MXPA99000383A - Device and method of divisi signal element - Google Patents

Abstract

A divisible signal element is described for use in devices and methods for quantitative and qualitative analysis. The binding of the analyte chosen simultaneously with a first and second analyte-specific lateral member of the separation signal element locks the portion that responds to the signal to the binding end of the substrate of the signal element, despite subsequent division at the site. of division that is intermediate to the first and second lateral members. Analytical devices comprising the elements of divisible signals are described, since they are analytical methods adapted to their use. The analytical devices of the present invention can be adapted for detection using conventional CD-ROM and DVD players

Description

DEVICE AND METHOD OF DIVISIBLE SIGNAL ELEMENT REFERENCE TO RELATED REQUESTS The present application is a continuation in part of the provisional application of the United States of America No. 60 / 021,367, filed on July 8, 1996 and of the provisional request of the United States of America No. 60 / 030,416 , presented on November 1, 1996, which are incorporated herein by reference. 1. INTRODUCTION The present invention relates to the field of diagnostics and the detection of small quantities of substances in liquids. More specifically, the invention relates to a dissociable signal element, particularly a dissociable signal reflector element for use in test devices. Testing devices employing these signal elements are adapted, in the preferred embodiments of the invention, to detection using standard laser-based detection systems such as compact disc read-only memory readers, video disc readers. digital, and similar. The invention further includes analytical methods for detecting analytes, using the assay devices of the present invention. The signaling element, what The test devices and test methods of the present invention are useful both for the detection of a large number of different analytes in a test sample, for the detection of a single analyte in a large number of samples. 2. BACKGROUND OF THE INVENTION 2.1 Small Scale Clinical Trials Until recently, most clinical diagnostic tests for the detection of small amounts of analytes in fluids had been conducted as individual tests; that is, as individual tests that led on single samples to detect individual analyte. More recently, economy efficiency has been obtained by designing devices for the preparation of multiple samples and the automatic addition of reagents, by designing devices for rapid analysis of large numbers of test samples, either in parallel series procession or fast. Frequently, this automatic reagent preparation devices and automatic multiplex analyzers are integrated in a single device. The large clinical laboratory analyzers of this type can perform hundreds of automatic tests semiautomatically efficiently, in one hour. However, these analyzers are expensive and can only be buy centralized laboratories and large hospitals. This centralization necessitates the transport of the sample, and frequently precludes urgent emergent analysis of the critical time samples. In this way, there is a strong need for simplified clinical trials that will reduce the cost of this dedicated analyzer as well as promote its distribution. E limit of this effort is the design of clinical tests suitable for use in the bedside of a patient or in the patient's home without dedicated detectors. Blood glucose and pregnancy tests are well-known examples. Although useful tests of this class have been offered for many years, one important discovery was the introduction of solid-phase immunoassays and other strip tests, since about 1980. The most notable are the Advance ™ test (Johnson &Johnson), the hC test of RAMPMR (Monoclonal Antibodies, Inc.), Clear Blue Easy (Unipath Ltd.) and ICON (Hybritech). Clear Blue Easy ^ has all the reagents in a laminated membrane and uses colored latte conjugated microspheres as the signal reagent. It uses a capillary migration immunoconcentration format. The ICON is an immunoconcentration assay of double monoclonal sandwich. This test has been produced quantitatively through the use of a small reflectance instrument. Otherwise, everything These methods are only qualitative. The migration distance can be used as a bas for quantitative tests. Those that are commercially available are the Quantab ™ (Environmental Test Systems) AccuLevel ™ (Syva), AccuMeter ™ (ChemTrak), Clinicmeter (Crystal Diagnostics) and Q.E.D ™ (Enzymatics). One of the new ones is the thermometer type test device (Ertinghausen G., US Patent No. 4,087,556) which is not commercially available yet. These systems can be used to test analytes of general chemistry, such as cholesterol, as well as blood levels or therapeutic drugs. A disadvantage, however, of each of these formats is that only one, or a very limited number of the tests, can conveniently be performed simultaneously. To fill the gap between mass analyzers and strips, some small instruments have been developed. The most notable is the ICAMR Eclipse (Biotope, Inc.). This device is an automatic centrifugal immunoassay and bench top system, random access. Patient samples are pipetted into a cartridge that is placed in a rotor. Sixteen tests can be run in approximately 17 minutes. The results are measured mediant visual spectrometry of ultraviolet or mediant rays fluorometry Four different types of cartridges are needed. Each cartridge has a relatively complicated structure. Despite these developments, there is still a need for a simple device that can be easily used for multiple quantitative tests, and preferably, that does not require specialized detection instrumentation. 2. 2 Airborne Probe Configurations of Spatial Makes Recently, spatially airborne configurations of different biomaterials have been manufactured on solid supports. These probe configurations allow simultaneous analysis of a large number of analytes. The examples are configurations of oligonucleotides or peptides that bind to a solid support and that capture supplementary analytes. One of these systems is described by Fodor et al., Nature, volume 364, August 5, 1993. Short oligonucleotide probes attached to a solid support bind the supplementary sequences containing the longer DNA strands in the liquid sample; then the nucleic acid sequence of the sample is calculated by computer, based on the hybridization data that was collected in this way.

In the test system described by Fodor, the configuration is reversed on a temperature-regulated flow cell against a container containing the labeled target molecules. In order to distinguish the molecules fixed to the surface, the system requires an extremely sensitive detector. In accordance with the foregoing, a need remains for an economic system to manufacture spatially steerable probe configurations in a simplified format, which provides both rapid detection and the ability to test large numbers of test substances (ie, analytes) in a one-step fluid test sample, or a minimum number of steps, or assay a single test substance or analyte in a large number of fluid test samples. 2. 3 Detection Systems Based on Spatially Directable Lase Rays Different devices for consumer electronic use allow spatially identifiable detection of digital information. In particular, different formats have been developed based on the registration potential of differential reflectance and transraitapci information. On compact audio or memory compact discs reading of conventional compact discs, the digital information - or the digitally encoded analogue information - is encoded on a circular plastic disc by means of disc indentations. TypicallyThese indentations are of the order of an eighth to a quarter of the incident beam wavelength of a laser beam that is used to read the information present in the disk. Indentations in the disc cause destructive interference within the beam that reflects, which corresponds to a bit that has a "zero" value. The flat areas of the disk reflect the light of the laser beam back to a detector and the detector gives a value of "one" to the corresponding bit. In another convention, a change in intensity of a reflected light has a value of one, while a constant intensity corresponds to zero. Since the indentations have been formed in a disc in. a regular pattern from a master copy containing a previously determined ".bit" of "one" bits, the resulting signal received by the detector can be processed to reproduce the same information that was encoded on the disk teacher. The standard compact disc is formed from a 12-centimeter polycarbonate substrate, a reflective metallized cap, and a protective varnish coating. The format of compact disks and memory d Read only current CDs are described in the ISO 9660 industry standard, which is incorporated into the present as a reference. The polycarbonate substrate is transparent polycarbonate of optical quality. In a compact disc pressed or copied in a standard mass, the data layer is part of the polycarbonate substrate, and the data is printed in the form of a series of slits by a printer during the injection molding process. During this process, the molten polycarbonate is injected into a mold, usually under high pressure, and then cooled so that the polycarbonate takes the form of a mirror image of the mold, "printing" or "printing"; therefore, the slits q representing the binary data in a disc substrate is created in, and maintained by the polycarbonate substrate with a mirror image of the print slits that are created during the comparison process. The main printed one is typically glass. The slits in the compact disc substrate are printed in a continuous spiral. The reflective metal layer that is applied thereon, typically aluminum, assumes the shape of the solid polycarbonate substrate, and differentially reflects the beam of the laser beam to the reading assembly depending on the presence or absence of the "slits". "An acrylic varnish is coated on a thin layer by rotation in the upper part of the metal reflection layer, to protect it from abrasion and corrosion. Although similar in concept and compatible with compact disc readers, the information is recorded differently on a compact disc that can be burned (CD R, for its acronym in English). In the compact disk that can be recorded, the data layer is separated from the polycarbonate substrate. Instead, the polycarbonate substrate has a spiral groove printed on it as a direction to rotate the incident laser beam. Although cyanine was the first material used for these discs, it generally uses a cyanin stabilized by metal instead of "crude" cyanin. An alternative material is phthalocyanine. A metalophthalocyanine compound is disclosed in U.S. Patent No. 5,580.69. In the compact disc that can be recorded, the organic ink layer is interposed between the polycarbonate substrate and the metallized reflection layer, usually 24 carat, but alternatively silver, of the medium. The information is recorded by a recording laser beam of the appropriate previously selected wavelength that selectively fuses the "slits" within the tint layer - rather than burning holes in the ink, this simply fuses it slightly, causing it to become non-translucent so that the read laser beam beam is refracted well that reflected back to the detectors of the reader as by means of a physical depression in the pressed compact standard disk. As in a standard compact disc, varnish coating protects the layers that carry the information. Other physical formats are being developed to record and store the information, based on the same concept of the compact disc: the creation of differential transmittance reflectance on a substrate that will read a laser beam. One of these formats is called Digital Video Disc (DVD). A Digital Video Disc looks like a standard compact disc: it is a 12-millimeter disc that looks like a silver tray, with a hole in the center to attach a rotating transmission mechanism Like a compact disc, the data is recorded on the disc in a spiral track of small slits, and the discs are read using a beam of 'laser beam. In contrast to a compact disc, which can store approximately 680 million bytes of digital data under the ISO 9660 standard, the Digital Video Disc can store from 4.7 billion to 1 billion bytes of digital data. The largest capacity of the Digital Video Disc is obtained by making smaller slits and the spiral more tight, that is, by means of reducing the inclination of the spiral, and by means of Record the data in as many as four layers, two on each disk lad. The smaller size of the depression and the tighter tilt requires that the wavelength of the reading laser beam be smaller. While the smaller wavelength is backward compatible with standard pressed compact discs, it is not compatible with the current versions of the compact disc that can be recorded based on ink. The following table compares the features of Digital Video Disc and Compact Disc.

Table 1: Comparison of the Characteristics of the DVD and CD DVD CD Diameter 120 mm 120 mm Disc thickness 1.2 mm 1.2 mm Thickness Substrate 0.6 mm 1.2 mm Inclination Track 0.74 μm 1 6 μm Minimum size depression 0.4 μm 0 83 μm Wavelength Laser 635/650 nm 780 nm Data Capacity 4.7 gigabytes / layer / side 0.68 gigabits Layers 1, 2, 0 4 1 In this way, a Digital Video Disc of a single side / single layer can contain 4.7 gigabytes of digital information. A single-sided / double-rap Digital Video Disc can contain 8.5 gigabytes of information. A two-sided / single-layer Digital Video Distro can contain 5. gigabytes of information, while a double-sided, double-layered Digital Video Disc contains up to 17 gigabyte of information. Each of the variations consists of two 0.6 mm substrates that are fixed. Depending on the capacity, the disk may have one to four layers of information. In the 8.5 gigabyte and 1 gigabyte options, a semi-reflector is used to access two layers of information from one side of the disk. For the options of 8.5 gigabytes and 17 gigabytes, the second information layer can be molded on the side of the second substrate, or it can be added as a photopolymer layer. In any case, a semireflector layer d is required to allow the two information layers to be read from one side of the disk. For the 17 Gigabyte Digital Vide Disc, it is necessary to produce two double layer substrates, and join them together. The laser beam reader of the Digital Video Disc is designed to adjust its focus to any layer depth, so that you can have fast and automatic access to both. All three formats described above require the tray to be rotated. The nominal constant linear velocity of a Digital Video Disc system is 3.5 to 4.0 meters per second (slightly more fast for the larger slits in the double cap versions), which is more than 3 times the speed of a standard compact disc, which is 1.2 mps. 3_j_ COMPENDIUM OF THE INVENTION It is an aspect of the present invention that provides a dissociable signal element for use in the devices and methods of quantitative and qualitative testing. The dissociable signal element comprises a dissociable spacer having a substrate fixing end, a responsive signal end, and an intermediate dissociated site at the substrate binding end and the signal responsive end. The dissociable signal element also includes a responsive signal fraction attached to the dissociable spacing at its signal responsive end. A first lateral member adapted to attach a first site to a chosen analyte, and a second lateral member adapted to join a second site of the same analyte, are present in the signal element. The first and second lateral members confer analit specificity on the dissociable signal element. The first lateral member is fixed at the dissociable spacing, intermediate at the responsive end of the signal and the dissociated site, and the second lateral member is attached to the dissociable spacer, intermediate at the dissociable site and end of substrate fixation. The fixation of the analyte chosen simultaneously to the first and second side members of a dissociable signal element, binds the responsive fraction of the signal to the substrate binding extremity of the signal element, despite the subsequent dissociation at the dissociation site. which is intermediate in the first and second lateral members; conversely, if the selected analyte is not set simultaneously to the first and second side members of a dissociable signal element, it allows the loss, through dissociation, of that responsive signal fraction of the signal element. The presence or absence of the signal after contact with the sample and contact with the dissociation agent indicates the presence or absence of the analyte, respectively. In another aspect, the invention provides a test device comprising a solid support substrate to which a plurality of dissociable signal elements are attached, in a spatially steerable pattern. In some embodiments of the test device, the solid support may be preferably a plastic, and in these embodiments, polycarbonate is most preferred. In some embodiments, solid support is molded as a disk, preferably in dimensions compatible with detection by detectors based on the reflection of existing laser beams, such as a compact disc (CD) player. audio, a compact disc read-only memory reader (CD-ROM), a digital video disc (DVD) reader, similar. In certain preferred embodiments of the assay device, the responsive signal fraction of the joined dissociable signal elements is adapted to reflect scattering incident light, particularly incident laser ray light. In these embodiments of the dissociable reflection signal element, the signal responsive fraction can be a metal microsphere, preferably a microsphere consisting essentially of gold, more preferably a gold microsphere of diameter between 1-3 microns. These modalities are suitable for detection in devices based on existing laser beam reflectance, such as compact disc (CD) audio, compact disc read only (CD-ROM), or disk disc readers. digital vide Another aspect of the present invention is to adapt the existing test methods to employ the test devices based on the dissociable signal element of the invention. Generally, a test adapted to use the test device based on the dissociable signal element of the present invention comprises the steps of: contacting the test device with a liquid sample contacting the test device with a test agent. dissociation adapted to dissociate the plurality of fixed dissociable signal elements, remove the responsive signal ends of the divided signal elements, and detect the presence of the signal responsive fraction of the restricted signal elements of the analyte, adhering to the substrate of solid support. The spatial direction capability of the signal elements in the assay device allows the identification of the analytes bound to different signal elements, including the identification of multiple analytes in a single assay. The invention thus provides, in one embodiment, nucleic acid hybridization assays, in which the first and second side elements of the dissociable signal elements include oligonucleotides. The simultaneous binding of a nucleic acid present in the test sample to the first and second side elements of the dissociable signal element prevents loss, through dissociation, of the signal responsive end of the signal element. In another aspect, the invention provides an assay device comprising dissociable signal elements responsive to a plurality of nucleic acid sequences. This aspect of the invention provides a suitable device and method for sequencing the acid nucleic through the capacity of spatial direction of the signals that are generated after contact with a sample that contains nucleic acid. The invention additionally provides immunoassays. In these embodiments, the laterale elements that confer specificity of the dissociable signal elements include antibodies, antibody fragments, or antibody derivatives. The simultaneous binding of an analyte to the antibody of the first side element and the antibody of the second side element prevents the loss, through dissociation, of the signal element signal responsive end. In another aspect, the invention provides test devices comprising a solid support substrate to which a plurality of dissociable signal elements are joined and over which there is also encoded digital information, in the form of computer software. 4. BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood by reference to the following drawings, in which: Figure IA is a schematic representation of a plurality of associative spacers covalently attached to their surface joining end, to a derivatized in the substrate of the test device.

