 figure 14. For example, scheme of access of rows electric power to some is illustrated in figure 15 a and columns to provide or all the electrodes. In this scheme, one of the electrodes 201, 202 in each column of the plurality of electrode / counter electrode pairs is connected to a common electrical conductor 205 in a part 200, and each of the counter electrodes in each row of the plurality of pairs Electrode / Cantraelectrode connects to a lead 207, 208 on bracket 200. The conductors 205, 206 are connected to the connections CI, C2, respectively, at the support edge 200 and the conductors 207, 2O8 are so cut to the connections Rl, R2, respectively. Each of these connections is then caneeted via a separate line to the waveform generator. As a result, in the configuration of Figure 15, the number of required connections and signal lines from the waveform generator are reduced from 8 to 4.
In order to allow rapid and sequential delivery of power to each pair of electrodes, a computer controlled switching device is beneficial. The configuration of Figure 16 shows a plurality of electrodes connected to a first multiplexer 310. Several counter electrodes are connected to a second multiplexer
320. The first multiplexer is also connected to a first
-73 pole of a 330 voltage source that typically supplies the electrical potential that varies over time, see below. The second mulex iplexor is also connected to a second pole of the valta je source. Using the A0-A3 direction lines electrically connected to each of the multiplexers and connected to a latch 340, a computer processor 350 can direct the multiplexers to selectively connect any of the first electrodes to the first pole of the voltage source and any or all of the second electrodes to the second pole of the voltage source
As shown in Figure 17, various voltage sources are connected through separate sets of multiplexers to each of the electrodes. If a first electrical potential or range of electrical potentials is required on a particular pair of electrodes, the mu 11iplexers 410, 420 associated with the providing voltage source 430, this potential are driven by the compute processor 350, typically by of a bolt 340, thus connecting this particular voltage source with the pair of electrodes in question. If a different electrical potential or a different electrical patent range is required for another pair of electrodes, the multiplexers
440, 450 associated with these different voltage sources
460 they are driven by the computer processor, thus connecting this voltage source through the matched 440, 450 muitiplexers to the pair of electrodes.
If the electrode array in this embodiment has at least a portion of the electrode pairs that can be driven independently, as shown in Figure 14 or 15, for example, one pair of electrodes can be powered by a voltage source while that another pair of electrodes can be driven simultaneously with another source of voltage sources in Figure 17 can be replaced with a single voltage source connected to both sets of parallel multiplexers or, allowing two pairs of e 1 ec t rods to be driven from the same voltage source
Rather than source a duplicate set of mu 11iplexers for each voltage as shown in Figure 17, a plurality of voltage sources 520, 530 can be provided as shown in Figure 18. These voltage sources can be connected via of a computer controlled electrical switch 510 or switches for a single set of 310, 320 multiplexers. Bed shown in Figure 18, the computer could direct switch 510 to connect a particular voltage source to the multiplexers, and could also direct the multiplexers (by penalizing their address lines A0-A3) to connect the selected sources voltage with torque
-7575 specific desired electrodes.
Alternatively, the electrical potential applied to each of the pairs of electrodes in one embodiment can be varied. This is especially beneficial when using a cassette that has a plurality of different binding domains. Such a cassette may refer to a different range of electrical potential applied in different bonding domains. Several different modalities capable of varying the electrical potential to just each electrode are contemplated.
A computer controlled voltage source can be used to advantage. A computer controlled voltage source is a voltage source that can be directed by a computer to select a particular electrical potential to supply. At the end, it can be programmed to sequentially apply a particular range of electrical potentials for a predetermined period of time. In such a system, the power lines electrically connected to the computer and the voltage source allow the computer to program the voltage source to produce the particular electrical potential for application to the pair of electrodes that must receive power.
Additional methods of supplying power to the plurality of pairs of electrodes can also be employed. By
For example, a plurality of reference electrodes can be placed in close proximity to each of the various pairs of electrode and counter electrode to detect the voltage applied there. In this way, additional control of the voltage waveform can be maintained.
Figure 36 shows another embodiment of the invention; Electrode assemblies (3600, 3601) are supported on each of two surfaces (3602, 3603) separated by a pattern of gaps in an insulator 3604 (eg, a sheet of plastic with 3605 drilled holes). Each electrode can pass in a plurality of holes. If a potential is applied between one electrode on each surface, the current can only pass through an interval that is in contact with both electrodes, thus limiting the location of any electrochemistry or ECL. that can happen. In the preferred embodiment shown in the figure, the electrodes (3600, 3601) are sets of 11 lines on a holder. The two sets of electrodes on the two surfaces are oriented perpendicular to each other. Intervals on the insulation sheet are located only at the intersection of the electrodes on each surface.
This modality has the advantage compared to individually directed electrode pairs that fewer electrical conductors are required.
In an alternative embodiment, the 3604 isolator is omitted and
-771 the surfaces are placed in close proximity such that there is only a narrow gap between the two surfaces. In this 1-mode mode, a power of 1 to 1 each between electrodes on each surface will preferably cause current to pass through the intersection of the electrode (for example, where the distance between the electrodes is minimal)
<td>1imitating like this</td><td>the</td><td>Location </td><td>of</td><td>any</td><td>phenomenon</td>
<td>elect roqu im i. co o</td><td>all right</td><td>ECL you can</td><td colspan="2">occur.</td><td></td>
<td></td><td> 5.5.</td><td>DETECTION OF</td><td>THE</td><td>LIGHT</td><td></td>
<td>The light generated</td><td>by</td><td>1st issue</td><td>of</td><td colspan="2">ECL triggers is</td>
<td>detected by a</td><td colspan="2">detect associate</td><td>of</td><td>light or</td><td>you will detect</td>
placed adjacent to the apparatus of the invention. The light detector may, for example, be a film, a photomulti tube, a photodiode, an avalanche photodiode, a charge coupled device (CCD), or another light detector or camera. The light detector may be a detector Simple to detect sequential emissions or it can be a plural detector to spatially detect and resolve simultaneous emissions at single or multiple wavelengths of emitted light. The emitted and detected light can be visible light or it can be emitted as non-visible radiation such as infrared or ultraviolet radiation. The detector or detectors can be stationary or mobile. The emitted light or other radiations can be directed towards the detector or
-7878
<td>detectors by</td><td>means of lenses, mirrors as well as guides</td>
<td>fiber light</td><td>optics or light conduits (single,</td>
<td>multiple, fixed</td><td>or mobile) positioned in either</td>
adjacent to the binding surface of the cassette or the
<td>detector can</td><td>directly receive the light. Furthermore, the</td>
<td>PMAMS supports</td><td>and electrode surfaces themselves can</td>
<td>be used to</td><td>guide or allow the transmission of the</td>
light.
PMAMS can be formed on the surface of a set of light detectors in such a way that each
<td>detector receive</td><td>only light coming from a domain</td>
link. The set of light detectors can be a CCD chip, and the binding domains can be fixed (using standard coupling chemical reactions - on the surface of the semiconductor device.
Drops deposited on the linking domains, or on a second nearby surface, can be used as microlenses to direct or control the light emitted. Alternatively, a light detector can be oriented directly in front of the cassette; and various light focusing devices such as parabolic reflectors or lenses may be used in place of a light conduit to direct light from any one of several linking domains to the detector. Light emitted from at least two discrete link domains
-79i can be measured simultaneously or sequentially.
The error caused by thermal displacement, aging of the apparatus, or the electrical noise inherent in the light detectors can be controlled by means of a cutter device.<sup>11</sup> between the light mea surement device and the 1 in 1 meter being measured. The cutter can be any of the common metal cutters well known to people with certain cognates in the art, such as a rotating disc with slits or cutouts that allow light to pass through. Alternatively, the light may be cut by an LCD shutter, a set of LCD shutters, a valve, or solid state light valves or the like. Alternatively, a flat set of LCD shutters or solid-state light valves such as those known in the art of optical computing can be used. These devices are preferably located between the plane of the cassette and the conduit. light (or conduits) or light focusing devices that direct light from the linking domains to the light detector. In one embodiment, a shutter is located above each of the linking domains. When an LCD shutter or light valve is used, the shutters can be modulated at different frequencies to simultaneously provide speeds
Different cut -80i for different light emission link domains. Using this technique, a plurality of different light signals can be overlaid and these signals measured simultaneously by means of a single detector. An electronic bandpass filter 1 electrically connected to the light detector can then be used to separate the single electrical signal 1 into various electrical components, each corresponding to one of several
Γ individual light components. By cutting off the light, as mentioned above, or by using another mechanism well known in the art, an AC light waveform is created that allows removal by electronic filtering of the DC noise component.
Likewise, the ECL signal can be calibrated by comparing the previously determined results with standard reagents to correct for signal modulation due to reagent exhaustion.
5.. ECL SIGNAL ANALYSIS
Signals from a given link domain can have a range of values, and these values are correlated with a quantitative measurement to provide an analog signal 1. In another technique, a digital signal is obtained from each domain to indicate that an analyte is present or not present.
Statistical analysis is used for both techniques, and is
-8181 speci cally useful for translating a plurality of digital signals to provide a quant i tat i ve result. Some analytes, however, require a present / absent digital signal 1 indicating a threshold concentration. Analog and / or digital formats can be used separately or in combination. Other statistical methods can be used with GEFS. For example, it is possible to create PMAMS concentration gradients on a surface (Chaudhury et al., 1992, Science 256: 1539-1541).
This technique is used to determine ion concentrations by statistical analysis of the bond in the concentration gradient. Multiple linear sets of FilAtlS with concentration gradients can be preduced with a variety of specific binding reagents 15 different. Ion concentration gradients can consist of discrete binding domains that have different concentrations of the binding reagents.
The presence of control test systems on the binding surface of the cassette is an important element 20 to ensure the uniformity of each analysis to control signal variation (eg, variations due to ion degradation, fluctuations, aging of cassettes and other components, thermal changes, noise in the electronic circuit and noise in the photodetection device, etc.). For example, linking domains
-82 multiple redundants (containing identical binding reagents or different binding reagents that are specific for the same analyte) for the same year can be used. In the other example, analytes of known concentration or control domains of one
PMAMS are either linked in a brave way with a known amount of an ECL marker or a known amount of ECL marker is used in solution.
t
Tests carried out in accordance with this
The invention quickly and efficiently collects large amounts of data that can be stored, for example, in the form of a database consisting of a compilation of chemical information or research. The collected data can also be used for rapid personal or forensic identification. For example, the use of a plurality of nucleic acid probes when exposed to a digital sample of human DNA identif ies does not result.
Signature DNA that can be clinical or
5.7 PREPARATION OF
TESTS OF being used for a footprint easily used for
MULTIPLE ELECTRODES
The electrodes can, in general terms, have a width or diameter of
0.01 to 10 mm. In a preferred range, the electrode dimension pairs are 0.01 to i mm in terms of their (width or diameter or widest dimension depending on the geometry of the electrode pairs).
-83ι
Preferably, the electrodes are made from suitable conductive materials, such as, for example, transparent metal or semiconductor metal films (for example, gold or tin-tin oxide, scraped, as is well known in the art, for example, for the manufacture of liquid crystal displays and the like. In the assembled form of the cassette, a sufficient space remains between the first part and the second support to contain
Γ an analytical sample such as a thin film 10 or a wetted surface.
The electrodes can be made from materials containing carbon, carbon fibers, carbon nanotubes, and / or aggregates thereof.
The electrodes can be made from 15 carbon fibers. One or more individual fibrils and / or one or more fibril aggregates can be precessed to form a larger aggregate (US Patent No. 5,124,075).
This larger aggregate is a mat or mesh (hereinafter referred to as a fibril mat) wherein the fibrils may be entangled or interwoven.
Fibril mats typically have a surface area of between 50 and 400 m2 / gram.
By way of example, a fibril mat can be used as a working electrode, a counter electrode or a reference electrode in analytical and / or analytical electrochemistry.
-84 preparation. In one example, the fibril mat is used as an electrode for electrochemiluminescence (ECL).
The PMAMS binding domains may be supported by a mat of fibrils.
The PMAMS of the present invention has a plurality of discrete link domains, between which two or more may be identical to each other or may be different. The fibril mat supports one or more binding domains.
One or more microfluidic guides can be used to prepare a plurality of binding domains on a fibril mat. Different or identical binding reagents may be present in a plurality of microfluidic guides and / or multiple distinct binding agent may be present in one microfluidic guide.
In Figures 22A and 22B, several microfluidic guides 2201, preferably an assembly, are used to supply, preferably concurrently, to the fibril mat regions 2200, droplets containing the desired binding reagents to form domains Discrete Binding 2202. Binding reagents bind to portions present on the fibril mat. Binding reagents can either be adsorbed nonspecifically on the mat or dried on the surface.
Desired binding reagents are supplied to the fibril mat while suction filtration is applied to ι
The part, all or mat lateral expansion of the fibrils are prepared by compressing suspensions of carbon fibrils in a substrate to allow the suspension liquid to pass (by filter)
Examples of filters that can form fibril mats include filter paper, filters formed from polymeric membranes (for example, nylon), metallic fibers, fiberglass filters, and / or a combination of two or more. filter materials
One of skill in the art of which these materials are presented by way of example only of the many suitable solid materials possible
Figures 23A and
23B illustrate a fashion in which they can be manufactured by suction filtration »A horse dispersion 2301 is filtered using a 2300 filter optionally equipped with a 2303 filter membrane and / or a holder
-86i applied by means of a vacuum source 2305 to the filter by means of, for example, a filter flange 2306. A fibrilla mat 2304 collects in either of the membranes or both filter membranes 2303 and / or filter holder 2302 Fiber mat 2304, with or without filter membrane 2303, can be removed from the filter.
In another embodiment, fibril suspensions are passed through a filter using pressure. In one example, pressure is exerted on a mock fibril suspension 1 by compressing a limited layer of air and / or liquid above the suspension with a disk. In a specific example, the suspension of fibrils is limited in a syringe, the piston is a syringe plunger, and the filter is a disposable syringe filter (many filters of this type are well known to a person skilled in the art).
Fibril suspensions are either pushed into a filter by capillary action or are filtered by wicking the suspension into or through a filter.
In another embodiment, individual fibrils or aggregates of fibrils are slowly renamed cova on mats. Derived fibrils with photosensitive portions polymerize when exposed to light and irradiate with light.
The filter can be used to trap fibrils in its pores to form a composite mat where the filter
-8787 acts as a support. In figure 24, a fibril mat
2400 It can be prepared by passing a fibril paste 2401, supplied by a source 2402, between two large rollers 2403. In this process, which can be analogous to processes found in the manufacture of sheets of paper or polymer, the Rollers push the liquid from the suspension and a large, continuous mat of fibrils is produced from which smaller mats can be cut.
The fibril mats can be either free-standing (eg unsupported) or supported.
The filtration speed can vary to achieve desired varieties on the mat. For example, properties can be varied including the uniformity or not the magnitude of the entanglement of the fibrils or fibril aggregates, the thickness, the porosity of the mat, and / or comb i. I was born and my friends
Carbon fibril suspensions are limited and the
The liquid in which the fibrils are suspended are removed. For example, the liquid in which the fibrils are suspended evaporates. In another example, the liquid is removed by heating. In another the suspension is subjected to centrifugation and the resulting liquid (for example, the supernatant) is removed. In another example, the liquid is removed by evacuation
-88i
The suspension can be placed on one or more of the filters described above, and the suspension dried by evaporation. The suspension can be dried by heating or baked in an oven or the liquid can be removed by freezing and removing the liquid. In another example, the liquid is removed by evacuation with a pump. Many other methods well known to a person skilled in the art are available for removing liquids from a suspension.
Fibril suspensions suitable for carrying out fibril mats by filtration can be formed by dispersing one or more carbon fibrils in a liquid, almost solid or appropriate gel. Examples of suitable liquids include, but are not limited to, water, ethanol, methanol, hexane, methylene chloride, buffered solutions, sur fac tan tes, organic solvents, solutions containing biological media (eg, protein, antibodies or fragments thereof, cells, subcellular particles, viruses, nucleic acids, antigens,
1ipoproteins, palisaccharides, 1ipides, glycoproteins, carbohydrates, peptides, hormones or pharmacological agents, solutions of small molecules, precursors of polymers, solutions of acids or bases, oils and / or combinations thereof ).
A suspension of fibrils can be prepared by
-8989 dispersion of carbon fibrils in an aqueous solution by sonication roed io. In another fashion, a surfactant and / or detergent can be added.
The fibril mat can have a thickness of about 0.01 pm to 10,000 pm.
In preferred embodiments, the fibril mat has a thickness of between 1 pm and 100 pm. Spices are preferred in fashion, in which the fibril mats are located from 10 mm to 200 mm in width or diameter »
The fibrilla mat can be repeatedly washed and re-filtered to remove residual materials that remain from the suspension »
The fiber mats prepared by filtration or evaporation are heated (for example, in an oven) to remove 1 i res of the suspension 1 not removed by fi 1 traction.
Successive fi 11 ration steps can be used to form fibrils made up of one or more different layers that are either in contact with one or several other layers or that are in very close proximity to one or various other layers. The layers can be distinguished by several properties, including, without limitation, the differences in porosity, density, thickness, decrease in size of individual fibrils 25 and / or microscopic aggregates of fibrils, type, number,
-90ι and / or size of the fibrils aggregates, the chemical derivation of the fibrils (see below /, and / or the presence of other matter fixed on the fibrils.
Figure 25 is a multi-layer fibrilla mat
2500 which is prepared by successive filtration steps.
A layer 0.5 to 100 pm 2501 thick of flat fibrils forms the first layer; a 0.5 to 10 pm thick layer of 2502 fibrils incorporating portions as per i
e example poly- (et ilenglicoles) that resists the adsorption of proteins and other molecules form the second layer; a layer of thickness 0.5 to 5 pm 2503 that incorporates one or more link domains (vide supra) forms the third layer. The binding domains contain one or more 2504 antibodies, which can bind to an anal i to 2505. This antibody / ana1 ito complex can bind a 2506 labeled antibody.
The marker can be an ECL marker. In other embodiments, the marker can be one or more markers described elsewhere in this application. Such a multi-layered mat may be independent or supported on one of several possible supports described herein.
Multi-layer mats can be formed in which there are combinations of layers, in which some or all of the layers may be different.
The filter used to form the fibril mat, the fibrils and / or the fibril mat can be * coated.
-9191
<td colspan="2">In modalities</td><td>individuals,</td><td>the</td><td>coatings</td><td>saint</td>
<td>metallic.</td><td>These</td><td>reves timi entos</td><td>they can</td><td colspan="2">be configured from</td>
<td>such way</td><td>that></td><td>certain parts</td><td>are</td><td>coated and</td><td>others</td>
<td>pa rtes na.</td><td>In</td><td>an example the</td><td colspan="2">coating se</td><td>apic</td>
by electrodeposit. In another example, the coating is applied by electrodeless deposit.
The filter is coated with a metal, and the fibril is derived with a chemical functional group that forms a bond with said metal. The filter is either a metal screen or a sheet of metal.
The fibril mat may be flat or deformed, regular or irregular, round, oval, rectangular, or any of many shapes, rigid or flexible, transparent, translucent, partially or totally opaque, and may have composite or regional properties. of different compound or individual properties.
The mat can be a disk or a piece taken from a sheet.
A plurality of fibril matting can preferably be manufactured concurrently, and preferably as a whole. In one example, a set of microfluidic guides forms a plurality of fibril mats on a support as described above. In another, a set of filters, or a configured filter (for example, with regions of different porosity) is used to prepare a
-9292 set of fibril mats.
□ A mask with a set of holes (for example, a screen> is used to cover certain parts of a filter or support, and a plurality of straight fibrile mats are made concurrently by either filtration and / or evaporation.
