Decoding of array sensors with microspheres

[0001] This application is a continuation of US Pat. No. 09 / 189,543 filed on November 10, 1998 and No. 60 / 090,473 filed on June 24, 1998. The present invention relates to compositions and methods for decoding microsphere array sensors.

[0002] There are several assays and sensors for the detection of the presence and / or concentration of specific substances in fluids and gases. Many of these rely on specific ligand / antiligand reactions as detection mechanisms. That is, it is known that a pair of substances (ie, a binding pair or a ligand / antiligand) binds to each other but binds slightly or not to other substances. This has been the focus of some techniques that utilize these binding pairs for complex detection. They generally label one component of the complex in a way to produce a whole complex that can be detected using, for example, radioisotopes, fluorescent and other optically active molecules, enzymes, etc. It is done by.

[0003] In these sensors, luminescence-based detection mechanisms are particularly used. In recent years, the use of fiber optics and fiber optic strands in combination with light absorbing dyes for chemical analytical measurements has evolved rapidly, especially over the last decade. The use of fiber optics and techniques for this purpose is described by Milanovich et al., "Novel Optical Fiber Techniques For Medical Application", Proceedings of the SPIE 28th Annual International Technical Symposium On Optics and Electro-Optics, Volume 494, 1980; Seitz, WR, "Chemical Sensors Based On Immobilized Indicators and Fiber Optics" in CRC Critical Reviews In Analytical Chemistry, Vol. 19, 1988, pp. 135-173; Wolfbeis, OS, "Fiber Optical Fluorosensors In Analytical Chemistry" in Molecular Luminescence Spectroscopy, Methods and Applications (SG Schulman), Wiley & Sons, New York (1988); Angel, SM, Spectroscopy 2 (4): 38 (1987); Walt et al., "Chemica Sensors and Microinstrumentation", ACS Symposium Series, Vol. 403, 1989, p. 252 and Wolfbeis, OS, Fiber Optic Chemical Sensors, Ed. CRC Press, Boca Raton, FL, 1991, Described by 2nd Volume.

[0004] When an optical fiber is used in an in vitro / in vitro sensor, one or more light absorbing dyes are placed near its distal end. Typically, light from a suitable light source is used to illuminate the dye through the proximal end of the fiber. Light propagates along the length of the optical fiber, and some of this propagated light exits the distal end and is absorbed by the dye. The light absorbing dye may or may not be fixed; it may or may not adhere directly to the optical fiber itself; in a fluid sample containing one or more subjects of interest. It may or may not be suspended in; it may or may not be retained for secondary use in the second optical measurement.

[0005] Once the light is absorbed by the dye, several lights of different wavelengths and intensities are re-emitted and transmitted by either the same fiber or the collected fiber to the observing and measuring detection system. The interaction between the light transmitted by the optical fiber and the properties of the light absorbing dye provides an optical basis for qualitative and quantitative measurements.

[0006] Of the many different classes of light-absorbing dyes conventionally used with fiber strands and bundles of optical fibers for various analytical purposes, a composition called "fluorophore" that emits light after absorption. Rather than absorbing an object and light and radiating it as light, a composition called a "chromophore" that internally converts the absorbed light into heat is more common.

[0007] Fluorescence is a physical phenomenon based on the ability of several molecules to absorb light (photons) at specific wavelengths and then emit light of longer wavelengths with lower energy. Substances that can fluoresce have several common characteristics: 1 wavelength λ_abAbsorbs light energy at; reaches the excited state of energy; then another light wavelength λ_emHas the ability to radiate light. The absorption and fluorescence spectra are unique to each fluorophore and are often graphically represented as two separate curves that slightly overlap. The same fluorescence emission spectrum is generally observed regardless of the wavelength of the excitation light, and therefore the wavelength and energy of the excitation light may vary moderately; the light emitted by the fluorophore is always the same emission spectrum. Will provide. Finally, the intensity of the fluorescent signal may be measured as the yield of emitted light. Fluorescence yield is the ratio of the number of emitted photons to the number of photons initially absorbed by the fluorophore. The following references are recommended for more detailed information on each of these features. Lakowicz, JR, Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983; Freifelder, D., Physical Biochemistry, 2nd Edition, WH Freeman and Company, New York, 1982, "Molecular Luminescence Spectroscopy Methods and Applications; Part I" (SG Schulman) in Chemical Analysis, vol. 77, Wiely & Sons, Inc., 1985, The Theory of Luminescence, Stepanov and Gribkovskii, lliffe Books, Ltd., London, 1968.

[0008] In contrast, substances that absorb light and do not fluoresce usually convert light into heat or kinetic energy. The ability to internally convert absorbed light identifies the dye as a "chromophore." Dyes that absorb light energy, such as chromophores, do so at individual wavelengths of energy and are characterized by a unique molar absorption coefficient at that wavelength. Chemical analysis using absorption spectroscopy with visible and ultraviolet wavelengths in combination with fiber light strands and the absorption coefficient allows spectral measurements to determine concentrations for the specific analysis of interest. The most common use of absorbance measurements over fiber optics is to determine the concentration calculated according to Beer's Law; therefore, a composition that absorbs light energy at a given wavelength with a single absorbance wavelength. The greater the amount of, the greater the optical density with respect to the sample. Thus, the total amount of directly absorbed light correlates with the amount of composition in the sample.

Much of the recent developments in the use of fiber optic sensors in qualitative and quantitative analytical measurements relate to the desire to place and / or fix various light absorbing dyes at the distal ends of the fiber optics. Thus, a variety of different fiber optic chemical sensors and methods have been reported for specific analytical measurements and applications such as pH measurement, oxygen detection and carbon dioxide analysis. These developments are illustrated by the publications below. Freeman et al., Anal. Chem. 53:98 (1983); Lippitsch et al., Anal. Chem. Acta. 205: 1, (1988); Wolfbeis et al., Anal. Chem. 60:2028 (1988); Jordan et al., Anal. Chem. 59: 437 (1987); Lubbers et al., Sens. Actuators 1983; Munkholm et al., Talanta 35: 109 (1988); Munkholm et al., Anal. Chem. 58: 1427 (1986); Seitz, WR, Anal. Chem. 56: 16A-34A (1984); Peterson et al., Anal. Chem. 52: 864 (1980); Saari et al., Anal. Chem. 54: 821 (1982); Saari et al., Anal. Chem. 55: 667 (1983) Zhujun et al., Anal. Chem. Acta. 160: 47 (1984); Schwab et al., Anal. Chem. 56: 2199 (1984); Wolfbeis, OS, "Fiber Optic Chemical Sensors", Ed. CRC Press, Boca Raton, FL, 1991, 2nd Volume; and Pantano, P., Walt, DR, Anal. Chem. 481A-487A, Vol. 67, (1995).

[0010] More recently, fiber optic light sensors have been constructed that allow the use of multiple dyes with a single separate fiber luminous flux. U.S. Pat. Nos. 5,244,636 and 5,250,264 of Walt et al. Disclose a system that attaches multiple different dyes to the distal ends of a bundle (the teachings of each of these patents are incorporated herein by reference). Will be). The disclosed form allows a bundle of separated optical fibers to optically approach individual dyes. This avoids the problem of decoding the separated signals in the reluminescence from each dye that occurs when two or more dye-derived signals combine, and there is significant overlap in the dye emission spectra.

U.S. Pat. Nos. 08 / 818,199 and 09 / 151,877 have microspheres or beads on the surface of a substrate, eg, on the ends of a fiber luminous flux (each individual fiber contains beads containing an optical sign). ) Describe the array configuration to be used. Since the beads fall randomly, a unique optical sign is needed to "decode" the array. That is, after the array is created, the arrangement of individual sites on the array can be correlated with beads or bioactive substances at specific sites. This means that the beads can be randomly distributed on the array, which means that it is a fast and inexpensive process compared to either the prior art in situ synthesis or spotting techniques. As outlined below, once the beads are loaded on the array, the array can be decoded or used with full or partial decoding that occurs after the test.

[0012] One drawback with previous systems is that they require a series of unique optical signs. Large numbers of such signs are available, for example, by the use of different dyes in different ratios, but a decoding system that does not rely on the use of a series of optical signs is preferred. Therefore, it is an object of the present invention to provide a method that allows decoding of a bead array without relying solely on unique optical signs.

[Disclosure of the Invention] According to the above object, the present invention provides an array construct comprising a substrate having a surface containing a discrete site. The construct is further identified to bind a decoder-binding ligand so that at least the first and second subpopulations (each subpopulation contains a bioactive substance); and the identity of the bioactive substance can be elucidated. Includes a population of microspheres containing an identifier binding ligand. Microspheres are distributed on the surface.

[0014] In a further aspect, the invention provides an array construct comprising a substrate having a surface comprising separated sites and a population of microspheres comprising at least the first and second subpopulations. Each subpopulation contains bioactive substances and does not contain optical signs.

[0015] In a further aspect, the present invention provides a method of manufacturing an array construct as described above. The method is characterized in that a surface containing individual portions is formed on the substrate, and the microspheres are distributed on the surface so that the individual portions contain microspheres. The microspheres contain at least the first and second subpopulations, each subpopulation containing a bioactive substance, and do not contain an optical sign.

[0016] In a further aspect, the present invention is characterized in that a surface containing individual portions is formed on the substrate, and the microspheres are distributed on the surface so that the individual portions contain microspheres. Provide a method of manufacturing. Microspheres contain at least the first and second subpopulations (each subpopulation contains a bioactive substance) and an identification binding ligand that binds a decoder binding ligand so that the identification of the bioactive substance can be elucidated.

[0017] In a further aspect, the invention provides an array construct as described above and adds a plurality of decoding binding ligands to the array construct to identify the location of at least a plurality of bioactive substances. Provided is a method for decoding a featured array configuration.

[0018] In a further aspect, the invention provides a method of determining the presence of a target subject in a sample. The method is characterized by contacting the sample with an array construct as outlined herein to determine the presence or absence of a target subject.

(Best Mode for Carrying Out the Invention) The present invention has separated beads, also referred to as microspheres, which generally carry different chemical functionality, capable of binding to individual microspheres. It is based on previous studies involving a bead-based analytical chemistry system distributed on a substrate containing a patterned surface of the (discrete) site. Since the beads are generally randomly placed on a substrate, conventional studies have been conducted in combination with unique optical signs, generally fluorescent dyes, that can be used to identify the chemical functionality of any individual bead. I was dependent. This allows candidate agents (ie, compounds and antibodies such as nucleic acids) to be synthesized far from their array placement, i.e., candidate agents can be synthesized on beads and then The beads can be randomly distributed on the patterned surface. The beads are first coded with an optical sign, which means that the array can be decoded later. That is, after the array is created, the position of individual sites on the array at a particular site can be correlated with the beads or candidate drug. This is the traditional in This means that beads can be randomly distributed on an array faster and cheaper than in situ synthesis and spotting techniques.

