CN116981944A

CN116981944A - Biochemical probe connected to epoxy resin

Info

Publication number: CN116981944A
Application number: CN202280018714.8A
Authority: CN
Inventors: A·B·什韦茨; D·L·拉姆斯德尔; M·M·斯特劳布; Y·V·S·N·穆尔蒂; P·S·史密斯; A·C·菲茨帕特里克
Original assignee: Idexx Laboratories Inc
Current assignee: Idexx Laboratories Inc
Priority date: 2021-03-02
Filing date: 2022-02-28
Publication date: 2023-10-31

Abstract

The present application relates to a method for preparing a solid substrate for performing biological and chemical assays and to a solid substrate prepared by the method.

Description

Biochemical probe connected to epoxy resin

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional application No. 63/288,018, filed on 1 month 17 of 2021, and U.S. provisional application No. 63/155,472, filed on 3 months 4 of 2021, both of which are incorporated herein in their entirety.

Statement regarding federally sponsored research or development

Is not suitable for

Incorporation of materials submitted on optical discs

Is not suitable for

Technical Field

The present application relates to a method for preparing a solid substrate for performing biological and chemical assays and to a solid substrate prepared by the method. In particular, solid substrates for multiplex bioassays.

Background

Arrays for biological and chemical analysis can be created by attaching probe molecules to a solid substrate having a surface comprising a resin material (e.g., a functionalized epoxy). The array allows rapid screening of large numbers of biomolecules, such as nucleic acids and proteins, in very small sample volumes. For example, particles called microspheres or microbeads (called barcoded microbeads) with identifiable tags and/or labels have been used in parallel multiplex assays to identify disease-related targets, toxin-related targets, gene-related targets, etc. The microbeads have a resin coating on their surface that is conjugated with one or more probe molecules that have affinity for and/or have the ability to interact with one or more specific target molecules. Each probe molecule is attached to a separate bead, which is encoded so as to be uniquely identifiable. In an assay, microbeads are contacted with a sample to bind different target molecules in the sample to microbeads to which the corresponding probe molecules are conjugated. The bar code enables identification of the target.

Microbead assay is now an important tool for biological product assays and diagnostics. Microbead-based technology represents a subtle and versatile method for performing highly parallel quantitative multiparameter assays. They form the basis of various techniques for detecting and quantifying nucleic acids and proteins in a sample.

Epoxy-based resins have been used as coating materials for attachment to probe molecules. However, attaching probe molecules to the surface of an epoxy-based resin requires an additional step of first functionalizing the resin surface. U.S. patent No. 9,255,922 discloses a substrate, such as a microbead or microsphere, coated with an epoxy-based resin attached to probe molecules. This patent teaches that epoxy resins are hydrophobic, which represents a limitation for many biological applications, and therefore, the epoxy resin must be modified with additional functional monomers before the probe molecule can be effectively attached to the resin. Thus, after (or simultaneously with) the formation of the epoxy, the epoxy needs to be contacted with additional functional monomers to functionalize the epoxy, thereby enabling the biomolecular probes to effectively attach to the surface of the epoxy. This additional step of contacting the epoxy resin with the functional monomer is laborious and time consuming.

There is a need in the art for simplified methods to prepare substrates having surfaces comprising epoxy-based resins that can be effectively attached to biomolecular probes. The inventors have surprisingly found that biomolecular probes can be effectively attached to a substrate coated with an epoxy resin without first functionalizing the epoxy resin with additional functional monomers.

These and other features and advantages of the present application will become apparent from the following detailed description of the preferred embodiments, which, by way of example, illustrates the principles of the application.

Citation of any reference in this application shall not be construed as an admission that such reference is prior art to the present application.

Disclosure of Invention

The present application relates to a substrate for bioanalytical analysis and a method of preparing a substrate for bioanalytical analysis. Substrates for bioassays include epoxy-based resins with biomolecular probes directly bonded to polymeric resins.

The substrate for bioassay was prepared by:

(i) Providing a substrate having a surface comprising an epoxy-based resin, and

(ii) The biomolecular probe is contacted with the epoxy resin such that the biomolecular probe is directly bound to the epoxy resin.

The invention also relates to a method for determining the presence of an analyte in a sample. The method comprises the following steps: contacting the sample with a substrate having a surface comprising an epoxy resin having biomolecular probes directly bound to the epoxy resin, wherein the biomolecular probes specifically bind to the analyte.

Drawings

FIG. 1 is a graph of signal intensity as fluorescence intensity on BMB measured as a single median result (MFI) for all beads in a well versus cell column position (i.e., 1-12) for a 96-well plate for the determination of antibodies specific for three Anaplasma-derived peptides designated "AP", "Aph" and "Apl" as designated on the X-axis of FIGS. 1A and 1B, as described in example 6. Fig. 1A depicts the signal intensity versus cell column position for a standard read buffer and fig. 1B depicts the signal intensity versus cell column position for a citrate read buffer, as described in example 6.

Detailed Description

The present invention relates to a substrate for bioanalytical analysis and a method of preparing a substrate for bioanalytical analysis. Substrates for bioassays include epoxy resins having biomolecular probes directly bonded to the epoxy resin.

As used herein, the phrase "biomolecular probe directly bound to an epoxy resin" and similar phrases mean that the biomolecular probe is attached to the resin by simply contacting the biomolecular probe with the resin without first contacting the epoxy resin with another agent that is covalently reactive with the epoxy resin. The biomolecular probe may be passively attached to the epoxy, or covalently attached to the epoxy.

The substrate for bioassay was prepared by:

(i) Providing a substrate having a surface comprising an epoxy-based resin, and

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

In one embodiment, the substrate is a solid support coated with an epoxy-based resin. Illustrative epoxy-coatable solid support materials include, but are not limited to, particles, beads, and surfaces, including glass, polymers, latex, elemental metals, metal composites, alloys, silicon, carbon, and mixtures thereof.

In one embodiment, a portion of the epoxy is polymerized prior to contacting the biomolecular probe with the epoxy.

Suitable epoxy-based resins include, but are not limited to, EPON SU-8, EPON 1001F, 1002F, 1004F, 1007F, 1009F, 2002, and 2005 (commercially available from Hexon Specialty Chemicals company of Fei Ye teville, north carolina, usa). EPON SU-8 and EPON 1002F are preferred resins.

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

SU-8 is commercially available from Hexion Specialty Chemicals company as EPON SU-8 as a solution containing SU-8 and a photoacid generator.

1002F is a photocurable epoxy (CAS: 25036-25-3). 1002F is a polymer of 4,4'- (1-methylethylene) bisphenol with 2,2' - [ (1-methylethylene) bis (4, 1-phenylenemethylene) ] bis (ethylene oxide) and is commercially available from Flexion Specialty Chemicals company under the trade name EPON 1002F as a solution containing 1002F and a photoacid generator.

Suitable biomolecular probes include, but are not limited to, lipids, polysaccharides, amino acids, polypeptides, oligopeptides, peptides, antibodies and fragments thereof, polynucleotides (including single-and double-stranded DNA and RNA), 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 and the like. Exemplary biomolecular probes include SDMA, ADMA, T4, cortisol, progesterone, and enzymes (e.g., lipases, such as pancreatic lipase, etc.).

Without wishing to be bound by theory, it is believed that the biomolecular probes bind to epoxy-based resins by reaction of amine, thiol, or hydroxyl groups on the biomolecules with epoxy groups on the resin. Biomolecular probes may also be passively bound to epoxy resins. As used herein, the phrase "passive binding" refers to binding by non-covalent interactions (such as van der waals interactions, hydrophobic interactions, hydrophilic interactions, or hydrogen bonding interactions, etc.).

The substrate having a surface comprising an epoxy-based resin may be, but is not limited to, a film alone or adhered to another solid surface; microbeads; particles; a microsphere; a microchip; paramagnetic beads; particles containing identifying features such as bar codes and the like; paramagnetic particles; paramagnetic particles containing barcodes; and beads containing nickel barcodes.

In some embodiments, the biomolecular probe is directly bound to the epoxy resin by simply contacting the epoxy resin with the biomolecule.

