Biochemical probes attached to epoxy resin

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 on January 17, 2021, and U.S. Provisional Patent Application No. 63 / 155,472, filed on March 4, 2021, the contents of which are hereby incorporated by reference in their entirety.

[0002] Declaration Regarding Federally Sponsored Research or Development Not applicable.

[0003] Incorporation by Reference of Materials Submitted on Compact Disc Not applicable.

[0004] The present invention is directed to a method for fabricating a solid substrate for performing biological and chemical assays, and to the solid substrate fabricated by that method. In particular, it is directed to a solid substrate for use in multiplex bioassays.

Background Art

[0005] Arrays for biological and chemical analysis may be created by attaching probe molecules to a solid substrate having a surface containing a resin material such as a functionalized epoxy resin. Using arrays allows for rapid screening of numerous biomolecules, such as nucleic acids and proteins, with very small sample volumes. For example, particles known as microspheres or microbeads with identifiable labels and / or markings, called barcoded microbeads, are used in parallel multiplex analysis to identify disease-related targets, toxin-related targets, and gene-related targets, among others. Microbeads have a resin coating on their surface conjugated with one or more probe molecules having affinity and / or interaction capabilities for one or more specific target molecules. Each probe molecule is attached to a separate bead that is uniquely coded for identification. In an assay, when microbeads are brought into contact with a sample, various target molecules in the sample bind to the microbeads to which the corresponding probe molecules are conjugated. The barcodes enable target identification.

[0006] Microbead assays are now a crucial tool in biological assays and diagnostics. Microbead-based techniques are sophisticated and versatile methods for performing highly parallel quantitative multi-parameter assays. Microbead-based techniques form the basis for various techniques for detecting and quantifying nucleic acids and proteins in samples. [Overview of the project] [Problems that the invention aims to solve]

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

[0008] In this technical field, there is a need for a simple method for producing substrates coated with epoxy resins that can efficiently adhere biomolecular probes. Unexpectedly, the inventors discovered that biomolecular probes can be efficiently adhered to epoxy resin-coated substrates without the need to first functionalize the epoxy resin with additional functional monomers.

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

[0010] Any reference cited in this application should not be interpreted as such reference being prior art to this application.

[0011] This invention relates to a substrate for biological analysis and a method for producing a substrate for biological analysis. The substrate for biological analysis includes an epoxy resin having a biomolecular probe directly bonded to a polymerized resin.

[0012] Substrates for biological analysis are (i) Prepare a substrate having a surface containing epoxy resin, and (ii) Contact the epoxy resin and the biomolecular probe so that the biomolecular probe directly bonds to the epoxy resin. It is prepared by [method].

[0013] The present invention also relates to a method for assaying the presence of an analyte in a sample. This method involves contacting a sample with a substrate having a surface containing an epoxy resin, the substrate having a biomolecular probe directly bonded to the epoxy resin, so that the biomolecular probe specifically binds to the analyte. [Brief explanation of the drawing]

[0014] [Figure 1A] Figures 1A and 1B plot the signal intensity as the fluorescence intensity (MFI) of BMB measured as a single median result for all beads in the wells, against the cell column position (i.e., 1–12) in a 96-well plate, for the assay described in Example 6 for antibodies specific to three Anaplasma-derived peptides labeled "AP," "Aph," and "Apl" on the X axis. As described in Example 6, Figure 1A shows the signal intensity versus cell column position in standard read buffer, and Figure 1B shows the signal intensity versus cell column position in citrate read buffer. [Figure 1B] Same as above. [Modes for carrying out the invention]

[0015] This invention relates to a substrate for biological analysis and a method for producing a substrate for biological analysis. The substrate for biological analysis includes an epoxy resin having a biomolecular probe directly bonded to the epoxy resin.

[0016] When used herein, “biomolecular probes directly bonded to epoxy resins” and similar phrases mean that the biomolecular probes are attached to the resin by simply bringing the biomolecular probes into contact with the resin, without first contacting the epoxy resin with another reagent that covalently reacts with the epoxy resin. The biomolecular probes may be passively attached to the epoxy resin or covalently attached to the epoxy resin.

[0017] Substrates for biological analysis are (i) Prepare a substrate having a surface containing epoxy resin, and (iii) Contact the epoxy resin and the biomolecular probe so that the biomolecular probe directly bonds to the epoxy resin. It is prepared by [method].

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

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

[0020] In one embodiment, the epoxy resin portion is polymerized before the biomolecular probe is brought into contact with the epoxy resin.

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

[0022] SU-8 is a photocurable epoxy 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 novolak resin having an average epoxide group functionality of about 8. The structure of the SU-8 epoxy resin is as follows.

[0023]

Chemical formula

[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 polymer of phenol, 4,4'-(1-methylethylidene)bis- 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]

Chemical formula

[0027] Suitable biomolecular probes include, but are not limited to, lipids, polysaccharides, amino acids, polypeptides, oligopeptides, peptides, antibodies and their fragments, polynucleotides (including single-stranded and double-stranded DNA and RNA), shorter oligonucleotides, aptamers, lectins, avidins, streptavidins, 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] While not bound by theory, biomolecular probes are thought to bond to epoxy resins through reactions between the amine, thiol, or hydroxyl groups of the biomolecules and the epoxy groups of the resin. Alternatively, biomolecular probes may bond passively to epoxy resins. As used herein, "passively bonded" refers to bonding via non-covalent interactions such as van der Waals interactions, hydrophobic interactions, hydrophilic interactions, or hydrogen bonding interactions.

[0029] Substrates having a surface containing epoxy resin may include, but are not limited to, a single thin film or a thin film bonded to another solid surface, microbeads, microparticles, micropellets, microwafers, paramagnetic beads, microparticles containing identification features such as barcodes, paramagnetic microparticles, paramagnetic microparticles containing barcodes, and beads containing nickel barcodes.

