Optical disk, apparatus for conducting an optical control, method for detecting the presence or the absence of an analyte in a sample and method for controlling a biological, chemical or biochemical sample

The invention relates, in general, to diagnostic tests and their methodology, in particular, to components for diagnostic tests, configured on a compact optical disk and to the methodology for their use.

The need for clinical trials to be faster, cheaper and simpler is huge. Ideally, patients can do their own analysis if they wish. A step towards this goal was made by miniaturizing and integrating different test operations. At this time, a number of bio - chip tests (so - called because some are made using silicon chip photolithography techniques) are in the market or are emerging. All of these approaches require a machine for reading results and a computer.

Also, commercially available disc-shaped cassettes used for clinical trials along with UV / Vis spectrometry. U.S. Patent 5,122,284 discloses a centrifugal rotor containing a number of interconnected fluid chambers connected to a plurality of small tanks. The rotor is adapted for use with a conventional laboratory centrifuge and is made of materials that allow photometric detection of the results of the analyzes that took place in small reaction tanks. A large number of rotor configurations and related devices have been described for the same tests or for similar analysis tests. See, for example, US Patents 5,472,603; 5 173 193; 5 061 381, 5 304 348; 5,518,930; 5,457,053; 5,409,665; 5 160 702; 5,173,262; 5,409,665; 5 591 643; 5 186 844; 5,122,284; 5 242 606 and the patents mentioned therein. The lyophilized reagents used in these systems are described in US Patent 5,413,732.

The principles of the centrifugal analyzer have been adapted to a disk that can be used in an instrument such as a CD driver (Mian et al., WO 97/21090 patent application). Mian has a modified CD driver with dual function: 1 is used to read the information stored on the disk, and 2 is used to rotate the disk. However, Mian does not show the use of the ability to read a CD driver for analysis of the tests.

Despite recent progress, there remains a need for a simpler test setup, which performs tests quickly, efficiently, accurately and at low cost. The technical problem, of the present invention, is the combination of diagnostic analysis with the technology of computers and compact discs. In its preferred embodiments, the only instrument required is a computer with a compact disc reader. All chemistry takes place inside a compact disc, which can be referred to as an integrated biocompact disk (BCDI). The same compact disc is also software-encoded, meaning control information and machine readable instructions that give information to a computer before, after and during the test.

CDs or DVDs are the most economical and, in many ways, the best environment for collecting information. It should be noted that CDs and DVDs are acronyms used at the moment, which may change in the future, even if the basic technology remains largely unchanged. A CD or DVD driver is in many ways the equivalent of a confocal scanning microscope. at the same time, these instruments are comparable to some good centrifuges, because in commercial drivers the frequency of rotation is between 200-12000 rpm and can be adjusted between certain limits. Combining these three traits, in the same analytical system, results in a great simplification in comparison with any other analytical technique, in addition, the performance is comparable or better than the other competing methods. Although this invention requires slightly modified CD or DVD drivers, it is possible to incorporate these changes in commercial drivers. This will make possible the existence of points for patient care (POPC) and the use, at home, of this invention. The use of CD or DVD drivers will allow the faithful digital analysis of any sample without any specific analytical tool:

In one of its aspects, the invention relates to an optical disk, adapted to be read 1 by an optical reading instrument, constituted by a first sector comprising a substantially autonomous means of analysis, for linking an analyte. whose presence in a sample is 3 suspected at least at a predetermined location in the first sector and, optionally, a second sector that contains a means of control for conducting the analysis and locating the information 5 about the analyte, compared to one or more analysts suspected of being in the sample, the information being accessible to a reading instrument, where the presence or absence of the analyte at the respective location can be determined by the reading instrument, using means of controlling and locating the information. Depending on the nature of the test, the disc may contain fluid storage elements, fluid transfer means, such as one or more capillaries, valves, batteries, dialyzers, columns, filters, electric field sources, wires. or other electroconductive elements, such as surface metal deposits and the like.

In another aspect, the invention relates to a method for detecting the presence or absence of an analyte suspected of being present in a sample, using an optical disc, separated into two sectors: one for conducting the analysis itself and another for storing the 15 computer program, which allows testing in the other sector.

There is also presented a method of optical control of a biological, chemical or biochemical sample, by driving an optical disk, adapted to be read by an optical reading device. 19

The disk may have one or more ports for sample entry to release the fluid sample into the test sector. Such ports, if any, are preferably 21 sealable so that, after applying the sample to the disk, the sealed disk including the sample comprises a hermetically sealed device, which can be easily removed by conventional means or other means. removal mechanisms, used for biological residues. Also, the disk testing sector is conveniently divided into several subsections for sample preparation and analyte separation. A subsection for the reception of residues may also be present. The test sector can be divided into a multitude of subsectors, each of which receives a sample. Each such subsector can analyze one or more analytics, depending on each particular application. 29 In another aspect, the invention relates to an apparatus for conducting a test comprising an optical disk, a disk reader and an information processor, the disk comprising a first 31 sector, having a substantially autonomous analysis means for locating an analyte suspected of being in a sample, at least in a predetermined location in the first sector and, optionally, 33 a second sector containing control information, for conducting the analysis and information on the location of the analyte, compared to one or more analytes suspected of being in the sample, 35 accessible to the reading instrument and processable by the information processor, in which the disk is adapted to be read by the instrument and the information processor is adapted to determine the presence or absence of the analyte in the respective location, using the control information and the location information. The device may include a 39-read instrument that has a CD-ROM or DVD player and an information processor, such as a personal computer. 41 In another aspect, the invention relates to an optical disk, adapted to be read by a CD-ROM or DVD reader, comprising a substantially autonomous disk-based analysis means for locating an analyte suspected to be it was in a sample, in at least one predetermined location on the disk and includes means at the respective location, for detecting the absence or presence of the analyte by the CD-ROM or DVD reader.

