MXPA99007981A - Laboratory in a disk - Google Patents

Abstract

An apparatus is described that includes an optical disk, adapted to be read by an optical reader, comprising a first sector having substantially self-contained assay means for localizing an analyte suspected of being in a sample to at least one, predetermined location in the first sector and a second sector containing control means for conducting the assay and analyte location information, with respect to one or more analytes suspected of being in a sample, accessible to the reader, wherein the presence or absence of the analyte at said location is determinable by the reader using the control means and the location information. Depending on the nature of the assay, the disk will include fluid storage means, fluid transfer means, such as one or more capillary ducts, valves, batteries, dialyzers, columns, filters, sources of electric fields, wires or other electrical conductive means such as metallic surface deposits and the like.

Description

LABORATORY IN A DISK FIELD OF THE INVENTION This invention is generally related to diagnostic tests and methodology for them. In particular, this relates to diagnostic test components configured in a compact optical disc and the methodology for its use. 10 BACKGROUND • There is a huge need to make clinical trials faster, cheaper and simpler to perform. Ideally, patients should be able to perform tests of them, if they wish. One way towards this goal has been through miniaturization and the integration of different testing operations. Currently, many trials by bio-chip (so-called because some are built using photolithography techniques of silicon chip). All these approaches require a reading machine and a computer. Disc-shaped cassettes are also commercially available that are used for clinical trials, in conjunction with UV / Vis spectrometry. The United States Patent of North America Number 5,122,284 describes a centrifugal rotor containing many interconnected fluid chambers, connected to a plurality of cuvettes. The rotor is adapted to be used with a conventional laboratory centrifuge, and is formed of materials that allow the photometric detection of the results of the tests that have been carried out in the reaction cells. A large number of rotor configurations and related apparatus have been described for the same analyzes or similar types of analysis. See for example Patents of the United States of North America Nos. 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 enlisted in them. US Pat. No. 5,413,732 describes lyophilized reagents for use in such systems. The principles of a centrifugal analyzer have been adapted into a disk that can be used in an instrument similar to a CD disk drive (Mian, et al., Application WO 97/21090). Mian teaches a modified CD drive with a dual function: 1. This is used to read information stored on the disk, and 2. This is used to spin the disk. However, Mian does not teach the use of the reading ability of a CD drive for real test analysis. Despite recent advances, there is still one need for a simpler test setup that performs tests quickly, efficiently, accurately and at low cost. The present invention combines diagnostic tests with computers and compact disk technology. In its most preferred mode, the only instrument 5 that is needed is a computer with a CD player. All the chemistry is done within a compact disc, which can be referred to as an integrated biocompact disc (IBCD). The same compact disc is also encoded with software, it is say, instructions and control information readable by • machine, which provides instructions to a computer before, during, or after the test. CDs or DVDs represent the most economical storage medium and in many ways the best. HE should note that CD and DVD are acronyms that are currently used, which may change in the future, even if the underlying technology remains basically the same. A CD or DVD disc drive is a many aspects equivalent to a confocal scanning microscope. At the same time these instruments can be compare good centrifuges, because in commercial disk drives the rotation frequency is between 200-12,000 revolutions per minute, and can be adjusted within certain limits. The combination of these three characteristics within the same analytical system results in a large simplification, compared to any other analytical technique. Even so, performance is comparable or better than in most competitive methods. Although this invention requires slightly modified CD or DVD disc drives, it is possible to incorporate these changes into the commercial disk drives. This will enable Care-in-the-Patient-Point (POPC) and home use of this invention. The use of CD or DVD disk drives will allow the exact digital analysis of any sample, without any specific analytical instrumentation. 10 • COMPENDIUM OF THE INVENTION In one aspect, the invention is directed to an optical disk, adapted for reading by an optical reader, which comprises a first sector that has a test element Substantially integrated to fix an analyte suspected of being in a sample to at least one predetermined location in the first sector, and optionally a second sector containing a control element to conduct the assay and the The location of the analyte, with respect to one or more analytes suspected of being in a sample, accessible to the reader, where the presence or absence of the analyte in that location can be determined by the reader, using the element of control and location information. Depending on In the nature of the test, the disc may include a fluid storage element, a fluid transfer element, such as one or more capillary ducts, valves, batteries, dialysers, columns, filters, electric field sources, wires or other electrical conductive elements, 5 such as metal surface tanks and the like. The disk may have one or more ports of entry of the sample to send sample fluid to the test sector. These ports, if present, can preferably be sealed, so that after the application of the shows the disc, the sealed disc that includes the sample • comprises a hermetically sealed device that can be disposed of conveniently, or by any conventional means or other disposal mechanisms to deal with biological waste. In addition, the disc's testing sector is fifteen . conveniently divided into different subsections for the preparation of the sample and the separation of the analyte. Similarly, a waste receptacle subsection may conveniently be provided. The test sector can be divided into a multiplicity of sub-sectors that receive, each, a sample. Each of these sub-sectors can analyze one or more analytes, depending on the immediate particular application. In another aspect, the present invention is directed to an apparatus for conducting an assay, comprising a disk Optical disk, a disk reader, and an information processor, the disk comprising a first sector having a substantially integrated assay element for locating an analyte suspected of being in a sample to at least one • previously determined location in the first sector, and optionally a second sector containing control information to conduct the assay and information on the location of the analyte, with respect to one or more analytes suspected of being in a sample, accessible to the reader, and that can process the information processor, in where the disc is adapted for the reader to read, and the • information processor is adapted to determine the presence or absence of the analyte at that location, using the control information and the location information. The device may include a reader that has a CD-ROM drive or DVD, and an information processor, such as a personal computer. In yet another aspect the invention is directed to an optical disk, adapted for read by a CD-ROM or DVD reader, comprising a substantially test element. integrated in the disk, to locate an analyte that is suspected of being in a sample until at least one previously determined location on the disk, and an element in said location for the detection of the absence or presence of the analyte, by means of of the CD-ROM or DVD reader.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of a disc of this invention. Figure 2 A is a more detailed schematic representation of a sample preparation and a disk test sector, illustrating the overall scheme of a typical test sector. Figure 2B is a schematic representation of a ubiquitous assay sector that is capable of performing immunoassays, DNA testing, cell counting, spectrophotometric assays and • electrolyte analysis. Figure 3 is a schematic representation of a disk of this invention, illustrating a multiplicity of test sectors, each sector having an individual sample input port 15. Figure 4 is a more detailed schematic representation of one of the test sectors illustrated in Figure 3. Figure 5 is a schematic representation of a chemically activated battery, useful in the present invention. Figure 6 is a schematic representation of a structure for providing a dialysis function in the disc of this invention. Figure 7 is a schematic representation of a column that can be included in the disk of this invention.

