Dual-scale multiplex array for detection of small proteins, inflammatory cytokines, and pathogen-specific antibodies in human serum

Abstract

A method and system for the simultaneous detection of low abundance and high abundance proteins using arrayed imaging reflectometry (AIR) are described. In particular, a method and system for increasing the sensitivity of AIR for detecting low abundance proteins while simultaneously detecting high abundance proteins are described.

Description

[0001] Attorney Docket No. 204606-0181-00WC)

[0002] DUAL-SCALE MULTIPLEX ARRAY FOR DETECTION OF SMALL PROTEINS, INFLAMMATORY CYTOKINES, AND PATHOGEN-SPECIFIC ANTIBODIES IN HUMAN SERUM

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 681,444 filed on August 9, 2024, incorporated herein by reference in its entirety.

[0005] FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0006] This invention was made with government support under AR072000 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND OF THE INVENTION

[0008] Detection of extremely low levels of a broad range of nanoscale targets remains a significant challenge, particularly when detecting extremely low levels of a first target while simultaneously detecting relatively high levels of a second target. Blood serum proteins known or suspected to have a critical role in human disease are targets of particular interest. While individual tests are available for some of these proteins, the dynamic range, sensitivity, and multiplex capability of these tests can be lacking. What is needed is a system for detecting multiple proteins simultaneously with high sensitivity and dynamic range. The methods and systems described herein satisfy this unmet need.

[0009] SUMMARY OF THE INVENTION

[0010] In some embodiments, the invention relates to a method for simultaneous detection of low abundance (pg / mL) and high abundance (>0. 1 pg / mL) proteins using arrayed imaging reflectometry (AIR), the method comprising: a) providing an AIR sensor chip comprising a first surface bound capture molecule that specifically binds a target low abundance protein, and a second surface bound capture molecule that specifically binds a target high abundance protein; b) incubating the sensor chip with a sample solution comprising the target low abundance protein and the target high abundance protein; c) incubating the sensor chip with a solution comprising a detection molecule that specifically binds the target low abundance protein; d) incubating the sensor chip with a solution comprising an enzyme-conjugated molecule that binds the detection molecule, thereby creating a mass-enhanced detection molecule complex comprising the first surface bound capture molecule, the target low abundance protein, the detection molecule, and the enzyme- conjugated molecule; e) incubating the sensor chip with a solution comprising an enzymatic substrate, wherein the enzyme-conjugated molecule generates a polymer from the substrate at the site of the mass-enhanced detection complex; and f) measuring the signal of the sensor chip using AIR for (1) the bound mass-enhanced detection molecule complex and polymer, and (2) the bound target high abundance protein.

[0011] In some embodiments, the method further comprises g) comparing the measured signal to an AIR response calibration of a known series of target low abundance protein concentrations after formation of a mass-enhanced detection molecule complex and incubation with a solution comprising an enzymatic substrate, and a known series of target high abundance protein concentrations; and h) determining the concentration of target low abundance protein and target high abundance protein in the sample.

[0012] In some embodiments, the detection molecule comprises at least one molecule selected from the group consisting of a protein, a protein complex, a polymer complex, a dextran complex, a peptide tag, a lanthanide element, a nucleotide tag, a nanoparticle, a polymer nanoparticle, a dendrimer, a large molecular complex, high optical density molecules, high optical density molecular complexes, and high optical density particles. In some embodiments, the detection molecule comprises a biotin-conjugated antibody that specifically binds the target low abundance protein.

[0013] In some embodiments, the enzyme-conjugated molecule comprises at least one molecule selected from the group consisting of streptavidin, horseradish peroxidase (HRP), alkaline phosphatase (AP), a metal nanoparticle, a polymer nanoparticle, and a dendrimer. In some embodiments, the enzyme-conjugated molecule comprises streptavidin conjugated to polymerized HRP.

[0014] In some embodiments, the enzymatic substrate comprises 3,3’- diaminobenzidine (DAB), 4-chloro-l-napthol (4CN), 3,3’,5,5’-tetramethylbenzidine (TMB), nitro blue tetrazolium (NBT), or 5-bromo-4-chloro-3-indolyl phosphate (BCIP). In some embodiments, the enzymatic substrate comprises DAB. In some embodiments, the sensor chip is incubated with the sample solution for 17-20 hours at about 4°C. In some embodiments, the sensor chip is incubated with the sample solution for 1 hour at about 22°C.

[0015] In some embodiments, the solution comprising the detection molecule comprises the detection molecule at a concentration between 0.1 pg / mL and 1 pg / mL. In some embodiments, the sensor chip is incubated with the solution comprising the detection molecule for between 10 and 60 minutes. In some embodiments, the solution comprising the detection molecule comprises the detection molecule at a concentration of 0.5 pg / mL and the sensor chip is incubated with the solution comprising the detection molecule for 30 minutes.

[0016] In some embodiments, the solution comprising the enzyme-conjugated molecule comprises the enzyme-conjugated molecule at a concentration between 1 pg / mL and 5pg / mL. In some embodiments, the sensor chip is incubated with the solution comprising the enzyme-conjugated molecule for between 5 and 15 minutes. In some embodiments, the solution comprising the enzyme-conjugated molecule comprises the enzyme-conjugated molecule at a concentration of 1 pg / mL and the sensor chip is incubated with the solution comprising the enzyme-conjugated molecule for 10 minutes.

[0017] In some embodiments, the solution comprising the enzymatic substrate comprises the enzymatic substrate at a concentration between 0.1 and 1 mg / mL. In some embodiments, the sensor chip is incubated with the solution comprising the enzymatic substrate for between 5 and 20 minutes. In some embodiments, the solution comprising the enzymatic substrate comprises the enzymatic substrate at a concentration of 0.5 mg / mL and the sensor chip is incubated with the solution comprising the enzymatic substrate for 15 minutes.

[0018] In some embodiments, the sample solution comprises the target low abundance protein at a concentration between 1 fg / mL and 100 ng / mL.

[0019] In some embodiments, the sample solution comprises the target high abundance protein at a concentration of at least 1 pg / mL.

[0020] In some embodiments, the signal is a differential reflectance signal.

[0021] In some embodiments, the sample solution comprises serum, plasma, wholeblood, saliva, urine, cerebral spinal fluid, tumor aspirate, infection site aspirate, Medium Enriched for Secreted Antibodies. In some embodiments, the sample solution comprises serum.

[0022] In some embodiments, the first surface bound capture molecule is an antibody that specifically binds the target low abundance protein. In some embodiments, the second surface bound capture molecule is an antigen that specifically binds the target high abundance protein.

[0023] In some embodiments, the target low abundance protein is a cytokine, chemokine, growth factor, T-cell exhaustion marker, a bacterial antigen, a viral antigen, or a cancer antigen. In some embodiments, the target low abundance protein is a cytokine selected from the group consisting of IL-6, IL-10, IL-17A, IL-17F, IL-27, and TNFa.