Fibril mats can have a density of 0.1 to 3.0 grams / cm2. The mat can have a variable density. For example, mechanical force or pressure can be applied to the mat at different times to increase or decrease the density »
Fibril mats may have pores. These pores can extend partially and / or totally through the mat or they can be part of a pore network. These 15 pores can have dimensions that are roughly 50 angstroms at 1000 pm. In a preferred embodiment, the fibril mat has pores with dimensions ranging from 200 angstroms to 500 angstroms. Porosity
<td></td><td>of the mat can</td><td>depend on</td><td>the density of</td><td>the</td><td>mat,</td>
<td> 20</td><td>Inter alia</td><td> •</td><td></td><td></td><td></td>
<td></td><td>The porosity of the</td><td>mat can</td><td>be constant</td><td>in</td><td>all the</td>
<td></td><td>mat or else can</td><td colspan="2">increase or decrease</td><td>in</td><td>i fón</td>
from the position on the mat. The fibril mat can have a wide variety of pores of different sizes distributed in a disorganized and / or random manner.
-9393
The fibril mat may contain different regions of different pores. For example, the fibril mat may have one or more layers, each having a different porosity. Fibril mats may have columns of different pores running through the mat.
The porosity of the mat can vary by including different amounts of aggregates of carbon fibrils, where the aggregates have sizes, shapes, r
different compositions or combinations. In a particular example, a mat is prepared from individual fibrils, CC fibrils (described above), and BN fibrils (described above), or different combinations. For example, the fibril mat may have some pores large enough to pass biological cell-sized objects, some pores may pass biological media the size of protein or antibodies, some pores may pass only small organic molecules (with a molecular weight less than 1000), and / or combinations thereof,
The porosity of the mat can be of such magnitude that one or several molecules,
11quids, solids, emulsions, suspensions, gases, gels and / or dispersions can be dispersed in, within, and / or through the mat. The porosity of the fibril mat is such that biological media can be dispersed (either actively or passively) or
-9494 may be forced by some means on, into and / or through the mat. Examples of biological media include, but are not limited to, whole blood, fractionated blood, plasma, serum, urine, protein solutions, antibodies or fragments thereof, cells, subcellular particles, viruses, nucleic acids, antigens,
1poproteins,
i.posaccharides, 1 Ipides, g 1 icoprotein 1 ñas, carbohydrates, peptides, hormones or pharmacological agents. The fibril mat can have one or several different pore layers in such a way that a material can pass through one or more layers, but not through other layers.
The fibrilla mat is supported by or on another material. By way of example, the support material may be a metal, plastic, polymer, elastomer, gel, paper, ceramic, glass, liquid, wax, oil, paraffin, organic solid, or a mixture of two or more of them. . The material can be solid or liquid. If it is in a solid state, it can contain one or more holes or pores. In specific examples, the support can be a metal mesh, a nylon filter membrane or a filter paper. The support can be a conductor, semiconductor and / or insulator.
In a fashion 1 nity presented in North American Patents
No. 5,304,306 and 5,098,771, fibrils can be dispersed in another material. For example, fibrils can be dispersed in
-9575 oils, waxes, paraffin, plastics (for example, ABS, polystyrene, polyethylene, acrylonitrile, etc.), ceramics, tef 1on, polymer, the atoms, gel, and / or coffibi na ions thereof. Fibril dispersions in other materials are conductive. Fibril dispersions in other materials can be molded, pressed, formed, cast, spun, woven, and / or thrown in such a way that objects of a desired shape and / or configuration are formed. The fibril mat can incorporate other material, for example thin fibers, fragments or meta balls 1 to increase the conductivity of the mat. In another example, the fibril mat can incorporate other carbon, glass, and / or metal fibers of varying sizes, shapes, and density to create a different porosity that can be achieved with fibrils alone. In another aspect, the mat may incorporate magnetic beads (eg, DYNAL beads). In this last example, the accounts can either be used to change a mat property, or they can themselves be used as supports to freeze binding domains.
Other carbon fibers (eg carbon nanostructures, non-carbon tubes, bu.ckminsterful le reindeer, buckytubes, fullerenes, or combinations thereof) can be used in place of carbon fibrils.
They can prepare carbon fibrils with fixed chemical functional groups cova slowly on their
-96superf ic ie. As described in the publicae ion internaciona1 these chemical functional groups include, without limitation, COOH, OH, NH2, Νh id rox isucc inimide (NHS) esters, poly- (ethylene glycols), alkyl groups <(CH2) n ), and / or cornb i nations of the same.
These and other chemical functional groups can be used
Some chemical functional groups (eg, COOH, NH2, can be used to connect other such chemical functional groups and small molecules.
In many modalities, aster groups of
NHS is used to bind other α-material molecules that carry a chemical nucelof ί1 ico fune group (eg, an amine). In a preferred embodiment, the nucleophilic chemical functional group is present in and / or within a biomolecule, either naturally and / or by chemical derivation.
Examples of suitable biomolecules include, but are not limited to, amino acids, proteins and functional fragments of, antibodies, nucleic linker fragments, and combinations thereof.
one of many possible techniques and is generally applied to the examples given here and many other analogous materials and / or biomolecules. In a fashion 1 ity ι
Preferred reacts that can be used for ECL can
NHS fixation on fibrillate fibril by means of one or more fibrils by means of cova lens bonds of an ECL test ester group may well be a mat of (for example, reaction with an NHS ester) by reaction with appropriate linker * (vide supra), by means of a non-specific link, and / or by a combination thereof. Nucleic acids and / or cells can be fixed on fibrils or fibril mats by means of cova lens bonds with NHS ethers fixed on fibrils.
It may be desirable to control the extent of non-specific binding of materials on fibrils and / or fibril mats. Merely by way of example # 1 imitates, it may be desirable to reduce or avoid non-specific adsorption of proteins, antibodies, antibody fragments, cells, subcellular particles, viruses, serum and / or one or more of their components, ECL markers (eg RuII (bpy) 2 and Rui 11 (bpy) 3 derivatives), oxalates, trialkylamines, antigens, ana 1 i tos, and / or combinations thereof. In another example it may be desirable to increase the binding of the biomolecules.
One or more chemical portions that reduce or prevent non-specific binding may be present within, in, or
-9898 either nearby gives one to several fibrils, one or more aggregates of fibrils, and / or a mat of fibrils. Nonspecific binding is controlled by cava fixation of PEG portions on one or more fibrils and / or fibril mat. Charged residues (eg phosphates, ammonium ions) can be covalently attached to one or more fibrils or to a fibril mat.
Materials used in the support, electrodes and / or domains t
Binding agents can be treated with surfactants to reduce nonspecific binding. For example, fibrils or fibril mats can be treated with surfactants and / or detergents well known to a person with certain skill in the art (eg, the Tween series,
Tri ton, Span, Bri j>. Fibril fibers or mats are washed, soaked, incubated, zoned and / or a combination thereof with patent solutions and / or detergents. Solutions of PESs and / or molecules that behave similarly to PE (for example, oligosaccharides or polysaccharides, other hydrophilic oligomers ('' Folyethy i lene glycol biomedical applications, api icae bi-technical ions
1992, Plenum Press) can be combined with surfactants
Nonspecific adsorption or hydrophilic polymers) chemistry: Bi otechnica1 and (Chemistry of polyethylene glycols: and biomedical) Harris, JM Editor, to be used instead of and / or in and / or detergents.
unwanted from certain entities
-9999 as for ex ample the aforementioned can be blocked by non-specific adsorption competitive. This species of competitive linkage may be bovine serum albumin immunoglobulin G (IgG) (BSA)
The non-specific ECL-MARKER link can be reduced by chemical modification of the MARKER.
For example, him
MARKER can mod i. fi caree to increase its hydrophilicity, (for example by linking hydrophilic, polar hydrogen and / or functional groups charged to the
1igandos d ip go id i lo en
Ru (bpy3)> and consequently reduces the non-specific binding of the MARKER on another surface
It may be desirable and mobilize biomolecules or other fix antibodies, fragments of antibodies, proteins, enzymes, substrates antigens, haptens,
1ipoproteins, 1iposaccharides, cells, subcellular components, cell receptor, viruses, nucleic acids, antigens,
1 liquids, glycoproteins, carbohydrates, peptides, amino acids, hormones, protein ligands, pharmacological agents, and / or combinations thereof
It may also be desirable to use non-biological entities such as, but not limited to, polymers, elastomers, gels, coatings, ECL markers, active ion-oxidation-reducing species <eg tripropylamine,
-100 100 oxa cough), inorganic materials, chelation agents, linkers, etc., on fibrils).
One or more of a plurality of species can be linked in a non-specific way (for example, adsorb) on the surface of the fibrils or fibril mats.
Biological molecules or other means can be fixed on fibrils or fibril mat by non-specific adsorption. The extent of non-specific adsorption
Γ For any given fibril, fibril mat and / or biomolecule will be determined by certain properties of each. Certain chemical functional groups or biological portions present in fibrils can reduce or increase non-specific binding. The presence of hydrophobic and / or hydrophilic parts on the surface of a protein can increase or decrease the non-specific binding of the protein on fibrils or fibril mats. Hydrophilic and / or hydrophobic parts are used to control non-specific binding in controlled areas.
Fibrils can be derived with alkyl chains (CH2) and / or carboxylic acid groups to increase the non-specific binding of biological molecules or media or other materials.
Figure 26 illustrates the above embodiment schematically in the case of a single fibril. A fibril
2600 is derived with alkyla 2601. Biomolecules
-101I
2602, 2603, and 2604 bind non-specifically to the alkylene chains. 2605 polymers / elastomer are also bonded.
Non-derived fibrils, fibril aggregates, and / or fibril mats are used for the immobilization of biomolecules, biological media, and other materials by specific binding.
The ECL MARKER contains charged debris. The ECL MARKER 'is made to be selectively attracted to a holder and / or electrode. For example, a derived ECL MARKER that has a net negative charge may have a relatively low affinity for an electrode at a lower potential and then have a higher affinity for an electrode as the electrode potential becomes more oxidizing. The affinity of the ECL marker and / or binding reagent with the electrode is made to modulate, for example, to decrease the affinity during binding and / or washing steps to increase the affinity of the ECL marker and / or binding reagents to increase the effective power felt by the ECL marker during an ECL reading.
In Figure 2B, molecules (both biological and non-biological) can be attached to fibrils by means of a cova lens bond. 2800 fibrils that carry NHS ester chemical functional groups 25 can form intolerant bonds
-102I
102
2801 with biomolecules or 2802, 2803 biological media. These biological media can employ an amine group to form a covalent bond by reaction with the NHS ester group. Polymer 2808 is immobilized.
A person with certain cognacs in the art will recognize the generality of NHS ester groups as coupling agents for molecules and will be able to select both the appropriate biomolecules and the appropriate reaction conditions to achieve immov i 1 izac i on.
A pair of portion and / or MI "and SI molecules, of which one or more is fixed on a fibril, have a mutual affinity or a binding capacity. Ml / Sl can be antibody / antigen, antibody / hapten, enzyme ima / substitute, enzyme ima / cofactor, enzyme / inhibitor, lect ina / carbohydrate, receptor / hormone, receptor / effector, nucleic acid / acid nucleic, protein / nucleic acid, viral / ligand, cell / cellular receptor, etc.
Many combinations of Ml / Sl link pairs and you can select appropriate combinations to achieve the desired link 20. Either MI and SI or both MI and SI can be fixed on one or more fibrils.
Figures 27 and 28 illustrate some of the many possible configurations with this embodiment. In the figure
27, a 2700 alkyl chain derived fibril 2700 binds nonspecifically to a 2702 molecule that
-103103 has either a mutual affinity or a binding capacity for another 2703 molecule. Molecule 2703 binds also to another 2704 molecule. A blocking molecule 2705 can adsorb non-specifically on fibrils. A blocking polymer 2706 and / or a polymer 2707 having a ligand (2708) that has an affinity for a 2709 molecule are adsorbed in a non-specific manner.
In FIG. 28, a 2800 fibril binds via 2801 to biomolecules 2802 and 2803, and a linker molecule 2804. The linker molecule 2804 has either a mutual affinity or binding capacity for another 2805 bi-organic molecule. Bi-DNA 2803 has either a mutual affinity or binding capacity for another 2806 linker molecule, which is covalently linked to 2807. Polymer 2808 with a 2812 ligand specific for a 2809 bonding partner covalently binds to a fibril. Blocking molecules (eg ESA) 2811 and 2810 blocking polymers are covalently attached.
A fibril can be derived with biotin and / or a biotinylated linker and avidin and / or streptavidin can bind to this linker. Avidin and / or streptavidin can bind to fibril, and a biotinylated antibody and / or protein can bind. Avidin and / or streptavidin 25 can be immobilized on fibrils by either nc binding
-104i
104 coupling pair either the same coupling pair, or a combination thereof. The use of (strep) av id i na and biot i na as “binding pairs is a widely applied method for fixing biomolecules □ biological media on other materials and is well known to experts in the field (Sprinke et al », 1993, Langmuir 9: 1821).
A binding pair can be a monoclonal antibody and an antigen that binds to this antibody.
Pairs of
M1 / S1 / M2) can be formed.
MI is a monoclonal antibody,
SI is an antigen for MI, and M2 is an antibody that binds to SI. This complex may constitute a sandwich complex ** antibody / antigen / antibody (such antibodies may or may not be monoclonal>. M2 may be an antibody labeled with an active marker for ECL (v ide supra), a fluorescent, a radioactive dizzy, an enzyme marker, and / or combinations thereof.
MI can be a portion that can form complexes with meta 1, methyl ion, or an orthomanomethyl compound a (a "chelating agent) and SI is a metal, well composed organometallic metal ion (a" chelate ") that forms a
MI, and M2 is a portion in a biological molecule that with the MI / SI complex (Sershon and
Khilko, 1995,
Journal of Immunologica1 Methods,
7371).
-105105
The manufacture of patterns of metal electrodes and elements conductive to such electrodes on a surface is carried out by means well known in
Leventis et al.,
North American Patent No. 5,189,549). The preparation of metal films on transparent surfaces is used to produce liquid crystal displays and is easily adapted for the preparation of electrodes in accordance with the invention. Haneko, 1987, '
Liquid Crystal
Reidel conformity electrodes
Chem. Soc.
Displays, KTK Scientific
Publishers, Tokyo,
Publish i ng
Transparent surfaces can also be prepared, for example, with the DiMilla et
116 <5) í2225—2226. Depos i tan al., 1994, J
A.M
0.5 nm titanium and 5 nm gold on transparent substrates (glass or plastic). A thin layer of gold as prepared by the DiMilla method, supra, can be used to prepare a transparent electrical structure in the middle of the Kumar method, supra. Modifications to this procedure to increase the thickness of the conductive layers for increased current carrying capacity while preferably maintaining transparency are desirable and apparent to the person with some knowledge in the art. Such techniques can
-106106 can be used to prepare electrode surfaces that are aligned with or in the vicinity of such discrete binding domains of a PMAMS.
In addition, the films and / or monolayers can be composed of pore ions that facilitate the transfer of electrical power 1 from the electrode surface to the ECL marker, instead of using insulating portions (eg, alkyl chains ) in accordance with that presented by Zhang and Bar rd. For ex ample, an orbital patterning of pi in exten tively conjugated systems can be used for th e re fe nty of the methods. Such transfer of orbital pi pi electrons is proposed by poly-pi role or other conjugated rings or double linked structures.
Oligonucleotides can be used to modulate electron transfer. For example, DNA splicing pi bonds from double-stranded DNA can be used to increase electron transfer rates. Oligonucleotides linked on an electrode surface 20 can be used as the binding agent in a binding domain. By linking a complementary sequence of oligonucleotides a double strand with an organized splice pi bonds are formed. In a particular embodiment, an immobilized first oligonucleotide or primary oligonucleotide is labeled for ECL (eg, covalently
-107107 linked to a support). In another embodiment, a secondary complementary oligonucleotide either ol or igonucleotide of sequence partially complementary to the primary ol igonucleotide is labeled tertiary complementary or partially complementary oligonucleotide of the secondary oligonucleotide is labeled (eg, a sandwich assay). Branched oligonucleotide chains can also be used. A R
A variety of oligonucleotides and / or mimics of oligonucleotides can be used (eg, oligonucleotides with modified bases and / or modified structures containing for example nitrogen and / or carried out. The variable stability of the pi junction in oligonucleotides and / or Oligonucleotide complexes can be monitored through electron transfer modulation (the and / or impedance measurements) generated by a pair of labeled double helix oligonucleotides for
ECL stabilized by can be correlated against expected from a more disordered single-stranded oligonucleotide. Variation in terms of the double-stranded label for fully complementary ECL and a double-stranded oligonucleotide for partially complementary ECL can be correlated. Further,
-108108 multi-oligonucleotide oligonucleotide complexes can be employed. For example, triple propellers can be used.
The modulation of the electrode transfer rate loops can be measured using ECL detection as well as electronic devices. Markers for ECL can be slowly cova linked to oligonucleotide strands and / or non-covalently associated (eg, r
interspersed). E.1 DNA can be coupled to the electrode without the use of a linker (for example, adsorption of 5 'of gold-thiolated DNA) or with a short linker (less than 10 atoms) to ensure a low resistance to the transfer of DNA electrons to the electrode »A bonding chain can be used that can efficiently transport 15 electrons from the electrode to the DNA strand (eg, a polyacetylene chain).
A mixed monolayer and / or film can be used in which at least one constituent of the monolayer or film, as appropriate, facilitates the transfer of the electric potential.
Alternatively, a molecule or particle that facilitates the transfer of electrical potential is adsorbed on the monolayer or film. As examples of the above, monolayers conjugated to and / or conductive microparticles that adsorb on and / or are close to the electrode surface 25 can be used. Regular spaces
-109i
109 configured are created in the monolayer and / or film. By using controlled space patterns in an ordered substantially perpendicular SAM composed of long-chain alkaniols (i.e. insulator) to which ECL markers have subsequently been attached, the effective potential imposed on ECL markers can be controlled. For example, Figure 11 shows a cassette 1200 formed of a single support 1202 with a metal layer f
1204, a SAM 1206 pattern and spacing ios 1208 between the SAM patterns.
Proteins labeled for non-covalent ECL with a surface can bind monolayer. Lina protein on the surface of a methyl surface. The gold derivative with gold surface terminated alkaniols can act as either the working electrode or the counter electrode. A plurality of link domain can be incorporated into a single support as illustrated in Figures 11-13. In preferred embodiments, the binding domains contain labeled and / or unlabelled proteins and / or nucleic acids and / or cells and / or chemical species.
Alternatively, the length of the monolayer components (for example, the length of the alkane chain in the alkantiol monolayers) can be varied to control the effective potential on the exposed surface of the
-110I
110 monolayer.