Figure 8 is a schematic representation of an electrically controlled valve, useful in the present invention. Figure 9 is a schematic representation of a reagent train configured in attached capillary ducts, which is useful in the present invention. Figure 10 is a schematic representation of a configuration of linear test sites that are conveniently located in a flow channel in the test sector 10 of the disk of this invention. • Figures 11 AC is a schematic representation of a variation of a test element that is particularly useful for the detection of viral and bacterial particles and cells, which uses the general methodology of site-specific localization of the substance to be tested. detect. Figures 12 A-C is a schematic representation of a variation of the detection methodology, in which opaque particles are used instead of reflection particles, 20 and fixed to a reflection surface. The zigzag lines represent oligonucleotides, but can be any recognition molecules, such as antibodies. In this example the particles are plastic spheres, but they can be liposomes, cells, and so on. Figure 13 is a schematic representation of a test element of the invention, illustrating the spacer molecule, with component side arms and dissociation site, fixed to a disk surface at one end, and to a reporter element (gold sphere) or latex) in its other 5 extreme. Figure 14 A is a schematic representation of a first test element of this invention, at an early stage during the test procedure. Figure 14B is a schematic representation of a second test element of this invention, in one step • early during the test procedure. Figure 14 C is a schematic representation of the test element in Figure 14 A, where the analyte molecules have. the lateral arms are fixed, forming a connection curve 15 between the sides of the dissociation site. Figure 14 D is a schematic representation of the test element in Figure 14 B, where the molecules of the analyte have not been attached to the side arms, and a connection curve between the sides of the analyte has not been formed. dissociation site. Figure 14 E is a schematic representation of the test element in Figure 14 C, after the spacing molecules have been dissociated. The reporter element remains attached to the surface of the disk at a discrete site.

Figure 14 F is a schematic representation of the test element in Figure 14 D, after the spacer molecules have been dissociated. The reporter element is separated from the surface of the disk, and is free to wash away from its discrete site. Figure 15 is a schematic representation of a cuvette assembly. In this example, four cuvettes and their associated reagent chambers and sample preparation are shown, as well as the light sources. Figure 16 is a schematic representation of a capillary configuration that can be used to perform the • isoelectric focus. Figure 17 is a schematic representation of an apparatus for measuring exact volumes.

DETAILED DESCRIPTION OF THE INVENTION In Figure 1 a global schematic representation of an integrated biocompact disk (IBCD) is established. The disc (Biocompact Disc, BCD) can be of virtually any shape and size. For the most practical applications This is circular, has a diameter of 10-1000 mm, more conveniently 20-200 mm, and a thickness of 0.1-20 mm, more conveniently 0.5-3 mm. Disk 10 contains two sectors: a test sector 11 and a software sector 12. A central hole 13 is provided for location on a CD player. The software to control the assay can be on a separate disk. However, it is preferred to have the software on the disk associated with an assay for a particular analyte or analytes, for • minimize the opportunity for human error when performing the 5 trial. In the following description, the possible components and unit operations of the integrated biocompact disk are presented. The disc typically rotates up to 16,000 revolutions per minute on conventional CD-ROM or DVD players. In all CD-ROM and DVD readers can adjust the speed • within certain limits (200-16,000 revolutions per minute). However, for some operations it may be convenient to use rotations at different speeds, for example 1000-10,000 revolutions per minute, and more preferably 2000-15,000 revolutions per minute. For any particular test, the control software dictates the rotation regime during the analysis. This regime, the speeds and the timing, including the times in which probably no rotation occurs to allow the incubation, the electrophoresis, isoelectric focusing, etc., are controlled to send reagents and samples to the appropriate sites in the assay sector, as indicated by the test protocols. The available rotary speeds allow a significant centrifugal force that can be used to move liquids.

