Biochemical probes attached to epoxy resins

Abstract

The present invention is directed to methods for fabricating solid substrates for performing biological and chemical assays, and to the solid substrates fabricated thereby.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 288,018, filed January 17, 2021, and U.S. Provisional Patent Application No. 63 / 155,472, filed March 4, 2021, the contents of which are incorporated herein in their entireties.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable.

[0003] INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC Not applicable.

[0004] The present invention is directed to methods for fabricating solid substrates for performing biological and chemical assays, and to solid substrates fabricated thereby, particularly solid substrates for use in multiplexed bioassays. [Background technology]

[0005] Arrays for biological and chemical analysis may be created by attaching probe molecules to a solid substrate having a surface that includes a resin material, such as functionalized epoxy resin. The use of arrays allows for rapid screening of a large number of biomolecules, such as nucleic acids and proteins, in a very small sample volume. For example, particles known as microspheres or microbeads with identifiable labels and / or markings, called barcoded microbeads, have been used in parallel multiplexed analysis to identify disease-related targets, toxin-related targets, gene-related targets, and the like. The microbeads have a resin coating on their surface that is conjugated to one or more probe molecules that have affinity and / or interaction capabilities for one or more specific target molecules. Each probe molecule is attached to an individual bead that is uniquely identifiable and coded. In an assay, the microbeads are contacted with a sample, and the various target molecules in the sample bind to the microbeads to which the corresponding probe molecules are conjugated. The barcode allows for target identification.

[0006] Microbead assays are currently important tools in biological assays and diagnostics. Microbead-based technology is an elegant and versatile approach for performing highly parallel quantitative multiparameter assays. Microbead-based technology forms the basis of a variety of techniques for detecting and quantifying nucleic acids and proteins in samples. Summary of the Invention [Problem to be solved by the invention]

[0007] Epoxy resins have been used as coating materials for attaching probe molecules. However, to attach the probe molecules to the surface of the epoxy resin, an additional step is required in which the surface of the resin must first be functionalized. U.S. Pat. No. 9,255,922 discloses substrates, such as microbeads or micropellets, coated with an epoxy resin having probe molecules attached thereto. The patent teaches that epoxy resins are hydrophobic, limiting many biological applications, and that the epoxy resin must be modified with additional functional monomers before the probe molecules can be efficiently attached to the resin. Therefore, after the epoxy resin is formed (or during the formation of the epoxy resin), it is necessary to contact the epoxy resin with additional functional monomers to functionalize the epoxy resin, so that the biomolecular probes can be efficiently attached to the surface of the epoxy resin. This additional step of contacting the epoxy resin with the functional monomer is laborious and time-consuming. [Means for solving the problem]

[0008] There is a need in the art for a simple method for fabricating epoxy-based resin-coated substrates to which biomolecular probes can be efficiently attached. The inventors have unexpectedly discovered that biomolecular probes can be efficiently attached to epoxy-based resin-coated substrates without the need to first functionalize the epoxy-based resin with additional functional monomers.

[0009] These and other features and advantages of the present invention will become apparent from the remainder of the disclosure, and in particular from the following detailed description of the preferred embodiments, all of which illustrate, by way of example, the principles of the present invention.

[0010] The citation of any reference in this application shall not be construed as an admission that such reference is prior art to the present application.

[0011] The present invention is directed to a biological analytical substrate and a method for fabricating a biological analytical substrate comprising an epoxy-based resin having biomolecular probes directly bonded to the polymerized resin.

[0012] Substrates for biological analysis include: (i) providing a substrate having a surface comprising an epoxy-based resin; and (ii) contacting the epoxy resin with the biomolecular probe such that the biomolecular probe is directly bound to the epoxy resin; It is prepared by:

[0013] The present invention is also directed to a method of assaying for the presence of an analyte in a sample, the method comprising contacting the sample with a substrate having a surface comprising an epoxy-based resin having a biomolecular probe directly bound to the epoxy-based resin, where the biomolecular probe specifically binds to the analyte. [Brief description of the drawings]

[0014] [Figure 1A] 1A and 1B are plots of signal intensity as BMB fluorescence intensity (MFI) measured as a single median result of all beads in a well versus cell column position (i.e., 1-12) of a 96-well plate for antibodies specific for three Anaplasma genus-derived peptides, labeled "AP," "Aph," and "Apl" on the x-axis of the assay described in Example 6. FIG. 1A shows signal intensity versus cell column position in standard read buffer and FIG. 1B shows signal intensity versus cell column position in citrate read buffer, as described in Example 6. [Figure 1B] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention is directed to a biological analytical substrate and a method for fabricating a biological analytical substrate comprising an epoxy-based resin having a biomolecular probe directly bonded to the epoxy-based resin.

[0016] "Biomolecular probes directly bonded to an epoxy-based resin" and similar phrases, as used herein, mean that the biomolecular probes are attached to the resin by simply contacting the biomolecular probes with the resin without first contacting the epoxy-based resin with another reagent that covalently reacts with the epoxy-based resin. The biomolecular probes may be passively attached to the epoxy-based resin or may be covalently attached to the epoxy-based resin.

[0017] Substrates for biological analysis include: (i) providing a substrate having a surface comprising an epoxy-based resin; and (iii) contacting the epoxy resin with the biomolecular probe such that the biomolecular probe is directly bound to the epoxy resin; It is prepared by:

[0018] In one embodiment, the substrate is an epoxy-based resin.

[0019] In one embodiment, the substrate is a solid support coated with an epoxy-based resin. Exemplary solid support materials that can be coated with an epoxy-based resin include, but are not limited to, particles, beads, and surfaces including glass, polymers, latex, elemental metals, metal composites, alloys, silicon, carbon, and hybrids thereof.

[0020] In one embodiment, a portion of the epoxy-based resin is polymerized before the biomolecular probe is contacted with the epoxy-based resin.

[0021] Suitable epoxy-based resins include, but are not limited to, EPON SU-8, EPON 1001F, 1002F, 1004F, 1007F, 1009F, 2002, and 2005 (available from Hexion Specialty Chemicals, Fayetteville, N.C.). EPON SU-8 and EPON 1002F are preferred resins.

[0022] SU-8 is a photocurable epoxy-based resin. SU-8 is a formaldehyde polymer of (chloromethyl)oxirane and 4,4-(1-methylethylidene)bisphenol (CAS: 28906-96-9). SU-8 is a polymeric solid epoxy novolac resin with an average epoxide group functionality of about 8. The structure of SU-8 epoxy resin is as follows:

[0023] [ka]

[0024] SU-8 is commercially available from Hexion Specialty Chemicals under the trade name EPON SU-8 as a solution containing SU-8 and a photoacid generator.

[0025] 1002F is a photocurable epoxy resin (CAS: 25036-25-3). 1002F is a phenolic, 4,4'-(1-methylethylidene) bis-polymer with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bis(oxirane) and is commercially available from Hexion Specialty Chemicals under the trade name EPON 1002F as a solution containing 1002F and a photoacid generator.

[0026] [ka]

[0027] Suitable biomolecular probes include, but are not limited to, lipids, polysaccharides, amino acids, polypeptides, oligopeptides, peptides, antibodies and fragments thereof, polynucleotides (including single-stranded and double-stranded DNA and RNA), shorter oligonucleotides, aptamers, lectins, avidin, streptavidin, biotin, and polyethylene glycol. Preferably, the biomolecular probe is a polypeptide, oligopeptide, peptide, polynucleotide, or shorter oligonucleotide. In one embodiment, the biomolecule is an antibody. In one embodiment, the biomolecular probe is a synthetic molecule, such as rhodamine. Exemplary biomolecular probes include SDMA, ADMA, T4, cortisol, progesterone, and enzymes (e.g., lipases, such as pancreatic lipase).

[0028] Without being bound by theory, it is believed that the biomolecular probe is bound to the epoxy-based resin by the reaction of the amine, thiol, or hydroxyl groups of the biomolecule with the epoxy groups of the resin. The biomolecular probe may also be passively bound to the epoxy-based resin. The phrase "passively bound" as used herein means binding by non-covalent interactions such as van der Waals interactions, hydrophobic interactions, hydrophilic interactions, or hydrogen bonding interactions.

[0029] The substrate having a surface comprising an epoxy-based resin may be, but is not limited to, a thin film alone or attached to another solid surface, a microbead, a microparticle, a micropellet, a microwafer, a paramagnetic bead, a microparticle comprising an identifying feature such as a barcode, a paramagnetic microparticle, a paramagnetic microparticle comprising a barcode, and a bead comprising a nickel barcode.

[0030] The biomolecular probes are directly attached to the epoxy resin by simply contacting the epoxy resin with the biomolecule.

[0031] In one embodiment, the biomolecular probes are contacted with the epoxy-based resin by adding a substrate having a surface comprising an epoxy-based resin to a solution of the biomolecular probes to prepare a contact mixture. In one embodiment, the solution of the biomolecular probes is an aqueous solution. In one embodiment, the solution of the biomolecular probes is a buffered aqueous solution. In one embodiment, the solution of the biomolecular probes is a dimethylsulfoxide (DMSO) solution.