RO 119751 Β1 in the following, are briefly shown, fig. 1 ... 17 companions:

FIG. 1 is a schematic representation of a disc of this invention;

FIG. 2A, a more detailed schematic representation of a sample preparation and analysis sector on the disk, illustrating the general arrangement of a typical test sector;

FIG. 2B, schematic representation of an existing test sector, which is capable of immunostaining, DNA testing, cell counting, spectrophotometric testing and electrolyte analysis;

FIG. 3, a schematic representation of a disk of this invention, illustrating a multiplicity of test sectors, each having an individual port for sample entry;

FIG. 4 is a more detailed schematic representation of one of the test sectors illustrated in FIG. 3;

FIG. 5 is a schematic representation of a chemically actuated battery useful in the present invention;

FIG. 6, schematic representation of a structure, to provide a dialysis function on the disc of this invention;

FIG. 7, a schematic representation of a column, which may be included on the disk of this invention;

FIG. 8 is a schematic representation of an electrically controlled valve useful in the present invention;

FIG. 9 is a schematic representation of a chain of reagents, configured in united capillary channels, which is useful in the present invention;

FIG. 10 is a schematic representation of a linear arrangement of test locations, which are conventionally located in a drainage channel in the test sector of the disc of this invention;

» ·

FIG. 11 AC, schematic representation of a variant of a test element, which is particularly useful for detecting particles and viral and bacterial cells, using the general methodology of the specific localization of the substance to be detected;

FIG. 12 AC, schematic representation of a variant of the detection methodology, in which opaque particles are used, instead of reflective particles and bound to a reflecting surface. The zigzag lines represent oligonucleotides, but they can be any recognition molecules, such as antibodies. The particles are, in this example, plastic spheres, but they can be liposomes, cells, etc.

FIG. 13, schematic representation of a test element of the invention, illustrating the spacer molecule, with component side arms and cleavage site, linked to a surface of the disk at one end and to a reporter element (gold or latex sphere) at the other. end;

FIG. 14A, schematic representation of a first test element of this invention, at an early stage during the test procedure;

FIG. 14 B, schematic representation of a second test element of this invention, at an early stage, during the test procedure;

FIG. 14 C, schematic representation of the test element in fig. 14A, wherein the annealing molecules have the lateral arms linked, forming a connecting loop between the sides of the cleavage site;

FIG. 14 D, schematic representation of the test element in fig. 14 B, wherein the analyte molecules do not have side arms linked and do not form a connecting loop between the sides of the cleavage site;

FIG. 14 E, schematic representation of the test element in fig. 14 C, after the distance molecules have been cleaved; the reporter elements remain attached to the surface of the disc in a discrete site;

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FIG. 14 F, schematic representation of the test element in fig. 14 D, after 1 distance molecules were cleaved; the reporter element detaches from the surface of the disc and is free, it can be removed by washing from its discrete place; 3

FIG. 15, schematic representation of an assembly of small tanks; four small tanks and their associated reagents and sample preparation chambers as well as light sources are presented in this example;

FIG. 16 is a schematic representation of an arrangement of capillary pipes, which can be used to achieve isoelectric focusing;

FIG. 17, schematic representation of an apparatus for measuring the exact volumes. 9

A schematic, global representation of an integrated bio-compact disc (BCDI) can be found in fig. 1. The disc (Bio-compact disc, DBC) can in fact have any shape and size. 11 For most applications, it is circular and has a diameter of 10-1000 mm, the most advantageous 20 - 200 mm and a thickness of 0.1 - 20 mm, the most advantageous 0.5 - 3 mm. Disk 10 contains 13 two sectors: a test sector 11 and a software sector 12. A central orifice 13 is provided for localization in a compact disc reader. The software for controlling the test may be on a separate disk. However, it is preferable for the software to be on disk, in combination with a test for an analyte or some particular analyzer to minimize the possibility of a human error when performing the test. The possible components and operations of the BCDI are presented in the following description. 19

