CZ306299A3 - Optical disk - Google Patents

Abstract

The device is made up of an optical disc for reading the optical reader includes a solid sector separate test means for determining the monitored The value of the analyzed sample control means for performing the test and location information of the observed values in the analyzed sample, with this sector is accessible by reading devices where presence or the absence of the observed value in this area can be determined reading equipment using control means and information location. Depending on the nature of the test, the disc may contain fluid storage means, transfer means liquids, one or more capillary channels, valves, batteries, dialysis units, columns, filters, sources electric fields, conductors or other electrical interconnecting means such as metal coatings surface, etc.

Description

Technical field

The invention relates generally to diagnostic tests and methods for carrying them out. Specifically, it relates to diagnostic test components configured on a compact optical disc and methods of using them.

BACKGROUND OF THE INVENTION

There is still a considerable need to accelerate clinical trials and make them cheaper and easier to perform. Ideally, patients should be able to perform these tests themselves if necessary. One way to achieve this was to miniaturize and integrate various test operations. At present, a number of biochip-based tests are commercially available or under development (some are called because some of them are produced by photolithographic methods on silicon chips. In all of these approaches, a reader and computer are required).

Disc-shaped cassettes used for clinical testing in conjunction with ultraviolet and visible spectroscopy are also available. U.S. Pat. No. 5,122,284 discloses a centrifuge rotor comprising a plurality of interconnected liquid chambers connected to a plurality of cuvettes. The rotor is adapted to be usable in conjunction with a conventional laboratory centrifuge and is made of materials that allow photometric detection of test results performed in reaction cuvettes. A large number of rotor configurations and related devices have been described for the same or similar types of assays, 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;

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5,591,643; 5,186,844; 5,122,284; 5,242,606; and patents cited therein. Lyophilized agents useful in these systems are described in US Patent 5,413,732.

The principles of the centrifugal analyzer have been adapted to a disc that can be used in a CD-like device (Mian et al., WO 97/21090). Mian describes a modified dual-function CD (CD-drive) drive: 1. used to read information stored on the disc, and 2. used to rotate the disc. However, the use of the CD reading capability for analysis and actual tests is not mentioned in this source.

Regardless of recent progress, there remains a need for a simpler test configuration that performs tests quickly, efficiently, accurately and at low cost. The present invention combines diagnostic tests with computer technology and compact disc technology. In a most preferred embodiment, the only necessary device is a computer with a CD reader. All chemical reactions are performed inside a compact disc, which may be referred to as an integrated biocompact disc (IBCD). Software is also encoded on the same CD.

machine-readable instruction and control information that provides instructions to the computer before, during, and after the test.

CDs or DVDs are the most economical and in many ways the best media for information storage. It should be noted that CDs and DVDs are commonly used abbreviations that may change in the future, even if the underlying technology used remains essentially the same. The CD or DVD drive is equivalent in many respects to a scanning confocal microscope. At the same time, these devices are comparable to good centrifuges because in commercial drives the rotation speed is between 200 and

12,000 rpm and can be adjusted within a certain range. Combining these three properties into the same analytical system leads to greater

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-3simplification compared to any other analytical technique. However, performance is comparable or better than most competing methods. Although the present invention requires slightly modified CD or DVD drives, it is possible to introduce these changes into commercial drives.

This will allow Point-Of-Patient-Care (POPC) care and home use of the invention. The use of CD or DVD drives enables accurate digital analysis of any sample without any specific analytical equipment.

SUMMARY OF THE INVENTION

One embodiment of the invention is directed to an optical disc that is adapted to be read by an optical reading device that comprises a first sector comprising substantially self-sustaining analyte binding means believed to be present in the sample to at least one predetermined region in the first sector, and optionally a second sector comprising assay control means and analyte location information relating to one or more analytes that are believed to be present in a sample accessible to the reader, the presence or absence of the analyte in said sector The location may be determined by the reader by means of control means and location information. Depending on the nature of the assay, the disc may include fluid storage means, fluid transfer means such as one or more capillary channels, valves, batteries, dialysis devices, columns, filters, electric field sources, conductors, or other electrical conduction means such as metals stored on the surface, etc.

The disc may include one or more sample ports for delivering a liquid sample to the test area. These inputs, if present, are preferably closable so that upon application of the sample onto the disc, a closed disc, including the sample, forms a hermetically sealed element.

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99 · 99 999 • 9 · 9 which can be conveniently handled by conventional methods or other mechanisms for handling bio-waste. The test sector on the disk is also suitably divided into different sub-compartments for sample preparation and analyte separation. It is also possible to conveniently include a waste collection sub-compartment. The test sector can be divided into a number of subdivisions, each of which receives a sample. Each such subdivision may analyze one or more analytes depending on the particular application used.

Another embodiment of the invention is directed to a test apparatus comprising an optical disc, a disc reader, and an information processor, wherein the disc comprises a first sector comprising substantially separate test means for locating an analyte that is believed to be present in the sample to at least one predetermined area in the first sector, and optionally a second sector containing control information for carrying out the assay and analyte location information relating to one or more analytes which are expected to be present in the sample, accessible to the reader and processable by the information processor, wherein the disc is adapted to be read by a reader and the information processor is adapted to determine the presence or absence of the analyte at said location using control and location information. The apparatus may comprise a reader equipped with a CD-ROM or DVD reader and an information processor, which may be a personal computer.