[0032] In a preferred embodiment, the substrate having a surface comprising an epoxy resin is washed with DMSO before the substrate having a surface comprising an epoxy resin is added to a solution of a biomolecular probe to prepare a contact mixture. In one embodiment, the substrate having a surface comprising an epoxy resin is washed with DMSO immediately before contacting the substrate with a solution of a biomolecular probe to prepare a contact mixture. Unexpectedly, it has been found that contacting the epoxy resin with DMSO before the epoxy resin is contacted with the biomolecular probe provides a substrate for biological analysis with less variability in the number of biomolecular probes bound to the epoxy resin and less variability in the signal used to detect the presence of an analyte in a sample that binds to the biomolecular probe. Unexpectedly, it has been found that contacting the epoxy resin with DMSO before the epoxy resin is contacted with the biomolecular probe provides a substrate for biological analysis that exhibits a better signal.

[0033] In this method, by directly attaching the biomolecular probe to the epoxy resin, advantageously avoids the additional step of having to functionalize the epoxy resin by (i) reacting the epoxy resin with another molecule before attaching the biomolecular probe to the epoxy resin or (ii) mixing another molecule into the epoxy resin before polymerization. By avoiding this additional step, the method is advantageously faster, less expensive, and eliminates a step where error or variability can potentially occur.

[0034] When a solution of a biomolecular probe is used to contact the biomolecular probe with a substrate having a surface containing an epoxy resin, the concentration of the biomolecular probe in the solution is in the range of about 0.05 mg / mL to about 5 mg / mL, preferably about 0.01 mg / mL to about 3.0 mg / mL, more preferably about 0.15 to about 2.5 mg / mL, for example, about 1.5 mg / mL.

[0035] The concentration of the substrate having a surface comprising an epoxy resin in the contact mixture is in the range of about 50,000 to about 5 million substrates / mL, preferably about 100,000 to about 3 million substrates / mL. In one embodiment, the biomolecular probe is a peptide, and the concentration of the substrate having a surface comprising an epoxy resin in the contact mixture is in the range of about 100,000 to about 3 million substrates / mL, for example, about 2 million substrates / mL. In one embodiment, the biomolecular probe is an antibody, and the concentration of the substrate having a surface comprising an epoxy resin in the contact mixture is in the range of about 100,000 to about 1.8 million substrates / mL, for example, about 1 million substrates / mL.

[0036] The solution of the biomolecular probe is typically contacted with a substrate having a surface comprising an epoxy-based resin for a time sufficient for the biomolecular probe to bind to the epoxy-based resin. Typically, the solution of the biomolecular probe is contacted with a substrate having a surface comprising an epoxy-based resin for at least about 4 hours, preferably at least about 8 hours, more preferably at least about 10 hours. In one embodiment, the molecular probe is contacted with a substrate having a surface comprising an epoxy-based resin for about 4 hours to about 18 hours.

[0037] The contact mixture (i.e., the substrate having a surface comprising an epoxy-based resin and the solution of biomolecular probes) is maintained at a temperature sufficient to bond the biomolecular probes to the epoxy-based resin. In one embodiment, the contact mixture is maintained at a temperature of about 4° C. to about 65° C., preferably about 15° C. to about 30° C., and more preferably about 18° C. to 27° C. In one embodiment, the contact mixture is agitated to ensure that the surface of the substrate having a surface comprising an epoxy-based resin is in sufficient contact with the solution of biomolecular probes.

[0038] In one embodiment, after contacting a substrate having a surface comprising an epoxy-based resin with a solution of a biomolecular probe to bind the biomolecular probe to the epoxy-based resin, the solution of the biological probe is removed and the resulting biomolecule-functionalized substrate is washed with a mixture of about 1% bovine serum albumin (BSA) (commercially available from Proliant Biologicals, Ankeny, IA), about 0.05% Tween-20 (commercially available from Sigma Aldrich, St. Louis, MO), and 0.05% Proclin 950 (commercially available from Sigma Aldrich, St. Louis, MO) in phosphate buffered saline (PBS) having a pH of about 7.4. A suitable PBS solution comprises about 1.8 mM sodium dihydrogen phosphate (commercially available from Sigma Aldrich, St. Louis, MO), about 8.4 mM sodium monohydrogen phosphate (commercially available from Sigma Aldrich, St. Louis, MO), and about 145 mM sodium chloride (commercially available from Amresco, Salon, OH). In one embodiment, the biomolecule-functionalized substrate is washed at least three times with at least about 200 μL of the wash solution. In one embodiment, the biomolecule-functionalized substrate is washed at least three times with about 200 μL to about 1,000 μL of the wash solution.

[0039] Suitable buffers include, but are not limited to, phosphate, TRIS, HEPES, MES, EPPS, Bis-TRIS, Bis-TRIS propane, PIPES, ADA, MOPS, MOPSO, ACES, BES, Tricine, TES, Gly-Gly, DIPSO, inorganic buffers, organic buffers, acetate-based and citrate-based buffers.

[0040] The resulting washed biomolecule-functionalized substrate can then be added to a solution of about 1% BSA, about 0.05% Tween-20, about 0.05% Proclin 950 in PBS at a pH of about 7.4 for use in the assay.

[0041] In a first aspect of the method, the biomolecular probe is a protein such as an antibody, an enzyme (e.g., streptavidin and avidin), or a portion of an antibody (e.g., Fc fragment and FAB fragment), and the solution of the biomolecular probe is an aqueous solution. In one embodiment of the first aspect of the method, the solution of the biomolecular probe is an aqueous solution buffered to about pH 5.5 with about 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) and about 140 mM guanidine-HCl. In one embodiment of the first aspect of the method, the solution of the biomolecular probe is an aqueous solution buffered to about pH 8 with about 100 mM 3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (EPPS) and 140 mM guanidine-HCl.

[0042] In one embodiment of the first aspect of the method, the substrate having a surface comprising an epoxy-based resin is washed with a PBS solution containing about 0.05% Tween-20 before contacting the substrate with a solution of biomolecular probes. In one embodiment of the first aspect of the method, the substrate having a surface comprising an epoxy-based resin is washed at least three times with at least about 200 μL of a PBS solution containing about 0.05% Tween-20 before contacting the substrate with a solution of biomolecular probes. In one embodiment of the first aspect of the method, the substrate having a surface comprising an epoxy-based resin is washed at least three times with about 200 μL to about 1,000 μL of a PBS solution containing about 0.05% Tween-20 before contacting the substrate with a solution of biomolecular probes.

[0043] In a preferred embodiment of the first aspect of the method, the substrate having a surface comprising an epoxy resin is then further washed with DMSO before contacting the substrate with a solution of biomolecular probes. In one embodiment of the first aspect of the method, the substrate having a surface comprising an epoxy resin is washed at least three times with at least about 200 μL of the washing solution. In one embodiment of the first aspect of the method, the substrate having a surface comprising an epoxy resin is washed at least three times with about 200 μL to about 1,000 μL of DMSO before contacting the substrate with a solution of biomolecular probes.

[0044] In one embodiment of the first aspect of the method, a substrate having a surface comprising an epoxy-based resin and a solution of a biological probe are contacted to attach the biological probe to the epoxy-based resin, and then the biological probe solution is removed and the resulting biologically functionalized substrate is washed with a mixture of about 1% BSA, about 0.05% Tween-20, and about 0.05% Proclin 950 in PBS having a pH of about 7.4. In one embodiment, the biologically functionalized substrate is washed at least three times with at least about 200 μL of the washing solution. In one embodiment, the biologically functionalized substrate is washed at least three times with about 200 μL to about 1,000 μL of the washing solution.

[0045] The resulting washed biomolecule-functionalized substrate can then be added to a solution of about 1% BSA, about 0.05% Tween-20, about 0.05% Proclin 950 in PBS at a pH of about 7.4 for use in the assay.

[0046] In one embodiment of the first aspect of the method, a substrate having a surface comprising an epoxy-based resin is a barcoded magnetic bead, such as a barcoded magnetic bead coated with SU-8 epoxy-based negative photoresist (commercially available from Applied BioCode, Santa Fe Springs, Calif.).

[0047] In a second aspect of the method, the biomolecular probe is a peptide having a cysteine ​​residue, and the solution of the biomolecular probe is a solution in DMSO. In one embodiment of the second aspect of the method, the solution is DMSO containing about 1% Tween-20. In one embodiment of the second aspect of the method, the peptide concentration is in the range of about 0.2 mM to about 1 mM peptide, for example about 0.5 mM.

[0048] In one embodiment of the second aspect of the method, the substrate having a surface comprising an epoxy resin is washed with a DMSO solution containing about 1% Tween-20 before contacting the substrate with a solution of biomolecular probes. In one embodiment of the second aspect of the method, the substrate having a surface comprising an epoxy resin is washed at least three times with at least about 200 μL of a DMSO solution containing about 1% Tween-20 before contacting the substrate with a solution of biomolecular probes. In one embodiment of the second aspect of the method, the substrate having a surface comprising an epoxy resin is washed at least three times with about 200 μL to about 1,000 μL of a DMSO solution containing about 1% Tween-20 before contacting the substrate with a solution of biomolecular probes.