[0020] However, the drawback of these methods is that for large arrays, the system requires a large number of different optical signs, which are difficult and time consuming to utilize. Therefore, the present invention provides some improvements to these methods and is generally directed to array coding and decoding methods. That is, as will be apparent to those of skill in the art, the placement of bioactive agents is generally random, thus requiring a code / decoding system to identify the bioactive agent at each location in the array. This can be done in a variety of ways, described in more detail below, a) binding to the bioactive agent or identifier binding ligands (IBLs) bound to the beads, generally labeled directly. Decoder or Decoding) Use of binding ligands (DBLs); b) Target bead placement (eg, use photoactivated or photodegradable moieties to position beads individually, as described in detail below. Decoding by using sub-bundle or selective loading of the site; c) Selective decoding that decodes only the beads that bind to the target. Or d) In any combination of these, optionally, this decoding can only occur on all beads or beads bound to individual target analysts (analyte), as described in detail below. Includes possible decoding. Similarly, this can occur before or after the addition of the target analyst.

[0021] Once the bioactive agent has been identified and located in the array, the array is exposed to a sample containing the target analyzer. As described below, this can be done before or during the analysis. The target analyzer binds to the bioactive agent and results in a change in the optical signal of the individual beads, as described in detail below. In the present invention, "decoding" does not depend on the use of optical signs, but on the use of decoder binding ligands added during the decoding process. The decoder binding ligand is a unique identification binding ligand located on the bead, for example, if the bead contains a single-stranded nucleic acid as a bioactive agent, it binds to the bioactive agent itself. Decoder-binding ligands are labeled directly or indirectly, and decoding occurs by detecting the presence of this label. The continuous use of a pool of decoder-binding ligands can significantly reduce the number of decoding steps.

[0022] Accordingly, the present invention provides an array composition containing at least a first substrate having a surface containing individual sites. As used herein, "array" means a plurality of candidate agents in a sequence format, and the size of the array depends on the structure and the intended use of the array. Arrays can contain from about two different bioactive agents (ie, different beads) to millions, and even very large fiber optic arrays are possible. In general, the array can contain from 2 to 1 billion or more, depending on the size of the beads and substrate, the intended use of the array, ie very high density, high density, medium density, low density, very low. It can be an array of densities. The preferred range for very dense arrays is about 10,000,000 to about 2,000,000,000 (all numbers are cm).²Per), preferably about 100,000,000 to about 1,000,000,000. The range of the high density array is from about 100,000 to about 10,000,000, particularly preferably from about 1,000,000 to about 5,000,000. The range of medium density arrays is from about 10,000 to about 100,000, particularly preferably from about 20,000 to about 50,000. Low density arrays are generally less than 10,000, preferably about 1,000 to about 5,000. Very low density arrays are less than about 1,000, preferably about 10 to about 1,000, especially about 100 to about 500. In certain embodiments, the structures of the invention do not have to be in array format, i.e. for certain embodiments, structures containing a single bioactive agent may be made as well. In addition, some arrays may use multiple substrates of different or identical structures. Thus, for example, a large array can contain multiple smaller substrates.

[0023] Also, one advantage of the structures of the present invention is the ability to create extremely high density arrays, especially through fiber optic technology. For example, beads of 200 μm or less (200 nm beads are also possible) can be used, and very small fibers are also known, 0.5 cm.²Individual beads or fibers per 15,000,000 or higher density (possibly 25-50 x 10)⁶Also), 1mm²It is also possible to obtain as many as 250,000 (and possibly millions) of different fibers and beads in the Fiber Optic Bundle.

[0024] The "base" or "solid support" or other grammatical equivalent used herein can be modified to include separate individual sites suitable for bead binding or association, at least one. It means any substance that reacts sensitively to one of the detection methods. As will be apparent to those skilled in the art, the number of possible substrates is very large. Possible substrates include, but are not limited to, glass, modified or functionalized glass, plastics (polymers of acrylic, polystyrene and styrene with other substances, polypropylene, polyethylene, polybutylene, polyurethane, teflon and the like. Includes), polystyrene-based materials including polysaccharides, nylon or nitrocellulose, resins, silica or silicon and modified silicon, carbon, metals, inorganic glass, plastics, optical fiber bundles and various other polymers. In general, the substrate allows optical detection and does not fluoresce to the extent that it is perceived. Generally, the substrate is flat, but as will be apparent to those skilled in the art, other arrangements of the substrate can be used as well, for example, allowing access to the beads of the sample and confocal microscopy for detection. By embedding the beads in a plastic porous block that can be used, a three-dimensional arrangement can also be used. Similarly, beads can be located on the inner surface of a tube for flow-through sample analysis to minimize sample volume. Preferred substrates include optical fiber bundles described below and planes such as glass, polystyrene and other plastics and acrylics.

[0025] In a preferred embodiment, the substrate is an optical fiber bundle as commonly described in US Patent Applications 08 / 944,850 and 08 / 519,062 and PCT / US98 / 05025 and PCT / US98 / 09163. Alternatively, it is an array, which is incorporated herein by explicit exhibitor. A preferred embodiment utilizes a preformed, centralized fiber optic array. As used herein, "preformed, centralized fiber optic array" means a coaxially arranged, integrated fiber optic bundle along the length direction. Fiber strands are generally individually cladding. However, one thing that distinguishes other fiber optic formats from preformed, unitary arrays is that the fibers cannot be physically manipulated individually, that is, in general, one strand is along the length direction. In any respect, it cannot be physically separated from other fair bar strands.

[0026] At least one surface of the substrate is modified to include separate individual sites for later association with microspheres. These sites are physically altered sites, that is, the use of wells or small depressions in the substrate that can hold the beads and support the microspheres, or the use of other forces (magnetic force or compression), or , Chemically altered or activated sites such as chemically functionalized sites, electrostatically altered sites, hydrophilic / hydrophobically functionalized sites, adhesive spots, etc. Can be included. The sites can be patterned, i.e., have a constant design or arrangement, or may be randomly distributed. In a preferred embodiment, a constant pattern of parts is used so that the address can be specified in the XY coordinate plane. The "pattern" in this sense preferably includes the repetition of unit cells that allow the beads on the substrate to have a high density. However, it should be noted that these sites do not have to be separate sites. That is, for example, it is also possible to use an adhesive or a chemically functional uniform surface that allows the beads to associate at any position. Thus, the surface of the substrate is modified to allow the microspheres to associate with individual sites, whether individual sites are continuous or discontinuous with other sites. For example, the surface of the substrate is modified to have separated sites with a single associated bead, or otherwise the surface of the substrate is modified so that the beads sink somewhere and enter the last separated portion. You can also make it.

[0027] In a preferred embodiment, the surface of the substrate is modified to include wells, i.e., recesses on the surface of the substrate. This can be done by methods generally known to those of skill in the art, using a variety of techniques including, but not limited to, photolithography, stamping techniques, molding techniques and microetching techniques. As will be apparent to those skilled in the art, the technique used will depend on the shape of the composition and the substrate. In a preferred embodiment, a physical change is created on the surface of the substrate to give rise to a site. In a preferred embodiment, the substrate is a fiber optical bundle and the surface of the substrate is one end of a fiber bundle commonly described in US patent applications 08 / 818,199 and 09 / 151,877. These are explicitly included in this specification. In this embodiment, wells are created in the end or distal ends of a fiber optical bundle containing individual fibers. In this embodiment, the cores of the individual fibers are etched with respect to cladding to form small wells or depressions at one end of the fibers. The required depth of the well depends on the size of the beads added to the well.

[0028] In general, in this embodiment, the well functions chemically as outlined below, but the microspheres are not covalently attached to the well and a cross-linking agent can be used on the beads, or Physical barriers, ie films or membranes, can also be used. In a preferred embodiment, the microspheres of the invention are to contain chemically modified sites that can be used to bind, either shared or unshared, to separate sites or locations on the substrate. Modify the surface of the substrate. As used herein, the term "chemically modified moiety" refers to an amino group, a carboxy group, which can be used to co-bond microspheres, which generally contain a corresponding reactive functional group. Addition of a type of functional group, including oxo and thiol groups; Addition of a type of sticky substance that can be used to attach microspheres (introduction of conventional functional groups to add a sticky substance) (By either by or by the direct addition of an adsorbent); when the microspheres have a charging group opposite to their site, the addition of one type of charging group for electrostatically coupling the microspheres (functionality). (Similar to group introduction); Under suitable experimental conditions, the site is specifically hydrophobic or hydrophilic so that the addition of hydrophobic or hydrophilic microspheres results in the binding of the microspheres to the site based on hydroaffinity. It includes, but is not limited to, the addition of one type of functional group. For example, in an aqueous system, when a hydrophobic site is used with hydrophobic beads, the beads predominantly bind to the site. As mentioned above, "mold" in this sense is used to uniformly treat the surface of a bead to bond it to a separate site, as well as to process the surface to obtain a separate site. Include. This can be done in a variety of ways, as will be apparent to those skilled in the art.

[0029] The composition of the present invention further comprises a population of microspheres. As used herein, the term "population" means a plurality of beads, as described above for an array. Within the population, there are separate subpopulations that can be one microsphere or multiple identical microspheres. That is, in one embodiment, the array may only contain one bead for each bioactive substance, as described in more detail below; a preferred embodiment contains multiple beads of each type. Use. As used herein, "microspheres" or "beads" or "particles" or grammatically equivalents mean small, distinct particles. The composition of the beads will vary depending on the type of bioactive substance and the method of synthesis. Suitable bead compositions are compositions used in the synthesis of peptides, nucleic acids and organics, such as plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic substances, triazole, carbon graphite, titanium dioxide, latex or Cross-linked dextran, including, but not limited to, cepharose, cellulose, nylon, cross-linked micelles and teflon, all of which can be used. Bangs Laboratories, Fishers IN's "Microspere Detection Guide" is a useful guidebook.

The beads need not be spherical; irregular particles can also be used. In addition, the beads may be porous to increase the surface area of the beads available for attachment of bioactive material or attachment of IBL. Bead sizes range from nanometers, i.e. 100 nm to millimeters, i.e. 1 mm, with beads of about 0.2 micron to about 200 microns being preferred, beads of about 0.5 to about 5 microns being particularly preferred, but in some cases. Can also be used with smaller beads. Important in the present invention is to use a substrate / bead pairing that allows the beads to bind or adhere to separate sites on the surface of the substrate so that the beads do not move during the assay process.

As will be apparent to those skilled in the art, each microsphere may contain a bioactive substance, and depending on the method of synthesis thereof, microspheres containing no bioactive substance may be present. The "candidate bioactive substance" or "biologically active substance" or "functional group substance" or "binding ligand" in the present specification can be attached to the microspheres of the present invention as used herein. Means, for example, proteins, oligopeptides, small organic molecules, coordination complexes, polysaccharides, ligands, and the like. It will be appreciated that the compositions of the present invention have two main uses. In a preferred embodiment, as described in more detail below, the presence of a particular target analytic agent; the composition, eg, for detecting the presence or absence of a particular nucleotide sequence or a particular protein, eg, an enzyme, antibody or antigen. Things are used. In another preferred embodiment, the composition can be used to screen a bioactive substance, i.e., a drug candidate, for binding to a particular target analysis substance.