[0030] By simply bringing the epoxy resin into contact with biomolecules, the biomolecular probe is directly bonded to the epoxy resin.

[0031] In one embodiment, a substrate having a surface containing an epoxy resin is added to a solution of the biomolecular probe to prepare a contact mixture, thereby bringing the biomolecular probe into contact with the epoxy resin. In one embodiment, the solution of the biomolecular probe is an aqueous solution. In one embodiment, the solution of the biomolecular probe is a buffered aqueous solution. In one embodiment, the solution of the biomolecular probe is a dimethyl sulfoxide (DMSO) solution.

[0032] In a preferred embodiment, the substrate having a surface containing an epoxy resin is washed with DMSO before it is added to a solution of biomolecular probes to prepare a contact mixture. In one embodiment, the substrate having a surface containing an epoxy resin is washed with DMSO immediately before it is brought into contact with a solution of biomolecular probes to prepare a contact mixture. Unexpectedly, it was discovered that contacting the epoxy resin with DMSO before it comes into contact with the biomolecular probes results in a substrate for biological analysis with less variability in the number of biomolecular probes that bind to the epoxy resin, and thus less variability in the signal used to detect the presence of analytes in the sample that bind to the biomolecular probes. Unexpectedly, it was found that contacting the epoxy resin with DMSO before it comes into contact with the biomolecular probes results in a substrate for biological analysis that exhibits a better signal.

[0033] This method, by directly bonding the biomolecular probe to the epoxy resin, advantageously avoids the additional step of functionalizing the epoxy resin by (i) reacting the epoxy resin with another molecule before bonding the biomolecular probe to the epoxy resin, or (ii) mixing another molecule into the epoxy resin before polymerization. By avoiding this additional step, this method is advantageously faster, less expensive, and eliminates a step that could potentially be prone to error or variability.

[0034] When a biomolecular probe solution is used to bring the biomolecular probe into contact 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 containing 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 containing 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 another embodiment, the biomolecular probe is an antibody, and the concentration of the substrate having a surface containing 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] A solution of a biomolecular probe is typically brought into contact with a substrate having a surface containing an epoxy resin for a period of time sufficient for the biomolecular probe to bond to the epoxy resin. Typically, the solution of the biomolecular probe is brought into contact with the substrate having a surface containing an epoxy resin for at least about 4 hours, preferably at least about 8 hours, and more preferably at least about 10 hours. In one embodiment, the molecular probe is brought into contact with the substrate having a surface containing an epoxy resin for about 4 to about 18 hours.

[0037] The contact mixture (i.e., a substrate having a surface containing an epoxy resin and a solution of the biomolecular probe) is maintained at a temperature sufficient for the biomolecular probe to bond to the epoxy 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 stirred to ensure that the surface of the substrate having a surface containing an epoxy resin is in sufficient contact with the solution of the biomolecular probe.

[0038] In one embodiment, a substrate having a surface containing an epoxy resin is brought into contact with a solution of a biomolecular probe to bond the biomolecular probe to the epoxy resin. After removing the biomolecular probe solution, the resulting biomolecularly functionalized substrate is washed with a mixture of approximately 1% bovine serum albumin (BSA) (commercially available from Proliant Biologicals, IA, Ankeny), approximately 0.05% Tween-20 (commercially available from Sigma Aldrich, MO, St. Louis), and 0.05% Proclin 950 (commercially available from Sigma Aldrich, MO, St. Louis) in phosphate-buffered saline (PBS) with a pH of approximately 7.4. A suitable PBS solution contains approximately 1.8 mM sodium dihydrogen phosphate (commercially available from Sigma Aldrich, MO, St. Louis), approximately 8.4 mM sodium monohydrogen phosphate (commercially available from Sigma Aldrich, MO, St. Louis), and approximately 145 mM sodium chloride (commercially available from Amresco, OH, Salon). In one embodiment, the biomolecular functionalized substrate is washed at least three times with at least about 200 μL of washing solution. In another embodiment, the biomolecular functionalized substrate is washed at least three times with about 200 μL to about 1,000 μL of washing solution.

[0039] Suitable buffering agents include, but are not limited to, phosphates, TRIS, HEPES, MES, EPPS, Bis-TRIS, Bis-TRIS propane, PIPES, ADA, MOPS, MOPSO, ACES, BES, trichine, TES, Gly-Gly, DIPSO, inorganic buffering agents, organic buffering agents, acetic acid-based and citrate-based buffering agents.

[0040] Next, the obtained washed biomolecular functionalized substrate can be added to a solution of approximately 1% BSA, approximately 0.05% Tween-20, and approximately 0.05% Proclin 950 in PBS at a pH of approximately 7.4 for use in the assay.

[0041] In a first aspect of this method, the biomolecular probe is a protein such as an antibody, an enzyme (e.g., streptavidin and avidin), or a part 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 this 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 this 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 this method, a substrate having a surface containing an epoxy resin is washed with a PBS solution containing about 0.05% Tween-20 before contacting it with a biomolecular probe solution. In one embodiment of the first aspect of this method, a substrate having a surface containing an epoxy resin is washed at least three times with at least about 200 μL of PBS solution containing about 0.05% Tween-20 before contacting it with a biomolecular probe solution. In one embodiment of the first aspect of this method, a substrate having a surface containing an epoxy resin is washed at least three times with about 200 μL to about 1,000 μL of PBS solution containing about 0.05% Tween-20 before contacting it with a biomolecular probe solution.