[0049] In one embodiment of the second aspect of the method, a substrate having a surface comprising an epoxy-based resin and a solution of a biological probe are contacted to bind the biological probe to the epoxy-based resin, and then the biological probe solution is removed and the resulting biological molecule-functionalized substrate is washed with a mixture of about 1% BSA, about 0.05% Tween-20, and about 0.05% Proclin 950 in PBS having a pH of about 7.4. In one embodiment, the biological molecule-functionalized substrate is washed at least three times with at least about 200 μL of the washing solution. In one embodiment, the biological molecule-functionalized substrate is washed at least three times with about 200 μL to about 1,000 μL of the washing solution.

[0050] The resulting washed biomolecule-functionalized substrate can then be added to a solution of about 1% BSA, about 0.05% Tween-20, about 0.05% Proclin 950 in PBS at a pH of about 7.4 for use in the assay.

[0051] In one embodiment of the second aspect of the method, the substrate having a surface comprising an epoxy-based resin is a barcoded magnetic bead, such as a barcoded magnetic bead coated with SU-8 epoxy-based negative photoresist (commercially available from Applied BioCode, Santa Fe Springs, Calif.).

[0052] Without wishing to be bound by theory, a second embodiment of the method involves reacting a thiol group of a biomolecular probe with an epoxide group of an epoxy-based resin. The cysteine ​​residue may be present anywhere in the peptide sequence. The cysteine ​​may be spaced from the peptide by a linker, such as a PEG linker. The PEG may be of a defined length, such as by using a discrete PEG. dPEG (commercially available from Quanta Biodesign, Plain City, OH) is a particularly suitable PEG. Methods for linking cysteine ​​residues to proteins using PEG linkers are known in the art. See, for example, I. Hamley, PEG-Peptide Conjugates, Biomacromolecules, vol. 15:1543-59, 2014. Other linkers and spacers include beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric acid (Ava), 6-aminohexanoic acid (Ahx), trioxatridecane-succinic acid (Ttds), and peptides.

[0053] In one embodiment, the peptide is N- and C-terminally capped by N-terminal acetylation (Ac) or C-terminal amidation. When the biomolecular probe is an antibody, the antibody can be conjugated to the epoxy-based resin through the thiol group by reducing the antibody using a reducing agent such as dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or β-mercaptoethanol (BME) to make the thiol group in the hinge region of the antibody accessible for conjugation with the epoxy-based resin.

[0054] In one embodiment, a substrate having a surface comprising an epoxy-based resin is prepared by coating a support with a solution of SU-8 resin and a photoacid generator, such as triphenylsulfonium hexafluoroantimonate, in a solvent, such as gamma-butyrolactone or cyclopentanone, e.g., EPON SU-8 (commercially available from Hexion Specialty Chemicals, Fayetteville, NC). The support is then heated to remove the solvent, leaving a coating of solid epoxy-based resin on the support. The thickness of the solid epoxy-based resin may be several hundred microns thick. Typically, the thickness of the solid epoxy-based resin ranges from about 1 nm to about 3 mm. Optionally, a portion of the solid epoxy-based resin is then polymerized by irradiation with UV light to obtain a polymerized epoxy-based resin. In one embodiment, a photomask is placed on top of the solid epoxy-based resin prior to irradiation with UV light, such that a pattern remains on the polymerized epoxy-based resin. In one embodiment, the support is separated from the solid epoxy-based resin.

[0055] In one embodiment, a substrate having a surface comprising an epoxy-based resin is prepared by coating a support with a solution of 1002F resin and a photoacid generator, such as triphenylsulfonium hexafluoroantimonate, in a solvent, such as gamma-butyrolactone or cyclopentanone, e.g., EPON 1002F (commercially available from Hexion Specialty Chemicals, Fayetteville, NC). The support is then heated to remove the solvent, leaving a coating of solid epoxy-based resin on the support. The thickness of the solid epoxy-based resin may be several hundred microns thick. Typically, the thickness of the solid epoxy-based resin ranges from about 1 nm to about 3 mm. Optionally, a portion of the solid epoxy-based resin is then polymerized by irradiation with UV light to obtain a polymerized epoxy-based resin. In one embodiment, a photomask is placed on top of the solid epoxy-based resin prior to irradiation with UV light, such that a pattern remains on the polymerized epoxy-based resin. In one embodiment, the support is separated from the solid epoxy-based resin.

[0056] In one embodiment, the sample is a fecal sample. In one embodiment, the molecule (i.e., analyte) being assayed is a protein produced by an intestinal parasite, such as, for example, a roundworm, a whipworm, a hookworm, a tapeworm, or a heartworm, or the parasite Giardia, and the biomolecular probe is an antibody to the protein. In one embodiment, the antibody is specific for a fecal antigen. In one embodiment, the biomolecular probe is selected from the group consisting of an antibody that specifically binds to a fecal antigen from a roundworm, an antibody that specifically binds to a fecal antigen from a whipworm, an antibody that specifically binds to a fecal antigen from a hookworm, an antibody that specifically binds to a fecal antigen from a tapeworm, an antibody that specifically binds to an antigen from a heartworm, and an antibody that specifically binds to a fecal antigen from Giardia.

[0057] Examples of antibodies that specifically bind to fecal antigens from hookworms are disclosed in U.S. Patent Nos. 9,239,326 and 8,895,294. Examples of antibodies that specifically bind to fecal antigens from roundworms are disclosed in U.S. Patent Nos. 8,097,261; 9,212,220; 9,103,823; 8,105,795; and 8,895,294. Examples of antibodies that specifically bind to fecal antigens from whipworms are disclosed in U.S. Patent Nos. 8,367,808 and 8,895,294. Examples of antibodies that specifically bind to fecal antigens from tapeworms are disclosed in U.S. Patent Application Publication No. 2020 / 0102378. Examples of antibodies that specifically bind to fecal antigens from Giardia are disclosed in H. Stibbs, Monoclonal antibody-based enzyme immunoassay for Giardia lamblia antigen in human stool, J. Clin. Microbiol., (Vol. 11): pp. 2582-2588, November 1989, and H. Stibbs et al., Identification of Giardia lamblia-specific antigens in infected human and gerbil feces by western immunoblotting, J. Clin. Microbiol., (Vol. 10): pp. 2340-6, October 1990.

[0058] In one embodiment, the sample is a blood sample from a subject, the analyte is an antibody produced by the subject's immune response to a protein produced by an infectious pathogen, and the biomolecular probe is a protein, polypeptide, or oligopeptide capable of specifically binding to the antibody. Proteins, polypeptides, or oligopeptides capable of specifically binding to circulating antibodies in animals infected with bacteria of the Ehrlichia genus, including Ehrlichia canis, Ehrlichia chaffeensis, and Ehrlichia ewingii, have been disclosed in U.S. Pat. Nos. 7,087,372; 7,407,770; 7,445,788; 7449,191; 7,842,473; 7,888,054; 8,980,274; 7,183,060; 7,744,872; 8,409,817; 9,850,295; 8,158,751, and 9,605,032. Proteins, polypeptides, or oligopeptides capable of specifically binding to circulating antibodies in animals infected with bacteria of the Anaplasma genus, including Anaplasma phagocytophylum and Anaplasma platys, are disclosed in U.S. Patent Nos. 6,964,855; 7,439,321; 8,303,959; 6,306,402; 6,204,252; 8,093,088, and 9,120,857. Proteins, polypeptides, or oligopeptides capable of specifically binding to circulating antibodies in animals infected with bacteria of the Borrelia genus, including Borrelia burgdorferi, are disclosed in U.S. Patent Nos. 6,719,983; 6,740,744; 6,475,492, and 6,660,274.

[0059] In one embodiment, the analyte is an antibody resulting from a subject's response to a protein produced by Borrelia burgdorferi, which causes Lyme disease, and the biomolecular probe is a protein or a portion of a protein produced by Borrelia burgdorferi.

[0060] In one embodiment, the sample is a blood sample from the subject, the analyte is a metabolite, and the biomolecular probe is an antibody capable of specifically binding to the metabolite.

[0061] In one embodiment, the analyte is symmetric dimethylarginine (SDMA).Antibodies that specifically bind to SDMA are disclosed in U.S. Patent No. 8,481,690.

[0062] In one embodiment, the sample is a blood sample from the subject, the analyte is an antigen of pathogen origin present in the blood of an infected subject, and the biomolecular probe is an antibody against the antigen. In one embodiment, the analyte is an antibody that specifically binds to a circulating antigen from Dirofilaria immitis. Antibodies that specifically bind to a circulating antigen from Dirofilaria immitis are disclosed in U.S. Pat. No. 4,839,275.