The bioactive substance is typically an organic molecule, preferably a small organic compound having a molecular weight greater than 100 daltons and less than about 2500 daltons, but includes many species. Bioactive substances have functional groups that are essential for structural interactions with proteins, especially hydrogen bonds, typically at least one amino, carbonyl, hydroxyl or carboxyl group, preferably at least two. Contains functional groups. Bioactive substances often include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the functional groups described above. Bioactive substances are also found in biomolecules, including peptides, nucleic acids, sugars, fatty acids, steroids, purines, pyrimidines, derivatives thereof, structural analogs or combinations. Nucleic acids and proteins are particularly preferred. Bioactive substances can be obtained from a variety of sources, including libraries of synthetic or natural compounds. Many means are available for random and directional synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, for example. Alternatively, a library of natural compounds in the form of bacterial, fungal, plant and animal extracts is also available or readily produced. In addition, naturally or synthetically produced libraries and compounds are readily modified via conventional chemical, physical and biochemical means. Known pharmacological materials may be subjected to directional or random chemical modifications such as acylation, alkylation, esterification and / or amidation to produce structural analogs.

[0033] In a preferred embodiment, the bioactive substance is a protein. As used herein, the term "protein" means a covalent bond of at least two amino acids, and includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptide pseudostructures. Thus, as used herein, "amino acid" or "peptide residue" means both naturally occurring amino acids and synthetic amino acids. For example, allogeneic-phenylalanine, citrulline and norleucine are considered amino acids for the purposes of the present invention. The side chains may be in either the (R) or (S) arrangement. In a preferred embodiment, the amino acid is the (S) or L-configuration. If non-naturally occurring side chains are used, for example, non-amino acid substituents may be used to prevent or prevent in vivo degradation.

[0034] In one preferred embodiment, the bioactive substance is a naturally occurring protein or a fragment of a naturally occurring protein. Thus, for example, random or directional digestion of protein-containing cell extracts or protein cell extracts can be used. In this regard, libraries of prokaryotic and eukaryotic proteins can be prepared for screening with the systems described herein. In this embodiment, bacterial, fungal, viral and mammalian proteins are particularly preferred, the latter being particularly preferred, and human proteins being most preferred. In a preferred embodiment, the bioactive substance is a peptide of about 5 to about 30 amino acids, preferably about 5 to about 20 amino acids, particularly preferably about 7 to about 15 amino acids. The peptide may be a digest of the naturally occurring protein described above, a random peptide or a "biased" random peptide. "Randomized" or grammatically equivalent herein means that nucleic acids and peptides consist essentially of random nucleotides and amino acids, respectively. In general, these random peptides (or the nucleic acids described below) are chemically synthesized so that they can be in any position of any nucleotide or amino acid. Synthetic methods are designed to produce randomized proteins or nucleic acids to form possible combinations of all or most of the length of the sequence, thus forming a library of randomized bioactive proteinaceous substances. can do.

[0035] In a preferred embodiment, a library of bioactive substances is used. The library provides a structurally diverse population of bioactive substances and is likely to bind to the target analyte to a sufficient extent. Therefore, the reactant library must be large enough so that at least one of its members has a structure that imparts affinity to the target analyte. It is difficult to measure the absolute size required by the reactant library, but the immune response characteristics provide a clue: 10⁷-10⁸Species antibody diversity provides at least one combination that is sufficiently compatible to interact with most of the antigens that an organism may face. Thus, in the preferred embodiment, at least 10⁶, Preferably 10⁷, More preferably 10⁸Species bioactive substances are simultaneously analyzed by the methods of the invention. The preferred method maximizes the size and variety of the library. In a preferred embodiment, the library is sufficiently randomized and there is no sequence priority or stationarity anywhere. In a preferred embodiment, the library is biased. That is, a location in the sequence remains stationary or is selected from a limited number of possibilities. For example, in a preferred embodiment, the nucleotide or amino acid residue is phosphorylated to a proline for the SH-3 domain in a predetermined type, eg, a hydrophobic amino acid, a hydrophilic residue, cross-linked to create a cysteine. Sites are randomized within serine, threonine, tyrosine, histidine, etc., or sterically polarized (small or large) residues to purines.

[0036] In a preferred embodiment, the bioactive substance is a nucleic acid (generally referred to herein as a "probe nucleic acid" or "candidate probe"). As used herein, "nucleic acid" or "oligonucleotide" or grammatically equivalent means a covalent bond of at least two nucleotides together. The nucleic acids of the invention generally contain phosphodiester bonds, but in some cases, for example, phosphoramides (Beaucage et al., Tetrahedron, 49 (10): 1925 (1993) and the like, as shown below. References in; Letsinger, J.Org.Chem., 35: 3800 (1970); Sprinzl et al., Eur.J.Biochem., 81: 579 (1977); Letsinger et al., Nucl.Acids Res., 14: 3487 (1986); Sawai et al., Chem.Lett., 805 (1984); Letsinger et al., J.Am.Chem.Soc., 110: 4470 (1988); and Paulels et al., Chemica Scripra, 26: 141 (1986)) , Phosphodiester (Mag et al., Nucleic) Acids Res., 19: 1437 (1991); and U.S. Pat. No. 5,644,408), phosphorodithioate (Briu et al., J. Am. Chem. Soc., 111: 2321 (1989)), o-methylphosphoramide. Linkage (Eckstein, Oligonucleotides and Analogs: A Practical Approach, Oxford University (See Press) and Peptide Nucleic Acid Skeletons and Linkages (Egholm, J.Am.Chem.Soc., 114: 1895 (1992); Meier et al., Chem.Int.Ed.Engl., 31: 1008 (1992); It may have another skeleton, including Nielsen, Nature, 365: 566 (1993); Carisson et al., Nature, 380: 207 (1996), all of which are incorporated herein by reference). Includes nucleic acid analogs. Other similar nucleic acids are positive skeletons (Denpcy et al., Proc. Natl. Acad. Sci. USA, 92: 6097 (1995)); nonionic skeletons (US Pat. No. 5,386,243; No. 5637684; No. 5602240; No. 5216141; and No. 4469863; Kiedrowski et al., Angew, Chem.Intl.Ed.English, 30:423 (1991); Letsinger et al., J.Am.Chem.Soc., 110: 4470 (1988); Letsinger et al. , Nucleosides & Nucleotides, 13: 1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research," edited by YSSanghui and P. Dan. Cook; Mesmaeker et al., Bioorganic & Medical Chem. Lett. , 4: 395 (1994); Jeffs et al., J. Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) and US Pat. Chapter 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research," YSSanghui and P. Dan. Includes nucleic acids with a non-ribose skeleton as described in Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acid (see Jenkins et al., Chem. Soc. Rev., (1995) pp. 169-176). Several nucleic acid analogs are described in Rawls, C. & E News, June 2, 1997, p. 35. All of these publications are incorporated herein by specifying the source. These modifications can also be made in the ribose-phosphate backbone to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in a physiological environment; for example, PNAs in particular. preferable. In addition, a mixture of naturally occurring nucleic acids and analogs can be prepared. Mixtures of various nucleic acid analogs and mixtures of naturally occurring nucleic acids and analogs can also be prepared. Nucleic acids may be either single-stranded or double-stranded, as specified, or may contain portions of both double-stranded or single-stranded sequences. The nucleic acid may be DNA, both genomic DNA and cDNA, RNA or hybrid; where the nucleic acid is a combination of deoxyribo- and ribo-nucleotides, uracil, thymine, cytosine, guanine, inosin, xantanine, hypoxanthanine, It may contain any combination of bases, including isocytosine, isoguanine and base analogs, such as nitropyrrole and nitroindole. For example, PNA is particularly preferred. In addition, a mixture of naturally occurring nucleic acids and analogs can be prepared. Mixtures of various nucleic acid analogs and mixtures of naturally occurring nucleic acids and analogs can also be prepared. Nucleic acids may be either single-stranded or double-stranded, as specified, or may contain portions of both double-stranded or single-stranded sequences. The nucleic acid may be DNA, both genomic DNA and cDNA, RNA or hybrid; where the nucleic acid is a combination of deoxyribo- and ribo-nucleotides, uracil, thymine, cytosine, guanine, inosin, xantanine, hypoxanthanine, It may contain any combination of bases, including isocytosine, isoguanine and base analogs, such as nitropyrrole and nitroindole. For example, PNA is particularly preferred. In addition, a mixture of naturally occurring nucleic acids and analogs can be prepared. Mixtures of various nucleic acid analogs and mixtures of naturally occurring nucleic acids and analogs can also be prepared. Nucleic acids may be either single-stranded or double-stranded, as specified, or may contain portions of both double-stranded or single-stranded sequences. The nucleic acid may be DNA, both genomic DNA and cDNA, RNA or hybrid; where the nucleic acid is a combination of deoxyribo- and ribo-nucleotides, uracil, thymine, cytosine, guanine, inosin, xantanine, hypoxanthanine, It may contain any combination of bases, including isocytosine, isoguanine and base analogs, such as nitropyrrole and nitroindole.

[0037] In a preferred embodiment, the bioactive substance is a library of cloned nucleic acids, including DNA and RNA. In this embodiment, individual nucleic acids are generally prepared using conventional methods (including, but not limited to, growth in plasmids or phage vectors, amplification methods including PCR, etc.). .. Preferably, the nucleic acids are arranged in a microtiter plate format and in a particular format, such as beads added to attach the library. Chemical or affinity capture (including, for example, inclusion of derivatized nucleotides such as AminoLink or biotinylated nucleotides that can be used later to attach nucleic acids to the surface, and affinity capture by hybridization), cross-linking, And various methods (not limited to these methods) understood in the art, including electrostatic attachment, etc., to attach the clone library (or any nucleic acid described herein). it can. In a preferred embodiment, affinity capture is used to attach the cloned nucleic acid to the beads. For example, one member of the binding pair can derivatize the cloned nucleic acid and the other member of the binding pair can derivatize the beads. Suitable binding pairs are those described herein with respect to IBL / DBL pairs. For example, the cloned nucleic acid may be biotinylated (eg, using enzymatic uptake of biotinylated nucleotides or by photoactivated cross-linking of biotin). The biotinylated nucleic acid can then be captured on streptavidin-coated beads, as is known in the art.Similarly, other hapten-receptor combinations such as digoxigenin and anti-digoxigenin antibodies can be used. Alternatively, the chemical groups used to add the nucleic acid to the surface can be added in the form of derivatized nucleotides. A preferred attachment is a covalent bond, but if there are multiple attachment sites per nucleic acid molecule, even a relatively weak interaction (ie, non-covalent bond) is sufficient to attach the nucleic acid to the surface. It is possible. Thus, for example, by using beads having a charge opposite to that of the bioactive substance, electrostatic interactions can be used for adhesion. Similarly, affinity capture using hybridization can be used to attach the cloned nucleic acid to the beads. For example, as is known in the art, polyA-added RNA is routinely captured by hybridization to oligo-dT beads. For this, oligo-dT capture and subsequent cross-linking steps (eg, psoralen cross-linking) may be used. When the nucleic acid of interest does not contain a poly A moiety, it can be attached by polymerization with a terminal transferase or ligation of an oligo A linker, as is known in the art. Alternatively, chemical cross-linking may be performed, for example, by photoactivating and cross-linking thymidine to a reactive group, as is known in the art.