Another energy source that can be easily used in the integrated biocompact disk is chemical energy. A more adequate form of chemical energy is released by a battery in the form of electrical energy. The mechanical and chemical energies • allow the operation of many kinds of components. The 5 important components of an integrated biocompact disk may include one or more of the following: capillary ducts, containers, filters, dialysis membranes, chromatographic columns, electrophoretic gels, and micromechanical or electronic components that include microprocessors, electrodes, especially enzyme electrodes, cuvettes, and • test elements. The possible unit operations performed by the components include the following: centrifugation, filtration, liquid transfer, liquid mixing, dialysis, column separations, heating, cooling, electroconvection, electrophoresis, and detection of analytes and signaling thereof. The integrated biocompact disk is conveniently made of two pieces comprising upper and lower halves. The lower half can contain almost all the components, while the upper half may be a flat cover containing only a few components, such as electrodes and wires. The number of layers in this invention can be more than two, and in addition many components can be manufactured as modules. Especially the containers of reagents, tray assemblies, columns, micromechanical components, light sources, and microprocessors are conveniently assembled as modules. Different characteristics can be printed on the soft plastic. Different components can be glued, either by means of thermal curing or ultraviolet rays, they can be fused together, they can be connected by means of complementary mechanical characteristics, they can be fastened mechanically or they can simply be enclosed within a larger component. Some areas can be treated, for example, with ammonia plasma to make these areas • hydrophilic. The surface can be further treated by different molecules that make the surface inert, or alternatively give it specific adsorption properties. Silylation is a general method for the treatment of surfaces (Virtanen, J.A., Kinnunen, P.K.J. and Kulo, A., "Organosilanes and their hydrolytic polymers as surface treatment agents for use in chromatography and electronics," U.S. Patent No. 4,756,971). The covalent adhesion of detergents will reduce the adsorption of proteins, such as albumin, and will also reduce the adsorption of soluble proteins. Metal electrodes and wires can be evaporated over desired areas. Masks or resistances can be used to locate plasma treatment or metal deposition. Ducts can be machined capillaries and compartments for storage and retention of fluids within the optical discs, or can be formed by chemical means, or in injection molding operations. As shown with reference to Figure 2, the test sector • may contain a port 14 of sample input. Preferably, the sample port can be sealed, such that the disc is effectively sealed, except for the ventilation necessary to allow the fluid to flow, to protect it from any biological hazards. By different means, for example, centrifugal force and the like, As is well known in the art, a portion is dosed • of the sample to a sample preparation site 15, which may contain reagents and the like, for the purpose of conducting the assay. Alternatively, or in conjunction with the reagents that are already in the preparation segment of the For example, a reagent train 16 may be provided to send, as necessary, the reagents in the order appropriate to the sample preparation segment. Figure 9 shows additional details of the reagent train. It may be necessary to separate the analyte from the shows, at least partially, and this can be done in a separation segment of the sample generally designated as 17. If electrical power is required for the separation process, a battery 18 is provided. Subsequently in Figure 5 are shown and additional details of the battery. The resulting sample is then transferred to the test site 19. In a preferred embodiment of the invention, the assay site contains an assay element, as described in greater detail below. The analyte is fixed to a predetermined location on the disk, if it is present in the sample, and the reader detects the presence of the analyte, from the information that identifies the particular analyte with the location in which it is fixed. . A waste compartment is provided to collect the overflow of reagents or the sample that exceeds the dosed quantities for use in the test, and the • Different compartments and fluid transfer channels are properly ventilated to allow fluid flow along the surface of the test area. In one embodiment of the invention, as shown in In the Figure, a multiplicity of sectors 21, 22, 23, etc. of test, each sector connected to a port 24, 25, 26, of individual sample input, respectively, can be provided. The operation of each sector is substantially as described above, although they can be conducted different trials at the same time in individual sectors, either for a multitude of analytes or a multitude of patients. Figure 4 shows in more detail the details of a particular sector, where the different possible components are identified with the same numbers that are used in the previous description.

Components As shown in Figure 5, a battery can be provided consisting simply of two layers of metal, such as copper and zinc, which are in the lower and upper half, respectively. 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, this may be diluted sulfuric acid, that contains copper ions, and the battery is activated. This • Battery generates a voltage of 1.5 volts for only about 1 hour. However, this is more than enough to finish the analysis. You can make batteries that last longer, if necessary, from other materials or metal layers thicker. It is important to know that allowing water to flow into the space between the layers of metal deactivates the battery. The activation and deactivation cycle can be repeated many times. Many batteries can be coupled in series, to increase the potential, if necessary.

Optionally, photodiodes can be included within the circuit system. In this case, the computer that controls the test with information about the active circuits is provided. In addition, you can use a miniaturized battery, previously manufactured, and activate it by closing the electric circuit with a salt, for example, sodium chloride solution. Capillaries are preferably used to transfer liquid and air. In addition, very small volumes of liquid can be stored in the capillary tubes. Preferably, the capillary air ducts are hydrophobic, while the capillary ducts that come into contact with water are hydrophilic. As necessary, the capillary conduits may have circular or rectangular cross sections. The typical depths are between 10 μm and 500 μm, while the widths are between 50 μm and 2 μm • μm. The capillary air ducts use the largest dimensions to avoid any formation of a pressure gradient, unless otherwise desired. The speed of the flow depends on the frequency of the disk rotation integrated biocompact, the dimensions of the capillary duct and the viscosity and 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 reader. In this way, the dimensions of the capillary duct are used to adjust the speed of the liquid transfer. The capillary duct system can be provided with "bottlenecks", i.e. restrictions in the transverse areas of the capillary duct, to control the velocity of the liquid as needed. Hydrophilicity and hydrophobicity with the same purpose.

The exact dimensions of the capillary network and the chambers can be designed by using the Navier-Stokes equation: pv = pb - + pt2v where p is the density, p is the pressure, v is the velocity, b is the force field, μ is the viscosity and T is the differential operator of the (Mase, Continuum Mechanics, McGraw-Hill, 1970).