Figure IB illustrates the union of a reflection signaling means, a metal microsphere, to the responsive signal ends of the plurality of dissociable spacers, creating elements of dissociable signals of reflection; Figure 2A is a schematic representation of a nucleic acid hybridization assay that is adapted to use the dissociable reflection signal elements of the present invention shortly after the introduction of a sample containing nucleic acids; Figure 2B is a schematic representation of a late stage of the assay procedure of Figure 2A, in which the oligonucleotides present in the sample have been fixed to the supplementary oligonucleotide side elements of a first dissociable signal element, but not have been attached to a second, different set of lateral elements of the oligonucleotide of a second dissociable signal element; Figure 2C is a schematic representation of a late stage of the test procedure of Figures 2B, after dissociation of the spacer molecules. The reflecting gold microsphere is removed, which is not blocked by the specific hybridization of the supplementary oligonucleotides from the test sample, from the surface of the test device, providing a signal dirigible in a spatial manner, in a differential way of reflection; Figures 2D-2E are schematic representations of an aspect of the invention, in which a soluble oligonucleotide that is added to the test sample, increases sensitivity in a nucleic acid hybridization assay; Figure 2F is a schematic representation, in a nucleic acid detection assay that is adapted to use the reflective dissociable signal elements of the present invention, of the use of DNA ligase to increase the resistance with which the specific binding of the analyte adheres to the responsive signal end of the dissociable spacer, to the derivatized substrate of the assay device, thereby allowing for an increased wash force and increased assay specificity; Figure 3A schematically depicts an immunoassay that is adapted to use the dissociable reflection signal element of the present invention. Figure 3A illustrates the antibodies, which are adapted to bind to an epitope site of an antigen suspected of being in a test sample, attached to the side elements of the dissociable spacers of a plurality of signal elements; Figure 3B is a schematic representation of a late stage in the assay process that is represented in the Figure 3A and illustrates the antigen binding from the sample of two antibodies of a dissociable signal element, but the antigen failure of the sample to be fixed to a second set of antibody side members, attached to a second signal element dissociable; Figure 3C is a schematic representation of the test of Figures 3A and 3B at a still later stage in the test process, after the dissociation of the spacers of the signal element. The reflecting gold microsphere is removed, which is not blocked by the association of specific antigen binding of the sample to the antibodies of the signal element, of the surface of the test device, providing a spatially steerable, reflection signal of differential way; Figures 4A to 4G illustrate schematically the preparation of the solid support substrate, on which the dissociable reflection signal elements are deposited in previously determined patterns, to create the spatially steerable test device of this invention; Figure 5A is a schematic representation of the chemical structure of an exemplary spacer molecule exemplary of the reflection dissociable signal element of this invention, subsequent to its attachment to the surface of the derivatized plastic substrate of the assay device, prior to derivatization with the lateral members of the oligonucleotide, in which piv denotes a pivaloyl protecting group, MMT denotes monomethoxytrityl, and n and m each independently represents an integer greater than, or equal to one; Figure 6 is a further schematic representation of a dissociable spacer molecule, which particularly illustrates the site in the spacer molecule that is susceptible to dissociation, and which further indicates the sites for attachment of the side members, which are shown protected by the pivaloyl and monomethoxytrityl groups; Figures 7A to 7C schematically illustrate a means for joining the molecules of the spacer dissociable to the activated surface of the substrate of the test device. In the illustrated example, the aminated surface of the substrate shown in Figure 7A is converted to active esters, as shown in Figure 7B. The dissociable spacer molecules are bound by activated esters to the solid support, as shown in Figure 7C; Figures 8A and 8B illustrate the intermediate steps during the attachment of a first lateral member of the oligonucleotide in the surface binding site of the dissociation site of a plurality of dissociable spacer molecules; Figures 9A and 9B are schematic representations illustrating the intermediate steps in joining a second oligonucleotide member on the signal responsive side of the dissociation site of a plurality of dissociable spacer molecules; Figure 10A is a schematic representation illustrating the substantially complete dissociable spacer molecule of the dissociable reflection signal element of the present invention, as bound to the solid substrate of the assay device, and before the attachment of the microspheres to the responsive end of signal of dissociable spacer molecules; Figure 10B illustrates the union of a single reflection particle with the responsive signal end of the dissociable spacers of Figure 10A, which complete the reflection dissociable signal element of the present invention; Figures HA to 11G illustrate different patterns of spatially steerable deposition of the dissociable reflection signal elements on circular, flat disk substrates, in which: Figure HA uniquely identifies a directional line, which can be encode on the disc substrate, from which the location of the dissociable spacers can be measured. In Figure HA, the dissociable spacer molecules are deposited in ring lanes; Figure 11B shows the spiral deposition of ios dissociable signal elements, and identifies in particular a central vacuum of the disk ring that is particularly adapted to engage the rotary transmission means; Figure 11C demonstrates the deposition of the dissociable signal elements in a suitable pattern for testing multiple samples in parallel, with the concurrent encoding of the interpretive software in the center lanes; Figure 11D schematically represents an embodiment in which the substrate of the test device has been further microfabricated, to segregate the individual test sectors, thereby allowing the rotation of the test device during the addition of the sample without mixing of the sample, - Figure HE schematically represents a modality in which the substrate of the test device has been further microfabricated, to compel the unidirectional sample flow during the rotation of the test device; Figure 11F demonstrates the deposition of the dissociable signal elements in a suitable spatial organization to test 20 samples for 50 different analytes each; Figure 11G shows the intersection pattern orthogonal that is created by superimposing the spiral patterns, with the spiral arms of opposite direction or chirality; Figure 12 is a schematic representation of the direction of the analyte-specific signals that are generated by the test device of Figure HA; Figure 13 is a schematic example of an impression to be used when printing the side members of the oligonucleotide on the dissociable spacers that are pre-bound to a solid substrate. The printing, as shown, is made of two pieces, a printing piece and a feeding piece. The print part contains holes, which are filled by the required chemicals through the feed piece containing channels. The channels, in turn, are connected to a capillary glass configuration. In this configuration, a row of holes is filled with the same chemical. Different patterns of hole and channel can be used as needed; Figure 14 is a schematic representation of the pattern of the side element of the oligonucleotide resulting from orthogonal two-step printing using the printout described in Figure 13. Numbers 1, 2, 3 and 4 represent different phosphoramidite sequences that they are used in the synthesis. In oligonucleotide syntheses using chronometers, for example, the number 1 can be AAA, the number 2 AAC, number 3 AAG and number 4 AAT. The first number in each place gives the building block of the oligonucleotides that is closest to the base structure of the dissociable spacer; the second member (if any) represents the next building block. Orthogonal printing is particularly convenient when depositing the dissociable reflection signal elements of the present invention, on a substrate in the form of a disc, - Figure 15 is a schematic representation of a supplementary concave printing process for printing large numbers of members laterals of oligonucleotides simultaneously on the dissociable spacers, previously bound to a solid substrate. Dissociable spacers themselves are not shown; Figure 16 demonstrates a geometry in which a single sample is channeled, in parallel into four different sectors of the test device. If either the density of the biobits or the affinity of the biobits differs in the four sectors, a large dynamic concentration range can be determined by detecting the position in each sector of the positive dissociable signal element furthest from the site of the biobits. application of the sample; Figure 17 demonstrates an alternative test device geometry that is distributed with the dissociable spacers, in which a first side element is attached analyte specific, directly to the substrate of the assay device, while a second analyte-specific side element binds directly to the signal responsive fraction, which is shown here as a plastic microsphere; Figure 18 demonstrates a further alternative geometry that is distributed with the dissociable spacers, in which a first side element is attached directly to the substrate of the test device, a second side element is directly attached to the signal responsive fraction, and the analyte causes the agglutination of the signal-responsive fractions.

