Low background multi-well plates for fluorescence measurements of biological and biochemical samples
Abstract
Device for spectroscopic measurements, comprising: a platform; and a polymer layer with low fluorescence and high transmittance, deposited separately on said platform, wherein said polymer layer produces approximately 400 percent or less of fluorescence compared to molten silica approximately 150 micrometers thick at wavelengths excitation between 300 to 400 nm and emission wavelengths between approximately 300 to 800 nm, and wherein said polymer layer comprises a polymer based on a cycloolefin.
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38 claims: 1 independent, 37 dependent
- 1ES 2 289 784 T3 REIVINDICACIONES 1. Dispositivo para mediciones espectroscópicas, que comprende:una plataforma;y una capa de polímero con baja fluorescencia y elevada transmitancia, depositada separadamente sobre dicha plataforma, en el que dicha capa de polímero produce aproximadamente 400 por ciento o menos de fluorescencia comparada con la sílice fundida de aproximadamente 150 micrómetros de espesor a unas longitudes de onda de excitación de entre 300 a 400 nm y a unas longitudes de onda de emisión de entre aproximadamente 300 a 800 nm, y en el que dicha capa de polímero comprende un polímero basado en una cicloolefina.
- 2Dispositivo según la reivindicación 1, en el que dicha plataforma es una placa de microvaloración con una huella de una placa de microvaloración de 96 pocillos estándar y que presenta pocillos de microvaloración.
- 3Dispositivo según la reivindicación 1, en el que dicha plataforma es una placa multipocillo para la detección de una señal a partir de una muestra y dicha capa forma por lo menos una porción de una superficie del fondo de un pocillo de dicha placa multipocillo.
- 4Dispositivo según la reivindicación 3, en el que dicha placa multipocillo presenta entre aproximadamente 96 y aproximadamente 3.456 pocillos.
- 5Dispositivo según la reivindicación 4, en el que dicha placa multipocillo comprende paredes del pocillo realizadas en un material distinto al polímero de cicloolefina.
- 6Dispositivo según la reivindicación 5, en el que dicha capa comprende una soldadura por fusión con calor con dichos pocillos.
- 7Dispositivo según la reivindicación 6, en el que dicha capa presenta un espesor de aproximadamente 50 a 300 micrómetros de espesor.
- 8Dispositivo según la reivindicación 6, en el que dicha capa produce aproximadamente 200 por ciento o menos de la fluorescencia comparada con el vidrio de sílice fundida de 100 micrómetros de espesor a las longitudes de onda de excitación de entre aproximadamente 300 a 400 nm y a unas longitudes de onda de emisión de entre aproximadamente 300 a 800 nm.
- 9Dispositivo según la reivindicación 4, en el que dicha placa multipocillo comprende pocillos realizados en un copolímero de cicloolefina.
- 10Dispositivo según la reivindicación 9, en el que dicha capa comprende una soldadura por fusión con calor con dichos pocillos.
- 11Dispositivo según la reivindicación 9, en el que dicha capa presenta un espesor de aproximadamente 50 a 200 micrómetros de espesor.
- 12Dispositivo según la reivindicación 9, en el que dicha capa produce aproximadamente 200 por ciento o menos de la fluorescencia comparada con el vidrio de sílice fundida de 100 micrómetros de espesor a unas longitudes de onda de excitación de entre aproximadamente 300 a 400 nm y a unas longitudes de onda de emisión de entre aproximadamente 300 a 800 nm.
- 13Dispositivo según la reivindicación 4, en el que dicha capa presenta un espesor de aproximadamente 20 a 300 micrómetros de espesor.
- 14Dispositivo según la reivindicación 13, en el que dicha placa multipocillo comprende además un pigmento para la reducción de fondo.
- 15Dispositivo según la reivindicación 13, en el que dicha capa comprende además un miembro de un par de enlace.
- 16Dispositivo según la reivindicación 13, en el que dicha capa comprende además una pluralidad de células vivas.
- 17Dispositivo según la reivindicación 15, en el que dicha capa comprende además un revestimiento para aumentar la unión de las células. ES 2 289 784 T3
- 18Dispositivo según la reivindicación 1, en el que dicha plataforma es un andamiaje para una matriz de sitios de ensayo determinados previamente espacialmente sobre dicha capa y dicho andamiaje no interfiere sustancialmente con la detección de una señal de dichos sitios de ensayo.
- 19Dispositivo según la reivindicación 18, en el que dicha capa presenta un espesor de aproximadamente 40 a 300 micrómetros de espesor.
- 20Dispositivo según la reivindicación 19, en el que dichos sitios de ensayo son de aproximadamente 10 micrómetros cuadrados a 200 micrómetros cuadrados de área.
- 21Dispositivo según la reivindicación 19, en el que dicha capa se derivatiza para la unión de entidades químicas.
- 22Dispositivo según la reivindicación 20, en el que dichos sitios de ensayo están impresos sobre dicha capa.
- 23Dispositivo según la reivindicación 20, en el que dichos sitios de ensayo comprenden un miembro de un par de enlace.
- 24Dispositivo según la reivindicación 1, en el que dicha plataforma comprende un copolímero de cicloolefina.
- 25Procedimiento para producir un dispositivo para mediciones espectroscópicas según la reivindicación 1, que comprende:depositar separadamente una capa de polímero con baja fluorescencia y elevada transmitancia sobre una plataforma de polímero, en el que dicha capa de polímero comprende un polímero basado en cicloolefina.
- 26Procedimiento según la reivindicación 25, en el que dicha plataforma es una placa de microvaloración con una huella de una placa de microvaloración de 96 pocillos estándar que presenta pocillos de microvaloración.
- 27Procedimiento según la reivindicación 25, en el que dicho polímero se selecciona de entre el grupo de copolímero de polietileno ciclopenteno, copolímero de polietileno y ciclohexano y copolímero de polietileno y ciclohepteno.
- 28Procedimiento según la reivindicación 25, que comprende además la etapa que consiste en exponer dicha capa y dicho polímero a una cantidad suficiente de energía de radiofrecuencia para promover el calentamiento interno de dicha capa y de dicho polímero.
- 29Procedimiento según la reivindicación 25, en el que dicho depósito comprende soldaduras por calor de dicha capa de polímero a dicha plataforma de polímero a una temperatura de 320 grados.
- 30Procedimiento según la reivindicación 25, que comprende además la etapa que consiste en aplicar entre aproximadamente 1.034 y 1.517 bar (15.0000 y 22.000 psi) de presión a dicha capa y a dicho polímero.
- 31Sistema para mediciones espectroscópicas, que comprende:unos reactivos para un ensayo, y un dispositivo según la reivindicación 1 en el que dicha plataforma es para la detección de una señal a partir de una muestra.
- 32Sistema según la reivindicación 31, en el que dicha plataforma presenta una huella de una placa de microvaloración de 96 pocillos estándar que presenta pocillos de microvaloración.
- 33Sistema según la reivindicación 31, que comprende además un detector.
- 34Procedimiento para la detección de una señal, que comprende:poner en contacto una muestra con el dispositivo según la reivindicación 1 en el que dicha plataforma es para la detección de una señal a partir de dicha muestra, y detectar una señal a partir de dicha muestra.
- 35Procedimiento según la reivindicación 34, en el que dicha plataforma es una placa de microvaloración con una huella de una placa de microvaloración de 96 pocillos estándar que presenta pocillos de microvaloración.
- 36Procedimiento según la reivindicación 34, en el que dicha detección comprende detectar la epifluorescencia de debajo de dicha plataforma. ES 2 289 784 T3
- 37Procedimiento según la reivindicación 34, en el que dicha plataforma es una placa multipocillo y dicho detector comprende la detección con una disposición óptica que corresponde a la densidad de pocillos en dicha placa multipocillo.
- 38Dispositivo según la reivindicación 1, en el que dicha capa de polímero produce de aproximadamente 100 a 50 por ciento o menos de la fluorescencia comparada con la sílice fundida de aproximadamente 150 micrómetros de espesor.
Independent claims38
260 paragraphs in 11 sections, as filed
ES 2 289 784 T3
DESCRIPTION
Low base fluorescence multiwell plates for fluorescence measurements of biological and biochemical samples.
Cross reference to related requests
This application claims the rights of an earlier filing date in United States patent applications: US application 08 / 867,567, filed June 2, 1997; US application 08 / 868,018, filed June 3, 1997; US application 08 / 867,584 filed June 2, 1997; and US application 08 / 868,049, filed June 3, 1997; each incorporated for reference.
Technical field
The present invention relates generally to multiwell plates and platforms composed of cycloolefins for use in spectroscopic measurements and procedures for producing such devices. Multiwell plates and platforms are particularly useful for fluorescence measurements of chemical or biological samples.
Introduction
A number of multiwell plates are commercially available for culturing cells or for conducting chemical or cellular assays. While many of these multiwell plates offer the desired characteristics of biocompatibility, ease of production, and substantial structural integrity, the inventors of the present invention have generally discovered that these plates, especially plates with polymeric bottoms, suffer from a high degree of fluorescence. substantially. The relatively high amount of base fluorescence inherent in commercially available plates with polymeric bottoms makes such plates generally unsuitable for highly sensitive fluorescence measurements associated with many assays, particularly assays of microliter or lower volumes.
The inventors of the present invention recognized a need in chemical and biological techniques for multiwell plates and platforms for chemical or biological cases, such as binding assays or cell-based assays. The present inventors prepared criteria for selecting materials suitable for the production of multiwell plates and platforms for such applications. As a key example of the selection criteria, which is fully described herein, the present inventors investigated the spectral properties of various polymers, including their fluorescence and transmittance, for compatibility with spectroscopic measurements of chemical and biological events. . Such materials would also desirably, but not necessarily depending on the application, possess biocompatibility, relative chemical inertness, and sufficient rigidity for application by hand and ease of production. The present inventors selected a variety of polymers for testing based, in part, on the structural characteristics of the polymers, which are fully described herein. The present inventors' search for polymers included search fields not associated with spectroscopic measurements, including techniques associated with cycloolefin polymers, such as electronic and audio recording techniques. The present inventors compared a variety of materials with sheets of fused silica (eg, glass) that possess relatively lower inherent fluorescence. From the number of films tested, the present inventors surprisingly found cycloolefin films that possessed fluorescence and transmittance properties approaching (or even improving) those of fused silica glass.
As described herein, the present inventors have for the first time developed new multiwell plates utilizing cycloolefins that offer excellent performance characteristics in assays. Such multiwell plates can be used in conventional 96-well plates or higher density formats. The present inventors also describe here for the first time new platforms that can be used for assays or reaction sites that are particularly corrected for cycloolefin production. Such plates and platforms can also be used for other applications such as diagnostics or chemical synthesis.
Summary
The present invention includes devices for spectroscopic measurements, such as multiwell plates and platforms. Typically such devices comprise a high transmittance, low fluorescence layer comprising a cycloolefin polymer and well (s) of a plate or multiwell platform to support the layer.
The multiwell plates of the present invention comprise a high transmittance, low fluorescence layer, comprising a cycloolefin polymer, and a well (s) to support, or form, the layer. The cycloolefin usually comprises at least a portion of a surface of a bottom of a well of the multiwell plate.