Pressure is a scalar field, while v and b are fields • vector. Commercial computer software is available for solving the Navier-Stokes equation in complicated geometries. The containers or compartments formed in the discs are used to enter the le, to store reagents, to perform reactions and to collect waste. Their depth is about 1-2000 μm, preferably about 10-800 μm, and they can have any possible shape, although cross sections are preferred circular or rectangular. The compartments are hydrophilic, except for one end of the waste container, which has a capillary air duct that is hydrophobic. The reaction compartments can be formed with electrodes for heating, electroconvection or electrochemical purposes. The electrodes are preferably evaporated gold films. The compartments may also have valves that are operated by electricity or chemically, as described below. The storage containers can be coated with metal, preferably coated with gold, to prevent the penetration of water into the plastic. Reagents can also be pre-packaged in cassettes, which are virtually impermeable. These cassettes can be closed during storage and opened manually, by means of drilling, or by means of open a valve or plug when the cassette of the • shows on the disk. The opening of the cassette can also be facilitated by means of centrifugal force when the integrated biocompact disc begins to rotate. In any case, an appropriate flow of liquid is maintained during the test. means of controlling the computer by means of a CD or DVD player. The flow of liquid during the test can be monitored by the use of a reflection element. The reflection element uses the laser that is in the CD player or DVD, and the fact that even when the liquid is transparent, its reflection rate is significantly different from that of air. In this way the laser light is reflected back to the CD or DVD player, in the presence of air, and in some other direction in the presence of liquid, or vice versa. Another method to monitor the flow of liquid is to use an active light source, such as an LED (light emitting diode) or a semiconductor laser. That light can be energized by the presence of an electrically conductive liquid, such as plasma or pH regulator, acting to close an electronic circuit. A visual display of liquid crystal can be used to transmit information from the integrated biocompact disk to the CD or DVD drive, and to the computer. The visual display of liquid crystal can have a large number of pixels (picture elements) that reflect the light when there is a • potential on the LC film. These pixels can be, for example, linearly organized, so that at one extreme low potential for light reflection is needed, while at the other end the potential must be much more. high to obtain the result. A CD or DVD drive is capable of locating the reflection pixels and, in accordance with this, the potential in the circuit can be measured. The potential change can be due to an electromechanical process in one of the electrochemical cells. By For example, an electrode coated with cholesterol oxidase will generate hydrogen peroxide in the presence of cholesterol. Hydrogen peroxide will change the potential of the circuit system, and cholesterol can be quantified. Filters can be used to remove particles large, such as cells, powder, etc. of the soluble le. In accordance with the foregoing, filters are most preferably included as part of the le entry compartment. The filters can be formed from porous plastic, glass, cross-linked cotton or cellulose, and so on. These 5 materials may be in the form of rods or similar shapes, depending on the particular use for which they are being placed. Plastics, such as Teflon, can be used as films. Since chaotropic agents are often used to denature the oligonucleotides during the preparation • From the sample, it is convenient to provide a dialysis element on the disc to remove the salt before the test is performed. As shown in Figure 6, a dialysis unit is prepared by placing a membrane 27 of dialysis either in one or both halves (upper and lower) of a compartment formed on disk 10. Taking into account • small volumes, the pH regulator that is already inside the dialysis membrane is usually sufficient, and typically no pH regulator is needed on the side of the dialysis membrane. membrane opposite the fluid layer. A column can be prepared, as shown in Figure 7, by filling a compartment 28 with a desired gel, adsorbent or ion exchanger, for example, silica gel, Sephadex, etc. (the particular material is choose for the particular application for which this is used), and placing a filter 29 at the other end. Examples of potential uses include separating smaller molecules from larger ones, and fractionating hydrophilic compounds and • hydrophobic. An ion exchange column is especially useful for the separation of nucleic acids from other biomolecules. The columns lend themselves to other uses that may be convenient or necessary to conduct any particular trial. Figure 8 illustrates a valve, designated usually like 30, which can be located at one end • of a column or a reaction container, which has two capillary ducts 31 and 32 outlet. In addition, there are two electrodes 33 and 34, which are initially not charged in the position illustrated, and a conductive metal foil 35, which is adapted to close one or the other of the capillary ducts, depending on its position in relation to each • capillary tube. The metal sheet is biase to close one of the capillary ducts when no current is flowing, and operates to open the capillary duct previously closed and close the other capillary when the current flows. As an example, the valve is made from a thin sheet of gold, which is pressed mechanically against the other outlet capillary, and is electrically connected to the nearest electrode. When the battery, the nearest electrode repels the gold foil, and the .other electrode joins it. As a result, the gold foil is pressed against the other exit. Other conductive metal foils can be used, but for most operations a metal that is conductive and does not corrode is preferred. The battery can be deactivated as explained above, and then the valve is changed back to its original position. The laser of the CD-R or CD-RW disk drives has an energy of up to 10 mW that can heat objects to high temperatures, up to 600 ° C. The energy is enough • Strong enough to drill holes in many materials, including plastics. The plastic must contain a dye that absorbs the laser light. Thermal expansion can be used for reversible valve actuation. For example, bending of the bimetallic sheets is extremely sensitive to temperature.

• Piezoelectric material can be used as a valve. Piezoelectricity can also be used to measure extremely small volumes of liquids, for example, can divide nanoliters of the sample between different tests. Valve-like operations can also be performed chemically, by deposition from the solution of a solid chemical compound and / or the dissolution of a deposited solid compound. The first outlet of that valve is closed by the deposition of a chemical compound inside the capillary. The compound can be, for example, silver chloride. The chloride ions may be in the main fluid stream, while in separate lateral capillary passages there is pure water and silver nitrate in water. The lateral capillary conduits are configured in such a way that the water is added first and then the silver nitrate to the main fluid stream containing the chloride. At the moment when silver ions arrive at the intersection, it becomes obstructed, acting in a • effective as a closed valve. Alternatively, a capillary conduit may be initially blocked by the solid form of a soluble compound, such as sodium chloride. The addition of any aqueous solution dissolves the cap of sodium chloride and the capillary conduit is opened. The test element is preferably used in the assay site of the present invention. Briefly, the assay element (Figure 13) includes a spacer 61 dissociable, covalently attached at one end 60 to the surface 59 of the disk, and at the other end 62 to a reporter element 65. Preferred embodiments of the reporter element described herein include reflective gold spheres or opaque latex spheres. Also included are two survey elements 63a, 63b, which are referenced hereinafter as side arms, which are covalently attached to each spacer, such that a side arm is connected to each side of the dissociation site 64 of the spacer. Preferred embodiments of the side arms described herein include oligonucleotides, antibodies and oligonucleotide-antibody conjugates. The test elements can be used to detect the presence of an analyte, and create a signal thereof through a positive or negative recognition event (Figure 14). An event of positive recognition (Figure 14A, C and E) occurs when an analyte 66 binds to both • lateral arms 63a, 63b, resulting in the termination of a connective cycle 67 between the two sides of the spacer divided into two parts by the dissociation site 64. A negative recognition event (Figure 14B, D and F) occurs when the analyte 66 is fixed to only one or none of the lateral arms 68a, 68b, and consequently no cycle is made that connects the two sides of the spacer. When to a • Positive recognition event is followed by the dissociation of the spacers, a connection without breaking remains intact from the disk to the reporter element (Figure 14E). On the other hand, the dissociation of the spacers in a test element after a negative recognition event, results in the report elements being disconnected from the disk (Figure 14F). In this way, negative recognition gives as a result, loose reporter elements that wash easily, while positive recognition results in reporter elements being retained in their discrete test sectors. In any case, the results can be observed immediately by means of the 5 CD-ROM or DVD reader. Herein further embodiments of the invention are described, which use both reflexive and opaque reporter molecules, and positive and / or negative recognition events to perform a wide range of possible assays.