In one embodiment, the biomolecular probe is contacted with the epoxy resin by adding a substrate having a surface comprising the epoxy resin to a solution of the biomolecular probe to provide a contact mixture. In one embodiment, the solution of the biomolecular probe is an aqueous solution. In one embodiment, the solution of biomolecular probes is a buffered aqueous solution. In one embodiment, the solution of the biomolecular probe is a Dimethylsulfoxide (DMSO) solution.

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 the solution of biomolecular probes to provide the contact mixture. In one embodiment, the substrate having a surface comprising an epoxy resin is washed with DMSO immediately before contacting the substrate having a surface comprising an epoxy resin with a solution of biomolecular probes to provide a contact mixture. Surprisingly, it was found that contacting an epoxy with DMSO prior to contacting the epoxy with a biomolecular probe provided a substrate for bioassay as follows: the substrate exhibits less variability in the number of biomolecular probes bound to the epoxy and less variability in the signal obtained in detecting the presence of analytes bound to the biomolecular probes in a sample. Surprisingly, it was found that contacting an epoxy with DMSO before contacting the epoxy with a biomolecular probe provided a substrate for bioanalytical analysis that exhibited better signals.

By directly binding the biomolecular probe to the epoxy resin, the method advantageously avoids the additional step of having to functionalize the epoxy resin by: (i) Reacting the epoxy with another molecule prior to binding the biomolecular probe to the epoxy, or (ii) mixing the other molecule into the epoxy prior to polymerization. By avoiding this additional step, the method is advantageously faster, cheaper, and eliminates steps that may be erroneous or variable.

When a solution of biomolecular probes is used to contact the biomolecular probes with a substrate having a surface comprising an epoxy resin, the concentration of biomolecular probes in the solution ranges from about 0.05mg/mL to about 5mg/mL, preferably from about 0.01mg/mL to about 3.0mg/mL, more preferably from about 0.15mg/mL to about 2.5mg/mL, for example about 1.5mg/mL.

The concentration of the substrate having the epoxy-containing resin surface in the contact mixture ranges from about 0.05x 10 ⁶ substrate/mL to about 5.0X10 ⁶ substrate/mL, preferably about 0.1X10 ⁶ substrate/mL to about 3.0X10 ⁶ substrate/mL. In one embodiment, the biomolecular probe is a peptide and the concentration of the substrate having the surface comprising the epoxy resin in the contact mixture ranges from about 0.1x 10 ⁶ substrate/mL to about 3.0X10 ⁶ substrate/mL, e.g., about 2.0X10 ⁶ substrate/mL. In one embodiment, the biomolecular probe is an antibody and the concentration of the substrate having the surface comprising the epoxy resin in the contact mixture ranges from about 0.1x 10 ⁶ substrate/mL to about 1.8X10 ⁶ substrate/mL, e.g., about 1x 10 ⁶ substrate/mL.

The solution of the biomolecular probe is typically contacted with a substrate having a surface comprising an epoxy resin for a time sufficient to allow the biomolecular probe to bind to the epoxy resin. Typically, the solution of biomolecular probes is contacted with a substrate having a surface comprising an epoxy 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 resin for about 4 hours to about 18 hours.

The contact mixture (i.e., the solution of the substrate having the surface comprising the epoxy resin and the biomolecular probe) is maintained at a sufficient temperature to allow the biomolecular probe to bind to the epoxy resin. In one embodiment, the contact mixture is maintained at a temperature between about 4 ℃ and about 65 ℃, preferably between about 15 ℃ and about 30 ℃, more preferably between about 18 ℃ and 27 ℃. In one embodiment, the contact mixture is agitated to ensure that the surface of the substrate having the surface comprising the epoxy resin is in sufficient contact with the solution of biomolecular probes.

In one embodiment, after contacting a substrate having a surface comprising an epoxy resin with a solution of biomolecular probes such that the biomolecular probes bind to the epoxy resin, the solution of biomrobes is removed and the resulting biomolecular functionalized substrate is washed with the following mixture: the mixture was a mixture of about 1% Bovine Serum Albumin (BSA) (commercially available from Proliant Biologicals company of Ankany, U.S.A.), about 0.05% Twain-20 (commercially available from Sigma Aldrich company of St.Louis, mitsui, U.S.A.), and about 0.05% Proclin 950 (commercially available from Sigma Aldrich company of St.Louis, mitsui, U.S.A.), at a pH of about 7.4. Suitable PBS solutions include about 1.8mM disodium hydrogen phosphate (commercially available from Sigma Aldrich, st.Louis, mitsui, U.S.A.), about 8.4mM sodium dihydrogen phosphate (commercially available from Sigma Aldrich, st.Louis, mitsui, U.S.A.), and about 145mM sodium chloride (commercially available from Amresco, salon, ohio, U.S.A.). In one embodiment, the biomolecule-functionalized substrate is washed at least three times with at least about 200 μl of a washing solution. In one embodiment, the biomolecule-functionalized substrate is washed at least three times with about 200 μl to about 1,000 μl of a washing solution.

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 buffers, and citrate-based buffers.

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 (pH about 7.4) for measurement.

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 and FAB fragments), 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 with about 100mM 2- (N-morpholinoethanesulfonic acid (MES) and about 140mM guanidine-HCl pH (pH about 5.5). In one embodiment of the first aspect of the method, the solution of the biomolecular probe is an aqueous solution buffered with about 100mM 3- [4- (2-hydroxyethyl) piperazin-1-yl ] propane-1-sulfonic acid (EPPS) and 140mM guanidine-HCl (pH about 8).

In one embodiment of the first aspect of the method, the substrate having a surface comprising an epoxy resin is washed with a PBS solution containing about 0.05% Tween-20 prior to contact with the 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 PBS solution containing about 0.05% Tween-20 prior to contacting with the 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 about 200 μl to about 1,000 μl of PBS solution containing about 0.05% Tween-20 prior to contacting with the solution of biomolecular probes.

In a preferred embodiment of the first aspect of the method, the substrate having the surface comprising the epoxy resin is then further washed with DMSO prior to contact with the solution of biomolecular probes. In one embodiment of the first aspect of the method, the substrate having a surface comprising the epoxy-based 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 the epoxy-based resin is washed at least three times with about 200 μl to about 1,000 μl of the washing solution.

In one embodiment of the first aspect of the method, after contacting the substrate having an epoxy-containing surface with a solution of biomolecular probes to attach the biomolecular probes to the epoxy, the solution of biomolecular probes is removed and the resulting biomolecular functionalized substrate is washed with a mixture of about 1% BSA, about 0.05% Tween-20, about 0.05% Proclin 950 in PBS (pH of about 7.4). In one embodiment, the biomolecule-functionalized substrate is washed at least three times with at least about 200 μl of a washing solution. In one embodiment, the biomolecule-functionalized substrate is washed at least three times with about 200 μl to about 1,000 μl of a washing solution.

In one embodiment of the first aspect of the method, the substrate having a surface comprising 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 company of san fepristine, california).

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 ranges from about 0.2mM to about 1mM peptide, for example about 0.5mM.

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 comprising about 1% Tween-20 prior to contact with the 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 DMSO solution comprising about 1% Tween-20 prior to contact with the solution of biomolecular probes. In an 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 DMSO solution comprising about 1% Tween-20 prior to contacting with the solution of biomolecular probes.

In one embodiment of the second aspect of the method, after contacting the substrate having a surface comprising an epoxy resin with a solution of biomolecular probes to attach the biomolecular probes to the epoxy resin, the solution of biomolecular probes is removed and the resulting biomolecular functionalized substrate is washed with a mixture of about 1% BSA, about 0.05% Tween-20 and about 0.05% Proclin950 in PBS (pH of about 7.4). In one embodiment, the biomolecule-functionalized substrate is washed at least three times with at least about 200 μl of a washing solution. In one embodiment, the biomolecule-functionalized substrate is washed at least three times with about 200 μl to about 1,000 μl of a washing solution.

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% Proclin950 in PBS (pH about 7.4) for measurement.