Generally speaking, alkantiols can have carbon chains ranging in length from 1 carbon atom to 100 carbon atoms. In a preferred embodiment, the carbon length of the alkantiol contains between 2 and 24 carbon atoms, the length of the carbon chain of the aIcant iol is between 2 and IB carbon atoms. The length of<sub>r</sub>the carbon chain is between 7 and 11 carbon atoms. Such acantiols may have several head groups exposed to the test media including methyl groups, hydroxy groups, amines, carbaxy-1-acids, oligoCetilenglicol), phosphate groups, phosphoryl groups, biotin, nitritotriacetic acid, glutathione ion, epox idos, di ni t rofeni 1o, and / or NHS esters. Other head groups include 1 igands frequently used for the puri fi cation of i nmov i 1 ization of recombinant fusion protein (eg Sassenfeld, 1990, T1BTECH BíBB-93). Binding domains can be derived to varying degrees to achieve varying densities of binding reagents. For example, different densities of activatable chemical agents can be employed and / or shunts can be carried out in various measures. Mixed chemical elements can be used to create desired bond densities. Mixed monolayers can be used to control the density of activatable groups and / or binding reagents. The density of
-111I
111 Link groups are controlled by optimizing the relationship between the ECL signal and noise - The total number of link sites within a link domain is controlled to optimize the light signal intensity of 5 ECL relative to Other ECL light signals from other link domains that such ECL light signals are detected sequentially or simultaneously and / or relative to 1 light detection device. t
The voltage waveform can be applied to activate ECL asoc i markers. Ados with a binding domain within a PMAMS one or more sufficient to activate an ECL light generation can be applied multiple times to the same alkantiol derived surface with multiple ECL light marker. An electronic potential is applied enough to generate an ECL reversibility. The potential is applied to generate an almost revers
<td></td><td>ECL. In</td><td>a</td><td>almost series</td><td>reversible shapes</td><td>of</td><td>wave of</td>
<td></td><td>vol ta je,</td><td>the</td><td>domain of</td><td>link within which</td><td>I know</td><td>associate the</td>
<td> 20</td><td>marker</td><td>of</td><td colspan="2">ECL (eg binds), you can</td><td colspan="2">be chemical</td>
and / or physically altered. The series of applied voltage waveforms can provide irreversible light generation from ECL.
Furthermore, an electrical potential sufficient to release the components of the monolayer can be applied. It is desirable
-112112
<td>release such</td><td>components</td><td>of</td><td>manocapa</td><td>where he</td><td>volume</td>
<td>above the</td><td>surface</td><td>of</td><td>electrodes</td><td colspan="2">is small (for</td>
<td>example another</td><td>support o</td><td>all right</td><td>plate that</td><td>leans</td><td>on</td>
electrode surface). In this way, as the handlayer is disrupted, even some ECL markers that are not efficiently excited can be excited by the electrode surface to generate the electrochemiluminescent signal and the ECL markers are restricted to a small volume which restricts infusion from of the electrode. Various monolayer compositions can be used to control the degree of monolayer disorder for a given potential. Monolayers with components with a strong intercomponent affinity resistant to 1 disorder of longer alkantiols effectively resist that short chains of will be more monolayer
Chains of the disorder in alkantiols. By further varying the length of the chain, the desired stability can be achieved
Modifying link domains within a
PMAMS can be used to modulate the ECL signal. A series of voltage waveforms is applied to generate a multiplicity of ECL signals. Such a multiplicity of ECL signals can be used to achieve extra and / or better results. A statistical analysis of the ECL signal modulation rate can be correlated with
-113I
113 the overall quality of a link domain or multiple link domains. In addition, such a multiplicity of ECL signals can be used to increase the ratio of signal 1 to noise by, for example, filtering out certain ECL signals from a series. Furthermore, multiple pulses of electronic potential waveform can be employed to reduce undesirable signal modulation due to non-specific binding. An electronic potential can be applied to avoid the non-specific bonding of certain charged spec ies. Furthermore, the electronic power 1 can be applied to promote the location close to? a binding domain of certain analytes or chemical species of interest. The applied voltage waveform supplies a large overpower1 (for example, a potential higher than what is required to generate ECLs). Overpatences can be used to modulate ECL signals in a series of voltage waves or in a single pulse wave of voltage. Additionally, overpotentials can be used to modulate the ECL reaction kinetics and / or modulate the binding potential chemically and / or physically. Furthermore, one or more voltage waveforms and / or other electronic probes known to those skilled in the art can be used to evaluate and / or correlate and / or extrapolate information regarding the quality and / or electronic properties of a
-114114 electrode or several electrodes.
Preferably, the efficiency of the ECL reaction can be increased by extending the surface area of the working electrode by ionizing an additional conduction device 1 in contact with the electrodes.
Projections or extensions from the electrode (for example, cables or fibers) of conductive materials or conductive particles can be used to increase the surface area of the electrodes, so that the electric field comes closer to 1 marker of
ECL. At the same time, indentations or wells in the electrode structures can serve the same purpose.
Particularly, conductive particles can either line the spaces on the electrode surface and / or cover the support 15 or monolayer in such a way that the electric field around the ECL marker is increased in terms of its absolute magnitude, as shown in the Figure 12. These conductive particles extend the surface area of the electrode and consequently increase the efficiency of the ECL reaction. Figure 12 shows a cassette 1300 that has a part 1302 that carries a SAM 1306 configured in a metallic layer 1304 and indicates conductive microparticles that fill the spaces (for example, 1208 in Figure 11) and that extend above the metal surface. among the 25 SAN configurations. In the house of conductive particles
-115115 Magnetic íj, a magnet or magnetic field can be used to attract particles to the surface. The conductive particles can also be used as described to extend the bonding electric potential of figure B, which has between the electrode surfaces and the domain of a PMAMS with two approximate supports. Cassette 900 consists of a first holder 902 a multiple set of electrodes, and a second holder 904 having a PMAMS.
Conductive microparticles
906 they are positioned between the opposite surfaces to extend the electrical potential towards the ECL marker in the binding domains (not illustrated).
At the same time, conductive copalimers grow from the exposed spaces on the electrode surface to facilitate the extension of the electrical potential around the sample ECL marker as presented in Figure 13
Figure 13 shows a 1400 cassette that
1402 bearing a 1404 metal layer on a surface
1406 SAM configured conductors 1408 extend the multipoint domains (not electric field of elect rods <not shown) on the surface of the supplied
SAM stops by an illustrated) to link surface of
SAM.
Conductive polymers can also be used as described to spread the electrical potential between
-116116 Electrode surfaces and link domains of a PMAMS with two approximate supports as illustrated in Figure 7. In Figure 7, cassette 800 consists of approximate 802 and 804 supports. Conductive copelimers 806 grow between the handsome surfaces to extend the electrical potential toward the ECL marker in the binding domain (not illustrated).
Figure 9 illustrates a cassette 1000 formed with a first »
holder 1002 having a multiple electrode array, a second holder 1004 having a PMAMS bonding surface, where conductive projections (1006) (eg thin wire or other protuberances) of the working electrode extend the electric field around the ECL marker in the PMAMS binding domains.
Electrode pairs can be created in various configurations. The simplest configurations, presented in the figures accompanying this presentation, are made of metal and / or metal oxide film and / or semiconductor films stacked on a non-conductive flat surface. The electrodes of these pairs of electrodes preferably define between them a region of relatively constant width thus providing a relatively constant electric field.
Other electrode configurations are provided.
Several of these settings appear in the lists in
-117117 plant in Figures 19 (a) - <e ?. Figure 19 (a) shows a pair of interdigitated comb-shaped electrodes. In this structure, each electrode has a plurality of fingers that extend from the conductor creating a comb shape. The pair of electrode and counter electrode can be positioned adjacent to a domain of can be positioned between an electrode and counter electrode. The pair of concentric electrodes, í
one circular and one semicircular (c) shows two semicircular electrodes with their straight edges facing each other. Figure 19 <d) shows a pair of rectangular electrodes. Figure (e) shows a pair of interdigitated elect rods that have complementary surfaces to form an unused route between them
Electrode / counter electrode pairs can also be formed into specific complementary shapes of shapes on the GEFS bonding surface for alignment purposes. Exemplary forms appear in Figure 68. A support 712 is shown carrying pairs of electrodes 714720. The pairs of electrodes may be, for example, circular
714, interdigitalized
716, triangular interdigitalization
718 or i nterd ig i ta1 i zac i on of multiple electrodes
-118118
In the modalities shown in Figures 14-19 presented above, the pairs of electrodes are located on a single support- Alternately, the pairs of electrodes are located on a first support and a second opposite support 5 as shown in 1 .a figure 2.
5.8. CASSETTES
Cassettes contain one or more supports of the invention. Cassettes can include a plurality of link domains and one or more working electrodes.
Figure 2 presents a cassette where each of several link domains 30 on holder 26 are adjacent to a different electrode from the various electrodes 32. Counter electrodes 38 are formed on a second holder 28. An ECL assay is carried out as previously described by placing a sample in the binding domain 30 and then by moving these supports together 28 so that the counter electrodes 38 are adjacent to each of the domains of link 30 and an ECL reaction can be triggered as described above by a waveform generating device 39, via a conductor 34, and an ECL signal detected and recorded by a light detecting device 40, cable 41, and digital computerized device 42.
Figure 3 illustrates a cassette where each of several link domains 48 have a different pair of several
-119119 electrode / counter electrode pairs 50 adjacent thereto on support 44. Support 46 may optionally be positioned adjacent to support 44 such that support 46 forms a sample containing d ispasitiv adjacent to several link domains 48 and various electrodes
fifty. Accordingly, an ECL reaction can be triggered by electrical connection 52 by a waveform generating device 54, and an ECL signal detected by light detecting device 56 and 10 recorded and analyzed by a digital computing device.
58.
A cassette containing one or more pairs of supports is provided as shown in Figure 21, each pair of support being positioned such that the surface of a first support 1501 containing binding domains faces the surface ie containing binding domains on second support 1502, where each surface contains electrodes 1504 and binding domains 1506; such that each binding domain on the first support faces and is aligned with one electrode on the second support, and each binding domain on the second support faces and is aligned with an electrode on the first support,
Figure 4 illustrates a cassette where ECL electrodes are optional. Link domains 64 in support 60 are in contact with a sample of 25 suspected to contain
-120I
120 an analyte. Regions 66 on support 62 contain reaction medium to either detect an analyte of interest or to carry out a desired reaction. Support 60 and support 62 are brought together so that binding domains 64 and regions 66 are in Contact and the presence of an analyte or reaction product is determined by means of a reporter system, for example, a colorimetric or fluorescent chemiluminescent signal that f
It can be detected by a photodetector device 60 and registered and analyzed by a digitized computing device 70.
In a preferred embodiment, a cassette or apparatus of the present invention comprises a device for supplying samples in the plurality of discrete binding domains (see, for example, item 1 in Figure 1 of the Patent
North American No. 5,147,806; item 1 in Figure 1 of US Patent 5,068,088; each of which is incorporated by reference in its entirety). The device for supplying the samples can be a stationary or mobile device 20 and can be any device known in the art, including without one or more inputs, orifices, pores, channels, ducts, microfluid guides ( for example, cap i lares), tubes, taps, etc.
Fluids can be displaced through the system by various well-known methods, for example: pumps, pipettes,
-121i
121 syringes, gravity flow, capillary action, wick, is electrophores, rasp, vacuum, etc. The device for fluid movement can be located on the cassette or on a separate unit. The sample can be placed in all linking domains together. Alternatively, a sample can be placed in particular binding domains by a means of capillary fluid transport. Alternatively, samples can be placed on the support ΐ
by means of an automatic pipette control device for supplying fluid samples directly to the PMAMS in a holder, or in a reserve in a cassette or cassette holder for its subsequent delivery directly to the connection surface .
Supports can be prepared from materials including without
1imi tac ion, glass, plastic, ceramic, polymeric materials, elastomeric materials, metals, carbon or materials that contain carbon, alloys, composite sheets, silicone and / or layered materials, supports can have a wide variety of properties chemical, and / or optical structures. They can be rigid or flexible, deformed planes, transparent, translucent, partially different properties, or totally reflective or composite properties, regions with and can be a composite of more than one material.
-122i
122
Reagents for carrying out tests can be stored in the cassette and / or in a separate container, React i vos can be stored in a dry and / or wet state. In one embodiment, dry reagents in the cassette are rehydrated by adding a test sample. In a different fashion, reagents are stored in solution in blister packs that open due to pressure from a moving roller or piston. The cassettes may contain either a waste compartment or a sponge for the storage of liquid waste after the completion of the test. In one embodiment, the cassette includes a device for preparing the biological sample to be tested. A filter may be included to remove cells from the blood. In another example, the cassette may include a device such as a precision capillary for measuring a sample.
The plurality of link domains and the plurality of electrodes / counter electrodes on the supports are typically placed in corresponding proximity to each other by mechanical means, using guide posts, alignment pins, hinges (between each support), or edges gluttony. The optical guide device can be used to position both supports and electronic devices using defined optical guide marks on the supports, □ other systems employing matching devices
-1231
123 Electric or magnetic are also available.
The cassette holders can be configured to protect the electrode pairs from contact with the sample until an ECL reaction is required to be triggered. For example, the electrodes can be kept separate from the bonding domain surface until contact of the electrodes with the sample is required through the use of various mechanical devices such as a removable electrode protection device.
A cassette or apparatus of the present invention comprises reference electrodes, for example, Ag / AgCl or a saturated calomel electrode (SCE).
The supports can be held together by clips, adhesives, rivets, dowels or any suitable mechanical fixation. It can also be held together by the surface tension of a liquid sample or by a compression device removably placed on opposite sides of the two supports.
The cassette may also comprise more than two supports with, for example, alternate layers of bonding domains and electrodes on multiple supports comprising both a bonding surface and an electrode surface on a single support. This will form a tr id imena iona1 set of ECL analysis cells. All the previous components 25 of the cassette are transparent, except, optionally,
-124124 Some areas between the bonding domains, eg, multiple transparent bonding surfaces, electrode surfaces, and supports may be stacked.
The first support and the second support can be flat and opposite to define a volume that contains the sample between them. Alternatively, the first support layer and the second support layer can be configured in other suitable shapes including spheroid, cuboid, cylindrical shape, provided that the two supports, and the other 10 components thereof, conform in shape.
For example, Figure 10 shows a cassette 1100 formed of two adjacent non-flat supports 1102 and 1104. Each support has a complementary surface of conformation. Each support can the other in having a multiple surface or they can be PMAMS or both elastomeric things for another support. The supports or also be prepared in a supplied in a set of electrodes
One or both supports conform to the shape of the well the cassettes form to pre-cut, a suitable length from can or from a supplier of dispositions for gratings »The cassette can also include receiving samples such as a volume that contains a sample and grooves, channels, indentations and the like for sample distribution.
Figure 37 shows a cassette in which domains of
-125i
125 bond (3702) within and / or on a matrix (3703) are presented on a surface (3701). A second surface (3700) supporting a working electrode (3704) and a counter electrode (3705) is positioned such that the binding domains are in close proximity to the working electrode. Under conditions that lead to light generation from the ECL marker bound to the binding domains, light can be detected through either or both surfaces. A set of light detectors <3706, for example a CCD set, an intensified CCD set, or an avalanche photodiode set) is used to simultaneously measure the plurality of light signals from each of the link domains . The light detector assembly provides an image of the light generated from the link domains. Lenses, reflectors and / or optical guides can be used to magnify the image. In other examples, detected light from light detector regions or regions (eg, light that detects pi xels (s)) is mapped to one or more link domains. Image analysis can be used to help correlate the detected light with the binding domains. In a preferred fashion, the surface is elastomeric or deformable and can therefore establish intimate contact with the
-126126 electrode surfaces. The binding domains bind to polymers capable of carrying ion currents from the counter electrode to the working electrode. In a more preferred embodiment, the objects are 5 water swollen polymers capable of carrying an ion current from the counter electrode to the working electrode.
Figure 38 shows a cassette where the binding domains <3805, 3806, 3807) are presented on the surfaces of different objects (3808, 3809, 3810) supported on the counter electrode (3800). A working electrode (3801) is placed near the surface of the objects. Under conditions that 1 lead to ECL from labeled groups linked to them; Bond domains, light can be detected through either or both electrodes (if one or both electrodes is transparent or semi-transparent) and / α on the side. A set of light detectors (3802) is used to simultaneously roam the plurality of light signals from each of the 20 link domains. The objects can be elastomeric and / or deformable and can therefore form intimate contact with the working electrode. The objects can be polymers capable of carrying ion currents from the counter electrode to the working electrode. The objects can be water-swollen polymers capable of carrying a
-127127 ion current from the counter electrode to the working electrode.
A transparent support containing one or more binding domains comes in contact with a fibril mat electrode. The reagents can flow either between the supports / binding domains and the fibril mat, or through the mat to the binding domains. Light can pass from the linking domains, through the *
transparent support to a detector.
In another preferred fashion, an electrode is coated with an optically translucent or transparent layer of carbon fibrils, to increase the effective surface area of the electrode.
To advantage, the PMAMS supports and / or cassettes of the present invention can be packaged in the form of item sets. The element set comprises one or more PMAMS supports prepared in accordance with the present invention for carrying out assays, controls and carrying out ECL reactions including the like »Reagents may optionally be included in the element set, including reagents from control, test
ECL and calibration reagents and the like. A reagent mix can be included which contains a plurality of linkers specific for a plurality of different analytes
-128i
128
5,9
DEVICE FOR
CARRY OUT ECL REACTIONS
In one embodiment, the
PMAMS on supports, and cassettes containing the same, an apparatus, containing a device for applying one or more test samples in the binding domains of
PMAMS and initiate a plurality of ECL reactions
Such an apparatus may be derived from conventional apparatus suitably modified in accordance with the present invention for
carrying out a plurality of ECL tests on the basis of a support or cassette. The invention provides various apparatus adapted to carry out ECL tests using each of the specific PMAMS modalities described in the previous sections. Zoski et al. (North American Patent
No. 5,061,445) have an apparatus for carrying out ECL reactions. Required modifications include supplying sopart and / or handling cassettes, supplying multiple samples, directing multiple electrodes by one source for a voltage waveform, and acquiring and processing various ECL signals.
Illustrative apparatus elements in accordance with the present invention appear in Figure 6A. Such apparatus 700 comprises upper and lower supports 702, 704 and an electrode guard 710. The upper part carries a plurality of electrode / counter electrode pairs (not illustrated). Bottom bracket 1 cam link domains
-129I
129
706. The apparatus can remove the electrode shield from the cassette and position the electrode / counter electrode pairs to contact the bound analyte in the binding domains. A fluid or reagent flow space 708 is adjacent to the support bearing the binding domains. The apparatus may also simultaneously or sequentially send an identical or individually determined individual voltage waveform to each of the plurality of electrode / counter electrode pairs 10 to trigger ECL reactions in the cassette and then measure the radiation of ECL emitted, by means of a photon detector, for example, a light detector device. The apparatus may further comprise a temperature control device for maintaining the temperature of the support and / or 15 cassettes, orienting the environment there and for adjusting the temperature as required to optimize the ECL reaction conditions. The temperature control devices are preferably a heating and cooling device, for example, electric resistance heating elements, cooling fans, cooling devices, and any other suitable source of heating or cooling. The temperature control device also includes temperature sensors, for example, a thermostat or thermocouple device 25, and devices for turning the unit on and off.
-130130 heating device or cooling device in response to detected temperature changes
The device also offers a device to hold, move and manipulate one or more supports or cassettes to carry out the ECL reactions. The apparatus may further comprise a stationary or mobile sample delivery device for placing a sample in the
PM PMAMS binding domains, as described 10 for the above cassettes.
The apparatus may comprise an electrode contact device capable of electrically connecting the set of steerable electrode connections separately from the cassette to an electronic voltage waveform generating device, eg, a potentiostat (see for example FIG. 5 of US Patent No. 5,068,088). The waveform generating device supplies signals in sequence 1 or simultaneously to
<td></td><td>trigger independent</td><td>a</td><td>plurality</td><td colspan="2">of reactions</td><td>of</td>
<td> 20</td><td>ECL on the cassette »</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td>During an ECL test,</td><td>a</td><td>stream</td><td>ionic</td><td>between</td><td>the</td>
<td></td><td>working electrode and the</td><td colspan="2">counter electrode</td><td>can</td><td>flow</td><td>to</td>
through ionically driven liquid (eg water containing ionic salts), through a thin film of such liquid, and / or through a solid matrix
-131V
131 ionically conductive.