[0024] In some embodiments, the target high abundance protein is an antibody. In some embodiments, wherein the target high abundance protein is an antibody against a bacterial antigen, a viral antigen, or a cancer antigen, or is an autoantibody. In some embodiments, the target high abundance protein is C-Reactive Protein (CRP).

[0025] In some embodiments, the invention relates to a system comprising a device for simultaneous detection of low abundance and high abundance proteins in a sample using arrayed imaging reflectometry (AIR), comprising: an AIR sensor chip comprising a first surface bound capture molecule that specifically binds a target low abundance protein, and a second surface bound capture molecule that specifically binds a target high abundance protein; a detection molecule that specifically binds the target low abundance protein; an enzyme-conjugated molecule that specifically binds the detection molecule; and an enzymatic substrate, wherein the enzyme-conjugated molecule generates a polymer from the substrate.

[0026] In some embodiments, the system further comprises at least one component selected from the group consisting of a light source, a spatial filter, a collimating lens, and a camera. In some embodiments, the light source is a HeNe laser. In some embodiments, the light source is polarized. In some embodiments, the camera is a charge-coupled device (CCD) camera.

[0027] In some embodiments, the detection molecule comprises at least one molecule selected from the group consisting of a protein, a protein complex, a polymer complex, a dextran complex, a peptide tag, a lanthanide element, a nucleotide tag, a nanoparticle, a polymer nanoparticle, a dendrimer, a large molecular complex, high optical density molecules, high optical density molecular complexes, and high optical density particles. In some embodiments, the detection molecule comprises a biotin-conjugated antibody that specifically binds the target low abundance protein.

[0028] In some embodiments, the enzyme-conjugated molecule comprises at least one molecule selected from the group consisting of streptavidin, horseradish peroxidase (HRP), alkaline phosphatase (AP), a metal nanoparticle, a polymer nanoparticle, and a dendrimer. In some embodiments, the enzyme-conjugated molecule comprises streptavidin conjugated to polymerized HRP.

[0029] In some embodiments, the enzymatic substrate comprises 3,3’- diaminobenzidine (DAB), 4-chloro-l-napthol (4CN), 3,3’,5,5’-tetramethylbenzidine (TMB), nitro blue tetrazolium (NBT), or 5-bromo-4-chloro-3-indolyl phosphate (BCIP). In some embodiments, the enzymatic substrate comprises DAB.

[0030] In some embodiments, the sensor chip is incubated with the sample solution comprising the low abundance target protein and the high abundance target protein for 17-20 hours at about 4°C. In some embodiments, the sensor chip is incubated with the sample solution comprising the low abundance target protein and the high abundance target protein for 1 hour at about 22°C.

[0031] In some embodiments, the solution comprising the detection molecule comprises the detection molecule at a concentration between 0.1 pg / mL and 1 pg / mL. In some embodiments, the sensor chip is incubated with the solution comprising the detection molecule for between 10 and 60 minutes. In some embodiments, the solution comprising the detection molecule comprises the detection molecule at a concentration of 0.5 pg / mL and the sensor chip is incubated with the solution comprising the detection molecule for 30 minutes.

[0032] In some embodiments, the solution comprising the enzyme-conjugated molecule comprises the enzyme-conjugated molecule at a concentration between 1 pg / mL and 5pg / mL. In some embodiments, the sensor chip is incubated with the solution comprising the enzyme-conjugated molecule for between 5 and 15 minutes. In some embodiments, the solution comprising the enzyme-conjugated molecule comprises the enzyme-conjugated molecule at a concentration of 1 pg / mL and the sensor chip is incubated with the solution comprising the enzyme-conjugated molecule for 10 minutes. In some embodiments, the solution comprising the enzymatic substrate comprises the enzymatic substrate at a concentration between 0.1 to 1 mg / mL. In some embodiments, the sensor chip is incubated with the solution comprising the enzymatic substrate for between 5 and 20 minutes. In some embodiments, the solution comprising the enzymatic substrate comprises the enzymatic substrate at a concentration of 0.5 mg / mL and the sensor chip is incubated with the solution comprising the enzymatic substrate for 15 minutes.

[0033] In some embodiments, the sample solution comprises the target low abundance protein at a concentration between 1 fg / mL 100 ng / mL.

[0034] In some embodiments, the sample solution comprises the target high abundance protein at a concentration of at least 1 pg / mL.

[0035] In some embodiments, the sample comprises serum, plasma, whole-blood, saliva, urine, cerebral spinal fluid, tumor aspirate, infection site aspirate, or Medium Enriched for Secreted Antibodies. In some embodiments, the sample comprises serum.

[0036] In some embodiments, the target low abundance protein is a cytokine, a bacterial antigen, a viral antigen, or a cancer antigen. In some embodiments, the target low abundance protein is a cytokine selected from the group consisting of IL-6, IL-10, IL-17A, IL-17F, IL-27, and TNFa.

[0037] In some embodiments, the target high abundance protein is an antibody. In some embodiments, the target high abundance protein is an antibody against a bacterial antigen, a viral antigen, or a cancer antigen, or is an autoantibody. In some embodiments, the target high abundance protein is C-reactive protein. (CRP).

[0038] In some embodiments, the first surface bound capture molecule is an antibody that specifically binds the target low abundance protein. In some embodiments, the second surface bound capture molecule is an antigen that specifically binds the target high abundance protein.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0041] Figure 1 depicts a flow diagram of an exemplary method of the present invention.

[0042] Figure 2 depicts a model of the low-abundance target-specific mass-building amplification strategy.

[0043] Figure 3 depicts the results of example experiments using dual array without HRP / DAB signal enhancement demonstrating the unique ability of label -free arrayed imaging reflectometry (AIR) technology to simultaneously detect small proteins, cytokines, and antibodies.

[0044] Figure 4 depicts the stepwise addition of sample, detection molecule, enzyme- conjugated molecule, and enzymatic substrate, with AIR images of actual probe spots at each step.

[0045] Figure 5, comprising Figure 5A and Figure 5B, depicts the results of example experiments demonstrating detection antibodies pre-conjugated to poly-HRP SA have no significant cross-binding to other cytokines or probes on the assay.

[0046] Figure 6 depicts standard curves of AIR response vs. known concentrations of target IL-6 cytokine. A linear fit for low concentrations (<25 pg / mL) of IL-6 enables interpolation of a lower limit of detection of 1.4 pg / mL. Also depicted is the full dilution range (1 pg / mL to 1 pg / mL) with an expected Langmuir binding curve with a 4-parameter logistic (4PL) fit.