For example, in some tests the arms can be connected • Laterals before a sample is added, and the fixation of the analyte acts to disconnect the lateral arms. In this case, a positive recognition event results in the disappearance of the reporter element, while an event of negative recognition results in the reporter element being retained. Other possible embodiments of the test element described herein do not include dissociable spacers with side arms. In one of those alternative schemes, the The integrated biocompact disk surface is coated with metal, preferably with gold, and the analyte connects the opaque particles, such as latex beads, or dye-loaded liposomes, to the metal surface.

Opaque Spheres as Test Elements The previous test elements are based on the fixation of the reflective particles to the transparent surface of the integrated biocompact disk. The situation can be reversed in such a way that the opaque particles are fixed to a reflective surface. This approach is especially convenient when testing large cells, and is generally illustrated in Figure 12. A film is deposited on the plastic surface. metal. You can encode information within this layer • Metal, as it is done in conventional CDs. This information may include spatial directions or other information related to the trial. The metal layer is additionally covered with a plastic layer. Then it is aminated this, as previously described, and in place of gold spheres, large latex spheres 58 (10-50 μm) are attached to the substrate. • diameter), which contain a dye, by means of the spacing molecules, as previously described. These latex spheres are partially coated with molecules of recognition, as described above for the gold spheres. The recognition of the cell fixes the latex spheres to the substrate even after the spacers are dissociated, and the dye in the spheres prevents the reflection of the laser light from the metal layer. Alternatively, if a fluorescent dye and a wavelength are used. If appropriate, the fluorescent emission of the spheres can be used to monitor the assay. This requires a specialized instrument, and will be facilitated by blue lasers when they become available for use on 5 CD-ROM or DVD players. In the simplest version of the cell detection assay, the latex spheres are not connected to the integrated biocompact disk before the assay, but are added after the cells are fixed to the biocompact disk integrated. The suspension of latex spheres, the • recognition molecules in the spheres are fixed to the appropriate cells, and these cells are immobilized. Later these latex spheres can be observed by reduced reflectance, using the CD-ROM or DVD reader. 15 Complementary Fixation of Spacers One drawback of the covalent fixation of spacers is that the disk does not regenerate easily after the spacers are dissociated. If the spacers are In addition to being connected to the substrate with complementary oligonucleotides, the disk can be regenerated after an assay is completed. The spacers or their residues are removed by heating, or by the use of chaotropic agents. The fixing duplexes are denatured spacers, and the disk can be cleaned. The disc retains the oligonucleotides that were fixing old spacers. All oligonucleotides in a test site are identical. These may be different in different test sites, or they may be identical throughout the integrated biocompact 3 disk. New spacers having oligonucleotides complementary to those in the integrated biocompact disk are added. After incubation, the complementary oligonucleotides of the spacer and the integrated biocompact disk are hybridized. The excess spacers are washed. In In this case, the lateral arms of the oligonucleotide can be • adhere to the spacers before the spacers adhere to the surface. Then the gold spheres are added, these are fixed by means of the thiol groups or the disulfide bridges of the spacers, and the disk is ready for used again. For the UV / Vis, fluorescence or chemiluminescence spectrometric tests, a cuvette is used. A • Biocompact disc tray is basically a capillary duct that is located between a light source and a photodetector. The light can be guided by means of mirrors and waveguides. The number of cuvettes in the biocompact disk varies between 0-10,000 and more conveniently between 0-50 per assay sector. The sample arrives inside most of the buckets by means of a sample preparation chamber.

These chambers may contain pre-charged reagents, or the reagents are stored in separate chambers and mixed with the sample as it arrives inside the sample preparation chamber. The sample and the reagents can be • Electrically heating by means of infrared radiation that is generated by means of a photodiode. After the incubation period, the sample is transferred into the cuvette. The transmitted or emitted light is measured by means of a photodetector. In this invention the photodetector is more conveniently inside the CD or DVD disk drive. 10 Light sources for trials • Spectrophotometers are more conveniently photodiodes or semiconductor lasers. It is possible to use the light source of the CD or DVD drive. However, these instruments currently use only a length of o.nda 'that . Corresponds to infrared or red light. If an internal light source of the CD or DVD drive is used, the • photodiode or laser in Figure 15 with a mirror. Although many tests can be performed by using infrared or red light, for most applications it is convenient to use additional light sources. For example, an array of photodiodes can be manufactured in such a way that red, yellow, green and blue light can be generated. It is possible to design a photodiode for any given wavelength and, accordingly, the number of photodiodes can be to 300, to cover the entire UV / visible spectral range. The laser generates more energy and focuses better than the photodiodes, and it is preferred. Especially the microcavity and the nanodont lasers are very small, and these can be manufactured to emit almost any wavelength. The light sources can be manufactured as a module that can be adhered on the disk before, and removed after the use of the biocompact disk.