The disc usually rotates up to 16,000 rpm in CD-ROM or DVD players. In all CD-ROM or DVD readers the speed can be adjusted between certain limits (200 -16,000 21 rpm). However, for certain operations it may be advantageous to use rotations at different speeds, for example, 1000-1000 rpm, and especially 2000-5000 rpm. For any particular test, 23 control software dictates the rotation regime during the analysis. This regime, speeds and times, including times when there may be no rotation to allow incubation, electro-force, isoelectric focusing, etc., is controlled to release the reactants and samples into the appropriate strains in the test sectors as require testing protocols. Available rotational speeds allow significant centrifugal force, which can be used to move liquids. Another energy source that can be easily used in BCDI is chemical energy. A more suitable form of chemical energy is released by a battery in the form of electricity. Mechanical and chemical energy allow the operation of many types of speakers. Important components of BCDI may include one or more of the following: capillaries, containers, filters, dialysis membranes, chromatography columns, electrophoretic gels, valves, any micromechanical or electronic components, including microprocessors, electrodes, especially enzyme electrodes. , small tanks and test items. Possible unit operations, 35 that the components may undergo, include centrifugation, filtration, fluid transfer, liquid mixing, dialysis, column separation, heating, cooling, electroconvection, electrophoresis, and analyte detection and signaling.

The BCDI is conveniently composed of two parts comprising the lower and upper halves. The lower half may contain almost all the components, while the upper half may be a flat coating, containing only a few components, such as electrodes and wires. The number of layers can be greater than two and many components can also be prefabricated as modules. As a module, the reagent containers, the tank assemblies, the columns, the micromechanical components, the light sources and the microprocessors are advantageously assembled. Different features can be printed on soft plastic 45. Different components can be bonded, either by heat treatment or by UV cross-linking, melted together, connected by complementary mechanical features, mechanically or simply fastened 47

EN 119751 Β1 included in a larger component. Some areas can be treated, for example, with ammonia plasma to make these areas hydrophilic. The surface can be further treated with different molecules that make the surface inert or, alternatively, give it specific adsorption properties. Silylation is a general method for surface treatment (Virtanen, JA, Kinnunen, PKJ and Kulo, A., Organosilanes and their hydrolytic polymers as surface treatment agents for use in chromatography and electronics, USP 4 756 971). Covalent attachment of detergents reduces the adsorption of proteins, such as albumin, and also reduces the adsorption of soluble proteins. The metal electrodes and wires can evaporate on the desired areas. Masks or resistors may be used to locate plasma treatment or metal deposition. Capillary pipes and fluid storage and retention compartments can be processed on optical discs or they can be formed by chemical means or by injection molding operations. As shown with reference to FIG. 2, the test sector may contain a port for sample entry 14. The sample port is preferably sealable so that the disc is effectively sealed except for ventilation, to allow fluid flow, to protect it from any biological events. By various means, for example, centrifugal force and the like, which are well known in the art, a portion of the sample is measured at a sample preparation site 15, which may contain reactants and the like to perform the test. Alternatively or together with the reactants already present in the sample preparation segment, a series of reactants 16 may be provided to deliver, if necessary, the necessary reactants in the order corresponding to the sample preparation segment. Additional details of the chain of reactants are shown in fig. 9. It may be necessary to separate the analyte from the sample, at least partially, and this can be done in a generally designated segment of the sample separation 17. A battery 18 is provided, if electricity is required for the separation process. Additional details of the battery are shown in fig. 5, as described below. The resulting sample is then transferred to the test site 19. In a preferred embodiment of the invention, the test site contains a test element as described in more detail below. The analyte is linked to a predetermined location on the disk, if present in the sample, and the presence of the analyte is detected by the reader, from the information that identifies the particular analyte with the location to which it is linked. A residue compartment is also provided to collect the surplus of reagents or sample which exceeds the quantities measured for use in the test and the various compartments and channels for fluid transfer are properly ventilated to allow fluid to flow through the surface of the test area.

In one embodiment of the invention, a plurality of test sectors 21, 22, 23 can be provided. as shown in FIG. 3, each sector being connected to an individual port for sample entry 24.25, respectively 26. The operation of each sector is similar to the one described above, although different tests can be conducted at the same time in individual sectors for a variety of analyzes. or for a multitude of patients. The details of a specific sector are shown in fig. 4, where the various possible components are identified by the same numbers as used herein.

As shown in FIG. 5, a battery can be provided that consists simply of two metal layers, such as copper and zinc, which are in the lower half and respectively in the upper half. During storage they are separated by air. When the disc is rotated, the space between these two metals is filled with dilute mineral acid, depending on the nature of the metal electrodes. In the case of copper and zinc, it can be dilute sulfuric acid, which contains copper ions and the battery is activated. This battery only generates a 1.5V voltage for one hour. However, it is more than necessary to carry out this analysis. Longer batteries can be manufactured, if necessary, from other materials or more layers

RO 119751 Β1 thick metal. It is important that if the water is allowed to flow through the space between 1 metal layers, the battery is deactivated. The on and off cycle can be repeated several times. Several batteries can be connected in series to increase the power, if necessary. Optionally, photodiodes may be included in the circuit. In this case, the computer that controls the test is provided with information about the active circuits. Also, a prefabricated miniaturized battery can be used and activated by closing the electrical circuit with a salt, for example, sodium chloride, solution. 7