In one embodiment of the second aspect of the method, the substrate having a surface comprising 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 company of san fessprins, california), and the like.

Without wishing to be bound by theory, the second aspect of the method involves the reaction of thiol groups on the biomolecular probe with epoxide groups on the epoxide resin. Such cysteine residues may be present at any position of the peptide sequence. Cysteine may also be separated from the peptide by a linker (such as a PEG linker, etc.). The PEG may have a defined length, such as by using discontinuous PEG. dPEG (Quanta Biodesign, prain, ohio) is a particularly suitable PEG. Methods for attaching cysteine residues to proteins using PEG linkers are known in the art. See, e.g., I.Hamley, PEG-Peptide Conjugates, biomacromolecules,15:1543-59,2014. Other linkers and spacers include beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminopentanoic acid (Ava), 6-aminocaproic acid (Ahx), trioxatridecane-succinic acid (Ttds) and peptides.

In one embodiment, capping is performed at the N-and C-terminus of the peptide by N-terminal acetylation (Ac) or C-terminal amidation. When the biomolecular probe is an antibody, the antibody may be bound to the epoxy resin via a thiol group by reducing the antibody using a reducing agent such as Dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), β -mercaptoethanol (BME), or the like to allow the thiol group in the hinge region of the antibody to be easily bound to the antibody.

In one embodiment, a substrate having a surface comprising an epoxy resin is prepared by coating a support with a solution comprising SU-8 resin and a photoacid generator (such as triphenylsulfonium hexafluoroantimonate, etc.) in a solvent (such as gamma butyrolactone or cyclopentanone, etc.), such as EPON SU-8 (commercially available from Hexion Specialty Chemicals of Fei Ye teville, north carolina, usa). The support is then heated to remove the solvent and leave a coating of solid epoxy-based resin on the support. The thickness of the solid epoxy resin may be several hundred microns thick. Typically, the thickness of the solid epoxy-based resin ranges from about 1nm to about 3mm. Optionally, a portion of the solid epoxy resin is then polymerized by ultraviolet light irradiation to provide a polymerized epoxy resin. In one embodiment, a photomask is placed atop the solid epoxy resin prior to irradiation with ultraviolet light to leave a pattern on the polymerized epoxy resin. In one embodiment, the support is spaced from the solid epoxy resin.

In one embodiment, a substrate having a surface comprising an epoxy resin is prepared by coating a support with a solution comprising a 1002F resin and a photoacid generator (such as triphenylsulfonium hexafluoroantimonate, etc.) in a solvent (such as gamma butyrolactone or cyclopentanone, etc.), such as EPON 1002F (commercially available from Hexion Specialty Chemicals company of Fei Ye teville, north carolina, usa), etc. The support is then heated to remove the solvent and leave a coating of solid epoxy-based resin on the support. The thickness of the solid epoxy resin may be several hundred microns thick. Typically, the thickness of the solid epoxy-based resin ranges from about 1nm to about 3mm. Optionally, a portion of the solid epoxy resin is then polymerized by ultraviolet light irradiation to provide a polymerized epoxy resin. In one embodiment, a photomask is placed atop the solid epoxy resin prior to irradiation with ultraviolet light to leave a pattern on the polymerized epoxy resin. In one embodiment, the support is spaced from the solid epoxy resin.

In one embodiment, the sample is a fecal sample. As used herein, the term "fecal sample" includes fecal matter, any fecal containing sample, as well as fecal fractions and extracts. The method of preparing the fecal extract is described in U.S. patent No. 8,367,808. In one embodiment, the molecule (i.e. analyte) to be determined is a protein produced by, for example, intestinal worms (such as roundworms, whipworms, hookworms, tapeworms or heartworms) or the parasite giardia, and the biomolecular probe is an antibody against 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 roundworm, an antibody that specifically binds to a fecal antigen from whipworm, an antibody that specifically binds to a fecal antigen from hookworm, an antibody that specifically binds to a fecal antigen from tapeworm, an antibody that specifically binds to an antigen from heartworm, and an antibody that specifically binds to a fecal antigen from giardia.

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. An example of an antibody that specifically binds to a fecal antigen from roundworm is in U.S. patent No. 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. An example of an antibody that specifically binds to a fecal antigen from tapeworm is disclosed in U.S. patent No. 11,001,626. Examples of antibodies that specifically bind to fecal antigens from giardia are disclosed in h.stibbs, monoclonal antibody-based enzyme immunoassay for Giardia lambda antigen in human stool, j.clin.microbiol., (11): 2582-2588, nov.1989 and h.stibbs et al, identification of Giardia lambda-specific antigens in infected human and gerbil feces by western immunoblotting, j.clin.microbiol., (10): 2340-6, oct.1990.

In one embodiment, the sample is a blood sample from a subject, the analyte is an antibody produced by an immune response of the subject to a protein produced by an infectious agent, 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 circulating antibodies of animals infected with bacteria of the genus erigeron, including canine erigeron (Ehrlichia canis), chafeerigeron (Ehrlichia chaffeensis) and eulerike (Ehrlichia ewingii), are described in U.S. patent No. 7,087,372;7,407,770:7,445,788;7,449,191;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 circulating antibodies of animals infected with Anaplasma bacteria, including phagocytic Anaplasma (Anaplasma phagocytophylum) and flattened Anaplasma plattys, are described in U.S. patent 6,964,855;7,439,321;8,303,959;6,306,402;6,204,252;8,093,008 and 9,120,857. Proteins, polypeptides or oligomers capable of specifically binding circulating antibodies of animals infected with borrelia bacteria, including borrelia burgdorferi (Borrelia burgdorferi), are described in U.S. patent No. 6,719,983;6,740,744;6,475,492 and 6,660,274.

In one embodiment, the analyte is an antibody produced by the subject's response to a protein produced by borrelia burgdorferi causing lyme disease, and the biomolecular probe is a protein or a portion of a protein produced by borrelia burgdorferi.

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. As used herein, the term "blood sample" includes whole blood or any portion or fraction of whole blood, including, but not limited to, serum and plasma.

In one embodiment, the analyte is symmetrical dimethyl arginine (SDMA). Antibodies that specifically bind SDMA are disclosed in U.S. patent No. 8,481,690.

In one embodiment, the sample is a blood sample from a subject, the analyte is an antigen derived from a pathogen present in the blood of the infected subject, and the biomolecular probe is an antibody to the antigen. In one embodiment, the analyte is an antibody that specifically binds to a circulating antigen from heartworm (heartworm canis (Dirofilarira immitis)). Antibodies that specifically bind to circulating antigens of heartworm (heartworm) are disclosed in U.S. patent No. 4,839,275.

In one embodiment, a substrate for bioassays is prepared by a method involving:

(i) Providing a silicon/aluminum sheet;

(ii) Coating the silicon/aluminum flakes with a first solution of a first epoxy-based resin dissolved in a first solvent to provide silicon/aluminum flakes coated with a first epoxy-based resin coating;

(iii) Heating the silicon/aluminum sheet coated with the first epoxy-based resin coating to remove at least a portion of the first solvent to provide a silicon/aluminum sheet coated with the first solid epoxy-based resin coating;

(iv) Depositing a nickel bar code on the first solid epoxy coating to provide a silicon/aluminum sheet having the first solid epoxy coating and the nickel bar code;

(v) Coating the nickel bar code with a second solution of a second epoxy-based resin dissolved in a second solvent to provide silicon/aluminum flakes coated with the first epoxy-based resin coating and the second epoxy-based resin coating;

(vi) Heating the silicon/aluminum flakes coated with the first and second solid epoxy-based resin coatings to remove at least a portion of the second solvent to provide silicon/aluminum flakes coated with the first and second solid epoxy-based resin coatings,

wherein the nickel bar code is located between the first solid epoxy coating and the second solid epoxy coating;

(vii) Optionally, polymerizing at least a portion of the first solid epoxy coating and the second solid epoxy coating to provide a silicon/aluminum sheet laminated with the first solid epoxy, the nickel bar code, and the second solid epoxy coating, wherein at least a portion of the first solid epoxy coating and the second solid epoxy coating have been polymerized;

(viii) Separating the silicon/aluminum flakes from the silicon/aluminum flakes coated with the first epoxy coating, nickel bar code, and second epoxy coating to provide bar coded magnetic beads;

(iv) The barcoded magnetic beads are contacted with biomolecules so that the biomolecules are directly combined with epoxy resin.