Another object of the invention is an optical disc that is adapted to be read by a CD-ROM or DVD reader comprising substantially separate test means on an analyte localization disc that is believed to be present in the sample for at least one predetermined area on the a disc and, in said area, means for detecting the presence or absence of the analyte by a CD-ROM or DVD reader.

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DETAILED DESCRIPTION OF THE INVENTION

An overall schematic representation of the integrated biocompact disc (IBCD) is shown in Figure 1. The disc (biocompact disc, BCD) can be of any shape and size. For most practical applications, it has a circular shape with a diameter of 10 to 1000 mm, most preferably 20 to 200 mm and a thickness of 0.1 to 20 mm, most preferably 0.5 to 3 mm. The disc 10 comprises two sectors: a test area H and a software area 12. A hole 13 is provided in the center for placement in the CD reader. The test management software may be on a separate disk. However, it is preferred that the software be on disk together with a test for a particular analyte or analytes to minimize the opportunity for human error in the tests. Possible components and unit operations on the IBCD are described below. The disc rotates in conventional CD-ROM or DVD readers typically at a speed of up to 16,000 rpm. In all CD-ROM and DVD readers, the speed is adjustable within certain limits (200 to 16,000 rpm. However, for some operations it may be advantageous to use different rotational speeds, for example 1000 to 10,000 rpm and most preferably 2000 to 5000 rpm. For any particular test, the control software determines the mode of rotation during analysis The rate and timing mode, including times at which rotation is not allowed to allow incubation, electrophoresis, isoelectric focusing, etc., is controlled to deliver reagents and sample to the appropriate assay. The available rotation speeds allow significant centrifugal force to be used to move liquids, and another energy source that can easily be used in IBCDs is chemical energy. in the form of electric en Mechanical and chemical energy allow the operation of many types of equipment Important components of IBCDs may contain one or more of the following components. Capillaries, reservoirs, filters, dialysis membranes, chromatographic columns, electrophoretic gels, valves, any micromechanical or electronic components including microprocessors, electrodes, in particular

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000 000 • 0 0 · 0 000 enzymatic electrodes, cells and test elements. These components allow the following individual operations to be performed: centrifugation, filtration, liquid transfer, liquid mixing, dialysis, column separation, heating, cooling, electro-convection, electrophoresis and analyte detection and indication.

The IBCD is conveniently made of two pieces containing upper and lower halves. The lower half may comprise almost all of the components, while the upper half may be a flat housing containing only a small number of components, such as electrodes and conductors. The number of layers 10 of the present invention may be more than two, and many components may be pre-fabricated as modules. In particular, reagent containers, cuvette assemblies, columns, micromechanical components, light sources and microprocessors are preferably assembled in the form of modules. The various elements can be molded into soft plastic. The various components can be glued either by heat treatment or UV-curing, melted, bonded to complementary mechanical elements, mechanically fastened or simply sealed into a larger component. For example, some regions may be treated with ammoniacal plasma to render them hydrophilic. The surface may further be treated with various molecules that render the surface inert or alternatively impart specific absorption properties to the surface. The general method for treating surfaces is silylation (Virtanen, J.A., Kinnunen, P.

K.J. and Kulo, A., 'Organositanes and their hydrolytic polymers as surface treatment agents for use in chromatography and electronics',

USP 4,756,971). Covalent binding of detergents will reduce the adsorption of proteins such as albumin and will also reduce the adsorption of soluble proteins. The metal electrodes and conductors can be steamed to the desired areas. Masks or resists can be used to localize plasma treatment or metal deposition. Capillary channels and fluid storage and containment spaces may be made on optical discs or formed by chemical means or injection molding. As shown in Fig. 2, the test sector may comprise a " 4 " " 4 " 444 " 444 " ·· 4 · ··

The sample inlet aperture 14. The sample inlet aperture may preferably be closed so that the disc is effectively closed except for the necessary ventilation to allow fluid to flow to protect against any biological hazard. By means of various methods, for example centrifugation force and similar means known in the art, a portion of the sample is metered into the sample preparation site 15, which may contain reagents and the like to perform the assay. Alternatively or together with reagents already present in the sample preparation compartment, a reagent reservoir 16 may be present which, as necessary, dispenses the necessary reagents in the correct order into the sample preparation compartment. Further details of the reagent container are shown in Figure 9. It may be necessary to separate the analyte from the sample, at least in part, as may be done in the sample separation compartment, generally designated 17. If electrical energy is required for separation, 18. Further details of the battery structure are shown in Fig. 5 and will be described below. The resulting sample is then transferred to a test site 19. In a preferred embodiment of the invention, the test site comprises a test element as described in more detail below. The analyte binds, if present in the sample, to a predetermined location on the disk, and the presence of the analyte is detected by a reader from information that identifies a particular analyte by the location to which it is bound. The space designated waste is designed to collect excess reagents or sample that exceeds the measured amounts for use in the test, and the various compartments and fluid transfer channels are vented to allow liquid to flow through the test sector surface.