[0047] Figure 7 depicts standard curves of AIR response vs. known concentrations of target IL-17A cytokine. A linear fit for low concentrations (<10 pg / mL) of IL-17A enables interpolation of a lower limit of detection of 1.4 pg / mL. Also depicted is a full dilution range (1 pg / mL to 1 pg / mL) with an expected Langmuir binding curve with 4PL fit.

[0048] Figure 8 depicts a summary of example experiments demonstrating Limit of the Blank, Lower limit of detection, and limit of quantitation with CV < 20% reported in target concentration in the dual scale AIR assay for IL-6, IL-17A, 1L-17F, IL-27, IL- 10, TNFa, Procalcitonin, and C-Reactive Protein. Figure 9 depicts the results of example experiments demonstrating the ability of the dual-scale array strategy to detect cytokines and small proteins as well as antibodies in a diabetic foot infection serum sample.

[0049] Figure 10 depicts the results of example experiments demonstrating the effect of DAB incubation time on AIR response at different target concentrations of cytokines (1000, 100, and 10 pg / mL). P-values were calculated from a one-way ANOVA assuming unequal variance.

[0050] Figure 11 depicts raw AIR images demonstrating the effect of HRP- conjugated detection antibody concentration and DAB incubation time on image quality and probe spot intensity.

[0051] Figure 12 depicts the results of example experiments demonstrating that preincubation of biotin-conjugated detection antibodies with Streptavidin poly-HRP does not achieve single-digit pg / mL detection.

[0052] Figure 13 depicts the results of example experiments demonstrating that the noise, measured as the standard deviation of technical replicate probes, increases with increased SA-poly-HRP concentration and incubation time.

[0053] Figure 14 depicts calculated lower limits of detection for each protein at each SA-poly-HRP incubation condition. The overall best condition that results in an acceptable lower limit of detection for all proteins is 1 pg / mL for 10 minutes. Linear Fits of AIR response for each protein at this condition are shown. Assay performance deteriorates at longer incubations and higher SA-poly-HRP concentrations for some proteins.

[0054] Figure 15 depicts the results of example experiments demonstrating that detection antibody concentrations of 0.1 pg / mL are not sufficient to reach LLOD goals, but detection antibody concentrations of 0.5 pg / mL do enable single-digit pg / mL LLODs.

[0055] DETAILED DESCRIPTION

[0056] The present invention relates to a system for the simultaneous detection of low abundance and high abundance proteins in a sample using AIR.

[0057] In some embodiments, the invention relates to a method for the simultaneous detection of low abundance and high abundance proteins. In some embodiments, the method comprises the steps of: a) providing an AIR sensor chip, b) incubating the sensor chip with a sample solution, c) incubating the sensor chip with a solution comprising a detection molecule, d) incubating the sensor chip with a solution comprising an enzyme-conjugated molecule, e) incubating the sensor chip with a solution comprising an enzymatic substate, and f) measuring the signal of the sensor chip using AIR.

[0058] In some embodiments, the invention relates to a method of diagnosing, determining the risk of developing, or monitoring a disease or disorder associated with the presence or relative level of at least one protein in a subject. Accordingly, the present invention provides methods for identifying subjects who have developed or are at risk of developing an infection, autoimmune disease, cancer, or another disease or disorder.

[0059] Definitions

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0061] As used herein, each of the following terms has the meaning associated with it in this section.

[0062] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0063] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate.

[0064] The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Tn: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0065] The term “antigen” or “Ag” as used herein is defined as a molecule that can bind to a specific antibody. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.

[0066] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0067] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0068] As used herein, the terms “fragment” or “functional fragment” refer to a fragment of a molecule, such as an antibody or antigen. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length molecule. The term “label” when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to a probe to generate a “labeled” probe. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition (e.g., HRP-DAB).

[0069] The “level” of one or more molecule means the absolute or relative amount or concentration of the molecule in the sample.

[0070] “Measuring” or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of a subject’s clinical parameters.

[0071] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0072] A “reference level” of a molecule means a level of the molecule that is indicative of a particular disease state, phenotype, or lack thereof, as well as combinations of disease states, phenotypes, or lack thereof. A “positive” reference level of a molecule means a level that is indicative of a particular disease state or phenotype. A “negative” reference level of a molecule means a level that is indicative of a lack of a particular disease state or phenotype.

[0073] “Sample” or “biological sample” as used herein means a biological material isolated from an individual. The biological sample may contain any biological material suitable for detecting the desired biomarkers and may comprise cellular and / or non-cellular material obtained from the individual.

[0074] “Standard control value” as used herein refers to a predetermined amount of a particular molecule that is detectable in a sample, such as a serum sample. The standard control value is suitable for the use of a method of the present invention, in order for comparing the amount of a molecule of interest that is present in a sample. An established sample serving as a standard control provides an average amount of the molecule of interest in the sample that is typical for an average, healthy person of reasonably matched background, e.g., gender, age, ethnicity, and medical history. A standard control value may vary depending on the molecule of interest and the nature of the sample.

[0075] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6 and any whole and partial increments therebetween. This applies regardless of the breadth of the range.

[0076] System

[0077] In some embodiments, the invention relates to a system for the simultaneous detection of low abundance and high abundance proteins in a sample using arrayed imaging reflectometry (AIR).

[0078] In some embodiments, the system comprises an AIR sensor chip. In some embodiments, the sensor chip includes a multilayer substrate comprising a surface comprising at least two surface-bound capture molecules. In some embodiments, the surfacebound capture molecule is an antibody, an antigen, an antibody fragment, a protein, an oligonucleotide, or a nucleotide aptamer.

[0079] In some embodiments, the chip comprises a first surface-bound capture molecule and a second surface-bound capture molecule. In some embodiments, the first surface-bound capture molecule specifically binds a target low abundance protein, and the second surface-bound capture molecule specifically binds a target high abundance protein. In some embodiments, the first surface-bound capture molecule is an antibody, an antigen, an antibody fragment, a protein, an oligonucleotide, a nucleotide aptamer. In some embodiments, the first surface-bound capture molecule is an antibody that specifically binds the target low abundance protein. In some embodiments, the second surface-bound capture molecule is an antibody, an antigen, an antibody fragment, a protein, an oligonucleotide, or a nucleotide aptamer. In some embodiments, the second surface-bound capture molecule is an antigen that specifically binds the target high abundance protein. In some embodiments, the second surface-bound capture molecule is an enterotoxin that specifically binds the target high abundance protein.

[0080] In some embodiments, the chip comprises a plurality of surface-bound capture molecules that specifically bind a plurality of low abundance proteins. In some embodiments, the chip comprises a plurality of surface-bound capture molecules selected from antibodies, antigens, antibody fragments, proteins, oligonucleotides, nucleotide aptamers, or a combination thereof that specifically bind a plurality of low abundance proteins. In some embodiments, the chip comprises a plurality of surface-bound antibodies that specifically bind a plurality of low abundance proteins.