ES 2 289 784 T3
The platforms of the present invention comprise a high transmittance, low fluorescence layer, or window, generally comprising a cycloolefin polymer or other low fluorescence material, and a scaffold or framework to support, or form, the layer. The window has predetermined dimensions and can be arranged as a plurality of windows on the frame in any geometric arrangement, including two-dimensional arrangements. In some embodiments the window allows the detection of spectroscopic events, in which light often passes through the window. In other embodiments the window is basically a reaction or assay site that can allow the detection of a chemical reaction or assay.
The present invention also includes methods for detection and production that relate to the multiwell plates and platforms of the present invention.
Detailed description of the present invention
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning commonly understood by any person skilled in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures of spectroscopy, drug discovery, cell culture, molecular genetics, plastics production, polymer chemistry, diagnostics, amino acid and nucleic acid chemistry, and Sugars described below are well known and commonly used in the art. Standard techniques are typically used for plastics preparation, signal detection, recombinant nucleic acid procedures, polynucleotide synthesis, and microbial cultivation and transformation (eg, electroporation, lipofection). The techniques and procedures are generally carried out according to conventional procedures of the art and according to various general references (see generally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2<sup>to</sup> ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Lakowicz, JR Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983) for fluorescence techniques, which are incorporated herein by reference) provided throughout the document. Standard techniques are used for chemical synthesis, chemical analysis, and biological testing. As used throughout the specification, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
"Fluorescent donor moiety" refers to the radical of a fluorogenic compound that can absorb energy and is capable of transferring energy to another fluorogenic molecule or to a part of a compound. Suitable donor fluorogenic molecules include, but are not limited to, coumarins and dyes related to xanthene dyes such as fluoresceins, rhodoles and rhodamines, resorrufins, cyanine dyes, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazides such such as luminol and isoluminol derivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans, aminoquinolines, dicyanohydroquinones and complexes of europium and terbium and related compounds.
"Quencher" refers to a chromophoric molecule or part of a compound that is capable of reducing the emission of a fluorescent donor when it binds to the donor. Quenching can occur by any of a number of mechanisms including fluorescence resonant energy transfer, photoinduced electron transfer, paramagnetic enhancement of system crossover, Dexter exchange coupling, and excitation coupling such as the formation of dark complexes.
"Acceptor" refers to a quencher that operates via the transfer of fluorescence resonant energy. Many acceptors can re-emit the transferred energy as fluorescence. Examples include coumarins and related fluorophores, xanthenes such as fluoresceins, rhodoles and rhodamines, resorrufins, cyanines, difluoroboradiazaindacenes, and phthalocyanines. Other chemical classes of acceptors generally do not re-emit the transferred energy. Examples include indigos, benzoquinones, anthraquinones, azo compounds, nitro compounds, indoanilines, di- and triphenylmethanes.
"Binding partner" refers to two moieties (eg, chemical or biochemical) that possess an affinity for each other. Examples of binding partners include antigen / antibody, lectin / avidin, target polynucleotide / probe oligonucleotide, antibody / antibody, receptor / ligand, enzyme / ligand, and the like. "A member of a binding pair" refers to one half of the pair, such as an antigen or a ligand.
"Dye" refers to a molecule or part of a compound that absorbs specific frequencies of light, including but not limited to ultraviolet light. The terms "dye" and "chromophore" are synonymous.
"Fluorophor" refers to a fluorescent chromophore.
"Membrane permeating derivative" refers to a chemical derivative of a compound that possesses increased membrane permeability compared to that of a non-derivatized compound. Examples include ester, ether, and carbamate derivatives. These derivatives are best compounded to cross cell membranes, that is, membrane permeable, because the hydrophilic groups are masked to provide more hydrophobic derivatives. Also, masking groups are designed to cleave from a precursor (e.g., precursor
ES 2 289 784 T3 fluorogenic substrate) within the cell to generate the derived substrate intracellularly. Since the substrate is more hydrophilic than the membrane permeating derivative it is now captured within cells.
"Alkyl" refers to aliphatic, linear, branched, and cyclic groups of generally 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms, and more preferably 1 to 4 carbon atoms. The term "lower alkyl" refers to straight and branched chain alkyl groups of 1 to 4 carbon atoms.
"Aliphatic" refers to saturated and unsaturated alkyl groups of generally 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, and more preferably 1 to 4 carbon atoms.
"Heat fusion welding" refers to heat induced welding. The heat source can be any source sufficient to promote some degree of bonding between the two portions (separately or otherwise) of a material (s), including a chemical reaction, an external heat source (e.g., a hot roll , ultrasonic or air) or internal heating (for example, radio frequency heating).
"Isolated polynucleotide" refers to a genomic, cDNA, or synthetic polynucleotide, or some combination thereof, which by virtue of its origin the "isolated polynucleotide" (1) does not associate with the cell in which the "isolated polynucleotide" is found in nature, or (2) is operably linked to a polynucleotide to which it is not linked in nature.
"Isolated protein" refers to a protein of cDNA, recombinant RNA, or of synthetic origin or some combination thereof, which by virtue of its origin the "isolated protein" (1) is not associated with proteins found with the which is normally associated in nature, or (2) is isolated from the cell in which it normally exists or (3) is isolated free of other proteins from the same cellular source, e.g. free of human proteins, or (4) it is expressed by a cell of a different species, or (5) it does not exist in nature. "Isolated naturally occurring protein" refers to a protein that by virtue of its origin the "isolated naturally occurring protein" (1) is not associated with proteins normally found in nature, or (2) it is isolated from the cell in which it normally exists or (3) it is isolated free of other proteins from the same cellular source, eg, free of human proteins.
"Polypeptide" as used herein as a generic term refers to a native protein, fragments, or analogs of a polypeptide sequence. Therefore, the native protein, fragments and analogs are species of the genus of the polypeptide.
"Naturally exists" as used herein, as applied to an object, refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is that it exists in a natural.
"Operably linked" refers to a juxtaposition in which the components so described are in a relationship that allows them to function in their intended form. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
"Control sequence" refers to polynucleotide sequences that are necessary to effect expression of the coding and non-coding sequences to which they are linked. The nature of such control sequences differs depending on the host organism; in prokaryotes, such control sequences generally include a promoter, ribosomal binding site, and the transcription termination sequence; in eukaryotes, such control sequences generally include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, components whose presence may influence expression, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
"Polynucleotide" refers to a polymeric form of nucleotides at least 10 bases in length, of ribonucleotides or deoxynucleotides, or of a modified form of each type of nucleotide. The term includes double-stranded and single-stranded forms of DNA.
"Corresponds to" refers to that a polynucleotide sequence is homologous (that is, it is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a sequence of reference polypeptide. In contrast, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. To illustrate this, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA".
The "polypeptide fragment" refers to a polypeptide that possesses an amino-terminal and / or carboxy-terminal deletion, but the remainder of the amino acid sequence is generally identical to corresponding positions in the deduced naturally occurring sequence. , for example, from a full-length cDNA sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, preferably by
ES 2 289 784 T3 at least 14 amino acids long, more preferably at least 20 amino acids long, generally at least 50 amino acids long, and more preferably even at least 70 amino acids long.
"Plate" refers to a multiwell plate, unless otherwise modified in the context of its use.
"Modulation" refers to the ability to increase or inhibit a biological activity property or process (eg, enzyme activity or receptor binding); such enhancement or inhibition may depend on the existence of a specific event, such as activation of a signal transduction pathway, and / or may manifest itself only in particular cell types.
The term "modulator" refers to a chemical compound (which exists naturally or unnaturally), such as a biological macromolecule (eg, nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials. such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Modulators are evaluated for their potential activity as inhibitors or activators (directly or indirectly) of a biological procedure or procedures (eg, agonist, partial antagonist, partial agonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitors of proliferation or neoplastic transformation cells, cell proliferation promoting agents and the like) by including the selection assays described herein. The activity of a modulator can be known, unknown or partially known.
The term "test chemical" refers to a chemical to be tested by one or more selection method (s) of the present invention as a putative modulator.
The terms "label" or "labeled" refer to the incorporation of a detectable label, for example, by incorporation of a radiolabeled amino acid or by binding to a biotinyl moiety polypeptide that can be detected by labeled avidin (for example , streptavidin containing a fluorescent marker or an enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of markers for polypeptides include, but are not limited to, the following: radioisotopes (eg,<sup>3</sup>H, <sup>14</sup>C, <sup>35</sup>Yes, <sup>125</sup>1,<sup>131</sup>1), fluorescent markers (eg, FITC, rhodamine, lanthanide phosphors), enzyme markers (or reporter genes) (eg, horseradish peroxidase, β-galactosidase, β-lactamase, luciferase, alkaline phosphatase), chemiluminescence, clumps biotinyl, previously determined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper partner sequences, binding sites for secondary antibodies, metal-binding domains, epitope markers). In some embodiments, the markers are attached by spacer arms of various lengths to reduce potential steric hindrance.
"Fluorescence marker" refers to the incorporation of a detectable marker, for example, by incorporation of a fluorescent moiety into a chemical entity that binds to a target or to a polypeptide binding of detectable biotinyl moieties. by avidin (eg streptavidin containing a fluorescent marker or an enzyme activity that can be detected by fluorescence detection methods). Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, dyes (eg, FITC and rhodamine), intrinsically fluorescent proteins, and lanthanide phosphors. In some embodiments, the markers are attached by spacer arms of various lengths to reduce potential steric hindrance.
The "reporter gene" refers to a nucleotide sequence that encodes a protein that is easily detected by its presence or activity, including, but not limited to, luciferase, green fluorescent protein, chloramphenicol acetyltransferase, β-galactosidase, secreted alkaline phosphatase. of placenta, β-lactamase, human growth hormone and other secreted enzyme reporters. Reporter genes generally encode a polypeptide that is not produced other than by a host cell that is detected by analysis of the cell (s), for example, by direct fluorometric, radioisotopic, or spectrophotometric analysis of the cell (s ) and preferably without the need to remove cells for signal analysis from one well. Preferably, the gene encodes an enzyme that produces a change in the fluorometric properties of a host cell that is detected by qualitative, quantitative or semi-quantitative transcriptional activation function. Exemplary enzymes include esterases, phosphatases, proteases (tissue plasminogen activator or urokinase), and other enzymes whose function can be detected by suitable chromogenic or fluorogenic substrates known to those of skill in the art. Reporter gene proteins, particularly enzymes, can also be used as probes in biochemical assays, for example after appropriate conjugation to the target or to a chemical entity that binds the target.
"Transmittance" refers to the fraction of incident light that passes through a medium at a given wavelength. One can also consider the ratio of radiant power transmitted through the medium to the radiant power incident on the medium at a particular wavelength.
Other chemical terms herein are used according to conventional use of the art, as exemplified in The McGraw-Hill Dictionary of Chemical Terms (ed. Parker, S., 1985), McGraw-Hill, San Francisco, incorporated herein by reference. present specification for reference).
ES 2 289 784 T3
Embodiments of the present invention
As a non-limiting introduction to the scope of the present invention, the present invention includes various general and useful aspects, including:
1) Multiwell plates with cycloolefin well bottoms that are useful in fluorescence measurements,
2) platforms with cycloolefin layers or windows that are useful in fluorescence measurements,
3) production procedures of (1) and (2), and
4) detection systems and procedures based, in part, on (1) and (2).