Accordingly, an apparatus for measuring electrochemiluminescence in a sample may comprise a plurality of cells to contain at least 1 sample, where a cell may be formed from one or more electrodes and one or more counter electrodes and a first support comprising a plurality of discrete link domains. The electrodes and counter electrodes can be provided on the surface of the first support or alternatively
<td> 10</td><td>the surface</td><td>of</td><td>one second</td><td>support where the</td><td colspan="2">second support</td>
<td></td><td>it's found</td><td>in</td><td colspan="2">a close closeness with</td><td>the domains</td><td>of</td>
<td></td><td>link in</td><td>the</td><td>first me r</td><td>support. The</td><td>electrodes</td><td>and</td>
Counter electrodes can occur in pairs. The cell may further comprise a plurality of sensor electrodes to detect the voltage adjacent to the working electrode. The cassette may further comprise a cell containing a reference electrode.
The apparatus further comprises a light detection device that can detect ECL reactions carried out on the. cassette, for example, by a detection device or by several detection devices. Such a detector device includes, simply by way of example, a set of fiber optic channels in correspondence with the set of electrodes and positioned adjacent there, connected to a set of photodetector devices, or to a single
-132132 light detector device that can scan the set of ECL signals as they are emitted.
The apparatus optionally comprises a digital computer or a microprocessor to control the functions of the various components of the apparatus.
The apparatus also comprises a signal processing device. In one embodiment, and simply by way of example, the signal processing device r comprises a digital computer for transferring, recording, analyzing and / or displaying the results of each ECL test.
Alternatively, the apparatus comprises an electrode translation device, for example, to scan one or more electrode / counter electrode pairs through the bonding surface to trigger ECL sequentially 1.
Size exclusion fi lters can be used in a parallel set of PMAMS.
5.10. ECL TESTS THAT MAY BE CONDUCTED
ECL markers for use in accordance with the present invention may be selected from ECL markers known in the art (see section 2.2, above, and US Patent No. 5,310,687). The ECL marker may comprise, for example, an organic metal-containing compound, where the metal is selected from within the
-133133 group consisting of ruthenium, osmium, tapeworm, iridium, radium, platinum, palladium, molybdenum, technetium and tungsten
Chemical bonding reactions suitable for preparing those for ECL markers are well known in the reagents.
<td> 5</td><td>technique,</td><td>and</td><td>filed,</td><td>by</td><td>example,</td><td>by Bard</td>
<td></td><td>(Patents</td><td colspan="2">North American Icanas</td><td>Us.</td><td> 5,310,687</td><td>and 5,221,</td>
<td></td><td colspan="2">device</td><td>Fixing</td><td>of the</td><td>marker</td><td>from ECL</td>
et over binding reagent can r
605)
The one being covalent and / or non covalent. A marker for a hydrophobic binding reagent does not
Binding ECLs can be non-covalently linked to (for example, by ionic interactions or depressions). In covalent, marker (s) of (covalently or not in turn cova binding reagent is found) on other examples of
ECL bind a complex that has non-covalently linked to a
A more specific example would be the covalent fixation of Ru (bpy) 3 through a linker with a Ni (11) -tri nitrilotriacetic acid complex. This molecule is fixed on binding reagents that include a sequence of peptides that contains a plurality of strains. Other pairs of receptor 1 ligands are known in the art and can be similarly employed (Sassenfeld, 1990, TIBTECH 8: 88-93). Also, a marker of
ECL can be used which contains a multiplicity of organometallic compounds (eg containing Ru) 25 configured as a branched network (eg via
-134I
134 of a network of hydrocarbon linkers). Such rami networks. Strings containing a multiplicity of ECL capable organometallic ions can be either fixed once or fixed at a plurality of positions on a molecule to be labeled for ECL. In another fashion, the marker of
ECL containing a multiplicity of organometallic compounds is a linear polymer with organometallic groups attached at a plurality of positions along the length of the polymer chain (eg, linear, branched, or cyclic polymers).
A plurality of binding domains can be employed in a variety of additional ECL assay formats well known in the art.
In quantitative assays, a known amount of labeled reagent for ECL is employed and the amount of ECL measured is correlated with known standards to calculate the amount of analyte present.
Direct, reverse, competitive, and sandwich tests can be carried out by methods well known to those skilled in the art. In the case of competitive assays, for example, a method for quantitatively determining the amount of an analyte of interest in a volume of multi-component liquid sample is performed as follows. The binding surface ie is concurrently in contact with (a) a known amount of an ECL-labeled ligand that can compete
-135135 with the analyte of interest for binding to a binding reagent present in the binding domains, and (b) sample suspected of containing the analyte of interest; contacting is effected under appropriate conditions such that the analyte of interest and the igand are competitively linked to the binding reagent. The presence of the analyte in the sample will reduce the amount of competitor ECL-labeled ligand that binds to the binding domain, thus reducing (compared to the level when no analyte is present in the sample) the resulting amount of ECL . The ECL in the resulting link domain is triggered and the amount of light emitted is determined quantitatively, thus quantitatively determining the amount of the year of interest present in the sample. Alternatively, the sample may be in contact with the binding surface before the surface will then compete with that from the sample for the 1 igand labeled analyte previously on the surface for bound ECL.
PMAMS will displace a certain part of the analyte previously linked
In an alternative embodiment, the sample may be treated in such a way as to contain substances / molecules that are labeled for ECL, and a standard amount of unlabelled analyte of interest may be in contact with the
-1361
136 Binding surface before or concurrently with the contacting of the binding surface with the sample to carry out a competition test.
In a sandwich assay, the labeled ligand for ECL is a binding element that specifically binds to a second binding moiety on the analyte of interest, therefore, when the analyte specifically binds to a binding reagent in the domain A PMAMS binding agent is present in a sample, a "sandwich" is thus formed, consisting of the binding reagent in the binding domain, bound to the analyte from the sample, linked to the corresponding link part marked for ECL. In another competitive sandwich assay, copies of the analyte itself are fixed on the binding domains of the binding surface of multiple pools prior to exposure to the sample.
The sample then contacts the bonding surface. A labeled partner link for ECL (which can specifically bind to the analyte)? It will go to the analyte in the absence of free analyte (from the sample) in the test solution, but will be competitively inhibited in the presence of free analyte (from the sample) in the test solution.
In alternative modalities, sequential marking is carried out. For example in a particular modality of a
-137137 sandwich assay, the analyte bound to the binding domain is sequentially contacted with a plurality of binding partners labeled for ECL of the analyte. ECL measurements and optional wash steps are carried out between contacts with each different liaison partner. In this way, an ECL measurement of several different binding portions of an analyte (eg CD8 +, T cell receptor positive for a cell antigen a, b) can be performed. Additionally, ECL markers. Multiples, each emitting light at a distinguishable wavelength can bind to a different binding reagent specific for a different portion on an analyte. Furthermore, a plurality of distinguishable reporting devices (eg, ECL marker, fluorescent marker, and enzyme-linked marker) each attached to a different binding reagent specific for a different binding portion of an analyte can be employed, for example , to distinguish a CD4 +, a cell is capable of receiving a T cell antigen receptor, b from a CD8, a cell capable of receiving T cell antigen a, b.
As two binding domains they contain labeled proteins and / or nucleic acids and / or cells and / or chemical species. Such labeled components (eg, markers for ECL can be added to 1 binding domain during manufacture, before the start of the assay, during an assay
-138138 and / or at the end of a trial. For example, multiple marked components can be added at various times and sequential readings can be taken. Such readings can provide cumulative information. In another fashion, the PMAMS binding domains can be reused multiple times. After carrying out a given test, the surface can be washed under conditions that rejuvenate the activity of one binding domain or several binding domains on the surface of PMAMS. By way of example ID, some binding reactions can be reversed by changing the ionic strength of the reaction solution. Alternatively, heat can be used to dissociate bond complexes. Some linking domains may be inherently self-renewing. Binding domains containing catalytic (eg, enzymatic) functionalities can be used more than once. Binding domains are used continuously, and therefore can be used in biocensor applications. Additionally, the assay can be formatted in such a way that the binding agent reacted on the multispecific mu-shaped surface is marked for ECL. By linking certain analytes of interest
<td></td><td>in a sample,</td><td>the</td><td>signal</td><td>ECL will be</td><td>modulated</td>
<td></td><td>quantitatively.</td><td>By</td><td>example,</td><td>the reactive</td><td>link</td>
<td> 25</td><td>marked for ECL</td><td>fi jado</td><td>on</td><td>the surface</td><td>can be</td>
-1391
139 specific for an analyte on a cell surface, eg, antigens such as alpha and beta T cell antigens, receptor antigens, or CD4 or CD8 antigens. Upon exposure to a mixture of cells, the cells bound on the surface will sterically impede the ability of an electrode surface, approached to a multi-array specific surface, to excite the ECL-labeled binding reagent by downregulating
Γ consequently descending the ECL signal.
Homogeneous and heterogeneous tests can be carried out. In heterogeneous assays, the labeled unbound reagent is separated from the labeled labeled reagent (for example, by
<td></td><td>one step</td><td>washing)</td><td>before the</td><td>exposition</td><td colspan="2">of the reagent</td>
<td></td><td>dizzy</td><td>linked or</td><td>not linked to</td><td>a potential</td><td>e1éc tri co.</td><td>In</td>
<td> 15</td><td>essays</td><td>homogeneous,</td><td>the reagent</td><td>marked no</td><td>bound and</td><td>the</td>
Reacted and linked markings are exposed together to an electrical potential. In homogeneous tests, the intensity or the spectral characteristics of the signal emitted by the bound labeled reagent is either greater than the intensity of signal 1 emitted by the unbound labeled reagent or less than said intensity. The presence or absence of linked and unlinked receptive components can be determined by measuring the intensity difference.
Once the desired steps of contacting
-1401
140 the binding reagents with the analyte or competitor of the same and any binding partner have been completed, it is ensured after that the ECL marker is subjected to an environment conducive to ECL. 5 suitable ECL test media are known in the art. Such an assay medium preferably includes a molecule that promotes the ECL of an ECL marker, including, but not limited to, oxalate, NADH, and most preferably tripropylamine. Such a promoter molecule may be supplied free in solution, or may be provided via a bond during production at (eg, as a product of a chemical reaction) the surface of PMAMS, a monolayer at the surface, the binding domain, the electrode surface, a binding reagent, and / or an ECL marker, etc. If the surrounding medium marks you
ECL attached to the linking domains that result from the contacting steps is conducive to ECL, no medium change is required. At ternati ve, the medium can be adjusted or replaced to provide an ECL-friendly medium. An electrode and counter electrode are already in the vicinity of the bonding domain, or are approaching or contacting the bonding domain, a voltage waveform is applied, and ECL is detected or measured.
In a preferred embodiment of the invention, the steps above
-1411 give rites of contacting the binding reagents with the analyte or a competitor of the same and any binding partner thereof are carried out in the absence of electrodes and counter electrodes, that is, in such a way that the sample is not in contact with the electrode or counter-electrode. Subsequent to these sample steps in contact, the electrodes and counter electrodes are brought close enough to the ECL marker attached t
ECL
A support that has a PMAMS can be used to sequence nucleic acid strands. For example, a PMAMS with a plurality of binding domain is prepared with different oligonucleotide probes of known nucleotide sequence as binding reagents in different binding domains. That is, different binding domains will contain binding reagents of different known nucleotide sequences. The oligonucleotide chain or fragments of the oligonucleotide chain can be sequenced and then left linked (hybridize) to the PMAMS binding domains. Nucleic acids to be sequenced are marked for ECL. Binding assays are carried out on PMAMS and the distribution of ECL signals from discrete binding domains on PMAMS is used to sequence the oligonucleotide chain.
The above method is based on the ability to
-142I
<img file="MX9706471A_D0003.tif" />
Oligonucleotides shortened to hybridize with their complementary or substantially complementary sequence in another nucleic acid molecule (see, eg, Strezoska et al., 1991, Proc. Nati. Acad. Sci. USA 38: 1089-
<td>1093; Ba i ns,</td><td> 1992,</td><td>Bi o / Tec h no1ogy</td><td colspan="2">10; 757-58, which</td><td>I know</td>
<td>incorporate here</td><td>by</td><td>reference).</td><td>Can be</td><td colspan="2">to select</td>
<td>conditions of</td><td>such</td><td>Way that</td><td>the grade</td><td>wanted</td><td>of</td>
<td>complimentarity</td><td>of</td><td>sequence is</td><td>necessary</td><td>for</td><td>a</td>
<td colspan="2">successful hybridization.</td><td>The h ibridac i ón</td><td colspan="2">of a molecule of</td><td>DNA</td>
of unknown sequence with a predetermined sequence probe detects the presence of the complementary sequence in the DNA molecule. The method is preferably practiced in such a way that the hybridization reaction is carried out with the oligonucleotide probes linked to the binding domains and the sample DNA in solution.
A PMAMS can also be used to isolate, screen and / or select a novel molecule or a complex of desired function (for example binding or catalysis). A PMAMS can be used to isolate compounds and / or carry compounds for therapeutic uses. A PMAMS containing a plurality of peptides, nucleic acids, viral vectors, or polymers, synthesized by a variety of combinatorial chemistries can be performed using the methods of the present invention. A wide variety of such PMAMS treated supports 25 can be used for rapid screening
-143143 to determine, for example, binding to a cellular receptor labeled for ECL. In one method, a PMAMS with a wide variety of unrelated peptide sequences is used to isolate the guide link peptide sequences. Then, a PMAMS is used with peptides of sequences related to those that showed binding with the molecule of interest (for example, a cellular receptor) in the first PMAMS. The process is repeated until a peptide with the desired binding characteristics is found.
An analyte of interest may be, for example, an entire cell, a subcellular particle, virus, prion, viroid, nucleic acid, protein, antigen, 1ipapratein, 1popolysaccharide, lipid, glycoprotein, portion of ca rboh idrate, derived from cellulose, antibody or fragment thereof, peptide, hormone, pharmacological agent, cell or cellular components, organic compounds, biological na polymer, synthetic organic molecule, arganometallic compounds or an inorganic molecule present in the sample.
The sample can be derived from, for example, a solid, emulsion, suspension, liquid, or gas. Furthermore, the sample can be derived from, for example, body fluids or body tissues, water, food, blood, serum, plasma, urine, faeces, tissue, saliva, oils, organic solvents, or air. The sample may comprise a reducing agent or
-144144 either an oxidizing agent.
Assays for detecting or measuring the following substances can be reacted by the present invention by incorporating a specific binding reagent for said substances in the binding domains of the binding surfaces of the invention. on: aIbumin, phosphate alkaline rate, alt / SGPT, ammonia, amylase, AST / SGOT, total bilirubin, used blood nitrogen, calcium, carbon dioxide, chloride, cholesterol total 1, creat i ni na, GGT, glucose, HDL cholesterol, iron, LDH, magnesium, phosphorus, potassium, total protein, sodium, triglyceride, uric acid, drugs of abuse, hormones, modulators of the cardiovascular system, tumor markers, disease antigens, drug antibodies, antigens that cause allergies, i nmunop rotate 1 ñas, ci toe: i ñas, anemia / metabolic markers 1 icos, carbamazepine, digoxin, gentamicin, lithium, phenobarbit1, feni toi na, procainamide, quinidine, teof i 1 ina, tobramycin na, va 1proic acid, amphetamines, antidepressants, barb and turates , benzod iacep inas, canab i noides, coca i na, LSD, methadone, metaca tarpaulin, op ia tes, phenelindine, f ropoxi feno, ethanol, sa1ic i lato, acetami nó feno, estradio1, p regesteroña, testosterone, hCG / bhCG , follicular stimulation hormone, luteinizing hormone, prolactin, thyroid hormones such as thyroid stimulating hormones, T4, TUP, total T3, free 25 T4, cortisol, creatine kinase-MB, creatine kinase
-145 145 total, PT, APTT / PTT, LD ISOs, creatinine nyase ISOs, myoglobin, myoluzine chain, troponin 1, troponin T, chlamydia, gonorrhea, herpes virus, Lyme disease, Epstein Barr virus, IgE , Rubella-G, Rubella-M, CMV-G, CMB-M, tOKO-G, toxo-M, HBsAg (hepatitis B virus surface antigen), HIV 1, HIV 2, anti-HBc, anti-HBs , HCV, anti-HAV IgM, anti-HEic, anti-HAV, HEfeAg, anti-HBeAg, TB, prostate specific antigen, CEA, AFP, PAP, CA125, CAI 5-3, CA19-9, microg1obul i na b2, hemoglobulin, red blood cells, HBcAb, HTLV, ALT, STS-syphilis, ABO blood type antigens and antigens of other blood types, cytomegalovirus, ferritin, B-12, folate, hemoglobin gly ica1ada, amphetamines , antidepressants and other psychotropic ps pharmaceuticals.
ECL measurements in different binding-domains can be carried out sequentially or simultaneously.
A PMAMS specific for an analyte of interest that is a cell surface protein is first exposed to a sample containing cells, where it is desired to count the cells in the sample. In a preferred embodiment, a known and / or diluted sample volume is exposed to a PMAMS having a specific binding domain multiplicity for at least one cell surface antigen. The linked cells can then be quantified by fixing a linked secondary linking group with a
-146i
146 marker
ECL. It is a group capable of a wide range of cell types, for example a marker of
ECL bound to a hydrophobic group capable of inserting into a cell membrane or onto a lec ti na directed against cell surface sugars binds to a secondary antibody directed against a cell surface antibody. In a more specific embodiment, various cell types linked on the same domain can be distinguished by the use of marker-labeled antibodies to ECL. It is preferred to ensure that the number of binding domains of the specific challenges for a given amalite on the surface of a cell exceeds the average number of cells to be linked that are present in the sample.
Statistical techniques can then be used to determine the number of cells per sample volume. This technique can also be used, for example, to count other particles such as viruses, where the binding reagent recognizes an antigen in the virus. The domains can be small compared to the size of a cell such that only one cell can be linked per domain, 1 thus raising each domain to one which can then be analyzed in addition to the domains using statistical methods
Sundays are large compared to the size of a cell of size 1
-147i
147 so that multiple cells can join with one domain »
In this case, the signal level from each domain can be calibrated for sample volume.
An image analysis using a set of light detectors (for example, a CCD camera or a set of avalanche photodiodes) could be used to count cells and determine cell morphologies
The invention preferably also offers methods for
1O carry out reaction is ECL, for example, tests, at a rate of 1000 ECL reactions in a period of 5 to 15 minutes »
5.11. PMAMS FOR USE WITH OTHER ANALYTICAL AND / OR ECL METHODS The techniques described for ECL-based detection can be used in screening with other assay techniques, for example, as domains where catalysis and other chemical reactions occur. Discrete binding domains in accordance with the present invention can be used in other test techniques such as chemical to chemical chemistry tests, for example of electro ion ter minations, enzyme determinations on ( unique, blood protein determinations, glucose, urea, and creatinine determinations, and the like Other test techniques that can be combined with ECL tests and / or used alone with the PMAMS of the present invention
-148I
148 They include chemiluminescence-based labeling, fluorescence-based assays, enzyme-linked assay systems, electrochemical assays (see, eg, Hickman et al., 1991, Science 252: 688-691) and / or detection assay systems by resonance (eg acoustic * techniques and surface plasmon).
Drop FMAS supports can be used in which there are a plurality of different chemical elements within the drop set. Each drop may contain 10 different binding reagents and / or different chemical assays (ie, reaction medium therefor). For example, the droplets may be hydrophilic, supported by hydrophilic surface binding domains surrounded by hydrophobic surface regions. The droplets are protected by an hydrophobic solution covering the surface. The hydrophilic solution to be tested is deposited in a second PMAMS with hydrophilic binding domains surrounded by hydrophobic regions. The two surfaces are correspondingly accepted to cause contact 20 of the hydrophilic domains on the opposite surfaces and a spectral analysis is carried out to detect the reaction products of the chemical tests.