[0081] In some embodiments, the chip comprises a plurality of surface-bound capture molecules that specifically bind a plurality of high abundance proteins. In some embodiments, the chip comprises a plurality of surface-bound capture molecules selected from antibodies, antigens, antibody fragments, proteins, oligonucleotides, nucleotide aptamers, or a combination thereof that specifically bind a plurality of high abundance proteins. In some embodiments, the chip comprises a plurality of surface-bound antigens that specifically bind a plurality of high abundance proteins. In some embodiments, the chip comprises a combination of at least one surfaced-bound antigen and at least one surfacebound enterotoxin that specifically bind a plurality of high abundance proteins.

[0082] In some embodiments, the capture molecules may be bound to the surface of the AIR sensor chip by a coating or an array of coatings on the AIR sensor chip. In some embodiments, the coating is a single layer of material. In some embodiments, the coating is a plurality of layers of material. In some embodiments, the material is selected from silicon dioxide (glass), silicon nitride, a polymer, and an evaporated molecular material.

[0083] In some embodiments, the surface of the AIR sensor chip forms a coating that results in destructive interference of polarized light illuminating the surface at an appropriate incident angle and wavelength in the absence of binding to the capture molecule. In some embodiments, exposure of the surface to a sample comprising the target low abundance and / or target high abundance protein produces a detectable change in reflectance at a location upon binding of the target low abundance or target high abundance protein.

[0084] The AIR sensor chip is further described in U.S. Pat. No 7,292,349, U.S. Pat. No. 7,692,798, and U.S. Pat. No. 10,209,254, the disclosures of which are incorporated herein by reference in its entirety.

[0085] In some embodiments, the sensor chip is incubated with the sample solution comprising a target low abundance protein and a target high abundance protein. In some embodiments, the sample solution comprises the target low abundance protein at a concentration between 1 fg / mL and 100 ng / mL. In some embodiments, the sample solution comprises the target high abundance protein at a concentration of at least 0.1 pg / mL.

[0086] In some embodiments, the sample solution comprises at least one biological sample. Biological samples can comprise, for example, serum, plasma, whole-blood, saliva, urine, cerebral spinal fluid, tumor aspirate, infection site aspirate, or medium enriched for secreted antibodies. In some embodiments, the biological sample is serum. In some embodiments, the serum comprises proteins or antibodies formed in response to an infection by a microorganism, bacteria, or virus, other foreign proteins, or proteins or antibodies derived from an autoimmune disease or cancer.

[0087] In some embodiments, the system comprises a detection molecule. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule. In some embodiments, the detection molecule comprises at least one of a protein, a protein complex, a polymer complex, a dextran complex, a peptide tag, a lanthanide element, a nucleotide tag, a nanoparticle, a polymer nanoparticle, a dendrimer, a large molecular complex, a high optical density molecule, a high optical density molecular complex, and a high optical density particle. In some embodiments, the detection molecule comprises an antibody or antibody fragment. In some embodiments, the detection molecule specifically binds the target low abundance protein. In some embodiments, the detection molecule comprises a biotin-conjugated antibody that specifically binds the target low-abundance protein.

[0088] In some embodiments, the system comprises an enzyme-conjugated molecule. In some embodiments, the enzyme-conjugated molecule binds the detection molecule. In some embodiments, the enzyme-conjugated molecule binds the detection molecule through a covalent bond. In some embodiments, the enzyme-conjugated molecule binds the detection molecule through a non-covalent bond.

[0089] In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule. In some embodiments, incubating the sensor chip with the solution comprising an enzyme-conjugated molecule creates a mass-enhanced detection molecule complex comprising the first surface-bound capture molecule, the target low abundance protein, the detection molecule, and the enzyme-conjugated molecule.

[0090] In some embodiments, the enzyme-conjugated molecule comprises a protein, a protein complex, a polymer complex, a dextran complex, a peptide tag, a lanthanide element, a nucleotide tag, a nanoparticle, a large molecular complex, a high optical density molecule, a high optical density molecular complex, or a high optical density particle. In some embodiments, the enzyme-conjugated molecule comprises at least one molecule selected from streptavidin, horseradish peroxidase (HRP), alkaline phosphatase (AP), a metal nanoparticle, a polymer nanoparticle, and a dendrimer. In some embodiments, the enzyme- conjugated molecule comprises streptavidin conjugated to polymerized HRP.

[0091] In some embodiments, the system comprises an enzymatic substrate. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate. In some embodiments, the enzyme-conjugated molecule generates a polymer from the substrate at the site of the mass-enhanced detection complex. In some embodiments, the enzymatic substrate comprises at least one molecule selected from 3,3 ’-diaminobenzidine (DAB), 4-chloro-l-napthol (4CN), 3,3’,5,5’-tetramethylbenzidine (TMB), nitro blue tetrazolium (NBT), or 5-bromo-4-chloro-3-indolyl phosphate (BCIP). In some embodiments, the enzymatic substrate comprises DAB.

[0092] In some embodiments, the system comprises a light source. In some embodiments, the light source is positioned to direct at least a portion of light from the light source toward the coating on the receptor in a manner effective to result in a condition of near perfect interference. By condition of “near perfect interference”, it is intended that the reflectivity of light (in the absence of bound target molecules) is less than about 104, or less than about I O5, or less than about 106or even less than about I O7. In some embodiments, the light source is tunable, collimated, and / or monochromatic. In some embodiments, the light source is polarized. In some embodiments, the light source is non-polarized. In some embodiments, the light source is non-polarized, and the system further comprises a polarizer. In some embodiments, the light source is a light-emitting diode, a laser, or a lamp with a narrow bandpass filter. In some embodiments, the light source is a HeNe laser.

[0093] In some embodiments, the system comprises a spatial filter. In some embodiments, the spatial filter comprises and objective. In some embodiments, the spatial filter comprises an optical pinhole. In some embodiments, the objective is a lens. In some embodiments, the optical pinhole is disposed at the focal point of the objective.

[0094] In some embodiments, the system comprises a collimating lens. The collimating lens can be any lens, lens system, or other optical device configured to calibrate the pointing and focus of an optical imaging system.

[0095] In some embodiments, the system comprises a detector. The detector can include any reflected beam measurement device known in the art. In some embodiments, the receptor is a spectrometer. In some embodiments, the detector is a camera. In some embodiments, the camera is a charge-coupled device.

[0096] Method of Detection

[0097] The present invention relates to a method for the simultaneous detection of low abundance and high abundance proteins. In some embodiments, the method comprises detecting one high abundance protein and one low abundance protein. In some embodiments, the method comprises detecting a plurality of high abundance proteins and a target low abundance protein. In some embodiments, the method comprises detecting a high abundance protein and a plurality of low abundance proteins. In some embodiments, the method comprises detecting a plurality of high abundance proteins and a plurality of low abundance proteins.