In one embodiment of the invention, a plurality of test sectors 21, 22, 23, etc. may be present, as shown in Fig. 3, each sector being connected to an individual sample inlet port 24, 25 or 26. Each sector operates substantially as described above, although different assays may be performed in different sectors, either for multiple analytes or multiple patients simultaneously. Sector-specific details are shown in more detail in Figure 4,

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Component

As shown in Fig. 5, a battery may be present which is simply composed of two metal layers such as copper and zinc, which are in the lower and upper half, respectively. They are separated by air during storage. When the disc is rotated, the space between the 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 diluted sulfuric acid, which contains copper ions and activates the battery. This battery produces a voltage of 1.5 V for only about one hour. However, this is more than enough to complete the analysis. Longer-lasting batteries can be made from other materials or thicker metal layers if needed. It is important that water inflow into the space between the metal layers deactivates the battery. The activation and deactivation cycle can be repeated several times. If necessary, it is possible to group several batteries in series to increase the voltage. For example, photodiodes can be included in the circuit. In this case, the computer controlling the test run receives information about the active circuits. It is also possible to use a miniaturized finished battery which is activated by closing the electrical circuit with a salt solution, for example sodium chloride.

Capillaries are preferably used for liquid and air transfer. Very small volumes of liquid can also be stored in capillaries.

Air-containing capillaries are preferably hydrophobic, while capillaries that come into contact with water are hydrophilic. If desired, the capillaries may have a circular or rectangular cross-section. Typical depths are between 10 µm and 500 µm, while the widths are between 50 µm and 2 mm. Air capillaries use larger diameters to prevent the formation of any pressure gradient unless otherwise required. The flow rate depends on the rotation speed of the IBCD, the dimensions

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9 · 9 99 · «capillaries and liquid viscosity and density. The physical properties of the liquid are determined by the test and the rotation speed is limited to a certain extent by a CD-ROM or DVD reader. Thus, the capillary dimensions are used to adjust the liquid transfer rate. The capillary ducts may be provided with constricted locations, i.e., a capillary cross-sectional restriction, to control fluid velocity as desired. Hydrophilicity and hydrophobicity can be used for the same purpose.

The exact dimensions of the capillary network and chambers can be designed using the Navier-Stokes equation:

io pv ~ pb - Vp + pV ² v where p is density, p is pressure, v is velocity, b is molecular force field, μ is viscosity and V is differential operator del (Mase, Continuum Mechanics, McGraw-Hill, 1970) . Pressure is a scalar field, while v and b are vector fields. For complicated geometric shapes, commercial computer software is available to solve the Navier-Stokes equation.

The cartridges or compartments formed on the disk are used for sample entry, reagent storage, reactions, and waste collection. Their depth is about 1 to 2000 µm, preferably about 10 to 800 µm, and can have any possible shape, although 20 circular or rectangular cross-sections are preferred. The compartments have a hydrophilic treatment in addition to one end of the waste container in which the air capillary is located, which is hydrophobic. Reaction compartments may be formed with electrodes for heating, electro-convection or electrochemical purposes. The electrodes are preferably steam-deposited gold films. The compartments may also be provided with valves which are controlled electrically or chemically as described below. The containers may be coated with a metal, preferably gold, to prevent water from penetrating into the plastic. The reagents may also be pre-packaged in cartridges that are actually impermeable. These cartridges may be closed during storage and opened manually by piercing or opening the valve or stopper when in place · 99 9 · · 9 9 · • 9 9 · 9 9 · * ··· 999

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- 10 sample cassettes in the disc. The cartridge can also be opened by centrifugal force when the IBCD starts to rotate. In any case, the appropriate fluid flow during the test is maintained by computer control using a CD or DVD reader.

The fluid flow rate of the test can be monitored using a reflector. The reflector uses a laser that is part of a CD or DVD reader and the fact that, although the liquid is transparent, its reflection index is significantly different from air. Thus, in the presence of air, the laser beam is reflected back into the CD or DVD reader and in the presence of the liquid it is reflected in another direction or vice versa. Another way to monitor fluid flow is to use an active light source such as an LED or semiconductor laser. Such a light source may be powered by the presence of an electrically conductive liquid, such as plasma or buffer, which causes the electrical circuit to close.

A liquid crystal display (LC) can be used to transfer information from the IBCD to a CD or DVD reader and to a computer. The LC display can have a large number of pixels that reflect light when it is embedded on the LC film potential. For example, these pixels can be organized in a straight line so that at one end a low potential for light reflection is needed, while at the other end the potential to achieve the same result must be substantially higher. CD or DVD drives are able to locate reflecting pixels and thus the potential in the circuit can be measured. The potential change may be caused by an electrochemical process in an electrochemical cell. For example, a cholesterol oxidase coated electrode will form hydrogen peroxide in the presence of cholesterol. Hydrogen peroxide alters the potential of the circuit and cholesterol can be quantified.

To remove large particles such as cells, dust, etc.

3o filters can be used from a soluble sample. Thus, filters are most preferably used as part of the inlet compartment. The filters can be »» · · · · * · · · · · · · · ·

11 made of porous plastic, glass, cross-linked cotton or cellulose, etc. These materials may be in the form of rods or similar shapes, depending on the particular application. Plastic films such as Teflon may be used as films.

Since chaotropic agents are often used to denature the oligonucleotides in sample preparation, it is preferable to provide dialysis means on the disk to remove salt prior to performing the assay. As shown in Fig. 6, the dialysis unit is made by inserting the dialysis membrane 27 into one or both halves (upper and lower) of the compartment formed on the disc 10. Taking into account the small volumes, usually the amount of buffer present inside the dialysis membrane is sufficient, and typically no buffer is needed on the membrane side opposite the liquid layer.