Capillaries are used to transfer liquid or air. Also, very small volumes of liquid can be stored in the capillaries. Preferably, the air capillaries are hydrophobic, while the capillaries that come in contact with the water are hydrophilic. If necessary, the capillaries may have cross-sections, circular or rectangular. Typical depths are between 10 pm and 500 pm, 11 while the widths are between 50 pm and 2 mm. The air capillaries use the largest dimensions to prevent the formation of a pressure gradient, unless this is the case. The speed of flow depends on the frequency of rotation of the BCDI, the size of the capillaries and the density of the liquid. The physical properties of the liquid are dictated by the test and the frequency of rotation is limited to a certain extent by the CD-ROM or DVD player. Thus, capillary dimensions are used to adjust the speed of fluid transfer. The 17 capillary pipes may have glass necks, that is to say, some drips in the cross section of the capillary, to control the speed of the liquid as needed. Hydrophilia and hydrophobia can be used for the same purpose.

The exact dimensions of the capillary network and the chambers can be given by the Navier 21 - Stokes equation:

pv = pb - Δρ + μΔ²ν, 23 where p is density, p is pressure, speed is reported, b is the force field of the body, μ 25 is viscosity and Δ is the delta differential operator (Mass, Continuum Mechanics, McGraw Hill, 1970). Pressure is a scalar field, while v and b are vector fields. Software for commercial computers is available to solve the Navier-Stokes equation in complicated geometries. 29

Containers or compartments formed on the disk are used for the introduction of the sample, for the storage of reactants, for carrying out reactions and for collecting waste. Their depth is about 1 - 2000 µm, preferably about 10 - 800 µm, and they can be of any shape, although those with a circular cross-section or rectangle are preferred. The compartments are hydrophilic, except for one end of the waste container that has an air capillary that is hydrophobic. The reaction compartments can be formed with 35 electrodes for heating, electroconvection for electrochemical purposes. Electrodes are preferably evaporated gold films. The compartments may also have valves that open 37 by electricity or chemically as described below.

The storage containers may be covered with metal, preferably with gold, to prevent the ingress of water into the plastic. Reagents can also be prepackaged in boxes, which are, in principle, impermeable. These cassettes can be closed during storage and opened manually, 41 by pricking or opening a valve or buffer, when the sample box is placed on the disk. The opening of the cassette can also be facilitated by the centrifugal force when the BCDI starts to rotate. In any case, the proper flow of liquid is maintained during the test, by computer control, via a CD or DVD player. 45

Fluid leakage during the test can be monitored by using a reflective element. The reflective element uses the laser that is in the CD or DVD player and even then 47

EN 119751 ,1 when the liquid is transparent, its reflection index is significantly different from that of air. Thus, the laser light is reflected back to the CD or DVD player in the presence of air and in another direction, in the presence of the liquid, or vice versa. Another method of monitoring fluid leakage is to use an active light source, such as an LED or a semiconductor laser. Such light can be powered by the presence of an electrically conductive liquid, such as a plasma or a buffer, which acts to close an electronic circuit.

An LC screen can be used to transmit information from the BCDI to the CD or DVD driver and to the computer. The LC screen can have a large number of pixels that reflect light, when there is potential on the LC film. These pixels can, for example, be organized linearly, so that at one end a small potential is needed for light reflection, while at the other end the potential must be much greater for the same result. A CD or DVD driver is able to locate the reflective pixels and consequently the potential in the circuit can be measured. The potential change may be due to an electrochemical process in one of the electrochemical cells. For example, an electrode covered with cholesterol oxidase generates hydrogen peroxide in the presence of cholesterol. Hydrogen peroxide changes the potential of the circuit and cholesterol can be quantitatively determined.

Filters can be used to remove large particles such as cells, dust, etc. from the soluble sample. Accordingly, the filters are preferably included as part of the sample inlet. The filters can be made of porous plastic, glass, cotton or cellulose, cross-linked etc. These materials may be in the form of a plug or similar forms, depending on the particular use they are given. As films, plastics such as Teflon can be used.

Because chaotropic agents are often used to denature the oligonucleotides, during the preparation of the sample, it is advantageous to provide a method of dialysis on the disc to remove the salt before the analysis takes place. As shown in FIG. 6, a dialysis unit is prepared by placing a dialysis membrane 27, either on one or both halves (up and down) of a disc compartment 10. Considering the small volumes, the buffer that is already inside the dialysis membrane is usually sufficient and, usually, no buffer is needed on the membrane side opposite the fluid layer.

A column can be prepared, as shown in FIG. 7, by filling a compartment 28 with a desired gel, adsorbent or ion exchanger, for example, silica gel, Sephadex etc (each material is chosen depending on the particular application for which it is used) and placing on the other end of a filter 29 Examples of possible uses include separation of larger molecules from smaller ones and fractionation of hydrophilic and hydrophobic compounds. An ion exchanger column is particularly useful for separating nucleic acids from other biomolecules. The columns may also have other uses that may be convenient or necessary for the conduct of any particular test.