[0098] In some embodiments, the method comprises the steps of: a) providing an AIR sensor chip, b) incubating the sensor chip with a sample solution, c) incubating the sensor chip with a solution comprising a detection molecule, d) incubating the sensor chip with a solution comprising an enzyme-conjugated molecule, e) incubating the sensor chip with a solution comprising an enzymatic substate, and f) measuring the signal of the sensor chip using AIR. In some embodiments, step a) comprises providing an AIR sensor chip. In some embodiments, the sensor chip comprises a substrate, one or more coating layers of the substrate, and at least one probe or capture molecule bound to the coating surface. In some embodiments, the surface-bound capture molecule is an antibody, an antigen, an antibody fragment, a protein, an oligonucleotide, a nucleotide aptamer.

[0099] In some embodiments, the sensor chip comprises at least two surface-bound capture molecules. In some embodiments, the chip comprises a first surface-bound capture molecule and a second surface-bound capture molecule. In some embodiments, the first surface-bound capture molecule specifically binds a target low abundance protein, and the second surface-bound capture molecule specifically binds a target high abundance protein. In some embodiments, the first surface-bound capture molecule is an antibody, an antigen, an antibody fragment, a protein, an oligonucleotide, or a nucleotide aptamer. In some embodiments, the first surface-bound capture molecule is an antibody that specifically binds the target low abundance protein. In some embodiments, the second surface-bound capture molecule is an antibody, an antigen, an antibody fragment, a protein, an oligonucleotide, or a nucleotide aptamer. In some embodiments, the second surface-bound capture molecule is an antigen that specifically binds the target high abundance protein. In some embodiments, the second surface-bound capture molecule is an enterotoxin that specifically binds the target high abundance protein.

[0100] In some embodiments, the chip comprises a plurality of surface-bound capture molecules that specifically bind a plurality of low abundance proteins. In some embodiments, the chip comprises a plurality of surface-bound capture molecules selected from antibodies, an antigens, antibody fragments, a proteins, oligonucleotides, nucleotide aptamers, or a combination thereof that specifically bind a plurality of low abundance proteins. In some embodiments, the chip comprises a plurality of surfaced-bound antibodies that specifically bind a plurality of low abundance proteins.

[0101] In some embodiments, the chip comprises a plurality of surface-bound capture molecules that specifically bind a plurality of high abundance proteins. In some embodiments, the chip comprises a plurality of surface-bound capture molecules selected from antibodies, antigens, antibody fragments, a proteins, oligonucleotides, nucleotide aptamers, or a combination thereof that specifically bind a plurality of high abundance proteins. In some embodiments, the chip comprises a plurality of surfaced-bound antigens that specifically bind a plurality of high abundance proteins. In some embodiments, the chip comprises a combination of at least one surfaced-bound antigen and at least one surfacebound enterotoxin that specifically bind a plurality of high abundance proteins.

[0102] The capture molecules may be bound to the surface of the AIR sensor chip by a coating or an array of coatings on the AIR sensor chip. In some embodiments, the coating is a single layer of material. In some embodiments, the coating is a plurality of layers of material. In some embodiments, the material is selected from silicon dioxide (glass), silicon nitride, a polymer, and an evaporated molecular material.

[0103] In some embodiments, the surface of the AIR sensor chip forms a coating that results in destructive interference of polarized light illuminating the surface at an appropriate incident angle and wavelength in the absence of binding to the capture molecule. In some embodiments, exposure of the surface to a sample comprising the target low abundance and / or target high abundance protein produces a detectable change in reflectance at a location upon binding of the target low abundance or target high abundance protein.

[0104] The AIR sensor chip is further described in U.S. Pat. No 7,292,349, U.S. Pat. No. 7,692,798, and U.S. Pat. No. 10,209,254, the disclosures of which are incorporated herein by reference in its entirety.

[0105] In some embodiments, step b) comprises incubating the sensor chip with a sample solution. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 0°C and 10°C. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 0°C and 10°C for 10 to 30 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 0°C and 10°C for 15 to 25 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 0°C and 10°C for 17 to 20 hours.

[0106] In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 2°C and 6°C. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 2°C and 6°C for 10 to 30 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 2°C and 6°C for 15 to 25 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 2°C and 6°C for 17 to 20 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 3°C and 5°C. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 3°C and 5°C for 10 to 30 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 3°C and 5°C for 15 to 25 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 3°C and 5°C for 17 to 20 hours.

[0107] In some embodiments, the sensor chip is incubated with the sample solution at about 4°C. In some embodiments, the sensor chip is incubated with the sample solution at a temperature of about 4°C for 10 to 30 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature of about 4°C for 15 to 25 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature of about 4°C for 17 to 20 hours.

[0108] In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 17°C and 27°C. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 17°C and 27°C for 15 minutes to 4 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 17°C and 27°C for 30 minutes to 2 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 17°C and 27°C for 45 minutes to 1.5 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 17°C and 27°C for 1 hour.

[0109] In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 20°C and 24°C. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 20°C and 24°C for 15 minutes to 4 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 20°C and 24°C for 30 minutes to 2 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 20°C and 24°C for 45 minutes to 1.5 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 20°C and 24°C for 1 hour.

[0110] In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 21°C and 23°C. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 21°C and 23°C for 15 minutes to 4 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 21°C and 23°C for 30 minutes to 2 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 21°C and 23°C for 45 minutes to 1.5 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature between 21°C and 23°C for 1 hour.

[0111] In some embodiments, the sensor chip is incubated with the sample solution at a temperature of about 22°C. In some embodiments, the sensor chip is incubated with the sample solution at a temperature of about 22°C for 15 minutes to 4 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature of about 22°C for 30 minutes to 2 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature of about 22°C for 45 minutes to 1.5 hours. In some embodiments, the sensor chip is incubated with the sample solution at a temperature of about 22°C for 1 hour.

[0112] In some embodiments, the sensor chip is incubated with a sample solution comprising a target low abundance protein and a target high abundance protein. In some embodiments, the sensor chip is incubated with a sample solution comprising the target low abundance protein at a concentration between 1 fg / mL and 100 ng / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target low abundance protein at a concentration between 1 fg / mL and 1000 fg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target low abundance protein at a concentration between 1 pg / mL and 1000 pg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target low abundance protein at a concentration between 1 ng / mL and 100 ng / mL.

[0113] In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 0.1 pg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 0.5 pg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 1 pg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 10 pg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 50 pg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 100 pg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 500 pg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 1000 pg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 10 mg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 50 mg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 100 mg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 500 mg / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 1 g / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 5 g / mL. In some embodiments, the sensor chip is incubated with a sample solution comprising the target high abundance protein at a concentration of at least 10 g / mL.

