WO2000029831A1

WO2000029831A1 - Labeling microparticles capable of absorbing infrared light and methods of making and using the same

Info

Publication number: WO2000029831A1
Application number: PCT/US1999/026985
Authority: WO
Inventors: Mary B. Frischkorn-Meza; Chad I. Owen; Janming Hou
Original assignee: Bangs Laboratories, Inc.
Priority date: 1998-11-13
Filing date: 1999-11-12
Publication date: 2000-05-25
Also published as: AU2149300A

Abstract

There is provided a labeling material capable of absorbing infrared light which comprises a plurality of polymeric microparticles. Each of the microparticles has a diameter of less than 500 νm and incorporates an infrared absorbing dye. The infrared absorbing dye absorbs radiation at a wavelength of from about 770 nm to about 10 νm.

Description

Labeling Microparticles Capable of Absorbing Infrared Light and Methods of Making and Using the Same

RELATED U.S. APPLICATION DATA This application claims the benefit of U.S. Provisional Application 60/108,294, filed November 13, 1998. FIELD OF THE INVENTION The present invention relates to labeling microparticles which absorb light at infrared wavelengths. The invention also relates to methods of making and using such labeling microparticles.

BACKGROUND OF THE INVENTION Microparticles impregnated with a fluorescent or visible-light colored dye have been used in a wide variety of applications. For example, fluorescent or colored microparticles used as labels in diagnostic assays for the purpose of indicating the presence of an analyte have been reported in the literature. Examples of such uses have been reported, for example, in U.S. Patent Nos. 5,786,219; 5,573,909; 5,723,218; 5,326,692; Molday, et al. J. CELL BIOL. 64, 75 (1975); Margel, et al. J. CELL SCI. 56, 157 (1982). Such microparticles may be conjugated to an antagonist of the analyte, such as an antibody or binding portion of a cell receptor. Typically, the microparticle-antagonist conjugate is contacted with a sample which is to be assessed for the presence of the analyte. The microparticle-antagonist conjugate can react with analyte if it is present in the sample. The presence or absence of analyte in the sample may then be assessed by determining the amount of microparticles which have formed a reaction product with the analyte. Fluorescent microparticles, to which antagonists have been attached, have been used for immunoassays (U.S. Pat. No. 4,808,524), for nucleic acid detection and sequencing (Vener, et al. ANALYT. BIOCHEM, 198, 308 (1991); Kremsky, et al. NUCLEIC ACIDS RES. 15, 2891 (1987); Wolf, et al., NUCLEIC ACIDS RES. 15, 2911 (1987)), as labels for cell surface antigens, FLOW C YTOMETRY AND SORTING, ch. 20 (2nd ed. (1990)), and as tracers to study cellular metabolic processes (J. LEUCOCYTE BIOL. 45, 277 (1989)). Since these dyed microparticles have a particular color or are fluorescent, the presence or absence of analyte may be detected by the naked eye or through the assistance of a device. However, the use of such microparticles in diagnostic assays can be limited, or even rendered impossible if, for example, one or more constituents in a test sample interfere with the detection of fluorescent or colored microparticles. For example, the presence of hemoglobin or lysed red blood cells in blood interferes with the detection of fluorescent or colored microparticles. For this reason, determining the presence of analyte in whole blood is difficult, and whole blood often must be pretreated to remove the interfering substances but not the analyte to be measured. Such a process increases the complexity and cost of diagnostic assays, as well as potentially diminishing the sensitivity of the assay. These same problems exist with respect to determining the presence of analyte in any substance which would interfere with the detection of fluorescent or colored microspheres. Previous attempts to address the interference effects in the visible light spectrum have required complicated and expensive equipment and methodologies.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a system for detecting the presence or absence of analyte in a sample through measurement of infrared light (IR) absorbance or reflectance. The IR absorbing labeling microparticles of the present invention may be detected using conventional IR absorbance or reflectance methods and equipment, thus providing a novel and efficient label. The labeling microparticles of the invention can minimize or even eliminate problems in the art connected with analyzing a test sample which contains constituents which would interfere with fluorescent or visible light colored microparticles, while permitting relatively simple, inexpensive analyses. Thus, the present invention provides a wide variety of diagnostic assays which are characterized by the use of IR absorbing microparticles as a label. In accordance with the demonstrating features and advantages of the present invention, there is provided a labeling material capable of absorbing infrared light which comprises a plurality of polymeric microparticles. Each of the microparticles has a diameter of less than 500μm and incorporates an infrared absorbing dye. The infrared absorbing dye absorbs radiation at a wavelength of from about 770nm to about lOμm. Methods of preparing and using the labeling material are also provided.