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

[0044] In one embodiment of the first aspect of this method, a substrate having a surface containing an epoxy resin is brought into contact with a solution of a biomolecular probe to adhere the biomolecular probe to the epoxy resin, the biomolecular probe solution is removed, and the resulting biomolecularly functionalized substrate is washed with a mixture of about 1% BSA, about 0.05% Tween-20, and about 0.05% Proclin 950 in PBS at a pH of about 7.4. In one embodiment, the biomolecularly functionalized substrate is washed at least three times with at least about 200 μL of the washing solution. In another embodiment, the biomolecularly functionalized substrate is washed at least three times with about 200 μL to about 1,000 μL of the washing solution.

[0045] Next, the obtained washed biomolecular functionalized substrate can be added to a solution of approximately 1% BSA, approximately 0.05% Tween-20, and approximately 0.05% Proclin 950 in PBS at a pH of approximately 7.4 for use in the assay.

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

[0047] In a second aspect of this 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 this method, the solution is DMSO containing about 1% Tween-20. In one embodiment of the second aspect of this 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 this method, a substrate having a surface containing an epoxy resin is washed with a DMSO solution containing about 1% Tween-20 before contacting it with a biomolecular probe solution. In one embodiment of the second aspect of this method, a substrate having a surface containing an epoxy resin is washed at least three times with at least about 200 μL of DMSO solution containing about 1% Tween-20 before contacting it with a biomolecular probe solution. In one embodiment of the second aspect of this method, a substrate having a surface containing an epoxy resin is washed at least three times with about 200 μL to about 1,000 μL of DMSO solution containing about 1% Tween-20 before contacting it with a biomolecular probe solution.

[0049] In one embodiment of the second aspect of this method, a substrate having a surface containing an epoxy resin is brought into contact with a solution of a biomolecular probe to bond the biomolecular probe to the epoxy resin, the bioprobe solution is removed, and the resulting biomolecularly functionalized substrate is washed with a mixture of about 1% BSA, about 0.05% Tween-20, and about 0.05% Proclin 950 in PBS at a pH of about 7.4. In one embodiment, the biomolecularly functionalized substrate is washed at least three times with at least about 200 μL of the washing solution. In another embodiment, the biomolecularly functionalized substrate is washed at least three times with about 200 μL to about 1,000 μL of the washing solution.

[0050] Next, the obtained washed biomolecular functionalized substrate can be added to a solution of approximately 1% BSA, approximately 0.05% Tween-20, and approximately 0.05% Proclin 950 in PBS at a pH of approximately 7.4 for use in the assay.

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

[0052] Although not bound by theory, a second aspect of this method involves reacting a thiol group of a biomolecular probe with an epoxide group of an epoxy resin. This cysteine ​​residue may be located at any position in the peptide sequence. The cysteine ​​may be separated from the peptide by a linker such as a PEG linker. The PEG can be of a specified length by using discrete PEG, for example. 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: pp. 1543-1559, 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 capped at its N-terminus and C-terminus by N-terminal acetylation (Ac) or C-terminal amidation. If the biomolecular probe is an antibody, the antibody can be conjugated to the epoxy resin via thiol groups by reducing the antibody using a reducing reagent such as dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or β-mercaptoethanol (BME) to make the thiol groups in the hinge region of the antibody accessible for binding to the epoxy resin.

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

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

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

[0057] Examples of antibodies that specifically bind to fecal antigens derived 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 derived 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 derived 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 derived from tapeworms are disclosed in U.S. Patent Application Publication No. 2020 / 0102378. Examples of antibodies that specifically bind to fecal antigens derived 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. 2340-2346, October 1990.

[0058] In one embodiment, the sample is a blood sample from the 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 that antibody. Proteins, polypeptides, or oligopeptides capable of specifically binding to circulating antibodies in animals infected with bacteria of the genus Ehrlichia, including Ehrlichia canis, Ehrlichia chaffeensis, and Ehrlichia ewingii, are disclosed in U.S. Patent 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 genus Anaplasma, 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 genus Borrelia, 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 the target's response to a protein produced by Borrelia burgdorferi, which causes Lyme disease, and the biomolecular probe is the protein or a portion of the 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). An antibody that specifically binds to SDMA is disclosed in U.S. Patent No. 8,481,690.

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

[0063] In one embodiment, the substrate for the biological assay is (i) Prepare a silicon / aluminum wafer. (ii) Prepare a silicon / aluminum wafer coated with a first epoxy resin coating by coating a silicon / aluminum wafer with a first epoxy resin coating by coating a silicon / aluminum wafer with a first epoxy resin coating, (iii) Heat the silicon / aluminum wafer coated with the first epoxy resin coating to remove at least a portion of the first solvent, thereby preparing a silicon / aluminum wafer coated with the first solid epoxy resin coating. (iv) Depositing a nickel barcode onto the first solid epoxy resin coating to prepare a silicon / aluminum wafer having the first solid epoxy resin coating and the nickel barcode. (v) Coat the nickel barcode with a second solution of the second epoxy resin dissolved in the second solvent to prepare a silicon / aluminum wafer coated with the first epoxy resin coating and the second epoxy resin coating. (vi) Heating a silicon / aluminum wafer having a first solid epoxy resin coating and a second epoxy resin coating to remove at least a portion of the second solvent, thereby preparing a silicon / aluminum wafer coated with the first solid epoxy resin coating and the second solid epoxy resin coating. The nickel barcode is located between the first solid epoxy resin coating and the second solid epoxy resin coating, to be prepared. (vii) Optionally, polymerize at least a portion of the first solid epoxy resin coating and the second solid epoxy resin coating to prepare a silicon / aluminum wafer layered with the first solid epoxy resin, nickel barcode, and second solid epoxy resin coating, wherein at least a portion of the first solid epoxy resin coating and the second solid epoxy resin coating are polymerized. (viii) Separating the silicon / aluminum wafer coated with the first epoxy resin coating, nickel barcode, and second epoxy resin coating to prepare barcoded magnetic beads. (iv) Contact the biomolecules with barcoded magnetic beads so that they directly bond to the epoxy resin. It is prepared by a method that includes [a specific method].