[0114] In some embodiments the target low abundance protein is a cytokine, chemokine, growth factor, T-cell exhaustion marker, bacterial antigen or other bacterial protein, viral antigen or other viral protein, cancer antigen or other cancer protein, inflammation marker proteins, antibody, antibody fragment, membrane protein, toxin, pollutant, nucleic acid, or drug molecule. In some embodiments, the target low abundance protein is a cytokine. In some embodiments, the target low abundance protein is a cytokine selected from the group consisting of IL-6, IL- 10, IL-17A, IL-17F, IL-27, and TNFa.

[0115] In some embodiments of the methods and system described herein, the target high abundance protein is an antibody or antibody fragment, cytokine, chemokine, growth factor, T-cell exhaustion marker, bacterial antigen or other bacterial protein, viral antigen or other viral protein, cancer antigen or other cancer protein, inflammation marker protein, antibody, antibody fragment, membrane protein, a toxin, pollutant, nucleic acid, or drug molecule. In some embodiments, the target high abundance protein is an antibody against a bacterial antigen, a viral antigen, a cancer antigen, or a fragment thereof, or is an autoantibody or a fragment thereof. In some embodiments, the target high abundance protein is C-Reactive Protein (CRP).

[0116] In some embodiments, the target high abundance protein is an antibody to S. aureus. In some embodiments, the target low abundance protein is selected from the group consisting of IL-6, IL- 10, IL-17A, IL-17F, IL-27, and TNFa.

[0117] In some embodiments, the sample solution comprises at least one biological sample. Biological samples can comprise, for example, serum, plasma, whole-blood, saliva, urine, cerebral spinal fluid, tumor aspirate, infection site aspirate, or medium enriched for secreted antibodies. In some embodiments, the sample solution comprises serum. In some embodiments, the serum comprises proteins or antibodies formed in response to an infection by a microorganism, bacteria, or virus, other foreign proteins, or proteins or antibodies derived from an autoimmune disease.

[0118] In some embodiments, step c) comprises incubating the sensor chip with a solution comprising a detection molecule. In some embodiments, the detection molecule comprises at least one of a protein, a protein complex, a polymer complex, a dextran complex, a peptide tag, a lanthanide element, a nucleotide tag, a nanoparticle, a polymer nanoparticle, a dendrimer, a large molecular complex, a high optical density molecule, a high optical density molecular complex, and a high optical density particle. In some embodiments, the detection molecule comprises an antibody or antibody fragment. In some embodiments, the detection molecule specifically binds the target low abundance protein. In some embodiments, the detection molecule comprises a biotin-conjugated antibody that specifically binds the target low-abundance protein.

[0119] In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.01 and 10 pg / mL. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.01 and 10 pg / mL for 1 minute to 2 hours. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.01 and 10 pg / mL for 10 minutes to 60 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.01 and 10 pg / mL for 20 minutes to 40 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.01 and 10 pg / mL for 30 minutes.

[0120] In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.05 and 5 pg / mL. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.05 and 5 pg / mL for 1 minute to 2 hours. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.05 and 5 pg / mL for 10 minutes to 60 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.05 and 5 pg / mL for 20 minutes to 40 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.05 and 5 pg / mL for 30 minutes.

[0121] In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.1 and 1 pg / mL. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.1 and 1 pg / mL for 1 minute to 2 hours. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.1 and 1 pg / mL for 10 minutes to 60 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.1 and 1 pg / mL for 20 minutes to 40 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration between 0.1 and 1 pg / mL for 30 minutes.

[0122] In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration of 0.5 pg / mL. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration of 0.5 pg / mL for 1 minute to 2 hours. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration of 0.5 pg / mL for 10 minutes to 60 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration of 0.5 pg / mL for 20 minutes to 40 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the detection molecule at a concentration of 0.5 pg / mL for 30 minutes.

[0123] In some embodiments, step d) comprises incubating the sensor chip with a solution comprising an enzyme-conjugated molecule. In some embodiments, the enzyme- conjugated molecule binds the detection molecule. In some embodiments, the enzyme- conjugated molecule binds the detection molecule through a covalent bond. In some embodiments, the enzyme-conjugated molecule binds the detection molecule through a non- covalent bond.

[0124] In some embodiments, incubating the sensor chip with the solution comprising an enzyme-conjugated molecule creates a mass-enhanced detection molecule complex comprising the first surface-bound capture molecule, the target low abundance protein, the detection molecule, and the enzyme-conjugated molecule.

[0125] In some embodiments, the enzyme-conjugated molecule comprises a protein, a protein complex, a polymer complex, a dextran complex, a peptide tag, a lanthanide element, a nucleotide tag, a nanoparticle, a large molecular complex, a high optical density molecule, a high optical density molecular complex, or a high optical density particle. In some embodiments, the enzyme-conjugated molecule comprises at least one molecule selected from streptavidin, horseradish peroxidase (HRP), alkaline phosphatase (AP), a metal nanoparticle, a polymer nanoparticle, and a dendrimer. In some embodiments, the enzyme- conjugated molecule comprises streptavidin conjugated to polymerized HRP.

[0126] In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration between 0.1 pg / mL and 10 pg / mL. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme- conjugated molecule at a concentration between 0.1 pg / mL and 10 pg / mL for 1 minute to 30 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration between 0.1 pg / mL and 10 pg / mL for 5 minutes to 15 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration between 0.1 pg / mL and 10 pg / mL for 7 minutes to 12 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration between 0.1 pg / mL and 10 pg / mL for 10 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration between 0.5 pg / mL and 5 pg / mL. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme- conjugated molecule at a concentration between 0.5 pg / mL and 5 pg / mL for 1 minute to 30 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration between 0.5 pg / mL and 5 pg / mL for 5 minutes to 15 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration between 0.5 pg / mL and 5 pg / mL for 7 minutes to 12 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration between 0.5 pg / mL and 5 pg / mL for 10 minutes.

[0127] In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration between 0.75 pg / mL and 2.5 pg / mL. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme- conjugated molecule at a concentration between 0.75 pg / mL and 2.5 pg / mL for 1 minute to 30 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration between 0.75 pg / mL and 2.5 pg / mL for 5 minutes to 15 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration between 0.75 pg / mL and 2.5 pg / mL for 7 minutes to 12 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration between 0.75 pg / mL and 2.5 pg / mL for 10 minutes.

[0128] In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration of 1 pg / mL. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration of 1 pg / mL for 1 minute to 30 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration of 1 pg / mL for 5 minutes to 15 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration of 1 pg / mL for 7 minutes to 12 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration of 1 pg / mL for 10 minutes.