DETAILED DESCRIPTION OF THE INVENTION The microparticles useful in the invention may be comprised of polymeric materials, such as are suitable for use in diagnostic assays. The microparticles may be spherical or irregular in shape. The microparticles may be any size suitable for use in diagnostic assays. The microparticles are preferably lOnm to 5mm in diameter, more preferably 20nm to 200μm in diameter, most preferably 20nm to 25μm. Suitable polymeric materials include a variety of polymerizable monomers, including styrenes, acrylates, esters, acetates, amides and alcohols. Preferred polymeric materials include polystyrene; polymethylmethacrylate; polyvinyltoluene; copolymers of various acrylates, such as methylmethacrylate, acrylic acid, methacrylic acid, hydroxyethylmethacrylate, glycidylmethacrylate, butylmethacrylate, ethylmethacrylate, copolymers of styrene and acrylates, such as methylmethacrylate, acrylic acid, methacrylic acid, hydroxyethylmethacrylate, glycidylmethacrylate, ethylenedimethacrylate, butylmethacrylate, ethylmethacrylate, copolymers of styrene and other monomers, such as maleic anhydride, paramethylstyrene, acrylamide, aminostyrene, methylstyrene, vinylbenzylchloride, bromostyrene, butadiene; single-component polymers or copolymers as previously listed cross-linked with compounds such as divinylbenzene and ethylenedimethacrylate. The cross-linking compounds provide increased physical rigidity and heat stability, as well as increased solvent resistance. Polymeric based materials suitable for microparticles have been reported, for example, in U.S. Patent Nos. 5,573,909, 4,415,700, 5,356,718, 4,358,388 (hereby each incorporated by reference). The microparticles are preferably polystyrene (or latex)-based or polymethylmethacrylate- based. Microparticles can be further modified by copolymerizing or coating with one or more secondary monomers, to alter the surface properties of the particles. The microparticles of the present invention may be optionally linked to various substances, such as bioreactive substances, including antagonists which target specific analytes. Such bioreactive substances include peptides; proteins (such as polyclonal and monoclonal antibodies, enzymes, binding portions of cell receptors, avidin, streptavidin, protein A, and protein G); nucleic acid compounds such as nucleotides, oligonucleotides, DNA and RNA; non-polymeric biomolecules such as biotin and digoxigenin; carbohydrates such as lectin and polysaccharides; viruses; toxins; metal chelates; and haptens including hormones, vitamins, and drugs. The bioreactive substances are attached to the surface of the microparticles by methods known in the art. See, e.g., U.S. Patent No. 5,573,909 (hereby incorporated by reference). Generally, the bioreactive substances are either covalently attached (for example, as described in Guilford, Chemical Aspects of Affinity Chromatography, CHEM. SOC. REV. 2:249 (1973) incorporated herein by reference) or non-covalently adsorbed to the surface of the microparticles (for example, by methods described in Nathan et al, Antitumor Effects of Hydrogen Peroxide in Vivo, J. EXP. MED. 154: 1539 (1981), incorporated herein by reference). These surface-attached or adsorbed molecules preferably are added to the microparticles after the IR light absorbing dyes have been incorporated, and can render the microparticles useful in a wide variety of diagnostic tests. Microparticles of the present invention may optionally contain additional materials for various purposes, including improving performance or ease of handling of the microparticles in the particular diagnostic assay being used. For example, magnetically responsive materials may be incorporated into the microparticles which would, for example, be capable of both detecting the presence of analyte and separating the analyte linked to the antagonist/microparticle conjugate from the sample through the application of a magnetic force. See, for example, U.S. Patent Nos. 5,374,531 and 5,340,719, each of which are incorporated herein by reference. Suitable magnetically responsive materials include iron oxides, such as magnetite, and chromium dioxide. The particles also can contain any other desired components which do not interfere significantly with its performance as a label, such as stabilizers, antioxidants, etc. The microparticles contemplated by the present invention are impregnated with a dye which absorbs light at IR wavelengths. The IR absorbing dye may be any dye which absorbs infrared light, preferably with minimal absorbance in the ultraviolet or visible regions, and which is suitable for