The column may be treated as shown in Fig. 7 by filling compartment 28 with the desired gel, adsorbent or ion exchange material such as silica gel, Sephadex and the like (the particular material being selected according to the application) and inserting the filter 29 into the other end. Examples of potential uses include separating smaller molecules from larger molecules and fractionating hydrophilic and hydrophobic compounds, and the ion exchange column is particularly useful for separating nucleic acids from other biomolecules. The columns are also suitable for other uses that may be suitable or necessary for performing a particular test.

Giant. 8 shows a valve, generally designated 30, that may be located at one end of a column or reaction vessel having two outlet capillaries 31 and 32. In addition, two electrodes 33 and 34 are present which are not initially charged in the indicated position, and a conductive metal foil 35 that is adapted to close one or the other capillary depending on the position of the foil relative to these capillaries. The metal foil is deflected to close one of the flowing streams without the flowing current. * * «* * * * Bez · bez bez bez bez bez bez bez bez bez bez bez bez bez ··· ··

12 of the capillaries, opening the previously closed capillary and closing the second capillary at current flow. As an example, the valve is made of a thin gold foil that is mechanically pushed against the second outlet capillary and electrically connected to the nearest electrode. When the battery is activated, the gold foil is repelled from the nearest electrode and snaps to the other electrode. As a result, the gold foil is pressed against the second exit. Other conductive metal foils may be used, but for most applications a metal that is conductive and does not corrode is preferred. The battery can be deactivated as explained above and the valve is then returned to its original position.

The CD-R or CD-RW laser has an output of up to 10 mW, which can heat objects up to high temperatures, even up to 600 ° C. The energy is strong enough to create openings in various materials including plastics. The plastic should contain a dye that absorbs laser light. Temperature expansion can be used for reversible valve closure. Extremely temperature sensitive are, for example, movements of bimetallic foils.

A piezoelectric material can be used as the valve. The piezoelectric effect can also be used to measure extremely small volumes of liquids, for example, nano-liter sample volumes can be divided between different tests.

Valve-like operations can be performed in a chemical manner by depositing a solid chemical compound from solution and / or dissolving the deposited solid compound. The first outlet of such a valve is closed by storing the chemical compound inside the capillary. For example, the compound may be silver chloride. Chloride ions may be present in the main liquid stream, while pure water and silver nitrate solution in water are present in separate secondary capillaries. The secondary capillaries are arranged so as to be in the main stream of the liquid containing them

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The test element is preferably used at the test site of the present invention. Briefly, the assay element (FIG. 13) includes a cleavable linker molecule 61, covalently attached at one end 60 to the surface of the disc 59 and at the other end 62 to the reporter element 65. Preferred embodiments of the reporter element described herein are gold reflective or opaque particles. latex particles. Also, two recognition elements 63a and 63b are present. which are referred to as side arms, and which are covalently attached to each linker molecule in such a way that one side arm is attached to each side of the cleavage site 64 of the linker molecule. Preferred embodiments of the side arms described in the present invention

Examples of the invention are oligonucleotides, antibodies, and oligonucleotide-antibody conjugates. Test elements can be used to detect the presence of an analyte and can generate a signal by either a positive or negative recognition event (Fig. 14). A positive recognition event (FIGS. 14a, c and e) occurs when the analyte 66 binds to both side arms 63a. 63b, resulting in a link loop 67 between two sides of the linker molecule divided into two portions by the cleavage site 64. A negative recognition event (FIGS. 14b, d and f) occurs when the analyte 66 binds to only one or does not bind to any side arm

68a. 68b. so that a loop connecting the two ends of the linker molecule is not formed. If the positive recognition event is followed by cleavage of the linker molecules, the disc is uninterrupted to the reporter

- 14 The element remains intact (Fig. 14e). On the other hand, cleavage of the linker molecules in the test element upon negative recognition results in the removal of the reporter element from the disk (Fig. 14f). Thus, negative recognition results in the release of reporter elements that are easily washed away, while positive recognition results in retention of the reporter elements in their specific areas of the assay. In any case, the results can be immediately observed using a CD-ROM or DVD reader.

Also described are other embodiments of the invention that utilize both light-reflecting reporter molecules and opaque reporter molecules and positive and / or negative recognition events to perform a wide range of possible assays. For example, in some assays, the side arms may be joined prior to sample addition and binding of the analyte will cause the side arms to disengage. In this case, positive recognition results in separation of the reporter element, while negative recognition result results in keeping the reporter element in place.

Other possible embodiments of the assay element described in the present application do not include fissile linker linker molecules. In one such alternative scheme, the surface of the IBCD is coated with a metal, preferably gold, and the analyte links opaque particles, such as latex beads or dye-filled liposomes, to the metal surface.

Opaque beads as test elements

The foregoing test elements are based on the binding of light-reflecting particles to the transparent surface of the IBCD. This situation can be reversed by attaching opaque particles to the reflecting surface. This approach is particularly advantageous when large cell determination is performed and is generally illustrated in Figure 12.

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A metal film is deposited on the plastic surface. Information can be encoded into this metal layer, as is the case with conventional CD carriers. This information may include a spatial address or other test-related information. The metal layer is further covered with a plastic layer. This is then aminated as described above, and instead of gold beads, large latex beads 58 (diameter 10 to 50 µm) that contain a dye are bound to the substrate using linking molecules. The latex beads are partially covered with recognition molecules as described above, in the case of both gold and gold beads. Cell recognition allows the latex beads to bind to the substrate even after cleavage of the linking molecules, and the dye in the particles prevents reflection of the laser beam from the metal layer. Alternatively, if the correct fluorescent dye and wavelength of laser light are used, fluorescent particle emission can be used to monitor the assay. This requires a special device and will be enabled by blue lasers when they become available for use in CD-ROM or DVD readers.