The fibril mats may such that there are a plurality of discrete hydrophobic and / or hydrophilic domains surrounded by hydrophilic and / or
-149I
149 hydrophobic. Drops of aqueous solutions containing binding reagents can lean on hydrophilic regions and be 1 mimicked by surrounding hydrophobic regions. These drops may contain, for example, fibrils, aggregates of 5 fibrils, binding reagents, ECL reagents, assay reagents, surfactants, PEGs, detergents, a plurality of biological molecules mentioned above for example, and / or combinations thereof. .
The hydrophobic solution covering the first GEFS is controllably removed (eg, evaporated, subjected to a wick process) to expose only part of the hydrophilic droplets in the upper parts to the environment. A hydrophilic solution to be tested for an optimal chemical reaction is then exposed to the surface of the PMAMS - the hydrophilic droplets and the solution to be tested is mixed and analysis carried out (eg emp1o, spec t ra1).
PMAMS binding domains can also be used as a pre-filter or filter. For example, a cell-specific GEFS 20 can be used in certain cases alone as a filter for certain cell types as well as in combination with a size exclusion filter. The resulting analyte solution is exposed to a PMAMS specific for a subcellular particulate matter (eg, virus). Subcellular GEFS in the form of particles
-1501
150 and / or a size extrusion filter is used to generate a small molecule solution of ana 1 i te (eg, protein, small chemical entities). By using a serial PMAMS assay system the analyte solution can be sequentially purified to decrease non-specific analyte interactions.
The optical opacity of a material used for a support, electrode, and / or binding domain can be varied to achieve r
the desired properties. Such a material can be transparent, transparent or subtly opaque, depending on the thickness, composition and / or optical density of the material.
The optical opacity of fibril mats increases with increasing mat thickness. Very thin mats are optically translucent. Thicker mats can be substantially opaque. In some examples, mats that have a thickness within the range of 0.01 pm to 0.5 pm are substantially translucent. In other examples, mats with a thickness greater than 20 pm are substantially opaque.
Mats with a thickness between 0.5 pm and 20 pm have an intermediate opacity, which increases with the increased thickness of the fibril mat. The optical opacity of a particular thickness of an ester may depend on the composition, density, derivation, number of layers, types and amounts of materials dispersed on the mat, and / or
-151i
151 a combination of them. It can also depend on the wavelength of the light used.
If a material is substantially translucent at a given thickness and substantially opaque at another thickness, light emitted from a certain depth in the material may exit the material while light emitted from another depth (for larger example) can be either substantially absorbed or dispersed by the material. In one example, the variable opacity of a material allows the material to be used as an optical filter,
Light emitted from a certain depth on a fibril mat can pass substantially through the mat and can be observed with or near a surface of the fibril mat. Light emitted from another depth can be substantially absorbed and / or scattered by the mat and not be observed by a detector placed on or near the mat surface. This property of fibril mat (and / or optically similar materials) can be used to distinguish between bound and unbound reagents in ECL assay.
Some reagents can be dispersed (actively or passively), pulled (for example, by suction filtration and / or capillary action), subjected to a wick process, or pushed by pressure to a
-1521
152 It is not enough depth that the light emission in a porous material is either 1 or 1 mat. In one example, physical and fi lter fi lter certain reagents certain reagents surface of in or through linked da nties already locate in such a way as absorbed a reactive mat is either dispersed by that of fibrils it acts as certain reagents are dragged and / or bind to a very thin layer either on the mat or in the vicinity of the mat.
the mat of au no that is in well in a reactive layer of species of fus i ¿n, bound to bound pulled, etc. one or more speci? es plus link domains (these domains are very thin near mat in PMAMS ). Reagents and / or others are either suspended on the fibrile surface and / or on such that the very thin ones on the reagents through fibril mat or the surface of the solutions flow or from the mat the surface of the reagent mat is bond only fibril to one surface layer of the mat. Mats can be washed once or several times in one or more directions
Reagents may bind on the reagent mats or fiber domains, one or more other binding domains, the same reagents linked to one or more than pass through the mat, or a combination of the
-153153 themselves.
Porous materials used in supports and / or electrodes may have more than one layer in which the top layer has binding domains and other layers within the mat have binding domains. In one example, a fibril mat (schematically illustrated in Figure 29), the top layer 2900 is thick enough to prevent the passage of light originating from layers 2901, 2902 r
coming from the mat under this layer. Light 2903 originating from sources 2904, 2905 linked to this top layer can be detected by means of a detector 2906 located on or near the surface of the mat. Light originating from sources 2907, 2908, 2909 in lower layers 2901, 2902 is absorbed and / or scattered by any of the layers or all layers and cannot be detected by detectors 2906 , 2910.
A prefi 1 traction step can be used to select sizes, types, fibril derivatives and / or aggregates of fibrile particle particles prior to mat fabrication. The fi lter agent used to fi ll a fibril suspension is a fibril mat of a certain porosity or of various porosities.
A porous material (eg, a fibril mat) can act as the support for the binding domains, a
-154I
154 an electrode that can be used for ECL or other electrochemical applications, a filter that can be used to control the supply of reagents, and / or an optical filter that? It can transmit, absorb and / or spread light in 5 v rivers.
5.12. ELECTROCHROME ECL PRESENTATION PANELS
The invention also provides for the production of isolated electrochemical pixels for use in flat panel displays. Topographic techniques 1 and have been proposed for use in flat panel presentations in e1ec t rochrom i cas and e1ec t roqu imi co1um i nescenci to create pixels that, when electronically directed, have effect
Limited on nearby pi xels (ie, limited diaphony) (see US Patent No. 5,189,549). A limitation of the lithographic technique to reduce such crosstalk is that the electrolyte material must be able to change its conductivity upon exposure *
to the light. It is a feature of the present invention to reduce the crosstalk between pixels without the need to use materials capable of modulating photoinduced conductivity, thus allowing the use of a wide range of solutions, gel.es or different films.
The two electrode surfaces that are the pixel active region meet on two surfaces that face each other in a sandwich configuration. The
-155i
155 The electrode surfaces are coated, for example, with complementary electrochromic materials. To reduce dissonance, a conductive electrolyte film is placed between the electrode surfaces with non-conductive regions between different pairs of electrodes (ie, between pixel elements). If the coated electrode surfaces are hydrophilic, then the surface areas around the electrodes are made to be hydrophobic (for example, by stamping or by depositing through a mask) and small droplets. Hydrophilic conductors are placed on the electrode on the first surface (for example, by means of a set of fluids) and then the second surface is automatically aligned and contacts the first surface in such a way that the electrodes are in correspondence. Small electrolytic drops can therefore be limited to the area within a pixel with no conductive material between the pixels. The pairs of one-pixel electrodes are side by side in close proximity on the same surface. If the pairs of coated electrodes are hydrophilic, the area encompassing both electrodes is made to be hydrophilic with a hydrophobic ring around the hydrophilic area of the electrode (for example, by stamping or by depositing through a mask). The 25 small drops described in the two previous modalities
-156I is stable i san using hydrophobic solutions. The viscosity of the solutions can be increased to increase the stability of the small droplet assemblies. The h idrof i 1 isiclad and the h idrof ob isity can be reversed. In 5 other ways, the small droplets may contain solutions capable of polymerization to increase conductivity (for example, polymer electrodes.
In addition, structured features can be used to limit the font size between pi; <els. For example, an elastomeric pattern (eg, poly (dimethylsiloxane)) with ring-shaped stamp protrusion characteristics capable of circling side-by-side pairs of electrode pixels on a surface can be used to isolate solutions, gels or elect role-playing movies between pi xels. At the same time, pairs of side-by-side electrode pipes can be placed in electrically insulating well-shaped structures on a surface, electrolyte solutions, gels, or films.
2O placed in the wells above the electrodes, and the entire surface covered or coated to isolate and contain the electrolytic components of each pixel.
5.13. PMAMS FOR USE IN OTHER CHEMICAL REACTIONS
The PMAMS of the present invention can also be used to carry out chemical reactions not in combination with
-157i
157
ECL »For example, all the non-ECL techniques and tests presented in section 5.11 above can be used.
A cassette is provided to detect or measure an analyte of interest in a sample, comprising: (a) a first support having a plurality of discrete link domains on the surface thereof to form at least one link surface, at least some of the discrete link domains are of different link specs ic than the others link domains, each of the plurality of link domains with challenges is hydrophical and surrounded by hydrophobic regions, and (b) a second support having a plurality of hydrophilic domains comprising suitable reaction media to carry out a chemical test there. to form a test surface, where the plurality of discrete binding domains and the plurality of reaction media can be contacted in such a way that a sample to each other present in each binding domain comes into contact with a reaction medium to detect or measure an analyte of interest. Alternatively, the binding domains can be hydrophobic, and the second support has a plurality of hydrophobic domains containing reaction media.
The invention also provides a method of detecting or measuring analytes of interest in a sample comprising:
-158J
158 (a) the droplet placement of a sample containing an ana 1 i to be detected or measured in a p lure 1 i ty of discrete link domains on a support surface, where 1 a pleness of domi Binding children of the challenges>: omp renders 1 minus one binding domain containing binding reagents that are identical to each other and differ in terms of specificity of the binding reagents contained within the other binding domains, each of the discrete domains dee η 1 ace is car at. cterizac om or hydrophobic or hydrophilic, provided that the region of the supporting surface surrounding each binding domain is (i) hydrophobic and the binding domain is hydrophilic, and (ii) hydrophilic and the binding domain is hydrophobic , to allow one or more analytes? of interest in the sample are linked to the 15 binding domains, and (b) contacting the drops in the first support with a surface of a second support that has ne. na ρ 1 ur at 1 idaddedo minios hidrof 11 icosdisc: challenges; what u e comprises suitable reaction means to carry out a chemical test there, and (c) determine the. presence of the 20 analysts of interest that are linked on the link domain.
There is also provided a method of detecting or measuring analytes of interest in a sample, comprising (a) dropping a sample containing an analyte to be detected or measured in a plurality of binding domains
-159ι
15?
discreet in one. support surface where the plurality of discrete binding domains comprises at least one binding domain containing binding reagents that are identical to each other and differ in terms of specificity of the binding reagents contained within other binding domains, each one of the discrete binding domains is characterized by being either hydrophobic or hydrophilic, provided that the region of the supporting surface surrounding each of the binding domains is (i) hydrophobic and the binding domain e is hydrophilic (ii) hydrophilic if the binding domain is hydrophobic h, to allow that one or more analytes of interest in the sample is bound on the binding domains, and (b) droplet placement of a reaction medium on the sample drops; and (c) determining the presence of analytes of interest that are bound on the binding domain.
In a particular example of this aspect of the present invention, binding domains, each of which incorporates a different enzyme that employs a sequential intermediate in a chemical reaction as its substrate, are located on a PMAMS surface, such that the product A given enzyme reaction, which is the reagent for a subsequent enzyme, flows to the next enzyme in the reaction path. The invention also provides the
-160ι
160 immov i 1 i bulk ionization of enzymes in self-assembling aionolayers, for example for industrial application, using methods described above.
For example, sheets with such enzymes immobilized on one or both sides can be stacked to achieve high proportions between surface area and volume of solution.
Alternatively, such immobilized enzymes can be fixed on porous materials. Additionally, such immobilized enzymes can be found in rods, stirring agents, in the walls of tubes or capillaries, or in the walls of containers such as an incubator chamber.
In an alternative aspect of the invention, non-ECL assays such as those described above can be carried out on 15 PMAMS analogs, said PMAMS analogs differ from the PMAMS linkages described above in that the PMAMS analogs contain discrete domains to carry. For non-ECL reactions, the discrete domains do not necessarily have a binding reagent incorporated and therefore are not necessarily binding domains. Such PMAMS analogs have discrete domains for carrying out reactions and are prepared to inhibit the expansion and / or diffusion of fluid applied to the discrete domains. In one embodiment, the domains are either hydrophobic or hydrophilic relative to the surrounding regions in the
-161161 support surface, to help contain the reaction medium and / or sample in the dyseto domains. The use of wells, depositing of reaction medium or sample in porous felts or porous materials, dissected deposit of reaction medium or sample in wales, films, etc., can be used to inhibit expansion or diffusion. Each one of
<td></td><td>such</td><td>domains</td><td>discrete has a</td><td>diameter</td><td>or</td><td>width</td>
<td>J i</td><td>I inferred</td><td>or at 1 mm,</td><td>from preferene ia dent ro</td><td>of the range</td><td>of</td><td>50 nm at</td>
<td>t 7</td><td>1 mm,</td><td>with more</td><td>degree of preference</td><td>inside of the</td><td>ra</td><td>ngo of 1</td>
look at 1 mm. The same reaction medium or different reaction medium can be deposited in each of the discrete domains before the application of the sample, or the application of the sample can precede the deposit of the reaction medium.
In a preferred aspect of using PMAMS analogs to conduct non-SL assays, drops of reaction medium are placed in a plurality of discrete domains, preferably supplied concurrently from a set of microfluidic guides; and then, optionally, to increase stability and / or protect the drop, a more viscous solution (eg, oil) is placed on top of the reaction medium or, alternatively, between the discrete domain; and then the sample containing an analyte to detect or measure is applied to each domain, either by discrete application to each
-162162 discrete domain or, in bulk, by exposing the entire surface of the PMAMS analog containing the domains to a fluid sample. Any resulting reaction in the binding domains is allowed to proceed, and the results are observed through the use of a reporter and detection system selected from those known in the art.
The invention is further described in the following t
Example the purpose of which is not in any way to limit the scope of the invention.
6. EXAMPLES
6.1. PREPARATION OF A SURFACE. OF MAB PMAMS THROUGH MICROSTAKING
A 1-2 micron thick exposed and developed photosetting substance matrix is prepared according to well known procedures in a square array pattern. A 10: 1 mixture of SYLGARD 164 silicone elastomer (poly <dimethylsiloxane) available from Dow Corning) and the corresponding curing agent SYL6ARD 184 is poured into the matrix and cured. Polymerized SYLGARD 184 is carefully removed from the silicon matrix. The resulting elastomeric stamping is inked by exposure to a hydrophilic OH-terminated alkantiol, SH (CH2) 11 ~ (0CH2CHJ60H, in an ethanolic solution (1-10 mM), which is automatically contacted accordingly
-163163 dowel with an aligned ring surface, and removed, the substrate is then washed for a few seconds (eg 1-20 seconds) with a solution of an alacanthiol with CH3 h idrof ób ico termination, SH (CH2) 10CH3 (1-1.0 mM in
<td> 5</td><td>eta no1) (Kumar et</td><td>al », supra</td><td>and Prime</td><td>et</td><td>to the.,</td><td>It starts</td>
<td></td><td>252: 1164-7). The</td><td>surface</td><td>resulting</td><td>I know</td><td>dry</td><td>then</td>
<td></td><td>gently go down a</td><td>flow of</td><td>no t rogen.</td><td>A</td><td>with j</td><td>side of</td>
Capillaries containing a hydrophilic solution automatically spike contact with the 'aligned surface' aligning the capillaries with the SH (CH2) 11- (0CH2CH2) 60H domains. Each capillary in the capillary pool contains monoclonal antibodies (MABs), specific for an analyte of interest, capable of covalently binding to reactive OH groups in the hydrophilic domains through an amide bond.
6.2. PREPARATION OF A MAB V NUCLEIC ACID PMAMS SURFACE BY MICRO-STAMPING
A matrix of exposed and revealed fatoendurecib substance of 2 microns thick is prepared according to 20 well known procedures in a square arrangement pattern. A 10: 1 blend of silicone elastomer 184
SYLGARD and the corresponding healing agent SYLGARD 184 is emptied into the womb and cured. The polymerized SYLGARD 184 is carefully removed from the silicon matrix. The resulting elastomeric print is inked by
-1641
164 exposure to a hydrophilic OH-terminated alkantiol, SH (CH2) 11 "(0CH2CH2) 60H, in an ethanolic solution <1-10 mM),; Automatically spike contact with a gold aligned surface, and removed. The substrate is then washed for a few seconds, (eg 2-10 seconds) with a solution of a hydrophobic CH3-terminated alkantiol, SH (CH2) 10CH3 (1-10 mM in ethanol) (Kumar et al., <Supra and Prime et al., Se ience 252: 1164-7). The resulting surface is then gently dried under a flow of nitrogen. A set of capillaries containing hydrophilic solutions is then automatically contacted in spikes with the surface aligned by aligning the capillaries with the SH (CH2) 11- (0CH2CH2) 60H domains. Each capillary in the capillary pool contains either antibodies or modified nucleic acids, specific for an analyte of interest, capable of covalently binding to the OH reactive groups in the hydrophilic domains via amide binding bonds.
6.3. PREPARING A PMAMS SURFACE BY ATTACK
CHEMICAL
A clean gold surface is exposed to a hydrophilic OH-terminated alkantiol, SHCCH2) 11- <0CH2CH2) 60H (Prime et al., Science 2521164-1167) in an ethanic solution (ΙΙΟmM). A linear set of fine-tip chemical attack utensils robotically comes into contact
-1651
165 automatically corresponding with an aligned gold surface, and the linear array is used to chemically attack both the X and ¥ dimensions of the surface creating a two-dimensional network joint of domain domains.
SH (CH2) 11- <OCH2CH2) 60H. The substrate is then washed for a few seconds (eg 2-10 seconds) with a solution of a hydrophobic SH (CH2) 11- (0CH2CH2) 6CH3 (1-10 mM in ethanol). The resulting surface is then gently dried under a nitrogen flux. A set of capillaries containing hydrophilic solutions then automatically come into corresponding pin contact with the surface aligning the capillaries with the domains
SH (CH2) 11- (0CH2CH2) 60H. Each capillary in the capillary pool contains antibodies or nucleic acids, specific for an analyte of interest, capable of covalently binding with the OH receptive groups in the hydrophilic domains.
6.4. WALL TESTING ON A PMAMS SURFACE
A transparent surface of PMAMS is made in accordance with the above, which is substantially transparent with a highly specific set of primary antibodies bound to the surface. The set of electrodes, monologue surface are selected in such a way that they are transparent. PMAMS surface exposes
-166166 then to a solution sample of pi which is suspected of containing an analyte of interest for testing. The sample is then washed leaving the antibody bound to the analytes on the surface. The PMAMS surface is then exposed to a solution containing dizzy antibodies to specific secondary ECLs for surface bound analytes. This solution is then washed from the surface of PMAMS leaving labeled secondary antibodies
Γ for ECLs bound to the domains where the analyte is present.
The electrode assembly is protected by a removable barrier to avoid premature contact of the sample with the electrode surface to avoid contamination defects. The barrier is then removed and the electrode assembly, if wetted with the test regulator, comes into aligned contact with the PMAMS surface. The electrode assembly is connected to an electronic potential waveform generator, and potential is applied to pairs of working / counter electrodes. A CCD then reads the emitted light and the signal is sent to a microprocessor which converts the signal into the desirable readable form.
The reading is compared to the reading obtained using controls in the form of known amounts of one year of interest to calculate the actual amount of analyte.
-167i
167
6.5. TESTING ON A SURFACE OF A FIRST AND SECOND PMAMS
PMAMS
A transparent PMAMS surface is made in accordance with the above rite with a configured set of primary antibodies bound to the surface. The surface of
PMAMS then exposes to a solution sample suspected of containing an ana 1 i to f after leaving the bound antibody sample is washed to analytes on the surface
A second PMAMS is provided, under protective cover, with an alternate hydrophobic / hydrophilic pattern where microdrops of a plurality of SL-labeled secondary antibodies are configured
The barrier protecting the second PMAMS in correspondence with the first PMAMS is removed and the microdrops correspond with the primary antibody binding domains in the first PMAMS. The second PMAMS is removed from the electrode assembly and comes into aligned contact with the first PMAMS surface. The electrode array is cognated to an electric potential waveform generator, and the potential is applied to the working / counter electrode pairs. A fatomul tipl icadcir tube then reads the emitted light and the signal is sent to a microprocessor which converts the signal into readable form
-168168 desired.