. DETAILED DESCRIPTION OF THE INVENTION The assay device and the assay method of this invention use a dissociable signal element for the detection of analytes in fluid test samples. The binding of the analyte previously selected for detection avoids the loss - through dissociation - of the signal responsive fraction of the signal element. The generation of a signal from the signal responsive fraction of the restricted signal element is then used to signal the presence of the analyte in the sample. In a preferred embodiment, the responsive fraction of the signal reflects or scatters the incident light, or is another light-steerable -aired. The fixation of the analyte that was selected previously for detection, it avoids the loss - through dissociation - of the light responsive fraction of the signal element. The reflection or scattering of the incident light, preferably incident laser beam light, is then used from the reflection fraction of the restricted signal element, to signal the presence of the analyte in the sample. The reflective dissociable signal elements of the present invention are particularly adapted for detection, using detectors based on the reflectance of already existing lasers, including compact disc (CD) sound players, read-only memory readers of compact discs (CD-ROM), laser beam disc readers, digital video disc (DVD) players, and the like. In this way, the use of the dissociable reflection signal elements of the present invention allows for the easy adaptation of the existing test chemistries and the existing test schemes for detection, using the installed wide base of the detectors based on the reflectance of existing laser beams. This leads to substantial cost savings per test on standard tests using dedicated detectors. In addition, the wide and universal distribution of the detection equipment based on laser beam reflection further allows the tests - how they adapt to use the dissociable reflection signal element of the present invention - tests that are to be carried out at locations determined by the presence of a dedicated detector are distributed for use at point-of-service. These assays include immunoassays, cell counting, genetic screening assays based on hybridization, genetic screening assays based on nucleic acid sequencing, nucleic acid sequencing itself, and the like. The current invention allows for the distribution of test devices for research laboratories, doctor's offices, and individual homes, which must currently be performed in centralized locations. Each of the detectors based on the laser reflectance mentioned hereinabove - which include the read-only memory readers of compact discs, the digital video disc players and the like - is adapted to detect, discriminate, and interpret the digital information dirigible in a spatial manner in their respective medium: the compact disc readers can specifically and separately address multiple binary files, including binary file coding computer programs (the ISO 9660, which is incorporated herein by reference, defines a common steerable file structure), - in the same way also the digital video disc readers can direct fromspecifies and separates binary files and digital video signals encoded by MPEG. The spatially steerable capabilities of the laser-based reflectance detectors currently used to detect and interpret information encoded on compact discs and the like confer particular advantages in trials that are adapted to use the dissociable signal elements. of reflection of the present invention. In this way, pattern deposition of multiple signal elements on a single support member or substrate, coupled with the use of a detector that can direct the spatial location of these individual signal elements, allows concurrent testing of a single sample for multiple different analytes. In this manner, the present invention is further directed to the test devices, commonly referred to herein as discs, bio-compact disks, bio-compact discs, or digital video discs, which comprise combinations spatially dirigible elements of dissociable signaling signals of different analyte specificity. Among these useful combinations are those that increase the prediction value or the specificity of each of the individual trials, the combinations that inculpate or excuse particular diagnoses in a differential diagnosis; combinations that provide broad general selection tools, and the like. The pattern deposition of multiple signal elements with identical specificity additionally allows the detection, using a single assay device, of large concentration ranges of a single analyte. It is therefore another aspect of the present invention to provide test devices comprising elements of spatially controllable, dissociable reflection signals of identical specificity, whose physical location can transmit the concentration information. The spatially steerable capabilities of digital detectors based on laser reflectance further allow the combination of interpretive software and the same test elements on a single test device. Another aspect of the present invention, therefore, is a test device on which the software is encoded in a spatially distinct area of the pattern deposition of the dissociable reflection signal elements. The software can include important information for the correct tracking by means of the incident laser beam, the interpretative algorithms of the test, the standard control values, the self-diagnostics, and the like. The software can include transmitters of the device and software that can load the information of the diagnosis to remote places. The software can include patient education information for clinical trials, and can be adapted for selected audiences. The substantially binary nature of the test data indicating the dissociable reflection signal elements of the present invention has the additional advantage of providing tests that are adapted to their use substantially resistant to instrument noise. For example, small variations in the reflection of light - such as small variations in the intensity of the light provided by the laser source and small variation in the size of the reflection particle do not generally affect the result of the test, since the detectors only record a signal when the reflection of the light reaches a threshold. Similarly, the electronic noise of the detection device itself and the noise associated with an analog-to-digital conversion does not affect the test results. This advantage is particularly appreciated in the design and manufacture of strong detection instruments useful for field testing or for testing under difficult environmental operating conditions. . 1 Elements of dissociable reflection signals, spatially steerable The general operation of the signal element This invention, which is also called a biobit, can be understood more particularly by means of reference to Figures 1-3, which schematize embodiments of the present invention. With reference to Figure 1, there is provided a substrate 20 with a derivatized surface 21 to which the dissociable spacer molecules 30 are attached, each dissociable spacer having, in addition to a surface joining end, a responsive signal extremity, which is sample close to the d metal microsphere 40. The substrate, which may be porous or solid, although currently solid is preferred, may be selected from a variety of materials such as plastics, glass, mica, silicon, and the like. However, plastics are preferred for reasons of economy, ease of derivatization to bind the sparger molecules to the surface, and compatibility with laser-based reflectance detectors, such as compact disc reading only readers. and digital video disc. Typical plastics that can be used are polypropylenes, polyacrylates, polyvinyl alcohols, polyethylenes, polymethylmethacrylates and polycarbonates. Polypropylene and polycarbonate are currently preferred, and polycarbonate is most preferred. The surface 21 of the substrate 20 can be conveniently derivatized to provide fixation. covalent to each of the dissociable spacer molecules 30. The metal spheres provide a convenient reflection signal generating means for detecting the presence of a spacer molecule binding to the substrate 20 of the assay device. Typical materials are gold, silver, nickel, chromium, platinum, copper, and the like, with gold being currently preferred for its ability to be fixed in a simple and narrow manner for example, by dative fixation to a free SH group in the signal responsive end of the spacer dissociable. The metal spheres can be solid metal or they can be formed of plastic, or glass beads or the like, on which a metal coating has been deposited. Also, other reflection materials can be used in place of the metal. The gold spheres that are currently preferred are fixed directly to the thio group of the signal responsive end of the spacer that can be dissociated. Each of the dissociable spacer molecules is attached to one end 31 to support the surface 21, for example, by an amide bond, and at the other end 32 to a signal generating means (which is also called a signal responsive fraction). , for example by means of a thio radical to a reflection metal microsphere 40. The spacer molecule has a dissociation site 33 which is susceptible to dissociation during the test procedure, by chemical or enzymatic means, heat, light or the like, depending from the nature of the dissociation site. Chemical means are presently preferred with a siloxane dissociation group, and a sodium fluoride solution, exemplary, respectively, of a chemical dissociation site and a chemical dissociation agent. Other groups susceptible to dissociation may also be used, such as ester groups or dithio groups. The dithio groups are especially convenient if gold spheres are added after dissociating the spacer. The dissociation site 33 is between the first surface junction end 31 of the dissociable spacer molecule 30, and the second. signal responsive end 32 of the dissociable spacer molecule 30. The spacers can contain two or more dissociation sites to optimize the complete dissociation of all spacers. The specificity of the analyte is conferred on the spacer dissociable by the side members 34a and 34b, which are also called lateral arms, placed on opposite sides of the dissociation site 33.; that is, placed close to the surface junction end and close to the signal responsive end of the dissociable spacer molecule 30, respectively. Side members 34a and 34b in their typical configuration include an oligonucleotide, typically 5- to 20-mers, preferably 8- to 17-mers, more preferably 8- to 12-mers, although oligonucleotides can be used longer. The side members also they may include, without limit and as required, peptides, organic linkers to peptides or proteins, or the like. A large number of dissociable spacer molecules will be present at any particular dissociation site on the solid surface 21 of the assay device, which is also called a disk, a biocompatible disk, or BCD. In one aspect of the invention, the lateral members of the oligonucleotide are adapted to fix additional unique chains of nucleic acids that may be present in a test sample. The supplementary oligonucleotides comprise members of a specific binding pair, ie, an oligonucleotide will be attached to a second supplementary oligonucleotide. As more particularly described in Figures 2A through 2C, which outline one embodiment of the invention, spacer molecules dissociable at different sites on the surface of the assay device will have different oligonucleotide side members. As shown in Figure 2A, such a dissociable signal element has oligonucleotide side members 34a and 34b, while the second dissociable signal element has oligonucleotide side members 35a and 35b. As further described in Figures 2A to 2C, when contacted with a test sample containing an oligonucleotide 36, the side members 34a and 34b of the supplementary oj-igonucleotide will be fixed with the oligonucleotide present in the sample, to form a double helix as shown in Figure 2B. Since there is no complementarity between the oligonucleotide 36 and the lateral members 35a and 35b of the oligonucleotide, there is no binding between those groups, as further illustrated in Figure 2B. When the dissociation site 33 is dissociated, but for fixation by the double helix coupled oligonucleotides, the metal microspheres 40 will be free of the surface and will be removed therefrom. This is illustrated more fully in Figure 2C. If it is desired to test multiple samples for a single oligonucleotide, the spacing molecules at different sites will generally have the same oligonucleotide side members. The presence and absence of the metal microsphere 40 can then be detected as the reflectance or the absence of reflectance of the incident light, particularly the incident laser beam light. Figure 2F is a schematic representation of the use of the DNA ligase in a further embodiment of the nucleic acid detection modality of the present invention to increase the force with which the specific binding of the analyte adheres to the signal responsive end of the spacer dissociated to the derivatized substrate of the test device, thereby allowing this mode, an increased wash force, producing an increased specificity of the test. Those skilled in the detection of nucleic acid will appreciate that the dissociable reflection signal elements of the present invention are particularly suitable for detecting amplified nucleic acids of defined size, in particular nucleic acids using different forms of the polymerase chain reaction (PCR ), the ligase chain reaction (LCR), amplification schemes using T7 and SP6 RNA polymerase, and the like. In a further embodiment of the invention described in Figures 3A to 3C, the lateral members 34a, 35a, and 35b of the oligonucleotide, are non-covalently coupled to the modified antibodies 38a, 38b, 38c, and 38d, to allow an immunoassay.The non-covalent binding of the modified antibodies to the lateral members is mediated through the complementarity of the oligonucleotides of the lateral member of the dissociable spacer and the oligonucleotides that are covalently bound to the antibodies. the use of the supplementary nucleic acid molecules to effect the non-covalent, combinatorial assembly of the supramolecular structures, in the co-proprietary and co-pending applications of US Pat. Nos. 08 / 332,514, filed on October 31:. : < 94 08 / 424,874, filed April 19, 1995, and 08 / 627,695, filed March 29, 1996, which are incorporated herein by reference. In another embodiment, the antibodies can be covalently linked to the cleavable spacer, using conventional crosslinking agents, either directly or through linkers. The antibodies comprise a first member of a first specific binding pair and a first member of a second specific binding pair. The second member of the first specific binding pair and the second member of the second specific binding pair will be epitopic sites different from an antigen of interest. More specifically, the lateral member 35a of the oligonucleotide binds to the antibody-oligonucleotide 38c and the lateral member 35b of the oligonucleotide binds to the antibody-oligonucleotide 38d. Antibodies 38c and 38d are adapted to fix the different epitope sites on an antigen that may be present in the test sample. By means of the different epitope sites in an antigen, different occurrences, spatially separated, of the same epitope or of different epitopes present in different sites are intended. In a second assay element, side members 34a and 34b of the oligonucleotide are linked to different antibodies 38a and 38b, each of these antibodies is again adapted to bind to an epitope site different from an antigen.