These aspects of the present invention, in addition to others described herein, can be accomplished using the methods and compositions of matter described herein. To obtain a full appreciation of the scope of the present invention, it will be further recognized that various aspects of the present invention may be combined to carry out the desired embodiments of the present invention.
Platforms and multiwell plates
The present invention includes devices for spectrophotometric measurements, such as multiwell plates and platforms. Typically, such devices comprise a high transmittance, low fluorescence layer comprising a cycloolefin polymer and well (s) of a multiwell plate or platform to support the layer. Both the plates and the multiwell platforms are described below.
Multiwell plates
The multiwell plates of the present invention comprise a high transmittance, low fluorescence layer, comprising a cycloolefin polymer, and a well (s) to support, or form, the layer. The cycloolefin generally comprises at least a portion of a bottom surface of a well of the multiwell plate. In many embodiments, to facilitate ease of production, the cycloolefin will comprise substantially the entire background. Cycloolefin can also be used to form plate walls, which is a second way to reduce the inherent fluorescence of a plate. In some shaped embodiments of the present invention, the cycloolefin will optionally comprise any portion of a plate, including the bottom of the plate, the walls of the wells, the interwell structural members that interconnect the wells, the sides of the plate, the upper or lower surfaces of the plate, in addition to the plate covers.
Multiwell plates can offer any number of wells in any well arrangement on any multiwell plate format or footprint. Typically, the wells will be arranged in two-dimensional linear arrangements and generally have between about 96 and 9600 wells, preferably the number of wells is a multiple of 96. Higher numbers of wells or increased well density can also be easily accomplished since cycloolefin polymers can be easily produced in a variety of well shapes and small volume and dimension shapes. Other commonly used number of wells includes 1,536, 3,456, and 9,600. Well volumes are typically between 500 nanoliters and greater than 200 microliters, depending on well depth and cross-sectional area. Well volumes of 1, 2, 5, 10, 20, 50, 100, 200, and 500 microliters are commonly used. The wells can be produced in any cross-sectional shape (in plan view) including square, round, hexagonal, and combinations thereof. The wells can be produced in any cross-sectional shape (in vertical view) including sliding vertical walls with round or smooth bottoms, tapered walls with smooth or round bottoms, and curved vertical walls with smooth or round bottoms and combinations thereof. In applications of the present invention that can use focused light, the cycloolefin can be used to form a lens that is part of the bottom of the well. The lens will vary in thickness and curvature depending on the application.
The materials for the production of the plate will typically be polymeric, as these materials lend themselves to mass production techniques. Polymeric materials can particularly facilitate plate production by molding processes known in the art and developed in the future. Polymers that are compatible with cycloolefin should be used in regions of the plate in physical contact with cycloolefin. In some embodiments, the wells of the plate may be produced with a material other than a cycloolefin polymer and the cycloolefin bonded, soldered, or otherwise fused to the second material. Polymers with glass transition temperature suitable for heat-induced melting with cycloolefin can be selected for production of wells and other portions of the plate. Preferably, the polymers are selected for low fluorescence or other properties described herein. The entire plate, except the bottom, can be produced from a second polymer and then heat welded to a cycloolefin film of suitable dimensions using procedures as known in the art or those developed in the future. It is also preferred to produce a substantial portion or the entire plate of cycloolefin. Such uses of second polymers can also be used as a guide to form other embodiments of the present invention.
Since most measurements will not typically require light to pass through the wall of the well, the polymers can include pigments that darken the walls of the well or absorb light. Such application of pigments
ES 2 289 784 T3 will help reduce base fluorescence. Pigments can be introduced by any means known in the art, such as coating or mixing during polymerization procedures. Pigment selection can be based on a mixture of pigments to wet the entire background inherent to the polymer, or a single pigment or a set of pigments selected to filter or absorb light at the desired wavelengths. Pigments can include carbon black. Such pigmentation is generally not desired in embodiments in which light is directed through the walls of the well as a method of illuminating the contents of the wells. Such techniques can also be applied to deck embodiments of the present invention.
The thickness of the cycloolefin comprising the bottom of the plate can vary depending on the set of required properties of the bottom of the plate that can be imposed by a particular application. Such properties include the amount of intrinsic fluorescence, stiffness, resistance to breakage, and throughput requirements related to the cycloolefin used in the plate. The cycloolefin layers at the bottom of the wells are typically between about 30 to 500 microns thick, and preferably about 50 to 300 microns thick. Such thickness values can also be used as a guide in forming other embodiments of the present invention.
One of the outstanding characteristics of the multiwell plates of the present invention is their low intrinsic fluorescence. The bottom layer comprising the cycloolefin typically produces about 400 percent to 200 percent or less of the fluorescence compared to 150 micron thick fused silica. Fused silica glass is typically used as a "gold standard" for comparing relative fluorescence. Fluorescence and relative fluorescence can be measured using any of the reliable techniques known or developed in the art, preferably the techniques described herein are used. Preferably, the fused silica standard used herein to display the surprisingly low fluorescence of cycloolefin is used as a standard. Preferably, the bottom layer comprising the cycloolefin typically produces about 100 to 50 percent or less of the fluorescence compared to fused silica about 150 microns thick. The amount of intrinsic fluorescence can be dictated, in part, by the thickness of the layer. In some applications that can tolerate particularly thin layers, such applications where the layer does not require significant structural strength, the thickness of the layer can be quite thin (for example, 20 to 80 microns) to reduce the fluorescence arising from the layer. The thinness of a layer is also generally quite balanced against the difficulty of uniform welding or the generation of thinner layers in production processes. The low relative fluorescence of cycloolefin devices is generally present at excitation wavelengths of between about 300 to 400 nm and at emission wavelengths of between about 300 to 800 nm. Such relative fluorescence values can also be used as a guide to form other embodiments of the present invention.
The multiwell plates of the present invention may comprise coatings or surface modifications to facilitate the various applications of the plate as described herein and those known or developed in the relevant art. Coatings can be introduced using any suitable method known in the art, including printing, spraying, radiant energy, ionization or dipping techniques. Surface modifications can also be introduced by suitably derivatizing a polymer before or after the production process and including a suitable derivatized polymer in the cycloolefin layer. The derivatized polymer can then be reacted with a chemical moiety that is used in a plate application. Before reaction with a chemical moiety, such a polymer can then provide covalent or non-covalent binding sites on the cycloolefin. Such sites on or on the surface of the cycloolefin can be used to bind moieties, such as test components (e.g., a member of a binding partner), chemical reactive components (e.g., solid synthesis components for synthesis of amino acids or nucleic acids) and the components of cell culture (for example, proteins that facilitate growth or adhesion). Examples of derivatized polymers include those described by US Patent No. 5,583,211 (Coassin et al.). Preferred embodiments are particularly based on polyethylene and polypropylene derivatives which can be included as cycloolefin copolymers.
The cycloolefin layer can also include a plurality of living cells. Such embodiments are useful for the cell-based assays described herein and for growing cells using culture procedures. The plates of the present invention may include a coating (eg, polylysine) to enhance cell attachment.
The uses of multiwell plates are known in the relevant arts and include diagnostic assays, chemical or biochemical binding assays, filtration assays, chemical synthesis sites, storage sites, and the like. Such uses can also be applied to the present invention. It will be recognized that some types of multiwell plates for spectroscopic measurements can often be used for other multiwell plate applications. Typically, a multiwell plate is used to detect a signal from a sample. Different types of signal measurements are described herein.
In another embodiment, the present invention provides a multiwell plate for spectroscopic measurements, comprising a plurality of wells and each well comprising a wall and a bottom with a low fluorescence and high transmittance portion comprising a cycloolefin copolymer, and a frame, in which the wells are arranged in a frame. The multiwell plate can be used to detect a signal from a
ES 2 289 784 T3 sample. The multiwell plate in some embodiments lacks the footprint of a standard 96-well microtiter plate (ie, "non-standard footprints"). The footprint of a standard 96-well microtiter plate is 12.7 cm long and 8.5 cm wide. The generally accepted standard footprint for a standard 96-well microtiter plate for robotic applications is 12.77 ± 0.25 cm in length and 8.55 ± 0.25 cm in width (see T. Astle, Standards in Robotics and Instrumentation, J. of Biomolecular Screening, vol. 1, pages 163-168 (1996)). In no case will the standard footprint be greater or less than the range of lengths and widths presented in Table 1, which is a maximum of 12.83 cm and a minimum of 12.63 cm for the length and a maximum of 8.63 cm and a minimum of 8.37 cm for the width. In non-standard fingerprints, the multiwell plate can have 864 or more wells (eg, 1,536, 3,456, and 9,600).