[0129] In some embodiments, step e) comprises incubating the sensor chip with a solution comprising an enzymatic substrate. In some embodiments, the enzyme-conjugated molecule generates a polymer from the substrate at the site of the mass-enhanced detection complex. In some embodiments, the enzymatic substrate comprises at least one molecule selected from 3,3 ’-diaminobenzidine (DAB), 4-chloro-l-napthol (4CN), 3, 3’, 5,5’- tetramethylbenzidine (TMB), nitro blue tetrazolium (NBT), or 5-bromo-4-chl oro-3 -indolyl phosphate (BCIP). In some embodiments, the enzymatic substrate comprises DAB.

[0130] In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.01 mg / mL and 10 mg / mL. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.01 mg / mL and 10 mg / mL for 1 minute to 30 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.01 mg / mL and 10 mg / mL for 5 minutes to 25 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.01 mg / mL and 10 mg / mL for 10 minutes to 20 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.01 mg / mL and 10 mg / mL for 15 minutes.

[0131] In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.1 mg / mL and 1 mg / mL. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.1 mg / mL and 1 mg / mL for 1 minute to 30 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0. 1 mg / mL and 1 mg / mL for 5 minutes to 25 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.1 mg / mL and 1 mg / mL for 10 minutes to 20 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.1 mg / mL and 1 mg / mL for 15 minutes.

[0132] In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.3 mg / mL and 0.8 mg / mL. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.3 mg / mL and 0.8 mg / mL for 1 minute to 30 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.3 mg / mL and 0.8 mg / mL for 5 minutes to 25 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.3 mg / mL and 0.8 mg / mL for 10 minutes to 20 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration between 0.3 mg / mL and 0.8 mg / mL for 15 minutes.

[0133] In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration of 0.5 mg / mL. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration of 0.5 mg / mL for 1 minute to 30 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration of 0.5 mg / mL for 5 minutes to 25 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration of 0.5 mg / mL for 10 minutes to 20 minutes. In some embodiments, the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration of 0.5 mg / mL for 15 minutes.

[0134] In some embodiments, step f) comprises measuring the signal of the sensor chip using AIR. In some embodiments, the signal from the arrayed imaging reflectometry sensor measures the optical thickness and refractive index of at least one bound target protein. In some embodiments, step f) comprises measuring the signal of the sensor chip using AIR for the bound mass-enhanced detection molecule complex and polymer. In some embodiments, step f) comprises measuring the signal of the sensor chip using AIR for the bound target high abundance protein. In some embodiments, step f) comprises measuring the signal of the sensor chip using AIR for (1) the bound mass-enhanced detection molecule complex and polymer, and (2) the bound target high abundance protein.

[0135] In some embodiments, the method further comprises the step of g) comparing the measured signal to an AIR response calibration. In some embodiments, step g) comprises comparing the measured signal to an AIR response calibration of a known series of target low abundance protein concentrations after formation of a mass-enhanced detection molecule complex and incubation with a solution comprising an enzymatic substrate. In some embodiments, step g) comprises comparing the measured signal to an AIR response calibration of a known series of target high abundance protein concentrations.

[0136] In some embodiments, the method further comprises the step of: h) determining the concentration of the target high abundance and target low abundance protein in the sample.

[0137] In some embodiments, the method comprises the steps of: a) providing an AIR sensor chip with bound capture molecules specific to an antibody to 5. aureus and at least one protein selected from IL-6, IL- 10, IL-17A, IL-17F, IL- 27, and TNFa, b) incubating the sensor chip with a sample solution comprising serum at a temperature of about 22°C for 1 hour or at a temperature of about 4°C for 17 to 20 hours, c) incubating the sensor chip with a solution comprising a detection molecule, wherein the detection molecule comprises a biotin-conjugated antibody that specifically binds the target low-abundance protein(s), and wherein the sensor chip is incubated with the solution comprising the detection molecule at a concentration of 0.5 pg / mL for 30 minutes, d) incubating the sensor chip with a solution comprising an enzyme- conjugated molecule, wherein the enzyme-conjugated molecule comprises streptavidin conjugated to polymerized HRP, and wherein the sensor chip is incubated with a solution comprising the enzyme-conjugated molecule at a concentration of 1 pg / mL for 10 minutes, e) incubating the sensor chip with a solution comprising an enzymatic substate, wherein the enzymatic substrate comprises DAB, and the sensor chip is incubated with a solution comprising the enzymatic substrate at a concentration of 0.5 mg / mL for 15 minutes, and f) measuring the signal of the sensor chip using AIR.

[0138] Method of diagnosis

[0139] In some embodiments, the invention relates to a method of diagnosing, determining the risk of developing, or monitoring a disease or disorder associated with the presence or relative level of at least one protein in a subject. Accordingly, the present invention provides methods for identifying subjects who have developed or are at risk of developing an infection, autoimmune disease, cancer, or another disease or disorder. Examples of infections include, but are not limited to, bacterial, viral, fungal, parasitic, bone and joint, sepsis, skin and soft tissue, ocular, and respiratory infections. Bacterial infections include, but are not limited to, infections of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus spp., Enterococcus spp., Cutibacterium acnes, Pseudomonas spp., Enterobacteriaceae spp., and Haemophilus spp., wherein spp. species pluralis), is defined as any species of the genus.

[0140] Examples of autoimmune diseases include, but are not limited to, systemic lupus erythematosus (SLE), dermatomyositis, type 1 diabetes, rheumatoid arthritis, multiple sclerosis, Graves’ disease, psoriasis, inflammatory bowel disease, Sjogren's syndrome, atopic dermatitis, and celiac disease.

[0141] Examples of cancers include, but are not limited to, breast, gastric, lung, colorectal, head, neck, pancreatic, prostate, and skin (e g., melanoma) cancers.

[0142] In some embodiments, the method of diagnosing, determining the risk of developing, or monitoring a disease or disorder comprises detecting a high abundance and / or low abundance protein in a sample using the methods described herein.

[0143] In some embodiments, the subject is a human subject and may be of any race, gender, and age. Information obtained from the methods of the invention described herein can be used alone or in combination with other information (e.g., disease status, disease history, demographic information, vital signs, blood chemistry, etc.) from the subject or from the biological sample obtained from the subject.

[0144] In some embodiments, a biological sample from a subject is assessed for the level of a target low abundance protein and / or a target high abundance protein. In some embodiments, the level of the target protein is determined to be increased when the level of said target proteins detected in a biological sample of a subject is increased when compared with a comparator control. In some embodiments, the comparator control is a positive control, a negative control, a historical control, a historical norm, or the level of a reference molecule in the biological sample.

[0145] In some embodiments, the level of the target protein is determined to be elevated when the level of the target protein in the sample is increased by at least 10%, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 175%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, by at least 500%, by at least 600%, by at least 700%, by at least 800%, by at least 900%, by at least 1000%, by at least 1500%, by at least 2000%, by at least 2500%, by at least 3000%, by at least 4000%, or by at least 5000%, when compared with a comparator control.