[0064] In one embodiment, the first solution of the first epoxy resin dissolved in the first solvent is the same as the second solution of the second epoxy resin dissolved 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 also relates to a method for assaying the presence of an analyte in a sample. This method involves contacting a sample with a substrate having a surface containing an epoxy resin, the substrate having a biomolecular probe directly bonded to the epoxy resin, so that the biomolecular probe specifically binds to the analyte.

[0067] The terms “specifically binds to the analyte,” “specifically binds,” “has specificity,” and “specific to the analyte,” and similar terms, as used herein, have the meaning recognized in the art, namely that a biomolecular probe recognizes and binds to the analyte (or class of analytes) with a higher affinity than other nonspecific molecules. For example, an antibody that arises against an antigen and binds to that antigen more efficiently than other nonspecific molecules can be described as specifically binding to that antigen. Binding specificity can be tested using methods known in the art, such as 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 the protein produced by the intestinal parasite. In one embodiment, the antibody against the protein produced by the intestinal parasite is selected from the group consisting of an antibody that specifically binds to fecal antigens derived from roundworms, an antibody that specifically binds to fecal antigens derived from whipworms, an antibody that specifically binds to fecal antigens derived from hookworms, an antibody that specifically binds to fecal antigens derived from tapeworms, an antibody that specifically binds to antigens derived from heartworms, and an antibody that specifically binds to fecal antigens derived from Giardia.

[0069] In one embodiment, the sample is a blood sample from the 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 that antibody.

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

[0071] In one embodiment, the sample is a blood sample from a 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 the infected subject, and the biomolecular probe is an antibody against that antigen. In another embodiment, the analyte is an antibody that specifically binds to a circulating antigen derived from Dirofilaria immitis. [Examples]

[0073] The present invention is not limited in scope by the specific embodiments disclosed in the examples intended to illustrate certain aspects of the invention, and any functionally equivalent embodiments are within the scope of the invention. Indeed, it will be apparent to those skilled in the art that there are various modifications to the invention beyond those illustrated and described herein, and such modifications are intended to be included within the scope of the appended claims. Such variations of the invention, including the substitution of all currently known or subsequently developed equivalents that are considered to be within the scope of the invention, as well as minor changes in formulation or experimental design, should be considered to fall within the scope of the invention as incorporated herein. [Examples]

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

[0075] Antibody-coated solutions were obtained by preparing a solution of monoclonal antibody with a final concentration of approximately 0.15–2.5 mg / mL (typically about 1.5 mg / mL) in an aqueous buffer with a pH of approximately 5.5 containing approximately 100 mM MES (commercially available from Sigma Aldrich, St. Louis, MO) and approximately 140 mM guanidine-HCl (commercially available from Sigma Aldrich, St. Louis, MO), or in an aqueous buffer with a pH of approximately 8 containing approximately 100 mM EPPS (commercially available from Sigma Aldrich, St. Louis, MO) and approximately 140 mM guanidine-HCl.

[0076] A sufficient amount of BMB was suspended in BMB washing buffer (approximately 1.8 mM sodium dihydrogen phosphate (commercially available from Sigma Aldrich, MO, St. Louis), approximately 8.4 mM sodium monohydrogen phosphate (commercially available from Sigma Aldrich, MO, St. Louis), approximately 145 mM sodium chloride (commercially available from Amresco LLC, OH, Salon), and approximately 0.05% Tween-20 (commercially available from Sigma Aldrich, MO, St. Louis), approximately pH 7.4) to obtain a final concentration of approximately 100,000 to 1,800,000 BMB / mL (typically about 1,000,000 BMB / mL for antibodies). The BMB was washed three times with BMB washing buffer. All BMB washings (in this step and subsequent steps) were carried out as follows:

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

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

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

[0080] Next, the antibody-coupled BMB is suspended in the assay buffer at the desired final concentration for use in the assay. [Examples]

[0081] Coupling of biomolecular probes and barcoded magnetic beads via thiol groups The coupling of a biomolecular probe, such as a monoclonal antibody or protein containing a thiol group, with barcoded magnetic beads (BMBs) coated with epoxy resin is achieved by absorbing the biomolecular probe onto the BMB surface according to the following procedure.

[0082] A peptide coating solution was obtained by preparing a peptide solution with a final peptide concentration of approximately 0.02 to approximately 1 mM (typically approximately 0.1 mM) in DMSO (commercially available from Sigma Aldrich, St. Louis, MO) containing approximately 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 discrete PEG12. The acetylated-Cys(dPEG12)[peptide]amide was supplied as a custom-component compound from New England Peptide, Gardner, MA.

[0083] The BMB suitable for use in this method is BMB manufactured from SU-8 (CA, commercially available from Applied BioCode Corp. in Santa Fe Springs), an epoxy-based negative photoresist.

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

[0085] First, the tube containing the BMB was placed on a magnetic stand, allowing the BMB to adhere to the magnet for 1 to 10 minutes. The supernatant was carefully aspirated and removed, and then the BMB was resuspended in a volume of washing 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] Immediately after washing the BMB with washing buffer, the washed BMB was combined with the peptide coating solution and incubated at room temperature (18-27°C) for approximately 4 hours while mixing. After 4 hours of incubation, the peptide-coupled BMB was washed three times with a fixed volume of assay buffer (approximately 1.8 mM sodium dihydrogen phosphate, approximately 8.4 mM sodium monohydrogen phosphate, and approximately 1% BSA, approximately 0.05% Tween-20, and approximately 0.05% Proclin 950 in approximately 145 mM sodium chloride, approximately pH 7.4).