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.

In one embodiment, the silicon/aluminum flakes are separated from the first solid epoxy, nickel bar code, and second solid epoxy coating by contacting the silicon/aluminum flakes with sodium hydroxide.

The invention also relates to a method of determining the presence of an analyte in a sample. The method comprises the following steps: contacting the sample with a substrate having a surface comprising an epoxy resin having a biomolecular probe directly bound to the epoxy resin, wherein the biomolecular probe specifically binds to the analyte.

As used herein, the phrases "specifically bind to an analyte," "specifically bind," "specifically to an analyte," and similar phrases have their art-recognized meaning, i.e., a biomolecular probe recognizes and binds an analyte (or class of analytes) with greater affinity than other non-specific molecules. For example, an antibody raised against an antigen binds to that antigen more effectively than other non-specific molecules, and such an antibody can be described as specifically binding to an antigen. Binding specificity may be tested using methods known in the art, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), surface plasmon resonance or immunoblot assays, and the like.

In one embodiment, the sample is a fecal sample. In one embodiment, the analyte is a protein produced by a helminth and the biomolecular probe is an antibody to the protein produced by the helminth. In one embodiment, the antibody directed against a protein produced by an intestinal worm is selected from the group consisting of: an antibody that specifically binds to a fecal antigen from roundworm, an antibody that specifically binds to a fecal antigen from whipworm, an antibody that specifically binds to a fecal antigen from hookworm, an antibody that specifically binds to a fecal antigen from tapeworm, an antibody that specifically binds to an antigen from heartworm, and an antibody that specifically binds to a fecal antigen from giardia.

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

In one embodiment, the analyte is an antibody raised by the subject's response to a protein produced by borrelia burgdorferi causing lyme disease, and the biomolecular probe is a protein or a portion of a protein produced by borrelia burgdorferi.

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 symmetrical dimethyl arginine (SDMA).

In one embodiment, the sample is a blood sample from a subject, the analyte is an antigen derived from a pathogen present in the blood of the infected subject, and the biomolecular probe is an antibody to the antigen. In one embodiment, the analyte is an antibody that specifically binds to a circulating antigen from heartworm (heartworm of dogs).

Examples

The scope of the invention is not limited by the specific embodiments disclosed in the examples intended to illustrate several aspects of the invention, and any functionally equivalent embodiments are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims. Such variations of the invention, including all now known or later developed alternatives and variations of the formulation or minor variations of the experimental design, as contemplated by those skilled in the art are considered to fall within the scope of the invention contained herein.

Example 1: passive coupling of biomolecular probes and barcoded magnetic beads

Barcoded Magnetic Beads (BMB) were made of SU-8, an epoxy negative photoresist (commercially available from Applied BioCode, st., calif.). Coupling of the biomolecular probes (such as monoclonal antibodies or proteins, etc.) to the BMB is accomplished by adsorbing the biomolecular probes to the surface of the BMB as follows.

Monoclonal antibody solutions were prepared at final concentrations of about 0.15-2.5mg/mL antibody (typically about 1.5 mg/mL) in aqueous buffer (pH about 5.5) containing about 100mM MES (commercially available from Sigma Aldrich, st.louis, miso) and about 140mM guanidine-HCl (commercially available from Sigma Aldrich, miso), or in aqueous buffer (pH about 8) containing about 100mM EPPS (commercially available from Sigma Aldrich, miso) and about 140niM guanidine-HCl to provide antibody coating solutions.

Sufficient BMB was suspended in BMB wash buffer (about 1.8mM disodium hydrogen phosphate (commercially available from Sigma Aldrich, st. Louis, mitsui, U.S.A.), about 8.4mM sodium dihydrogen phosphate (commercially available from Sigma Aldrich, st. Louis, mitsui, U.S.A.), about 145mM sodium chloride (commercially available from Amresco, salon, ohio, U.S.A.), and about 0.05% Tween-20 (commercially available from Sigma Aldrich, st. Louis, mitsui, U.S.A.), at a pH of about 7.4, to provide about 0.1X 10 ⁶ BMB/mL to 1.8X10 ⁶ The final concentration of BMB/mL (typically about 1X 10 for antibodies) ⁶ BMB/mL). BMB was washed three times with BMB wash buffer. All BMB washes (for this step and subsequent steps) were performed as follows:

the tube containing the BMB was first placed on a magnetic rack and the BMB was attached to the magnet for 1-10 minutes. The supernatant was then carefully aspirated and the BMB resuspended in wash buffer having a volume approximately equal to the original suspension volume of BMB (about 200. Mu.L to about 1,000. Mu.L). These steps were repeated three times in total to provide BMB pellets.

After washing the BMB with the washing buffer, the BMB was washed three times with DMSO (commercially available from Sigma Aldrich company of st.

Coating reaction: immediately after DMSO washing, the antibody coating solution (about 1.5mg/mL final concentration) was combined with DMSO-washed BMB (about 1x 10 ⁶ BMB/mL final concentration) were combined and incubated at room temperature (18-27 ℃) for 4-18 hours with mixing. After completion of incubation, antibody-conjugated BMB was washed three times with assay buffer (about 1.8mM disodium hydrogen phosphate (commercially available from Sigma Aldrich, st. Louis, mich.), about 8.4mM sodium dihydrogen phosphate (commercially available from Sigma Aldrich, st. Louis, st. Jobi), about 145mM sodium chloride (commercially available from Amresco, salon, ohio), about 1% BSA (commercially available from Proliant Biogicals, ankany, st. Jobi), about 0.05% Tween-20 (commercially available from Sigma Aldrich, st. Louis, mimo), and about 0.05% Proclin 950 (commercially available from Sigma Aldrich, st. Louis, mimo, USA), at a pH of about 7.4).

The antibody-conjugated BMB is then suspended in assay buffer at the desired final concentration for assay.

Example 2: coupling biomolecular probes to barcoded magnetic beads via thiol groups

Coupling of biomolecular probes containing thiol groups (such as monoclonal antibodies or proteins, etc.) to Barcoded Magnetic Beads (BMB) coated with epoxy resin is performed by adsorbing the biomolecular probes onto the surface of the BMB by the following steps.

A solution of the peptide in DMSO (commercially available from Sigma Aldrich company of St.Louis, mitsui) containing about 1% Tween-20 (commercially available from Sigma Aldrich company of St.Louis, mitsui, USA) was prepared to a final peptide concentration of about 0.02mM to about 1mM (typically about 0.1 mM) to provide a peptide coating solution. The peptide used in this example was acetyl-Cys (dPEG 12) [ peptide ] -amide, where the peptide was a 25 amino acid residue oligopeptide and dPEG12 was discontinuous PEG12. The acetyl-Cys (dPEG 12) [ peptide ] -amide was custom synthesized by company New England peptide of Gardner, ma:

BMB suitable for this process is BMB prepared from SU-8, an epoxy negative photoresist (commercially available from Applied BioCode Inc. of St.Fei Prins, calif.).

Will be sufficient to provide about 0.1x10 in the coating reaction described below ⁶ BMB/mL to 3x10 ⁶ BMB/mL (typically about 2x 10 ⁶ BMB/mL) the final concentration of BMB was suspended in a volume of BMB wash buffer (about 1% Tween-20 in DMSO) of about 200. Mu.L to about 1,000. Mu.L. BMB was washed three times with about 1% Tween-20 in DMSO as described below. All BMB washes (for this step and subsequent steps) were performed as follows:

The tube containing the BMB was placed on a magnetic rack and the BMB was attached to the magnet for 1-10 minutes. The supernatant was carefully aspirated and the BMB was resuspended in a volume of wash buffer approximately equal to the original suspension volume of BMB. These steps were repeated three times in total to provide BMB pellets.