The reading is compared to the reading obtained using controls in the form of known amounts of an analyte of interest to calulate the amount of area 1 of year 1.
6.6. NUCLEIC ACID TEST ON A PMAMS SURFACE
A transparent PMAMS surface is made in accordance with the above described with a configured multispecific set of single-stranded nucleic acid probes bound to the surface. The probes are complementary to the 5 'region of a nucleic acid analyte of interest. The PMAMS surface is then exposed to a solution sample suspected of containing a hybridizable nucleic acid analyte of interest to be tested, the sample has been previously denatured, ie, treated to return the analyte single-threaded interest. The sample is then washed leaving hybridized analytes on the surface. The PMAMS surface is then exposed to a solution containing specific secondary ECL-labeled nucleic acid probes 20 for the 3 'end of the bound nucleic acid analytes on the surface. This solution is then washed from the surface of PMAMS leaving ECL-labeled nucleic acid probes attached to the domains where the analyte is present.
The barrier that protects the second corresponding PMAMS
-1691
169 with the first PMAMS it is removed and the microdrops correspond to the primary antibody binding domains in the first PMAMS. The second PMAMS is removed and the electrode assembly comes into contact aligned with the surface of the first PMAMS
The electrode assembly is connected to an electronic potential waveform generator, and the pairs of working electrodes / counter electrodes. A
CCD mi c rop rocesador light that converts the signal is sent to in the form of a desired reading
Reading controls in the interest of
6.7. TEST is compared with the form of quantities to calculate the amount of reading obtained using known analyte of analytes.
COMPETITIVE ON A PMAMS SURFACE WITH A
PHOTOMULTIPLIER DETECTOR
A surface conformance to specifics for PMAMS as above configured a transparent analyte described with antibodies produced from a primary set, of interest, linked to the surface
The surface of
PMAMS is then exposed to a sample of solution to be tested which is a mixture of a sample of interest and a known amount of a molecule labeled for
Competitive ECL with the analyte of interest for its link to
-170ι the antibodies. The sample is then washed leaving analytes bound to antibodies and / or competitive linkers labeled on the surface.
The set of electrodes is protected by a removable barrier to avoid contact of the sample with the surface of the electrodes in order to avoid contamination effects. The barrier is then removed and the electrode assembly, which is moistened with the test regulator, comes into aligned contact with the PMAMS surface. The electrode assembly is connected to an electronic potential waveform generator, and a potential is applied to the working electrode / counter electrode pairs. An IP photomult tube then reads the emitted light and signal 1 is sent to a microprocessor that converts signal 1 into the desired readable form.
The reading is compared to the reading obtained using controls in the form of known amounts of an analyte of interest to calculate the actual amount of analytes.
6.8. COMPETITIVE TEST ON A PMAMS SURFACE WITH A
CCD DETECTOR
A transparent GEFS surface is made in accordance with the above described with a configured mu 11 i speci fi c set of surface bound primary antibodies. GEFS surface is exposed
-171i
171.
then to a sample of solution of cu.a 1 it is suspected that it contains the analyte of interest to be tested. The sample is then washed leaving antibodies bound to analytes on the surface.
A second PMAMS, under protective cover, is provided with an alternative hydrophobic / hydrophilic pattern in which microdroplets of a plurality of a known amount of a competitive ECL-labeled molecule with an analyte of interest are configured.
The protective barrier of the second PMAMS corresponding to the first PMAMS is removed and the microdrops correspond to the primary antibody binding domains in the first PMAMS. The second PMAMS is removed and the electrode assembly comes in aligned contact with the PMAMS surface. The electrode assembly is connected to an electronic potential waveform generator, and a potential is applied to the working electrode / counter electrode pairs. A CCD then reads the emitted light and the signal is sent to a microprocessor that converts the signal into the desired readable form.
The reading is compared to the reading obtained using controls in the form of known amounts of an analyte of interest to calculate the actual amount of analyte.
6.9. PREPARATION OF A MAB PMAMS SURFACE BY MICROSTAKING WITH A SH (CH2) 10CH3 ALCANTIOL
-1721
A 1-2 micron thick exposed and developed photo-hardening matrix is prepared according to well known procedures in a square array pattern. A 10si blend of silicone elastomer 184
SYLGARD <polyidimethylsiloxane); available from Dow Corning) and SYLGARD 184 Curing Agent is poured onto the matrix and cured. SYLGARD 184 pd i ripple is carefully removed from the silica matrix »The resulting elastomeric stamp is inked by exposure to a hydrophilic DH-terminating alkantiol, SH <CH2) 110H, in an ethanolic solution <1-10 mM), which Automatically contacts corresponding tang with a gold aligned surface, and is removed. The substrate is then washed for a few seconds (eg 2-10 15 seconds) with a hydrophobic CH3-terminated alkantiol solution, SH (CH2) 10CH3 il-10mM in ethanol) (Kumar et al., Supra). The resulting surface is then gently dried under a flow of nitrogen. »A set of capillaries containing hydrophilic solutions then automatically comes into corresponding contact with the pin with the surface at 1 line aligned by aligning the capillaries with the domains.
SH <CH2) 110H to place specific antibodies in each domain. Each capillary in the capillary pool contains monoclonal antibodies, specific for a year of interest, capable of slowly binding to the groups.
-1731
173 OH reagents in hydrophilic domains.
6.10. PREPARATION OF A MAB AND A PMAMS SURFACE OF
NUCLEIC ACID BY MICRO-STAMPING WITH AN ALCANTIOL
SHCCH2) 10CH3
A 1-2 micron thick exposed and developed photosetting substance matrix is prepared according to well known procedures in a square array pattern. A 10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding SYLGARD 184 curing agent 10 is poured into the matrix and cured. The polymerized SYLGARD 184 is carefully removed from the silicon matrix. The resulting elastomeric stamp is inked by position to a hydrophilic OH determination alkantiol,
SH (CH2) 11OH, in an ethanolic solution (1-10 mM), automatically contacted with spike correspondence with an aligned gold surface, and removed. The substrate is then washed for a few seconds (eg, 2-10 seconds) with a hydrophobic CH3-terminated alkantiol solution, SH (CH2) 10CH3 (1-10 mM in ethanol) (Kumar et al., Supra). The resulting surface is then gently dried under a flow of nitrogen. A set of capillaries containing hydrophilic solutions are automatically contacted correspondingly in a spike with the aligned surface aligning the capillaries with the SH <CH2) 11OH domains, to
-174I
174 place specific antibodies and / or hybridizable nucleic acids in each domain. Each capillary in the capillary pool contains antibodies or modified nucleic acids, specific for an analyte of interest, capable of covalently binding with reactive OH groups in the hydrophilic domains through amide binding bonds.
6.11. PREPARING A PMAMS SURFACE USING A
STREPTAVIDINE-BIOTIN LINKER
An exposed and developed 1-2 mm thick matrix of light-setting substance is prepared according to bi-cognac procedures in a square arrangement pattern. A 10: 1 mixture of SYLGARD 1Θ4 silicone elastomer and the corresponding SYLSARD 184 curing agent is poured into the matrix and cured. The polymerized SYLSARD 184 is carefully removed from the silicon matrix. The resulting elashomeric print is injected by exposure to a mixture of mercaptoundecanol and 12 "me reap to <8-b iot i nam i da - 3,6 - d ioxaoc ti 1) dodecanamide where the 20 molar fraction of thiol biotinylated is 0.1 (see Spinke et al., 1993, Langmuir 9s1821-5 and Spinke et al., 1993,
J.Chem.Phys. 99 (9): 7012-9). The substrate is then washed for a few seconds (eg, 2-10 seconds) with a solution of alkantiol terminated in hydrophobic CH3, 25 HS (CH2) 10CH3 alkantiol (1-10 mM in ethanol) (see Kumar et
-175175 al. sapra, Biebuyck, Whi tesi des>. The resulting surface is then gently dried under flux of ni trogen. A capillary assembly containing a solution of est rep tav id ina in each capillary is then contacted automatically in a corresponding manner in the ear with the surface aligned. Each capillary in the capillary pool is aligned and contacts a biotinylated domain and the capillary array is removed and the surface is washed. A second capillary array containing a multiplicity of bioti ni ni antibodies and bioti.nilate nucleic acid solutions then automatically comes in contact with spikes with the aligned surface to place specific antibodies and specific nucleic acids on each domain.
6.12. PREPARATION OF A UNIQUE SEA SURFACE
A set of working electrode pairs and interdigitalization counter electrodes on a silicon gold surface is manufactured by methods known in the art (eg see Kumar et al supra). In this example, the electrode array and the discrete link domain array exist on the same surface of a support. A 1-2 micron thick exposed exposed photocurable substance matrix is prepared according to procedures well known in the art for working electrode patterns. A 10: 1 mix of
-1761
176 silicone elastomers
SYLGARD (Poly (DimethyIsilaxane (PDMS)); available from Dow Corning) and the corresponding SYLGARD 184 curing agent are poured onto the matrix and cured. The polymerized SYLGARD 184 is carefully removed from the silicon matrix. The resulting elastomeric stamp is inked by exposure to a hydrophilic OH determination alkantiol, SH (CH2) 11C0CH2CH2) 60H, in an ethanolic solution tl-10 mM), contacted automatically in correspondence of spike with the working electrodes at i drawn on the surface of gold electrode arrays, and then removed A capillary array containing hydrophilic solutions is then automatically contacted with spike correspondence by aligning the 15 capillaries with the SH (CH2) 11- (0CH2CH2) 60H domains on the electrode array surface to place antibodies specific in each domain. Each capillary in the capillary array contains monee canvas antibodies, specific for one year of interest, capable of covalently binding to the reactive OH groups in the hydrophilic domains through a binding of amide.
6.13. TEST CARRIED OUT ON THE UNIQUE SURFACE OF MAB
A bracket is manufactured in accordance with that described in
6'12, supra, A PDMS stamp is manufactured in accordance with the previously described 25 from a matrix of
-177177 photo-hardening substance configured as rings each independently circumscribing a pair of locking electrode / counter-electrode. The surface of the electrode array is then exposed to a sample to be analyzed, washed with a mixture of ECL-labeled secondary antibodies, and then washed with a test buffer containing triproylamine. The PDMS stamp is then aligned and contacts correspondingly aligning
Γ PDMS stamp rings to circumscribe and define individual test regulator volume elements above each pair of electrodes. An overpower1 is applied to the pairs of electrodes such that the monolayer is released from the surface exposing the working electrode to ECL-labeled secondary antibodies. A photomultip1icator tube then reads the light emitted through the transparent PDMS and the signal is sent to a microprocessor which converts the signal into the desired readable form.
The reading is compared to the reading obtained using controls in the form of known amounts of an analyte of interest to calculate the actual amount of analyte.
6.14. PREPARATION OF A SINGLE SURFACE WITH WORKING ELECTRODES AND COUNTER-ELECTRODES
An arrangement of 25 working gold electrode pairs electrodes and interdigitation counter electrodes with
-1781
178 Gold bonding domains between the interdigitating electrodes on a gold silicon support is manufactured by methods known in the art (eg see Kurnar et al. supra). In this example, the set of 5 electrodes and the set of discrete link domains exist on the same surface. A 1-2 micron thick exposed and developed photocurable substance matrix is prepared according to well known procedures on the pattern of bonding domains between pairs of 10 electrodes of interdigi ta1 i zac i¿n. A 10s1 mixture of SYLGARD silicone elastomer 184 (poly (dimethylsiloxane (PDMS)); available from Dc »w Corning) and the corresponding SYL8ARD 184 curing agent is poured into the matrix and cured. The polymerized SYLGARD 184 is carefully removed from the silicon matrix. The resulting elastomeric print is inked '<sup>1</sup> by exposure to a hydrophilic alkantiol, OH, SH (CH2) 11- (0CH2CH2) 60H determination, in an ethanolic solution (1-10 mM), automatically contacting corresponding spike with the aligned gold 20 binding domains. the electrode assembly surface, and then removed. A set of capillaries containing hydrophilic solutions then automatically come into corresponding pin contact, aligning the capillaries with the SH (CH2) 11- (OCH2CH2) 60H domains on the surface of electrode sets to place
-179ι
179 specific antibodies in each domain. Each capillary in the capillary pool contains antibodies specific for an analyte of interest, capable of binding slowly digs with reactive OH groups in the hydrophilic domains through an amide bond.
6.15 TEST CARRIED OUT ON A SINGLE SURFACE WITH WORKING ELECTRODES AND COUNTER-ELECTRODES
A support surface in accordance with that described in
6.14, supra, is manufactured by the standard methods. The prepared surface is exposed to a sample to be analyzed, washed with a mixture of ECL-labeled secondary antibodies, and then washed with a test buffer containing tr iprop i lamina. The electrode array is connected to an electronic potential waveform generator, and a potential is applied to the working electrode / counter electrode pairs. A photomultiplier tube then reads the emitted light and signal 1 is sent to a microprocessor that converts the signal into the desired reading form.
The reading is compared to the reading obtained using controls in the form of known amounts of an analyte of interest to calculate the amount of analyte area 1.
6.16. PREPARATION OF A SURFACE WITH COUNTERELECTRODES A 1-2 micron thick exposed and developed photo-hardening substance matrix is prepared in accordance with
-180I
180 well-cognated procedures in a square arrangement pattern. A 10sl mixture of 5YLBARD silicone elastomer 184 and the corresponding SYLGARD 184 curing agent is poured into the matrix and cured. Polymerized SYLGARD 1Θ4 5 is carefully removed from the silicon matrix. The resulting elastomeric stamp is inked by exposure to a hydrophilic alkantiol determination DH, SH <CH2) 1 Γ- (DCH2CH2 / 60H, in an ethanolic solution <1-10 mM), it is automatically put into corresponding contact with a Counter electrode configured aligned, and a square link domain on a gold surface, and removed. The configured gold surface consists of steerable ring electrodes that circumscribe the binding domains where SHich?) 1115 (0CH2CH2) 60H has been stamped. A gap or gap exists between each gold counter electrode and each square gold substrate for each monolayer binding domain. A set of capillaries containing binding reagent solutions is then correspondingly contacted with spike 20 automatically with the aligned surface matching the capillaries with the SH (CH2) 11- (0CH2CH2) 6DH domains. to place specific antibodies or nucleic acids in each domain. Each capillary in the capillary pool contains antibodies or nucleic acids, specific for an analyte of interest, capable
-181ι
1Θ1 to bind cova slowly with reactive OH groups in the hydrophilic domains.
6.17. TEST CARRIED OUT ON A SINGLE SURFACE WITH WORKING ELECTRODES AND COUNTER-ELECTRODES ON SURFACES
DIFFERENT
The upper die support surface in 6.16 is exposed to a sample solution to be analyzed. The surface of the substrate is then washed and exposed to a solution containing a plurality of Lú-Strewn monoclonal antibodies to ECL or ECL-labeled nucleic acids of different specificity and then washed with a test regulator containing tripropylamine. A set of steerable transparent working electrodes is manufactured with each working electrode in the set corresponding to a discrete link / counter electrode domain region on the holder as described in section 6.16 above. The two supports are moistened with the test regulator and contacted with formal aligning automatically. The electrode assemblies are connected to an electronic potential waveform generator, and a potential is applied to the aligned pairs of working electrodes / counter electrodes creating
<td></td><td>a field of</td><td>power 1</td><td>between the</td><td>two</td><td>supports.</td><td>A</td><td>CCD</td><td>read</td>
<td></td><td>then the</td><td>light emitted</td><td>through</td><td>of the</td><td>elect rodo</td><td>of</td><td colspan="2">job</td>
<td> 25</td><td>transparent</td><td>and the signal</td><td>sent to</td><td>a</td><td colspan="3">microprocessor</td><td>than</td>
-182I
182 converts the signal into the desired reading form.
The reading is compared to the reading obtained using controls in the form of known quantities of an analyte of interest to calculate the area amount 1 of ana 1 i to.
<td> 5</td><td> 6. 18</td><td>MANUFACTURING</td><td>OF</td><td colspan="2">A MAT OF FIBRILLES</td><td colspan="2">(SCATTERED) CC POR</td>
<td></td><td></td><td></td><td></td><td>VACUUM FILTRATION</td><td></td><td></td><td></td>
<td></td><td>A</td><td>aqueous paste</td><td>of</td><td>DC fibrils, with</td><td>a</td><td>solution of</td><td>1 mg</td>
<td></td><td>of</td><td>f ibr ilas / mL</td><td>I know</td><td>prepared by</td><td>the</td><td>mix of</td><td> 0. 17.</td>
w / w of CC Fibrils / Deionized Water »DC Fibrils were dispersed (the largest aggregates, at the miera scale were dispersed into small aggregates or individual fibers) in the pulp by immersing a 400 sonification horn wats in the pasta for a period of 10 minutes to 1 hour. The extent of the dispersion was monitored by light microscopy.
A naylon filter membrane (pore size 0.47 pm, 25mm diameter) was placed on a 25mm diameter glass frit filter. The dispersed fibril paste was filtered through the membrane / filter facility by suction filtration (Figure 23A9.) Aliquots of the paste (5 ml) were diluted with 20 ml deionized water, then filtered through an instalac membrane ion / fi 1 tro. For an average mat of approximately 0.25-0.3 grams / cc, a mat of approximately 100 required aliquots.
-183i
183
The suction filtration continued until all the water from the dispersion was removed in the area (with visual inspection). The mat was peeled (by hand) directly from the filter membrane.
The mat was dried in an oven for approximately LO15 minutes at a temperature of 60 ° C. The mat was cut, poached, or otherwise sectioned for use.
6.19. MANUFACTURE OF A FIBER MAT IN A METAL MESH SUPPORT THROUGH EVAPORATION
An aqueous paste of CC fibrils, with a solution of 1 mg of fibrils / mL was prepared by mixing 0.1% w / w of fiber and CC / deionized water. The DC fibrils were dispersed (the largest aggregates, at the mine scale were dispersed into small aggregates or individual fibers) in the pulp by immersing a 400 watt sonification horn in the pulp over a period of time of 10 minutes to 1 hour. The extent of the dispersion was monitored by light microscopy.
A 1 cm2 section of stainless steel mesh (count 400) was placed in a 25 mm diameter paper filter. A 5 ml aliquot of the paste was pipetted onto the surface of the screen / filter paper assembly. The water in the paste was allowed to evaporate either at room temperature and pressure, or in a heated oven.
Once the fibril mat was dry, aliquots were added
-1841
184 additional. Fibrils and screens were detached as a single unit from the filter paper.
The mat was cut, pinched, or otherwise sectioned for use.
6.20 IMMOBILIZATION OF AVIDINE IN FIBRILS CARRYING GROUPS
NHS ESTER FUNCTIONALS
COOH-derived fibrils (supplied by Hyperion
Catalysts Inc.) were suspended in anhydrous diaxane at
Γ approximately 10 mg / ml under constant stirring. Added a molar excess allowed to dissolve, 20-fold of N-hydroxusuccinimide
Then, a molar excess ethyl diami πο-p ropi 1-ca rbod iimi da was added.
<td></td><td>mixture Then</td><td>It was of</td><td>hectic for the agitation, the</td>
<td> 15</td><td>solids</td><td colspan="2">three were washed</td>
<td></td><td>time with</td><td colspan="2">anhydrous methanol, and</td>
<td></td><td colspan="2">palisulfone</td><td>from 0.45 pm. The</td>
and was 20 (EDAC), and the hours at room temperature, supernatant was aspirated and the times with anhydrous dioxane, one was filtered on a filter membrane, washed with additional methanol and placed in a glass bottle under vacuum until no further weight reduction will be observed.