use in diagnostic assays. In accordance with the invention, such dyes should absorb radiation at a wavelength within the range of about 770nm to about lOμm and, more preferably about 770nm to about 1200nm. The IR absorbing dye should be chosen so that its absorbance is greatest in the infrared spectrum, with minimal absorbance outside that spectrum, and so that the microparticles of the invention absorb sufficient amounts of infrared radiation to act as a label in diagnostic assays, as described herein. Dyes which primarily generate fluorescence or visible color, with minimal infrared absorbance, are disfavored and are not utilized in the present invention. Suitable IR absorbing dyes include those which are soluble in solvents of polystyrene, polymethylmethacrylate or the polymer of which the microparticles are composed, and which have limited or no solubility in water (e.g., hydrophobic dyes). Dyes capable of absorbing radiation in the infrared spectrum are available commercially from suppliers such as Exciton (Dayton, Ohio), the Aldrich Chemical Company, and Epolin (New Jersey). Exciton dyes include those sold under the trade designations NP-800, IRA 800, IRA 885 or IRA 955. Aldrich dyes include those sold under the trade designation IR-27, which contains 4-[2-[2-chloro-3-[(2-phenyl-4H-l-benzopyran-4-ylidene)ethylidene]-l-cyclohexen-l-yl] ethenyl]-2-phenyl-l-benzopyrylium perchlorate, the trade designation IR-140, which contains (5,5 €-dichloro-l 1-diphenylamino- 3,3^E-diethyl- 10, 12-ethylenethiatricarbocyanine perchlorate, and the trade designation IR- 1040, which contains 4-[2-[3 [(2,6-diphenyl-4H-thiopyran-4- ylidene)ethylidene]-2-phenyl - 1 -cyclohexen- 1 -yl]-ethenyl]-2,6-dephenylthiopyrylium tetrafluoroborate. Epolin dyes are available under the trade designation Epolight, and include such dyes as Epolight 111-57, Epolight III- 117, Epolight IV-62A, Epolight IV- 62B, Epolight V-63, Epolight V-72, and others. Impregnation of the dye within the microparticle may take place in any manner so long as the amount of IR absorbing dye within and/or on the surface of the microparticle absorbs sufficient IR light for the purposes of the diagnostic assay being used. Thus, the microparticle may contain the IR absorbing dye on the surface of the microparticle, or the dye may be present in the interior of the microparticle, or both. The IR absorbing dyes are incorporated into the microparticles by any suitable methods known in the art, such as copolymerization of a monomer and a dye-containing comonomer, or by covalent attachment of dye to the surface of chemically functionalized microparticles as described in Molday, et al., New Immunolatex Spheres: Visual Markers of Antigens On Lymphocytes for Scanning Electron Microscopy, J. CELL BIOL., 64, 75-88 (1975), or, most preferably, by addition of a suitable dye derivative in a suitable organic solvent to an aqueous suspension of polymer microparticles. For example, IR absorbing microparticles may be produced by free radical initiated, anaerobic copolymerization of an aqueous suspension of a mono-unsaturated monomer that may or may not contain a covalent bonding group such as carboxyl, amino or hydroxyl and at least 10% by weight of an IR absorbing monomer, according to the methods set forth in U.S. Patent No. 4,326,008. The IR absorbing dyes may be attached covalently to the surface of functionalized microparticles, for example carboxyl functionalized microparticles, using the methods described previously for attachment of a bioreactive substance. The IR absorbing microparticles may also be produced by addition of polymer particles to a dye-containing solution or by gradual addition of a solution of the appropriate IR absorbing dye in an appropriate solvent to a stirred aqueous suspension of microparticles, as described by Bangs, UNIFORM LATEX PARTICLES (Seragen, Inc. 1984), and as detailed below. Random internal incorporation of dye into the microparticle can allow surface functional groups of the microparticle to be attached to bioreactive substances. To dye relatively large (>5μm diameter) particles or particles which are cross- linked to prevent dissolution in the dyeing solvent and which are easily dried and easily redispersed in another medium (e.g., poly(styrene/20%methylmethacrylate/20%methacrylic acid/2%divinylbenzene)), the procedure can be as follows. An oil-soluble IR absorbing dye can be dissolved in xylene, methyl ene chloride or other appropriate solvent. Dry microparticles can be added to the dye solution and stirred at room temperature (or higher) overnight. The solvent and excess dye can be removed by