Immediately after washing the BMB with the wash buffer, the washed BMB was combined with the peptide coating solution and incubated with mixing at room temperature (18-27 ℃) for about 4 hours. After 4 hours of incubation, peptide-conjugated BMB was washed three times with a volume of assay buffer (about 1% BSA in about 1.8mM disodium hydrogen phosphate, about 8.4mM sodium dihydrogen phosphate, and about 145mM sodium chloride, about 0.05% Tween-20, and about 0.05% Proclin 950, pH about 7.4).

Example 3: coupling of biomolecular probes to barcoded magnetic beads via sulfhydryl groups generated by reduced disulfide-linked cysteines

Macromolecules such as monoclonal antibodies and the like can be coupled via thiol groups that are generated by: monoclonal antibodies are reacted with a reducing agent such as Dithiothreitol (DTT), tris (2-carboxyethylphosphine) (TCEP), or 2-mercaptoethanol (BME), etc., to reduce the disulfide-linked cysteine side chains to facilitate binding to the surface of BMB.

For covalent coupling of the reduced antibody to the epoxy group on BMB, a reduced antibody coating solution was prepared by diluting the antibody to a concentration of about 5mg/mL in a reduction buffer (50 mM sodium phosphate (commercially available from Sigma Aldrich, st.lou, miso, usa), 75mM sodium chloride (commercially available from amesco, salon, ohio), 2mM EDTA (commercially available from Sigma Aldrich, st.lou, miso, usa) and 5mM DTT (commercially available from Thermo Fisher Scientific, walsepham, ma, usa) at a pH of about 7.4. The resulting solution was incubated at 18-27℃for about 0.5 hours.

After reduction, the reduced antibodies were exchanged into the following solutions using a G25 Zeba Spin desalting column (commercially available from Thermo Fisher Scientific company of waltham, ma) according to the manufacturer's instructions: the solution contained 50mM sodium phosphate (commercially available from Sigma Aldrich company of St.Louis, mitsui, USA), 75mM sodium chloride (commercially available from Amresco company of Salon, ohio), and 2mM EDTA (commercially available from Sigma Aldrich company of St.Louis, mitsui, USA), at a pH of about 7.4. The antibody solution was adjusted to a concentration of about 0.5mg/mL and the resulting solution was immediately added to the DMSO-washed BMB.

BMB washed with DMSO was prepared by: will first be sufficient to provide about 0.1x10 in the coating reaction described below ⁶ BMB/mL to 1.8X10 ⁶ BMB/mL (typically about 1X 10 for antibodies) ⁶ BMB/mL) final concentration BMB was suspended in a volume of about 200. Mu.L to about 1,000. Mu.L of wash buffer (about 1.8mM disodium hydrogen phosphate, about 8.4mM sodium dihydrogen phosphate, about 145nM sodium, and about 0.05% Tween-20). BMB was washed three times with wash buffer as follows.

All BMB washes (for this and subsequent steps) were performed as follows: the tube containing the BMB was first placed in a magnetic rack and the BMB was attached to the magnet for 1-10 minutes. The supernatant was then carefully aspirated and the BMB resuspended in wash buffer having a volume approximately equal to the original suspension volume of BMB. These steps were repeated three times to provide BMB pellets.

After washing the BMB with the washing buffer, the BMB was washed three times with DMSO (commercially available from Sigma Aldrich company of st. The BMB was then suspended in DMSO at a volume approximately equal to the original suspension volume and incubated at 18-27℃for about 4 hours with mixing. After incubation, the tube containing the BMB was placed on a magnetic rack and the BMB was attached to the magnet for 1-10 minutes. The supernatant was then carefully aspirated to provide DMSO-washed BMB pellets.

Immediately after DMSO washing, the antibody coating solution (about 0.5mg/mL final concentration) was combined with DMSO-washed BMB (about 1x 10 ⁶ BMB/mL final concentration) were combined and incubated with mixing at room temperature (18-27 ℃) for about 18 hours.

After incubation was completed, the tube containing BMB was placed on a magnetic rack and BMB was attached to the magnet for 1-10 minutes. The supernatant was then carefully aspirated, the BMB resuspended in a volume of wash buffer approximately equal to the original suspension volume (about 1.8mM disodium hydrogen phosphate, about 8.4mM disodium phosphate, about 145mM sodium chloride, and about 0.05% Tween, pH about 7.4), and the resulting solution incubated at 18-27℃for about 15 minutes.

After incubation was completed, the tube containing the BMB was placed on a magnetic rack and the BMB was attached to the magnet for 1-10 minutes. The supernatant was then carefully aspirated and the BMB resuspended in assay buffer having a volume approximately equal to the original suspension volume. Assay buffer: about 1% BSA, about 0.05% Tween-20, about 0.05% Proclin 950, in about 1.8mM disodium hydrogen phosphate, about 8.4mM sodium dihydrogen phosphate, and about 145nM sodium chloride, pH is about 7.4. The resulting suspension was mixed incubated at 18-27℃for about 30 minutes. The antibody-conjugated BMB was then washed three times with assay buffer at a volume approximately equal to the original suspension volume.

Example 4: coupling rhodamine to barcoded magnetic beads:

materials:

DMSO washing of BMB:

to a 1mL centrifuge tube on a magnetic rack, 0.5mL of BMB suspended in storage buffer was added to provide a concentration of 100,000BMB/mL.

BMB suitable for this process is BMB prepared from SU-8, an epoxy negative photoresist (commercially available from Applied BioCode Inc. of St.Fei Prins, calif.). BMB is provided in a storage buffer containing sodium chloride (0.8%), potassium chloride (0.02%), disodium hydrogen phosphate (0.144%), monopotassium hydrogen phosphate (0.024%), tween-20 (0.05%), and ProClin 950 (0.1%).

Liquid was removed from each centrifuge tube with a pipette, and then about 0.5mL DMSO (commercially available from Sigma Aldrich corporation of st. The tube was vortexed vigorously for 10 seconds, placed back on a magnetic rack, left to stand for 1 minute, and the DMSO removed with a pipette. The washing process was repeated 2 more times. Then, about 0.5mL DMSO was added to each tube, the tube was vortexed vigorously, and left to stand on a mixer at room temperature for 4 hours.

Coating with rhodamine-lissamine:

after mixing for 4 hours, the tube was removed, placed on a magnet holder for 1 minute, DMSO was removed with a pipette, and about 0.5mL of about 150mM EPPS buffer, pH about 9.0, was added. The tube containing BMB in EPPS buffer was then vortexed vigorously for 10 seconds, placed back on the magnet holder, and the EPPS buffer removed with a pipette. The washing process was repeated 2 more times. After washing was completed, about 0.5mL EPPS buffer was added to each tube and the tube was vortexed. Then, about 10.0mM rhodamine-Liylamine (9. Mu.L, 0.3mg/mL solution in DMSO) or sulforhodamine (control, 1.2. Mu.L, 2.8mg/mL solution in DMSO) was added to the epoxy BMB in EPPS buffer. The BMB suspension was turned upside down at room temperature for about 22 hours in the dark. After this time, the coated BMB was placed on a magnetic stent and the solvent was removed with a pipette. The coated BMB was washed with about 1.0mL of a solution of about 1.8mM disodium hydrogen phosphate, about 8.4mM sodium dihydrogen phosphate, about 145nM sodium chloride and about 0.05% Tween-20 by brief vortexing, and then the solvent was removed. This procedure was repeated 5 times. Finally, about 1.0mL of a solution of about 1.8mM disodium hydrogen phosphate, about 8.4mM sodium dihydrogen phosphate, about 145nM sodium chloride, and about 0.05% Tween-20 was added to the coated BMB to a final concentration of about 50,000BMB/mL. About 5.0 μl of the resulting BMB suspension was then added to the wells of a 96-well plate, with a final count of about 250 BMB/well. About 200. Mu.L of a solution of about 1.8mM disodium hydrogen phosphate, about 8.4mM sodium dihydrogen phosphate, about 145nM sodium chloride, and about 0.05% Tween-20 was added to each well to provide a suspension for determining BMB.

Fluorescence of each well was determined using a microplate reader (commercially available from Applied BioCode company of san feiprism, california).