TABLE 1
<td>Mfrs cat. =</td><td>Mfrs name</td><td colspan="3">External dimensions</td><td colspan="5">Wells</td>
<td></td><td></td><td>Length</td><td>Width</td><td>Height</td><td>Wells</td><td>Shape</td><td>Colour</td><td>Material</td><td>Background</td>
<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td>AGTC</td><td> 128,118</td><td> 85,319</td><td> 41,148</td><td></td><td></td><td></td><td>styrene</td><td>1 ml</td>
<td></td><td>AIM</td><td> 127,762</td><td> 85,598</td><td> 41,504</td><td></td><td></td><td></td><td>styrene</td><td>1 ml</td>
<td></td><td>AIM</td><td> 127,635</td><td> 85,141</td><td> 40,945</td><td></td><td></td><td></td><td>propylene</td><td>1 ml</td>
<td></td><td>Beckman</td><td> 127,93</td><td> 85,55</td><td> 41,84</td><td> 96</td><td>round</td><td>Sure</td><td>styrene</td><td>round</td>
<td></td><td>Beckman</td><td> 127,93</td><td> 85,55</td><td> 41,84</td><td> 96</td><td>round</td><td>translucent</td><td>propylene</td><td>round</td>
<td> 373660</td><td>Beckman</td><td> 127,787</td><td> 85,573</td><td> 14,224</td><td></td><td></td><td>Sure</td><td>styrene</td><td>smooth</td>
<td> 25870</td><td>Coming / Costar</td><td> 127,68</td><td> 85,12</td><td> 14,2</td><td> 96</td><td>round</td><td>Sure</td><td>styrene</td><td>smooth (bevel)</td>
<td> 35207</td><td>Corning / Costar</td><td> 127,61</td><td> 85,166</td><td> 14,224</td><td></td><td></td><td>Sure</td><td>styrene</td><td>smooth</td>
<td> 35205</td><td>Coming / Costar</td><td> 127,33</td><td> 85,014</td><td> 14,224</td><td></td><td></td><td>Sure</td><td>styrene</td><td>background U</td>
<td></td><td>Coming / Costar</td><td> 127,6</td><td> 85,2</td><td> 14,3</td><td> 96</td><td>round</td><td>Sure</td><td>styrene</td><td>conical</td>
<td> 7000003</td><td>Corning / Costar</td><td> 127,1</td><td> 85,3</td><td> 14,3</td><td> 96</td><td>round</td><td>black</td><td>styrene</td><td>smooth</td>
<td> 7000004</td><td>Coming / Costar</td><td> 127,6</td><td> 85,47</td><td> 14,2</td><td> 96</td><td>round</td><td>black</td><td>styrene</td><td>smooth</td>
<td> 7000008</td><td>Coming / Costar</td><td> 126,7</td><td> 84,62</td><td> 14,45</td><td> 96</td><td>round</td><td>translucent</td><td>propylene</td><td>round</td>
<td> 7000010</td><td>Coming / Costar</td><td> 127,83</td><td> 85,42</td><td> 14,53</td><td> 96</td><td>round</td><td>Sure</td><td>styrene</td><td>smooth</td>
<td> 35203</td><td>Corning / Costar</td><td> 127,508</td><td> 85,319</td><td> 14,224</td><td></td><td></td><td>Sure</td><td>styrene</td><td>smooth</td>
<td> 35202</td><td>Corning / Costar</td><td></td><td> 85,42</td><td> 14,326</td><td></td><td></td><td>Sure</td><td>styrene</td><td>smooth A / 2</td>
<td> 35190</td><td>Dynatech</td><td> 127,889</td><td> 85,649</td><td> 14,173</td><td></td><td></td><td>Sure</td><td>styrene</td><td>smooth</td>
<td> 35189</td><td>Dynatecb</td><td> 127,838</td><td> 85,522</td><td> 14,097</td><td></td><td></td><td>Sure</td><td>styrene</td><td>background V</td>
<td> 35194</td><td>Evergreen</td><td> 127,483</td><td> 85,344</td><td> 14,376</td><td></td><td></td><td>Sure</td><td>styrene</td><td>smooth</td>
<td> 35192</td><td>Evergreen</td><td> 127,483</td><td> 85,217</td><td> 14,275</td><td></td><td></td><td>Sure</td><td>styrene</td><td>background U</td>
<td> 35191</td><td>Evergreen</td><td> 127,432</td><td> 85,268</td><td> 14,3</td><td></td><td></td><td>Sure</td><td>styrene</td><td>background V</td>
<td> 35197</td><td>Falcon</td><td> 127,381</td><td> 85,471</td><td> 14,351</td><td></td><td></td><td>Sure</td><td>styrene</td><td>smooth</td>
<td> 7000017</td><td>Genetix</td><td> 128,28</td><td> 86,31</td><td> 10,17</td><td> 384</td><td>round</td><td>Sure</td><td>styrene</td><td>smooth</td>
<td> 35188</td><td>Immulon</td><td> 127,406</td><td> 85,344</td><td> 14,402</td><td></td><td></td><td>Sure</td><td>styrene</td><td>smooth</td>
<td> 35716</td><td>Interlab</td><td> 127,914</td><td> 85,852</td><td> 13,665</td><td></td><td></td><td>Sure</td><td>styrene</td><td>background V</td>
<td></td><td>Iwaki</td><td> 127,279</td><td> 85,065</td><td> 14,021</td><td></td><td></td><td></td><td>styrene</td><td>smooth</td>
<td> 35181</td><td>LabSystems</td><td> 127,838</td><td> 85,598</td><td> 15,291</td><td></td><td></td><td>black</td><td>propylene</td><td>smooth</td>
<td> 35187</td><td>MicroFluor</td><td> 127,406</td><td> 85,217</td><td> 12,224</td><td></td><td></td><td>White</td><td>propylene</td><td>smooth</td>
<td> 35184</td><td>MicroFluor</td><td> 127,508</td><td> 85,42</td><td> 14,275</td><td></td><td></td><td>black</td><td>propylene</td><td>smooth</td>
<td> 35183</td><td>MicroFluor</td><td> 127,533</td><td> 85,42</td><td> 14,224</td><td></td><td></td><td>White</td><td>propylene</td><td>smooth A / 2</td>
<td> 35185</td><td>MicroLite</td><td> 127,584</td><td> 85,369</td><td> 14,148</td><td></td><td></td><td>White</td><td>propylene</td><td>smooth</td>
<td> 35186</td><td>MicroLite 2</td><td> 127,635</td><td> 85,471</td><td> 14,199</td><td></td><td></td><td>White</td><td>propylene</td><td>smooth</td>
<td></td><td>Millipore</td><td> 128,016</td><td> 85,75</td><td> 14,859</td><td></td><td></td><td>White</td><td>propylene</td><td>smooth</td>
<td></td><td>Millipore</td><td> 127,813</td><td> 85,598</td><td> 14,605</td><td></td><td></td><td>Sure</td><td>styrene</td><td>smooth</td>
<td> 35177</td><td>NBT</td><td> 127,838</td><td> 85,598</td><td> 14,3</td><td></td><td></td><td>Sure</td><td>styrene</td><td>background U</td>
<td> 7000001</td><td>Nunc</td><td> 127,6</td><td> 83,7</td><td> 14,4</td><td> 96</td><td>round</td><td>Sure</td><td>styrene</td><td>smooth</td>
<td> 7000006</td><td>Nunc</td><td> 127,7</td><td> 85,6</td><td> 14,5</td><td> 384</td><td>square</td><td>Sure</td><td>styrene</td><td>smooth</td>
<td> 63765</td><td>Nunc</td><td> 127,559</td><td> 85,573</td><td> 14,351</td><td></td><td></td><td>Sure</td><td>styrene</td><td>smooth</td>
<td> 35201</td><td>Nunc</td><td> 127,432</td><td> 85,344</td><td> 14,097</td><td></td><td></td><td>Sure</td><td>styrene</td><td>background U</td>
<td> 35200</td><td>Nunc</td><td> 126,314</td><td> 84,379</td><td> 14,351</td><td></td><td></td><td></td><td>propylene</td><td>background U</td>
<td> 35199</td><td>Nunc</td><td> 127,305</td><td> 85,395</td><td> 14,402</td><td></td><td></td><td>Sure</td><td>styrene</td><td>background V</td>
<td> 35210</td><td>Packard</td><td> 127,762</td><td> 85,471</td><td> 14,275</td><td></td><td></td><td>White</td><td>propylene</td><td>GF / B</td>
<td> 35209</td><td>Packard</td><td> 127,965</td><td> 85,776</td><td> 14,351</td><td></td><td></td><td>White</td><td>propylene</td><td>GF / C</td>
<td> 35203</td><td>Pall</td><td> 127,635</td><td> 85,598</td><td> 14,325</td><td></td><td></td><td>White</td><td>propylene</td><td>smooth</td>
<td> 7000005</td><td>Polyfiltronics</td><td> 127,5</td><td> 85,8</td><td> 44,03</td><td> 96</td><td>square</td><td>translucent</td><td>propylene</td><td>round</td>
<td> 7000009</td><td>Polyfiltronics</td><td> 127,09</td><td> 85,12</td><td> 30,43</td><td> 96</td><td>round</td><td>translucent</td><td>propylene</td><td>filter</td>
<td> 7000011</td><td>Polyfiltronics</td><td> 127,3</td><td> 85,25</td><td> 16</td><td> 96</td><td>round</td><td>translucent</td><td>propylene</td><td>conical</td>
<td> 7000012</td><td>Polyfiltronics</td><td> 127,8</td><td> 85,69</td><td> 9,56</td><td> 384</td><td>round</td><td>translucent</td><td>propylene</td><td>conical</td>
<td> 35175</td><td>Polyfiltronics</td><td> 127,787</td><td> 85,552</td><td> 15,24</td><td></td><td></td><td>White</td><td>propylene</td><td>smooth</td>
<td> 35174</td><td>Polyfiltronics</td><td> 127,483</td><td> 85,547</td><td> 15,189</td><td></td><td></td><td>black</td><td>propylene</td><td>smooth</td>
<td> 35173</td><td>Polyfiltronics</td><td> 127,991</td><td> 85,7</td><td> 15,24</td><td></td><td></td><td>White</td><td>propylene</td><td>smooth clear</td>
<td> 35179</td><td>Polyfiltronics</td><td> 127,559</td><td> 85,344</td><td> 14,351</td><td></td><td></td><td>White</td><td>propylene</td><td>GF / B</td>
<td> 35180</td><td>Polymetrics</td><td> 127,533</td><td> 85,369</td><td> 14,097</td><td></td><td></td><td>translucent</td><td>propylene</td><td>depth V</td>
<td></td><td>Sumilon</td><td> 127,33</td><td> 85,395</td><td> 14,503</td><td></td><td></td><td></td><td>styrene</td><td>smooth</td>
<td> 35178</td><td>Tilertek</td><td> 127,381</td><td> 85,319</td><td> 14,224</td><td></td><td></td><td>Sure</td><td>styrene</td><td>smooth</td>
ES 2 289 784 T3
Typically, the multiwell plate has wells with a well center to well center distance of less than about 9 to 6mm, preferably less than 3mm and sometimes less than about 1mm. Smaller center to well center distances are preferred for smaller volumes. Such plates typically possess a cycloolefin polymer thickness of between about 20 and 200 microns thick, preferably about 30 to 80 microns. Preferably, the cycloolefin polymer has low fluorescence from an excitation light of about 300 to 500 nm and the low fluorescence, high transmittance portion is substantially the entire background. Often the wells and optionally the scaffold are composed of a cycloolefin copolymer, which helps reduce fluorescence.
The present invention optionally includes the caveat that when the multiwell plate is a microtiter plate with a footprint of a standard 96-well microtiter plate and having microtiter wells, the number of microtiter wells shall not exceed 864 microtiter wells. .
Platforms
The platforms of the present invention comprise a high transmittance, low fluorescence plate, or window, generally comprising a cycloolefin polymer or other low fluorescence material, and a scaffold or framework to support, or form, the layer. . The window has a predetermined dimension and can be arranged as a plurality of windows in the frame in any geometric arrangement, including two-dimensional arrangements. In some embodiments, the window allows the detection of spectroscopic events, in which light often passes through the window. In other embodiments, the window is basically a reaction or test site that can allow detection of a chemical test or reaction, for example by measuring the refraction or reflectance of light. When the window is a test or reaction site, light does not necessarily have to pass through the window.
The cycloolefin generally comprises at least a portion of the window. In many embodiments, to facilitate ease of production, the cycloolefin will comprise substantially the entire window and platform. Cycloolefin can also be used to form the scaffold or scaffold that forms the platform, which is a second way to reduce the inherent fluorescence of a plate. In some molded embodiments of the present invention, the cycloolefin will optionally comprise any portion of a platform, including the bottom of the platform, walls, structural members between windows that interconnect the wells, sides of the platform, top or bottom surfaces of the platform, in addition to the platform caps. Other polymers may be substituted for cycloolefin depending on spectroscopic or other requirements of the assay or reaction.
In one embodiment, the platform comprises a scaffold for an array of spatially predetermined test sites on the layer, and the scaffold does not substantially interfere with the detection of a signal from the test sites. The layer is typically about 40 to 300 microns thick. Assay sites are typically about 10 square microns to 200 square microns in area, although sites larger than 200 microns (e.g. 500 to 2,000 microns or larger) and sites smaller than 10 microns (e.g. , 5 to 0.5 microns or less). The layer can be derivatized for the binding of chemical entities as described herein. Test sites can also be printed onto the layer. Such platforms can be used and produced in many cases as the multiwell plates described herein. Preferably, the platform comprises a cycloolefin copolymer.