filtration. Preferably, the particles are not washed with more solvent, as this might remove some of the dye. Residual solvent can be removed from the IR absorbing dyed particles using a vacuum desiccator. Surface adsorbed dye can be removed by repeated washes with a water/surfactant solution, or other solvent which will dissolve surface dye, but which will not swell the polymer particles, until the supernatant does not contain detectable dye. To dye relatively smaller particles, i.e. less than 5 m in diameter or particles which are not cross-linked, the particles could be temporarily swelled with dye/solvent solution to permit the dye to enter the interstices of the particles. Solvent can then be driven off, such as by distillation, leaving the dye entrapped in the particles. The IR absorbing dye used in this procedure preferably is oil-soluble, with minimal water- solubility, since diffusion from the emulsion droplet through the aqueous phase to the particle is desirable. The dye can be dissolved, at a concentration below saturation, in xylene, methylene chloride, or other appropriate solvent. The solvent preferably is a solvent of the polymer, and has a significantly lower vapor pressure than the dye. The amount of dye used preferably is adjusted so substantially all of it can be absorbed by the particles. Excess dye can precipitate and be difficult to remove from the dyed particles. While the aqueous particle suspension stirs, dye/solvent solution can be slowly added. Phase separation should be checked periodically to ensure that the proper rate of dye addition is maintained. Solvent with water can be removed by distillation to form an azeotrope, and dye can remain entrapped in the polymer particles. The dye process may be repeated if a higher concentration of dye inside the particles is desired. Any suitable device, such as a spectrometer or spectrophotometer, set to detect absorbance or reflectance at infrared wavelengths, may be used to detect the presence of the microparticles. In this manner, a qualitative determination of the presence of analyte in a sample may be made. Moreover, a quantitative determination of the amount of analyte in a sample may be made by measuring the amount of IR light absorbance or reflectance due to the microparticles linked to the analyte. The following illustrations describe the incorporation of an infrared dye into the microparticles and are by way of example and not by way of limitation. EXAMPLE 1 10 mg of Epolight IV-62A infrared absorbing dye available from Epolin, Inc. of Newark, NJ was dissolved in 1 ml of a 30% methylene chloride, 70% ethanol mixture to form a dye solution. The ethanol component of the solvent contained 0.3%) (by weight) of the surfactant, sodium dodecyl sulfate. 1 gram of 10%> (by weight ) 0.94 micron diameter microparticles of a copolymer of styrene and acrylic acid with carboxylate functional groups (in liquid suspension) was put into a 1.5 ml centrifuge tube. The liquid was removed by centrifugation of the microparticles and decantation of the supernant, and an equal volume of methanol was added to the tube. The methanol contained 0.2 %> (by weight) sodium dodecyl sulfate. The aqueous suspension was sonicated to disperse the microparticles. The microparticle suspension was again centrifuged and the suernatant decanted. This washing process was repeated three times and the methanol was removed after the final centrifugation. 1 ml of the dye solution was then added to the microparticles. The suspension was sonicated and incubated at room temperature for approximately 10 minutes. The suspension was centrifuged and the liquid removed. An equal amount of water was then added. This washing process was repeated three times with an equal volume of water added after the final centrifugation. The suspension was sonicated to disperse the microparticles. Spectrophotometric measurement of the microparticles in water was conducted and it was determined that an infra-red absorbance peak occurred at a wavelength of lOOOnm. EXAMPLE 2 10 mg of Epolight IV-62A infrared absorbing dye available from Epolin, Inc. of Newark, NJ was dissolved in 1 ml of 2-ethoxy methanol to form a dye solution. 1 ml of 2% (by weight) 2.6 micron diameter polystyrene microparticles (with 2%> by weight divinylbenzene) was put into a 1.5 ml centrifuge tube. The liquid was removed by centrifugation and an equal volume of water was added. The aqueous suspension was sonicated to disperse the particles. This washing process was repeated three times and the water was removed after the final centrifugation. 