The epoxy BMB coated with rhodamine-lissamine showed significant fluorescence on the microplate reader, indicating that the rhodamine probes were effectively coated on the epoxy BMB surface. In contrast to sulforhodamine without reactive amine functionality, epoxy BMB incubated with epoxy BMB did not show any observable fluorescence. This demonstrates that the non-specific binding of the rhodamine probe to the epoxy BMB surface is very low, indicating that rhodamine-lissamine specifically reacts with the epoxy surface via primary amine functionality. It can be concluded that amine functionalized small molecules are capable of covalently binding to the epoxy BMB surface.

Example 5: coupling of amine-containing peptides to barcoded magnetic beads:

materials:

DMSO washing of BMB:

approximately 0.2mL of BMB suspended in storage buffer was added to a 1mL centrifuge tube on a magnet rack to provide a concentration of 1,00,000BMB/mL.

BMB suitable for this process is BMB prepared from SU-8, an epoxy negative photoresist (commercially available from Applied BioCode Inc. of St.Fei Prins, calif.). BMB is provided in a storage buffer containing sodium chloride (0.8%), potassium chloride (0.02%), disodium hydrogen phosphate (0.144%), monopotassium hydrogen phosphate (0.024%), tween-20 (0.05%), and ProClin-950 (0.1%).

Liquid was removed from each centrifuge tube with a pipette, and then about 0.2mL DMSO (commercially available from Sigma Aldrich corporation of st. The tube was vortexed vigorously for 10 seconds, placed back on a magnetic rack, left to stand for 1 minute, and the DMSO removed with a pipette. The washing process was repeated 2 more times. About 0.2mL of DMSO was added to each tube, the tube was vortexed vigorously and allowed to stand on a mixer for 4 hours at room temperature.

After mixing for 4 hours, the tube was removed, placed on a magnet holder for 1 minute, DMSO was removed with a pipette, and about 0.2mL of about 150mM EPPS buffer was added to the tube at a pH of about 9.0. Then, the tube containing BMB in EPPS buffer was vortexed vigorously for 10 seconds, placed back on the magnet holder, and the EPPS buffer was removed with a pipette. The washing process was repeated 2 more times.

A solution of about 0.1mM biotin-Lyme peptide and Lyme-Alexafluor555 was prepared from a 1.0mM stock solution. Each peptide has multiple lysine residues, but no cysteine residues or other sulfhydryl groups. Approximately 200. Mu.L of biotin-Lyme peptide solution, lyme-Alexafluor555 solution or control containing EPPS buffer alone was added to the BMB containing tube. The resulting BMB suspension was turned upside down at room temperature for about 2 hours in the dark. After this period of 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 0.2mL of a solution of about 1.8mM disodium hydrogen phosphate, about 8.4mM sodium dihydrogen phosphate, about 145nM sodium chloride, and about 0.05% Tween-20 by brief vortexing, and then the solvent was removed. This procedure was repeated 2 times.

About 200. Mu.L of SA-PE (streptavidin phycoerythrin) containing solution (8. Mu.g/mL SA-PE) was obtained by diluting SA-PE (commercially available as a 1mg/mL solution from Moss company of Pasadena, malaran) with a multiplex assay buffer of 1.8mM disodium hydrogen phosphate, 8.4mM sodium dihydrogen phosphate, 145nM sodium chloride, 0.05% Tween-20, 1% bovine serum albumin, 0.05% ProClin 950) was added to BMB and the beads were incubated for about 10 minutes. After incubation, the supernatant was removed, BMB was washed with about 0.2mL of a solution of about 1.8mM disodium hydrogen phosphate, about 8.4mM disodium phosphate, about 145nM sodium chloride and about 0.05% Tween-20 (pH of about 7.4) by brief vortexing, and then the solvent was removed. This procedure was repeated 5 times. Finally, about 0.4mL of a solution (pH about 7.4) of about 1.8mM disodium hydrogen phosphate, about 8.4mM sodium dihydrogen phosphate, about 145nM sodium chloride, and about 0.05% Tween-20 was added to the coated BMB to provide a final concentration of about 50,000BMB/mL. 5.0. Mu.L of the resulting BMB suspension was added to wells of a 96-well plate, with a final count of about 250 BMB/well, and about 200. Mu.L of a solution of about 1.8mM disodium hydrogen phosphate, about 8.4mM sodium dihydrogen phosphate, about 145mM sodium chloride, and about 0.05% Tween-20 was added to each well to provide a BMB suspension for assay.

The fluorescence of each well was determined using a microplate reader (commercially available from Applied BioCode company of san fepristine, california)

BMB coated with biotin-Lyme peptide and Lyme-Alexafluor555 peptide showed significant fluorescence on the microplate reader, indicating that the peptides were effectively coated on the BMB surface. The lack of fluorescence observed in the control indicated that the SA-PE analyte bound very poorly to the BMB surface, indicating that the biotin-Lyme peptide and Lyme-Alexafluor555 peptide reacted specifically with the epoxy surface via the primary amine functionality contained in the lysine residues of the peptide. It can be concluded that peptides containing lysine (amine functionality) are capable of covalently binding to the epoxy BMB surface.

Example 6: assay using barcoded magnetic beads followed by washing with citrate buffer: coupling of biomolecular probes with barcoded magnetic beads:

about 0.2mL (volume may vary from about 0.1mL to about 500 mL) of BMB suspended in a storage buffer was added to a 1mL centrifuge tube on a magnet rack to provide a concentration of about 1,00,000BMB/mL (the concentration may range from about 1,00,000BMB/mL to about 3x 10) ⁶ BMB/mL)。

Liquid was removed from each centrifuge tube with a pipette, and then about 0.2mL DMSO (commercially available from Sigma Aldrich corporation of st. The tube was vortexed vigorously for 10 seconds, returned to the magnet holder, allowed to stand for 1 minute, and the DMSO removed with a pipette. The washing process was repeated 2 more times. In one embodiment, about 0.2mL DMSO is then added to each tube, the tube vortexed vigorously, and left to stand on the mixer for 4 hours at room temperature. Mixing at room temperature for 4 hours is optional.

After mixing, the tube was removed and placed on a magnet holder, allowed to stand for 1 minute, DMSO was removed with a pipette, and about 0.2mL of about 150mM EPPS buffer (pH about 9.0) was added to the tube. Then, the tube containing BMB in EPPS buffer was vortexed vigorously for 10 seconds, placed back on the magnet holder, and the EPPS buffer was removed with a pipette. The washing process was repeated 2 more times. After the final wash with EPPS, BMB was coated with peptides or antibodies, respectively, as described in sections (a) and (B) below, such that each peptide and antibody bound to BMB with a different barcode.

(A) Coating BMB with peptide

Peptides (biochemical probes) using antibodies (e.g., peptides derived from the protein sequence of phagocytic anaplasma, platy anaplasma, canine anaplasma, especially elctrocera or borrelia, as described above) capable of specifically binding to antibodies of the patient against anaplasma, elctrocera or borrelia are synthesized using cysteines attached to the N-terminus of each peptide via a PEG12 linker, except that peptides derived from borrelia with N-terminal cysteines do not use a PEG linker. About 200. Mu.L of about 0.1mM solution of each peptide was added to the tube containing the EPPS washed BMB. The resulting BMB suspension was turned upside down at room temperature for about 2 hours in the dark. After this time, the tube containing BMB was placed on a magnetic rack and the solvent was removed with a pipette. BMB was then washed by brief vortexing with about 0.2mL of a solution of about 1.8mM disodium hydrogen phosphate, about 8.4mM sodium dihydrogen phosphate, about 145nM sodium chloride, and about 0.05% Tween-20, followed by removal of the solvent. This procedure was repeated 2 times.