[0038] In general, special methods are required to decode an array of clones, as described in more detail below. As generally described above for proteins, the nucleic acid bioactive substance may be a naturally occurring nucleic acid. For example, prokaryotic and eukaryotic genome digests may be used as described above for proteins. In general, the probes of the invention are designed to be complementary to the target sequence (either the target analyte sequence of the sample or any of the other probe sequences described herein) with the target. Allow hybridization with the probe of the present invention to occur. This complementarity does not have to be perfect. There may be several base pair mismatches that interfere with hybridization between the target sequence and the single-stranded nucleic acids of the invention. However, if the number of mutations is too high for hybridization to occur even under minimal stringency hybridization conditions, the sequence is a non-complementary target sequence. Thus, as used herein, "substantially complementary" means that the probe is sufficiently complementary to the target sequence and hybridizes under selected reaction conditions. High stringency conditions are known in the art, see, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989 and Short Protocols in Molecular Biology, ed. Ausbel, et al. Also integrated herein by reference). Stringent conditions are sequence dependent and will differ in different situations. Longer sequences specifically hybridize at higher temperatures. For further guidance on nucleic acid hybridization, Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, "Overview of principles of Hybridization and the strategy of nucleic acid assays" (1993). In general, stringent conditions are chosen to be about 5-10 ° C below the melting temperature (Tm) of the specific sequence at a constant ionic strength and pH. Tm hybridizes 50% of probes complementary to the target sequence to the target sequence in equilibrium (if the target sequence is in excess, 50% of the probes in equilibrium at Tm are occupied). Temperature (at constant ionic strength, pH and nucleic acid concentration). Stringent conditions are lower than sodium ions with a salt concentration of about 1.0 M, typically about 0.01 to 1. at pH 7.0 to 8.3. Sodium ion concentration of 0M, temperature is at least about 30 ° C for short probes (eg 10-50 nucleotides) and at least about 60 for long probes (eg longer than 50 nucleotides) ° C. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. In another embodiment, less stringent hybridization conditions are used. For example, moderate to low stringency conditions as known in the art may be used. See the above references in Maniatis and Ausbel and the above references in Tijssen. As used herein, the term "target sequence" or grammatical synonym means a nucleic acid sequence that lies on a single strand of nucleic acid. The target sequence may be a gene, regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or part of others. It can be of any length, and it is understood that longer sequences are more specific. As will be appreciated by those skilled in the art, complementary target sequences may be in many forms. For example, it may be included in a larger nucleic acid sequence, i.e., the whole or part of a gene or mRNA, a restriction fragment of a plasmid or genomic DNA, and the like. As will be described more fully later, probes are created to hybridize to the target sequence to determine the presence or absence of the target sequence in the sample. Generally speaking, this term will be understood by those skilled in the art. Will be created. Generally speaking, this term will be understood by those skilled in the art. Will be created. Generally speaking, this term will be understood by those skilled in the art.

[0039] In a preferred embodiment, the bioactive substance is an organic chemical moiety, and various organic chemical moieties are available in the literature. In a preferred embodiment, each bead comprises a single type of bioactive substance, preferably a plurality of individual bioactive substances are attached to each bead. Similarly, a preferred embodiment is to use more than one microsphere containing a unique bioactive substance, i.e., there is a degeneration constructed in the system by the use of a subpopulation of microspheres. Each microsphere in the subpopulation contains the same bioactive substance. As will be appreciated by those skilled in the art, the bioactive substance may be synthesized directly on the beads or attached to the beads after synthesis. In a preferred embodiment, a linker is used to attach the bioactive substance to the beads to allow for good attachment, sufficient flexibility to allow good interaction with the target molecule, and an undesired binding reaction. Try to avoid. In a preferred embodiment, the bioactive substance is synthesized directly on the beads. As is known in the art, many classes of compounds, such as peptides, organic moieties and nucleic acids, have been synthesized on beads-encapsulating supports. In a preferred embodiment, the bioactive substance is first synthesized and then covalently attached to the beads. Do this depending on the composition of the bioactive substance and beads, as will be appreciated by those skilled in the art. Functionalization of the surface of a solid support, such as certain polymers, using chemically reactive groups such as thiols, amines, carboxyls, etc. is widely known in the art. Therefore, "blank" microspheres with surface chemical groups that facilitate the attachment of desired functional groups by the user may be used. Some examples of these surface chemical groups for blank microspheres are, but are not limited to, amino groups including aliphatic and aromatic amines, carboxylic acids, aldehydes, amides, chloromethyl groups, hydrazides, hydroxyls. Includes groups, sulfonates and sulfates. Some types of functional groups are used, generally using known chemical methods. Different candidate agents can be added to the beads. For example, a candidate agent containing a carbohydrate can be attached to an amino-functionalized support. Carbohydrate aldehydes are made using standard methods and then the aldehydes are reacted with amino groups on the surface. In another embodiment, a sulfhydryl linker may be used. There are many sulfhydryl reactive linkers known in the art, such as SPDP, maleimide, α-haloacetyl, and pyridyl disulfide (see, eg, the 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200). (Integrated herein by reference)), they can be used to attach cysteine-containing proteinaceous agents to the support. Alternatively, the amino groups on the candidate agonist may be used for attachment to the amino groups on the surface. For example, many stable bifunctional linkers are well known in the art and include homologous and heterologous bifunctional linkers (see Pierce Catalog and Handbook pp. 155-200). In a further embodiment, well-known linkers (see Pierce's catalog) may be used to derivate the carboxyl groups (either those on the surface or those of candidate agents). For example, carbodiimide activates the carboxyl group to allow it to be attacked by good nucleophiles such as amines (Torchillin et al., Critical Rev. Therapeutic Drug Carrier Systems, 7 (4): 275-308 (1991). ) Reference (especially integrated herein)). The protein candidate agonist may be attached using other methods known in the art, for example, methods for attaching the antibody to the polymer. For example, Slinkin et al., Bioconj. Chem. 2: 342-348 (1991); Torchillin et al., Supra; Trubetskoy et al., Bioconj. Chem., 3: 323-327 (1992); King et al., Cancer Res. 54: 6176-6185 (1994); and Wilbur et al., See Bioconjugate Chem. 5: 220-235 (1994) (integrated herein by reference). It should be understood that the candidate agonist may be attached by a variety of methods, including those described above. Preferably, the attachment mode does not significantly change the functionality of the candidate agent. That is, the candidate agonist should be attached in a flexible manner that allows it to interact with its target.

[0040] Individual methods of immobilizing enzymes on microspheres are known in the art. In case of 1, NH₂Surface chemical group microspheres are used. Surface activation is performed with 2.5% glutaraldehyde in phosphate buffered Sayline (10 mM) (138 mM NaCl, 2.7 mM KCl) to a pH of 6.9. Stir this on a stirring bed for about 2 hours at room temperature. The microspheres are then rinsed with ultrapure water supplemented with 0.01% Twin 20 (surfactant) and then rinsed again with PBS at pH 7.7 supplemented with 0.01% Twin 20. Finally, the enzyme is added to the solution after prefiltration, preferably with a 0.45 μm amicon micropure filter.

[0041] In some embodiments, the microspheres further comprise an identity fire binding ligand for use in certain decoding systems. As used herein, the term "identity fire binding ligand" or "IBL" facilitates the elucidation of the identity of a bioactive substance attached to a bead by specifically binding to a corresponding decoder binding ligand (DBL). Means a compound. That is, the IBL and the corresponding DBL form a binding pair. As used herein, "specifically binding" means that an IBL distinguishes between a corresponding DBL and another DBL (ie, a DBL relative to another IBL) or another component or contaminating component of the system. It means that it binds to the DBL with sufficient specificity. The bond should be sufficient to continue to bond under the conditions of a decoding step, including a washing step to remove non-specific bonds. In some embodiments, for example, if the IBL and the corresponding DBL are proteins or nucleic acids, the dissociation constant of the IBL for that DBL is about 10.^-4~10^-6M^-1Less than, about 10^-5~10^-9M^-1Less than preferably about 10^-7~10^-9M^-1Is particularly preferable.

IBL-DBL binding pairs are known or can be easily found using known descriptions. For example, if the IBL is a protein, the DBL may include a protein (specifically, an antibody or fragment thereof (such as FAb)) or a small molecule, or vice versa (IBL is an antibody). , DBL is a protein). Metal ion-metal ion ligands or chelating agent pairs are also useful. Antigen-antibody pairs, enzymes and substrates or inhibitors, other protein-protein interaction pairs, receptor-ligands, complementary nucleic acids (including nucleic acid molecules that form triple spirals), and carbohydrates and their binding partners. It is also a suitable binding pair. Nucleic acid-nucleic acid binding protein pairs, including single-stranded or double-stranded nucleic acid binding proteins and small molecule nucleic acid binding agents, are also useful. Similarly, as outlined in US Pat. Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337, and related patents (specifically, this specification). The nucleic acid "aptamers" (as part of the book) can be developed to actually bind to any target; such aptamer-target pairs can be used as IBL-DBL pairs. Similarly, there is an extensive set of literature related to the development of bond pairs based on combinatorial chemistry methods.

[0043] In a preferred embodiment, IBL is a molecule whose color or luminescence properties change in the presence of selective binding DBL. In one embodiment, the DBL may carry a label such as a fluorophore, or may be attached to beads, i.e., "decoder beads".

[0044] In a preferred embodiment, the IBL-DBL pair comprises a substantially complementary single-stranded nucleic acid. In this embodiment, the binding ligand can be referred to as an "identity fire probe" and a "decoder probe". In general, the identity fire and decoder probes have a base pair length in the range of about 4 to about 1000, preferably about 6 to about 100, and particularly preferably about 8 to about 40. Importantly, the probe is long enough to be specific, i.e. long enough to distinguish between different IBL-DBL pairs, and a) optionally under experimental conditions. Short enough to allow dissociation, and b) effective hybridization.

[0045] In a preferred embodiment, the IBL does not bind to the DBL, as outlined below. Rather, IBL is used as the identity fire moiety (IM) that is directly identified, for example, through the use of mass spectroscopy.

Alternatively, in a preferred embodiment, the IBL and the bioactive substance are the same moiety; thus, as outlined herein, bioactivity, especially without the use of optical signs. The substance can be provided as both an identity fire and the bioactive substance. For example, in the case of nucleic acids, bead-binding probes (provided as bioactive substances) can also bind decoder probes to identify the sequence of probes on the beads. Thus, in this embodiment, the DBL binds to the bioactive substance. This is especially useful if this embodiment can provide information about the array or assay in addition to decoding. For example, the use of DBL allows array calibration curve preparation and assay development, as described in more detail below. This is even done without DBL alone; for example, in non-random arrays, the use of these probe sets can be used for array calibration curve creation and assay development even when decoding is not required. Can be made possible.

[0047] In a preferred embodiment, the microspheres do not contain an optical sign. That is, as outlined in U.S. Patent Applications 08 / 818,199 and 09 / 151,877, conventional treatments are uniquely used to identify unique bioactive substances in a partial population of microspheres. Had each partial population of microspheres containing an optical sign or tag of. That is, the decoding utilizes the optical properties of the beads, which distinguishes the beads containing the unique optical signature from the beads in other positions having different optical signatures. Thus, conventional processing imparts a unique optical sign to each bioactive substance, whereby microspheres containing this bioactive substance are identified based on this sign. These optical signs include dyes, usually chromogenic groups or phosphors, which are incorporated or adhered to the beads themselves. The variety of optical signs utilized different fluorescent dyes, different proportions of fluorescent dye mixtures, and different concentrations (intensities) of fluorescent dyes.