With further reference to the immunoassay which is schematized in Figures 3A to 3C, after application of the test solution containing the antigen 39, to the collection of the dissociable reflection signal elements which are illustrated in Figure 3A, the antigen 38 binds the antibodies 34a and 34b, thereby preventing uncoupling of the metal sphere 40 from the surface of the test device 20, when the dissociation site 33 is dissociated, such as, for example, by contacting an agent dissociation chemical. In contrast, the second dissociable signal element, which was not fixed by antigen 39 due to lack of binding affinity of antibodies 35a and 35b with antigen 39, allows the metal microsphere 40 to separate from the surface solid and that is removed from the sample. The presence or absence of the metal microsphere 40 can then be detected as the reflectance or the absence of reflectance of the incident light, in particular the incident laser beam light. As should be apparent, the coupling of the antibodies as described allows rapid adaptation of the immunoassay chemistries and standard immunoassay geometries for use with the dissociable reflection signal elements of the present invention. In the United States of America Patent Number 5,168,057, filed on December 1, 1992, which is incorporated herein by reference, some of these classical immunoassay geometries are further described. Thus, it should be apparent that the direct detection of the analyte that is schematized in Figure 3 is but one of the immunoassay geometries that can be adapted to the dissociable reflection signal elements and to the assay device of the present invention. . The present invention will prove to be particularly valuable in the selection of immunoassays for human immunodeficiency viruses, hepatitis A virus, hepatitis B virus, hepatitis C virus, and human herpeviruses. It will be further appreciated that the antibodies are exemplary of the broader concept of specific binding pairs, where the antibody can be considered the first member of the specific binding pair, and the antigen to which the second member of the specific binding pair is attached. . In general, a specific binding pair can be defined as two molecules whose mutual affinity is of sufficient strength and specificity to allow the practice of the present invention. In this way, the dissociable reflection signal elements of the present invention can include other members of specific fixing pairs as side elements. In these embodiments, the first lateral member of the dissociable signal element includes a first member of a first specific fixation pair, the second lateral member of the dissociable spacer includes a first member of a second specific fixation pair, wherein the second member of that first specific fixation pair and the second member of that second specific fixation pair, they are joined in a connective manner with each other, allowing the formation of a closed loop circuit of the general formula: the first member of the first specific fixing pair-second member of the first specific fixing pair-first member of the second fixing pair specific. Specific binding pairs well known in the art include biological receptors and their natural agonist and antagonist ligands, proteins and cofactors, biotin and either avidin or streptavidin, alpha spectrin and beta spectrin monomers. , and the Fc portions of antibody and Fc receptors. Although the modalities exemplified above - the direct detection of nucleic acid analytes and the direct immunoassay - have been described with the reflecting metal spheres attached to the spacer molecules that can be dissociated prior to conducting the assay, it is contemplated in this and in other embodiments that are further described herein, that the spacer molecules that are dissociable lacking a signal generating means, are they can first expose the sample, then dissociate, and then add the metal spheres in order to join only those spacer molecules that remain on the surface. After the addition of the metal spheres, the surface can then be read with an appropriate detector to identify the spacer molecules and the fixed analytes. In each of the embodiments of the test method of the invention, a sample to be tested must first be introduced. In one aspect, the test device is rotated and a sample of fluid, preferably diluted, is applied near the center of the substrate of the circular test device. Centrifugal forces associated with the rotation of the disc of the test device, distribute the fluid sample through the flat face of the solid substrate. In this way, the surface of the substrate is uniformly covered with a constant and evenly distributed fluid sample. In this method of application of the sample, the test sample is diluted, initially 100 μl, for processing to approximately 1 milliliter. This solution is added by dripping near the center of the rotating disc. The test sites and possibly the surface of the disc are hydrophilic and a fluid will form a very thin layer on the disc of the rotating test device. You can regulate the thickness of the fluid layer by the frequency of the addition of the drop and the frequency of the disk rotation. A preferred thickness is less than 10 micrometers, because all the molecules in the sample can then interact with the stationary molecules fixed by the spacers. Approximately 100 μl of the sample solution is needed to cover the disc. Other methods of sample application may be used with the dissociable reflection signal element and the assay device of the present invention. In particular, it should be appreciated that the rotary application described above is suitable principally for the application of a single sample per test device. In other aspects of the present invention, separate samples may be applied in discrete areas of a stationary disk. In this aspect, the test system can test approximately one thousand different samples. Approximately one million gold spheres can be placed on the disk, for each sample. Figure 11D shows a test device of the present invention, which has 16 separate test sectors. Figure HE shows a possible direction for the flow of the sample, with barriers to the flow of the fluid shown as lines. Thus, in one embodiment of the invention, design the assay device to test, for example, 1024 patient samples simultaneously, one analyte per assay device (i.e. per disc, each disc comprising a plurality of dissociable spacers with identical side members that confer identical analyte specificity ). In this embodiment, each of the spacer molecules on the disk must be identical, so that the same analyte can be tested; The spacer molecules at specific places on the disk will be identical to the spacer molecules elsewhere on the disk. This application is particularly useful in mass analyzes conducted in clinical laboratories, where a large number of patient samples are analyzed at the same time, to see the presence or absence of a single analyte. It will also be appreciated that multiple samples can be assayed by multiple analytes in a single assay device comprising elements of dissociable reflection signals with different analyte specificities. Figure 11F shows a test device that can be used to select 20 samples for 50 different biomolecules. In the latter case, it is possible to assay for a limited number of the same analytes in a multiplicity of test samples. Patient samples can be applied to the disc at specific locations by known methods, such as inkjet printing and configurations of micropipette with disposable tips, or a combination thereof. For large performance operations, the test disks can be loaded into a cartridge and the test samples loaded airtight, either directly on the disk or into the wells on a circular plate. After an appropriate incubation period, which can be only a few seconds to allow the sample to cross the surface of the support, it can be done, although in some modalities it is not necessary to do it, a washing step, to remove the sample. fixed. The strength of the wash can be adjusted as in conventional tests, to adjust sensitivity and specificity. For example, in the nucleic acid detection modalities, the salt concentration of the wash solution can be decreased to increase the wash force - thereby reducing the inequality between the analyte and the side members conferring specificity - - or it can be increased, to decrease the force of the washing, allowing by the same thing that an inequality occurs. The adjustment of the washing force in the hybridization of the nucleic acid and in the "immunoassay" embodiments of the present invention is completely within the skill in the art In one aspect, the surface of the circular disc is washed, when necessary , by adding a wash solution near the center of the rotating disc! Remove the sample solution as it leaves the periphery of the disc and collect. Due to the rotation of the disc, the washing step can be eliminated if the disc fluid sample is properly removed by normal centrifugal forces and no adjustment in the wash force is required. After the washing step, if any, a solution including a dissociating agent is added and distributed again on the surface of the disk. With reference to Figures 1-3, the spacer molecule has a dissociation site 33 that is susceptible to dissociation during the assay procedure, by chemical or enzymatic means, heat, light or the like, depending on the nature of the dissociation site. . At present, the chemical means with the siloxane cleavage group are preferred, and a solution of sodium fluoride is exemplary as a chemical dissociation agent for the siloxane group. Other groups susceptible to dissociation may be used, such as ester groups or dithio groups. The dithio groups are especially convenient if gold spheres are added after dissociating the spacer. In the case where the dissociation site is a siloxane fraction, which can be made stable against spontaneous hydrolysis but which dissociates easily under benign conditions by means of a fluoride ion, it is introduced a fluoride solution, with a concentration of 1 mM to 1 M, preferably 50 mM to 500 mM, more preferably 100 mM (0.1 M). The dissociation step will only last a few seconds. Although all spacers are dissociated during this step, the amide fixation between the dissociable spacer and the derivatized substrate of the test device remains stable under these conditions. After the application of the sample and the dissociation of the spacers, "the generating fractions of the signal must be separated, preferably a fraction of reflection, more preferably a metal sphere, more preferably a gold sphere, to provide The differential signal during the detection The removal step can include a second washing step, which can include the introduction of washing solutions There are different means by which differential washing forces can be developed in this stage of the test, allowing by the same the variation in the specificity and the sensitivity of the different test methods In one aspect, the separated reflection fractions can be removed by rotating the test device, with or without the addition of the washing solution. In this aspect, three parameters can be varied to provide the differential force: gold particle size, ve l ci dad rotational, and the valence of the spacer joint. Gold spheres suitable for use in the dissociable reflection signal element and the assay device of the present invention are readily available in variable diameters with the Aldrich Chemical Company, British BioCell International, Nanoprobes, Inc., and others, which range from 1 nanometer to, and including, 0.5-5 micrometers in diameter. The creation of gold spheres of smaller or larger diameter, as needed in the present invention, is included within the skill of the art. At a given rotational speed, the larger gold spheres experience centrifugal forces (relative to r3) and larger drag forces (relative to r) and are removed earlier than smaller spheres with similar fixation. This provides a basis for the differential force of the wash and also of the quantitative analysis. The centrifugal force affecting the gold spheres can also be adjusted by the rotation frequency so that the loose and weakly fixed spheres are removed. Only the spacers that have attached themselves to a supplementary molecule from the sample will continue to fix the gold spheres to the substrate. In addition, although the embodiments of the invention have been described with a single metal sphere attached to the signal responsive end of a single dissociable spacer, it should be appreciate that when gold is used in a preferred embodiment of the invention, thousands of spacers can fix a gold sphere, depending on its diameter. In this way, the strength of the test wash can be adjusted, at any given rotary speed, by varying the diameter of the gold sphere, and by additionally varying the relative density of the spacer dissociable to the gold spheres. . In this way, if practically all the spacers under a certain sphere of gold are connected by supplementary molecules, the fixation is very strong. If the spacers are only partially fixed under a certain sphere of gold, the sphere can remain or be removed depending on the radius of the sphere and the frequency of the rotation. In extreme cases, all spheres are either fixed or removed. These are alternatives that are expected for DNA analysis. In immunoassays, intermediate cases are preferred. In accordance with the above, the system must be optimized so that the normal control level corresponds to 50 percent of the fixation of the gold spheres. The highest or lowest fixation corresponds to the highest or lowest concentrations of the analyte, respectively, when two antibodies are used to fix, as illustrated in Figure 3. A strong centrifugal force can be used to remove the gold spheres fixed weakly. The centrifugal force that pulls a gold sphere will be of the order of 0.1 nN, although this force can vary within wide limits, depending on the mass of the gold sphere and the frequency of rotation of the disk. The force is strong enough to break the non-specific binding of the antibodies and to mechanically denature the unequal oligonucleotides. This is a very strong factor for increasing the strength of the interaction between the analyte and the dissociable signal elements of the present invention. In the embodiments of the present invention in which the reflection fraction of the dissociable spacer is ferromagnetic, as, for example, in which the reflection fraction is an iron pellet covered with gold or an iron alloy, those reflection fractions that are separated by dissociation and that are not secured to the substrate of the test device by the analyte, by the application of a magnetic field. In these modalities, those signal elements that remain attached to the substrate of the test device (disk), will also be responsive to the metal field, but their movement will be restricted by the length and flexibility of the cycle that forms the first lateral-analyte member -second side member. The ability to change the position of all signal elements linked through the application of a external magnetic field, although that change will necessarily be restricted by the length and flexibility of the cycle of the first lateral member-analyte-second lateral member, can add, in this modality, additional information. In particular, the brief application of a magnetic field will facilitate the discrimination of the signal induced by analyte from random noise, the noise being non-responsive to the application of an external magnetic field. After the removal of fractions of dissociable reflection signals that are not protected by the specific binding of the analyte, the disc can be read directly. In yet another embodiment, the disc can be covered by the transparent plastic coating in an optical manner to prevent further reaction of the gold spheres through the spin coating with a polymerizable varnish that is polymerized with ultraviolet light. The spinning coating of the compact disk is well established in the art: The test disk is expected to have a shelf life of more than ten years. Subsequently, the disk can be scanned by means of a laser beam reader which will detect, through reflection, the presence of a microsphere or gold reflection element in the different places previously determined in a spatial manner. Based on the distance of the microsphere from the axis of rotation of the disk and the angular distance from a Address line that forms a radial line on the disk, can be determined specifically the location of a specific metal sphere. Based on the specific location and previously determined locations of the specific binding pairs as compared to a main distribution map, the identity of the fixed material can be identified. In this way, in the above manner it is possible to analyze in a fluid sample thousands, or even larger numbers, of analytes simultaneously. . 2 Derivatization of the substrate Figures 4A to 4G illustrate schematically the preparation of the solid support substrate on which the dissociable reflection signal elements are deposited, to create the assay device of this invention. A portion of a generally flat solid support is illustrated in Figure 4A. As illustrated in Figure 4B, the surface of the support is coated with a protective layer 22, for example, a high melting point wax or the like. Next, a pattern of indentations or holes 25 is created in the protective layer by means of printing with the form 23 containing projections 24, as illustrated in Figure 4C. The pattern is quite regular and the indentations are made in all the places in which the separable spacer molecules would be conveniently located on the surface of the substrate. bearing. Any protective layer that remains on the bottom of the indentations, as illustrated in Figure 4D, is removed, as shown in Figure 4E. The exposed areas of the substrate 21 are activated or derivatized, as illustrated in Figure 4E, to provide binding of the fixing groups (eg, amino groups) to the surface of the substrate and to any remaining protective layer 22. , as shown in Figure 4F. Finally, the remaining protective layer is removed to expose the original surface of the substrate to which the amino groups are attached at certain previously determined sites, as illustrated in Figure 4G. Blank discs are available with Disc Manufacturing Inc. (Wilmington, Delaware). Derivatization of the amino acid can be performed by ammonia plasma using a radio frequency plasma generator (ENI, Rochester, NY). . 3 Synthesis and union of the dissociable spacers With reference to Figure 1 and Figures 5 and 6, a dissociable spacer molecule is described. The majority of the spacer, called the base structure, is poly (alkylene glycol), for example, polyethylene glycol, which has a molecular weight of 400-10,000, preferably 400-2000. The base structure has a first end 31 which is adopted to couple to a derivatized amino group present in the surface 21 of the substrate 20, and a second end 32, which is adapted to mate with the surface 41 of the metal micro sphere 40, by a link 51. In addition, between the end 31 and the site of dissociation 33, there is a side member 34a, which is commonly constructed from an oligonucleotide. Alternatively, these side members may be peptides-two or other organic molecules. More than two lateral members can be provided, but it is only necessary that two members can form a connective, molecular cycle around the dissociation site to fix the spacer molecule to the surface of the substrate, after dissociation into the substrate. the dissociation site. These side members can be joined to the base structure of the spacer by the linkers, such as polyethylene glycol. A mode of synthesis of the dissociable spacer molecule 30 which is illustrated in Figure 5 is substantially and generally as follows: chlorodimethylsilane is coupled to both ends of a polyethylene glycol molecule. The silane group that is incorporated into the molecule reacts in the presence of the catalytic amounts of the chloroplatinic acid within the N-acryloyl serine. The hydroxyl groups of the two serine fractions in the synthesis will be used later for the construction of the oligonucleotide side members. A hydroxyl group is first protected by a monomethoxytriphenylmethyl group and the product is purified by liquid chromatography. The other hydroxyl group is then protected with a pivaloyl or fluorenylmethyloxycarbonyl group (FMOC). Serine carboxyl groups are coupled with the amino-terminated poly (ethylene glycol). The amino group is further derivatized at the other end by the hydroxysuccinimide ester of 3- (2-pyridyl-dithio) propionic acid. The other amino group is not reacted, but is free to react later with the derivatized substrate. Further and in the Preparations that follow, an alternative, but substantially similar, and more detailed description of the synthesis of the spacer molecule is provided. The structure of the spacer molecule is shown schematically in Figure 5. The synthesis is started by first constructing the central portion of the spacer molecule. The two ends of the poly (ethylene glycol) are then silanized, for example, with chlorodimethylsilane to produce a compound of the formula of Compound 1. The silane groups are then derivatized with straight or branched chain alkenoic acid (for example, CH = CH (CH2) nC00H, n = l-ll, although the number of carbon atoms is insubstantial, such as vinylacetic acid, acrylic acid and the like), which have a terminal double bond, such as vinylacetic acid, to form a compound having the structural formula of Compound II, and reacting to provide a protected hydroxyl group on each side of the silane, to provide a later binding of the oligonucleotides, as illustrated by the compound having the structural formula of Compound III. Different common reagents can be used for this purpose, and the N-acryloyl serine and the methyl ester of TMT-serine when allowed to react in the presence of a catalyst, such as chloroplatinic acid, are exemplary of the preferred reagents. The resulting ester is partially hydrolysed, by the addition of an alkali metal hydroxide, such as sodium hydroxide, in an alcohol solvent, and preferably hydrolyzes the adjacent protected hydroxyl group to produce a compound representing the formula Structure of Compound IV The amino-terminated poly (ethylene glycol) at one end is derivatized with a thioether, such as the hydroxy-succinimide ester of 3- (2-pyridyl-dithio) propionic acid, and coupled with Compound IV to produce a compound which represents the structural formula of Compound VI. The terminal ester group is hydrolyzed to produce the acid, which is further reacted with the methoxyacetic acid, to produce the compound representing the structural formula of Compound VIII. This compound is treated with the aminated poly (ethylene glycol) to form the finished spacer molecule, substantially as illustrated in Figure 5.

Preparation 1: Compound IA a mixture of poly (ethylene glycol) (10 grams, 10 mmol, av. Molecular weight of 1,000 Aldrich Chemical Company) and triethylamine (TEA) (2.1 grams, 21 mmol) in 100 milliliters of dichloromethane (DCM), 2.0 grams of chlorodimethylsilane in 20 milliliters of dichloromethane are added dropwise, with cooling in an ice bath. After 10 minutes, the reaction mixture is filtered, and the filtrate is applied to a 200 gram silica column. The column is leached with dichloromethane / MeOH 19: 1, and the leaching gives poly (ethylene glycol), di (dimethylsilyl) ether, the compound represented by the structural formula of Compound I.

Preparation 2: Compound II Compound I (10 grams, 9 rrrr.ci) and vinylacetic acid (1.72 grams, 20 mmol) are dissolved in 60 milliliters of ethyl acetate (EtOAc). A catalytic amount (40 milligrams) of chloroplatinic acid is added, and the mixture is heated to boiling, and boiled for 1 hour. After cooling, the solution is applied directly on a 200 gram silica column. The column is leached with EtOAc and EtOAc / MeOH 9: 1, and leaching gives poly (ethylene glycol), di (2-carboxyethyldimethylsilyl) ether, the compound represented by the structural formula of Compound II.

Preparation 3: Compound III Compound II (9.5 grams, 8 mmol) and trimethoxytritylserine methyl ester (7.0 grams, 16 mmol) are dissolved in 100 milliliters of dichloromethane. Dicyclohexylcarbodiimide (DCC) (3.25 grams, 16 mmol), in 30 milliliters of dichloromethane, is added dropwise at room temperature. After 1 hour the reaction mixture is filtered. The filtrate is applied directly to a 300 gram silica column. The column is leached with dichloromethane / triethylamine 99: 1, and then with dichloromethane / MeOH / triethylamine 94: 5: 1. The leaching gives the compound represented by the structural formula of the Preparation 4: Compound IV Compound III (10 grams, 5 mmol) is dissolved in 100 milliliters of EtOH, and partially hydrolyzed by the addition of 10 milliliters of 0.5 M NaOH in EtOH. The mixture is acidified slightly by the addition of 300 milligrams (5 mmol) of acetic acid, and preferably the TMT group close to the carboxylate group is hydrolysed. After 30 minutes, the mixture is made slightly basic by the addition of 0.5 milliliter of tetraethylamine (TEA). The EtOH solution is fractionated by high performance liquid chromatography, using a reverse phase column leached with EtOH / water / TEA 90: 9: 1. The leaching gives the compound represented by the structural formula of Compound IV.