Unit Operations The following describes the operations of the unit: centrifugation, filtration, liquid transfer, • liquid mixing, dialysis, column separations, heating, cooling, electroconvection, and electrophoresis. Centrifugal force is the main force that used to transfer liquids into the integrated biocompact disk. This could also be used for centrifugation, which is • important when cells are separated from the plasma. In this case, it is convenient to include a filter with the admission container of the sample. 20 In the transfer of liquids, order and timing are important. In order to ensure the proper sequence of arrival at a certain reaction site, liquid streams can be created, such as those illustrated in Figure 9. In one embodiment, two conduits are provided. main capillaries 36 and 37, which are in fluid communication with one another by means of capillary connecting conduits 38, 39 and 40. One of the main capillary ducts is an air channel to allow fluid flow, and typically occurs hydrophobic. The other main channel 5 carries the reagents in liquid form, and is typically hydrophilic. The capillary connection conduits and the associated cavities can serve to store the reagents, generally designated 41, 42 and 43, and maintain their relative locations with respect to each other. He fluid compartment to which they are directed, and their • Shipping timing are controlled by their respective locations, the size of the capillary ducts, the density and viscosity of the fluids, and the rotational velocity of the disc. The liquids are separated by small bubbles of air, to avoid mixing, unless mixing is desired. In order to avoid pressure gradients, the capillary air ducts are connected upstream with all capillary liquid ducts. To further prevent liquids from entering the air capillary ducts, these are hydrophobic. The mixing of two solutions is done by fusing two capillary ducts in a Y-shaped formation. This only provides good mixing. To ensure more efficient mixing, a capillary duct can have small periodic elongations after fusion. Must be . Note that the rotation of the integrated biocompact disk results in efficient mixing in the containers. In dialysis the liquid is in contact with the membrane that contains the pH regulator. The molecular weight cutoff of the membrane can be selected to be between 300-500,000 Daltones. Because only a very thin layer of the liquid is in contact with the dialysis membrane, dialysis is very fast. However, the ratio of the liquid to the pH regulator is only between 1:10 and 1: 100, such that dialysis is not quantitative. For • Most purposes this is enough. Gel chromatography, adsorption and ion exchange are possible. The different molecular species are fractionated by the chromatographic medium and leave the capillary conduit separately, as in conventional chromatography. Using a valve, certain fractions can be selected, and guided within the test element. The heating is done better electrically. The upper and lower electrodes are separated by approximately 500 μm. If the solution contains ions, the system is virtually 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 achieved by to include a thermostat inside the circuit system. A bimetal element is a very simple thermostat that can close a circuit below a previously set temperature, and open it to a higher temperature. Another heating mechanism is provided by means of the laser 5 of the CD or DVD disc unit. Especially, the CD-R disk drives have powerful lasers. Either the upper part or the lower part of the cavity can have a liquid crystalline film that is insulated by means of a transparent layer, if necessary. On the other side of the cavity there is a reflective layer. When the temperature of the cavity • it is below the main transition temperature, the liquid crystal will scatter the light, and no reflection is observed. Above the main transition temperature the light is reflected back, and it can be discontinued. heating, and this is less effective in any way. The cooling is preferably provided by dissolution • endothermic, that is, the absorption of heat through the presence of a dissolving substance. The cooling solution and the solution to be cooled should be separated by means of a thin film of aluminum, copper, silver or gold. Cooling can also be produced by means of passive air cooling. This method cools only at room temperature, but for most purposes this is sufficient. You can also toggle cooling and heating in a cyclic manner, either in a cavity or in a serially alternating sequence of heating and cooling cavities. This allows polymerase chain reaction amplifications to be performed inside the integrated biocompact disk. 5 In particular applications, each electroconvection, electrophoresis and isoelectric focusing can be used. In the electroconvection the material is transferred without trying to separate it into components. In electrophoresis, separation is the main purpose. The separation is facilitated by using a gel that prevents convection. Because • the distances are very short, the available field resistance is sufficient for the appropriate electrophoresis. For the same reason the time required for separation is quite short, and may be in the order of 1-5 minutes, or .Up to 1 minute. In a few seconds the useful electroconvection can be realized. The isoelectric focus is basically • Electrophoresis in a pH gradient. A pH gradient can be created by an array of parallel capillary ducts, each of which contains a pH regulator different, in such a way that the pH changes gradually. Figure 16 demonstrates this. A large part of the pH regulator will remain in the capillary ducts, and this will ensure the existence of the pH gradient during isoelectric focusing. After the focus is over, you can Move the components along the capillary ducts by means of centrifugal force, or an orthogonal electrophoresis can be performed. This method allows the almost complete fractionation of human plasma proteins (Anderson, Tracy and Anderson, "The Plasma Proteins", 2nd Edition, Volume 4, 5 Academic Press, Inc., 1984). A particularly convenient configuration of a test site is illustrated in Figure 10. The test element contains the sparger molecules and the reflecting spheres as described above, but contains them in a linear array that can be conveniently • located in one or more of the capillary channels at the disc's test site. As described, the analyte binds to the sparger molecules that have lateral arms receptive to, or complementary to, the analyte (as illustrated in A), and After washing the analyte that has been fixed, it is located in specific locations of the array (as illustrated in B).

• The presence of fixed analytes is determined by conventional direction determination, such as with conventional CD players and associated software, such as has described.