[0048] Thus, the present invention does not rely solely on the use of optical properties to decode the array. However, as will be appreciated by those skilled in the art, in some embodiments it is possible to utilize optical signatures as an additional coding method with the systems of the invention. Thus, for example, as outlined below, the size of the array can be effectively increased, but if a set of decoding parts is used in some methods, one of them will , The use of beads in combination with optical signs. Thus, for example, when using a "set" of decoding molecules, the use of two groups of beads, one with and one without an optical sign, can effectively double the size of the array. Similarly, the use of multiple optical signs increases the possible size of the array.

[0049] In a preferred embodiment, each partial population of beads comprises a plurality of different IBLs. Using multiple different IBLs to encode a nuclear bioactive substance substantially increases the number of unique codes that can occur. That is, by using one unique IBL per bioactive substance, the size of the array is the number of unique IBLs (assuming that "reuse" did not occur, as outlined below. To do). However, when using the presence or absence of each IBL as an indicator, the size of the array can be increased by using multiple different IBLs per bead.ⁿCan be increased to. For example, granting 10 IBLs per bead yields a 10-bit binary code, where each bit is "1" (IBL present) or "0" (IBL absent). Can be shown. 10-bit binary sign is 2¹⁰It has a number of possible variants. However, the size of the array can be further increased if other parameters such as concentration or intensity are included, as discussed in more detail below; thus, for example, two concentrations of IBL were used. If the size of the array is 3ⁿIncrease. Thus, in this embodiment, each individual bioactive substance in the array is conferred with a combination of IBLs that can be added to the beads before, after, or during the synthesis of the bioactive substance. , IBL and co-addition of bioactive substance components).

[0050] Alternatively, if the bioactive substance is a polymer of various residues, i.e. the bioactive substance is a protein or nucleic acid, different IBL combinations are used to elucidate the sequence of the protein or nucleic acid. be able to.

Thus, for example, when two IBLs (IBL1 and IBL2) are used, the position of the nucleic acid can be elucidated: for example, adenosine can be represented by the presence of both IBL1 and IBL2; Thymidine can be represented by the presence of IBL1 but the absence of IBL2, cysteine can be represented by the presence of IBL2 but the absence of IBL1, and guanosine can be represented by the absence of both. .. Nucleic acid 2-position can be performed similarly with IBL2 and IBL4; thus, the presence of IBL1, IBL2, IBL3 and IBL4 gives the sequence of AA, and IBL1, IBL2 and IBL3 indicate the sequence AT, IBL1, IBL3 and IBL4 give the sequence TA and so on. IBL5 and IBL6 are used for the 3rd place. In this way, the use of 20 identity fires gives a unique code for every possible demer.

[0052] The system is similar to a protein, but requires a number of different IBLs to identify each position, depending on the possible diversity at each position. Thus, for example, if all amino acids are possible at all positions, then five IBLs are needed for each position. However, as outlined above, for example, when using random peptides as bioactive substances, there may be biases constructed in the system; not all amino acids can be present at all positions. Several positions can be preset and therefore it is possible to use four different IBLs for each amino acid.

[0053] In this way, one type of "barcode" can be constructed for the nuclear sequence; the presence or absence of different IBLs allows the identification of each bioactive substance.

[0054] In addition, the use of various concentrations or densities of IBL allows some degree of "reuse". For example, if the beads containing the first drug have a 1-fold concentration of IBL and the 2nd beads containing the second drug have a 10-fold concentration of IBL, then by using the saturated concentration of the corresponding labeled DBL, Let the user distinguish between the two beads.

[0055] As soon as microforms containing candidate agents and unique IBLs are produced, they are added to the substrate to form an array. Although most of the methods described herein add beads to the substrate prior to the assay, the order of array preparation, use and decoding can vary. For example, an array can be prepared, decoded, and then assayed. Alternatively, the array can be prepared, used in the assay, and then decoded; this can find unique use when only a small amount of beads need to be decoded. Alternatively, the beads can be added to the assay mixture, i.e., the sample containing the target analyte, before the beads are added to the substrate; after the addition and assay, the array can be decoded. This is especially preferred when the sample containing the beads is agitated or mixed; this can increase the amount of target analyte bound to the beads per unit time, thus hybridization (in the case of a nucleic acid assay). The speed becomes faster. This can find peculiar use when the concentration of the target analyte in the sample is low; in general, long binding times must be used at low concentrations.

[0056] In addition, the addition of beads to the assay mixture can allow classification or sorting. For example, beads from a large library can be added to the sample, and only these beads that bind the sample can be added to the substrate. For example, if the target analyte is fluorescently labeled (eg, by incorporating the label into a nucleic acid amplification reactant) or indirectly (eg, by using a sandwich assay), fluorescence will result from binding of the target analyte. The indicated beads can be classified via Fluorecence Activated Cell Sorting (FACS), and only these beads can be added to the array and then decoded. Similarly, classification can be done via affinity techniques; affinity columns containing the target analyte can be prepared and only these beads to be bound can be used in the array. Similarly, two bead systems can be used; for example, magnetic beads containing the target analyte can be used to "pull" these beads that bind to the target. It can be out) and then the magnetic beads can be disengaged (eg, due to increased temperature) and added to the array.

[0057] In general, array preparation and array decoding methods are performed to maximize the number of different candidate agents that can be uniquely encoded. The compositions of the present invention are prepared by various methods. Generally, arrays are prepared by adding a bead-containing solution or slurry to the surface containing the site for bead association. This is done in a variety of buffers, including aqueous and organic solvents and mixtures. The solvent can be evaporated and excess beads are removed.

[0058] In a preferred embodiment, when the beads are associated with an array using a non-covalent method, a novel method of loading the beads onto the array is used. The method involves exposing the array to a solution of particles (including microspheres and cells) and then energizing, eg, stirring or vibrating the mixture. This results in an array containing more tightly associated particles when agitated with sufficient energy to drop (or, in the case of wells, remove) weakly associated beads. These sites are then available to bind different beads. In this way, beads that show high affinity for the site are selected. Arrays prepared in this way have two major advantages over static loading; firstly, a high percentage of sites can be easily filled, and secondly, thus loaded assays It is shown that the bead loss during the assay is substantially reduced. Thus, in a preferred embodiment, these methods are used to obtain an array filled with at least about 50%, preferably at least about 75%, and particularly preferably at least about 90%. Similarly, the array obtained by this method preferably loses less than about 20%, preferably less than about 10%, particularly preferably less than about 5% of beads during the assay.

[0059] In this embodiment, a substrate having a surface with discrete sites is immersed in a solution containing particles (beads, cells, etc.). The surface has other types of sites on a patterned surface that have wells or have a discriminatory affinity for the sites as described herein. This discriminatory affinity becomes a competitive process in which particles that bind more tightly are selected. It is preferable that the entire surface on which the beads should be "supplied" is in fluid contact with the solution. This solution is generally a slurry with a bead: solution (volume: volume) in the range of about 10,000: 1 to 1: 1. In general, the solution can contain a number of reagents, such as aqueous buffers, organic solvents, salts, other reagent components and the like. In addition, the solution preferably contains an excess of beads; that is, there are more beads than the sites on the array. A preferred embodiment utilizes 2 to 1 billion times excess beads.

Immersion can be similar to assay conditions; for example, if the array should be "immersed" in a microtiter plate containing the sample from above, this configuration is repeated throughout the feed, thus gravity. Minimize beads that are likely to fall.

[0061] Once the surface is immersed, the substrate and / or solution are subjected to a competitive process. Thereby, the particles having a low affinity can be dissociated from the substrate and replaced with the particles having a high affinity for the site. This competing process is carried out by introducing energy in the form of heat, sonication, solution and / or substrate, in the form of stirring and mixing, vibrating and stirring.

[0062] In a preferred embodiment, stirring or vibration is utilized. In general, the amount of manipulation of the substrate is minimized to prevent damage to the array; thus, preferred embodiments utilize solution agitation rather than array, but either. As will be appreciated by those skilled in the art, this agitation can take many forms. A preferred embodiment utilizes a microtiter plate containing the bead solution, which is agitated using a microtiter shaker.

[0063] Stirring continues for a time sufficient to supply the array in the desired amount. Depending on the size and concentration of the beads and the size of the array, this time may range from about 1 second to several days, preferably about 1 minute to about 24 hours.

It should be noted that not all parts of the array may contain beads; that is, there may be empty parts on the surface of the substrate. In addition, there may be sites containing one or more beads, which is not preferred.

[0065] In some embodiments, the beads can be bound in a non-random or regular manner, for example, when chemical bonding is performed. For example, using a photoactivated binding linker or a photoactivated adhesive or masking agent, selected sites on the array are sequentially suitable for binding so that a predetermined population of beads is placed. It may be.

[0066] The arrays of the invention allow information about the identity of the candidate drug to be incorporated into the array, i.e., the random attachment of beads in the fiber wells to be "decoded" to identify the candidate drug at all positions. It is built to be. This may be done in a variety of ways before, during or after the detection of the molecule of interest using the array.

[0067] Thus, after creating the array, it is "decoded" to identify the location of one or more bioactive agents, i.e., each subpopulation of beads, on the surface of the substrate.

[0068] In a preferred embodiment, a selective decoding system is used. In this case, only microspheres that show a change in the optical signal as a result of the coupling of the objects of interest are decoded. This is usually done when the number of "hits", i.e. the number of sites to decode, is generally low. That is, the array is first scanned under experimental conditions in the absence of the target analyte. A sample containing the target analyte is added and only the positions that indicate changes in the optical signal are decoded. For example, beads at positive or negative signal positions are selectively labeled or released from the array (eg, using a photocleavable linker) followed by a screener of fluorescently activated cells (eg, using a photocleavable linker). It may be sorted or concentrated by FACS). That is, after releasing all the negative beads, the positive beads are released or analyzed on the spot. Alternatively, release and analyze all positives. Alternatively, the label may contain a halogenated aromatic compound, and detection of the label is performed using, for example, gas chromatography, chemical labeling, isotope labeling, or mass spectrum labeling.

As will be appreciated by those skilled in the art, this may be done in a system in which the array is not decoded; i.e., in a system where no correlation between bead composition and position is required. In this embodiment, beads are fed into the array and assayed. The "positive", ie beads that exhibit changes in the optical signal (more fully outlined below) are then "labeled" to distinguish and separate them from the "negative" beads. This can be done in several ways, preferably using fiber optic arrays. In a preferred embodiment, each bead contains a fluorescent dye. After assaying and identifying "positive" or "active beads", light is generally only on positive or negative fibers in the presence of photoactivated reagents (typically dissolved oxygen). Hit. In the former case, all active beads are photobleached. Thus, non-fluorescent active beads are removed from the fluorescent negative beads by non-selectively releasing all beads and subsequently sorting using, for example, a fluorescently activated cell sorter (FACS) machine. Can be sorted. Alternatively, when the negative fibers are exposed to light, all negatives are non-fluorescent and positives are fluorescent and sorting can continue. The characterization of the bound bioactive agent may be done directly, for example, using mass spectrometry.