[0063] In one embodiment, the substrate for the biological assay comprises: (i) providing a silicon / aluminum wafer; (ii) coating a silicon / aluminum wafer with a first solution of a first epoxy-based resin dissolved in a first solvent to provide a silicon / aluminum wafer coated with a first epoxy-based resin coating; (iii) heating the silicon / aluminum wafer coated with the first epoxy based resin coating to remove at least a portion of the first solvent to provide a silicon / aluminum wafer coated with a first solid epoxy based resin coating; (iv) depositing a nickel barcode on the first solid epoxy based resin coating to provide a silicon / aluminum wafer having a first solid epoxy resin coating and a nickel barcode; (v) coating the nickel barcode with a second solution of a second epoxy-based resin dissolved in a second solvent to provide a silicon / aluminum wafer coated with a first epoxy-based resin coating and a second epoxy-based resin coating; (vi) heating the silicon / aluminum wafer having the first solid epoxy based resin coating and the second solid epoxy based resin coating to remove at least a portion of the second solvent to provide a silicon / aluminum wafer coated with the first solid epoxy based resin coating and the second solid epoxy based resin coating; preparing a nickel bar code between a first solid epoxy based resin coating and a second solid epoxy based resin coating; (vii) optionally polymerizing at least a portion of the first solid epoxy based resin coating and the second solid epoxy based resin coating to provide a silicon / aluminum wafer layered with the first solid epoxy based resin, the nickel barcode, and the second solid epoxy based resin coating, wherein at least a portion of the first solid epoxy based resin coating and the second solid epoxy based resin coating are polymerized; (viii) separating the silicon / aluminum wafer from the silicon / aluminum wafer coated with the first epoxy-based resin coating, the nickel barcode, and the second epoxy-based resin coating to provide barcoded magnetic beads; (iv) contacting the barcoded magnetic beads with the biomolecule such that the biomolecule is directly bound to the epoxy resin; It is prepared by a method comprising:

[0064] In one embodiment, the first solution of the first epoxy based resin in the first solvent is the same as the second solution of the second epoxy based resin in the second solvent.

[0065] In one embodiment, the silicon / aluminum wafer is separated from the first solid epoxy resin, nickel barcode, and second solid epoxy resin coating by contacting the silicon / aluminum wafer with sodium hydroxide.

[0066] The present invention is also directed to a method of assaying for the presence of an analyte in a sample, the method comprising contacting the sample with a substrate having a surface comprising an epoxy-based resin having a biomolecular probe directly bound to the epoxy-based resin, where the biomolecular probe specifically binds to the analyte.

[0067] The phrases "bind with specificity to an analyte," "specifically bind," "having specificity," "specific for an analyte," and similar phrases, as used herein, have their art-recognized meaning, i.e., that a biomolecular probe recognizes and binds to an analyte (or class of analytes) with higher affinity than it binds to other non-specific molecules. For example, an antibody raised against an antigen and that binds to that antigen more efficiently than other non-specific molecules can be described as specifically binding to that antigen. Binding specificity can be tested using methods known in the art, such as, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blot assay.

[0068] In one embodiment, the sample is a fecal sample. In one embodiment, the analyte is a protein produced by an intestinal parasite and the biomolecular probe is an antibody against a protein produced by an intestinal parasite. In one embodiment, the antibody against a protein produced by an intestinal parasite is selected from the group consisting of an antibody that specifically binds to a fecal antigen from Ascaris worms, an antibody that specifically binds to a fecal antigen from Trichuris trichiura, an antibody that specifically binds to a fecal antigen from Hookworms, an antibody that specifically binds to a fecal antigen from Tapeworms, an antibody that specifically binds to an antigen from Heartworms, and an antibody that specifically binds to a fecal antigen from Giardia.

[0069] In one embodiment, the sample is a blood sample from a subject, the analyte is an antibody produced by the subject's immune response to a protein produced by an infectious pathogen, and the biomolecular probe is a protein, polypeptide, or oligopeptide capable of specifically binding to the antibody.

[0070] In one embodiment, the analyte is an antibody resulting from a subject's response to a protein produced by Borrelia burgdorferi, which causes Lyme disease, and the biomolecular probe is a protein or a portion of a protein produced by Borrelia burgdorferi.

[0071] In one embodiment, the sample is a blood sample from the subject, the analyte is a metabolite, and the biomolecular probe is an antibody capable of specifically binding to the metabolite. In one embodiment, the analyte is symmetric dimethylarginine (SDMA).

[0072] In one embodiment, the sample is a blood sample from the subject, the analyte is an antigen of pathogen origin present in the blood of an infected subject, and the biomolecular probe is an antibody to the antigen, hi one embodiment, the analyte is an antibody that specifically binds to a circulating antigen from Dirofilaria immitis. EXAMPLES

[0073] The present invention is not limited in scope by the specific embodiments disclosed in the examples, which are intended to illustrate some aspects of the invention, and all functionally equivalent embodiments are within the scope of the present invention. Indeed, those skilled in the art will recognize various modifications of the present invention in addition to those shown and described herein. Such modifications are intended to be included within the scope of the appended claims. Such modifications of the present invention, including the substitution of all equivalents now known or later developed that are considered to be within the knowledge of those skilled in the art, and minor changes in formulation or experimental design, should be considered to be within the scope of the present invention incorporated herein. EXAMPLES

[0074] Passive coupling of biomolecular probes to barcoded magnetic beads Barcoded magnetic beads (BMBs) are fabricated from SU-8, an epoxy-based negative photoresist (commercially available from Applied BioCode Corp., Santa Fe Springs, CA). Coupling of biomolecular probes, such as monoclonal antibodies or proteins, to the BMBs was achieved by absorbing the biomolecular probes onto the BMB surface according to the following procedure.

[0075] A solution of monoclonal antibody at a final concentration of about 0.15-2.5 mg / mL antibody (typically about 1.5 mg / mL) was prepared in an aqueous buffer containing about 100 mM MES (commercially available from Sigma Aldrich, St. Louis, MO) and about 140 mM guanidine-HCl (commercially available from Sigma Aldrich, St. Louis, MO) at a pH of about 5.5, or in an aqueous buffer containing about 100 mM EPPS (commercially available from Sigma Aldrich, St. Louis, MO) and about 140 mM guanidine-HCl at a pH of about 8 to provide an antibody coating solution.

[0076] A sufficient amount of BMB was suspended in BMB wash buffer (about 1.8 mM sodium dihydrogen phosphate (commercially available from Sigma Aldrich, St. Louis, MO), about 8.4 mM sodium monohydrogen phosphate (commercially available from Sigma Aldrich, St. Louis, MO), about 145 mM sodium chloride (OH, commercially available from Amresco LLC, Salon, MO), and about 0.05% Tween-20 (commercially available from Sigma Aldrich, St. Louis, MO), at about pH 7.4) to obtain a final concentration of about 100,000 to 1.8 million BMB / mL (typically about 1 million / mL for antibodies). The BMB were washed three times with BMB wash buffer. All BMB washes (for this step and subsequent steps) proceeded as follows:

[0077] First, place the tube containing BMB on a magnetic stand and allow the BMB to adhere to the magnet for 1-10 min. Then, carefully aspirate off the supernatant and then resuspend the BMB in a volume of wash buffer approximately equal to the original suspension volume of BMB (~200 µL-~1,000 µL). Repeat these steps a total of three times to obtain a BMB pellet.

[0078] After washing the BMB with wash buffer, the BMB were washed three times with a volume of DMSO (commercially available from Sigma Aldrich, St. Louis, MO) approximately equal to the original suspension volume to obtain a pellet of DMSO-washed BMB.

[0079] Coating reaction: After DMSO washing, the antibody coating solution (final concentration of approximately 1.5 mg / mL) was immediately combined with the DMSO-washed BMB (final concentration of approximately 1 million BMB / mL) and incubated with mixing at room temperature (18-27°C) for 4-18 hours. After incubation was completed, the antibody-coupled BMBs were washed three times with assay buffer (about 1% BSA (commercially available from Proliant biologicals, Ankeny, IA), about 0.05% Tween-20 (commercially available from Sigma Aldrich, St. Louis, MO), and about 0.05% Proclin 950 (commercially available from Sigma Aldrich, St. Louis, MO) in about 1.8 mM sodium dihydrogen phosphate (commercially available from Sigma Aldrich, St. Louis, MO), about 8.4 mM sodium monohydrogen phosphate (commercially available from Sigma Aldrich, St. Louis, MO), and about 145 mM sodium chloride (commercially available from Amresco LLC, Salon, OH), at about pH 7.4).

[0080] The antibody-coupled BMB is then suspended in assay buffer at the desired final concentration for use in the assay. EXAMPLES

[0081] Coupling of biomolecular probes to barcoded magnetic beads via thiol groups Coupling of biomolecular probes, such as monoclonal antibodies or proteins containing thiol groups, to barcoded magnetic beads (BMBs) coated with epoxy-based resin is achieved by adsorbing the biomolecular probes onto the BMB surface according to the following procedure.

[0082] A peptide coating solution was prepared with a final peptide concentration of about 0.02 to about 1 mM (typically about 0.1 mM) in DMSO (commercially available from Sigma Aldrich, St. Louis, MO) containing about 1% Tween-20 (commercially available from Sigma Aldrich, St. Louis, MO). The peptide used in this example was acetylated-Cys(dPEG12)[peptide]-amide, which is a 25 amino acid oligopeptide and dPEG12 is a discrete PEG12. Acetylated-Cys(dPEG12)[peptide]-amide was provided as a custom synthesis by New England Peptide, Gardner, MA.

[0083] A suitable BMB for use in the present method is one made from SU-8 (commercially available from Applied BioCode Corp., Santa Fe Springs, Calif.), an epoxy-based negative photoresist.