EXAMPLE 1 Assay Sector for Oligonucleotide Analysis (Figure 2, Assay Sector) A sample containing DNA is mixed with sodium dodecylsulfate to use the cells. This solution is transferred into the container denoted "Shows in" and the disk is rotated. The sample is filtered and mixed with a mixture of complementary oligonucleotides. These 5 oligonucleotides are complementary to those to be analyzed, and they also have a thiol group at one end. The hybridization is allowed to proceed in the container labeled "Sample Prep". Optionally, this container can be heated (not shown in the Figure). After the appropriate incubation, the disc is rotated. While it is • transfers the sample inside the denoted container "Sep. Sample" it is mixed with a solution of nuclease S sent from a lateral capillary conduit. The mixture is allowed to incubate in the "Sep. Sample" container that has two electrodes gold and a valve, as illustrated in Figure 8. The lower electrode is coated with spacers that have • isocyanate extreme groups. These bind to the thiol-containing oligonucleotides, many of which hybridize with the sample. All non-hybridized parts of DNA is digested and washed. Then the battery becomes operational. This is adjusted by the speed at which the acid and copper ions flow into the empty battery. The container is heated, the oligonucleotides fixed and released, and the valve is switched. The oligonucleotides are poured into the test area. After proper incubation the ligase arrives within the test area and the two lateral arms are connected in the spacer molecule, if the sample contains the appropriate oligonucleotide. The labile spacers are cut. If the spacers contain siloxane groups the cutting is done by the addition of fluoride ions. The loose gold spheres are washed by spinning the integrated biocompact disk at high speed. The reading can be done immediately.

EXAMPLE 2 Element of. Test for Detection of Cells and Viruses The alternative embodiments of the assay element described elsewhere herein are useful for the detection of viral and bacterial particles, cells and other particles that are larger than the oligonucleotides, antibodies, antigens and the like that have been previously described. Viruses are typically nearly spherical particles that have a diameter smaller than 0.5 μm. The bacteria are commonly either spherical or rod-shaped. Its largest dimension is less than 2 μm, excluding flagella and other similar external fibers. These pathogens are smaller or almost the same size as the gold spheres that are used to detect them, and their interaction with two arms side of the spacer may be limited. For this reason, these side arms are connected to the surface of the integrated biocompact disk and the gold sphere, instead of the spacer as illustrated in Figure 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 is bound to the surface of the substrate 46. The spacer molecule is provided with a typical dissociation site 47, for example a fraction of siloxane, as previously described. In contrast to the modalities described above, wherein the side arms are attached to the • spacer molecule between the substrate and the dissociation site and the gold sphere and the dissociation site, the lateral arms are attached to the gold sphere and to the surface of the substrate. For purposes of illustration, in Figure 11 Oligonucleotides 48 and 49 are bound to the surface of the substrate, and oligonucleotides 50 and 51 are • attached to the surface of the gold sphere. As illustrated, the complementary oligonucleotides are then conjugated with members of a specific binding pair, designated as 52, 53, 54, and 55, and bind to the oligonucleotides on the substrate and the gold sphere. This gives much more room for the cells to bind with antibodies or other recognition molecules. Each of the spacers has still when minus a dissociation site. These are, in all respects, identical to those previously described, except that they do not have any attached lateral arm molecule. When the cell arrives, for example, at the test site, if it contains fractions that form specific binding pairs with their respective complementary members, a connective cycle is formed between the gold sphere and the substrate. When the spacer molecule is dissociated, the gold sphere is retained in the substrate, and the presence of the cell can be detected, as previously described. However, if no specific fixing pair is formed, after the dissociation of the spacer, the gold sphere does not remain attached to the substrate and is removed. Antibodies or other recognition molecules can adhere to the substrate in a manner similar to that with which the spacers adhere. All the spacers in the integrated biocompact disk are identical and adhere at the same time to the amino groups or analogous active groups on the surface. Approximately half of the amino groups are used for the union of the spacers. The other half is used to couple the recognition molecules to the substrate. If all the recognition molecules on the surface of the integrated biocompact disk are similar, they can adhere at the same time as the spacers. Alternatively, if the recognition molecules are specific to each assay site, they can be dosed locally by means of contact printing, ink jet printing or microcapillary deposition. After the gold spheres adhere to the thiol groups in the spacers, the other recognition molecules, also by thiol groups, adhere to the gold spheres. For this purpose, these recognition molecules are first conjugated with a spacer containing a protected thiol or amino group. The amino group can be derivatized such that a thiol group is introduced. The different recognition molecules that are going to adhere to the gold spheres are dosed in a manner similar to that with which the other recognition molecules adhered with the integrated biocompact disk surface. The recognition molecules can be oligonucleotides. These oligonucleotides can be further hybridized with complementary oligonucleotide-biomolecule conjugates. This approach allows the binding of sensitive and reactive biomolecules, for example, proteins containing many amino or thiol groups. 20 The recognition molecules attached to the gold spheres are free to propagate around the sphere, although they are firmly attached. The cell that is recognized by both recognition molecules completes a connection cycle that fixes the gold sphere to the surface of the integrated biocompact disk. After dissociating the spacer, the gold sphere is retained and detected by the CD-ROM or DVD reader. A multiplicity of different recognition molecules can be used in the same assay site. The advantage of this approach is that all known mutants of certain pathogenic species can be detected at a test site. The different mutants can also be characterized in different test sites containing specific recognition molecules. 10 The integrated biocompact disc is a uni¬ analyzer • versal. This is easy to use, and in its most advanced form it contains all the reagents, and only the sample is added. It can be used in clinical laboratories, hospitals, doctors' offices, and at home. In the use at home you can upload the information to a doctor's office through the Internet. You can design the integrated biocompact disk • such that the genetic signature of each patient is measured at all times. Approximately 35 polymorphism points are sufficient to give every person a unique "bar code".