[0070] Alternatively, identification may be performed using an identification moiety (IM) that is similar to IBL but does not necessarily have to bind to DBL. That is, the composition of IM may be used as an identification rather than directly elucidating the structure of the bioactive drug. Thus, for example, a particular combination of IMs can be utilized to encode the beads, which can be used to release from the beads and then subsequently analyzed, for example, using gas chromatography or mass spectrometer. This allows the drug on the beads to be identified.

Alternatively, rather than having each bead contain a fluorescent dye, each bead contains a fluorescent precursor of the fluorescent dye. For example, a photocleavable protecting group such as an ortho-nitrobenzyl group on a fluorescent molecule can be used to photoactivate the fluorescent dye. After the assay, the "positive" or "negative" fibers are re-lighted to distinguish between these populations. The irradiated precursor is then chemically converted to a fluorescent dye. All beads are then sorted and released from the array to form a population of fluorescent and non-fluorescent beads (positive and negative, or vice versa).

[0072] In another preferred embodiment, the bead binding site (eg, well) contains a photopolymerizable reagent or a photopolymerizable agent is added to the assembled array. After performing the test assay, the "positive" or "negative" fibers are re-lighted to distinguish between these populations. As a result of irradiation, all positives or all negatives polymerize and are captured or bound to the site, while the remaining bead population can be released from the array. ..

[0073] In a preferred embodiment, the location of the bioactive agent is determined using a decoder binding ligand (DBL). As outlined above, a DBL is an identification binding ligand (if present) or, preferably a binding ligand that binds to the bioactive agent itself, if the bioactive agent is a nucleic acid or protein.

In a preferred embodiment, DBL binds to IBL, as outlined above.

[0075] In a preferred embodiment, the bioactive agent is a single-stranded nucleic acid, and DBL is a substantially complementary single-stranded nucleic acid that binds (hybridizes) to the biologically active agent. It is called a decoder probe. A decoder probe that is substantially complementary to each candidate probe is created and used to decode the array. In this embodiment, the candidate probe and decoder probe must be long enough to allow specificity (and the decoding process is performed under suitable conditions); that is, each candidate probe is Each candidate probe is coupled to its corresponding decoder probe with sufficient specificity to allow distinction.

[0076] In a preferred embodiment, the DBL is labeled directly or indirectly. As used herein, "labeled" means that a compound has at least one element, isotope or chemical compound attached to allow detection of this compound. Generally, there are three types of labels: a) isotope labels (radioisotopes or high mass number isotopes); b) magnetic, electrical, thermal; and c) colored or fluorescent dyes. However, particles such as enzymes and magnetic particles are also included in the label. Preferred labels include fluorescent labels. In a preferred embodiment, the DBL is directly labeled; that is, the DBL contains the label. In another embodiment, the DBL is indirectly labeled; that is, a labeling binding ligand (LBL) that binds to the DBL is used. In this embodiment, the labeling binding ligand-DBL pair can be as described above for the IBL-DBL pair. Suitable labels include fluorescent lanthanide complexes such as European and terbium complexes, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrocin, coumarin, methyl-coumarin, pyrene, malachite green, stillben, lucifer yellow. (Lucifer Yellow), Cascade Blue^TM), Texas Red, FITC, PE, cy3, cy5, as well as the 6th edition of the Molecular Probes Handbook by Richard P. Haugland ( In particular, by indicating the source, those described in (particularly as a part of the present specification) are included, but are not limited thereto.

[0077] In one embodiment, a label is a molecule whose color or luminescence properties change in the presence of IBL due to local environmental changes. For example, the label is (1) a fluorescent pH indicator whose emission intensity changes depending on pH; (2) a fluorescent ion indicator whose emission properties change depending on the ion concentration; or (3) ethidium whose fluorescence intensity increases in a hydrophobic environment. It may be a fluorescent molecule such as a salt.

Therefore, identification of the location of individual beads (or a subpopulation of beads) is the binding between a labeled DBL and an IBL or bioactive agent (ie, if the bioactive agent is a nucleic acid). Is performed using one or more decoding steps (hybridization between the candidate probe and the decoder probe). After decoding, the DBL can be removed and an array can be used; however, in some cases, for example, if the DBL binds to IBL and not to a bioactive drug, then removal of DBL is not necessary (if). Depending on the situation, it may be desirable). Further, as outlined here, decoding may be performed before, during, or after the assay the array is used in the assay.

[0079] In one embodiment, a single decoding step is performed. In this embodiment, each DBL is labeled with a unique label so that the number of unique labels is equal to or greater than the number of bioactive substances (in some cases, described herein). Similarly, if the variants are encoded in a different dimension (ie bead size or label), then a small number of variants of the candidate probe are the same. Decoders can be shared). For each bioactive substance or IBL, it specifically binds to it to create a DBL containing a unique label (eg, one or more fluorescent dyes). In this way, the identity of each DBL (both its composition (ie, its sequence, if it is at the nucleic acid) and its label) is known. The DBL then comprises the bioactive substance under conditions that allow the formation of a complex between the DBL and either the bioactive substance or the IBL (referred to as a hybridization complex if the component is a nucleic acid). The location of each DBL can be clarified by adding it to the array. This allows the location of each bioactive substance to be identified and a random array is decoded. The DBL can then be removed and the target sample applied if necessary.

[0080] In a preferred embodiment, the number of unique labels is less than the number of unique bioactive substances, and thus a continuous series of decoding steps is used. For ease of discussion, this embodiment is exemplified for nucleic acids, but other types of bioactive substances and DBLs are equally useful. In this embodiment, the decoder probe is divided into n pairs for decoding. The number of pairs corresponds to the number of unique tags. Each decoder probe is labeled with n different tags in n separate reactions. All decoder probes share the same n tags. Each pool of decoders contains only one of the n tag versions of each decoder, and no two decoder probes have the same array of tags across all pools. The number of pools required for this to be true is determined by the number of decoder probes and n. Hybridization of each pool to the array produces a signal at all addresses, including IBL. Continuous hybridization of each pool then yields a unique sequence-specific code for each candidate probe. It identifies candidate probes at each address in the array. For example, when using 4 tags, ideally 4 by 4 × n continuous hybridization.ⁿIndividual sequences can be identified, but in some cases more steps may be required. After hybridization of each pool, the hybrid is denatured and the decoder probe is removed to make the probe single-strand for the next hybridization (hybridizing a limited amount of targets to the available probe). It is also possible to prevent saturation. It can be analyzed by performing continuous hybridization and subtracting existing signals from previous hybridization).

[0081] An example is shown. Assume an array of 16 probe nucleic acids (numbers 1-16) and 4 unique tags (eg, 4 different fluorescence; labels A-D). Make decoder probes 1 to 16 corresponding to the probes on the beads. The first step is to label the decoder probes 1 to 4 with the tag A, the decoder probes 5 to 8 with the tag B, the decoder probes 9 to 12 with the tag C, and the decoder probes 13 to 16 with the tag D. The probes are mixed and the pool is contacted with the array containing the beads to which the candidate probe is attached. The position of each tag (and thus each decoder and candidate probe pair) is then determined. The first set of decoder probes is then removed. Add a second set, but this time tag decoder probes 1, 5, 9 and 13 with tag A, decoder probes 2, 6, 10 and 14 with tag B, and decoder probes 3, 7, 11 and 15 with tags. At C, the decoder probes 4, 8, 12 and 16 are labeled with tag D. Therefore, the beads containing the tag A in both decoding steps contain the candidate probe 1; the beads containing the tag A in the first decoding step and the tag B in the second decoding step contain the candidate probe 2; Beads containing tag A in the decoding step 1 and tag C in the second step contain candidate probe 3, and so on. As will be apparent to those skilled in the art, decoder probes can be made in any order and added in any order.

[0082] In one embodiment, the decoder probes are labeled in situ; that is, they do not need to be labeled prior to the decoding reaction. In this embodiment, the decoder probe to be inserted is shorter than the candidate probe and a 5'"protrusion" is made on the decode probe. The addition of labeled ddNTPs (each labeled with a unique tag) and polymerase allows for the addition of sequence-specific tags, thus creating a sequence-specific pattern of the signal. Similarly, other modifications (including concatenation, etc.) can be made.

In addition, since the size of the array is set by the number of unique decode-binding ligands, a set of unique DBLs can be "reused" to allow for a larger number of test sites. This can be done in several ways, for example by using several subpopulations that include optical features. Similarly, the use of positional code schemes within an array; different sub-bundles can reuse a set of DBLs. Similarly, one embodiment utilizes the bead size as a coding modality, thus allowing the reuse of a set of unique DBLs for each bead size. Alternatively, a continuous partial load of beads on the array also allows reuse of the DBL. In addition, "code sharing" can occur as well.

[0084] In a preferred embodiment, the DBL can be reused by allowing some subpopulation of beads to have optical characteristics. In a preferred embodiment, the optical feature is generally a mixture of reporter dyes (preferably fluorescent). By varying both the composition of the mixture (ie, the ratio of one dye to the other) and the concentration of the dye (which leads to the difference in signal intensity), a matrix of distinctive optical features can be produced. This can be done by covalently bonding the dye to the surface of the beads or by trapping the dye within the beads. The dye may be a luminescent group or a firefly phosphor, but is preferably a fluorescent dye, which provides a good signal-to-noise ratio for decoding due to its strong signal. Suitable dyes for use in the present invention include those listed above for DBL labeling.

[0085] In a preferred embodiment, encoding can be achieved with a ratio of at least two dyes, but more encoding dimensions may be added to the bead size, for example. Moreover, the labels are distinguishable from each other; therefore, two different labels can contain different molecules (ie, two different fluorescence), or can be one label with two different concentrations or intensities.

[0086] In a preferred embodiment, the dye is covalently attached to the surface of the beads. This can be done using functional groups on the surface of the beads, as generally outlined for binding of bioactive substances. As will be apparent to those of skill in the art, these bonds are made to minimize the effects of the dye.

[0087] In a preferred embodiment, the dye is non-covalently associated with the bead, generally by trapping the dye in the pores of the bead.

[0088] Furthermore, encoding with a ratio of two or more dyes rather than a single dye concentration is preferred. This is because it provides insensitivity to the intensity of light used to examine the characteristics of the reporter dye and the sensitivity of the detector.

[0089] In a preferred embodiment, a spatial or positional coding system is performed. In this embodiment, subbundles or subarrays (ie, parts of the entire array) are utilized. By analogy with the telephone system, each subarray is a "region code", which can have the same sign (ie, telephone number) of another subarray, which is separated by the location of the subarray. Thus, for example, the same unique indicator can be reused for each bundle. Therefore, the use of 50 unique labels in combination with 100 different sub-arrays can form an array of 5000 different bioactive substances. In this embodiment, it becomes important that one bundle can be identified from the other; this is generally manual or marker beads, which are beads containing a unique tag for each subarray. ), Through the use of different amounts of the same marker beads, or through the use of two or more marker beads in different proportions.

[0090] In another embodiment, additional encoding parameters such as microsphere size can be added. For example, the use of beads of different sizes can also allow reuse of a set of DBLs; that is, it is possible to use microspheres of different sizes to expand the encoding dimension of the microspheres. Fiber optic arrays containing pixels with different fiber diameters or cross sections can be manufactured; or two or more fiber optical bundles, each with a different cross section of the individual fiber, can be added together to create a larger bundle. It can be formed; or fiber optic bundles of fibers of the same size cross section can be used with beads of different sizes. Different diameters can be used to fill the largest well with the largest microspheres and then gradually move to smaller microspheres in smaller wells until wells of all sizes are filled. Thus, the same dye ratio can be used to encode microspheres of different sizes, thereby increasing the number of different oligonucleotide sequences or chemical functional groups present in the array. Although the fiber optic substrate has been outlined, other substrates and other binding modes can be used as well for this method and for the other methods outlined herein.