In the simplest version of the cell detection assay, the latex particles are not attached to the IBCD prior to the assay, but are added after the cells have bound to the IBCD. A suspension of latex beads is added, the recognition molecules on the beads are bound to the respective cells and immobilized. Latex beads may then be observed due to reduced reflectance by the CD-ROM or DVD reader.

Complementary binding of linker molecules

One disadvantage of covalent bonding of linker molecules is that the disk cannot be easily regenerated after cleavage of linker molecules. If, instead, the linker molecules bind to the substrate with complementary oligonucleotides, the disc can be regenerated after the assay is complete. Linking molecules or their

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Residues are removed by heating or by using chaotropic agents. The duplex molecules that bind the linker molecules are denatured and the disc can be cleaned. Oligonucleotides that bind the old linker molecules are retained on the disk. All oligonucleotides in one assay site are identical. They may be different at different assay sites or may be identical throughout the IBCD. New linker molecules with oligonucleotides complementary to the oligonucleotides bound to IBCD are added. After incubation, complementary oligonucleotides linker molecules and IBCD hybridize. Excessive linkers and molecules are washed away. In this case, the oligonucleotide side arms may be attached to the linker molecules prior to attachment of the linker molecules to the surface. Gold spheres are then added which are bound by thiol groups or disulfide bridges of the linker molecules and the disk is again ready for use.

Tests using ultraviolet or visible spectrophotometry, fluorescence or chemiluminescence are performed in cuvettes. The cuvette on the BCD is essentially a capillary that is located between the light source and the photodetector. Light can be guided by mirrors and waveguides. The number of cells on the BCD is between 0 to 10,000 and most preferably between 0 to 50 per test sector. The sample arrives in most cuvettes through the sample preparation chamber. These chambers may contain pre-loaded reagents or may be stored in separate chambers and admixed with the sample as it enters the sample preparation chamber. The samples and reagents can be heated by electrically infrared radiation, which is generated by a photodiode. After the incubation period, the sample is transferred to a cuvette. Transmitted or emitted light is measured by a photodetector. In the present invention, the photodetector is most preferably within a CD or DVD drive.

The light sources for spectrophotometric tests are most preferably photodiodes or semiconductor lasers. You can use a CD or DVD drive light source. However, these devices currently use only one wavelength, which corresponds to the infrared »·» - - W »w

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- 17 or red radiation. If the internal light source of the CD or DVD drive is used, the photodiode or laser in Fig. 15 is replaced by a mirror. Although several tests can be performed using infrared or red light, the use of light sources is preferred for most applications. For example, it is possible to fabricate a photodiode array in such a way as to provide red, yellow, green, and blue light. It is possible to design a photodiode for a specific wavelength and thus the number of photodiodes may be up to 300 to cover the entire spectral range of ultraviolet and visible radiation. The laser provides more power and better focusing than photodiodes and is therefore advantageous. In particular, micro cavity and nanodot lasers are very small and can be made to emit almost any wavelength. The light sources can be made as a module that can be attached to the disc before use and removed again after use of the BCD.

Unit operations

Unit operations are described below. Centrifugation, filtration, liquid transfer, mixing of liquids, dialysis, separation on columns, heating, cooling, electroconvection and electrophoresis.

The main force used to transfer liquids on an IBCD is the centrifugal force. It can also be used for centrifuges, which is important when cells are separated from plasma. In this case, it is advantageous to use a filter with a sample receiving container.

Sequence and timing are important when transferring fluids. Multiple liquid reservoirs are used to ensure the correct order of arrival at a particular reaction site, as illustrated in Fig. 9. In one embodiment, two main capillaries, 36 and 37, are present which are interconnected for liquid by interconnecting capillaries 38, 39 and 40. One of the major capillaries is an air channel to allow fluid flow and is typically rendered hydrophobic. The second main channel conducts the agents in liquid form and is typically hydrophilic. Connecting

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Capillaries and associated cavities can serve to store reagents and are generally designated 41, 42 and 43 and allow the relative relative placement of reagents to be maintained. The liquid compartment into which the liquid is fed and the timing of the delivery of each reagent are controlled by the location of the wells, the size of the capillaries, the density and viscosity of the liquids, and the rotation speed of the disc. The liquids are separated by small air bubbles to prevent mixing until the desired time. To prevent the formation of a pressure gradient, the capillaries are connected upstream with all air capillaries. To further prevent the entry of liquids into the air capillaries, the capillaries are hydrophobically treated. The mixing of the two solutions is accomplished by joining two capillaries in a Y-shape. This in itself allows good mixing. To ensure more efficient mixing, the capillaries may contain small, repetitive enlargements. It should be noted that the rotation of the IBCD leads to efficient mixing in the containers.

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

Gel, adsorption and ion exchange chromatography is also possible. The different kinds of molecules are fractionated by chromatographic supports and exited through the capillary separately, as is the case with conventional chromatography. Using a valve, some fractions can be selected and fed to the test element.