[0084] A sufficient amount of BMB to provide a final concentration of about 100,000-3 million BMB / mL (typically about 2 million / mL) for the coating reaction described below was suspended in a volume of about 200 μL to about 1,000 μL of BMB wash buffer (about 1% Tween-20 in DMSO). The BMB was washed three times with about 1% Tween-20 in DMSO as described below. All BMB washes (for this step and subsequent steps) proceeded as follows:

[0085] First, the tube containing BMB was placed on a magnetic stand and allowed to adhere to the magnet for 1-10 min. The supernatant was carefully aspirated and then resuspended in a volume of wash buffer approximately equal to the original suspension volume of BMB. These steps were repeated a total of three times to obtain a BMB pellet.

[0086] Once the BMB was washed with wash buffer, the washed BMB was combined with the peptide coating solution and incubated with mixing for approximately 4 hours at room temperature (18-27°C). After the 4 hour incubation, the peptide-coupled BMB was washed three times with a constant volume of assay buffer (approximately 1% BSA, approximately 0.05% Tween-20, and approximately 0.05% Proclin 950 in approximately 1.8 mM sodium dihydrogen phosphate, approximately 8.4 mM sodium monohydrogen phosphate, and approximately 145 mM sodium chloride, pH 7.4).

[0087] The antibody-coupled BMB is then suspended in assay buffer at the desired final concentration for use in the assay. EXAMPLES

[0088] Coupling of biomolecular probes to barcoded magnetic beads via thiol groups generated from reduced disulfide-linked cysteines Macromolecules such as monoclonal antibodies can be coupled through thiol groups by reacting the monoclonal antibody with a reducing agent such as dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoethanol (BME) to reduce the disulfide-linked cysteine ​​side chains so that they are accessible for binding to the surface of the BMB.

[0089] To covalently couple the reduced antibody to the epoxy groups of the BMB, a reduced antibody coating solution was prepared by diluting the antibody to a concentration of approximately 5 mg / mL in reduction buffer (50 mM sodium phosphate (commercially available from Sigma Aldrich, St. Louis, MO), 75 mM sodium chloride (commercially available from Amresco LLC, Salon, OH), 2 mM EDTA (commercially available from Sigma Aldrich, St. Louis, MO), and 5 mM DTT (commercially available from Thermo Fisher Scientific, Waltham, MA), pH approximately 7.4). The resulting solution was incubated at 18-27 °C for approximately 0.5 h.

[0090] After reduction, the reduced antibody was exchanged into a solution containing 50 mM sodium phosphate (commercially available from Sigma Aldrich, St. Louis, MO), 75 mM sodium chloride (commercially available from Amresco, Salon, OH), and 2 mM EDTA (commercially available from Sigma Aldrich, St. Louis, MO) at a pH of approximately 7.4 using a G25 Zeba spin desalting column (commercially available from Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. The antibody solution was adjusted to a concentration of approximately 0.5 mg / mL, and the resulting solution was immediately added to the DMSO-washed BMB.

[0091] DMSO-washed BMBs were prepared by first suspending a sufficient amount of BMBs to provide a final concentration of about 100-1.8 million BMBs / mL (typically about 1 million / mL for antibodies) in the coating reaction described below in a volume of about 200 μL to about 1,000 μL of wash buffer (about 1.8 mM sodium dihydrogen phosphate, about 8.4 mM sodium monohydrogen phosphate, about 145 nM sodium, and about 0.05% Tween-20, about pH 7.4). The BMBs were washed three times with wash buffer as described below.

[0092] All BMB washes (this step and the steps following) proceeded as follows: first, place the tube containing BMB on a magnetic stand and allow the BMB to adhere to the magnet for 1-10 min. Then, carefully aspirate off the supernatant and then resuspend the BMB in a volume of wash buffer approximately equal to the original volume used to suspend the BMB. These steps are repeated three times to obtain a BMB pellet.

[0093] After washing the BMB with wash buffer, the BMB was washed three times with a volume of DMSO (commercially available from Sigma Aldrich, St. Louis, MO) approximately equal to the original suspension volume to obtain a pellet of DMSO-washed BMB. The BMB was then suspended in a volume of DMSO approximately equal to the original suspension volume and incubated at 18-27 °C with mixing for approximately 4 hours. After incubation, the tube containing the BMB was placed on a magnetic stand and allowed the BMB to adhere to the magnet for 1-10 minutes. The supernatant was then carefully aspirated to obtain a DMSO-washed BMB pellet.

[0094] A suitable BMB for use in the present method is one made from SU-8 (commercially available from Applied BioCode Corp., Santa Fe Springs, Calif.), an epoxy-based negative photoresist.

[0095] After the DMSO wash, the antibody coating solution (final concentration of approximately 0.5 mg / mL) was immediately combined with the DMSO-washed BMBs (final concentration of approximately 1 million BMBs / mL) and incubated with mixing at room temperature (18-27 °C) for approximately 18 h.

[0096] After incubation was completed, the tube containing BMB was placed on a magnetic stand and allowed to adhere to the magnet for 1-10 min. The supernatant was then carefully aspirated and the BMB was resuspended in a volume of wash buffer (approximately 1.8 mM sodium dihydrogen phosphate, approximately 8.4 mM sodium monohydrogen phosphate, approximately 145 mM sodium chloride, and approximately 0.05% Tween-, pH 7.4) approximately equal to the original suspension volume, and the resulting solution was incubated at 18-27 °C for approximately 15 min.

[0097] After the incubation was completed, the tube containing the BMB was placed on a magnetic stand and the BMB was allowed to adhere to the magnet for 1-10 minutes. The supernatant was then carefully aspirated off and the BMB was resuspended in a volume of assay buffer approximately equal to the original suspension volume. Assay buffer: about 1% BSA, about 0.05% Tween-20, and about 0.05% Proclin 950 in about 1.8 mM sodium dihydrogen phosphate, about 8.4 mM sodium monohydrogen phosphate, and about 145 nM sodium chloride at pH 7.4. The resulting suspension was incubated at 18-27°C for about 30 minutes with mixing. The antibody-coupled BMB was then washed three times with a volume of assay buffer approximately equal to the original suspension volume.

[0098] The antibody-coupled BMB was then suspended in assay buffer at the final desired concentration for use in the assay. EXAMPLES

[0099] Coupling of rhodamine to barcoded magnetic beads: material:

[0100] [Table 1]

[0101] DMSO wash of BMB: To a 1 mL centrifuge tube on a magnetic rack, 0.5 mL of BMB suspended in storage buffer was added to give a concentration of 100,000 BMB / mL.

[0102] A suitable BMB for use in the present method is one made from SU-8, an epoxy-based negative photoresist (commercially available from Applied BioCode Corp., Santa Fe Springs, Calif.) The BMB is prepared in a storage buffer containing sodium chloride (0.8%), potassium chloride (0.02%), disodium hydrogen phosphate (0.144%), potassium dihydrogen phosphate (0.024%), Tween-20 (0.05%), and Proclin-950 (0.1%).

[0103] The liquid was removed from each of the centrifuge tubes with a pipette, then approximately 0.5 mL of DMSO (commercially available from Sigma Aldrich, St. Louis, MO) was added to each tube. The tubes were vortexed vigorously for 10 seconds, returned to the magnetic rack, allowed to sit for 1 minute, and the DMSO was removed with a pipette. This washing procedure was repeated two more times. Approximately 0.5 mL of DMSO was then added to each tube, the tubes were vortexed vigorously, and left on the mixer at room temperature for 4 hours.

[0104] Rhodamine-Lissamine Coating: After mixing for 4 hours, the tubes were removed and placed on a magnetic stand, allowed to sit for 1 minute, the DMSO was removed with a pipette, and approximately 0.5 mL of approximately 150 mM EPPS buffer, pH 9.0, was added to the tubes. The tubes containing BMB in EPPS buffer were then vortexed vigorously for 10 seconds, returned to the magnetic stand, and the EPPS buffer was removed with a pipette. This washing procedure was repeated two more times. After the washes were complete, approximately 0.5 mL of EPPS buffer was added to each tube and the tubes were vortexed. Approximately 10.0 μM rhodamine-lissamine (9 μL of a 0.3 mg / mL solution in DMSO) or sulfo-rhodamine (control, 1.2 μL of a 2.8 mg / mL solution in DMSO) was then added to the epoxy-based BMB in EPPS buffer. The suspension of BMB was rotated upside down at room temperature for approximately 22 hours, protected from light. After this time, the coated BMB was placed on a magnetic stand and the solvent was removed with a pipette. The coated BMBs were washed with about 1.0 mL of a solution of about 1.8 mM sodium dihydrogen phosphate, about 8.4 mM sodium monohydrogen phosphate, about 145 nM sodium chloride, and about 0.05% Tween-20 by brief vortexing, followed by removal of the solvent. This process was repeated five times. Finally, about 1.0 mL of a solution of about 1.8 mM sodium dihydrogen phosphate, about 8.4 mM sodium monohydrogen phosphate, about 145 nM sodium chloride, and about 0.05% Tween-20 was added to the coated BMBs to obtain a final concentration of about 50,000 BMBs / mL. About 5.0 μL of the BMBs in the resulting suspension were then added to the wells of a 96-well plate to obtain a final count of about 250 BMBs / well. About 200 μL of a solution of about 1.8 mM sodium dihydrogen phosphate, about 8.4 mM sodium monohydrogen phosphate, about 145 nM sodium chloride, and about 0.05% Tween-20 was added to each well to provide a suspension of BMB for use in the assay.