The embodiments of the present invention do not include optical, information recording media, typically used for the storage and selective searching of information (eg, CDs (compact discs) for computer data or audio recordings). Such optical recording media have a recording layer. The recording layer generally comprises either 1) predetermined physical deformations (e.g. pits or concavities) on the surface of the recording layer formed by irradiating the recording layer at the precise predetermined locations or 2) predetermined locations previously altered refractive index or reflectance on the surface of the recording layer formed by irradiating the recording layer at the previously determined sites. Recording layers are typically composed of a low melting point metal (eg Te) and may contain other elements for the desired properties (eg Cr, C and H). The recording layer is generally deposited on the substrate comprising a polymer, such as a cycloolefin. In contrast, the present invention is directed toward non-optical information recording media and the use of low fluorescence, high transmittance cycloolefins for use in chemical and biological techniques, as opposed to computer or audio techniques. In many of these embodiments the light will often pass through the cycloolefin layer as opposed to optical information recording media, in which the light "bounces" off the recording layer. Conventional optical information recording media are also different from the present invention in that the recording layers of such media contain a soft metal layer. The soft metal layer is normally a disadvantageous feature for measuring chemical or biological events. In many cases, such a recording layer will not be compatible with many chemical reactions or biological moieties. It is understood, however, that some embodiments of the present invention may contain or store information, in addition to providing that the sites of chemical or biological events take place in a CD format or in a modified one, the CD format being according to the dimensions and other features of the present invention. In some cases, such a chemical of biological events can provide a signal that can be measured as a change in refraction or reflection of light. It is also understood that some
ES 2 289 784 T3 embodiments of the present invention may include chemical or biological event sites that may be capable of selectively storing and searching information in a format that appears to be conventional compact disc format. Neither the present invention includes cycloolefins produced as blister bags nor as packaged material.
Materials, selection and testing criteria
This section describes materials, selection criteria, and rapid assays to facilitate the choice of a cycloolefin for multiwell plates and platforms described herein.
Materials
The present inventors carried out extensive research on different polymers in search of polymers that offer the suitable properties for the detection of spectroscopic signals, particularly fluorescence signals. The materials used in the present invention have not been used in the commercially available plates listed in Table 1. Surprisingly, these materials offer exceptional properties, including low intrinsic fluorescence, which has been demonstrated here for the first time. "Cycloolefins" generally refer to cycloolefin polymers, unless otherwise modified in the context of their use, and include copolymers such as those specified herein. "Cycloolefin copolymers" generally refer to cycloolefin copolymers, unless otherwise modified in the context of their use.
Typically, cycloolefins are used as films or resins to produce various embodiments of the present invention. Cycloolefin polymer-based resins and films can be used in various production procedures known in the relevant art and described herein. The selection criteria for cycloolefin films or resins are described very fully below. Cycloolefin films or resins containing UV absorbers, aromatic moieties, and styrene moieties are generally not preferred.
Suitable cycloolefins for many embodiments of the present invention include those described in US Patent Nos. 5,278,238 (Lee BL et al); No. 4,874,808 (Minami et al); No. 4,918,133 (Moriya et al); No. 4,935,475 (Kishimura et al); No. 4,948,856 (Minchak et al); No. 5,115,052 (Wamura et al); No. 5,206,306 (Shen); No. 5,270,393 (Sagane et al); No. 5,272,235 (Wakatsuru et al); No. 5,278,214 (Moriya et al); No. 5,534,606 (Bennett et al); No. 5,532,030 (Hirose et al); No. 4,689,380 (Nahm et al); and No. 4,899,005 (Lane et al). The cycloolefins available from Hoechst are preferred, especially cycloolefin (eg, cyclopentene, cyclohexane and cycloheptene) and their polyethylene copolymers, in addition to thermoplastic olefin polymers of amorphous structure (TOPAS line).
Multilayer laminates are preferred when multiple functional requirements are difficult to obtain from a single laminate (eg, layer or film). The properties of transmittance, stiffness, heat sealing, fluorescence, wet penetration can be mixed by using films of different resins. Blended resins known in the art and developed in the future can be used where multilaminate films or blended resins possess properties consistent with those of the present invention. For example, US Patent No. 5,532,030 (Hirose et al) describes the production of certain cycloolefin films, both single and multilaminate, that can be adapted for use in the devices described herein.
Selection and testing criteria
The properties desired for the cycloolefin films and resins used in the present invention will vary depending on the type of plate or multiwell platform desired. Materials are generally selected to provide an end product with low fluorescence, high transmittance, sufficient stiffness to resist deformity and to allow substantially single plane (especially for spectroscopic embodiments), good chemical inertness, relatively low cytotoxicity , low water absorption, heat / deflection resistance up to about 150 ° C and resistance to acids and bases. Starting materials with good molding properties are particularly desired.
The fluorescence of the films or the final product can be easily measured. Such measurements are carried out quickly and a number of films (eg 20 to 80 films) or prototype products can be tested rapidly in a matter of hours or days, typically less than a person week. Consequently, the films or resins used to make the final products can be quickly selected for the desired properties that are important in a particular application. Fluorescence measurements can be used as described above or those known in the art, provided that the measurements are comparable (or better) in sensitivity to the measurements previously described herein. A standard reference point for relative fluorescence, such as the standard described herein, is particularly useful for the comparison of different different cycloolefins and for determining their applicability to certain applications. The relative fluorescence properties described herein are particularly desired. Similarly, transmittance can be measured using techniques known in the relevant art.
ES 2 289 784 T3
In the final product, layer thicknesses of generally about 20 to 500 microns are most likely to impart the properties desired for use in the devices described herein above, especially low fluorescence and high transmittance. Although thinner or thicker films, such as about 10 to 1,500 microns, can be used in applications where the demands for extremely low fluorescence and high transmittance films are less severe, or where there is little loss in the desired properties as a function of film thickness. Preferably, the film thickness is between about 30 and 200 microns for multiwell plate applications, and more preferably between about 80 and 200 microns and more preferably between 80 to 200 microns. Preferably, the film thickness is between about 30 and 600 microns for scaffold applications where the films typically contribute to a structural function in the device that generally demands more strength or stiffness, more preferably between about 100 and 500 microns and more preferably between about 120 and 200 microns. Preferably, the film thickness is between about 75 to 600 microns for the thinnest regions of injection molded applications where the film typically contributes to structural function and more preferably between about 100 to 500 microns and most preferably between about 120 to 200 microns. Film thickness refers to the thickness of the film used (or material thickness). The layer thickness is generally about 100 to 200 percent of the film thickness, preferably about 100 to 150 percent of the film thickness, and more preferably about 100 to 125 percent of the film thickness. .
In the final product, the tensile stresses (Kg / cm<sup>2</sup> at 22 ° C) usually about 400 to 3,000 Kg / cm<sup>2</sup> they are more likely to impart the properties desired for use in the devices described herein, especially high transmittance, low fluorescence rigid devices. Although weaker or stronger films, such as approximately 200 to 3,500 Kg / cm<sup>2</sup> They can be used in different applications based on the demands for the breaking strength of the device. For example, the tear strength of the film generally does not need to be as great for the bottoms of multiwell plates as for applications where the film is part of the frame, on a multiwell plate or on a platform. reaction mixture substantially composed of the film itself. Preferably, the breaking stress is between approximately 500 to 2,000 Kg / cm<sup>2</sup> for multiwell plate applications, and more preferably between about 800 to 1,600 Kg / cm<sup>2</sup> and more preferably between approximately 900 to 1,400 Kg / cm<sup>2</sup>. Preferably, the ultimate stress for platform / scaffold applications is approximately 15 to 60 percent higher than for multiwell plate applications. Ultimate stresses can be measured by standard techniques known in the art.
Production procedures
The present invention includes a process for producing cycloolefin-based multiwell plates and platforms. A variety of procedures can be used including hot welding, insert molding, injection molding, and other procedures described herein and known in the art. One process comprises hot welding to a high transmittance, low fluorescence single layer polymer platform comprising a cycloolefin copolymer. The processes typically utilize a cycloolefin copolymer selected from the group consisting of cyclopentene polyethylene copolymer, cyclohexane polyethylene copolymer, and cycloheptene polyethylene copolymer. The method may alternatively, or optionally, comprise the step of exposing the layer and polymer to a sufficient amount of radio frequency energy to promote internal heating of the layer and polymer, or ultrasonic welding.
Alternatively, the process may involve heating the layer and the polymer forming the wells to about 320 ° C for a sufficient amount of time to allow the polymers to melt. Pressure can be applied to increase the welding process (for example, approximately 6.89-68.9 bar (100 and 1,000 psi) pressure to the layer and polymer for low pressure processes using low viscosity monomer solutions and about 689-1,724 bar (10,000 to 25,000 psi) for high pressure processes such as insert molding).
In another embodiment, the present invention provides a process for producing the multiwell plates by injection molding or insert molding. Injection molding techniques known in the art or developed in the future can be applied. The method comprises insert molding of at least one well into a bottom of the well of the multiwell plate, wherein the bottom is a cycloolefin copolymer. Using this procedure the cycloolefin films can be primarily heat melted to support the structure (eg, the walls of the well) to produce a plaque. The entire well or plate can also be produced from a cycloolefin. Insert molding can be carried out between about 195 and 350 ° C degrees, preferably the resins are heated from 260 ° to 320 ° C. The pressures used are typically between 689-1,724 bar (10,000 and 25,000 psi) and preferably between about 1,034-1,517 bar (15,000 to 22,000 psi).
Procedures for the preparation of cycloolefins and their polymers have been described. Older processes and cycloolefins were described in US Patent Nos. 4,002,815; No. 4,069,376; No. 4,110,528; Nos. 4,262,103 and 4,380,617 (by Robert J. Minchak et al.). A number of catalysts can be used in the production of
ES 2 289 784 T3 cycloolefins as known in the art or those developed in the future and can be used for the production of materials for various embodiments of the present invention. Such catalysts include those described in US Patent Nos. 5,278,238 (Lee et al) and 5,278,214 (Moriya et al). Despite the exact type of catalyst system used, cycloolefin monomers can be polymerized in the presence of a catalyst and ethylene-based functional copolymers to produce embodiments of the present invention suitable for injection molding. The polymerization can preferably be carried out in bulk. Bulk polymerization includes reaction injection molding (RIM), liquid injection molding (LIM), reinforced reaction injection molding RRIM, and resin transfer molding (RTM) and combinations thereof are known in the art. in addition to those developed in the future. Bulk polymerization is a polymerization that is carried out in the absence of a solvent or diluent. Reaction injection molding is a type of bulk polymerization in which a monomer in a liquid state is transferred or injected into a mold in which polymerization of the monomer takes place in the presence of a catalyst system. RIM is not a conventional injection molding for molten polymers and is easily distinguished from them.
RIM is a mixing and injection of two or more liquid components at low pressure, in one stage or in a single time, in a closed mold in which rapid polymerization occurs resulting in a molded plastic product. RIM differs from a conventional injection molding in a number of important ways. Conventional injection molding is carried out at pressures of approximately 689-1,379 bar (10,000 to 20,000 psi) in the mold cavity by melting a solid resin and transporting it to a mold maintained at a temperature below the melting temperature of the resin. . At an injection temperature of about 150 ° to 350 ° C, the viscosity of the molten resin in a conventional injection molding process is generally in the range of 50,000 to 1,000,000 and is typically about 200,000 cps. In the injection molding process, solidification of the resin occurs in approximately 10 to 90 seconds, depending on the size of the molded product, and then the molded product is removed from the mold. No chemical reaction occurs in a conventional injection molding procedure when the resin is introduced into a mold.