Heating is best done electrically. The distance between the upper and lower electrodes is approximately 500 µm. If the solution contains ions, the system is actually short-circuited and heated. Heating may be terminated by removing the ions from either the battery or the reservoir. A constant temperature can be achieved by placing the thermostat in

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99 99 circuit. A very simple thermostat is a bimetallic element that can close the circuit at a temperature below the set temperature and open it at a lower temperature. Another heating mechanism is provided by the laser of the CD or DVD drive. Especially powerful lasers contain mechanics

CD-R. Either at the top or at the bottom of the cavity, a liquid crystal film may be present on the surface, which is, if necessary, insulated with a transparent layer. On the other side of the cavity is a reflective layer. If the temperature in the cavity is lower than the main transition temperature, the liquid crystal scatters light and no reflection is observed. Above the main temperature and transition temperature, light is reflected back and heating can be interrupted; in any case, it has lower efficiency. The cooling is preferably carried out by endothermic dissolution, i.e. by heat absorption by the presence of the dissolving agent. The cooling solution and the cooled solution may be separated by a thin aluminum, copper, silver or gold film. Cooling can also be achieved by passive air cooling. This method only cools to ambient temperature, but is sufficient for most purposes. Cooling and heating can also be alternated in a cyclic manner, either in a single cavity or in alternating alternating heating and cooling cavities. This allows PCR amplification to be performed on the IBCD.

Electroconvection, electrophoresis and isoelectric focusing may be used in particular applications. In electrical convection, the material is transferred without any effort to divide it into individual components. In electrophoresis, separation is the main purpose. Separation is accomplished by using a flow-preventing gel. Since the distances are very short, the available field strength is sufficient for a working electrophoresis. For the same reason, the time required for separation is relatively short, which can be of the order of 1 to 5 minutes or even less than one minute. Usable electro-convection can be done in seconds. Isoelectric focusing is basically pH gradient electrophoresis. The pH gradient can be formed by a set of parallel capillaries, each containing a different buffer so that it gradually changes

444

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44 * 44 44 pH. This is shown in Figure 16. A large portion of the buffer remains in the capillaries and this ensures the existence of a pH gradient during isoelectric focusing. Upon completion of the focusing, the components are moved along the capillaries by centrifugal force or orthogonal electrophoresis can be performed.

This method allows almost complete fractionation of human plasma proteins (Anderson, Tracy and Anderson, "The Plasma Proteins",

2nd edition, volume 4, Academic Press Inc, 1984).

A particularly preferred configuration of the test site is shown in Figure 10. The test element consists of coupling molecules 10 and reflective beads as previously described, which are arranged in a linear array that can be conveniently located in one or more capillary channels at the test site at the test site. disk. As already described, the analyte binds to linker molecules having side arms receiving or complementary to the analyte (as shown in A) and after washing the analyte that has bound, the analyte is located at specific sites in the region (as shown in B). The presence of bound analytes is determined by routine address determination as with conventional CD-ROM readers using the attached software as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

Giant. 1 is a schematic representation of a disc according to the present invention.

Giant. 2A is a more detailed schematic representation of the sample preparation and disk testing sector showing the overall distribution of a typical test sector.

Giant. 2B is a schematic representation of a universal test sector capable of performing immunoassays, DNA testing, cell counting, spectrophotometric assays, and electrolyte analyzes.

FIG. 3 is a schematic representation of a disc according to the invention which illustrates a plurality of test sectors having each individual sample input.

Giant. 4 is a more detailed diagram of one of the test sectors illustrated in FIG. 3.

Giant. 5 schematically illustrates a chemically triggered battery usable in the present invention.

Giant. 6 schematically illustrates a structure providing a dialysis function on a disc according to the present invention.

FIG. 7 illustrates schematically a column that may be included on a disc according to the present invention.

Giant. 8 is a schematic illustration of an electrically operated valve useful in the present invention.

Giant. 9 is a schematic illustration of a series of reagents arranged 15 in interconnected capillary channels useful in the present invention.

Giant. 10 is a schematic illustration of a region of linear test sites that are conveniently located in the flow channel of the disk test sector of the present invention.

Giant. 11A-C is a schematic representation of various assay elements that are useful for detecting viral and bacterial particles and cells using a general methodology of site-specific localization of the detected substance.

Giant. 12A-C is a schematic representation of variations in detection methodology in which radiation-opaque particles are used instead of radiation-reflecting particles that bind to the radiation-reflecting surface. The zigzag lines represent oligonucleotides, but may be any recognition molecules, such as antibodies. The particles in this example are plastic beads, but may be liposomes, cells, and the like.

-22 ♦ 9 9 9 9 9 9 99 99 fr 9 9

Giant. 13 is a schematic illustration of a test element of the invention illustrating a spacer molecule with side arms and a cleavage site attached to the disc surface at one end and to a reporter element (gold or latex ball) at the other end.

Giant. 14A schematically illustrates a first test element of the present invention at an early stage of the test procedure.

Giant. 14B is a schematic representation of a second test element of the present invention at an early stage of the test procedure.

Giant. 14C is a schematic representation of the assay element of FIG. 14A, wherein analyte molecules bind to the side arms to form a link loop between the ends of the cleavage site.

Giant. 14D is a schematic representation of the assay element of FIGS. 14B, 15 where analyte molecules did not bind to the side arms and did not form a link loop between the ends of the cleavage site.

Giant. 14E is a schematic representation of the assay element of FIG. 14C after cleavage of linker molecules. The reporter element remains bound to the disc surface at a particular location.

Giant. 14F is a schematic representation of the assay element of FIG. 14D after cleavage of the linker molecules. The reporter element is released from the surface of the disc and may be washed away from a certain location.