[0091] In a preferred embodiment, coding and encoding are achieved by continuous loading of an array of microspheres. As outlined above for spatial codes, optical features can be "reused" in this embodiment. In this embodiment, a library of microspheres, each containing a different bioactive substance (or a subpopulation each containing a different bioactive substance) is divided into multiple sub-libraries; for example, the size and specificity of the desired array. Depending on the number of tags in, each can create 10 sub-libraries, each containing about 10% of the total library, and each sub-library contains approximately the same unique tags. The first sublibrary is then added to the fiber optic bundle containing the wells and the location of each bioactive substance is generally determined via the use of DBL. A second sublibrary is then added and the location of each bioactive substance is repositioned. In this case, the signal contains signals from the "first" DBL and the "second" DBL; by including two matrices, the position of each bead in each sublibrary can be determined. Similarly, the array is filled by continuously adding third, fourth, and other sub-libraries.

[0092] In a preferred embodiment, the code can be "shared" in several ways. In the first embodiment, a single code (ie, IBL / DBL pair) can be assigned to two or more substances if the binding strengths of the target analytes are sufficiently different. For example, two nucleic acid probes used in an mRNA quantification assay can share the same code if their hybridization signal intensity ranges do not overlap. This can occur, for example, if one of the target sequences is always present at a much higher concentration than the other. Alternatively, the two target sequences can always be present at similar concentrations, but with different hybridization efficiencies.

Apart from this, if the substances are functionally equivalent, only one code can be assigned to many substances. For example, when a set of oligonucleotide probes is designed for the common purpose of detecting the presence of a particular gene, the probes are functionally equivalent even if their sequences are different. Similarly, if a class or "family" of analytes was desired, all probes for individual members of the class, such as kinases or G-protein-coupled receptors, could share the code. Similarly, this type of array could be used to detect homologues of known genes. In this embodiment, each gene is indicated by a set of heterologous probes and hybridizes to different gene regions (and therefore different sequences). The probe set has a common code. If a homologue is present, it can hybridize to some, but not all, of the probe. The level of homology can be indicated by the fraction of the hybridizing probe as well as the average hybridization intensity. Similarly, multiple antibodies against the same protein could all share the same code.

[0094] In a preferred embodiment, decoding of self-assembled random arrays is based on pH titration. In this embodiment, in addition to the bioactive substance, the beads contain optical features, which are by the use of a pH-reactive dye (sometimes referred to herein as "pH dye") such as fluorophore. Occurs. This aspect is PCT Identical to those outlined in US98 / 05025 and USSN09 / 151,877, when the pH of the solution is adjusted from below pKa to above pKa (or vice versa), the dye used in the present invention has fluorescence intensity (or vice versa). Both references are specifically incorporated by citation, except to show changes in (other properties). In a preferred embodiment, pH dye sets, each having a different pKa, preferably selected in at least 0.5 pH units are used. A preferred embodiment is a pH dye set of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11 and 11.5 pKa. Use. Each bead can contain any subset of pH dyes, in which a bioactive substance specific code is generated. Decoding of the array is achieved by titrating the array at pH 1 to pH 13 in this way and measuring the fluorescence signal from each bead as a function of solution pH.

[0095] Thus, the present invention provides an array composition consisting of a substrate having a surface consisting of separate portions. The microsphere population is distributed in that part, and the population consists of at least the first and second subpopulations. Each subpopulation comprises a bioactive substance, plus at least one optical dye having a given pKa. The pKa of different optical dyes is different. In a preferred embodiment, there are several methods that can be used to decode the array, for example if the array contains cloned nucleic acids. In a preferred embodiment, if some sequence information about the cloned nucleic acid is known, a particular decoding probe can be made, as broadly outlined herein.

[0096] In a preferred embodiment, a "random" decoding probe can be created. As outlined earlier, the use of a series of hybridizations or multiple labels can result in unique hybridization patterns for each sensor element. This allows all beads representing a given clone to be considered to belong to the same group. In general, this is done with random or partially denatured decoding probes that bind in a sequence-dependent but less highly sequence-specific manner. The process can be repeated many times, each with a different labeling substance, to produce different signal patterns based on, in a sense, specific interactions. In this method, unique optical features are established for each sensor element. By applying pattern recognition or clustering algorithms to optical features, beads can be grouped into sets that share the same features (ie, hold the same probe).

[0097] Additional methods are needed to identify the actual sequence of the clone itself, for example, direct sequencing can be performed. By using an ordered array containing clones, such as a spotted cDNA, a "key" that associates a hybridization pattern with a particular clone whose location within the set is known. Can occur. In this way, clones can be recovered and further characterized. Separately, the clone array can be decoded using dual decoding with vector tags. For example, a partially randomized oligo is cloned into a nucleic acid vector (eg, plasmid, phage, etc.). Each oligonucleotide sequence consists of a limited set of subsets of sequences. For example, if a limited set contains 10 sequences, each oligonucleotide may have several subset (or all 10) sequences. That is, each of the 10 sequences may or may not be present in the oligonucleotide. Therefore, 2¹⁰Or there are 1,024 possible combinations. The sequences may overlap and may show secondary variants (eg, A, C, T and G substitutions) to increase the number of possible combinations. Clone the nucleic acid library into a vector containing a random coding sequence. Alternatively, tags can be added using other methods such as PCR. In this method, a small number of oligocoding probes can be used to decode the sequence of the clone.

[0098] Once made, the compositions of the present invention can be used in many applications. In a preferred embodiment, the composition is used to test the sample solution for the presence or absence of the target analyte, including quantifying the amount of the target analyte present. By "target analyte" or "analyst" or grammatically equivalent herein, it means any atom, molecule, ion, molecular ion, compound or particle whose binding partner is detected or evaluated. As will be apparent to those skilled in the art, many analytes can be used in the present invention, essentially any analyte that binds a bioactive substance, or a binding partner (ie, a drug candidate). Any of the required analytes can be used. Suitable analytes include organic and inorganic molecules, including biomolecules. When detecting target analytes, suitable target analytes include, but are not limited to, environmental pollutants (including pesticides, pesticides, toxins, etc.), chemicals (solvents, polymers, organic substances, etc.) (Including), therapeutic molecules (including therapeutic and abusive drugs, antibiotics, etc.), biomolecules (hormones, cytokines, proteins, nucleic acids, lipids, carbohydrates, cell membrane antigens and receptors (nerves, hormones, nutrients and cell surface receptors)) Or their ligands, etc.), whole cells (including prokaryotic cells (such as pathogenic bacteria) and eukaryotic cells, mammalian tumor cells), viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, etc.) and Includes spores and the like. Particularly preferred analytes are nucleic acids and proteins.

[0099] In a preferred embodiment, the target analyte is a protein. As will be apparent to those of skill in the art, there are a number of potential proteinaceous target analytes for which binding partners can be detected or evaluated using the present invention. Suitable protein-targeted analysts include, but are not limited to, (1) immunoglobulins; (2) enzymes (and other proteins); (3) hormones and cytokines, many of which serve as ligands for cell receptors. ) And (4) other proteins are included. In a preferred embodiment, the target analyte is nucleic acid. These analyzes are used in a wide range of applications. In a preferred embodiment, the probe is used for genetic diagnosis. For example, probes were prepared using the methods disclosed herein to generate non-polypoid colon cancer, the BRCA1 breast cancer gene, the various cancer-related genes P53, and the ApoE4 gene, which indicates a greater risk of Alzheimer's disease. Target sequences such as can be detected and easily presymptomatically screened for mutations in patients, cystic fibrosis genes, cytochrome p450s or any other well known to those of skill in the art. It becomes possible to screen).

[0100] In a further embodiment, detection of viruses and bacteria is performed using the complex of the present invention. In this embodiment, probes are designed to detect target sequences from various bacteria and viruses. For example, the latest blood screening tests are based on the detection of anti-HIV antibodies. The methods disclosed herein allow direct screening of clinical samples to detect HIV nucleic acid sequences, especially highly conserved HIV sequences. In addition, like an improved method for assessing the efficacy of antiviral treatment, this allows direct measurement of the virus circulating in the patient's body. Similarly, viruses associated with leukemia, HTLV-I and HTLV-II can be detected in this way. Bacterial infections such as tuberculosis, chlamydia and other sexually transmitted diseases can also be detected.

[0101] In a preferred embodiment, the nucleic acids of the invention are used as probes for toxic bacteria in screening water and food samples. For example, the sample is processed to lyse the bacterium and release its nucleic acid, followed by Salmonella, Campylobacter, Vibrio cholerae, Leishmania, E. coli enterotoxin strains and present. Probes can be designed to recognize strains that include, but are not limited to, pathogenic strains such as Salmonella cholera. Similarly, the bioremediation method can be evaluated using the compositions of the present invention. In a further embodiment, the probe is used for forensic "DNA fingerprinting" to match criminal case DNA (crime-scene DNA) to samples taken from victims and suspects. In a further embodiment, the probes in the array are used for sequencing by hybridization.

The present invention is also used as a method for detecting mutations or mismatches in target nucleic acid sequences. For example, analysis of the relationship between gene mutations and phenotypes by using polymorphic DNA markers has attracted attention in recent years. Previous studies have used short tandem repeats (STRs) as polymorphic position markers, but in recent years the use of single nucleotide polymorphisms (SNPs) has attracted attention. Common SNPs occur with an average frequency of 1 or more per kilobase of human genomic DNA. Some SNPs, especially those in and around the coding sequence, appear to be the direct cause of therapeutically appropriate phenotypic variants. There are many well-known polymorphisms that cause clinically significant phenotypes, such as apoE2 / 3/4 in Alzheimer's and other diseases (Cordor et al.,, Related to different relative risks (see Science 261 (1993)). Multiplex PCR amplification of the SNP position using sequence hybridization to an oligonucleotide array has been shown to be a rapid and reliable method for genotyping at least hundreds of SNPs simultaneously: Wang et al., See Science, 280: 1077 (1998); see also Schafer et al., Nature Biotechnology 16: 33-39 (1998). The compositions of the present invention can easily replace the prior art arrangements.

[0103] In a preferred embodiment, the composition of the present invention is used to screen for bioactive substances to find substances that bind to a target molecule and preferably alter its function. As mentioned above, a wide variety of different assay formats may be activated, as will be apparent to those of skill in the art. Generally, the binding partner labels the desired target analyte, the target analyte is bound to the bioactive agent to replenish the label on the beads, followed by detection. In a preferred embodiment, the binding of the bioactive agent to the target analyte is specific; that is, the bioactive agent specifically binds to the target analyte. As used herein, "specific binding" means that the substance binds to the analyte specifically enough to separate the analyte from other components or contaminants of the test sample. However, as will be apparent to those of skill in the art, the analyte could be detected using less specific bindings. For example, the system may use different binding ligands, such as arrays of different ligands, and detection of any particular analyte is due to its "feature" of binding to a panel of binding ligands, "electronic nose". It is similar to the way "nose)" works. This is especially useful in the detection of chemical analytes. Binding involves a washing step to remove non-specific binding (although in some embodiments no washing step is required) to maintain binding under assay conditions, i.e. a binding partner with low affinity. Should be sufficient to detect. In some embodiments, for example the detection of certain biomolecules, the separation constant of the analyte into the binding ligand is 10.^-4-10^-5M^-1Smaller, about 10^‐⁵-10^-9M^‐¹Smaller is preferred, 10^-7-10^-9M^-1Smaller is especially more preferred.