Fig. 8 illustrates a valve, generally designated 30, which can be located at one of the ends of a column or reaction vessel, which has two outlet capillaries 31 and 32. In addition, there are two electrodes, 33 and 34, which initially a conductive metal foil 35, which is adapted to close one or the other of the capillaries, according to its position relative to each capillary, is not placed in the illustrated position. The metal foil is determined to close one of the capillaries when no current passes and acts to open the closed capillary earlier and close the other capillary when the current passes. For example, the valve is made of a thin sheet of gold, which is mechanically pressed against the other outlet capillary and is electrically connected to the nearest electrode. When the battery is activated, the gold foil is rejected by the nearest electrode and is drawn by the other electrode. As a result, the gold foil is pressed on the other outlet. Other metal foils can be used

EN 119751 Β1 conductor of electric current, but for most operations, a metal is preferred which 1 is a good conductor and which is not corrosive. The battery can be deactivated, as explained earlier and the valve is then again in its original position. 3

The laser in the CD - R or CD - RW drivers has an energy of up to 10mW that can heat objects up to high temperatures, up to 600'C. The energy is large enough to make several holes in several materials, including plastic. The plastic must contain a paint that absorbs the laser light. Thermal expansion can be used for valve reversal. For example, bending of bimetallic foils is extremely temperature sensitive. 9

A piezoelectric material can be used as a valve. Piezoelectricity can also be used to measure extremely small volumes of liquids, for example, sample nanoliths 11 can be divided between different tests.

Valve-type operations may also occur chemically, by depositing 13 of a solution of a solid chemical compound and / or dissolving a solid compound, deposited. The first outlet of such a valve is closed by depositing a chemical compound inside the capillary. The compound may be, for example, silver chloride. Chloride ions can be in the main stream of fluid, while in separate side capillaries there is pure water and silver nitrate 17 in water. The side capillaries are configured so that, first, water is added and then silver nitrate is added to the main fluid stream, which contains chloride. When silver ions 19 reach the intersection, it stops, effectively acting as a closed valve. Alternatively, a capillary may first be blocked by the solid form of a soluble compound, such as sodium chloride. Addition of any aqueous solution dissolves the sodium chloride plug and the capillary opens. 2. 3

The test element is preferably used instead of the test of the present invention.

Briefly, the test element (Fig. 13) includes a sliding spacer 61 covalently attached with one end 60 on the surface of the disc 59 and the other end 62 on a reporting element 65. Preferred constructions of the reporting element described here include reflective gold spheres or 27 latex spheres of latex. Also included are two recognition elements 63a, 63b, hereinafter referred to as side arms that are covalently attached to either spacer 29 so that each side arm is connected to each side of the seat. Spacer splitting 64. Preferred embodiments of the side arms described herein include oligonucleotides, 31 cleotides, antibodies, and antibody-conjugated oligonucleotides. The test elements can be used to detect the presence of an analyte and to create a signal thereof through manifestation 33 of positive or negative recognition (fig. 14). A poignant event of recognition (fig. 14

A, C and E) occurs when an analyte 66 binds to both side arms 63a, 63b which 35 results in the completion of a connecting arc 67 between the two sides of the spacer bisected by the splitting site 64. A recognition event a negative (Fig. 14B, D and F) appears 37 when the analyte 66 binds only to one or to none of the side arms 68a, 68b and consequently no arc is made that connects the two parts of the spacer. 39 When a positive recognition event is followed by the splitting of the spacers, a disc connection remains with the reporter element (fig. 14E). On the other hand, the splitting of the distances from a test element followed by a negative recognition event results in reporter elements disconnecting from the disk (Fig. 14F). Thus, negative recognition results in the loss of rapporteur elements, which is easily removed by washing, while positive recognition results in rapporteur elements, which are retained in their 45 discrete test sectors. In each case, the results can be observed immediately through the CD - ROM or DVD player. 47

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Other embodiments of the invention are described herein, using both reflection and opaque reporter molecules, and positive and / or negative recognition events to perform a large number of possible assays. For example, in some tests the side arms can be connected before adding a sample and binding the analyte leads to the disconnection of the side arms, in this case, the positive recognition event results in the disappearance of the reporter element, while a negative recognition event results in retaining the rapporteur element.

Other possible variants of the test element described here do not include split arms with side arms. In such an alternative scheme, the surface of the BCDI is covered with metal, preferably gold, and the analyte connects the opaque particles, such as latex pearls, or liposomes loaded with paint, onto the metal surface.

The previous test elements are based on the binding of reflective particles on the transparent surface of the BCDI. The situation can be reversed so that the opaque particles bind to the reflective surface. This approach is particularly advantageous when subjected to large cell testing and is generally illustrated in FIG. 12.

On the plastic surface, a metal film is stored. The information can be encoded in the metal layer and can be made on conventional CDs. This information may include spatial addresses or other test related information. The metal layer is then covered with a plastic layer. It is then thinned, as described above, and instead of the gold spheres on the substrate, large latex spheres 58 (diameter 10-50 pm) are attached, which contain a paint, with the help of spacer molecules such as described above. These latex spheres are partially coated with recognition molecules, as described above, for gold spheres. The recognition cells attach the latex spheres to the substrate, even after the spheres are split and the paint from the spheres prevents the reflection of the laser light from the metal layer. Alternatively, if a suitable fluorescent dye and a corresponding laser wavelength are used, the fluorescence emission of the spheres can be used to monitor the test. For this, a specialized tool is needed and will be facilitated by blue lasers, when they can be used for CD - ROM or DVD players.