[0087] Next, the antibody-coupled BMB is suspended in the assay buffer at the desired final concentration for use in the assay. [Examples]

[0088] Coupling of biomolecular probes and barcoded magnetic beads via thiol groups generated from reduced disulfide-linked cysteine Macromolecules such as monoclonal antibodies can be coupled via 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 chain so that it can approach the surface of BMB.

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

[0090] After reduction, the reducing antibody was replaced with a solution containing 50 mM sodium phosphate (MO, commercially available from Sigma Aldrich, St. Louis), 75 mM sodium chloride (OH, commercially available from Amresco, Salon), and 2 mM EDTA (MO, commercially available from Sigma Aldrich, St. Louis), at a pH of approximately 7.4, using a G25 Zeba spin desalination column (MA, commercially available from Thermo Fisher Scientific, Waltham) 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 BMB washed with DMSO.

[0091] BMB washed with DMSO was first prepared by suspending a sufficient amount of BMB to provide a final concentration of approximately 100,000–1,800,000 BMB / mL (typically about 1,000,000 BMB / mL for antibodies) in approximately 200 μL–1,000 μL of washing buffer (approximately 1.8 mM sodium dihydrogen phosphate, approximately 8.4 mM sodium monohydrogen phosphate, approximately 145 nM sodium, and approximately 0.05% Tween-20, approximately pH 7.4) for the coating reaction described below. The BMB was washed three times with the washing buffer as described below.

[0092] All BMB washing (this step and subsequent steps) was carried out as follows: First, the tube containing the BMB was placed on a magnetic stand, allowing the BMB to adhere to the magnet for 1 to 10 minutes. Then, the supernatant was carefully aspirated and removed, and the BMB was resuspended in a washing buffer of approximately the same volume as the original volume used to suspend the BMB. These steps were repeated three times to obtain a BMB pellet.

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

[0094] The BMB suitable for use in this method is BMB manufactured from SU-8 (CA, commercially available from Applied BioCode Corp. in Santa Fe Springs), an epoxy-based negative photoresist.

[0095] After washing with DMSO, the antibody coating solution (final concentration approximately 0.5 mg / mL) was immediately combined with BMB (final concentration approximately 1 million BMB / mL) that had been washed with DMSO, and incubated at room temperature (18-27°C) for approximately 18 hours while mixing.

[0096] After incubation was complete, the tubes containing BMB were placed on a magnetic stand to allow the BMB to adhere to the magnet for 1–10 minutes. The supernatant was then carefully aspirated and the BMB was resuspended in a volume of washing 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-, approximately pH 7.4) approximately equal to the original suspension volume, and the resulting solution was incubated at 18–27°C for approximately 15 minutes.

[0097] After incubation was complete, the tube containing the BMB was placed on a magnetic stand to allow the BMB to adhere to the magnet for 1–10 minutes. The supernatant was then carefully aspirated and the BMB was resuspended in an assay buffer of approximately equal volume to the original suspension volume. 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 nM sodium chloride at pH 7.4. The resulting suspension was incubated at 18–27°C for approximately 30 minutes with mixing. The antibody-coupled BMB was then washed three times with an assay buffer of approximately equal volume to the original suspension volume.

[0098] Next, the antibody-coupled BMB was suspended in the assay buffer at the final desired concentration for use in the assay. [Examples]

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

[0100] [Table 1]

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

[0102] The BMB suitable for use in this method is BMB prepared from SU-8 (CA, commercially available from Applied BioCode Corp. in Santa Fe Springs), an epoxy-based negative photoresist. 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 centrifugation tube using a pipette, and then approximately 0.5 mL of DMSO (MO, commercially available from Sigma Aldrich in St. Louis) was added to each tube. The tubes were vortexed vigorously for 10 seconds, returned to the magnetic rack, allowed to stand for 1 minute, and the DMSO was removed with a pipette. This washing procedure was repeated two more times. Then, approximately 0.5 mL of DMSO was added to each tube, the tubes were vortexed vigorously, and the tubes were left on a mixer at room temperature for 4 hours.

[0104] Coating with rhodamine-lysamine: After mixing for 4 hours, the tubes were removed and placed on a magnetic stand. After standing for 1 minute, the DMSO was removed by pipette, and approximately 0.5 mL of approximately 150 mM EPPS buffer, approximately pH 9.0, was added to the tubes. The tubes containing BMB in the EPPS buffer were then vigorously vortexed for 10 seconds, returned to the magnetic stand, and the EPPS buffer was removed by pipette. This washing procedure was repeated two more times. After washing was complete, approximately 0.5 mL of EPPS buffer was added to each tube, and the tubes were vortexed. Approximately 10.0 μM rhodamine-lysamine (9 μL of 0.3 mg / mL solution in DMSO) or sulfo-rhodamine (control, 1.2 μL of 2.8 mg / mL solution in DMSO) was then added to the epoxy-based BMB in the EPPS buffer. The BMB suspension was rotated upside down at room temperature for approximately 22 hours to protect it from light. After this time, the coated BMB was placed on a magnetic stand, and the solvent was removed by pipette. The coated BMB was briefly vortexed and washed with approximately 1.0 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, followed by removal of the solvent. This process was repeated five times. Finally, approximately 1.0 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 was added to the coated BMB to obtain a final concentration of approximately 50,000 BMB / mL. Then, approximately 5.0 μL of the resulting suspension of BMB was added to the wells of a 96-well plate to obtain a final count of approximately 250 BMB / well. Approximately 200 μL of solutions containing 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 were added to each well to obtain suspensions of BMB for use in the assay.