1 ml of the dye solution was added to the particles. The suspension was sonicated and incubated at room temperature for approximately 10 minutes. The suspension was centrifuged and the liquid removed. An equal volume of water was added. This washing process was repeated three times. After the third centrifugation, the liquid was removed and an equal volume of water was added. The suspension was sonicated to disperse the particles. Spectrophotometric measurement of the particles in water was taken and it was determined that an infrared absorbance peak was obtained at a wavelength of 900nm. It has been found that hydrophobic, IR absorbing dyes with a small amount of water solubility are particularly suitable for incorporation into polymeric microparticles in a process where the microparticles are added to a dye containing solution (e.g. Examples 1 and 2). The relatively low solubility of the dye is believed to allow the dye to penetrate the interstices of the polymer molecules when the solvent is added. The use of dyes with no solubility in water may lead to the occurrence of microparticle clumping since both the dye and polymeric microparticles are hydrophobic, and flocculation may occur when the polymeric microparticles are added to solvent-containing dye solution. It is also believed that heat may be used to enhance dye uptake into the microparticles. The solvent choice is also important. Preferred solvents are a low swelling coefficient to minimize undesirable clumping. Further, the addition of a surfactant, e.g., sodium dodecyl sulfate can be used to reduce microparticle aggregation. The IR absorbing microparticles of the present invention may be used in a variety of known applications and assay formats. See, e.g., Hesterberg, et al., Point-of-Care Testing for Infectious Disease, J. CLIN. LIGAND ASSAY, 18(1). 34-42 (Spring 1995); Bangs, New Developments in Particle-Based Immunoassays: Introduction, PURE & APPL. CHEM., 68(10 . 1873-1879 (1996), each of which are incorporated herein by reference. For example, the IR absorbing beads of the present invention may be used as labels in any assays where fluorescent or colored microparticles, or other labels such as colloidal gold, have been used in the past. Such assays include the use of IR light absorbing microparticles as labels for specific cells, proteins, DNA or RNA, or other analytes. The IR absorbing particles of the invention also may be used in immunochromatographic strip assays, such as are described generally in U.S. Patent Nos. 5,591,645; 5,656,503; 5,622,871; 5,141,850; and 5,569,608, each of which are incorporated herein by reference. Such assays, for example, would comprise a test strip containing a bioreactive substance, such as an antagonist for the analyte which is the subject of the assay, and IR absorbing microparticles of the present invention also attached to an antagonist for the analyte which is the subject of the assay. Such antagonists, for example, could be monoclonal antibodies specific for different epitopes of the same analyte. When the sample to be tested is contacted with the test strip and microparticles, analyte present in the sample will react both with the antagonist attached to, or anchored on, the test strip, and to the antagonist attached to the IR absorbing microparticle, forming a "sandwich" of test strip/antagonist/analyte/antagonist/IR absorbing microparticle. In such a test, the sample may be contacted with strip and microparticles in any suitable order, or simultaneously. The test strip may then be analyzed using a device to detect IR light absorbance or reflectance in order to determine the presence or absence of analyte, and/or the amount of analyte in the tested sample. There are many variations of such strip tests which can be employed with the invention. For example, where an analyte, such as an antibody, has a limited number of epitopes, a competitive assay may be performed where the test strip contains analyte and the IR absorbing microparticle contains a bioreactive substance, such as an antagonist for the analyte. A test sample can be contacted with IR absorbing microparticles and then this mixture contacted with the test strip. If there is no analyte in the test sample, the IR absorbing microparticle will attach to the test strip due to binding between the analyte contained by the test strip and the antagonist attached to the microparticle. If there is analyte present in the sample, this analyte will react with the antagonist in the microparticle, thus preventing binding between the analyte on the test strip and the antagonist on the microparticle. The test strip may then be analyzed using a device to detect IR