[0105] The fluorescence of each well was measured using a microplate reader (commercially available reader, Applied BioCode Corp., Santa Fe Springs, Calif.).

[0106] The epoxy BMB coated with rhodamine-lissamine showed significant fluorescence in a microplate reader, indicating that the rhodamine probe was efficiently coated onto the epoxy BMB surface. The epoxy BMB incubated with the sulfo-rhodamine control, which does not have a reactive amine functional group, did not show any observable fluorescence. This demonstrates that the nonspecific binding of the rhodamine probe to the epoxy BMB surface is very low, indicating that rhodamine-lissamine reacts specifically with the epoxy surface via the primary amine functional group. It can be concluded that amine-functionalized small molecules can be covalently attached to the epoxy BMB surface. EXAMPLES

[0107] Coupling of amine-containing peptides to barcoded magnetic beads: material:

[0108] [Table 2]

[0109] DMSO wash of BMB: Approximately 0.2 mL of BMB suspended in storage buffer was added to a 1 mL centrifuge tube on a magnetic rack to give a concentration of 100,000 BMB / mL.

[0110] A suitable BMB for use in the present method is one made from SU-8, an epoxy-based negative photoresist (commercially available from Applied BioCode Corp., Santa Fe Springs, Calif.) The BMB is prepared in a storage buffer containing sodium chloride (0.8%), potassium chloride (0.02%), disodium hydrogen phosphate (0.144%), potassium dihydrogen phosphate (0.024%), Tween-20 (0.05%), and Proclin-950 (0.1%).

[0111] The liquid was removed from each of the centrifuge tubes with a pipette, then approximately 0.2 mL of DMSO (commercially available from Sigma Aldrich, St. Louis, MO) was added to each tube. The tubes were vortexed vigorously for 10 seconds, returned to the magnetic rack, allowed to sit for 1 minute, and the DMSO was removed with a pipette. This washing procedure was repeated two more times. Approximately 0.2 mL of DMSO was then added to each tube, the tubes were vortexed vigorously, and left on the mixer at room temperature for 4 hours.

[0112] After 4 hours of mixing, the tube was removed and placed on the magnetic stand, allowed to sit for 1 min, the DMSO was removed with a pipette, and approximately 0.2 mL of approximately 150 mM EPPS buffer, pH 9.0, was added to the tube. The tube containing BMB in EPPS buffer was then vortexed vigorously for 10 s, returned to the magnetic stand, and the EPPS buffer was removed with a pipette. This washing procedure was repeated two more times.

[0113] Approximately 0.1 mM solutions of both biotin-Lyme peptide and Lyme-Alexafluor555 were prepared from 1.0 mM stock solutions. Each peptide has multiple lysine residues but no cysteine ​​residues or other thiol groups. Approximately 200 μL of the biotin-Lyme peptide solution, the Lyme-Alexafluor555 solution, or a control containing only EPPS buffer was added to the tube containing BMB. The resulting BMB suspension was rotated upside down at room temperature for approximately 2 hours and protected from light. After this time, the tube containing BMB was placed on a magnetic rack and the solvent was removed with a pipette. The BMB was then washed with approximately 0.2 mL of a solution of approximately 1.8 mM sodium dihydrogen phosphate, approximately 8.4 mM sodium monohydrogen phosphate, approximately 145 nM sodium chloride, and approximately 0.05% Tween-20 by brief vortexing, followed by removal of the solvent. This process was repeated twice.

[0114] About 200 μL of SA-PE (streptavidin phycoerythrin)-containing solution (8 μg / mL SA-PE, obtained by diluting SA-PE (commercially available as a 1 mg / mL solution from Moss Inc., Pasadena, MD) with multiplex assay buffer: 1.8 mM sodium dihydrogen phosphate, 8.4 mM sodium monohydrogen phosphate, 145 nM sodium chloride, about 0.05% Tween-20, 1% bovine serum albumin, 0.05% Proclin 950) was added to the BMB and the beads were incubated for about 10 minutes. After incubation, the supernatant was removed and the BMB was washed with about 0.2 mL of a solution of about 1.8 mM sodium dihydrogen phosphate, about 8.4 mM sodium monohydrogen phosphate, about 145 nM sodium chloride, and about 0.05% Tween-20, about pH 7.4 by brief vortexing, followed by removal of the solvent. This process was repeated five times. Finally, about 0.4 mL of a solution of about 1.8 mM sodium dihydrogen phosphate, about 8.4 mM sodium monohydrogen phosphate, about 145 nM sodium chloride, and about 0.05% Tween-20, at about pH 7.4, was added to the coated BMBs to obtain a final concentration of about 50,000 BMBs / mL. 5.0 μL of the resulting BMB suspension was added to wells of a 96-well plate to obtain a final count of about 250 BMBs / well, and about 200 μL of a solution of about 1.8 mM sodium dihydrogen phosphate, about 8.4 mM sodium monohydrogen phosphate, about 145 nM sodium chloride, and about 0.05% Tween-20 was added to each well to obtain a BMB suspension for use in the assay.

[0115] The fluorescence of each well was measured using a microplate reader (commercially available reader, Applied BioCode Corp., Santa Fe Springs, Calif.).

[0116] Both BMBs coated with Biotin-Lyme peptide and Lyme-Alexafluor555 peptide showed significant fluorescence in the microplate reader, indicating that the peptides were efficiently coated onto the BMB surface. The lack of fluorescence observed in the controls demonstrates that non-specific binding of SA-PE analytes to the BMB surface is very low, indicating that Biotin-Lyme peptide and Lyme-Alexafluor555 peptide react specifically with the epoxy surface via the primary amine functional groups contained in the lysine residues of the peptides. It can be concluded that it is possible to covalently attach lysine (amine functional group)-containing peptides to the epoxy BMB surface. EXAMPLES

[0117] Assay using barcoded magnetic beads after citrate buffer washing: Coupling of biomolecular probes to barcoded magnetic beads To a 1 mL centrifuge tube on a magnetic rack, approximately 0.2 mL (volume can vary from approximately 0.1 mL to approximately 500 mL) of BMB suspended in storage buffer was added to obtain a concentration of approximately 100,000 BMB / mL (concentration may range from 100,000 BMB / mL to approximately 3 million BMB / mL).

[0118] A suitable BMB for use in the present method is one made from SU-8, an epoxy-based negative photoresist (commercially available from Applied BioCode Corp., Santa Fe Springs, Calif.) The BMB is prepared in a storage buffer containing sodium chloride (0.8%), potassium chloride (0.02%), disodium hydrogen phosphate (0.144%), potassium dihydrogen phosphate (0.024%), Tween-20 (0.05%), and Proclin-950 (0.1%).

[0119] The liquid was removed from each of the centrifuge tubes with a pipette, and then about 0.2 mL of DMSO (commercially available from Sigma Aldrich, St. Louis, MO) was added to each tube. The tubes were vortexed vigorously for 10 seconds, returned to the magnetic rack, allowed to sit for 1 minute, and the DMSO was removed with a pipette. This washing procedure was repeated two more times. In one embodiment, about 0.2 mL of DMSO was then added to each tube, the tubes were vortexed vigorously, and placed on a mixer at room temperature for 4 hours. This 4 hour mixing at room temperature is optional.

[0120] After mixing, the tube was removed and placed on a magnetic stand, left for 1 min, the DMSO was removed by pipette, and about 0.2 mL of about 150 mM EPPS buffer, pH 9.0 was added to the tube. The tube containing the BMB in EPPS buffer was then vortexed vigorously for 10 s, returned to the magnetic stand, and the EPPS buffer was removed by pipette. This washing procedure was repeated two more times. After a final wash with EPPS, the BMB was coated with peptides or antibodies as described in parts (A) and (B) below, respectively, with each peptide and antibody being combined with a BMB of a different barcode.

[0121] (A) Coating of BMB with peptides Peptides (biochemical probes) capable of specifically binding to patient antibodies against Anaplasma, Ehrlichia, or Borrelia genera (e.g., peptides derived from the protein sequences of Anaplasma phagocytophylum, Anaplasma platys, Ehrlichia canis, Ehrlichia ewingii, or Borrelia burgdorferi described above) were synthesized with a cysteine ​​linked to the N-terminus of each peptide via a PEG12 linker as described above, except for the peptides derived from Borrelia that have an N-terminal cysteine ​​but no PEG linker. Approximately 200 μL of a 0.1 mM solution of each peptide was added to a tube containing EPPS-washed BMB. The resulting BMB suspension was rotated end-over-end at room temperature for approximately 2 hours, protected from light. After this time, the tube containing BMB was placed on a magnetic rack and the solvent was removed with a pipette. The BMB was then washed with about 0.2 mL of a solution of about 1.8 mM sodium dihydrogen phosphate, about 8.4 mM sodium monohydrogen phosphate, about 145 nM sodium chloride, and about 0.05% Tween-20 by brief vortexing, followed by removal of the solvent. This process was repeated twice.