[0104] In general, a sample containing a target analyte (for detection of a target analyte or for screening a binding partner of a target analyte) is used to bind the target analyte to at least one bioactive agent. Add to the sequence under suitable conditions, i.e., generally physiological conditions. The presence or absence of the target analyte is then detected. As will be apparent to those skilled in the art, this can be done in a variety of ways, generally using changes in optical signals. This change can be caused by many different mechanisms. A few examples include the binding of pigmented analytes to beads, the production of pigment species on or near the beads, the destruction of existing pigment species, and changes in optical signs in analytical interactions with pigments on the beads. , Or other events that can optically transmit a response command signal.

[0105] In a preferred embodiment, the change in optical signal is the binding of a target specimen labeled with a label that is directly or indirectly detectable (preferably an optical label such as a fluorescent dye). The result is. Thus, for example, when using a target sample such as a protein, it can be labeled directly or indirectly with fluorescence, for example through the use of a labeled antibody. Similarly, nucleic acids are readily labeled, for example through PCR amplification, as known to those of skill in the art. Alternatively, a hybridization indicator can be used as a label based on the binding of the target sequence. Hybridization indicators usually reversibly bind preferably to double-stranded nucleic acids. Hybridization indicators include intercalators as well as sub-grooves and / or main-groove joints. In one preferred embodiment, an intercalator can be used; since intercalation usually occurs only in the presence of double-stranded nucleic acids, the label brightens only in the presence of target hybridization. Therefore, a new optical signal is generated and can be detected at this site based on the binding of the target sample to the bioactive agent.

Alternatively, in some cases, as described above, target specimens such as enzymes give rise to species that can be detected optically, either directly or indirectly.

[0107] Moreover, in some embodiments, the change in optical sign can be based on an optical signal. For example, the interaction of a chemical target sample with a fluorescent dye on a bead can alter the optical sign and thus produce different optical signals.

As will be appreciated by those skilled in the art, in some embodiments, the presence or absence of a target specimen may be an optical or non-optical signal (surface-enhanced Raman spectroscopy, surface plasmon resonance, radioactivity, etc.). Can be made using changes in (but not limited to).

As will be appreciated by those skilled in the art, the assay can be performed under a variety of experimental conditions. Various other reagents may be included in the screening assay. These are like salts, neutral proteins (eg albumin, detergents, etc.) that can be used to promote optimal protein-protein binding and / or reduce non-specific or background interactions. Includes various reagents. Reagents that improve the efficiency of the assay in other ways, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., can also be used. The mixture of ingredients can be added in any order to give the required binding. As known to those of skill in the art, various blocking and cleaning steps can be used.

In one preferred embodiment, a two-color antagonistic hybridization assay is performed. These assays can be based on conventional sandwich assays. The beads contain a capture sequence located on one side (upstream or downstream) of the SNP and can capture the target sequence. Two SNP allele-specific probes, each labeled with a different fluorophore, are hybridized to the target sequence. Usually, a calibration array showing better binding can be used to genotype from the ratio of the two signals. In addition, the probes are antagonistic, which means that there is no need to optimize binding conditions. Matched probes can be further replaced under conditions where the mismatched probes are stably bound. Therefore, antagonistic assays can give better discrimination under those conditions. Since many assays are performed in parallel, the conditions cannot be optimized for all probes at the same time. Therefore, an antagonistic assay system can be used to assist in compensating for non-optimal conditions for mismatch identification.

[0111] In one preferred embodiment, the dideoxynucleotide chain using the compositions of the present invention. Determine the termination sequence. In this embodiment, the primers are extended with ddNTPs fluorescently labeled with DNA polymerase. The 3'end of the primer is located near the SNP site. In this way. The single base extension is complementary to the sequence of the SNP site. The sequence of SNPs can be derived by comparing four base-specific signals using one, four different fluorophores for each base. This can be done in several ways. In the first embodiment, the capture probe can be extended; in this approach, the probe must be synthetic 5'-3'on the beads or bound to the 5'end to give a free 3'end for polymerase extension. Must be. Alternatively, a sandwich assay can be used; in this embodiment, the target is captured on the beads by a probe, followed by annealing and extension of the primer. Also, in the latter case, the target sequence may be unlabeled. In addition, the sandwich assay requires two specific interactions, so this probe increases specificity that is particularly beneficial for the analysis of composite samples.

[0112] Furthermore, if both the target sample and the DBL bind to the reagent, it is also possible to detect the unlabeled target sample through decoding antagonism.

[0113] In one preferred embodiment, the methods of the invention are useful in array quality control. Prior to the present invention, no method has been disclosed that provides a positive test of the performance of all probes on all arrays. Decoding the array not only gives this test, but also by utilizing the data generated by the decoding process itself. Therefore, no further experimental work is required. The present invention requires only one set of data analysis algorithms that can be coded in software.

Quality control measures can identify a wide range of systematic and random problems in the array. For example, a random point of dust or other contamination can cause some sensors to give an incorrect signal-this can be detected during decoding. Leakage of one or more agents from multiple arrays can also be detected; i.e., the arrays formulated herein self-assemble randomly and thus include different statistical distributions of each sensor element. Arrays can be made; for "sorting" arrays with good distribution from "sorting" arrays that are not ideally distributed, allow for actual QC on the array. These procedures also allow the determination of bead-free sites.

The advantage of this quality control procedure is that it can be performed immediately before (or immediately after, in some cases) the assay itself and is a true functional test of the individual sensor. Therefore, any problems that may arise between assembly and actual use can be detected. Decoding and quality control can be performed both before and after actual sample analysis, in applications where very high levels of reliability are required and / or where there is a significant potential for sensor failure during the course of the experimental procedure. ..

[0116] In one preferred embodiment, reagent quality control can be performed using an array. In many cases, biological macromolecules are used as reagents and must be quality controlled. For example, a large set of oligonucleotide probes can be provided as reagents. Performing quality control on a large number of different biological macromolecules is usually difficult. For example, when synthesizing a large number of different random oligonucleotides, it is desirable to check what each population shows. This can be done by treating a variable reagent (formulated as DBL) instead of an array using the approach described herein.

[0117] In preferred embodiments, the methods described herein are used for array calibration. For many applications such as mRNA quantification, the target sample has a signal that is a linear response to the concentration of the target sample, or otherwise determines the relationship between the concentration and the signal, even if it is non-linear. It is desirable to be able to evaluate the concentration of. For example, it is desirable that different probes show differences in hybridization efficiency and the like, and an appropriate probe can be selected under experimental conditions. Thus, for example, when performing expression profiling, and when quantification is desirable, create a calibration curve to study how individual probes react and get the best response (ie, at the desired concentration and conditions). A probe that provides (straightness) can be selected for further assay. Therefore, the present invention provides a method of creating calibration curves in parallel for a plurality of beads in an array. Calibration curves can be created under conditions that simulate the complexity of the sample to be analyzed. Each curve can be constructed independently of other curves (eg, for different concentration ranges) at the same time as all other curves for the array.

[0118] Therefore, different types of experiments can be performed in these embodiments. For example, sequential decoding schemes can be performed at different concentrations that are used as code "labels" rather than different fluorophores. In this method, the signal as a response to concentration can be measured for each bead. This calibration can be performed immediately prior to use of the array and all probes on the entire array can be individually calibrated as needed. Alternatively, different concentrations of decoding probes can have different labels.

It should be noted that assay calibration methods likewise provide use in non-random arrays; ie, these methods can also be used to calibrate other types of support-binding nucleic acid arrays. Thus, for example, sequential addition of probes from different pools to the biochip with different concentrations of probes allows calibration of any assay system. This type of analysis can also be performed in non-random arrays for quality control, ensuring the integrity and sequence of support-bound probes and identifying good probes in assay development.

[0120] In one preferred embodiment, the methods of the invention can be used for assay development as well. Thus, for example, the method allows identification of good and bad probes; as will be appreciated by those skilled in the art, some probes will not hybridize well or will cross-hybridize with one or more sequences. It doesn't work. These problems are easily detected during decoding. The ability to rapidly assess probe performance has the potential to significantly reduce assay development time and cost. Thus, probes that react linearly with concentration, show low non-specific binding, or signal in a particular range can be selected and added to a new array for the assay.

Similarly, in a preferred embodiment, the methods of the invention are useful in quantification in assay development. The main challenge of many assays is the ability to detect differences in sample concentration between samples, the ability to quantify these differences, and the measurement of absolute concentrations of samples, all in the presence of a complex mixture of related samples. is there. An example of this task is the quantification of specific mRNA in the presence of whole cell mRNA. One approach developed as the basis for mRNA quantification utilizes multiple matched and mismatched probe pairs (Lockhart et al., 1996) (completely incorporated herein by source). This approach is simple but requires a relatively large number of probes. In this approach, a quantitative response to concentration is obtained by averaging signals from a set of different probes for the gene or sequence of interest. This is essential because only some probes respond quantitatively and these probes cannot be predicted reliably. Without prior knowledge, only the average response of a properly selected collection of probes is quantitative. However, in the present invention, as with other assays, general application to assay-based nucleic acids is possible. In short, the approach is to identify probes that respond quantitatively in a particular assay, not on average with other probes. This is done by the array calibration scheme described above using a concentration-based code. The advantage of this approach is that each and all sequences can be tested in a valid way; less probes are needed; measurement accuracy is less dependent on the number of probes used; And the response of the sensor may be known for its high degree of accuracy. It is important to note that well-functioning probes are empirically selected to avoid the difficulty and uncertainty of predicting probe performance, especially in complex sequence mixtures. In contrast, in the experiments described so far using ordered arrays, a relatively small number of sequences are checked by performing a quantitative spike experiment in which known mRNA is added to the mixture.

[0122] In a preferred embodiment, a cDNA array is created for RNA expression profiling. In this embodiment, individual cDNA clones are amplified from the cDNA grown in the host vector system (eg, using PCR). Each amplified DNA is bound to a population of beads. Different populations are mixed together to create a collection of beads showing a cDNA library. The beads are arrayed, decoded as described above and used in the assay (as described herein, but decoding may occur after the assay as well). Assays are performed using RNA samples (whole cells or mRNA) that have been extracted, labeled as needed, and hybridized to the array. Antagonistic analysis allows the detection of differences in the expression levels of individual RNAs. Comparison with a suitable set of calibration standards allows quantification of the absolute amount of RNA.

The cDNA array can also be used for mapping, eg, mapping deletions / insertions, or, for example, changes in copy number in the genome from tumors or other tissue samples. This can be done by hybridization of genomic DNA. Instead of cDNA (or EST, etc.), other STSs (sequence tag sites) containing random genomic fragments can also be arrayed for this purpose.