(B) Coating BMB with antibodies

About 200 μl of an antibody (biochemical probe) capable of specifically binding to an antigen from heartworm (heartworm canitis) circulating in the blood of an infected animal at a concentration of about 10mg/mL (the concentration of the antibody may range from about 1.5mg/mL to about 12.0 mg/mL) was added to the tube containing the BMB. The resulting BMB suspension was turned upside down at room temperature for about 2 hours in the dark. After this time, the tube containing BMB was placed on a magnetic rack and the solvent was removed with a pipette. BMB was then washed by brief vortexing with about 0.2mL of a solution of about 1.8mM disodium hydrogen phosphate, about 8.4mM sodium dihydrogen phosphate, about 145nM sodium chloride and about 0.05% Tween-20, followed by removal of the solvent. This procedure was repeated 2 times.

And (3) measuring:

BMB coated with biomolecular probes described in (a) or (B) above (65,000BMB/mL in assay buffer (1.0% BSA, 0.05% Tween, 0.05% proclin 950 in PBS)) were mixed to create a multiplex BMB mixture. The multiplex BMB mixture was further diluted in assay buffer to achieve a concentration of 500BMB/mL for each biomolecular probe.

Approximately 100 μl of this diluted multiplex BMB mixture (i.e., approximately 50 beads per biomolecular probe) was added to each well of a 96-well plate using an integral automatic pipette (commercially available from Integra Biosciences company of Hudson, new hampshire, usa). BMB was washed in a 405-TS plate washer (commercially available from Vanuus, buddha) ) The top was washed with 300. Mu.L of 0.05% Tween-20 in PBS and soaked for 10 seconds, and 5 washes were performed. After the last wash, excess supernatant remained on the plate after the wash.

mu.L of sample (serum or plasma, pure) was added to BMB in each well on a 96-well plate. The plate was then placed on a flat plate mixer and mixed at 1,000rpm for 30 minutes. After 30 minutes of incubation, BMB was washed with 300. Mu.L of 0.05% Tween-20 in PBS and soaked for 10 seconds.

50. Mu.L of each biotinylated peptide (2.0. Mu.g/mL, i.e., the same peptide used as a biomolecular probe) or biotinylated anti-heartworm antibody (1.0. Mu.g/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 plates were then placed on a flat plate mixer and mixed for 15 minutes. After 15 minutes of incubation, BMB was washed with 300. Mu.L of 0.05% Tween-20 in PBS and soaked for 10 seconds.

mu.L of SA-PE (8.0. Mu.g/mL, commercially available from MOSS Inc. of Pasadena, mallotus, USA, catalog number: SAPERP 01) was added to each well of the 96-well plate. The plates were then placed on a flat plate mixer and mixed for 10 minutes. After 10 minutes of incubation, BMB was washed with 300. Mu.L of 0.05% Tween-20 in PBS and soaked for 10 seconds.

The resulting BMB is then treated in either of two ways.

In the first method, to each well of a 96-well plate, are added:

(i) About 200. Mu.L of buffer (commercially available from Applied BioCode Inc., st. Fei Springs, calif., catalog number 44-D0004-500) (standard read buffer).

In the second method, to each well of a 96-well plate, are added:

(ii) About 200. Mu.L of a citric acid buffer solution (citrate read buffer) containing trisodium citrate dihydrate (0.485M), citric acid (0.015M), sodium chloride (0.1M), proclin 950 (0.5 mL/L) at a pH of 6.1-6.3.

Ionic strength is an important factor in allowing the stability of immune complexes formed on the bead surface. Without wishing to be bound by theory, it is believed that the stabilization is due to the high salt concentration making the solution undesirably dissociable. Salts other than citrate can also work at high ionic strength, but their minimum concentration needs to be around 0.5M. However, other salts are undesirable in terms of production or transportation because they may precipitate from solution at temperatures below ambient. Advantageously, the mixture of citric acid buffer is kept in solution during storage or transport under refrigerated conditions.

Fluorescent use of each well2500 analyzer (BioCode company commercially available from san fei spellins, california).

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. Fig. 1 illustrates the signal intensity observed with a read buffer containing standard (fig. 1A) and with a citrate read buffer (fig. 1B). In the experiment, each well of a 96-well plate (i.e., a plate having 12 columns (1-12) and 8 rows (a-H)) was filled with the same BMB (e.g., each item bound to a bead, as described above), and each well read from column 1 (i.e., wells 1A through 1H) to column 12 (i.e., wells 12A through 12H). The time required to read the fluorescence of all wells in a 96-well plate (i.e., wells 1A through 12H) was about 37 minutes. As shown in fig. 1A, when the standard read buffer (i.e., the citrate-free buffer) is used, i.e., method (i), the signal intensity gradually decreases from the start of the read cycle to the end of the read cycle (about 37 minutes). In contrast, as shown in fig. 1B, when the citrate read buffer, i.e., method (ii), was used, no decrease in signal intensity was observed from the start of the read cycle to the end of the read cycle (about 37 minutes). In the assay for antibodies specific to each of the anaplasma derived peptides (designated "AP", "Aph" and "Apl" on the X-axis of fig. 1A and 1B), a decrease in signal intensity occurs during the read cycle when citrate-free buffers (e.g., standard read buffers) are used, but not when citrate-containing buffers (e.g., citrate read buffers) are used.

Fig. 1 shows that contacting BMB with citrate buffer prior to reading fluorescence is advantageous in avoiding fluorescence decrease over time as compared to other buffers.

In assays directed against antibodies specific for peptides derived from canine ehrlichia, eulizidine, and in assays directed against borrelia burgdorferi, similar effects of standard and citric acid read buffer on signal intensity over time were observed (not shown).

The entire disclosure of all documents cited is incorporated herein by reference.

Claims

1. A method of preparing 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 the substrate having the surface comprising an epoxy resin such that the biomolecular probe is directly bound to the epoxy resin.

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, avidins, streptavidin, biotin, and polyethylene glycol.

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

4. The method of claim 1, wherein the substrate having a surface comprising an epoxy resin is selected from the group consisting of: a membrane; microbeads; particles; a microsphere; a microchip; paramagnetic beads; microparticles containing a bar code; paramagnetic particles; microparticles containing a bar code; paramagnetic particles containing barcodes; and beads containing nickel barcodes.

5. The method of claim 1, wherein the biomolecular probe is contacted with the epoxy resin by contacting the substrate having a surface comprising the epoxy resin with a solution of the biomolecular probe to provide a contact mixture.

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

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

8. The method of claim 6, wherein the aqueous solution is buffered with about 100mM EPPS and about 140mM guanidine-HCl at a pH of about 8.

9. The method of claim 5, wherein the solution is a DMSO solution.

10. The method of claim 9, wherein the DMSO solution contains about 1% tween-20.

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

12. The method of claim 5, wherein the substrate having a surface comprising an epoxy resin is present in the contact mixture at a concentration ranging from about 0.05x 10 ⁶ substrate/mL to about 5.0X10 ⁶ substrate/mL.

13. The method of claim 1, wherein the biomolecular probe is contacted with the substrate having the surface comprising an epoxy resin for at least about 4 hours.

14. The method of claim 13, wherein the biomolecular probe is contacted with the substrate having the surface comprising an epoxy resin for about 4 hours to about 18 hours.

15. The method of claim 5, wherein the contact mixture is maintained at a temperature between about 15 ℃ and about 30 ℃.

16. The method of claim 1, wherein the substrate having a surface comprising an epoxy resin is washed with DMSO prior to contacting the substrate having a surface comprising an epoxy resin with the biomolecular probe.

17. The method of claim 16, wherein the DMSO contains about 1% tween-20.

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

19. A substrate for biological analysis comprising a substrate having a surface comprising an epoxy resin having biomolecular probes directly bound to the epoxy resin.