Compound IV Preparation 5: Compound V O, O '-bis (aminopropyl) polyethylene glycol (9.5 grams, 5 mmol, av. Molecular weight of 1900), triethylamine (0.5 grams, 5 mmol) and N-hydroxysuccinimide acid ester are dissolved. - (2-pyridyldithio) propionic (0.77 grams, 2.5 mmol), in 150 milliliters of dichloromethane. The mixture is stirred for 1 hour at room temperature, concentrated to half the volume, and fractionated on a 200 gram silica column. The column is leached with dichloromethane / MeOH 95: 5 to give the compound represented by the structural formula of Compound V.

Compound V Preparation 6: Compound VI Compound IV (3.5 grams, 2 mmol) and compound V (4.4 grams, 2 mmol) are dissolved in 100 milliliters of dichloromethane, and 450 milligrams (2.2 mmol) of dicyclohexylcarbodiimide are added in 5 milliliters of dichloromethane. After one hour the mixture is filtered, and fractionated on a 150 gram silica column. The column is leached with dichloromethane / MeOH / TEA 94/5/1, to give the compound represented by the structural formula of Compound VI.

Compound VI Preparation 7: Compound VII Compound VI (6.0 grams, 1.5 mmol) is dissolved in 50 milliliters of EtOH, and 3 milliliters of 0.5M NaOH in EtOH are added. After 30 minutes the product is purified by reverse phase high performance liquid chromatography, using EtOH / water / TEA 90: 9: 1 as a leach, to give the compound represented by the structural formula of Compound VII.

Compound VII Preparation 8: Compound VIII Compound VII (4.0 grams, 1 mmol) is dissolved in 80 milliliters of dichloromethane. The mixture of 320 milligrams (2 mmol) of methoxyacetic acid anhydride and 202 milligrams (2 mmol) of triethylamine in 5 milliliters of dichloromethane is added. The mixture is evaporated by means of a rotary evaporator until it is dried. The residue is purified by reverse phase high performance liquid chromatography, using EtOH / water / TEA 90: 9: 1 as a leach, to give the compound represented by the structural formula of Compound VIII.

Compound VIII Preparation 9: Compound IX Compound VIII (4.0 grams, 1 mmol) and O, O '-bis (aminopropyl) polyethylene glycol (4.8 grams, 2.5 mmol, av. Molecular weight of 1900) are dissolved in 100 milliliters of dichloromethane. , and 230 milligrams (1.1 mmol) of dicyclohexylcarbodiimide in 5 milliliters of dichloromethane are added. After 1 hour the mixture is filtered, and the mixture is fractionated on a 100 gram silica column, using dichloromethane / MeOH / TEA 94/5/1 as a leach, to give the compound represented by the structural formula of Compound IX , substantially as depicted schematically in Figure 5.

Compound IX .4 Union of Spacers Dissociable to the Substrate Each of the spacer molecules is joined at one end 31 to support the surface 21, for example, by an amide bond. In order to bind the spacer molecules to the amino-activated substrate, glutaric anhydride is reacted with the amino groups to expose a carboxylate group, which is shown more particularly in Figures 7A and 7B. The carboxylate groups can be esterified with pentafluorophenol. The free amino group in the spacer molecule will be coupled with this active ester. Particularly shown in Figure 7C are the spacer molecules and their junctions at the discrete sites to the solid support surface 21. At this stage in manufacturing, the hydroxyl groups remain protected. Although the side members of the oligonucleotide can be pre-synthesized in the spacers prior to binding to the solid surface support 21, it is preferred that they bind after the spacer molecule is attached to the solid support. . 5 Design and binding of fractions responsive to signals A feature of the present invention is the detection of signal-responsive fractions, associated with the dissociable spacer molecules deposited in spatially steerable patterns, previously determined, in the surface of the test device. In accordance with the foregoing, this invention provides methods, compositions and devices for joining fractions responsive to signals, and for detecting signals associated with spacer molecules that can be dissociated. . 5.1 Gold Particles as Fractions Responding to Signals In some preferred embodiments of the present invention, particles that reflect or scatter light are used as signal-responsive fractions. A particle that reflects and / or scatters light is a molecule or a material that causes the incident light to be reflected or elastically scattered, that is, substantially without absorbing the energy of the light. These particles that reflect and / or scatter light include, for example, metal particles, colloidal metal such as colloidal gold, colloidal non-metal labels such as colloidal selenium, dyed plastic particles made of latex, polystyrene, polymethylacrylate, polycarbonate or similar materials. The size of these particles varies from 1 nanometer to 10 micrometers, preferably from 500 nanometers to 5 micrometers, and more preferably from 1 to 3 micrometers. The larger the particle, the greater the effect of light scattering. Since this will be true of the both fixed and crude solution particles, however, the background can also be increased with the size of the particle that is used for scattering signals. Metal microspheres from 1 nanometer to 10 μm (micrometers) in diameter, preferably 0.5-5 micrometers, more preferably 1-3 micrometers in diameter, are preferred in the light reflection / light scattering mode of the present invention. The metal spheres provide a fraction that responds to signals convenient for the detection of the presence of a spacer molecule, dissociated, but restricted by analytes, fixed to the disk. Typical materials are gold, silver, nickel, chromium, platinum, copper, and the like, or alloys thereof, gold being currently preferred. The metal spheres may be solid metal or they may be formed of plastic, or glass globules or the like, after which a metal coating has been deposited. Similarly, the metal surface reflecting the light can be deposited on a metal microsphere of different composition. The metal spheres can also be alloys or aggregates. Gold spheres suitable for use in the dissociable reflection signal element and the assay device of the present invention can be easily obtained in variable diameters with Aldrich Chemical Company, British BioCell International, Nanoprobes, Inc., and others, varying from 1 nanometer to, and including 0.5 micrometers (500 nanometers) - 5 micrometers in diameter. It is within the skill in the art to create gold spheres of smaller diameter, as needed in the present invention. Conveniently, much smaller waits can be used when reading with near field optical microscopy, ultraviolet light, electron beam scanning probe microscopy. In these latter embodiments, smaller spheres are preferred because more dissociable spacers can be discriminated in a given area of a substrate. Although spherical particles are currently preferred, n-spherical particles are also useful for some embodiments. In biological applications, the fractions which respond to signals - particularly latex gold microspheres - will preferably be coated with detergents, or they will be derivatized, so that they have a surface charge. This is done to prevent the binding of these particles non-specifically with the surfaces, or with one another. The preferred gold spheres are currently fixed directly to the thio group of the end that responds to the dissociable spacer signal, producing a very strong fixation After the synthesis of the bracelet is completed At the side of the oligonucleotide, as will be described later, the pyridyldithium group present at the end which responds to signals from the spacer molecule, with dithioerythritol or the like, is reduced. The reaction is very rapid quantitative, and the resulting reduced thio groups have a high affinity for gold. Accordingly, the gold spheres are spread as a suspension in a liquid (eg, distilled water), by adding the suspension to the surface of the solid support 21. The gold sphere will only be attached to the sites that cover the spacers terminated in uncle, and will not join the remaining surface of the substrate.On the other hand, although the previous embodiments of the present invention have been described with a sun metal sphere attached to the end that responds to signals of u only dissociative spacer, it will be noted that when gold is used in a preferred embodiment of the invention, thousands of spacer can be attached to a gold sphere, depending on its diameter, It is estimated that approximately 1,000-10,00 spacer can be set dissociable to one 1-3 micrometer sphere As a result, the wash stringency of the test can be adjusted at any given rotational speed by varying not only the diameter of the test gold sphere, but also of the relative density of the spacer dissociable to the gold spheres.