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

[0106] Epoxy BMB coated with rhodamine-lysamine showed significant fluorescence in a microplate reader, indicating efficient coating of the rhodamine probe onto the epoxy BMB surface. Epoxy BMB incubated with a sulfo-rhodamine control lacking reactive amine functional groups showed no observable fluorescence. This demonstrates very low nonspecific binding of the rhodamine probe to the epoxy BMB surface, indicating that rhodamine-lysamine specifically reacts with the epoxy surface via primary amine functional groups. It can be concluded that amine-functionalized small molecules can be covalently bonded to the epoxy BMB surface. [Examples]

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

[0108] [Table 2]

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

[0110] The BMB suitable for use in this method is BMB prepared from SU-8 (CA, commercially available from Applied BioCode Corp. in Santa Fe Springs), an epoxy-based negative photoresist. 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 centrifugation tube using a pipette, and then approximately 0.2 mL of DMSO (MO, commercially available from Sigma Aldrich in St. Louis) was added to each tube. The tubes were vortexed vigorously for 10 seconds, returned to the magnetic rack, allowed to stand for 1 minute, and the DMSO was removed with a pipette. This washing procedure was repeated two more times. Then, approximately 0.2 mL of DMSO was added to each tube, the tubes were vortexed vigorously, and the tubes were left on a mixer at room temperature for 4 hours.

[0112] After mixing for 4 hours, the tubes were removed and placed on a magnetic stand. After standing for 1 minute, the DMSO was removed with a pipette, and approximately 0.2 mL of approximately 150 mM EPPS buffer, approximately pH 9.0, was added to the tubes. The tubes containing BMB were then vortexed vigorously in the EPPS buffer 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.

[0113] Approximately 0.1 mM solutions of both biotin-lime peptide and lime-Alexafluor 555 were prepared from 1.0 mM stock solutions. Each peptide contained multiple lysine residues but no cysteine ​​residues or other thiol groups. Approximately 200 μL of biotin-lime peptide solution, lime-Alexafluor 555 solution, or a control containing only EPPS buffer was added to a 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 by pipette. The BMB was then washed by short vortexing with approximately 0.2 mL 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 solution, followed by removal of the solvent. This process was repeated twice.

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

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

[0116] Both BMBs coated with biotin-lime peptide and lime-Alexafluor555 peptide exhibited significant fluorescence in a microplate reader, indicating efficient coating of the peptides onto the BMB surface. The lack of fluorescence observed in the control demonstrated very low nonspecific binding of the SA-PE analyte to the BMB surface, suggesting that the biotin-lime peptide and lime-Alexafluor555 peptide react specifically with the epoxy surface via the primary amine functional group contained in the lysine residue of the peptide. It can be concluded that lysine (amine functional group)-containing peptides can be covalently bound to the epoxy BMB surface. [Examples]

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

[0118] The BMB suitable for use in this method is BMB prepared from SU-8 (CA, commercially available from Applied BioCode Corp. in Santa Fe Springs), an epoxy-based negative photoresist. 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 centrifugation tube using a pipette, and then approximately 0.2 mL of DMSO (MO, commercially available from Sigma Aldrich in St. Louis) was added to each tube. The tubes were vortexed vigorously for 10 seconds, returned to the magnetic rack, allowed to stand for 1 minute, and the DMSO was removed with a pipette. This washing procedure was repeated two more times. In one embodiment, approximately 0.2 mL of DMSO was then added to each tube, the tubes were vortexed vigorously, and the mixture was left on a mixer at room temperature for 4 hours. This 4-hour mixing at room temperature is optional.

[0120] After mixing, the tubes were removed and placed on a magnetic stand, allowed to stand for 1 minute, the DMSO was removed with a pipette, and approximately 0.2 mL of approximately 150 mM EPPS buffer, approximately pH 9.0, was added to the tubes. The tubes containing the BMBs were then vortexed vigorously in the EPPS buffer 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 final wash with EPPS, the BMBs were coated with peptides or antibodies as described in parts (A) and (B) below, respectively, and each peptide and antibody was paired with BMBs of a different barcode.

[0121] (A) Coating of BMB with peptides Peptides (biochemical probes) specifically capable of binding to patient antibodies against Anaplasma, Ehrlichia, or Borrelia species (e.g., peptides derived from the protein sequences of Anaplasma phagocytophylum, Anaplasma platys, Ehrlichia canis, Ehrlichia ewingii, or Borrelia burgdorferi, as described above) were synthesized by linking cysteine ​​to the N-terminus of each peptide via a PEG12 linker, as described above, with the exception of the Borrelia-derived peptide which has an N-terminal cysteine ​​but lacks a PEG linker. Approximately 200 μL of approximately 0.1 mM solution of each peptide was added to a tube containing EPPS-washed 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 the BMB was placed in a magnetic rack and the solvent was removed by pipette. Next, the BMB was briefly vortexed 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, followed by removal of the solvent. This process was repeated twice.

[0122] (B) Coating of BMB with antibody Approximately 200 μL of an antibody (biochemical probe) at a concentration of approximately 10 mg / mL, specifically capable of binding to antigens derived from Dirofilaria immitis circulating in the blood of infected animals (the antibody concentration may range from approximately 1.5 mg / mL to approximately 12.0 mg / mL), was added to a tube containing BMB as described above. 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 by pipette. The BMB was then washed by short vortexing 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, followed by removal of the solvent. This process was repeated twice.

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

[0124] Approximately 100 μL of this diluted multiplexed BMB mixture was added to each well of a 96-well plate using an Integra automated pipette (commercially available from Integra Biosciences Corp., NH, Hudson) (i.e., approximately 50 beads per biomolecular probe). The BMB was immersed in 0.05% Tween-20 in 300 μL of 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 in the plate.