light absorbance or reflectance in order to determine the presence or absence of analyte, and/or the amount of analyte in the tested sample. Other competitive or noncompetitive sandwich assays useful in the invention include binding assays, such as those which may be performed in a well or in solution, as described in Hadfield et al., J. IMMUN. METH., 97, 153-158 (1987); U.S. Patent Nos. 4,419,453; 5,043,289; 5,491,095; 5,500,187; 5,583,054; and PCT Publ. WO 93/19367, each of which are incorporated herein by reference. Such assays can be performed by using plastic wells or beads in solution coated with a bioreactive substance, such as an antagonist of an analyte which is the subject of the assay. The assay also can use IR absorbing microparticles of the present invention, also attached to an antagonist for the analyte. Such antagonists, for example, could be monoclonal antibodies specific for different epitopes of the same analyte. When the sample to be tested is contacted with (1) the well walls or beads and (2) the IR absorbing microparticles, analyte present in the sample will react both with the antagonist attached to the well wall or bead and to the antagonist attached to the microparticle, thereby forming a "sandwich" of well wall or bead/ antagonist/analyte/antagonist/microparticle. The sample may be contacted with the well wall or beads and microparticles in any suitable order, or simultaneously. The well wall or beads may then be analyzed using a device to detect IR light absorbance or reflectance in order to determine the presence or absence of analyte, and/or the amount of analyte in the tested sample. Competitive assays may also be performed as described above with respect to immuno-chromotographic strip tests. Turbidimetric agglutination assays may be performed by using IR absorbing microparticles containing one or more antagonists specific for one or more different epitopes of the analyte which is the subject of the assay. Upon being contacted with the sample, different microparticles would bind to a single analyte at different analyte epitopes, thus causing an agglutination of microparticles when analyte is present in the sample. The sample can be analyzed using a device to detect IR light absorbance or reflectance in order to determine the presence or absence of analyte, and/or the amount of analyte in the tested sample. Competitive assays also may be performed using this format. The invention also includes "flow-through" assays, where IR absorbing beads can be captured on a surface such as a membrane, such as are generally described in U.S. Patent Nos. 5,395,754; 5,501,949; and 4,853,335, each of which are incorporated herein by reference. In such assays, for example, a sample can be contacted with IR absorbing microparticles, which are attached to a bioreactive substance, such as an antagonist specific for an analyte to be tested. If analyte is present, it will form a reaction product with the microparticles. The presence of this reaction product is established by ascertaining whether there is "flow through" a surface such as a membrane. In one embodiment, the membrane can be coated with a substance which is bioreactive with the analyte, so as to capture the IR absorbing microparticle/analyte reaction product. In another embodiment, a capture particle is contacted with the sample, in addition to the IR absorbing particle. If analyte is present, both the IR absorbing particles and the capture particles will react with analyte, and the resulting sandwiches will be retained by the membrane. In this embodiment, each capture particle individually can be large enough to be retained by the membrane, or the sandwiches can be large enough to be retained by the membrane, or the capture particles can be selected such that they will aggregate so that the aggregates will capture multiple analyte/IR absorbing reaction products and be retained by the membrane. In these embodiments, if analyte is not present, the IR absorbing particles will flow through the membrane. The membrane may then be analyzed using a device to detect IR light absorbance or reflectance in order to determine the presence or absence of analyte, and/or the amount of analyte in the tested sample. Other assays where an appropriately coated IR absorbing bead can act as a label to indicate the presence of an analyte include latex or particle agglutination assays on a solid surface, where analyte in the sample causes particles to agglutinate. It is believed that the infrared absorbance of these agglutinated particles would be different from the absorbance of non-agglutinated particles, and thereby would indicate the presence and/or concentration of analyte. Other assays which may employ IR microparticles can include nucleic acid-based assays, in which the IR absorbing particle, coated with a complementary strand of DNA or RNA indicates the presence of a target strand of DNA or RNA; and biosensor-type applications, such as is described in Morgan et al., Immunosensors: technology and opportunities in laboratory medicine, CLIN. CHEM., 42(2). 