In accordance with the above, if virtually all the spacers under a certain gold sphere are connected by complementary molecules, the fixation is very strong. If the spacers are only partially fixed under a certain gold sphere, the sphere can remain or be removed depending on the radius of the sphere, and the frequency of the rotation. . 5.2 Other Fractions Responding to Light-Responding Signals In some other embodiments of the dissociable signal element and the test device of the present invention, a light-absorbing material may be used rather than a light-reflecting material, such as a fraction that responds to signals. In this modality, the absence of reflected light from a directed location, rather than its presence, indicates the capture of analytes. The approach is analogous to, although in some way different from that used in compact discs that can be recorded. Although similar in concept and compatible with compact disc players, the information is recorded differently on a compact disc that can be burned (CD-R), compared to the information encoded by slits on a standard compact disc, compressed. On the compact disks that can be read the data layer is separated from the polycarbonate substrate. The polycarbonate substrate rather has a continuous spiral groove printed thereon, as a reference alignment guide for the incident laser. An organic dye is used to form the data layer. Although cyanine was the first material used for these discs, a cyanine compound stabilized by metal is generally used instead of "raw" cyanine. An alternative material is phthalocyanine. In U.S. Patent No. 5,580,696 describes one such metallo-phthalocyanine compound. In the compact discs that can be read, the organic dye layer is sandwiched between the polycarbonate substrate and the metallized reflection layer, usually 24 carat gold, but alternatively silver, of the medium. The information is recorded by an appropriately selected pre-selected wavelength laser beam that selectively fuses the "slits" in the dye layer - rather than burn holes in the dye, it simply fuses it slightly, causing it to return non-translucent, such that the laser beam is refracted instead of being reflected back to the reader's detectors, such as by a physical slit in the compressed standard compact disc. As in a standard CD, a varnish coating protects the layers that carry the information. In this embodiment of the present invention, Use a greater number of light-absorbing dyes, of which you can use them on the CD-R. The light absorbing dyes are any compounds that absorb energy from the electromagnetic spectrum, ideally at wavelength (s) corresponding to the wavelength (s) of the light source. As is known in the art, the dyes generally consist of conjugated heterocyclic structures exemplified by the following kinds of dyes: azo dyes, diazo dyes, triazine dyes, food dyes, biological dyes. Specific dyes include: Coomasi Brilliant Blue R-250 Dye (Biorad Labs, Richmond, Calif.) Reactive Red 2 (Sigma Chemical Company, St. Louis, Mo.), Bromophenol azu (Sigma); xylene cyanol (Sigma); phenolphthalein (Sigma). The Sigma-Aldrich Handbook of Stains, Dyes and Indicators, by Floyd J. Green, published by the Aldric Chemical Company, Inc., (Milwaukee, Wis.), Provides an abundance of data for others dyes With these data, dyes can be selected with the appropriate light absorption properties, which match the wavelengths emitted by the light source. In these embodiments, particles containing opaque dye can be used, instead of reflection particles as a signal fraction that responds to light, reversing by the same the phase of the encoded information. The Latex spheres can vary from 1 to 100 micrometers in diameter, preferably 10-90 micrometers in diameter, and most preferably are 10-50 micrometers in diameter. The dye will prevent reflection of the light from the laser beam from the metal layer of the disc substrate. In yet other embodiments, the signal-responsive element can be a fluorescer, such as fluorescein, propidium iodide or phycoerythrin, or a chemiluminescent, such as luciferin, that respond to incident light, or an indicator enzyme that dissociates soluble fluorescent substrates. to insoluble form. Other fluorescent dyes useful in this embodiment include tile red, rhodamine, green fluorescent protein, and the like. Fluorescent dyes will prove particularly useful when blue laser beams become widely available. The light reflecting, light scattering, and light absorbing embodiments of the present invention preferably employ a circular test device such as the substrate for pattern deposition of the elements of the dissociable signal. In a particularly preferred embodiment, the test device is compatible with existing optical disc readers, such as a compact disc player (CD), or a digital video disc (DVD) player, and therefore preferably a disk of approximately 120 millimeters d diameter, and approximately 1.2 millimeters thick. With disc it is also meant a ring .. It will be noted, however, that the elements of the dissociable reflection signal of the present invention can be deposited in spatially directed patterns on substrates that are not circular and essentially flat, and that These test devices are necessarily read with detectors suitably adapted to the shape of the substrate. The maximum number of separable signal elements, or biobits, that can be discriminated spatially on an optical disk, is a function of the wavelength and the numerical aperture of the objective lenses. A known way to increase the memory capacity in all kinds of optical memory discs, such as compact disc read-only memory, WORM (Write Once Many Write) discs, and magneto-optical discs, is decrease the wavelength of the light emitted by the laser beam of the diode that illuminates the data lanes of the optical memory disk. The smaller wavelength allows the discrimination of smaller data points on the disk, that is, higher resolution, and thus improved data densities. The read-only memories of current CDs employ a laser beam with a wavelength of 780 nanometers (nm). Current digital video disc readers employ a laser beam with a wavelength of between 635 and 650 nanometers. The new diode laser beams that emitted, for example, blue light (about 481 nanometers), would increase the number of signal elements that could be spatially directed on a single disk of the test device of the present invention. Another way to achieve blue radiation is by doubling the frequency of the infrared laser beam by nonlinear optical material. The read-only memory readers of current compact discs use both read-by-reflection and read-by-transmission. Both data access methods are compatible with the present invention. The gold particles are especially suitable for use as a fraction that responds to signals for CD-only memory readers of the reflection type. The light absorbing dyes are more suitable for the type of transmission readers, such as those described in United States Patent Number 4,037,257. . 5.3 Other Fractions Responding to Signals It will be apparent to those skilled in the art that the signal-responsive fractions suitable for adaptation to the dissociable spacer of the present invention are not limited to light reflecting or absorbing metal particles or dyes. light. Suitable fractions that respond to signals include, but are not limited to, any composition that can be detected by means of spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical elements. In some preferred embodiments, the fractions that respond to suitable signals include colorimetric labels, such as colloidal gold or colored glass or plastic globules (eg, polystyrene, polypropylene, latex, etc.), biotin for staining with labeled streptavidin conjugate, globules magnetic (eg, Dynabeads ™), radiolabels (eg, 3H, 125I, 35S, 14C, or 32P), and enzymes (eg, horseradish peroxidase, alkaline phosphatase, and others that are commonly used in a linked immunosorbent assay with enzyme). It will be apparent to those skilled in the art that numerous variations of signal-responsive fractions can be adapted to the dissociable spacers of the present invention. Many patents, for example, provide extensive teaching of a variety of techniques to produce detectable signals in biological assays. Those signal-responsive fractions are generally suitable for use in some embodiments of the present invention. As a non-limiting illustration, the following is a list of the Patents of the United States of America that teach the many signal-responsive fractions, suitable for some embodiments of the present invention: United States of America Numbers 3,646,346, element d generation of radioactive signal; 3,654,090, 3,791,932 3,817,838, element of generation of signal linked with enzyme; 3,996,345, fluorescent-cooling related signal generation element; 4,062,733, fluorescent or enzyme signal generating element; 4,104,029, element d generating chemiluminescent signal; 4,160,645, element d non-enzymatic catalyst generation; 4,233,402, element d generation of enzyme pair signal; 4,287,300, label d anionic charge of enzyme. All of the patents of the United States of America cited above are incorporated herein by reference, for all purposes. Other signal generation elements are also known in the art, for example, U.S. Patent Nos. 5,021,236 and 4,472,509, both incorporated herein by reference for all purposes. A meta-chelate complex can be employed to bind the signal generating element to the spacer-dissociable molecules, or to an antibody attached as a side member to the spacer molecule. In U.S. Patent No. 4,472,509, incorporated herein by reference for all purposes, s described methods that use an organic chelating agent ta as a DTPA bound to the antibody. In still other modalities, dial can be used magnetic fields instead of reflection spheres, and can be oriented by treating the disk with a magnetic field that is of sufficient strength. Since the empty sites will not have any magnetic material present, the location of the remaining spacer molecules can be detected and the information processed to identify the materials in the test sample. Additionally, reflective or magnetic material can be added after hybridization of the sample, to provide the signal generating element. Paramagnetic ions can be used as a signal generating element, for example, ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III), with gadolinium being particularly preferred. Useful ions in other contexts, such as X-ray imaging, include, but are not limited to, lanthanum (III), gold (III), lead (II), and especially bismuth (III). The elements for detecting those labels are well known to those of skill in the art. In this way, for example, radiolabels can be detected using photographic film or scintillation counters, the fluorescent labels can be detected using a photodetector to detect the emitted light. Enzymatic labels are detected typically by means of providing the enzyme with a substrate, and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply displaying the colored label. The colloidal gold label can be detected by measuring the scattered light. A preferred non-reflective signal generating element is biotin, which can be detected using an avidin or streptavidin compound. The use of such labels is well known to those of skill in the art and is described, for example, in U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein as a reference for all purposes. . 6 Union of the lateral members of the dissociable spacer The lateral members of the dissociable spacers confer specificity for analytes. In a preferred embodiment, the side members are oligonucleotides. Oligonucleotides can be added by step synthesis in the dissociable spacers, before the attachment of the spacers to the derivatized substrate of the test device (disk). Alternatively, fully prepared oligonucleotides can be attached in a single step, directly to the spacer molecules, before the attachment of the spacer molecule to the substrate of the assay device. In such circumstances, the spacer molecule has amino- and / or thiol-protected groups in place of two protected hydroxyl groups. A protecting group is removed, and an oligonucleotide having, for example, an isocyanate group at one end is added. In a similar manner a second oligonucleotide is attached as a second side member to the spacer molecule which can be separated. Alternatively, lateral member oligonucleotides can be synthesized after attachment of the dissociable spacers on the substrate, either in a single step using fully-prepared oligonucleotides, or by stepwise addition. The last alternative is expected to be the preferred one when a large number of assays with different specificity for analytes are incorporated into a single test device substrate. The general process by which the lateral members are joined to the dissociable spacers previously immobilized on the substrate, either in a single step or by stepwise addition, is called in the present stamping. Phosphoramidite chemistry is preferred to prepare the side members of the oligonucleotide, although they can be use other chemicals In conventional solid phase synthesis, oligonucleotides are prepared by the use of monomeric phosphoramidites. After conventional synthesis, the oligonucleotides are stripped from the resinous support, and purified by liquid chromatography to remove the reagents, including solvents and unreacted mononucleotides, and to remove the shorter oligonucleotides resulting from incomplete synthesis. In certain cases, the oligonucleotides can not be purified in this way, and the shorter oligonucleotides contaminate the desired oligonucleotide. This leads to unwanted hybridization. Oligonucleotide contaminants that omit only one nucleotide relative to the desired product are the most labor-intensive to treat, because their binding is almost equal in strength to that of the oligonucleotide having the correct sequence. In the preparation of the oligonucleotides to be used as side members in the dissociable reflection signal elements of the present invention, the use of trimeric or tetrameric phosphoramidites in the synthesis is convenient and preferred. Using tetrameric starting materials, for example, 12 -mers can be synthesized in three steps. In this case the inevitable products of the incomplete synthesis will be 8 -mers and 4 -mers, representing the failure of 1 or 2 steps of the synthesis, respectively. Since the fixation of the 8- Groupers are much weaker than the fixation of 12 -mers, these contaminants do not cause any significant interference. In the application of the lateral members to dissociable spacers by stepwise addition to spacers immobilized on the substrate surface of the assay device, the oligonucleotides can be conveniently attached to the dissociable spacers by chemical printing, which utilizes the formation of the solution desired oligonucleotide chemistry in a printed pattern that is complementary to the distribution of the spacer molecule in the solid support. Printing is fast and economical. This can also provide very high resolution. For example, in Science, Volume 269, pages 664-664 (1995), a simple printing method is described. In this printing method, one of the protective groups of the spacer molecule is removed on the substrate of the test device. The desired oligonucleotides are applied to the stamping surface, in a manner that will provide specific oligonucleotides at specific locations, previously determined in the stamping, and then the stamping surface is applied to the supporting surface of the substrate covered by the spacer, depositing by the same as the desired oligonucleotides in the discrete areas in which the spacer molecules reside. Subsequently, the second protective group is removed, and Apply a. oligonucleotide different from the activated area, again by chemical stamping. Those steps are particularly illustrated in Figures 8A, 8B, 9A, 9B, 13 and 14. Alternatively, the respective oligonucleotides can be applied by inkjet printing, such as by the methods described in U.S. Pat. of North America Nos. 4,877,745 and 5,429,807, the descriptions of which are incorporated herein by reference. Either of these direct printing methods is fast. When using trimers or tetramers to build oligonucleotides, two print cycles allow one to create a configuration of all possible oligos from 6-mer to 8-mer. To contain all 8 -mers, the test device must contain 256 x 256 different oligos. Additional printing cycles increase the length of the oligonucleotides rapidly, although not all combinations can be adapted on reasonably sized surfaces, and many test devices may have to be used to represent all such combinations. In Figure 15 an alternative printing process useful in the present invention, concave complementary printing, is shown. Although only two steps are shown, very large numbers of oligonucleotides can be printed at Same time. A mixture of oligonucleotides is synthesized; for example, 12 -mers can be synthesized using a mixture of four phosphoramidites in each step, and as a last step of the synthesis, a very long spacer is attached to each oligonucleotide. At the other end a reactive group is provided, such as an isothiocyanate. The mixture of oligonucleotides is incubated with the stamping that will fix complementary oligonucleotides at defined sites. During the printing process, the spacer will bond with the substrate. The double helices are denatured, for example, by heating, and the stamping and the substrate can be separated. Many other methods have been developed for the synthesis of oligonucleotides, and in particular, for the synthesis that can be directed spatially of oligonucleotides on solid surfaces, and are known to those skilled in the art. The methods that are proved particularly useful in the present invention are described in U.S. Patent Nos. 4,542,102; 5,384,261; 5,405,783; 5,412,087; 5,445,934; 5,489,678; 5,510,270; 5,424,186; 6,624,711; whose descriptions are incorporated herein by reference. Other methods that can be proved useful in the present invention generally include: (1) Photochemical synthesis by steps, (2) Chemical synthesis by stepwise jet and (3) Fixation of prepared oligonucleotides. previously. A glass capillary configuration system can also be used. In the latter case, the parallel synthesis can be performed in all the capillaries, as is done in an automated DNA synthesizer. Although lateral elements of the oligonucleotide have been described herein as DNA oligonucleotides synthesized using standard deoxyribonucleotide phosphoramidites, it is known that certain oligonucleotide analogs, such as RNA-pyranosyl (E. Szathmary, Nature 387: 662-663 (1997)) ) and peptide nucleic acids, form stronger duplexes, with higher fidelity than natural oligonucleotides. In accordance with the foregoing, these artificial analogs can be used in the construction of side elements of the oligonucleotide. Although the side members of the oligonucleotide are adapted to bind to complementary oligonucleotides, and consequently are directly useful in a nucleic acid probe assay, it is another aspect of the invention to conjugate these side members of the oligonucleotide, members of specific binding pairs with utility in other tests. In these latter embodiments, non-covalent attachment of the members of binding pairs, such as antibodies, to side-member oligonucleotides is mediated through the complementarity of the side-member oligonucleotides and the oligonucleotides that are covalently bound to the member of fixing pair. In the patent applications of the United States of North America Nos. 08 / 332,514, filed on October 31, 1994, 08 / 424,874, filed on April 19, 1995, and 08 / 627,695, filed on March 29, 1996, filed with the - possessions and slopes, incorporated herein by reference, describe in more detail the use of complementary nucleic acid molecules to effect the non-covalent, combinatorial assembly of supramolecular structures. As outlined in Figures 3A to 3C, the side members of oligonucleotide 34a, 34b, 35a, and 35b are non-covalently coupled to the modified antibodies 38a, 38b, 38c, and 38d, to allow an immunoassay. The non-covalent binding of the modified antibodies to the lateral members is mediated through the complementarity of the side member oligonucleotides and the oligonucleotides that are covalently bound to the antibodies. Although antibodies are exemplified in Figure 3, it will be noted that useful antibody fragments and derivatives such as Fab fragments, single chain antibodies, chimeric antibodies, and the like will also be tested. In general, members of binding pairs useful in this modality will generally be the first members of the first and second specific binding pairs, exemplified by antibodies, receptors, etc., which will be set respectively to antigens, ligands, and so on. . 7 Deposition by Patterns of Dissociable Reflection Signal Elements on the Test Device From the above description it will be noted that the spatial distribution of the dissociable reflection signal elements, which respond to analytes, can be determined on the test device ( disc substrate), in two levels: at the junction level of the dissociable spacer itself, and additionally at the junction level of the lateral spacer members. It will also be noted that the spatial distribution of analyte sensitivity can also be determined by a combination of the two. A method to control the distribution of dissociable spacers in the first of these steps is through the formation by substrate patterns, with hydrophilic and hydrophobic domains. First the hydrophobic surfaces are activated, and the hydrophilic surfaces are deactivated, in such a way that a hydrophilic and functional staining pattern is created, separated by a non-reactive hydrophobic network. If the substrate material is glass, mica, silicon, hydrophilic plastic or analogous material, the entire surface is first produced reactive by treatment with acid or base. The intermediate space between spots is silanized. This is best done by using a grid as a stamp. If on the other hand the substrate is u hydrophobic plastic, this can be activated by plasma treatment, in the presence of ammonia, and then silanized as a hydrophilic substrate. The use of resistant material in conjunction with lithographic or mechanical printing to remove resistance at desired sites, activation can be performed at those sites. A hydrophilic spacer such as polyethylene glycol (PEG) is preferably attached to the reaction stains. If the substrate contains an amino group or a thiol group, the polyethylene glycol can be pre-activated at the other end with a variety of functional groups, which are known to be coupled with an amino or thiol group. These include isocyanate, maleimide, halogenacetyl and succinimidoester groups. A photoresist can also be usefully used to pattern the deposition of dissociable signal elements. The resist is partially depolymerized by incident laser light, during manufacture, and can be dissolved from these areas. The exposed plastic or plastic is chemically treated, for example, aminated by ammonia plasma. After the resistant is removed, the spacer, the side members, and the signaling fraction in the treated area are connected, as necessary. The use of photoresists for pattern formation of master discs is well known in compact disc manufacturing techniques.