[0125] 50 μL of sample (serum or plasma, neat) was added to 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 incubation for 30 minutes, the BMB was washed by immersion in 300 μL of PBS with 0.05% Tween-20 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-filariasis antibody (1.0 μg / mL) in 1.0% BSA, 0.05% Tween, and 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 minutes. After incubation for 15 minutes, BMB was washed by immersion in 300 μL of 0.05% Tween-20 in PBS for 10 seconds.

[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 incubation for 10 minutes, the BMB was washed by immersion in 300 μL of PBS with 0.05% Tween-20 for 10 seconds.

[0128] Next, the obtained BMB was processed using one of two procedures.

[0129] In the first step, in each well of the 96-well plate, (i) Approximately 200 μL of buffer solution (commercially available from Applied BioCode Inc., Santa Fe Springs, CA. Catalog number 44-D0004-500) (standard reading buffer solution) Added.

[0130] In the second step, in each well of the 96-well plate, (ii) Approximately 200 μL of citrate buffer solution 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), with a pH of 6.1-6.3 (citrate reading buffer). Added.

[0131] Ionic strength is a crucial factor enabling the stability of the immune complexes formed on the surface of the beads. While not theoretically bound, the stabilizing effect is thought to be due to the high salt concentration that makes the solution unfavorable for dissociation. Other salts with high ionic strength besides citrate are also effective, but a minimum concentration of approximately 0.5 M is required. However, other salts are not ideal for manufacturing or transport because they precipitate from the solution at temperatures below ambient temperature. Advantageously, citrate buffer mixtures remain dissolved in the solution even when stored or transported 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, CA).

[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 intensities observed when using standard reading buffer (Figure 1A) and when using citrate reading buffer (Figure 1B). In the experiment, each well of the 96-well plate (i.e., a plate with 12 columns (1-12) and 8 rows (A-H)) was filled with the same BMB (i.e., all bound to beads as described above), and the signals were 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 in the 96-well plate (i.e., cells 1A-12H) is approximately 37 minutes. As can be seen from Figure 1A, when using a standard reading buffer (a citrate-free buffer), i.e., procedure (i), the signal intensity gradually decreases from the start to the end of the reading cycle (approximately 37 minutes). In contrast, as can be seen from Figure 1B, when using a citrate reading buffer, i.e., procedure (ii), no decrease in signal intensity was observed from the start to the end of the reading cycle (approximately 37 minutes). This decrease in signal intensity occurred during the reading cycle when using a citrate-free buffer, such as a standard reading buffer, but not when using a citrate-containing buffer, such as a citrate reading buffer, in assays of antibodies specific to each of the three Anaplasma peptides labeled "AP," "Aph," and "Apl" on the X axis in Figures 1A and 1B.

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

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

[0136] All cited references are incorporated herein by reference in their entirety.

Claims

1. A method for preparing substrates for biological analysis, (i) Prepare a substrate having a surface containing epoxy resin, and (ii) Bringing the biomolecular probe into contact with a substrate having a surface containing the epoxy resin, such that the biomolecular probe directly bonds to the epoxy resin. Including; A method comprising washing a substrate having a surface containing an epoxy resin with dimethyl sulfoxide (DMSO) before the biomolecular probe is brought into contact with the substrate having a surface containing an epoxy resin.

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

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

4. The method according to claim 1, wherein the substrate having a surface containing the epoxy resin is selected from the group consisting of thin films, microbeads, microparticles, micropellets, microwafers, paramagnetic beads, microparticles containing barcodes, paramagnetic microparticles, microparticles containing barcodes, paramagnetic microparticles containing barcodes, and beads containing nickel barcodes.

5. The method according to claim 1, wherein the biomolecular probe is brought into contact with the epoxy resin by bringing a substrate having a surface containing the epoxy resin into contact with a solution of the biomolecular probe to prepare a contact mixture.

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

7. The method according to claim 6, wherein the aqueous solution is buffered to pH 5.5 or pH 8 with 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 140 mM guanidine-HCl.

8. The method according to claim 5, wherein the solution is a DMSO solution which may contain 1% Tween-20.

9. The method according to claim 5, wherein the concentration of the biomolecular probe in the solution is in the range of 0.05 mg / mL to 5 mg / mL.

10. The method according to claim 5, wherein the concentration of the substrate having a surface containing the epoxy resin in the contact mixture is in the range of 50,000 to 5,000,000 substrates / mL.

11. The method according to claim 1, wherein the biomolecular probe is brought into contact with a substrate having a surface containing the epoxy resin for at least 4 hours, or 4 to 18 hours.

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

13. The method according to claim 1, wherein the substrate having a surface containing the epoxy resin is washed with DMSO containing 1% Tween-20 before being brought into contact with the biomolecular probe.

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

15. A substrate for biological analysis, comprising a substrate having a surface containing an epoxy resin having biomolecular probes directly bonded to the epoxy resin.

16. A method for preparing substrates for biological analysis, (i) Prepare a solution of the biomolecular probe in a solvent selected from the group consisting of (a) an aqueous solution buffered to pH 5.5 with 100 mM MES and 140 mM guanidine-HCl, and (b) an aqueous solution buffered to pH 8 with 100 mM EPPS and 140 mM guanidine-HCl. The concentration of the biomolecular probe is in the range of 0.05 mg / mL to 5 mg / mL. (ii) Prepare a substrate having a surface containing epoxy resin. (iii) Wash the substrate having a surface containing the epoxy resin with a phosphate-buffered saline (PBS) solution containing 0.05% Tween-20 to prepare a substrate having a surface containing epoxy resin that has been washed with PBS. (iv) Wash the substrate that has been washed with PBS having a surface containing the epoxy resin with DMSO to prepare a substrate that has been washed with DMSO having a surface containing the epoxy resin. (v) The substrate having a surface containing the epoxy resin, which has been washed with DMSO, is combined with the solution of the biomolecular probe, wherein the substrate having a surface containing the epoxy resin is prepared such that the biomolecular probe is directly bound to the epoxy resin, and the concentration of the substrate having a surface containing the epoxy resin, which has been washed with DMSO, is in the range of 50,000 to 5,000,000 substrates / mL, and the mixture is combined with the solution of the biomolecular probe, (vi) Washing a substrate having a surface containing the epoxy resin, wherein the biomolecular probe is directly bonded to the epoxy resin containing PBS with a pH of 7.4 containing 1% BSA, 0.05% Tween-20, and 0.05% Proclin 950. A method that includes this.