Giant. 15 is a schematic representation of a cuvette arrangement. In this example, four cuvettes and associated reagent and sample preparation chambers as well as light sources are shown.

Giant. 16 is a schematic representation of a capillary region that can be used to perform isoelectric focusing.

Giant. 17 is a schematic illustration of an accurate volume measuring apparatus.

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DETAILED DESCRIPTION OF THE INVENTION

Example 1

Test Sector for Oligonucleotide Analysis (Figure 2, Test Sector)

The DNA-containing sample is mixed with sodium dodecyl sulfate to lyse the cells. The solution is transferred to a cartridge labeled "sample" and the disc begins to rotate. The sample is filtered and mixed with a mixture of complementary oligonucleotides. These oligonucleotides are complementary to the nucleotides analyzed and have a thiol group at one end. Hybridization continues in the reservoir labeled "sample preparation". This container may optionally be heated (not shown). After a suitable incubation period, the disc begins to rotate. When transferring the sample to a container labeled "sample separation", it is mixed with the nuclease S solution supplied by the side capillary. The mixture is allowed to incubate in a "sample separation" container having two gold electrodes and a valve as shown in Figure 8. The lower electrode is coated with linking molecules with isothiocyanate end groups. These bind to oligonucleotides containing a thiol group, some of which hybridize to the sample. All unhybridized DNA portions are cleaved and washed. Then the battery is put into operation. This is achieved by the rate at which acid and copper ions flow into the empty battery. The container is heated, the released oligonucleotides are released and the valve is switched,

The oligonucleotides are rinsed into the test area. After suitable incubation, ligase is introduced into the test region and the two side arms of the linker molecule are joined if the sample contains the correct oligonucleotide. The labile linker molecules are cleaved off. If the linker molecules contain siloxane groups, cleavage is accomplished by the addition of fluoride ions. Loose balls of gold with

-24 I «

Wash the IBCD at high speed by rotating the IBCD. Readings can be made immediately.

Example 2

Testing element for detection of cells and viruses

Alternative embodiments of the test element described elsewhere herein are suitable for detecting viral and bacterial particles that are larger than the oligonucleotides, antibodies, antigens, and the like described above. Viruses are typically almost spherical particles with a diameter of less than 0.5 µm. The bacteria are normally either spherical or rod-shaped. The largest dimension is less than 2 µm if flagella and other similar external fibers are excluded. The pathogens are smaller or approximately the same size as the gold beads used to detect them, and their interaction with the two side arms of the linker molecule may be limited.

For this reason, the side arms are attached to the surface of the IBCD and the gold bead instead of bonding to the linker molecule, as shown in Fig. 11. The gold bead is attached to linker molecule 45 at one end of the linker molecule and the other end of the linker is bonded to the substrate surface. 46. The linker molecule is provided with a typical 47 cleavage site, for example, a siloxane group, as mentioned previously. Contrary to the previously described embodiments, where the side arms are attached to the bonding molecule between the substrate and the cleavage site and the gold ball and the cleavage site, the side arms are attached to the gold ball and the substrate surface. For purposes of illustration, in Figure 11, oligonucleotides 48 and 49 are attached to the surface of the substrate, and oligonucleotides 50 and 51 are bound to the surface of the gold bead. Complementary oligonucleotides conjugate to members of the specific binding pair designated 52, 53, 54 and 55 attached to the oligonucleotides on the substrate and the bead of gold, as shown in the figure. It provides

-25 • 0 0 · ·

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The cells have much more space for binding to antibodies or other recognition molecules.

The linker molecules always have at least one cleavage site. They are in all respects the same as the molecules described above except that they do not have side arm molecules attached. For example, when a cell reaches the assay site if it contains groups forming specific binding pairs with appropriate complementary members, a link loop is formed between the gold bead and the substrate. When the linker molecule is cleaved, the gold bead 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, the gold bead will not remain attached to the substrate after cleavage of the linker molecule and is removed.

Antibodies or other recognition molecules can be coupled to the substrate in a manner similar to the attachment molecules. All linker molecules on the IBCD are identical and are simultaneously bound to the amino groups of analogous active groups on the surface. About half of the amine groups are used to bind linker molecules. The other half is used to bind recognition molecules to the substrate. If all recognition molecules on the IBCD surface are similar, they can be bound simultaneously with the linker molecules. Alternatively, if the recognition molecules are specific to each site of the assay, they may be applied locally by contact printing, inkjet printing or microcapillary storage.

When the gold beads bind to the thiol groups of the linker molecules, additional recognition molecules are also attached to the gold beads by thiol groups. To this end, the recognition molecules are first conjugated to a linker molecule containing a protected thiol or amine group. Aminová

The group may be derivatized in such a way as to introduce a thiol group. The different recognition molecules to be bound to the gold beads are deposited in a similar manner as they were bound to the surface of the IBCD of the other recognition molecule.

The recognition molecules may be oligonucleotides. These oligonucleotides can further hybridize with complementary oligonucleotide-biomolecule conjugates. This approach allows the binding of sensitive and reactive biomolecules, such as proteins containing several amine or thiol groups.

The recognition molecules bound to the beads of gold may freely diffuse through the beads of the beads, although they are tightly bound. The cell, which is recognized by both recognition molecules, creates a connection loop that binds the gold ball to the surface of the IBCD. After cleavage of the linker molecule, the gold bead remains bound and can be detected by a CD-ROM or DVD reader.