193-209 (1996), where the IR absorbing beads change the characteristics of light such as to indicate the presence or absence of a specific substance. The test sample can also be any suitable vehicle for testing for the desired analyte, such as human or animal whole blood, plasma, serum, urine, saliva, fecal matter, or mucus, as well as plant material, food products, water, and soil. The IR absorbing labeling microparticles of the present invention are particularly effective in assays where the sample to be tested contains materials which, due to their interference with fluorescent or colored microparticles in the visible light spectrum, would normally require modification or pretreatment of the sample. Such materials can include whole blood, plant matter, soil, animal tissue, or any other substance which may provide interference in the visible light spectrum. For example, the red blood cells in whole blood contain hemoglobin which would cause the sample to turn red and make accurate determination of the presence of fluorescent or colored microparticles difficult and costly. In contrast, the IR absorbing microparticles of the present invention could be used in conjunction with whole blood or hemolyzed blood serum samples to detect a specific analyte, without interference from the blood cells or soluble hemoglobin, since hemoglobin does not absorb significant amounts of infrared light. The analyte to be detected using the IR absorbing microparticles may be a material of biological or synthetic origin that is present as a molecule or as a group of molecules, including, but not limited to, antibodies, amino acids, proteins, peptides, polypeptides, enzymes, enzyme substrates, hormones, lymphokines, metabolites, antigens, haptens, lectins, avidin, streptavidin, toxins, poisons, environmental pollutants, carbohydrates, oligosaccarides, polysaccharides, glycoproteins, glycolipids, nucleotides, oligonucleotides, nucleic acids and derivatized nucleic acids (including deoxyribo- and ribonucleic acids), DNA and RNA fragments and derivatized fragments (including single and multi-stranded fragments), natural and synthetic drugs, receptors, virus particles, bacterial particles, virus components, biological cells, cellular components (including cellular membranes and organelles), natural and synthetic lipid vesicles. Typically the analyte is present as a component or contaminant of a sample taken from a biological or environmental system. Accordingly, the IR light absorbing microparticles of the present invention may be useful in diagnostic assays for determining the presence of an analyte in a variety of classes, such as fertility/sex hormones (including human chorionic gonadotropin hormone, luteinizing hormone, follicle stimulating hormone, prolactin, progesterone, estradiol, testosterone, DHEA-S), adrenal/pituitary (such as cortisol and growth hormones including human and bovine growth hormone), anemia (including erythropoietin, ferritin, B12, folate, RBC folate, intrinsic factor), bone and mineral metabolism (including calcitonin, intact PTH, osteocalcin, deoxypyridinoline), cardiovascular (including CK-MB, digoxin, myoglobin, and troponin), allergy (including total IgE, specific IgE, and theophylline), infectious diseases (such as chlamydia, toxoplasmosis, rubella, CMV, Group A streptococci, Group C streptococci, and rotavirus), blood viruses such as HIV and hepatitis, tumor markers, diabetes, thyroid, hypertension, therapeutic drugs (such as gentamicin, phenytoin, tobramycin, and carbamazepine), drugs of abuse (such as THC), veterinary applications (such as LH, FLV, FIV, rotavirus), plant diseases (including turf diseases and fungal root diseases), water or food-borne pathogens (such as mycotoxin, E. coli, Listeria, cryptosporidium, ghiardia, Salmonella, campy lobacter, shigella, chloramphenicol, and antibiotic residues in milk), leads for high-throughput screening, and molecular diagnostics/DNA testing (including Apo E and BCRAl). The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and accordingly reference should be made to the appended claims rather than the foregoing specification as indicating the scope of the invention.