Alternatively, instead of using a resistor, a solid mask having small holes and other features necessary during the ammonia plasma treatment can be used. The holes have a diameter of approximately 1 to 3 micrometers. The holes are located circularly in the mask, forming a spiral rail, or a pattern that is a combination of spiral and circular trajectories. The mask can be made of metal or plastic. Many metals can be used, such as aluminum, nickel or gold. Polycarbonate is a preferred plastic, because it will retain the shape well. The plastics are reactive with the ammonia plasma, however, and a preferred method for using plastic masks, therefore, involves depositing a metal layer on the plastic, by evaporation, electron deposition, or other methods known in the art. You can make holes in the mask using a laser beam. Those skilled in the art will notice that it is possible to create 1000 holes of size of 1 micrometer in one second, in a thin metal or plastic plate. Alternatively, holes can be corroded by acid, by using conventional methods known in the semiconductor industry. In the approach of the mask to form by pattern the deposition of the elements of the signal, the mask is pressed against the substrate and the ammonia plasma is applied. It can Repeatedly use the mask. As it should be noted, the spatial distribution of sensitivity to analytes can also be conferred by application by lateral spacer arm patterns. With reference to the printing method described above, the schematic of a possible oligonucleotide stamp is shown in Figure 13. The stamp has holes that are filled with a certain chemical that will be used to provide the desired building block of the oligonucleotide being synthesized. In Figure 13 each row is filled with the same chemical, and in accordance with the above, four different chemicals can be used during a stamping cycle in the example given in Figure 13. In commercial systems the number of rows it will be considerably higher, typically 64-256, although lower and higher row numbers can be used in special cases. The linear stamp is desirable if all possible oligonucleotides of a certain size are to be fabricated on the substrate of the test device. In this way, all possible hexameric combinations of a given set of oligonucleotide building blocks can be prepared. For example, trimer phosphoramidites can be formed by two reaction cycles, by using a 64 row linear die. Each of the 64 different trimer phosphoramidites is Feeds inside a row of holes. After printing the phosphoramidites, the oxidant, the deblocker and the stop reagent are printed. Since these chemicals are the same in each stain, the stamper can be a flat plate, or you can simply submerge the entire surface within the reagent solution. The substrate is turned 90 °, and the same cycle is repeated. In this way, all possible combinations of trimers have been manufactured. Analogously, all combinations of any set of oligonucleotide amidites can be made. Figure 14 is an example showing the fabrication of all possible combinations of four different oligonucleotide amidites. After the first printing cycle, all the spots in each horizontal row contain the same oligonucleotide, but each row has a different oligonucleotide, these oligonucleotide fragments are denoted by numbers 1, 2, 3 and 4, in Figure 14. When the stamp is rotated 90 °, and the printing cycle is repeated, all combinations of four oligonucleotides are formed. The above orthogonal printing process is particularly convenient in the production of signal elements of this invention, in the disk mode. Orthogonal printing facilitates the distribution of the configuration of the spacer molecules in a pattern of concentric circles, similar to the information that is placed on audio compact discs or read-only memory of compact discs in ring patterns. A preferred variation of an orthogonal printing process employs the superimposition of two sets of spiral stamps with opposite chirality. The positioning of the stamp must be accurate within approximately 1 micrometer. This can be accomplished mechanically by using two to four pairs of bolt holes, or by an optoelectronically guided microtrailer. A removable reflection coating can be deposited on two perpendicular sides of the substrate, and the stamp and its relative positioning can be measured by means of an interferometer. The substrate and the stamp can also have a pair of micro-prisms, which must be perfectly aligned, in order that the light passes inside the photodetector. Figures HA to 11G illustrate different useful patterns of spatially steerable deposition of the dissociable reflection signal elements in circular, flat disk substrates. Figure HA particularly identifies a directional line, which can be encoded on the disc substrate, from which the location of the dissociable spacers can be measured. In Figure HA, the dissociable spacer molecules are deposited in lanes annular Figure 11B demonstrates the spiral deposition of the dissociable signal elements, and particularly identifies a central void of the disk ring, particularly adapted to engage rotational impulse elements. Figure HC demonstrates the deposition of the dissociable signal elements in a suitable pattern for the testing of multiple samples in parallel, with the concurrent coding of interpretation software in the central lanes. Figure HD schematically represents an embodiment in which the substrate of the test device has been further microfabricated to segregate the individual test sectors, thereby allowing the rotation of the test device during the addition of the sample, without mixing the samples. Figure HE schematically represents an embodiment in which the substrate of the test device has been further microfabricated to compel the flow of the unidirectional sample during the rotation of the test device. Techniques for microfabricating solid surfaces are well known in the art, and are particularly described in U.S. Patent Nos. 5,462,839; 5,112,134; 5,164,319; 5,278,048; 5,334,837; 5,345,213, which are incorporated herein by reference. Figure HF demonstrates the deposition of Dissociable signal elements in a spatial organization suitable for testing 20 samples for 50 different analytes. Figure 11G demonstrates the orthogonally intersecting pattern created by superimposing spiral patterns with lateral arms of direction or opposite chirality. The spatial distribution of the dissociable reflection signal elements, or biobits, can be designed on the surface of the test device to facilitate the quantification of the concentration of analytes. In this way, in some modalities, the capture of analytes is used for quantification. In one implementation, the assay device with a uniform density of biobits dedicated to each selected analyte is patterned. A test sample is placed on the disk, in the center of the disk. Through the application of rotational force, the test sample is spread radially to the periphery. In the dissemination process, analytes are captured by the respective cognate side element of the dissociable signal element, reducing the concentration of analytes in the front of the sample. With sufficient density of biobits in relation to the incident concentration, all the analytes are captured before the front of the sample reaches the periphery of the test device. Then you can determine the concentration of each analyte, in accordance with the location of the positive biobit that is further away from the sample introduction site. It will be noted that a greater dynamic range of analyte concentration will be detectable if more biobits are dedicated to the detected analyte. In the modality just described, the uniform density of biobits would increase. It will also be noted, however, that the density of biobits does not need to be constant, and that a linear or exponentially changing biobits density, as measured from the center of the disk to the periphery, can be used to change the dynamic range of the concentration detection. In other embodiments and aspects of the present invention, biobits with different affinities for the selected analyte can be linked to the assay device for a similar effect, that is, to increase the dynamic range of the concentration detection. It is also contemplated that other geometries may be used to convey the concentration information. Figure 16 demonstrates a geometry in which a single sample is piped in parallel, within four distinct sectors of the test device. If either the density of biobits, the affinity of the biobits, or both the density and the affinity of the biobits in the four sectors differ, a large dynamic range of concentration can be determined by the position detection in each sector of the positive biobit more distant from the sample application site. In other modalities, equilibrium tests are contemplated. In this way, the concentration is determined by sampling the entire disk ", and the determination of the percentage of positive biobits per analyte In each of these modalities, a number of biobits are generally dedicated for the detection of positive and negative controls. In other embodiments, the dissociable reflection signal elements (biobits) specific for multiple different analytes are formed in patterns in a number of different formats, for example, biobits of different specificity are mixed in each sector of a disk. These can be separated into different sectors.The ability to pattern specific biobits in previously defined locations, and the ability to decipher the identity of biobits through detectors such as a CD read-only memory reader, makes possible flexible designs One of experience in the art will notice that you must test and adjust the of the standards, using test samples containing known analytes of different concentrations. . 8 Alternative Test Device Geometries Viruses are typically nearly spherical particles that have diameters less than 0.5 micrometers. Bacteria are commonly either spherical or rod-shaped; its largest dimension is usually less than 2 micrometers, excluding flagella and other similar external fibers. These pathogens are somewhat smaller, or approximately the same size, as the gold spheres that are used in the dissociable signal elements of the present invention. Their interaction simultaneously with two lateral members of the dissociable signal element described above can, therefore, be spherically inhibited. In this way, an alternative geometry is prepared completely with the dissociable spacers. A side member specific to the analyte binds directly to the substrate surface of the assay device in a spatially steerable manner. The second lateral member, specific for a second site of the selected analyte, binds directly to the fraction that responds to signals. In the preferred embodiments, that fraction is a gold sphere. In this alternative geometry, the recognition of the analyte creates a direct sandwich of the formula: substrate-first lateral member-analyte-second lateral member -fraction that responds to signals. It can be said that this geometry is a limiting case in which "m" in the formula for the spacer dissociable. is zero. As shown in Figure 17, this particular geometry can also be proven useful in the detection of nucleic acid hybridization. In this alternative geometry, if the fraction that responds to signals is reflective, the information coding is similar to that in the geometries presented above - the presence of analytes is signaled by reflection. Alternatively, if the fraction responding to signals is opaque, for example, through the incorporation of dye, the coding is inverted: the presence of analytes is signaled by the absence of reflection from the metal layer of the device substrate. Magnetic plastic spheres can provide particular advantages in this alternative geometry. Because they contain magnetic particles inside, they are less transparent than latex spheres. On the other hand, magnetism can be used to remove weakly fixed spheres, which are otherwise difficult to remove, such as, for example, latex spheres, because their density is close to that of water, and centrifugal force will be tested. ineffective As shown in Figure 18, another variant of this alternative geometry takes the advantage of agglutination in a reflection test. In this alternative, the fractions that Responses to signals are preferably microspheres. These microspheres are relatively small (30-600 nanometers), so that one does not efficiently block light. The invention can be better understood by reference to the following non-limiting examples. 6. EXAMPLE I: INCREASING THE SPECIFICITY OF U NUCLEIC ACID HYBRIDIZATION TEST in a direct nucleic acid hybridization assay, the lateral elements of the dissociable signal element are oligonucleotides designed to hybridize with different sites in a previously determined nucleic acid to be detected in the sample. For many applications of this methodology, cross-reactivity with sample oligonucleotides having up to one mismatched single nucleotide should be minimized. In particular, nucleic acid hybridization assays, adapted to use the dissociable reflection signal element of the present invention, for the detection of point mutations, as, for example, for the detection of point mutations in the BRCAl genes. and BRCA2 that predispose for breast and ovarian cancers, must be able to discriminate between nucleic acid samples that contain a single mismatched nucleotide. The longer the lateral elements of the oligonucleotide of the dissociable signal element - and in this way the longer the sequence that is complementary between the lateral elements and the nucleic acid sample - the greater the possibility of mistakenly recognizing an incorrectly coupled sample, since the Hybridization strength, even given the presence of a bad coupling, will be reasonably high. Therefore, one way to reduce the mis-recognition of mis-coupled nucleic acid sequences is to reduce the length of the side element oligonucleotides. The specificity is increased by shortening the lateral arms to 8 -mer or even 6-mer. These will still hybridize at room temperature, depending on the stringency of the wash, the conditions of which are well known in the art. Poorly coupled oligonucleotides would use five or fewer nucleotides to form in pairs, and will form highly unstable binding at room temperature. This solution, however, presents its own problem: the relatively short overall length, of 12-16 nucleotides, which is used for recognition, leads to a concomitantly reduced overall strength of the hybridization that is required to constrain the fraction that responds to signals of the dissociable signal elements. As illustrated in Figures 2E-2F, the use of ligase partially solves this problem. The ligation will not only provide a fixation stronger, but will also act to ensure selectivity, since the DNA ligase will not bind to the oligonucleotides if there is poor coupling near the terminus of the oligonucleotides. Because the oligonucleotides are short, no mismatched base pairs are accepted. Ligase serves as a very strict double verification for the coupling of oligos. An alternative, and complementary, solution uses the triple recognition principle illustrated in Figures 2D-2E in a constructive manner to shorten the sequence of test samples available for hybridization to the side elements of the dissociable signal element. A soluble specificity-enhancing oligonucleotide, for example an 8-mer, which is complementary to the central part of the oligonucleotide in the sample, is added to the sample solution before contacting the test device with the fluid sample. This 8 -mer hybridizes well under the test conditions. The lateral elements of the dissociable signal elements recognize six nucleotides in the immediate vicinity of the previously formed duplex. The ligation will ensure the selectivity and will also provide a strong fixation. The ligase will not bind to the oligonucleotides if there is a bad coupling near the end of the oligonucleotides. Because the oligonucleotides are short, no base pairs are accepted poorly coupled. Ligase serves as a very strict double verification for the coupling of oligos. It will be apparent that the specificity enhancing soluble oligonucleotide, which is shown here as an 8-mer, can be designed to be added to the test sample, to position the potential mis-coupling near the ends of the sample, where the bad coupling will be more disadvantaged to be fixed to the side elements. On the other hand, because the addition of the ligase ensures a covalent cycle, the stringency of the wash can be increased by the addition of chaotropic agents and / or by heating, to remove any non-selective oligonucleotides. The "blocked" sample oligonucleotide suitable for, and capable of correctly fixing the side elements, can be mimicked, however, by a sample nucleic acid possessing the required terminal hexanucleotide sequences, connected directly to each other without the sequence of 8 -merms of intervention. As shown in Figure 2D, the ulterior addition to the sample of a 10-mer with sequence equally extracted from the first lateral element oligonucleotide sequence the second lateral element oligonucleotide sequence, will avoid that fixation after contact with the device d test of the present invention.