In a RIM process, the viscosity of materials fed to a mixing chamber is from about 1 to 10,000 cps, preferably 1 to about 1,500 cps, at injection temperatures ranging from room temperature to about 100 ° C, for different cycloolefin monomer systems. Molding temperatures in a RIM process are in the range of about 50 ° C to 150 ° C and pressures in the mold are generally in the range of about 3.45-10.3 bar (50 to 150 psi). . At least one component in the RIM formulation is a monomer that polymerizes to a polymer in the mold. The main distinction between conventional injection molding and RIM resides in the fact that in RIM, a chemical reaction is initiated in mixing, with optional heating, and completed in the mold to transform monomers to a polymeric state. For practical purposes, the chemical reaction should take place rapidly in less than about 2 minutes. Conventional injection molding can also be used to carry out various embodiments of the present invention. The term "injection molding" refers to both conventional injection molding and other types of injection molding described herein and known or developed in the art.
A LIM procedure is similar to a RIM system except that a shock source is not generally used. Instead, a simple mixer such as a static mixer, a stirring mixer and the like is used. Furthermore, in a LIM system, the injection molding cycle is carried out for a longer period of time and thus the chemical reaction can take place in a period of up to about 5 or 10 minutes.
Various reinforcing particles can also be used, which are injected with the solution when using the RIM or LIM procedure. In a practical way, the RIM process is not always suitable and therefore the reinforcing particles are generally used only in a LIM process, which is a reinforced liquid injection molding process. Another alternative is to use a material that already exists in a mold, for example a fiberglass material, or the like. Consequently, such systems are called RMRIM, RMLIM, or RTM. Due to reaction cure times in addition to injection molding times, the RMLIM system is generally preferred for some operations, and RMRIM and RTM for others.
Therefore, suitable blends or alloys of cycloolefins and copolymers can be used in any of the bulk polymerization systems described above in addition to variations thereof. As the above systems are generally conventional or known in the art as well as in the literature, they have not been described in detail herein, but have been briefly described briefly.
US Patent No. 4,426,502 to Minchak describes bulk polymerization (e.g., RIM) of modified cycloolefins using a catalyst modified cocatalyst whereby polymerization of cycloolefin monomers can be carried out in the absence of a solvent or diluent. The alkylaluminum halide cocatalyst is modified by prior reaction with an alcohol or an active hydroxyl-containing compound to form an alkyloxyalkylaluminum halide or an aryloxyalkylaluminum halide which is then used in the polymerization reaction. The pre-reaction can be carried out using oxygen, an alcohol or a phenol. Such a modification of the cocatalyst results in a decrease in the reducing potential of the catalyst.
Regardless of whether the halogen-free metathesis or halide metathesis catalyst system is used, the reaction rate is generally lowered by using the alcohols described above. In this way,
Depending on whether little or no alcohol is used, the halide metathesis catalyst system can cure the various cycloolefins in a matter of minutes and even seconds. If large amounts of alcohol are used, the cure can be a matter of hours or even days.
It is important to decrease the reducing power of the cocatalyst of the metathesis system to make such bulk polymerization reactions practical. When a monomer diluted with an unmodified alkylaluminum cocatalyst is mixed with a catalyst diluted with a monomer to polymerize a cycloolefin, the reaction is very rapid. In such systems, polymerization is generally unacceptable because polymer formed at the interfaces or in the two streams during intermixing prevents complete mixing and results in poor conversions. Modification of the cocatalyst by prior reaction with hydroxyl-containing materials reduces the activity of the cocatalyst to the point where proper mixing of liquid components can occur and acceptable polymeric products can be produced. Sometimes a cycloolefin monomer will contain various impurities that will naturally reduce the activity of the cocatalyst. In such cases, it is not necessary to add the active hydroxyl-containing materials to reduce the activity of the cocatalyst. With the modified cocatalyst, mixing of the cycloolefins, and other components, can be carried out at lower temperatures, such as room temperature, without immediately initiating polymerization. The cocatalyst can be formulated to allow reasonable shelf life at room temperature and in-mold thermal activation of the mixed liquid components. The cocatalyst can also be formulated to provide mixing initiated RIM systems.
When using a bulk polymerization process, the mixture of the cycloolefin monomers and the functional ethylene-based copolymers in addition to the catalyst and any other optional additives thereof can be added to the bulk polymerization mold having a good lower temperature. to the Tg of the polymerized cycloolefin polymers. This is especially desired as the reaction is generally exothermic and can result in a mold temperature increase of up to about 120 ° C. The final mold temperature is thus from about 50 ° C to about 200 ° C, generally from about 50 ° C to about 150 ° C, and preferably from about 50 ° C to about 90 ° C. Of course, such temperatures will vary depending on the specific type of catalyst system used, the specific type of cycloolefin monomers, and the like. When using the catalyst systems described hereinabove the mixture of the cycloolefin monomer and the ethylene-based functional copolymer has a good shelf life which is up to about 24 hours. Longer times would be desirable, the catalyst system is not added to the mixture but is kept separate. Thus, from the point in time of carrying out the polymerization of the cycloolefin monomers, the catalyst system is added to the mixture and polymerized in bulk. A preferred polymerization procedure includes the RIM procedure noted above.
Procedures for the detection of signals from samples
The present invention also provides a method for detecting a signal, comprising contacting a sample with a device for spectroscopic measurements, comprising: a layer with low fluorescence and high transmittance, comprising a cycloolefin copolymer and a platform to support the layer in which the platform is for the detection of a signal from a sample and provided that the platform is a plate microtiter plate with a footprint of a standard 96-well microtiter plate containing microtiter wells, the number of microtiter wells shall not exceed 864 microtiter wells and detect a signal from the sample. Preferably the detection comprises the detection of epifluorescence from below the multiwell plate or platform. The detection step can also use an optical arrangement that corresponds to the density and arrangement of the wells in the multiwell plate. Various markers can be used in assays using the present invention. It will often be desired to provide the binding sites on a multiwell plate or platform for use as part of the assay system. Such markers can be attached directly or indirectly to the surface of the polymer. Different spectroscopic techniques can be used with the present invention, such as colorimetric, spectrophotometric, luminescence and fluorescence procedures. Non-spectroscopic, light-based procedures such as refraction and reflectance procedures can be used.
Fluorescence measurements
It is recognized that different types of fluorescence monitoring systems can be used to practice the present invention with fluorescent probes, such as fluorescent dyes or substrates. Preferably, specialized systems are used in a high throughput screen, eg, 96-well microtiter plates or larger. Procedures for conducting assays on fluorescent materials are well known in the art and are described for example in Lakowicz, JR, Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance Energy Transfer Microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, DL & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, NJ, Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Col, Inc. (1978), pp. 296-361 and the Molecular Probes Catalog (1997), OR, US.
Fluorescence in a sample can be measured using a detector described herein or known in the art for multiwell plates. In general, excitation radiation, from an excitation source having a first wavelength, optionally passes through the excitation optics. Excitation optics
ES 2 289 784 T3 causes excitation radiation to excite the sample. In response, the fluorescent probes in the sample emit radiation having a wavelength that is different from the excitation wavelength. The collection optics then capture the light emitted from the sample. The device may include a temperature controller to maintain the sample at a specific temperature while scanning. According to one embodiment, a multiaxial translation step (eg, a specialized X, Y positioner) moves a microtiter plate containing a plurality of samples to position different wells to be exposed. The multiaxial translation stage, the temperature controller, the autofocus feature, and the electronics associated with imaging and data collection can be carried out by a suitably programmed digital computer. The computer can also transform the data collected during the test into another format for presentation.
Preferably, FRET (fluorescence resonance energy transfer) is used as a way to monitor probes in a sample (cellular or biochemical). The degree of FRET can be determined by any fluorescence or spectral lifetime characteristics of the excited construct, for example, by determining the intensity of the donor fluorescence signal, the intensity of the acceptor fluorescence signal. , the ratio of the fluorescence amplitudes near the maximum emission of the acceptor to the fluorescence amplitudes near the maximum emission of the donor or the life time of the excited state of the donor. For example, cleavage of the linker increases donor fluorescence intensity, decreases acceptor fluorescence intensity, decreases the ratio of acceptor to donor fluorescence amplitudes, and increases the donor's excited state lifetime.
Preferably, the changes in the signal are determined as the ratio of fluorescence at the two different emission wavelengths, a procedure referred to as "matching." Differences in the absolute amount of probe (or substrate), cells, excitation intensity and turbidity, and other base absorbances between addressable wells can affect the fluorescence signal. Therefore, the ratio of the two emission intensities is a more robust and preferred measure of activity than the intensity of the emission alone.
A ratio metered fluorescent probe system can be used in the present invention. For example, the reporter system described in PCT publication WO / 30540 (Tsien) possesses significant advantages over existing reporters for gene integration analysis, as it allows sensitive detection and isolation of both single-expressed and non-expressed living cells. . This assay system uses a non-toxic, non-polar fluorescent substrate that is easily loaded and then trapped intracellularly. Cleavage of the fluorescent substrate by jd-lactamase provides a fluorescent emission shift as soon as the substrate is converted to product. Since the reporter readout of β-lactamase is measured in proportions, it is unique among reporter gene assays in that it controls for variables such as the amount of substrate loaded in individual wells. The easily detected, stable intracellular leakage reading simplifies assay procedures by eliminating the need for wash steps, facilitating well selection utilizing the present invention.
Detector
In one embodiment the present invention provides a detector for monitoring spectroscopic events with the multiwell plates or platforms. Preferably the detector is a fluorescence detector and more preferably of the type that can be used for epifluorescence. For some embodiments of the present invention, particularly for plates with 96, 192, 384 and 864 wells per plate, a number of detectors are available. Such detectors are described in US Patent No. 5,589,351 (Harootunian), US Patent No. 5,355,215 (Schroeder) and PCT Patent Application WO 93/13423 (Akong). Alternatively, an entire plate can be "read" using an imager, such as a Molecular Dynamics Fluor-Imager 595. Conventional optical disc readers such as those known in the relevant art for spectroscopic analysis can be adapted for the platform applications of the present invention, as well as for the measurement of refracted or reflected light.
The detector is preferably capable of carrying out fluorescence emission measurements that are in the range of 400 to 800 nm. Typically, the detector comprises a means for excitation of fluorescence in the range of 350 to 800 nm. The detector is often capable of many different modes of operation that facilitate drug discovery assay requirements. These modes of operation may include: single excitation wavelength with single emission wavelength detection, single excitation wavelength, dual emission wavelength detection, sequential dual excitation wavelength with dual emission wavelength detection and the ratio measurement determination, sequential dual excitation wavelength with four emission wavelength detections and ratio measurement determinations, homogeneous time-resolved fluorescence with single excitation wavelength and single emission wavelength detection, homogeneous time-resolved fluorescence with single excitation wavelength and dual emission wavelength detection and measurement of the proportion, Homogeneous time-resolved fluorescence with sequential dual excitation wavelength and dual emission wavelength detection and ratio determination measurement, dual sequential excitation wavelengths, and single emission wavelength detection with measurement of proportion determination, luminescence measurement at a single wavelength with luminescence measurement at dual wavelengths, luminescence measurement at dual wavelengths with a ratio determination, and
ES 2 289 784 T3 time resolved fluorescence emission (intrinsic dye properties with or without a binding event). The detector preferably operates in the epifluorescence mode where the preferred illumination is from the bottom of the plate and the preferred capture is from the bottom of the plate as well. The detector can operate in all the modes mentioned above with the view of the bottom of the plate.