In the simplest version of the cell detection assay, latex spheres do not connect to the BCDI before the test, but are added after the cells bind to the BCDI. The suspension of latex spheres is added, the recognition molecules on the sphere bind to the corresponding cells and these cells are immobilized. These latex spheres can then be observed through reduced reflection, using CD - ROM or DVD players.

A disadvantage of the covalent bonding of the spacers is that the disk does not regenerate easily after the spacers split. If instead the spacers are connected to the substrate with complementary oligonucleotides, the disc can be regenerated after the completion of a test. The spacers or their residues are removed by heating or by the use of chaotropic agents. Duplexes connecting the distorted spacers and the disk can be cleaned. The disk holds the oligonucleotides that were linked to the old spacer. All oligonucleotides in a test area are identical. They may be different in different test areas, or they may be identical throughout the BCDI. New spacers are added that have complementary oligonucleotides on the BCDI. After incubation, the complementary oligonucleotides of the spacer and BCDI hybridize. Excess spacing is removed by washing. In this case the lateral arms of the oligonucleotides can be attached to the spacer, before the spacers are attached to the surface. Then add gold spheres, bind through thiol groups or disulfide bridges of the spacers, and the disc is ready for reuse.

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For the spectrophotometric, fluorescence or UV / Vis 1 chemiluminescence tests, a small tank is used. A small bin on BCDI is essentially a capillary that is located between a light source and a photodetector. The light can be guided through 3-wave mirrors and guides. The number of small tanks on the BCDI ranges from 0-10 000 and the most advantageous between 0-50 / test sector. In most small tanks, the sample reaches through a sample preparation chamber. These chambers may contain preloaded reagents or the reagents are stored in separate chambers and mixed with the sample, while it reaches the sample preparation chamber. The sample and the reactants can be heated electrically by the infrared radiation that is generated through a photodiode. After the incubation period, the sample is transferred 9 to the small tank. The light transmitted or emitted is measured by a photodetector. In this invention, the photodetector is more advantageously in CD or DVD drivers. 11

The light sources for spectrophotometric tests are advantageously photodiodes or semiconductor lasers. It is possible to use the light source of the CD or DVD driver. 13

However, these instruments usually use a single wavelength, which corresponds to red or infrared light. If an internal light source of the CD or 15 DVD driver is used, the photodiode or laser in fig. 15 is replaced by a mirror. Although multiple tests can be performed using red or infrared light, it is advantageous for most applications to use additional light sources. For example, a range of photodiodes can be manufactured, which can generate red, yellow, green and blue light. It is possible to design a photodiode 19 for any given wavelength and, consequently, the number of photodiodes can be up to 300, to cover the entire UV / visible spectrum range. The laser generates more energy 21 and focuses better than photodiodes and is preferred. Especially microcavity lasers and nano-point lasers are very small and can be manufactured to emit almost any 23 wavelength. Light sources can be manufactured as a module that can be attached to the disk before and can be removed after using the BCDI. 25 Below, the operations performed by the unit are described: centrifugation, filtration, liquid transfer, liquid mixing, dialysis, column separation, heating, cooling, electro-convection and electrophoresis.

The centrifugal force is the main force used for the transfer of liquids to the BCDI. It can also be used for centrifugation, which is important when the cells separate from the plasma. In this case, it is advantageous to include a filter in the sample taking container.

When transferring liquids, order and timing are very important. In order to provide a proper sequence of arrival at a certain reaction site, liquid carriers can be created as shown in FIG. 9. In one embodiment, two main capillaries, 36 and 37 are provided, which are in fluid communication with each other through the connecting capillaries 38, 39 and 40. One of the main capillaries is an air duct that allows fluid to flow 37 and is typically hydrophobic. The other main channel carries liquid-reactants and is usually hydrophilic. The connecting capillaries and associated cavities may serve for the storage of reactants, generally referred to as 41, 42 and 43.

The fluid compartment, to which the timing of delivery is directed, is controlled by their locations, capillary dimensions, fluid density and viscosity and the speed at which the disc rotates. The liquids are separated by small air bubbles to prevent mixing, unless mixing is desired. To prevent pressure gradients, the air capillaries are connected upstream to all liquid capillaries. To further prevent the entry of liquids into the air capillaries, they are hydrophobic.

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Mixing two solutions is done by joining two capillaries in a Y-shaped formation, so only a good mixing can be done. To ensure more efficient mixing, a capillary may undergo small periodic enlargements after joining. It should be noted that the rotation of the BCDI results in efficient mixing in containers.

In dialysis, the fluid is in contact with the membrane containing the buffer. The molecular weight of the membrane can be chosen to be between 300 - 500 000 Daltons. Because only a very thin layer of fluid is in contact with the dialysis membrane, dialysis is very fast. However, the ratio of fluid to buffer is only between 1:10 and 1: 100, so dialysis is not quantitative. For most purposes it is enough.