This eliminates possible errors due to the mixing of tubes or labels. The assays that can be performed include, but are not limited to immunoassays, DNA testing, cell counting and measurement of cell shape, detection of cancer cells in tissue samples, blood chemistry and electrolyte analysis. Other applications include mass screening of drug candidates, food and environmental safety analysis, and monitoring of pathogens and toxins in a battlefield.

EXAMPLE 3 Turbidimetric assay of lipase activity. The reagent cavity contains 15 μL of stabilized triolein emulsion (250 μM) containing sodium deoxycholate (30 mM) and CaCl2 (100 μM) at a pH of 9.0 in the regulator. pH TRIS (25 mM). The sample preparation chamber • contains lyophilized porcine colipase (0.5 μg). Two microliters of serum are taken into the sample preparation chamber (using the apparatus shown in Figure 17), together with stabilized triolein and other reagents. Part of the The mixture (5 μL) is transferred additionally into a cuvette. Because the outlet capillary goes to the • center of the disc, the back pressure will prevent more flow. The absorbance at 340 nm is read at one minute intervals. The? A / min is a measure of lipase activity. Although this invention has been described with respect to some specific embodiments, it is understood that modifications to the same and equivalents and variations will be apparent to one skilled in the art, and are intended to be and be included within the scope of the appended claims. to the present.

Claims (19)

1. An optical disk, adapted for a laser reader to read, comprising a first sector that has a 5 substantially integrated test element for fixing or reacting an analyte suspected of being in a sample to at least one predetermined location in the first sector, and optionally a second sector containing a control element for conducting the 10 assay and the location information of the analyte, with • with respect to one or more analytes suspected of being in a sample, accessible to a reader, and where the presence or absence of the analyte in that location can be determined by the reader, using the control element and the 15 information of the location and a port of entry of the sample.

2. The optical disk of Claim 1, wherein the sample inlet port is in fluid communication with the test element.

3. An apparatus for conducting a test, comprising an optical disk, a laser reader, and an information processor, wherein the disk comprises an input port of the sample, a first sector having a substantially integrated test element. to fix an analyte that is 25 suspect that it is in a sample to at least one previously determined location in the first sector, and optionally a second sector containing control information to conduct the assay and information on the location of the analyte, with respect to one or more analytes of 5 those that are suspected of being in a sample, accessible to the reader, and that can process the information processor, where the disc is adapted for the reader to read it, and the information processor is adapted to determine the presence or absence of the analyte at that location, using the control information and the location information.

4. The apparatus of Claim 3, wherein the reader is adapted to be coupled to an information processor.

5. The apparatus of Claim 4, wherein the information processor is a personal computer.

6. The disc of Claim 1, wherein the test element comprises a storage element of • Fluid and a fluid transfer element formed on a disc surface.

7. The disc of Claim 6, wherein the fluid transfer member comprises a capillary duct.

8. The disc of Claim 6, wherein the fluid transfer member comprises a valve.

9. The disc of Claim 6, wherein the disk comprises an electromechanical energy element.

10. The disk of Claim 1, wherein the test element comprises a sample port, a sample preparation sector, an analyte separation sector, and a test sector in which the analyte is located.

11. The disc of Claim 6, wherein the fluid transfer element is responsive to the centrifugal force or to an electric field.

12. The disc of Claim 1, wherein the disk comprises a multiplicity of first sectors, adapted to analyze a multiplicity of analytes.

13. The disk of Claim 1, characterized • because it also comprises a multiplicity of first sectors adapted to be analyzed by the same analyte or different analytes, wherein each of said multiplicity of sectors is adapted for fluid communication to a port of 15 sample.

14. A test element comprising a substrate, • a first oligonucleotide attached to the substrate, a spacer molecule attached at a first end to the first oligonucleotide by means of a second oligonucleotide, which is 20 complementary to the first oligonucleotide, wherein the spacer molecule also comprises an element for attachment to an analyte in a sample, and has a second end that can detect a detection element, the spacer molecule also comprising an intermediate site to the first and second 25 ends, which is dissociable, the fixation element having a first fraction between the first end of the spacer molecule and the dissociation site for attachment to a first part of the analyte, and a second fraction between the second end of the spacer molecule and the dissociation site for attachment to a second part of the analyte, wherein the dissociation site can be dissociated without chemically affecting the binding region of the analyte.

15. A test component, adapted to be read by a CD-ROM or DVD reader, comprising an optical disk 10 having a sample inlet port, and a test element substantially integrated in the disk to fix an analyte suspected of being in a sample to at least one predetermined location on the disk, and an element in that location to enable 15 detection of the absence or presence of the analyte by means of the CD-ROM or the DVD reader. •

16. An optical disk, adapted to be read by a CD-ROM or DVD reader, comprising a sample input port, a substantially integrated test element for 20 locate an analyte suspected of being in a sample to at least one previously determined location on the disk, and an element at that location to detect the absence or presence of the analyte via the CD-ROM or DVD reader .

17. The optical disk of Claim 1, wherein . The laser reader is a CD-ROM or a DVD disc reader.

18. The apparatus of Claim 3, wherein the laser reader is a CD-ROM or a DVD disc reader.

19. A test element comprising a substrate, a first oligonucleotide attached to the substrate, a spacer molecule attached at a first end to the first oligonucleotide by means of a second oligonucleotide, which is complementary to the first oligonucleotide, wherein the spacer molecule comprises also an element for fixing to a 10 analyte in a sample, and has a second end that can • detecting a detection element, the spacer molecule also comprising an intermediate site to the first and second ends, which is dissociable, the fixation element having a first fraction between the first end of the molecule 15 spacer and the dissociation site for attachment to a first part of the analyte, and a second fraction between the • second end of the spacer molecule and the dissociation site for attachment to a second part of the analyte, where the dissociation site can be dissociated 20 independently of the analyte.