10.4 mg of NHS ester fibrils were washed with PBS-1 (approximately 70 mM phosphate, 150 mM NaCl) (reagent ORIGEN 402-130-01, pH 7.8, IGEN, Inc.). The washed fibrils were suspended in 2.3 ml of avidin solution (8.3 mg avidin / ml PBS-1).
The suspension was allowed to cool at room temperature for
-1851
185
1.5 hours, with constant bottle rotation to provide shaking.
After 1.5 hours, the suspension was stored for hours at a temperature of 4 * C, and then was brought to room temperature and washed with PBS-1 and stored at a temperature of 4 * C as a suspension in PBS-1. .
6.21. IMMOBILIZATION OF MONOCLONAL ANTIBODY (ANTI-AFP) IN
<td></td><td>CARBON FIBRILLES</td><td></td><td></td><td></td>
<td></td><td>Hoisted carbon fibers 1</td><td>with</td><td>asters</td><td>NHS</td>
<td> 10</td><td>were prepared in accordance with</td><td>the</td><td>described in</td><td>the</td>
<td></td><td>Example 6.20.</td><td></td><td></td><td></td>
<td></td><td>14 mg of fibrils ~ ester were mixed</td><td>of</td><td>NHS with 500 mi</td><td>of</td>
PBS regulator 1. The mixture was toned for 20 minutes until it became a viscous paste. An additional 15,500 ml of PBS-1 buffer was added.
A total of 1.6 mg of anti-AFP antibody (alpha-fetal protein) in 80 ml of PBS-1 was added to the above paste.
The reaction was allowed to stand at room temperature for
2.5 hours.
6 ml of PBS-1 buffer were added and the reaction mixture was centrifuged at a temperature of 4 ° C for 5 minutes. The supernatant was removed by pipetting. This procedure was repeated 9 times.
After the final wash, the supernatant was removed, and the fibril-antiAFP product was stored at a temperature of
-186ι
186
4 * C.
6.22 CYCLE VOLTAMOGRAMS OF FIBER MATS:
COMPARISON OF FIBER MATS WITH LEAF ELECTRODES
GOLD
Cyclic vol t-amograms of 6 mM da Fe +++ / ++ <CN) 6 were measured in 0.5 M K2S04. In Figure 30A, CV was measured for a single DC fibrillary mat (dispersed) at 0.10 mA / cm at 10, 25, and 50 mV / sec. The mat was made of r
in accordance with what is described in Example 6.18. In Figure 30B, the CM was measured for a gold foil electrode at 0.05 mA / cm at 10, 25, and 50 mV / sec. All potentials are in volts Vs. Ag / AgCl.
6.23. ELECTROCHEMICAL PROPERTIES OF MAT ELECTRODES
OF FIBRILLES: COMPARISON OF ANODIC PEAK CURRENT WITH
MAT THICKNESS
Cyclic voltamograms of 6 mM Fe +++ / ++ (CN) 6 in 0.5 M K2S04 were measured for fibril mats from the same geographic area (0.20 cm2), but with different thicknesses. The peak anode current (Figure 31) increased with increasing mat thickness for thicknesses ranging from 24 pm to 425 pm. For each thickness, the anodic peak current also increased with increasing scanning speed (for speeds between 10 mV / sec and 150 mV / sec). The rate of increase of the pi anodic current, as a function of the thickness, is
-187i
197 it also increased as the thickening increased. Fibril mats that were 24 pm thick behaved comparably to gold leaf electrodes.
6.24. NON-SPECIFIC LINK OF PROTEINS ON FIBRILLES
Nonspecific binding of proteins on carbon fibrils (cc) was measured as follows; i) a solution of Ru (bipy) +++ ++ labeled proteins was exposed (MARKER 1)
Γ to a known amount of carbon fibrils to reach equilibrium;
i i. ) the labeled protein / fibri solution was centrifuged, and the supernatant was collected, and iii) the amount of labeled protein remaining in the supernatant was assayed using electrochemistry collision (ECL)
To generate the curve shown in figure 32, anti-CEA antibodies bound on the derived MARKER 1 (carcinoembryonic antigen antibody fixed on an ECL MARKER
MARKER 1) fibrils at 3 pg / mL, added at various dilutions of
CC (single) in potassium phosphate buffer pH 7
Fibrils were removed by centrifugation after vortexing them for 20 minutes. ECL assays measuring the amount of protein (unbound) remaining in the supernatant were performed on an ORIGEN 1.5 analyzer (IGEN, Inc.) in aliquots of the supernatant from the reaction mixture diluted 5-fold with
-188ι
188 ORIGIN test regulator. A decrease in signal. ECL (relative to ECL signal 1 for a reaction mix object that had not been exposed to fibrils) resulted from an increased binding of protein labeled with a 5-MARKER 1 derivative when a higher concentration of fibrils was present carbon.
6.25. REDUCTION OF NON-SPECIFIC LINK OF PROTEINS WITH
FIBRILLES WITH DETERGENTS / SURFACTANTS
Using the method described in Example 6.2.4, the effect of
IC surfactant about? the protein binding on fibrils is ana 1 i zó. Triton X-100 was added to the anti-i-CEA fixed to derive MARKER 1 / fibril mixtures, the solution was incubated for 20 minutes, the tubes were centrifuged, and diluted soaked rite quotas were added to the i 5 times with ORIGIN test regulator
The results appear in
<td>the board</td><td>next and</td><td>in the figure</td><td> 33.</td><td></td>
<td>Number of</td><td>(T-X1OO),</td><td>Intensity</td><td>Prot-MARKER 1</td><td><GF)</td>
<td>tube</td><td>ppm</td><td>peak</td><td>pg / ml</td><td>ppm</td>
<td> 19</td><td> 1674</td><td> 1611</td><td> 2.65</td><td> 52</td>
<td> 18</td><td> 837</td><td> 1634</td><td> 2.65</td><td> 52</td>
<td> 17</td><td> 418</td><td> 1697</td><td> 2.65</td><td> 52</td>
<td> 16</td><td> 209</td><td> 1583</td><td> 2.65</td><td> 52</td>
<td> 15</td><td> 105</td><td> 1772</td><td> 2.65</td><td> 52</td>
<td> 14</td><td> 52</td><td> 1463</td><td> 2.65</td><td> 52</td>
<td> 13</td><td> 26</td><td> 627</td><td> 2.65</td><td> 52</td>
-189I
189
13 23 2.65 52
A curve resulting from the graphing of the ECL intensity of a protein marked with a MARKER. 1 derivative in solution versus concentration of Triton X-100 appears in figure 33. A higher ECL signal corresponds to more prote 1na dizzy with e 1 MARKER 1 derived in the supernatant, which corresponds to less prote 1 na marked with MARKER 1 derivative linked to fibrils. The t
Triton X-100 concentrations ranging from 10 ppm
100 ppm reduced link length? increasing the concentration from 100 to 2000 ppm did not further reduce the magnitude of the bond.
6.26. FREE MARKER ECL IN SOLUTION WITH FIBER MAT MAT ELECTRODE
A fibril mat prepared in accordance with Example 6.18 was installed in the mounting area 3403 of the working electrode holder 3401 of the "Fibril Cell" facility shown in Figure 34. The holder 3401 was slid into the bottom from compartment 3400 of the 20 electrochemical cell. The 3M Ag / AgCl reference electrode (Cyprus No. EE008) was installed in the electrochemical cell compartment through reference cell hole 3402. The cell was filled with an assay regulator (IGEN No. 402-005-01 lot No. 5298) and fixed on the PMT 3404 holder. Using a universa 1 programmer
-190 EG&B PARC model 175 and an EG&G Model 175 Potent iostat / Ga1 vanostat, the potential was swept from 0 V to + 3 V versus Ag / AgCl at IDO mV / sec. ECL was measured by
Hamamatsu R5600U-01 that received 900V power through a Pacific Instrumentes Model 126 photometer »Analog data was digitized at 10 Hz by a CIODAS-1601 A / D board powered by HEM Sriap-Master. The Fibril Cell was drained, rinsed with 1000 pM of MARKER 1 (IGEN No. 402-004 — C lot No. 4297), and filled with 1000 pM of
MARKER 1. The potential was swept as with the test regulator. Figure 35 shows the ECL traces (measured at 24.0 +/- 0.2 C) for test regulator 3501 and 1000 pM of REGULATOR 1 3502. The dark corrected ECL peak area was 22.10 nAs for the test regulator and of
46.40 nAs for 1000 pM MARKER 1.
6.27. ABSORBED MARKED ANTIBODY ECL WITH
FIBRILE MAT
Fibril mats were made to a thickness of 0.D0B89 cm from single cc scattered fibrils in the manner described in Example 6.18. The dried mats were then punched into 3mm discs and mounted on supports. The supports used in this experiment were manufactured from a 0.0762 cm polyester sheet configured by conductive gold ink printed on pants.
-1911
191
This conductive spider ink formed the counter electrode, reference electrode, and provided conductors for the working electrodes and other electrodes. Two fiber mat discs were mounted on each support configured using conductive tape containing carbon on both sides (Research Adhesives). After assembly, the discs were speckled with 0.5 μΐ of 10 μΐ / ml of antibody. anti-TSH fixed on MARKER 1 dar.i dipped in deionized water (Ru-TSH mono 1: 2 June 26, 1995, ISEN,
Inc.) or 0.5 μΐ of 10 pg / ml of capture antibody dyed with 1 'MARKER anti-i-TSH in deionized water (TSH poly June 25, 1995, IGEN, Inc.) and allowed to dry. After drying, the mats were soaked with Test Regulator ΙΘΕΝ. The support soaked mats were placed on an IGEN Origin 1.5 based instrument and the ECL was read using a 500mV / s scan rate from 0 to 4500mV. Figure 43 compares the signals from
Peak ECLs from the 4301 mats containing the MARKER 1 antibody and the 4302 mats contained the MARKER 1 'labeled capture antibody.
6.28. ECL USING FIBER MAT MAT ELECTRODE FOR
SANDWICH TESTS
Anti-AFP capture antibody was immobilized on fibrils as described above. The anti-AFP fibrils were washed in deionized (di) water and resuspended in
-192I a density of 1 mg / ml. A 4-layer fibril mat was produced using vacuum filtration as described in Example ¿.18. Two milligrams of anti-AFP fibrils will be added to 3 mg of simple DC 5 dispersed fibrils and the mixture is diluted to a total volume of 20 ml in di. The diluted mixture was filtered on a 0.45 pm na Ion filter. This layer of ionic layer 1 was then followed by two core layers, each consisting of 5 mg of single DC scattered fibrils. The mat core was then covered with a layer of mixed fibrils identical to the initial layer. This resulted in a fiber row that had approximately 40% anti-AFP fibrils on the upper and lower surface and approximately 100% single fibrils in the core. This mixed mat was air dried under vacuum and pozied on 3mm discs. These discs were then mounted on supports as described in Example 6.27. Dry, supported anti-AFP mats were soaked with AFP Calibrators A, C, and F (ICEN, Inc.) and allowed to incubate for 15 minutes at room temperature on top of the bench. After incubation, supported electrodes were washed with a flow of deionized water for 10 seconds and then dried with a lint-free dryer. The fibril mats were then soaked with anti-AFP bound to labeled antibody
-1931 with derived MARKER 1 (ICEN, Inc.) and allowed to incubate for 15 minutes at room temperature on top of the bench. After incubation, the supported electrodes were washed with deionized water and dried with a dryer. The fibril mats were then soaked with an IGEN assay regulator and read as described in Example 6.27.
6.29. DETECTION OF AVIDINE ECL MARKED WITH MARKER 1 ON A POLYACRYLAMIDE SURFACE
A covalently linked biotin containing cross-linked poly ac ri lamide gel was prepared by copolymerizing acryl lamide, bis-acrylamide, and Nacr iloi1-N'-biot ini 1-3, 6-d ioxaoc tan-1, 9-d iam ina (b iot i na linked to an acrylamide moiety through a tri (ethylene glycol) linker) using well known conditions (initiation with ammonium persulfate and TEMED). In this experiment, the concentrations of the three monomer species were 2.6 M, 0.065 M, and 0.023 M respectively (these concentrations of acr i lamide and bis-acrylamide are reported to result in gels with pore sizes smaller than the most proteins). Polymerization of the monomer containing solution between two glass plates spaced at a distance of approximately 0.07 mm led to the formation of a gel plate of the same thickness. After the completion of the reaction of
-194194 polymerization, any unincorporated biotin from the gel was pooled in four PBS changes. Avidin labeled with a derived MARKER 1 (where avidin refers to neutravidin, a modified avidin designed to present a reduced NSB, was used in this experiment) was bound to the surface of the gel by immersing the gel in a solution containing the protein at a concentration of 50 pg / mL in PBS for 20 minutes. Avidin marked with r
MARKER. 1 in excess was removed by washing by
1O immersion of the gel in four changes of ECL assay regulator <200 mM sodium phosphate, 100 mM tripropylamine, 0-02% (w / v) Tween-20, pH 7.2). As shown in Figure 39, the gel (3900) was placed in contact with gold working electrodes (3901) and counter electrodes (3902) configured on a glass support (3903). Increasing the potential between the two electrodes from 0.0 to 3.0 V and returning it to 0.0 V at a speed of 500 mV / s produced an ECL light signal 1 in accordance with what was measured in a PMT (3904) placed above the gel { figure
40). A gel prepared without including the biotin containing acrylamide derivative did not provide an ECL signal (Figure 41). This signal 1 obtained from the biotin-containing polymer was indicative of the presence of a complete cas i protein monolayer on the gel surface,
-195i
6.30. IMMUNO ASSAY IN ECL SIDEWALL ON A SURFACE OF P0l.IACRILAMT.DA
A crosslinked biotin containing crosslinked polyacrylamide gel was prepared as described in Example 6.29. Streptavid i na adsorbs on the gel surface to form a binding doininium capable of capturing biotin-labeled species. The surface is treated with a solution containing tripropylamine, an unknown concentration of an analyte, a biotin-labeled antibody against the analyte, and a different ECL MARKER 1-labeled antibody against the analyte. The presence of the analyte causes the formation of a complex of the analyte and the two antibodies that are later captured on the surface of this replicate. The ECL MARKER 1 attached to the secondary antibody present on the surface is measured as described in Example 6.29.
6.31 MULTIPLE ECL SANDWICH IMMUNO ASSAYS ON POLYACRYLAMIDE SURFACES SUPPORTED IN AN ELECTRODE A matrix of photoendurecib substance exposed and developed at 1 ~ 2 microns thick is prepared according to procedures well known in the art to provide a pattern of circular depressions arranged in a set. A 10: 1 mixture of SYLGARD silicone si-1B4 elastomer and the corresponding SYLGARD 184 curing agent is poured into the matrix and cured. The SYLGARD
-196ι polymerized is carefully removed from the silicon matrix. The resulting elastomeric print is inked by exposure to a solution containing the hydrophilic thiol HS- (CH2) 11- (0CH2CH2) 3-OH (1-10 · roM) 5 in ethanol, contacted with a uro substrate aligned and removed. The substrate is washed for several seconds with a solution containing thiol H8- (CH2) 10; CH3 (1-10 mM ethanol). The resulting surface is then ♦ r rinsed with ethanol and dried under a flow of nitrogen. The treatment of the surface has a solution that contains 1 and 1 acryl chloride and triethylamine in dioxane leads to the operation of the ones with acrylate groups. A mixture of ac ri 1iolsucc inimids, hydrophilic-terminated acid-conjugated domains of capillaries containing b is-ac ri lamide, a zo ~ bis-c iavaleromeric, antibodies presenting amino groups then comes into contact with the surface aligned to 1ineanda the capillaries with the acrylate-terminated domains to place prepolymer solutions containing specific antibodies in each domain, Each capillary in the capillary pool contains specific antibodies for one year of different interest. Exposure of the small drops of configured prepolymer to ultraviolet light causes the formation of crosslinked gels in the substrate, each presenting a binding domain on the surface. Essay
-1971
197 It was carried out by treating the substrate with a mixture of analytes capable of binding one or more of the binding domains presented on the gel surfaces in a regulated solution containing tripropylamine and 5 secondary antibodies labeled with MARKER 1 of ECL. The binding domains (4200, 4201, 4202) (in polyacrylamide droplets (4203) on an hour electrode (4232) are then placed in the vicinity of a working electrode
IT0 4204 (as shown in Figures 42A-B. The lO light emitted from each of the link domains is quantified using a CCD camera (4205) and camped with the link domains for internal standards included in the solution shows.
6.32. MULTIPLE ECL COMPETITIVE IMMUNO ASSAYS IN
POLYACRYLAMIDE SURFACES SUPPORTED IN AN ELECTRODE
A 1-2 micron thick exposed and developed fatoendurecib substance matrix is prepared according to well known procedures to give a pattern of circular depressions arranged in a set. A mix
<td> 20</td><td>1Use of</td><td>elastomer</td><td>184 by silleona</td><td>SYLGARD</td><td>and the agent of</td>
<td></td><td>cu raeión</td><td>SYLGARD 184</td><td>correspondent</td><td>empties</td><td>in the matrix and</td>
<td></td><td>It cures.</td><td>The SYLGARD</td><td>polymerisation</td><td>stir</td><td>carefully</td>
of the silicon matrix. The resulting elastomeric print is inked<sup>1</sup>'by exposure to a solution containing the hydroxyl terminated thiol HS- (CH2> 11-198ι
198 (□ CH2CH2J3-0H <1-10 is contacted with a gold-aligned substrate and removed. The substrate is washed for several seconds with a resulting containing solution, then rinsed with ethanol and dried under a stream of ni trogen. Treatment of the surface with a solution containing acryloyl chloride and triethylamine in dioxane leads to the functionalisation of the r
Hydroxyl terminated domains with acrylate groups. A set of capillaries containing mixtures of acrylamide, bis-acrylamide, N-acryl iIsuccinimide, azo-biscyanovaleric acid, and antibodies then comes into contact with the surface at 1 i attached to 1 i by aligning the capillaries with the finished domains in acrylate to place prepolymer solutions containing specific antibodies in each domain. The capillaries in the capillary pool contain specific antibodies for different analytes of interest. Exposure of the small drops of configured prepolymer to ultraviolet light causes the formation of crosslinked gels in the substrate, each presenting a binding domain on the surface. The assay is carried out by treating the substrate with a mixture of analytes capable of binding one or more of the binding domains presented on the gel surfaces in a regulated solution containing tr iprop i lamina and analogous
-199199 marked with MARKER. 1 of ECL establishing a competition between analytes marked with
MARKER 1 for ECL and ana 11 unmarked for the link on domains <4200,
4201, 4202) (in cats on an ORA electrode ITD working electrode
4204 (as shown in figure
42. The light emitted from each of the binding domains is quantified using a CCD camera (4205) and compared to the binding domains for internal standards included in the sample solution.
6.33. MULTIPLE ECL TESTS FOR BINDING CELLS ON POLYACRYLAMIDE SURFACES SUPPORTED IN AN ELECTRODE
A 1-2 micron thick exposed and developed matrix of your photoendurec ib stains is prepared according to well-known procedures to provide a pattern of circular depressions arranged in a joint. A 10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding SYLGARD 184 curing agent is poured into the matrix and cured. The polymerized SYLGARD is carefully removed from the silicon matrix. The resulting elastomeric print is inked by exposure to a solution containing the hydroxy-terminated thiol HS- (CH2) 11 (DCH2CH2) 3-0H (1-10 mM) in ethanol, and contacted with a gold substrate aligned and removed. The substrate is
-200i
<img file="MX9706471A_D0004.tif" />
with a solution containing mM in et al). The protruding surface is then rinsed with ethanol and dried under a flow of nitrogen. Treatment of the surface with one containing acryloyl chloride and trieti laminates in dioxane cam to the hydroxyl terminated with acrylate groups. A set of contains mixtures of acrylamide, bis acrylamide,
Na c: ri 1 oi 1 succ inimi da, cyanovaleric acid, and antibodies directed against cell phones then comes into contact with the surface by iridescenting the capillaries with the acrylate-terminated domains to place prepolymer solutions in each domain. Exposure of the small drops of configured prepolymer to ultraviolet light causes the formation of crosslinked gels in the substrate, each presenting a binding domain on the surface. The assay is carried out by treating the binding domains first with a suspension of cells, then with a mixture of binding reagents capable of binding one or more of the bound cells to the gel surfaces in a buffered solution containing tripropylamine and MARKER 1-labeled secondary antibodies to ECL and / or other analyte-specific binding reagents. The binding domains (4200, 4201, and 4202) (in drops of
-201I
201 Polyacrylamide (4203) on a gold electrode (4232) are then placed very close to a T.TO working electrode
4204 (As shown in Figure 42. The light emitted from each of the binding domains is quantified using a CCD camera (4205) and compared to the binding domains for internal standards included in the sample solution.