20. A method of preparing a substrate for bioassays, comprising:

(i) Providing a solution of a biomolecular probe in a solvent selected from the group consisting of: (a) An aqueous solution buffered with about 100mM MES and about 140mM guanidine-HCl at a pH of about 5.5, and (b) an aqueous solution buffered with about 100mM EPPS and about 140mM guanidine-HCl at a pH of about 8,

wherein the concentration of the biomolecular probe ranges from about 0.05mg/mL to about 5mg/mL;

(ii) Providing a substrate having a surface comprising an epoxy-based resin;

(iii) Washing the substrate having a surface comprising an epoxy resin with a Phosphate Buffered Saline (PBS) solution containing 0.05% tween-20 to provide a PBS-washed substrate having a surface comprising an epoxy resin;

(iv) Washing the PBS-washed substrate having a surface comprising an epoxy resin with DMSO to provide a DMSO-washed substrate having a surface comprising an epoxy resin;

(v) Combining the DMSO-washed substrate having an epoxy-containing surface with a solution of the biomolecular probe, wherein the DMSO-washed substrate having an epoxy-containing surfaceIs in a concentration range of about 0.05x 10 ⁶ substrate/mL to about 5.0X10 ⁶ A substrate/mL to provide a substrate having a surface comprising an epoxy resin in which the biomolecular probe is directly bound to the epoxy resin; and is also provided with

(vi) The substrate having the surface comprising epoxy resin in which the biomolecular probe is directly bound to the epoxy resin is washed with PBS containing about 1% bsa, about 0.05% tween-20 and about 0.05% proclin 950 at a pH of about 7.4.

21. The method of claim 20, wherein the biomolecular probe is an antibody.

22. A method of preparing a substrate for biological analysis, comprising:

(i) Providing a solution of a biomolecular probe in DMSO containing about 1% tween-20, wherein the concentration of the biomolecular probe ranges from about 0.05mg/mL to about 5mg/mL;

(ii) Providing a substrate having a surface comprising an epoxy-based resin;

(iii) Washing the substrate having a surface comprising an epoxy resin with DMSO containing about 1% tween-20 to provide a DMSO-washed substrate having a surface comprising an epoxy resin;

(iv) Combining the DMSO-washed substrate having an epoxy-containing surface with a solution of the biomolecular probe, wherein the DMSO-washed substrate having an epoxy-containing surface has a concentration in the range of about 0.05x 10 ⁶ substrate/mL to about 5.0X10 ⁶ A substrate/mL to provide a substrate having a surface comprising an epoxy resin in which the biomolecular probe is directly bound to the epoxy resin; and is also provided with

(v) The substrate having a surface comprising an epoxy resin in which the biomolecular probe is directly bound to the epoxy resin is treated with PBS containing about 1% BSA, about 0.05% Tween-20, and about 0.05% Proclin 950 at a pH of about 7.4.

23. The method of claim 22, wherein the biomolecular probe comprises a cysteine residue.

24. 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 roundworm, an antibody that specifically binds to a fecal antigen from whipworm, an antibody that specifically binds to a fecal antigen from hookworm, an antibody that specifically binds to a fecal antigen from tapeworm, an antibody that specifically binds to an antigen from heartworm, and an antibody that specifically binds to a fecal antigen from giardia.

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

26. The method of claim 2, wherein the biomolecular probe is a protein expressed by an infectious source, a portion of a protein expressed by an infectious agent, a peptide or recombinant protein derived from a protein expressed by an infectious source, or a variant of a protein expressed by an infectious source.

27. The method of claim 28, wherein the biomolecular probe is capable of specifically binding to an antibody raised by the subject against a bacterium from the genus selected from the group consisting of the genus erigeron, the genus anamorpha, and the genus borrelia.

28. The method of claim 29, wherein the biomolecular probe is capable of specifically binding to an antibody raised by the subject against a bacterium selected from the group consisting of canine, chalcone, eugenone, phagocytophilic anaplasma, platyphagous anaplasma, and borrelia burgdorferi.

29. The method of claim 28, wherein the biomolecular probe is capable of specifically binding to antibodies raised against heartworm of the subject.

30. The method of claim 3, wherein the biomolecular probe is an antibody that specifically binds to a heartworm antigen.

31. The method of claim 28, wherein the biomolecular probe is capable of specifically binding an antibody to a metabolite.

32. The method of claim 33, wherein the metabolite is SDMA.

33. A method for determining the presence of an analyte in a sample, comprising contacting the sample with a substrate having a surface comprising an epoxy resin, the substrate having a surface comprising an epoxy resin having biomolecular probes directly bound to the epoxy resin, wherein the biomolecular probes specifically bind to the analyte.

34. The method of claim 35, wherein the sample is a fecal sample.

35. The method of claim 35, wherein the analyte is an antigen expressed by a intestinal worm and the biomolecular probe is an antibody to the antigen produced by the intestinal worm.

36. The method of claim 37, wherein the antibody to the antigen produced by the intestinal worm is selected from the group consisting of: an antibody that specifically binds to a fecal antigen from roundworm, an antibody that specifically binds to a fecal antigen from whipworm, an antibody that specifically binds to a fecal antigen from hookworm, an antibody that specifically binds to a fecal antigen from tapeworm, an antibody that specifically binds to an antigen from heartworm, and an antibody that specifically binds to a fecal antigen from giardia.

37. The method of claim 35, wherein the sample is a blood sample from a subject.

38. The method of claim 35, wherein the analyte is an antibody produced by an immune response of a subject to a protein produced by an infectious source, and the biomolecular probe is the protein, a portion of the protein produced by an infectious source, or a variant of the protein produced by an infectious source.

39. The method of claim 40, wherein the biomolecular probe is capable of specifically binding to antibodies raised by a subject against bacteria from the genus: the genus is selected from the group consisting of the genus erigeron, the genus intangium, and the genus borrelia.

40. The method of claim 41, wherein the biomolecular probe is capable of specifically binding to an antibody raised by the subject against a bacterium selected from the group consisting of canine, chalcone, eugenone, phagocytophilic anaplasma, platyphagous anaplasma, and borrelia burgdorferi.

41. The method of claim 40, wherein the biomolecular probe is capable of specifically binding to antibodies raised against heartworm of a subject.

42. The method of claim 40, wherein the biomolecular probe is capable of specifically binding an antibody to a metabolite.

43. The method of claim 44, wherein the metabolite is SDMA.

44. The method of claim 39, wherein the analyte is a pathogen-derived antigen, a fragment of the antigen, or a variant of the pathogen-derived antigen, and the biomolecular probe is an antibody specific for the antigen.

45. The method of claim 46, wherein the antigen is heartworm.

46. A method of determining the presence of an analyte in a sample, comprising:

(a) Forming a complex between the analyte and a biochemical probe in a first buffer solution, wherein the complex provides a signal;

(b) Replacing the first buffer solution with a second buffer solution; and is also provided with

(c) The intensity of the signal is read.

47. The method of claim 48, wherein the first buffer solution has a first ionic strength, the second buffer solution has a second ionic strength, and the second ionic strength is higher than the first ionic strength.

48. A method as in claim 49, wherein the second ionic strength is at least 0.4M.

49. A method as in claim 49, wherein the second ionic strength is at least 0.5M.

50. A method as in claim 49, wherein the second ionic strength is at least 0.6M.

51. The method of claim 48, wherein the second buffer is a citrate buffer.

52. The method of claim 53, wherein the citrate buffer has an ionic strength of at least 0.5M.

53. The method of claim 53, wherein the citrate buffer comprises trisodium citrate dihydrate, citric acid, sodium chloride, proclin 950, and has a pH ranging from about 5.5 to about 7.0.

54. The method of claim 55, wherein the pH is in the range of about pH 6.0 to about 6.5.

55. A process as set forth in claim 56 wherein said pH ranges from about pH 6.1 to about 6.3.

56. The method of claim 55, wherein the trisodium citrate dihydrate is at a concentration of about 0.485M, the citric acid is at a concentration of about 0.015M, the sodium chloride is at a concentration of about 0.1M, and the proclin 950 is at a concentration of about 0.5mL/L.

57. The method of claim 48, wherein the analyte is a peptide derived from a protein sequence of the genus Anemone, erickettsia or borrelia.

58. The method of claim 57, wherein the peptide is a peptide derived from the protein sequence of phagocyte anaplasma, flate anaplasma, canine, especially elctrolyte and borrelia burgdorferi.

59. The method of claim 48, wherein the analyte is an antibody capable of specifically binding to an antigen from heartworm (heartworm canis).

60. The method of claim 48, wherein the biomolecular probe is bound to the epoxy.

61. The method of claim 48, 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.

62. The method of claim 48, 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.