The ratio mode of the detector enables changes in signal levels from relative signal levels to be observed without complex calibration. The detector's ratio mode tolerates differences in the amounts of isolated targets, cells, or dye loading on cells. Therefore, differences between wells may exist for cells and levels of dyes, but within a single well, these differences can be normalized to a relative change in intensities. Without detection of the ratio measurement, the absolute signal levels can obscure the slight changes within each well.
The selection of different detector operating modes is often based on the type of test to be carried out. Thus, the detector is generally designed with numerous modes of operation to provide flexibility in detection. Each mode is selected based on its compatibility with a particular set of fluorescent probes and reagents. Detection is then tailored to meet probe and assay requirements.
The present invention also provides a system for spectroscopic measurements. The system comprises reagents for 1) an assay, 2) a device, comprising a layer with low fluorescence and high transmittance, comprising a cycloolefin copolymer and a platform to support the layer. The system may further comprise a detector.
Examples
Example 1
Fluorescence properties of cycloolefins compared to glass and other polymeric materials
To investigate the fluorescence properties of different selected films, different polymeric films were tested with fluorescence emission at predetermined excitation wavelengths and compared to the two types of fused (standard) silica glass sheets. These experiments were carried out using a SPEX Fluorolog 111 fluorometer with excitation wavelengths between 315 and 425 nm. The films and glassware were arranged on a support. The sample was placed with the excitation beam perpendicular to the face of the sample. The fluorescence emission from the sample was captured at an angle of approximately 12.5 degrees. The fluorescence emission of the material was reflected on a mirror and on a monometer. Emission radiation was selected by a monochromatic diffraction grating and detected by a photomultiplier tube of the instrument. The SPEX Fluorolog 111 fluorimeter uses water Raman radiation lines to calibrate and correct the baseline of day-to-day instrument measurements. This baseline correction was carried out each day prior to use of the instrument for calibration. The calibration file is stored with the measurements taken that day and subsequent measurements with the SPEX instrument can then be directly compared and corrected for instrument fluctuation.
The materials tested were 1) fused silica sheets (Corning Glass Works coverslip # 1 (catalog number 2935/583331), 2) polystyrene films (ps1, ps2 (from Plastic Suppliers) and ps3 (from Dow Chemical Company), 3) polycarbonate films (pc1 (from General Electric Corporation) and pc2 (from Plastic Suppliers); 4) alkyl, non-aromatic polymers (nap; obtained from Mobil Oil Company), 5) cycloolefin copolymer film (coc; obtained from Hoechst, Topas) and 6) Aclar (a fluorocarbon material from Allied Signal).
Table 2 shows the normalized fluorescence emission data for 400 to 650 nm at three different excitation wavelengths. Data is normalized to fused silica and to correct for instrumentation fluctuation. Polystyrene, which is often used as a component of multiwell plates (see Table 1), generated high levels of base fluorescence, consistent with its aromatic structure. Surprisingly, polycarbonate, which is often a compatible polymer, was generally better than polystyrene, especially at higher wavelengths. Surprisingly, the non-aromatic alkyl polymer was generally the second best polymer over the entire wavelength range tested. Also surprisingly, the cycloolefin copolymer produced the best results and nearly approximated the extremely low fluorescence levels of fused silica.
ES 2 289 784 T3
TABLE 2
<td colspan="9">Material</td>
<td>Ex = 315</td><td>Em = 400</td><td>Em = 425</td><td>Em = 450</td><td>Em = 475</td><td>Em = 500</td><td>Em = 550</td><td>Em = 600</td><td>Em = 650</td>
<td>glass</td><td> 0,22513</td><td> 0,25824</td><td> 0,26817</td><td> 0,30459</td><td> 0,33107</td><td> 0,38735</td><td> 0,51316</td><td></td>
<td>pc1-5m</td><td> 3,31071</td><td> 2,10230</td><td> 2,01953</td><td> 1,78778</td><td> 1,41036</td><td> 0,66876</td><td> 0,60586</td><td></td>
<td>pc2-5m</td><td> 11,04128</td><td> 7,04943</td><td> 6,11517</td><td> 5,18091</td><td> 3,79367</td><td> 1,70432</td><td> 1,05317</td><td></td>
<td>ps1-2m</td><td> 2,45986</td><td> 1,96447</td><td> 1,93714</td><td> 1,78340</td><td> 1,52374</td><td> 1,02494</td><td> 1,18893</td><td></td>
<td>ps2-2m</td><td> 2,20826</td><td> 1,72697</td><td> 1,69866</td><td> 1,64204</td><td> 1,48633</td><td> 1,07582</td><td> 1,18906</td><td></td>
<td>ps3-2m</td><td> 4,55807</td><td> 3,29823</td><td> 3,00096</td><td> 2,72352</td><td> 2,34132</td><td> 1,57409</td><td> 1,98743</td><td></td>
<td>Nap-1.5m</td><td> 1,01919</td><td> 0,75307</td><td> 0,62850</td><td> 0,52942</td><td> 0,50110</td><td> 0,56622</td><td> 1,12111</td><td></td>
<td>Nap-1.5m</td><td> 0,52658</td><td> 0,48978</td><td> 0,42466</td><td> 0,37654</td><td> 0,38220</td><td> 0,50960</td><td> 1,00787</td><td></td>
<td>Coc-clean</td><td> 0,40485</td><td> 0,40485</td><td> 0,34256</td><td> 0,31142</td><td> 0,31142</td><td> 0,41617</td><td> 0,83234</td><td></td>
<td>Clear-, 75m</td><td> 0,08473</td><td> 0,08875</td><td> 0,07864</td><td> 0,07368</td><td> 0,07503</td><td> 0,09701</td><td> 0,22497</td><td></td>
<td>Clear-3m</td><td> 0,27245</td><td> 0,26586</td><td> 0,27367</td><td> 0,26522</td><td> 0,29309</td><td> 0,44479</td><td> 1,03199</td><td></td>
<td>Ex = 350</td><td>Em = 400</td><td>Em = 425</td><td>Em = 450</td><td>Em = 475</td><td>Em = 500</td><td>Em = 550</td><td>Em = 600</td><td>Em = 650</td>
<td>glass</td><td> 0,30790</td><td> 0,20526</td><td> 0,23837</td><td> 0,17547</td><td> 0,16222</td><td> 0,17878</td><td> 0,25492</td><td></td>
<td>pc1-5m</td><td> 0,77802</td><td> 0,62572</td><td> 0,60586</td><td> 0,50323</td><td> 0,42708</td><td> 0,31452</td><td> 0,33769</td><td></td>
<td>pc2-5m</td><td> 3,96354</td><td> 2,74616</td><td> 2,20826</td><td> 1,61373</td><td> 1,24568</td><td> 0,75024</td><td> 0,62284</td><td></td>
<td>ps1-2m</td><td> 1,28801</td><td> 1,44858</td><td> 2,22754</td><td> 2,06013</td><td> 1,78340</td><td> 1,06594</td><td> 0,84387</td><td></td>
<td>ps2-2m</td><td> 1,01919</td><td> 1,34477</td><td> 1,85437</td><td> 1,84021</td><td> 1,64204</td><td> 1,08997</td><td> 0,89180</td><td></td>
<td>ps3-2m</td><td> 2,13182</td><td> 2,68388</td><td> 3,47092</td><td> 3,14252</td><td> 2,68388</td><td> 1,57692</td><td> 1,29381</td><td></td>
<td>Nap-1.5m</td><td> 0,95408</td><td> 0,80120</td><td> 0,81536</td><td> 0,59170</td><td> 0,53508</td><td> 0,58321</td><td> 0,79554</td><td></td>
<td>Nap-1.5m</td><td> 0,53791</td><td> 0,48695</td><td> 0,55206</td><td> 0,39918</td><td> 0,39918</td><td> 0,48129</td><td> 0,69079</td><td></td>
<td>Coc-clean</td><td> 0,42466</td><td> 0,38220</td><td> 0,43033</td><td> 0,31142</td><td> 0,31142</td><td> 0,38503</td><td> 0,56056</td><td></td>
<td>Clear-, 75m</td><td> 0,08689</td><td> 0,08710</td><td> 0,08669</td><td> 0,07327</td><td> 0,07224</td><td> 0,08050</td><td> 0,10733</td><td></td>
<td>Clear-3m</td><td> 0,24045</td><td> 0,23323</td><td> 0,24974</td><td> 0,21981</td><td> 0,23375</td><td> 0,31373</td><td> 0,43756</td><td></td>
<td>Ex = 400</td><td>Em = 400</td><td>Em = 425</td><td>Em = 450</td><td>Em = 475</td><td>Em = 500</td><td>Em = 550</td><td>Em = 600</td><td>Em = 650</td>
<td>glass</td><td></td><td></td><td> 0,29134</td><td> 0,21520</td><td> 0,25492</td><td> 0,18540</td><td> 0,26817</td><td> 0,43039</td>
<td>pc1-5m</td><td></td><td></td><td> 0,38073</td><td> 0,30459</td><td> 0,32114</td><td> 0,22844</td><td> 0,31783</td><td> 0,48667</td>
<td>pc2-5m</td><td></td><td></td><td> 0,65115</td><td> 0,59736</td><td> 0,62284</td><td> 0,43033</td><td> 0,53791</td><td> 0,77855</td>
<td>ps1-2m</td><td></td><td></td><td> 0,55347</td><td> 0,55347</td><td> 0,67646</td><td> 0,43731</td><td> 0,61155</td><td> 0,91561</td>
<td>ps2-2m</td><td></td><td></td><td> 0,49544</td><td> 0,50960</td><td> 0,60869</td><td> 0,46996</td><td> 0,65115</td><td> 1,00221</td>
<td>ps3-2m</td><td></td><td></td><td> 0,75873</td><td> 0,80120</td><td> 0,97107</td><td> 0,63417</td><td> 0,86065</td><td> 1,24568</td>
<td>Nap-1.5m</td><td></td><td></td><td> 0,57754</td><td> 0,59170</td><td> 0,67663</td><td> 0,50110</td><td> 0,72476</td><td> 1,08431</td>
<td>Nap-1.5m</td><td></td><td></td><td> 0,41900</td><td> 0,39635</td><td> 0,50394</td><td> 0,42466</td><td> 0,66248</td><td> 1,05883</td>
<td>Coc-clean</td><td></td><td></td><td> 0,32558</td><td> 0,33407</td><td> 0,41900</td><td> 0,37087</td><td> 0,55489</td><td> 0,87198</td>
<td>Clear-, 75m</td><td></td><td></td><td> 0,06295</td><td> 0,06295</td><td> 0,07121</td><td> 0,06966</td><td> 0,10010</td><td> 0,15686</td>
<td>Clear-3m</td><td></td><td></td><td> 0,14138</td><td> 0,14654</td><td> 0,17750</td><td> 0,20433</td><td> 0,32405</td><td> 0,47988</td>
Example 2
Fluorescence properties of cycloolefins compared to glass and other polymeric materials
To further investigate the fluorescence properties of different selected films, different polymeric films were tested with fluorescence emission at previously determined excitation wavelengths and compared to the two types of fused (standard) silica glass sheets. These experiments were carried out to simulate biochemical or cell-based assays involving an aqueous medium. Therefore, the films were mounted on a horizontal plastic support to allow the addition of a drop of aqueous medium. Three milliliters of water were distributed on the film and the fluorescence was recorded using a Zeiss inverted fluorescence microscope. The baseline was recorded in the absence of a film and subtracted from the signals in the presence of a film.