6.34. MULTIPLE ECL TESTS TO LINK ANALYZES ON r
CELLS ON POLYACRYLAMIDE SURFACES SUPPORTED IN A
ELECTRODE
An exposed and developed 1-2 micron thick exposed photorealistic substance matrix is prepared in accordance with well known procedures to provide a pattern of arranged cireular depressions in a set. A 10: 1 mixture of SYLGARD silicone elastomer 184 and the corresponding SYLGARD 184 curing agent is poured into the matrix and cured. The polymerization SYLGARD is carefully removed from the silicon matrix. The resulting elastomeric print is inked by exposure to a solution containing the hydroxyl terminated thiol
HS- (CH2) ll- (0CH2CH2) 3-0H (1-10 mM) in ethanol, placed in contact with an aligned gold substrate and removed. The substrate is washed for several seconds with a solution containing the thiol HS- (CH2) 10-CH3 (1-10 mM in ethanol). The resulting surface is then rinsed with ethanol and
-202I dries under you a solution containing acryloyl chloride trieti lamina in leads to
1st functionalization of the d-hydroxy-terminated domains with acrylate groups. A set of capillaries containing mixtures of acrylamide, bis-acrylamide,
NacriloiIsucci ni mida, azo-bis-cyanovaleric acid, and cells are then contacted with an aligned surface aligning the capillaries containing capillaries with the finished domains for the types in which to place solutions of p repo 1 i me ros cells res set of capillaries contains cells that
Those with different surface structures that are linked to different ana 1 i tos. Exposure of the small drops of prepolymer configured to ultraviolet light causes the formation of ret i gels. cu sides on the substrate, each presenting a bonding domain on the surface. The test is carried out by treating the gels with a sample that contains a mixture of analytes capable of binding to one or more of the binding domains, a regulated solution that contains markers 1 for ECL and / or other reagents of (4200, 4201, and 42O2) (in polyacrylamide drops (4203) in
-203a gold electrode (4232) is then placed in close proximity to an ITÜ working electrode (4204R) as shown in Figure 42. The light emitted from each of the binding domains is quantified using a CCD 5 camera (4205) and compared to the binding domains for internal standards included in the sample solution.
6.35. MULTIPLE ECL COMPETITIVE HYBRIDIZATION TESTS IN
POLYACRYLAMIDE SURFACES SUPPORTED IN AN ELECTRODE
An exposed and developed 1 »2 micron thick photosetting support matrix is prepared in accordance with well known procedures to provide a pattern of arranged circular depressions in an assembly. A mix
<td></td><td>10: 1 of</td><td>elastomer</td><td>184 silicone</td><td>SYLGARD</td><td>and the agent of</td>
<td></td><td>curation</td><td>SYLGARD 184</td><td>correspondent</td><td>empties</td><td>in the matrix and</td>
<td> 15</td><td>It cures.</td><td>The SYLGARD</td><td>polymerized it</td><td>stir</td><td>carefully</td>
from the matrix of si 1ic io. The protruding elastomeric print is "inked" by exposure to a solution containing the hydroxyl terminated thiol HS- (CH2) 11 (0CH2CH2) 3-OH (1-10 mM) in ethanol, it is contacted with a substrate of gold lined and removed. The substrate is washed for several seconds with a solution containing thiol HS— (CH2) 10 — CH3 (1-10 mM in ethanol). The resulting surface is then rinsed with ethanol and dried under a flow of nitrogen. Treating the surface with a solution containing acryloyl chloride and triethylamine in
-2041 di oxa does not cause funnelation of hydrophilic terminated domains with acrylate groups. A set of capí i lares que? Contains those of acr i lamide, b isacrylamide, N-acryloiIsuccinimide, azo-bis5c innovaleric, and nucleic acid probes functionalized with amino groups then come into contact with the aligned surface, aligning the capillaries with the finished domains with acrylate to place pre-polymer solutions containing specific probes in each domain. The capillaries in the capillary pool contain probes specific for a nucleic acid sequence of interest. Exposure of the small drops of configured prepolymers to ultraviolet light causes the formation of crosslinked gels on the substrate, each presenting a binding domain on the surface. The assay was carried out by treating the substrate with a mixture of samples that may contain sequences capable of binding to one or more of the binding domains presented on the gel surfaces in a regulated solution containing tripropylamine.
ECL MARKER 1 that can compete with the target year 1 to link on the surface. The binding domains (4200, 4201, and 4202) (in polyacrylamide droplets (4203) on a gold electrode (4232) are then placed in the vicinity of a working IT0 electrode (4204) as
-205ι
205 shown in fi gure 42. The emi light. t from each of the linking domains is quanti fied using a camera
CCD (4205) and compares to the binding domains for internal standards included in the sample solution.
6.36. MULTIPLE TESTS OF ECL HYBRIDIZATION SANDWICH
ON SURFACES
OF POLYACRYLAMIDE SUPPORTED IN AN ELECTRODE
A 1-2 micron thick exposed and developed photo-hardening matrix is prepared according to well-known procedures to give a pattern of arranged circular depressions in an assembly. A mix
Silicana e1astomero 184 10: 1
SYLGARD and the healing agent
SYL.GARD empties on mat rizy heals.
The SYLGARD pal imer i zed is carefully removed from the silicon matrix. The resulting elastomeric stamp is inked by exposure to a solution containing the hydroxyl terminated thiol HS- (CH2) 11- (0CH2CH2) 3-OH (110 mM) in ethanol, contacted with a gold substrate at 1 inate and it is removed. The substrate is washed for several seconds with a solution
CH3 (1-10 mM in ethanol). The resulting surface is then rinsed with ethanol and dried under a flow of nitrogen. Surface treatment has a solution containing acryloyl chloride and triethylamine in dioxane that results in the functionalization of hydroxyl-terminated domains with acrylate groups. A set of capillaries containing
-206i mixtures of acri 1 a ni ida, bi s-acrylamide,
<img file="MX9706471A_D0005.tif" />
ac ri 1 oi 1 succ i π imi gives azobis-cyanovaleric acid, and nucleic acid probes fused i ana 1 izadas with amino groups then come into contact with the aligned surface, aligning the capillaries with the domains dreaded with acrylate to place prepolymer salts containing specific probes in each domain.
The capillaries in the capillary array contain probes specific for a nucleic acid sequence of interest. Exposure of the configured small prepolymer droplets to ultraviolet light causes the formation of crosslinked gels in the substrate, each exhibiting a binding domain. on the surface.
The assay is carried out by treating the substrate with a mixture of samples that may contain sequences capable of binding one or more of the binding domains presented on the gel surfaces in a regulated solution containing tripropylamine and MARKER-labeled sequences. 1 of ECL, which can bind the analyte in non-complementary sequences on the probes attached to the surface. The binding domains (4200, 4201, and 4202) (in polyacrylamide droplets (4203) on a gold electrode (4232) are then placed near a working ITO electrode (4204) as shown in Figure 42. The light emitted from each of the domains is quantified using a CCD camera (4205) and
-207207 compares to the binding domains for internal standards included in the sample solution.
6.37. MULTIPLE TESTS OF DIFFERENT TYPES IN SOME
POLYACRYLAMIDE SURFACES SUPPORTED IN AN ELECTRODE
A matrix of exposed and developed thick photoenduring substance procedures is prepared in accordance with well-known procedures to provide a pattern of arranged circular depressions in one set. A mix
51 of curae ion is cured.
elastomer 184 si 1icone
SYLGARD 184 corresponding
SYLGARD leaves
THE GYLGARD pol imeri. zado is removed and the agent of in the matrix and cu i. dice of the silicon matrix. The resulting elastomeric print is inked containing the <0CH2CH2) 3 "0H by exposure to a solution that thiol <1-10 a gold substrate washes for several hours finished with hydrophilic HS- (CH2) 11mM) in ethanol, is placed in contact with the line and remove. The substrate is seconds with a solution containing the thiol HS- (CH2) 10-CH3 (1-10 mM in ethanol). The resulting surface is then rinsed with ethanol and dried under a flow of nitrogen. Treatment of the surface with a solution containing acryloyl chloride and triethylamine in dioxane causes functionalization of the hydroxyl-terminated domains with acrylate groups. A set of capillaries containing mixtures of acrylamide, bis25 acrylamide, N-acryloiIsuccinimide, azo-bis-2081 anovaleric acid, and any of the binding reagents described in Examples 6.31-6.36 then contact the aligned surface, aligning capillaries with acrylate-terminated domains for placing prepolymer solutions that contain specific probes are of interest.
domain. Each capillary in the set of capillaries domi nías of specific links for ana 1 itos de
Exposure of the small drops of prepolymer configured to ultraviolet light causes the formation of on the substrate, each presenting a binding domain on the surface. The assay is carried out by treating the substrate with a sample mixture that may contain analytes capable of binding one or more of the binding domains presented on the 15 gel surfaces in a buffered solution containing tripropyl lamina i good. Dizzy analogues with MARKER 1 compete with analytes for linkage over analytes from reluctant domains of binding interest and / or reactive with MARKER 1 of linkage ECLs on
4202) (in drops of polyacrylamide (4203) on a gold electrode (4232) are then placed near an ITO working electrode (4204) as shown in Figure 42. The light emitted from each of the binding domains is quantified using a CCD camera (4205) and compared with
-209ι
209 Link domains for internal standards included in the sample solution.
6.38. HIGHLY REVERSIBLE ECL
Polycrystalline gold electrodes (purchased in manual polishing medium 1) were cleaned sequentially
0.03 pm, followed by binding that chemical at 1: 3
H202 / H2S04
Ag / AgCl e1ec clean cycles were then used overnight in ethanol
Protein adsorption was carried out on C8SH-modified electrodes with 20 pl of 1-mark labeled bovine serum albumin (BSA) eluted in a phosphate buffered saline solution (PBS, 0.15 M NaCl / 0.1 M NaPi, pH 7.2) and extensive washing of the surface with the same regulator after a 10 minute incubation.
An ECL was performed in a three-electrode cell with an Ag / AgCl reference electrode, a platinum cable counter electrode, and an EG&G 283 potentiostat. The light intensity was measured with a Pacific Intruments photometer. and a Hamama tsu photomodule ip1 icator placed at the bottom of the electrochemistry cell. The protein adsorbed electrode was immersed in a 0.1 M TPA and 0.2 M phosphate solution, pH 7.2. An ECL response was observed
-210ι
210 highly reversible (gives substantially similar intensity on forward and backward scans f when the electrode potential was cycled between
0.0 V v 1.2
V, as shown in Figure 44A, indicating that of the thiol and protein layers on the electrode.
Cyclic voltammetric experiments were performed on the same instruments as for ECL, without the use of PMT and covered with
CSSH (not protein) in a 1 niM solution of potassium ferricyanide (in PBS) and the electrode was scanned at a cycle between + 0.5 V and -0.3
V. It is indicative that the monolayer is still intact at 1.2 V, since there was only capacitive current in the voltamogram between +0.5 V and -0.3
V and no pharadaic ferricyanide current (figure
44B).
6.39
ALMOST REVERSIBLE ECL
They were made the same way as described above. In the experiments of
ECL, the patena ia 1 was scanned between 0.0 V
V, the corresponding light intensity. As illustrated in Figure 45A, some loss of ECL was observed between scans forward and backward through the same cycle, as well as between scans.
-211i
Cyclical valtamograms of the oxidation at 1.5 showed a pharadaic current indicating pa re ia 1 of the thiol monolayer at 1.5
45E>
6.40. IRREVERSIBLE ECL experiments, protein modi ication and adsorption were carried out in the same way as in
0.0 V. Intense light was observed on the scan towards which it was observed under example conditions
6.38), but fell to the bottom on the reverse scan, as shown in Figure 46A. Voltamograms oxidation a
z.
V indicated that most of the thiol monolayer was desorbed (Figure 46B)
6.41. AN ECL SANDWICH IMMUNO ASSAY USING A
PRIMARY ANTIBODY IMMOBILIZED IN A GOLD ELECTRODE
CONFIGURED
In this example, an antibody against prostate specific antigen (PSA) is immobilized on a gold electrode configured for use in a PSA immunoassay.
A matrix of photoendurscib substance exposed and revealed from one to two microns thick is prepared in accordance with
-212i
<img file="MX9706471A_D0006.tif" />
well-known procedures to provide a layer of photocurable substance on a silicon holder with a 1mm x 1mm square patch where the sustain is removed
A lOsl mix of silicone elastomer
SYL.GARD
184 v the corresponding curing agent is poured onto it and cured. The polymorphized SYLGARD is removed cu i, damagingly from the matrix of si 1 ici o. The resulting elastomeric stamping is "inked by exposure to a solution containing the hydroxyl terminated thiol HS- <CH2) 11- (0CH2CH2J3-DH and the thiol terminated with nitrilliacetic acid (NT A) (CH2111 (OCH2CH2 ) 3OC (D) NH (CH2) 4CH (CO2H) N <CH2C02H2) in ethanol.
The inked stamp contacts a gold substrate and is removed to form a SAM x 1mm. The substrate is washed for several seconds with a solution containing only the hydroxyl-terminated thiol in ethanol, to avoid nonspecific protein binding on regions outside of the stamped characteristic
The resulting surface ie is then rinsed with ethanol dried under a stream the surface with a solution of NIC12 followed by treatment with a solution containing a fusion protein presenting the binding sites of an anti-PSA mouse monoclonal and the IHis peptide) ¿>, Leads to the immobilization of the protein. fus ion on the surface so
-213I
717 Ζ. one -?
controlled »This process provides a reproducible and predetermined amount of protein immobilized on the surface. The orientation of the protein on the surface is controlled by the location of the sequence (His) 6 in the primary structure of the> fusion protein. The absolute amount of immobilized p ratein is controlled by the proportion between the NEA-terminated thiol and the hydroxy-terminated thiol in the stamped SAM and by surface area 1 of the stamped terrestrial layer. A ca1 ibrae curve for PSA is determined by preparing solutions containing known concentrations of serum PSA ions (at concentrations ranging from 1 fM to 1 uM. Numerous surfaces prepared in accordance with the above described are treated with the PSA calibration standards and then with a solution containing a secondary antibody against PSA (marked with a derivative of MARKER
1) in an optimized concentration. The calibration curve is determined by immersing the surfaces in a
7.2), and through the emission measurement when the gold potential is cycled between
0.0 v a scan speed of
0.5 V / sec. The determination of unknown concentrations of PSA in the serum in the same procedure except that the concentration of PSA is
-214ι
<img file="MX9706471A_D0007.tif" />
calculated from the peak ECL signal by reference to the
INCORPORATION OF REFERENCES.
The present invention is not limited in its scope to the specific modalities of the invention herein, in addition to those described herein, will be apparent to a person skilled in the art from the and annexes.
Modifications fall within the scope of claims. Various publications are mentioned herein, their submissions are incorporated by reference in their entirety.
-2151
Contents104
71 members in 18 offices
Priority claims2
| Document | Office | Kind | Date |
|---|---|---|---|
| 40207695 | United States of America | A | |
| 40227795 | United States of America | A |
Members71
| Document | Office | Kind | |
|---|---|---|---|
| CA2213854A1 | Canada | A1 | |
| CA2704228A1 | Canada | A1 | |
| WO9628538A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU5420596A | Australia | A | |
| ZA961925B | South Africa | B | |
| BR9607193A | Brazil | A | |
| EP0821726A1 | European Patent Office (EPO) | A1 | |
| EA199700219A1 | Eurasian Patent Organization (EAPO) | A1 | |
| CA2265828A1 | Canada | A1 | |
| WO9812539A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU4649597A | Australia | A | |
| ZA978380B | South Africa | B | |
| CN1186513A | China | A | |
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| CZ284497A3 | Czechia | A3 | |
| HUP9801679A2 | Hungary | A2 | |
| MX9706471AThis record | Mexico | A | |
| JPH11502617A | Japan | A | |
| EP0821726A4 | European Patent Office (EPO) | A4 | |
| EP0944820A1 | European Patent Office (EPO) | A1 | |
| NZ306051A | New Zealand | A | |
| EP0944820A4 | European Patent Office (EPO) | A4 | |
| US6066448A | United States of America | A | |
| AU720625B2 | Australia | B2 | |
| KR20000036176A | Republic of Korea | A | |
| US6090545A | United States of America | A | |
| US6140045A | United States of America | A | |
| EA001198B1 | Eurasian Patent Organization (EAPO) | B1 | |
| HUP9801679A3 | Hungary | A3 | |
| JP2001503856A | Japan | A | |
| US6207369B1 | United States of America | B1 | |
| US2001021534A1 | United States of America | A1 | |
| AU743567B2 | Australia | B2 | |
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| US6673533B1 | United States of America | B1 | |
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| KR100564269B1 | Republic of Korea | B1 | |
| US2006068499A1 | United States of America | A1 | |
| US2006172340A1 | United States of America | A1 | |
| JP2008076407A | Japan | A | |
| CZ299135B6 | Czechia | B6 | |
| JP2008170449A | Japan | A | |
| IL117422A | Israel | A | |
| BR9607193B1 | Brazil | B1 | |
| JP2009115828A | Japan | A | |
| JP4365362B2 | Japan | B2 | |
| JP4365434B2 | Japan | B2 | |
| JP4486684B2 | Japan | B2 | |
| JP4491071B2 | Japan | B2 | |
| CA2213854C | Canada | C | |
| US7824925B2 | United States of America | B2 | |
| EP2280268A1 | European Patent Office (EPO) | A1 | |
| EP2284536A2 | European Patent Office (EPO) | A2 | |
| US2011124116A1 | United States of America | A1 | |
| EP2284536A3 | European Patent Office (EPO) | A3 | |
| JP4754003B2 | Japan | B2 | |
| HK1151588A1 | Hong Kong, China | A1 | |
| US8541168B1 | United States of America | B1 | |
| US8541174B2 | United States of America | B2 | |
| CA2704228C | Canada | C | |
| EP0821726B1 | European Patent Office (EPO) | B1 | |
| US8722323B2 | United States of America | B2 | |
| EP2280268B1 | European Patent Office (EPO) | B1 | |
| US2014274805A1 | United States of America | A1 |
Numbers
- Application
- 9706471
Titles2
- English
- MULTI-ARRAY, MULTI-SPECIFIC ELECTROCHEMILUMINESCENCE TESTING.
- Spanish
- PRUEBA DE ELECTROQUIMICOLUMINISCENCIA MULTIESPECIFICA, DE ARREGLOS MULTIPLES.
Classification
- CPC, 10
- G01N21/66
- C12Q1/68
- B82Y30/00
- G01N21/76
- G01N33/5438
- G01N33/582
- Y10S977/957
- Y10S436/806
- C12M1/00
- B82Y5/00
- IPC, 10
- C12M1 00
- C12Q1 68
- G01N21 66
- G01N21 76
- G01N21 78
- G01N33 543
- G01N33 548
- G01N33 566
- G01N33 58
- G01N37 00