Currently, the combination of 8 + 10 + 8 of the soluble specificity enhancing oligonucleotides is preferred, but other combinations can be used, such as 7 + 9 + 7 and 8 + 8 + 8. Another method to increase specificity includes the use of the so-called padlock probes, in which the oligonucleotides with a circular shape are concatenated, allowing extensive washing to remove weakly fixed probes. Padlock probes can achieve a 50: 1 discrimination between complementary and mismatched oligonucleotides individually (Nilsson et al., Science 265: 2085 (1994)), whereas with conventional probes this ratio is typically between 2: 1 and 10. :1. The side members of the oligonucleotide having the following sequences are prepared by automated synthesis, such that each of them contains a terminal thio (or aliphatic amino) group, depending on the binding site with the spacer molecule dissociable (extreme 5 'end). 3 ' ) . la: 5'-CGGGTGTGG Ib: CGGCCGCGG-3 'lía: 5' - CGGGTGTGA Hb: CGGCCGCGG-3 ' Illa: 5 '- CGGGTGTGC 11Ib: CGGCCGCGG-3' IVa: 5 '- CGGGTGTGT IVb: CGGCCGCGG-3' Dissociable spacer molecules are synthesized with two aliphatic amino groups, in place of the protected hydroxy groups described above, and one group is protected by monomethoxytrityl (MMT, unstable to acid), and the other group is protected by fluorenyloxycarbonyl (FMOC, unstable to base) . After removal of the fluorenyloxycarbonyl group, the amino function is allowed to react under aqueous conditions, with 4- (N-maleimidomethyl) -cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMMC). The derivatized thiol is added to the spacer molecule, and allowed to attach to the spacer molecule. Subsequently, the monomethoxytrityl is removed by treatment with acetic acid, and after washing with pH regulator, pH of 8, 4- (N-maleimidomethyl) -cyclohexane-1-carboxylic acid N-hydroxy-succinimide ester is added, and the Hb of the oligonucleotides is allowed to couple with the spacer molecule. The spacer molecules prepared above are attached to a polycarbonate substrate. A test sample containing 5'-GCCCACACCGCCGGCGCC-3 'is prepared, and allowed to have contact with the dissociable signal element at a temperature approaching Tm of the lateral members la and Ib. The temperature of the sample solution is heated to approximately 20 degrees Centigrade above the Tm. Subsequently, the signal element it is treated with 0.1 M sodium fluoride solution, and washed. The spacer molecules that remain attached to the surface signal the presence of, and are blocked by 5'-GCCCACACCGCCGGCGCC-3 '. The above processes are applied to the analysis of 5 'GCCCACACTGCCGGCGCC-3', 5'-GCCCACACGGCCGGCGCC-3 'and 5'-GCCCACAGCCGGCGCC-3', using, respectively, spacer molecules that incorporate the lateral members lia and IIb, Illa and IHb, and IVa and IVb. 7. EXAMPLE II: HIV-1 DETECTION The HIV-1 proviral DNA from the clinical samples is amplified as follows, essentially as described in US Pat. No. 5,599,662, incorporated herein by reference. Peripheral blood monocytes are isolated by standard Ficoll-Hypaqu density gradient methods. After isolation of the cells, the DNA is extracted as described in Butcher and Spadoro, Clin. Immunol Newsletter 12: 73-76 '(1992), incorporated herein. A polymerase chain reaction is carried out in a 100 microliter reaction volume, of which 50 microliter is contributed by the sample. The reaction contains the following reagents at the following initial concentrations: M Tris-HCl (pH 8.4) 50 mM KCl 200 μM each of dATP, dCTP, dGTP, and dUTP 25 pmoles of primer 1, of the sequence shown below 25 pmoles of primer 2, of the sequence which shows below 3.0 mM of MgCl2 10% glycerol 2.0 units of Taq DNA polymerase (Perkin Elmer) 2.0 units of UNG (Perkin-Elmer) Primer 1: 5 '-TGA GAC ACC AGG AAT TAG ATA TCA GTA CAA TGT -3 'Primer 2: 5' -CTA AAT CAG ATC CTA CAT ATA AGT CAT CCA TGT-3 'The amplification is done in a thermal cycler d TC9600 DNA (Perkin-Elmer, Norwal, Connecticut), using the following temperature profile: (1) pre-incubation - 50 ° for 2 minutes; (2) initial cycle - denaturation 94 ° C for 30 seconds, annealing at 50 ° C for 30 seconds, extension at 72 ° C for 30 seconds; (3) cycles 2 to 4 - denaturation at 94 ° C for 30 seconds, annealing for 30 seconds, extension at 72 ° C for 30 seconds increasing the annealing temperature in 2 ° increments (at 58 ° C) compared to the cycle 1; (4) cycles 5 to 39 - denaturation at 90 ° C for 30 seconds, annealing at 60 ° for 30 seconds, extension at 72 ° C for 30 seconds. After the temperature cycle, the reaction mixture is heated at 90 ° C for 2 minutes, and diluted to 1 milliliter. Alternatively, the sample is stored at -20 ° C, and after thawing, it is heated at 90 ° C for 2 minutes and then diluted to 1 milliliter. The spacers dissociable with the siloxane fraction are synthesized and bound in a uniform density to a derivatized 120-millimeter polycarbonate disk substrate, essentially as stated in sections 5.2 and 5.3 above. The following side members are then stamped on the dissociable spacers: first side member: 5 '-TAG ATA TCA GTA CAA-3' second side member: 3 '-TAT TCA GTA GGT ACA-5'. A suspension of gold microspheres, 1-3 micrometres in diameter, is added dropwise to the disk, which is gently rotated to distribute the gold particles. The gold particles are added until the effluent contains the same particle density as the initial suspension, thereby ensuring the saturation of the dissociable spacers. The sample is applied at room temperature, dripping, near the center of the test device, which is rotated at a continuous speed. The rotation is interrupted after the front of the sample reaches the periphery, The disc is incubated stationary at room temperature for 3-5 minutes. A milliliter of sample pH regulator is added dropwise as a wash, while the disk is rotated. One milliliter of 100 mM sodium fluoride is added, and it is distributed by rotating the disk. The disc is incubated stationary for 1-2 minutes, then 5 milliliters of sample pH regulator are added dropwise during vigorous rotation of the test disk. The disc is dried, then read directly into a compact disc read-only memory reader programmed to test at each previously determined site, after which the dissociable spacers are deposited. The present invention should not be limited in scope by the exemplified embodiments and examples, which are intended as illustrations of the individual aspects of the invention. In fact, for those skilled in the art, various modifications thereto, and equivalents and variations thereof, in addition to those which are shown and described herein, will be apparent from the above description and accompanying drawings. that those modifications are and are included within the scope of the appended claims All the publications cited herein are incorporated by reference in their entirety.

Claims (47)

1. CLAIMS 1. A dissociable signal element, comprising: a dissociable spacer, the dissociated spacer having a binding end to the substrate, an end that responds to signals, and an intermediate dissociation site to the substrate binding end and the signal-responsive end, - a fraction responding to signals, - a first lateral member adapted to be fixed to the first site in a selected analyte; and a second side member adapted to be fixed to a second site of said selected analyte; wherein the signal-responsive fraction is attached to a dissociable spacer at the signal-responsive end, the first lateral member is attached to the intermediate dissociative spacer at the end that responds to signals and the dissociation site, and the second lateral member is attached a dissociative spacer intermediate to the dissociation site and at the end of attachment to the substrate.
2. 2. The dissociable signal element of claim 1, wherein said signal-responsive fraction is adapted to reflect or disperse incident light.
3. 3. The dissociable signal element of claim 2, wherein the fraction that responds to signals It is a metal microsphere.
4. 4. The dissociable signal element of claim 3, wherein the metal microsphere consists essentially of a metal selected from the group consisting of gold, silver, nickel, platinum, chromium and copper.
5. 5. The dissociable signal element of claim 4, wherein said metal microsphere consists essentially of gold.
6. 6. The dissociable signal element of claim 5, wherein said gold microsphere has a diameter between 1 nanometer - 10 micrometers.
7. 7. The dissociable signal element of claim 6, wherein said gold microsphere has a diameter between 0.5-5 micrometers.
8. 8. The dissociable signal element of claim 7, wherein the gold microsphere has a diameter between 1-3 micrometers.
9. 9. The dissociable signal element of claim 1, wherein the dissociation site is susceptible to chemical dissociation.
10. 10. The dissociable signal element of claim 9, wherein the dissociation site susceptible to chemical dissociation includes at least one siloxane group.
11. 11. The dissociable signal element of claim 1, wherein the first lateral member and second side member include oligonucleotides.
12. 12. The dissociable signal element of claim 11, wherein the first and second side member oligonucleotides are 5mer-20mers. The dissociable signal element of claim 12, wherein the first and second side member oligonucleotides are 8mers-17mers. 14. The dissociable signal element of claim 12, wherein the first and second side member oligonucleotides are 8mer-12mers. The dissociable signal element of claim 1, wherein the first side member includes a first member of a first specific attachment pair, the second side member includes a first member of a second specific attachment pair, and the second member of said first specific fixing pair and said second member of the second specific fixing pair are each present on the surface of only one analyte. 16. The dissociable signal element of claim 15, wherein the first member of the first specific binding pair includes a first antibody, antibody fragment, or antibody derivative, and the first member of the second specific binding pair includes a second antibody, antibody fragment, or antibody derivative. The dissociable signal element of claim 15, wherein said first lateral member includes a first side member oligonucleotide, said second side member includes a second side member oligonucleotide, the first member of the first specific attachment pair includes a first fixing pair oligonucleotide, the first member of the second specific binding pair includes a second binding pair oligonucleotide, and the first side member oligonucleotide includes the sequence complementary to the sequence that is included in the first binding pair oligonucleotide, the second member oligonucleotide The side sequence includes the sequence complementary to the sequence that is included in the second binding pair oligonucleotide, and the complementary sequences are non-covalently associated. 18. An assay device, comprising: a solid support substrate, and a plurality of dissociable signal elements, according to claim 1, wherein the dissociable signal elements are attached through. its binding ends to the substrate, said solid support substrate in a spatially steerable pattern. 19. A test device, comprising: a solid support substrate, and a plurality of dissociable signal elements, according to any of claims 2-17, wherein said dissociable signal elements are joined through their ends of binding to the substrate, to dich substrate of solid support in a spatially dirigible pattern. 20. The testing device of claim 18, wherein the solid support substrate is a plastic which is selected from the group consisting of polypropylenes, polyacrylates, polyvinyl alcohols, polyethylenes, polymethylmethacrylates and polycarbonates. 21. The test device of claim 20, wherein the solid support substrate is polycarbonate. 22. The test device of claim 18, wherein said solid support substrate is formed as a disk. 23. The test device of claim 22, wherein said disk has an external diameter of about 120 millimeters and a thickness of about 1.2 millimeters. 24. The claim testing device 18, where the fraction that responds to signals from each of the dissociable signal elements is ferromagnetic. 25. The assay device of claim 18, wherein the first side member includes a first antibody, antibody fragment, or antibody derivative, And the second side member includes a second antibody, antibody fragment, or antibody derivative. 26. The testing device of the claim 25, wherein the first antibody and the second antibody are specific for different epitopic sites of a virus selected from the group consisting of human immunodeficiency virus, hepatitis A, hepatitis B, hepatitis C, and human herpes virus. 27. The testing device of the claim 26, wherein the first antibody and the second antibody are specific for epitopes of a human immunodeficiency virus. 28. The testing device of the claim 27, wherein said immunodeficiency virus is HIV-1. 29. The assay device of claim 27, wherein the immunodeficiency virus is HIV-2. 30. The assay device of claim 26, wherein the virus is hepatitis C. 31. The test device of claim 18, which includes, in said plurality, signal elements adapted to recognize different analytes. 32. The assay device of claim 18, wherein the spatial pattern of the signal elements indicates the concentration of one or more analytes. 33. The testing device of claim 18, characterized in that it also comprises computer software encoded on the support substrate. 34. A test method comprising the steps of: contacting the test device of claim 18 with a liquid sample; dissociating the signal elements of said test device with a dissociating agent; and detecting the presence of the fraction that responds signals of the dissociated signal elements, restricted by analytes. 35. A test method comprising the steps of: contacting the test device of claim 19 with a liquid sample; dissociating the signal elements of said test device with a dissociating agent; and detecting the presence of the fraction that responds signals of the dissociated signal elements, restricted by analytes. 36. The test method of claim 34, wherein the signal elements comprise one or more siloxane moieties, and the dissociating agent includes sodium fluoride. 37. The assay method of claim 36, wherein the dissociating agent includes 1 mM to 1 M sodium fluoride. 38. The test method of claim 36, wherein the dissociation agent includes from 50 mM to 500 mM sodium fluoride. 39. The assay method of claim 36, wherein the dissociating agent includes sodium fluoride at about 100 mM. 40. The test method of claim 34, characterized in that it also comprises one or more washing steps. 41. The test method of claim 34, characterized in that it also comprises the step of rotating said test device. 42. A nucleic acid hybridization assay, comprising the steps of: contacting the assay device of claim 18 with a liquid sample containing nucleic acids, wherein the first side member and the second side member of when minus one of said joined signal elements, include an oligonucleotide; dissociating the signal elements from the test device; and detect dissociated signal elements, restricted by analytes. 43. A method of nucleic acid sequencing, comprising the steps of: contacting said assay device of claim 18 with a liquid sample containing nucleic acids, wherein the first side member and the second side member of each one of the linked signal elements, include an oligonucleotide; dissociating the signal elements from the test device; and detecting dissociated signal elements, restricted by analytes, wherein the spatially steerable pattern of the lateral member oligonucleotide sequences allows for the calculable reconstruction of the contiguous sequence of the signal response. 44. An immunoassay, comprising the steps of: contacting the assay device of claim 18 with a liquid sample containing antigenic analytes, wherein the first side member and the second side member of at least one of the elements of bound signals, include an antibody, antibody fragment, or antibody derivative; dissociating the signal elements from said test device; and detect dissociated signal elements, restricted by analytes. 45. The immunoassay of claim 44, wherein the first and second antibodies each recognize an epitopic site of a virus selected from the group consisting of human immunodeficiency virus, hepatitis A, hepatitis B, hepatitis C, and human herpes virus. 46. The immunoassay of claim 45, wherein the virus is a human immunodeficiency virus. 47. The immunoassay of claim 46, wherein the virus is HIV-1. 49. The immunoassay of claim 46, wherein the virus is HIV-2. 50. The immunoassay of claim 45, wherein the virus is hepatitis B. 51. The immunoassay of claim 45, wherein the virus is hepatitis C. 52. The immunoassay of claim 45, wherein the virus is a human herpes virus. 53. An assay device for detecting analytes, comprising: an optical disk having analyte-specific signal elements, legibly disposed on the same. 54. The assay device of claim 53, wherein the analyte-specific signal elements are dissociable. 55. A test device for detecting analytes, comprising: an optical disk having analyte-specific signal elements, legibly disposed thereon, wherein the analyte-specific signal elements are dissociable signal elements, in accordance with any of claims 1-17. 56. An assay device for detecting analytes, comprising: an optical disk having analyte-specific side members disposed thereon. 57. The assay device of claim 56, wherein analyte-specific side members include antibodies, antibody fragments, or antibody derivatives. 58. The assay device of claim 56, wherein analyte-specific side members include nucleic acids. 59. A method for performing assays for detecting analytes, comprising the steps of: contacting the assay device of claim 53 with a sample, and detecting analyte-specific signals from the same. 60. A method for performing assays for detecting analytes, comprising the steps of: contacting the assay device of claim 54 with a sample; dissociating the signal elements from the test device; and detect dissociated signal elements restricted by analytes on it. 61. A method for performing assays for detecting analytes, comprising the steps of: contacting the assay device of claim 56 with a sample; and detect specific signals of analytes therefrom. 62. A method to use an optical disc reader to practice tests to detect analytes, comprising the steps of detecting, with said reader, specific signals of analytes from the assay device of claim 53. 63. A method for using an optical disc reader to practice assays for detecting analytes, comprising the steps of detecting , with said reader, the dissociated signal elements restricted by analytes from the assay device of claim 54. 64. A method for using an optical disc reader to practice assays for detecting analytes, comprising the steps of detecting, with the reader, analyte-specific signals from the assay device of claim 56. 65. A method for making an analytical device. assay for detecting analytes, which comprises arranging analyte-specific signal elements, which can be read on an optical disk. 66. A method for making a test device for detecting analytes, comprising arranging dissociable signal elements, specific to analytes, that can be read on an optical disk. 67. A method for making a test device for detecting analytes, comprising disposing analyte-specific side members, on an optical disk.