Claims

What is claimed is: 1. A labeling material capable of absorbing infrared light comprising: a plurality of polymeric microparticles, each of the polymeric microparticles having a diameter of less than 500μm and having an infrared absorbing dye incorporated therewith, the microparticles being detectable in a diagnostic assay.

2. The labeling material of claim 1 wherein each of the microparticles has a diameter of less than 200μm.

3. The labeling material of claim 1 wherein each of the polymeric microparticles has a diameter of about lOnm to about 25 μm.

4. The labeling material of claim 1 wherein the infrared absorbing dye absorbs radiation at a wavelength of from about 770nm to about lOμm.

5. The labeling material of claim 1 wherein the infrared absorbing dye absorbs radiation at a wavelength of from about 770nm to about 1200nm.

6. The labeling material of claim 1 wherein the infrared absorbing dye is hydrophobic.

7. The labeling material of claim 1 wherein each of the polymeric microparticles is comprised of polymerized monomers selected from a group consisting of styrene, acrylate, ester, acetate, amide and alcohol.

8. The labeling material of claim 7 wherein the infrared absorbing dye is soluble in a solvent for the polymerized monomers.

9. The labeling material of claim 7 wherein the polymeric microparticles are cross-linked with a compound selected from the group consisting of divinylbenzene and ethylenedimethacrylate.

10. The labeling material of claim 1 further comprising a bioreactive substance attached to the surface of the microparticles.

11. The labeling material of claim 10 wherein the bioreactive substance is selected from the group consisting of an amino acid, a carbohydrate, a peptide, a protein, a nucleic acid, a virus, a toxin, a metal chelate and a hapten.

12. The labeling material of claim 1 wherein the microparticles have a magnetically responsive material incorporated therewith.

13. A method of applying an infrared label to a polymeric microparticle comprising the steps of: forming an aqueous suspension of the polymeric microparticle; providing a quantity of an infrared dye; dissolving the infrared dye in a solvent to form a dye solution; admixing the dye solution to the suspension of the polymeric microparticle, and removing the solvent and excess dye from the suspension.

14. The method of claim 13 further comprising the step of attaching a bioreactive substance to the surface of the microparticle via covalent attachment or non- covalent adsorption.

15. The method of claim 14 wherein the bioreactive substance is selected from the group consisting of an amino acid, a carbohydrate, a peptide, a protein, a nucleic acid, a virus, a toxin, a metal chelate and a hapten.

16. The method of claim 13 further comprising the step of incorporating a magnetically responsive material to the polymeric microparticle.

17. The method of claim 13 further comprising the step of cross-linking polymeric microparticle with a compound selected from the group consisting of divinylbenzene and ethylenedimethacrylate.

18. A method of applying the labeling material of claim 1 to an analyte comprising the steps of: associating an antagonist of the analyte with microparticles of the labeling material via a covalent or non-covalent bond to form an antagonist-microparticle conjugate, and combining the microparticle-antagonist conjugate with the sample of the analyte to associate the conjugate to the analyte.

19. The method of claim 18 wherein the antagonist is selected from the group consisting of an amino acid, a carbohydrate, a peptide, a protein, a nucleic acid, a virus, a toxin, a metal chelate and a hapten.

20. A method of detecting an analyte in a test sample comprising the steps of: providing a labeling material capable of absorbing infrared light, the labeling material comprising a plurality of polymeric microparticles, each of the polymeric microparticles having a diameter of less than 500μm and having an infrared absorbing dye incorporated therewith, the microparticles being detectable in a diagnostic assay; associating an antagonist of the analyte with microparticles of the labeling material to form an antagonist-microparticle conjugate; combining the microparticle-antagonist conjugate with the test sample to allow the analyte to attach to the antagonist-microparticle conjugate if the analyte is present; collecting the antagonist-microparticle conjugate, and analyzing the antagonist-microparticle conjugate with means for detecting IR light absorbance or reflectance in order to determine the presence of the analyte.