[0122] (B) Coating of BMB with antibodies About 200 μL of an antibody (biochemical probe) at a concentration of approximately 10 mg / mL (the antibody concentration may range from about 1.5 mg / mL to about 12.0 mg / mL) capable of specifically binding to an antigen derived from the dog heartworm (Dirofilaria immitis) circulating in the blood of infected animals was added to the tube containing the BMB, as described above. The resulting BMB suspension was rotated upside down for about 2 hours at room temperature and protected from light. After this time, the tube containing the BMB was placed on a magnetic rack and the solvent was removed with a pipette. The BMB was then washed with about 0.2 mL of a solution of about 1.8 mM sodium dihydrogen phosphate, about 8.4 mM sodium monohydrogen phosphate, about 145 nM sodium chloride, and about 0.05% Tween-20 by brief vortexing, followed by removal of the solvent. This process was repeated twice.

[0123] Assay: A multiplex BMB mixture was constructed by mixing BMBs coated with the biomolecular probes described in (A) or (B) above at 65,000 BMBs / mL in assay buffer (1.0% BSA, 0.05% Tween, 0.05% Proclin 950 in PBS). The multiplex BMB mixture was further diluted with assay buffer to achieve a concentration of 500 BMBs / mL of each biomolecular probe.

[0124] Approximately 100 μL of this diluted multiplex BMB mixture was added to each well of a 96-well plate (i.e., approximately 50 beads per biomolecular probe) using an Integra automated pipette (commercially available from Integra Biosciences Corp., Hudson, NH). The BMB was immersed in 300 μL of 0.05% Tween-20 in PBS for 10 seconds and washed five times using a 405-TS plate washer (commercially available from BioTek®, Winooski, VT). After the final wash, excess supernatant (approximately 30 μL) was left on the plate.

[0125] 50 μL of sample (serum or plasma, neat) was added to the BMB in each well of a 96-well plate. The plate was then placed on a plate mixer and mixed at 1000 rpm for 30 minutes. After 30 minutes of incubation, the BMB was washed by immersion in 300 μL of 0.05% Tween-20 in PBS for 10 seconds.

[0126] 50 μL of each biotinylated peptide (2.0 μg / mL, i.e., the same peptide used as the biomolecular probe) or biotinylated anti-Heartworm antibody (1.0 μg / mL) in 1.0% BSA, 0.05% Tween, 0.05% Proclin 950 in PBS was added to each well of a 96-well plate. The plate was then placed on a plate mixer and mixed for 15 min. After the 15 min incubation, the BMB was washed by immersion in 300 μL of 0.05% Tween-20 in PBS for 10 s.

[0127] 50 μL of SA-PE, 8.0 μg / mL (commercially available from MOSS Inc., Pasadena, MD, catalog number SAPERP01) was added to each well of a 96-well plate. The plate was then placed on a plate mixer and mixed for 10 minutes. After the 10 minute incubation, the BMB was washed by immersion in 300 μL of 0.05% Tween-20 in PBS for 10 seconds.

[0128] The resulting BMBs were then treated in one of two ways.

[0129] In the first step, each well of a 96-well plate is (i) Approximately 200 μL of buffer (commercially available from Applied BioCode Inc., Santa Fe Springs, Calif., Catalog No. 44-D0004-500) (standard read buffer) was added.

[0130] In the second step, each well of the 96-well plate is (ii) Approximately 200 µL of a solution of citrate buffer containing sodium citrate tribasic dihydrate (0.485 M), citric acid (0.015 M), sodium chloride (0.1 M), and Proclin 950 (0.5 mL / L), pH 6.1-6.3 (citrate reading buffer). was added.

[0131] Ionic strength is an important factor that allows for the stability of the immune complexes formed on the surface of the beads. Without being bound by theory, it is believed that the stabilizing effect is due to the high salt concentration that makes the solution unfavorable for dissociation. High ionic strength salts other than citrate are also effective, but a minimum concentration of about 0.5M is required. However, other salts precipitate out of solution at temperatures below ambient temperature, making them less than ideal for manufacturing or shipping. Advantageously, the citrate buffer mixture remains dissolved in solution even when stored or shipped under refrigerated conditions.

[0132] The fluorescence of each well was determined using a BioCode® 2500 Analyzer (commercially available from Applied BioCode Corp., Santa Fe Springs, Calif.).

[0133] The fluorescence intensity of each sample in each well of a 96-well plate (i.e., a plate with 12 columns (1-12) and 8 rows (A-H)) was determined. Figure 1 shows the signal intensity observed when using standard read buffer (Figure 1A) and when using citrate read buffer (Figure 1B). In the experiment, each well of a 96-well plate (i.e., a plate with 12 columns (1-12) and 8 rows (A-H)) was filled with identical BMB (i.e., all bound to beads, as described above) and the signal was read from column 1 (i.e., wells 1A-1H) to column 12 (i.e., wells 12A-12H). The time to read the fluorescence of all cells of a 96-well plate (i.e., cells 1A-12H) takes approximately 37 minutes. As can be seen from FIG. 1A, when using the standard read buffer (a citrate-free buffer), i.e., procedure (i), the signal intensity gradually decreases from the start of the read cycle to the end of the read cycle (approximately 37 minutes). In contrast, as can be seen from FIG. 1B, when using the citrate read buffer, i.e., procedure (ii), no decrease in signal intensity was observed from the start of the read cycle to the end of the read cycle (approximately 37 minutes). This decrease in signal intensity occurred during the read cycle when a citrate-free buffer, such as the standard read buffer, was used, but not when a citrate-containing buffer, such as the citrate read buffer, was used, in assays of antibodies specific to each of the three Anaplasma genus-derived peptides, labeled "AP," "Aph," and "Apl" on the X-axis of FIG. 1A and FIG. 1B.

[0134] FIG. 1 shows that contacting BMB with citrate buffer before reading the fluorescence advantageously avoids the fluorescence decrease as a function of time compared to other buffers.

[0135] In assays with antibodies specific for peptides derived from Ehrlichia canis, Ehrlichia ewingii, and in assays with Borrelia burgdorferi (not shown), the effects of the standard and citrate read buffers on signal intensity as a function of time were observed to be similar.

[0136] The disclosures of all cited references are incorporated herein by reference in their entireties.

Claims

1. 1. A method of fabricating a substrate for biological analysis, comprising: (i) providing a substrate having a surface comprising an epoxy-based resin; and (ii) contacting a biomolecular probe with a substrate having a surface comprising the epoxy-based resin such that the biomolecular probe is directly bound to the epoxy-based resin; The method includes:

2. 2. The method of claim 1, wherein the biomolecular probe is selected from the group consisting of lipids, polysaccharides, amino acids, polypeptides, oligopeptides, peptides, antibodies and fragments thereof, polynucleotides, oligonucleotides, aptamers, lectins, avidin, streptavidin, biotin, and polyethylene glycol.

3. The method of claim 2 , wherein the biomolecular probe is an antibody.

4. 2. The method of claim 1, wherein the substrate having a surface comprising an epoxy-based resin is selected from the group consisting of a thin film, a microbead, a microparticle, a micropellet, a microwafer, a paramagnetic bead, a microparticle comprising a barcode, a paramagnetic microparticle, a microparticle comprising a barcode, a paramagnetic microparticle comprising a barcode, and a bead comprising a nickel barcode.

5. 2. The method of claim 1, wherein the biomolecular probes are contacted with the epoxy-based resin by contacting a substrate having a surface comprising the epoxy-based resin with a solution of the biomolecular probes to provide a contact mixture.

6. The method of claim 5 , wherein the solution is an aqueous solution.

7. 7. The method of claim 6, wherein the aqueous solution is buffered to about pH 5.5, or to about pH 8, with about 100 mM MES and about 140 mM guanidine-HCl.

8. The method of claim 5, wherein the solution is a DMSO solution that may contain about 1% Tween-20.

9. The method of claim 5, wherein the concentration of the biomolecular probe in the solution ranges from about 0.05 mg / mL to about 5 mg / mL.

10. 6. The method of claim 5, wherein the concentration of substrates having a surface comprising the epoxy-based resin in the contact mixture ranges from about 50,000 to about 5 million substrates / mL.

11. The method of claim 1, wherein the biomolecular probe is contacted with a substrate having a surface comprising the epoxy-based resin for at least about 4 hours, or from about 4 hours to about 18 hours.

12. The method of claim 5, wherein the contact mixture is maintained at a temperature of from about 15°C to about 30°C.

13. 2. The method of claim 1, wherein the substrate having a surface comprising the epoxy-based resin is washed with DMSO, which may contain about 1% Tween-20, before being contacted with the biomolecular probe.

14. A substrate for biological analysis prepared by the method of claim 1.

15. 1. A substrate for biological analysis comprising a substrate having a surface comprising an epoxy-based resin having a biomolecular probe directly bonded to said epoxy-based resin.