The materials tested were 1) fused silica sheets (Fisher cover slip number 1 (Fisher catalog number 12-542B (1996)), 2) polystyrene films (ps1, ps2 (from Plastic Suppliers) and ps3 (from Dow Chemical Company), 3) polycarbonate films (pc1 (from General Electric Corporation) and pc2 (from Plastic Suppliers); 4) alkyl, non-aromatic polymers (obtained from Mobil), 5) cycloolefin copolymer film (coc; obtained from Hoechst, Topas), 6) Aclar (a fluorocarbon material from Allied Signal) and 7) Syran Wrap.
Table 3 shows the normalized fluorescence emission data at 460 nm, 350 nm and 405 nm (excitation wavelengths). The data was normalized to fused silica. Polystyrene, which is often used as a component of multiwell plates (see Table 1), generated high levels of base fluorescence, consistent with its aromatic structure as in Example 1. Unlike Example 1, polycarbonate, which is often a biocompatible polymer, fared worse than polystyrene, especially at higher wavelengths. Consistent generally with Example 1, the non-aromatic alkyl polymer was generally better than polystyrene over the entire wavelength range tested. Consistent generally with Example 1, the cycloolefin copolymer produced the best results and surprisingly improved the extremely low fluorescence levels of fused silica. The Aclar film produced surprisingly low or extremely low fluorescence values relative to fused silica.
ES 2 289 784 T3
TABLE 3
Material
350ex / 460em Range
Material
405ex / 460em range
Fisher # 1 Polycarbonate Coverslip 5 mil
Polystyrene 2 mil NAP 1.5 ml NAP 1.5 ml coc # 2 2 mil coc # 1 2 mil clear sample (> 2 years of age)
Fisher # 1 coverslip polycarbonate 5 mil polystyrene 1 mil coc # 2 A 2 mil coc # 2 B 2 mil coc # 1 2 mil clarify 3 mil (> 1 year old) clarify 0.75 mil (> 1 year old ) syran wrap
1.02 1 Fisher # 1 Coverslip 1.03 1
6.91 6 polycarbonate 5 mil 19.79 6
3.57 5 polystyrene 2 mil 3.36 4
2.06 3 NAP 1.5 ml 5.76 3
1.33 3 NAP 1.5 ml 3.51 3
1.58 2 coc # 2 2 thousand 2.60 2
1.22 2 coc # 12mil 1.59 2
2.62 4 shows clarity of 2 years of 9.08 5 age)
1.00 5 Fisher # 1 Coverslip 1.00 1
5.15 9 polycarbonate 5 thousand 17.75 8
2.01 7 polystyrene 1 mil 2.53 7
1.09 6 coc # 2A2mil 1.71 4
0.89 4 coc # 2 B 2 thousand 1.65 3
0.86 3 coc # 12mil 1.47 2
0.71 1 clear 3 mil (> 1 year of 2.34 6 age)
0.64 1 clear 0.75 mil (> 1 year of 2.14 5 age)
4.18 8 syran wrap 22.12 9
Example 3
Cycloolefins are not cytotoxic to cultured cells
Cytotoxicity of cycloolefin was evaluated by incubating cells in cycloolefin multiwell plates for 60 hours at 37 ° C. 1.8 µl volumes of medium containing approximately 90 Chinese Hamster Ovaries (CHO) were plated on cycloolefin multiwell plates using a graduated pipette. A glass lid was placed over the wells to prevent evaporation. Cells were incubated for 60 hours at 5% CO<sub>2</sub>, at 37 ° C and in a 90% RH incubator. The cells were then tested for viability by loading with the vital dye calcein. CHO cells were loaded by incubation in a solution containing 4 pM calcein / AM for 30 minutes at room temperature. Cells were inspected using both phase contrast microscope to determine the total number of cells and fluorescence microscopy to determine the number of living cells. Approximately, more than 95% of cells were alive as indicated by loading with the calcein dye (approximately 200 cells / well).
Example 4
Cycloolefins are not cytotoxic to cultured cells and can be used for drug screening assays.
To investigate the cytotoxic properties of cycloolefins, cycloolefin films were tested using a cell viability assay. CCF2, a vital dye, as described in PCT publication WO96 / 30540 (Tsien), diffuses into cells and is captured by living cells possessing esterase activity that cleaves ester groups on molecules resulting in into a negatively charged molecule that is trapped inside the cell. Captured dye appears green within living cells. CCF2 was incubated with Jurkat cells for 1 hour in a 1 microliter well having black walls and a cycloolefin background, and fluorescence was adequately monitored. These Jurkat cells were constitutively expressing β-lactamase. Cells were cultured for 60 hours under the conditions of Example 3. After 60 hours, β-lactamase activity was measured using CCF2. The cells appeared blue indicating that β-lactamase was indeed active in these cells, which normally do not contain β-lactamase. These results demonstrate that cycloolefins can be used with sensitive fluorescence assays because the films provide low fluorescence backgrounds. This is particularly beneficial because it allows for smaller assay volumes (eg, 2 microliters or less) and the measurement of smaller signals (eg, from few cells or from a smaller number of isolated biochemical targets).
Publications
All publications, including patent documents and scientific articles, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. .
All titles are for the convenience of the reader and should not be used to limit the meaning of the text that follows the title, unless otherwise specified.
Contents11
68 members in 12 offices
Priority claims20
| Document | Office | Kind | Date |
|---|---|---|---|
| 19970867567 | United States of America | – | |
| 19970867584 | United States of America | – | |
| 86756797 | United States of America | A | |
| 86756797 | United States of America | A | |
| 86758497 | United States of America | A | |
| 86758497 | United States of America | A | |
| 19970868018 | United States of America | – | |
| 19970868049 | United States of America | – | |
| 86801897 | United States of America | A | |
| 86801897 | United States of America | A | |
| 86804997 | United States of America | A | |
| 86804997 | United States of America | A | |
| 867584 | – | – | – |
| 868018 | – | – | – |
| 868049 | – | – | – |
| 98925058867567 | – | – | – |
| US19970867567 | – | – | – |
| US19970867584 | – | – | – |
| US19970868018 | – | – | – |
| US19970868049 | – | – | – |
Members68
| Document | Office | Kind | |
|---|---|---|---|
| CA2290505A1 | Canada | A1 | |
| WO9852047A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU7478398A | Australia | A | |
| CA2262739A1 | Canada | A1 | |
| WO9855231A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU7709198A | Australia | A | |
| US5910287A | United States of America | A | |
| EP0921857A1 | European Patent Office (EPO) | A1 | |
| CA2271373A1 | Canada | A1 | |
| WO9942608A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU2787099A | Australia | A | |
| US5985214A | United States of America | A | |
| US6063338A | United States of America | A | |
| EP1010009A1 | European Patent Office (EPO) | A1 | |
| USD428157S | United States of America | S | |
| EP0921857A4 | European Patent Office (EPO) | A4 | |
| US6171780B1 | United States of America | B1 | |
| EP1066400A1 | European Patent Office (EPO) | A1 | |
| US6229603B1 | United States of America | B1 | |
| US6232114B1 | United States of America | B1 | |
| US6254833B1 | United States of America | B1 | |
| JP2002500759A | Japan | A | |
| AU743507B2 | Australia | B2 | |
| US2002012611A1 | United States of America | A1 | |
| USD454202S | United States of America | S | |
| US6372185B1 | United States of America | B1 | |
| JP2002515125A | Japan | A | |
| US6426050B1 | United States of America | B1 | |
| US2002119077A1 | United States of America | A1 | |
| US6468800B1 | United States of America | B1 | |
| US2002155617A1 | United States of America | A1 | |
| US6472218B1 | United States of America | B1 | |
| US6517781B1 | United States of America | B1 | |
| US2003039591A1 | United States of America | A1 | |
| CA2262739C | Canada | C | |
| US6678577B1 | United States of America | B1 | |
| US6685884B2 | United States of America | B2 | |
| US6730520B2 | United States of America | B2 | |
| EP1066400A4 | European Patent Office (EPO) | A4 | |
| US2004202582A1 | United States of America | A1 | |
| US6825042B1 | United States of America | B1 | |
| US2005019221A1 | United States of America | A1 | |
| US6861035B2 | United States of America | B2 | |
| US6890485B1 | United States of America | B1 | |
| US2005191670A1 | United States of America | A1 | |
| US2005214174A1 | United States of America | A1 | |
| US2005271551A1 | United States of America | A1 | |
| US7105132B2 | United States of America | B2 | |
| EP1010009A4 | European Patent Office (EPO) | A4 | |
| US2007009883A1 | United States of America | A1 | |
| EP0921857B1 | European Patent Office (EPO) | B1 | |
| CA2290505C | Canada | C | |
| AT367203T | Austria | T | |
| ATE367203T1 | Austria | T1 | |
| DE69838090D1 | Germany | D1 | |
| PT921857E | Portugal | E | |
| DK0921857T3 | Denmark | T3 | |
| ES2289784T3This record | Spain | T3 | |
| DE69838090T2 | Germany | T2 | |
| US2008226498A1 | United States of America | A1 | |
| US7459130B2 | United States of America | B2 | |
| JP2009080119A | Japan | A | |
| US2009148350A1 | United States of America | A1 | |
| JP4303793B2 | Japan | B2 | |
| JP4477085B2 | Japan | B2 | |
| US7854898B2 | United States of America | B2 | |
| CY1106874T1 | Cyprus | T1 | |
| CA2271373C | Canada | C |
Numbers
- Publication
- 2289784
- Publication, DOCDB
- 2289784
- Publication, EPODOC
- ES2289784T
- Application
- 98925058
- Application, DOCDB
- 98925058
- Application, EPODOC
- ES19980925058T
Titles2
- Spanish
- PLACAS MULTIPOCILLO DE BAJA FLUORESCENCIA DE BASE PARA LAS MEDICIONES DE FLUORESCENCIA DE MUESTRAS BIOLOGICAS Y BIOQUIMICAS.
- English
- BASE FLUORESCENCE LOW MULTIPOYCIL PLATES FOR FLUORESCENCE MEASUREMENTS OF BIOLOGICAL AND BIOCHEMICAL SAMPLES.
Classification
- CPC, 10
- B01L3/5085
- B01J2219/00317
- B01J2219/00659
- B01J2219/00707
- B01J2219/0072
- B01L2200/12
- B01L2300/0829
- B01L2300/12
- C40B60/14
- G01N21/6452
- IPC, 8
- G01N21 64
- B01L3 00
- B01L9 00
- C08G61 08
- C40B60 14
- G01N21 03
- G01N33 483
- G01N33 52