Multiple different recognition molecules may be used at the same test site. This approach has the advantage that all known mutants of certain pathogenic species can be detected at one test site. Different mutants can also be characterized at different assay sites that contain specific recognition molecules.

The IBCD is a versatile analyzer. It is easy to use and contains all reagents in its most advanced form, so only the sample is added. It can be used in clinical laboratories, hospitals, medical offices and at home. For home use, information can be transmitted to the doctor via the Internet. IBCD can be designed so that each patient's genetic fingerprint is measured. Approximately 35 polymorph points are sufficient for each person to receive a unique “barcode”. This eliminates possible errors caused by swapping tubes or stickers. Among the tests it is

- 27 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - detection of tumor cells in tissue samples, blood chemistry and electrolyte analysis. Other applications include massive systematic search for drug candidates, food and environmental safety analyzes, and monitoring of pathogens and toxins in the field in a military conflict.

Example 3

Turbidimetric Lipase Activity Test io The reagent cartridge contains 15 µl of stabilized triolein emulsion (250 μΜ) containing sodium deoxycholate (30 mM) and CaCl 2 (100 μΜ) at pH 9.0 in TRIS buffer (25 mM). The sample preparation chamber contains lyophilized porcine colipase (0.5 µg). Transfer 2 μΙ of serum into the sample preparation chamber (using the device shown in Figure 17) along with stabilized triolein and other reagents. Transfer part of the mixture (5 μΙ) to the cuvette. As the outlet capillary faces the center of the disc, the back pressure prevents further flow. Absorbance at 340 nm is read at 1 minute intervals. ΔΑ / min is a measure of lipase activity.

While the invention has been described in several specific embodiments, it will be apparent to one skilled in the art that modifications, equivalents, and variations of the invention are to be included within the scope of the appended claims.

Claims (6)

An optical disc adapted for reading by an optical reading device, characterized in that it comprises a first sector comprising substantially separate test means for binding or reacting an analyte believed to be present in the sample to at least one predetermined region in the a first sector, and optionally a second sector including test control means and analyte location information relating to one or more analytes that are expected to be present in a sample accessible to the reader, wherein the presence or absence of the analyte in said analyte the location may be determined by a reading device 15 using control means and location information. An optical disc according to claim 1, characterized in that it comprises a closable sample inlet for the liquid associated with the test means. 3. A test apparatus comprising an optical disc, a disc reader, and an information processor, wherein the disc comprises a first sector comprising substantially separate analyte binding means; 25 which is expected to be present in the sample, to at least one predetermined area in the first sector, and optionally a second sector that contains test information for carrying out the assay and analyte location information regarding one or more analytes that are expected to be present, that they will 30 present in the sample, readable and processable 99 » -29 • 9 * 99 • 9 ··· 999 • 9 9 • ·· 9 9 9 9 an information processor, wherein the disk is adapted to be read by a reader and the information processor is adapted to determine the presence or absence of an analyte at said location using control information and information 5 about the location. 4. The apparatus of claim 3, wherein the reader is a CD-ROM or DVD reader and the reader is adapted to communicate with the information device. 10 processor. The apparatus of claim 4, characterized by that the information processor is a personal computer. 15 Dec The disc of claim 1, characterized by test means they contain resources for preservation and liquid transfer means formed in the disc surface. 20. The disc of claim 6, wherein the liquid transfer means comprises a capillary channel. The disc of claim 7, wherein: 25 wherein the fluid transfer means comprises a valve. 9. The disc of claim 6 including means for obtaining electrochemical energy. • 99 -30 * 9 ·· 9 9 99 99 9 9 9 9 9,999,999 9 9 9 9 · 99 ·· The disc of claim 1, wherein the test means comprises a sample input, a sample preparation sector, an analyte separation sector, and a test sector, wherein 5 where the analyte is located. The disc of claim 6, wherein the fluid transfer means is responsive to a centrifugal force or electric field. 12. The disc of claim 1 comprising a plurality of first sectors adapted to analyze the plurality of analytes. 15. The disc of claim 1, further comprising a plurality of first sectors adapted to analyze the same analyte or different analytes, each of said plurality of sectors being adapted to be coupled to the sample inlet liquid. 14. A test element comprising a substrate, a first oligonucleotide bound to the substrate, a linker molecule bound to the first end of the first oligonucleotide via a second 25 of an oligonucleotide that is complementary to the first oligonucleotide, wherein the linker molecule further comprises means for binding to the analyte in the sample and comprises a second end that is detectable by means of detection, wherein the linker molecule further comprises a cleavage site between 30 with first and second ends, first group binding means -31 • 9 9 9 9 ϊ ··· ··· 9 9 between the first end of the linker molecule and the cleavage site for binding the first part of the analyte and the second group between the second end of the linker molecule and a cleavage site for binding the second part of the analyte. An assay component adapted to be read by a CDROM or DVD reader comprising an optical disc and substantially separate test means on an analyte binding disc, which is also believed to be present in the sample, for at least one in advance. a designated area on the disc and means therein for enabling the absence or presence of the analyte to be detected by a CD-ROM or DVD reader. An optical disc adapted to be read by a CDROM or DVD reader comprising substantially separate test means for locating an analyte believed to be present in the sample at least one predetermined area on the disc. 20 and means in said region on the disc for detecting the absence or presence of an analyte by a CDROM or DVD reader.