16. 1. A method of fabricating a substrate for biological analysis, comprising: (i) providing a solution of biomolecular probes in a solvent selected from the group consisting of: (a) an aqueous solution of about 100 mM MES and about 140 mM guanidine-HCl buffered to about pH 5.5; and (b) an aqueous solution of about 100 mM EPPS and 140 mM guanidine-HCl buffered to about pH 8; providing a concentration of said biomolecular probe ranging from about 0.05 mg / mL to about 5 mg / mL; (ii) providing a substrate having a surface comprising an epoxy-based resin; (iii) washing the substrate having a surface comprising the epoxy-based resin with a phosphate buffered saline (PBS) solution containing 0.05% Tween-20 to prepare a PBS-washed substrate having a surface comprising an epoxy-based resin; (iv) washing the PBS-washed substrate having a surface comprising an epoxy-based resin with DMSO to prepare a DMSO-washed substrate having a surface comprising an epoxy-based resin; (v) combining the DMSO-washed substrate having a surface comprising an epoxy-based resin with the solution of the biomolecular probe, to provide a substrate having a surface comprising an epoxy-based resin, to which the biomolecular probe is directly bound, the concentration of the DMSO-washed substrate having a surface comprising an epoxy-based resin being in the range of about 50,000 to about 5,000,000 substrates / mL; and (vi) washing a substrate having a surface comprising the epoxy-based resin, wherein the biomolecular probe is directly bound to the epoxy-based resin with PBS containing about 1% BSA, about 0.05% Tween-20, and about 0.05% Proclin 950 at a pH of about 7.4; The method includes:

17. The method of claim 16 , wherein the biomolecular probe is an antibody.

18. 1. A method for fabricating a substrate for biological analysis, comprising: (i) preparing a solution of the biomolecular probe in DMSO containing about 1% Tween-20; providing a concentration of said biomolecular probe ranging from about 0.05 mg / mL to about 5 mg / mL; (ii) providing a substrate having a surface comprising an epoxy-based resin; (iii) washing the substrate having a surface comprising the epoxy-based resin with DMSO containing about 1% Tween-20 to prepare a DMSO-washed substrate having a surface comprising an epoxy-based resin; (iv) combining the DMSO-washed substrate having a surface comprising an epoxy-based resin with the solution of the biomolecular probe, to provide a substrate having a surface comprising an epoxy-based resin, to which the biomolecular probe is directly bound, the concentration of the DMSO-washed substrate having a surface comprising an epoxy-based resin being in the range of about 50,000 to about 5,000,000 substrates / mL; and (v) washing a substrate having a surface comprising the epoxy-based resin, wherein the biomolecular probe is directly bound to the epoxy-based resin with PBS containing about 1% BSA, 0.05% Tween-20, and 0.05% Proclin 950 at a pH of about 7.4; The method includes:

19. The method of claim 18 , wherein the biomolecular probe comprises a cysteine ​​residue.

20. 4. The method of claim 3, wherein the antibody is selected from the group consisting of an antibody that specifically binds to a fecal antigen from a roundworm, an antibody that specifically binds to a fecal antigen from a whipworm, an antibody that specifically binds to a fecal antigen from a hookworm, an antibody that specifically binds to a fecal antigen from a tapeworm, an antibody that specifically binds to an antigen from a heartworm, and an antibody that specifically binds to a fecal antigen from a Giardia.

21. 18. The method of claim 17, wherein the antibody is selected from the group consisting of an antibody that specifically binds to a fecal antigen from a roundworm, an antibody that specifically binds to a fecal antigen from a whipworm, an antibody that specifically binds to a fecal antigen from a hookworm, an antibody that specifically binds to a fecal antigen from a tapeworm, an antibody that specifically binds to an antigen from a heartworm, and an antibody that specifically binds to a fecal antigen from a Giardia.

22. 3. The method of claim 2, wherein the biomolecular probe is a protein expressed by an infectious pathogen, a portion of the protein expressed by the infectious pathogen, a peptide or recombinant protein derived from a protein expressed by the infectious pathogen, or a mutant of the protein expressed by the infectious pathogen.

23. 23. The method of claim 22, wherein the biomolecular probe is capable of specifically binding to antibodies produced by a subject against a bacterium of a genus selected from the group consisting of Ehrlichia, which may be Ehrlichia canis, Ehrlichia chaffeensis, or Ehrlichia ewingii, Anaplasma, which may be Anaplasma phagocytophylum, or Anaplasma platys, and Borrelia, which may be Borrelia burgdorferi.

24. 23. The method of claim 22, wherein the biomolecular probe is capable of specifically binding to antibodies produced by a subject against Dirofilaria immitis.

25. The method of claim 3 , wherein the biomolecular probe is an antibody capable of specifically binding to an antigen of Dirofilaria immitis.

26. 23. The method of claim 22, wherein the biomolecular probe is capable of specifically binding to an antibody against a metabolite, which may be SDMA.

27. 1. A method for assaying for the presence of an analyte in a sample, comprising contacting the sample with a substrate having a surface comprising an epoxy-based resin having a biomolecular probe directly bound to the epoxy-based resin, the biomolecular probe binding with specificity to the analyte.

28. 28. The method of claim 27, wherein the sample is a fecal sample.

29. 28. The method of claim 27, wherein the analyte is an antigen expressed by an intestinal parasite and the biomolecular probe is an antibody against the antigen produced by the intestinal parasite.

30. 30. The method of claim 29, wherein the antibody against the antigen produced by the intestinal parasite is selected from the group consisting of an antibody that specifically binds to a fecal antigen from a roundworm, an antibody that specifically binds to a fecal antigen from a whipworm, an antibody that specifically binds to a fecal antigen from a hookworm, an antibody that specifically binds to a fecal antigen from a tapeworm, an antibody that specifically binds to an antigen from a heartworm, and an antibody that specifically binds to a fecal antigen from a Giardia.

31. 28. The method of claim 27, wherein the sample is a blood sample from the subject.

32. 28. The method of claim 27, wherein the analyte is an antibody produced by a subject's immune response to a protein produced by an infectious pathogen, and the biomolecular probe is the protein produced by the infectious pathogen, a portion of the protein, or a mutant of the protein produced by the infectious pathogen.

33. 33. The method of claim 32, wherein the biomolecular probe is capable of specifically binding to antibodies produced by the subject against bacteria from the group selected from the genus Ehrlichia, which may be Ehrlichia canis, Ehrlichia chaffeensis, or Ehrlichia ewingii, the genus Anaplasma, which may be Anaplasma phagocytophylum, or Anaplasma platys, and the genus Borrelia, which may be Borrelia burgdorferi.

34. 33. The method of claim 32, wherein the biomolecular probe is capable of specifically binding to antibodies produced by the subject against Dirofilaria immitis.

35. 33. The method of claim 32, wherein the biomolecular probe is capable of specifically binding to an antibody against a metabolite, which may be SDMA.

36. 32. The method of claim 31, wherein the analyte is an antigen of pathogen origin, a fragment of said antigen, or a variant of said antigen of pathogen origin, and the biomolecular probe is an antibody specific for said antigen.

37. 37. The method of claim 36, wherein the pathogen is Dirofilaria immitis.

38. 1. A method of assaying for the presence of an analyte in a sample, comprising: (a) forming a complex between the analyte and the biochemical probe in a first buffer solution, the complex providing a signal; (b) replacing the first buffer solution with a second buffer solution; and (c) reading the intensity of said signal. The method includes:

39. 39. The method of claim 38, wherein the first buffer solution has a first ionic strength and the second buffer solution has a second ionic strength, the second ionic strength being greater than the first ionic strength.

40. 40. The method of claim 39, wherein the second ionic strength is at least 0.4 M, at least 0.5 M, or at least 0.6 M.

41. 39. The method of claim 38, wherein the second buffer is a citrate buffer.

42. 42. The method of claim 41, wherein the citrate buffer has an ionic strength of at least 0.5 M.

43. 42. The method of claim 41, wherein the citrate buffer comprises sodium citrate tribasic dihydrate, citric acid, sodium chloride, Proclin 950, and has a pH in the range of about 5.5 to about 7.0, in the range of about 6.0 to about 6.5, or in the range of about 6.1 to about 6.

3.

44. 44. The method of claim 43, wherein the concentration of sodium citrate tribasic dihydrate is about 0.485 M, the concentration of citric acid is about 0.015 M, the concentration of sodium chloride is about 0.1 M, and the concentration of Proclin 950 is about 0.5 mL / L.

45. 39. The method of claim 38, wherein the analyte is a peptide derived from a protein sequence of the genus Anaplasma, which may be Anaplasma phagocytophylum or Anaplasma platys, the genus Ehrlichia, which may be Ehrlichia canis or Ehrlichia ewingii, or the genus Borrelia, which may be Borrelia burgdorferi.

46. 39. The method of claim 38, wherein the analyte is an antibody capable of specifically binding to an antigen from Dirofilaria immitis.

47. 40. The method of claim 38, wherein the biomolecular probe is bonded to an epoxy-based resin.

48. 39. The method of claim 38, wherein the biochemical probe is a polypeptide or protein, the analyte is an antibody capable of specifically binding to the biochemical probe, and the sample is obtained from a patient.

49. 40. The method of claim 38, wherein the biochemical probe is an antibody, the analyte is an antigen capable of specifically binding to the biochemical probe, and the sample is obtained from a patient.

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