Adsorption, gel and ion exchange chromatographs are all possible. The different molecular species are fractionated by the chromatography medium and leave the capillary separately as in conventional chromatography. Using a valve, certain fractions can be selected and guided to a test element.

heating is best done by electric means. The upper and lower electrodes are separated by approximately 500 pm. If the solution contains ions, the system is, in principle, short-circuited and heated. The heating can be completed by removing the ions either from the battery or from the container. A constant temperature can be obtained by including a thermostat in the circuit. A bimetallic element is a very simple thermostat, which can close a circuit under a preset temperature and open it at a higher temperature. Another heating mechanism is provided by the laser of the CD or DVD driver, in particular, the CD - R drivers with powerful lasers. Either the top or bottom of the cavity may have a crystalline liquid film that is insulated through a transparent layer if necessary. On the other side of the cavity is a reflective layer. When the cavity temperature is below the main transition temperature the liquid crystal spreads the light and no reflection is observed. Above the main transition temperature, the light is reflected back and the heating can be switched off and is still less efficient. Cooling is preferably done by endothermic dissolution, that is, the absorption of heat by the presence of a dissolving substance. The cooling solution and the solution to be cooled must be separated by a thin film of aluminum, copper, silver or gold. Cooling can also be done by passively cooling the air. This method only cools down to room temperature, but in most cases this is sufficient. Cooling and heating can also be cyclically alternated, either in a cavity or in an alternate series of cooling and heating steps of the cavities. This allows PCR amplifications to take place within the BCDI.

Electroconvection, electrophoresis and isoelectric convergence can each be used in particular applications. In electroconvection the material is transferred, without trying to separate it into components. In electrophoresis, separation is the main purpose. The separation is facilitated by the use of a gel that prevents convection. Because the distances are very short, the available field strength is sufficient for the appropriate electrophoresis. For the same reason, the time required for separation is quite short and can be 1-5 minutes, or even less than one minute. Useful electroconvection can be done in seconds. The isoelectric convergence is, in fact, electrophoresis with a pH gradient. A pH gradient can be created by a parallel capillary mass, each of which contains a different buffer, so that the pH changes gradually. This is shown in FIG. 16. Much of the buffer remains in the capillary and this guarantees the existence of the pH gradient during isoelectric convergence. After the convergence is complete, the components can be moved along the capillaries by centrifugal force or orthogonal electrophoresis can be performed. This method allows complete fractionation of human plasma proteins (Anderson, Tracy and Anderson, The Plasma Proteins, 2^when Ed., Vol. 4, Academic Press, Inc., 1984).

RO 119751 Β1

A particularly advantageous configuration of an analysis area is illustrated in FIG. 10. 1

The analysis element contains spacer molecules and spheres of reflection, as described above, but does so in a linear arrangement, which can conveniently be located in 3 one or more capillary channels at the site of analysis. on disk. As described, the analyte binds to the distal cells, which have lateral arms receptive to or complementary to the analyte (as illustrated in A) and after washing the analyte that has been bound is located in specific locations of the arrangement (such as is illustrated in B). The presence of linked analytes is determined 7 by the conventional determination of the address, such as with conventional compact disc readers and the associated software as described. 9

Example 1. The analysis sector for oligonucleotide analysis is shown in FIG. 2.0 sample containing DNA is mixed with sodium dodecyl sulfate to lysate the cells. 11 This solution is transferred to the container called Sample Input and the disk is rotated. The sample is filtered and mixed with a complementary oligonucleotide mixture. These 13 nucleotide oligos are complementary to those to be analyzed and also have a thiol group at one end. Hybridization is allowed to take place in the container called Test preparation. Optionally, this container can be heated (not shown). After proper incubation, the disc is rotated. While the sample is transferred to the container called Sample Separation, it is mixed with a nuclease solution S delivered through a side capillary. The mixture is left to incubate in the container. Separation of the sample, which has two gold electrodes and a valve, as shown in FIG. 8. The lower electrode is covered with spacers that have final isothiocyanate groups. They bind to thiol-containing oligonucleotides, some of which are hybridized with the sample.

All non-hybridized DNA parts are digested and removed by washing. The battery of 23 comes then operational. This can be controlled by the speed at which the acid and copper ions enter the empty battery. The container is heated, the bound oligonucleotides are released and valve 25 is closed.

The oligonucleotides are introduced into the jet in the analysis area. After proper incubation, the ligase arrives in the test area and the two lateral arms of the spacer molecule are connected, if the sample comprises the corresponding oligonucleotides. The sensitive spacers are 29 cut. If they contain siloxane groups, the cutting is done by adding fluorine ions. The free gold spheres are removed by washing, by rotating the BCDI at high speed. The reading can be done immediately.