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

18. A method for preparing substrates for biological analysis, (i) Prepare a solution of the biomolecular probe with DMSO containing 1% Tween-20. The concentration of the biomolecular probe is in the range of 0.05 mg / mL to 5 mg / mL. (ii) Prepare a substrate having a surface containing epoxy resin. (iii) Wash the substrate having a surface containing the epoxy resin with DMSO containing 1% Tween-20 to prepare a substrate having a surface containing epoxy resin that has been washed with DMSO. (iv) The substrate having a surface containing the epoxy resin, which has been washed with DMSO, is combined with the solution of the biomolecular probe, wherein the biomolecular probe is directly bound to the epoxy resin, the substrate having a surface containing the epoxy resin is prepared, and the concentration of the substrate having a surface containing the epoxy resin, which has been washed with DMSO, is in the range of 50,000 to 5,000,000 substrates / mL, and (v) Washing a substrate having a surface containing the epoxy resin, wherein the biomolecular probe is directly bonded to the epoxy resin containing PBS with a pH of 7.4 containing 1% BSA, 0.05% Tween-20, and 0.05% Proclin 950. A method that includes this.

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

20. The method according to claim 3, wherein the antibody is selected from the group consisting of an antibody that specifically binds to fecal antigens derived from roundworms, an antibody that specifically binds to fecal antigens derived from whipworms, an antibody that specifically binds to fecal antigens derived from hookworms, an antibody that specifically binds to fecal antigens derived from tapeworms, an antibody that specifically binds to antigens derived from heartworms, and an antibody that specifically binds to fecal antigens derived from Giardia.

21. The method according to claim 17, wherein the antibody is selected from the group consisting of an antibody that specifically binds to fecal antigens derived from roundworms, an antibody that specifically binds to fecal antigens derived from whipworms, an antibody that specifically binds to fecal antigens derived from hookworms, an antibody that specifically binds to fecal antigens derived from tapeworms, an antibody that specifically binds to antigens derived from heartworms, and an antibody that specifically binds to fecal antigens derived from Giardia.

22. The method according to claim 2, wherein the biomolecular probe is a protein expressed by an infectious pathogen, a part of the protein expressed by the infectious pathogen, a peptide or recombinant protein derived from the protein expressed by the infectious pathogen, or a variant of the protein expressed by the infectious pathogen.

23. The method according to claim 22, wherein the biomolecular probe can specifically bind to antibodies produced by a target against bacteria of a genus selected from the group consisting of the genus Ehrlicia (which may be Ehrlicia canis, Ehrlicia chaffeensis, or Ehrlicia ewingii), the genus Anaplasma (which may be Anaplasma phagocytophyllum, or Anaplasma platys), and the genus Borrelia (which may be Borrelia burgdorferi).

24. The method according to claim 22, wherein the biomolecular probe can specifically bind to an antibody produced by a target against Dirofilaria immitis.

25. The method according to claim 3, wherein the biomolecular probe is an antibody that can specifically bind to the antigen of Dirofilaria immitis.

26. The method according to claim 22, wherein the biomolecular probe can specifically bind to an antibody against a metabolite that may be symmetric dimethylarginine (SDMA).

27. A method for assaying the presence of an analyte in a sample, comprising contacting the sample with a substrate having a surface containing an epoxy resin, the substrate having a biomolecular probe directly bound to the epoxy resin, wherein the biomolecular probe specifically binds to the analyte, and the contact is performed accordingly. The method for producing the substrate comprises a step of washing the substrate having a surface containing an epoxy resin with dimethyl sulfoxide (DMSO) before the biomolecular probe is brought into contact with the substrate having a surface containing an epoxy resin.

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

29. The method according to 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. The method according to 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 derived from roundworm, an antibody that specifically binds to a fecal antigen derived from whipworm, an antibody that specifically binds to a fecal antigen derived from hookworm, an antibody that specifically binds to a fecal antigen derived from tapeworm, an antibody that specifically binds to an antigen derived from heartworm, and an antibody that specifically binds to a fecal antigen derived from Giardia.

31. The method according to claim 27, wherein the sample is a blood sample from the subject.

32. The method according to claim 27, wherein the analyte is an antibody produced by the target immune response to a protein produced by an infectious pathogen, and the biomolecular probe is the protein produced by the infectious pathogen, a part of the protein, or a variant of the protein produced by the infectious pathogen.

33. The method according to claim 32, wherein the biomolecular probe can specifically bind to antibodies produced by the target against bacteria from a group selected from the genus Ehrlicia, which may be Ehrlicia canis, Ehrlicia chaffeensis, or Ehrlicia ewingii; the genus Anaplasma, which may be Anaplasma phagocytophyllum, or Anaplasma platys; and the genus Borrelia, which may be Borrelia burgdorferi.

34. The method according to claim 32, wherein the biomolecular probe can specifically bind to the antibody produced by the target against Dirofilaria immitis.

35. The method according to claim 32, wherein the biomolecular probe can specifically bind to an antibody against a metabolite that may be symmetric dimethylarginine (SDMA).

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

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

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