Example 2. Alternative variants of the assay element described in this paper 33 are useful for detecting viral and bacterial particles, M cells, and other particles that are larger than the oligonucleotides, antibodies, antigens and the like, which have been described above. . Viruses are usually near-spherical particles with a diameter of less than 0.5 µm. Bacteria are usually either spherical or plug-shaped. Their largest size is less than 2 pm, except for flagella and other similar outer fibers. These pathogens are smaller or have about the same size as the gold spheres used, to detect them and their interaction with two lateral arms of the spacer may be limited. For this reason, these side arms connect to the surface of the BCDI and the golden sphere, instead of 41 connecting with the spacer as shown in fig. 11. The gold sphere is attached to a spacer molecule 45 at one end of the spacer molecule and the other end of the spacer 43 is attached to the surface of the substrate 46. The spacer molecule is provided with a typical cleavage site 47, for example a siloxane radical, such as has been described above. In contrast to the 45 variants described above where the side arms are attached to the spacer molecules between the substrate and the cleavage site, the side arms are attached to the gold spheres and to the surface 47

RO 119751 Β1 substrate. For illustration, in FIG. 11, the oligonucleotides 48 are attached to the surface of the substrate and the oligonucleotides 47 are attached to the surface of the gold sphere. Then the complementary oligonucleotides are conjugated with members of the specific binding pair, named 50 and 51, are attached to the oligonucleotides on the substrate and to the gold sphere as illustrated. This leaves much more room for cells to bind to antibodies or other recognition molecules. For a discussion of the cleavable sequence, see publication WO 98/01533, the disclosure of which is included herein by reference.

The spacers each have at least one splitting place. They are, from all points of view, identical to those described above, except that they do not attach molecules with side arms. When the cell, for example, reaches the site of analysis, if it contains radicals that form specific binding pairs with their respective complementary members, an arc of connection between the gold sphere and the substrate is formed. When the spacer molecule is cleaved, the gold sphere is retained on the substrate and the presence of the cell can be detected as described above. However, if no specific binding pairs are formed, after splitting the spacer, the gold sphere does not remain attached to the substrate and is removed.

Antibodies or other recognition molecules can be attached to the substrate in a manner similar to that in which the spacers are attached. All the distances on the BCDI are identical and are attached at the same time to the amino groups or analogous active groups on the surface. About half of the amino groups are used for attaching the spacer. The other half is used to couple the recognition molecules to the substrate. If all the recognition molecules on the BCDI surface are similar, they can be attached at the same time as spacers. Alternatively, if the recognition molecules are specific to each analysis site, they can be distributed locally by contact printing, inkjet printing or microcapillary deposition.

After the gold spheres are attached to the thiol groups of the spacers, the other recognition molecules are also attached, with the help of the thiol groups, to the gold spheres. For this purpose, these recognition molecules are first conjugated to a spacer containing a protected thiol or amino group. The amino group can be derivatized so that a thiol group is introduced. The different recognition molecules that need to be attached to the gold spheres are distributed in a similar way to that in which the other recognition molecules attach to the surface of the BCDI.

The recognition molecules can be oligonucleotides. These oligonucleotides can be further hybridized with complementary oligonucleotide conjugates - biomolecules. This approach allows the attachment of sensitive and reactive biomolecules, for example, proteins containing several amino or thiol groups.

The recognition molecules linked to the gold spheres are free to diffuse around the sphere even though they are closely linked. The cell that is recognized by both recognition molecules completes a connection loop that links the gold sphere to the surface of the BCDI. After the spacer splits, the gold sphere is retained and detected by the CD - ROM or DVD player.

A variety of different recognition molecules can be used from the same analysis site. The advantage of this approach is that all known mutants of a particular species of pathogens can be detected at one analysis site. Different mutants can also be characterized at different test sites containing specific recognition molecules.

The BCDI is a universal analyzer. It is easy to use and in its most advanced form, it contains all the reactants and only the sample is added. It can be used in clinical laboratories, hospitals, doctors' offices and at home. When used at home, the information can be transmitted to

EN 119751 Β1 a doctor's office via the internet. The BCDI can be created in such a way that the genetic signature of each patient is measured every time. About 35 points of polymorphism are enough to give each person a unique bar code. This 3 eliminates possible mistakes due to mixing tubes or labels. The assays that can be done include, but are not limited to immunoassays, DNA testing, cell counting and cell shape measurement, cancer cell detection in tissue samples, blood chemistry and electrolyte analysis. Other applications include mass testing of candidates for 7 drugs, analyzing food and environmental safety, and monitoring pathogens and toxins in a battlefield. 9

Example 3. This example presents a turbidimetric test of lipase activity. The reagent cavity contains 15 µl of emulsion of 250 μΜ of stabilized triolein, which contains 30 mM of 11 sodium deoxycholate and 100 μΜ of CaCl₂ at pH 9.0 in 25 mM TRIS buffer. The sample preparation chamber contains 0.5 µg of lyophilized porcine colipase. Two microliters of serum are taken into the sample preparation chamber (using the apparatus as shown in Fig. 17) together with stabilized triolein and other reagents. Part of the 5 μΙ mixture is further transferred into a small bowl 15. As the output capillary goes toward the center of the disc, the back pressure prevents the flow from continuing. The absorbance at 340 nm is read at intervals of 1 min. ΔΑ / min 17 is a measure of lipase activity.

While this invention is described, with respect to some specific variants, it is understood that modifications thereof and equivalents as well as variants thereof are obvious to one skilled in the art and are included in the scope of the appended claims. 21