[0146] In some embodiments, the level of the target protein is determined to be decreased when the level of the target protein in the sample is decreased by at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least

[0147] 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least

[0148] 2.9 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 75 fold, at least 100 fold, at least 200 fold, at least 250 fold, at least 500 fold, or at least 1000 fold, when compared with a comparator control.

[0149] Embodiments

[0150] The following causes describe particular Embodiments of the invention.

[0151] Embodiment 1 is a method for simultaneous detection of low abundance (pg / mL) and high abundance (>0.1 pg / mL) proteins using arrayed imaging reflectometry (AIR), the method comprising: a. providing an AIR sensor chip comprising a first surface bound capture molecule that specifically binds a target low abundance protein, and a second surface bound capture molecule that specifically binds a target high abundance protein; b. incubating the sensor chip with a sample solution comprising the target low abundance protein and the target high abundance protein; c. incubating the sensor chip with a solution comprising a detection molecule that specifically binds the target low abundance protein; d. incubating the sensor chip with a solution comprising an enzyme-conjugated molecule that binds the detection molecule, thereby creating a mass-enhanced detection molecule complex comprising the first surface bound capture molecule, the target low abundance protein, the detection molecule, and the enzyme-conjugated molecule; e. incubating the sensor chip with a solution comprising an enzymatic substrate, wherein the enzyme-conjugated molecule generates a polymer from the substrate at the site of the mass-enhanced detection complex; and f. measuring the signal of the sensor chip using AIR for (1) the bound mass- enhanced detection molecule complex and polymer, and (2) the bound target high abundance protein.

[0152] Embodiment 2 is the method of embodiment 1, further comprising: g. comparing the measured signal to an AIR response calibration of a known series of target low abundance protein concentrations after formation of a mass-enhanced detection molecule complex and incubation with a solution comprising an enzymatic substrate, and a known series of target high abundance protein concentrations; and h. determining the concentration of target low abundance protein and target high abundance protein in the sample.

[0153] Embodiment 3 is the method of embodiment 1 or 2, wherein the detection molecule comprises at least one molecule selected from the group consisting of a protein, a protein complex, a polymer complex, a dextran complex, a peptide tag, a lanthanide element, a nucleotide tag, a nanoparticle, a polymer nanoparticle, a dendrimer, a large molecular complex, high optical density molecules, high optical density molecular complexes, and high optical density particles.

[0154] Embodiment 4 is the method of any one of embodiments 1-3, wherein the detection molecule comprises a biotin-conjugated antibody that specifically binds the target low abundance protein.

[0155] Embodiment 5 is the method of any of embodiments 1-4, wherein the enzyme-conjugated molecule comprises at least one molecule selected from the group consisting of streptavidin, horseradish peroxidase (HRP), alkaline phosphatase (AP), a metal nanoparticle, a polymer nanoparticle, and a dendrimer. Embodiment 6 is the method of embodiment 5, wherein the enzyme- conjugated molecule comprises streptavidin conjugated to polymerized HRP.

[0156] Embodiment 7 is the method of any of embodiments 1-6, wherein the enzymatic substrate comprises 3,3 ’-diaminobenzidine (DAB), 4-chloro-l-napthol (4CN), 3,3’,5,5’-tetramethylbenzidine (TMB), nitro blue tetrazolium (NBT), or 5-bromo-4-chloro-3- indolyl phosphate (BCIP).

[0157] Embodiment 8 is the method of embodiment 7, wherein the enzymatic substrate comprises DAB.

[0158] Embodiment 9 is the method of any of embodiments 1-8, wherein the sensor chip is incubated with the sample solution for 17-20 hours at about 4°C.

[0159] Embodiment 10 is the method of any of embodiments 1-8, wherein the sensor chip is incubated with the sample solution for 1 hour at about 22°C.

[0160] Embodiment 11 is the method of any of embodiments 1-10, wherein the solution comprising the detection molecule comprises the detection molecule at a concentration between 0.1 pg / mL and 1 pg / mL.

[0161] Embodiment 12 is the method of any of embodiments 1-11, wherein the sensor chip is incubated with the solution comprising the detection molecule for between 10 and 60 minutes.

[0162] Embodiment 13 is the method of any of embodiments 1-12, wherein the solution comprising the detection molecule comprises the detection molecule at a concentration of 0.5 pg / mL and the sensor chip is incubated with the solution comprising the detection molecule for 30 minutes.

[0163] Embodiment 14 is the method of any of embodiments 1-13, wherein the solution comprising the enzyme-conjugated molecule comprises the enzyme-conjugated molecule at a concentration between 1 pg / mL and 5pg / mL.

[0164] Embodiment 15 is the method of any of embodiments 1-14, wherein the sensor chip is incubated with the solution comprising the enzyme-conjugated molecule for between 5 and 15 minutes.

[0165] Embodiment 16 is the method of any of embodiments 1-15, wherein the solution comprising the enzyme-conjugated molecule comprises the enzyme-conjugated molecule at a concentration of 1 pg / mL and the sensor chip is incubated with the solution comprising the enzyme-conjugated molecule for 10 minutes.

[0166] Embodiment 17 is the method of any of embodiments 1-16, wherein the solution comprising the enzymatic substrate comprises the enzymatic substrate at a concentration between 0.1 and 1 mg / mL.

[0167] Embodiment 18 is the method of any of embodiments 1-17, wherein the sensor chip is incubated with the solution comprising the enzymatic substrate for between 5 and 20 minutes.

[0168] Embodiment 19 is the method of any of embodiments 1-18, wherein the solution comprising the enzymatic substrate comprises the enzymatic substrate at a concentration of 0.5 mg / mL and the sensor chip is incubated with the solution comprising the enzymatic substrate for 1 minutes.

[0169] Embodiment 20 is the method of any of embodiments 1-19, wherein the sample solution comprises the target low abundance protein at a concentration between 1 fg / mL and 100 ng / mL.

[0170] Embodiment 21 is the method of any of embodiments 1-20, wherein the sample solution comprises the target high abundance protein at a concentration of at least 1 pg / mL.

[0171] Embodiment 22 is the method of any of embodiments 1-21, wherein the signal is a differential reflectance signal.

[0172] Embodiment 23 is the method of any of embodiments 1-22, wherein the sample solution comprises serum, plasma, whole-blood, saliva, urine, cerebral spinal fluid, tumor aspirate, infection site aspirate, Medium Enriched for Secreted Antibodies.

[0173] Embodiment 24 is the method of any of embodiments 1-23, wherein the sample solution comprises serum.

[0174] Embodiment 25 is the method of any of embodiments 1-24, wherein the first surface bound capture molecule is an antibody that specifically binds the target low abundance protein.

[0175] Embodiment 26 is the method of any of embodiments 1-25, wherein the second surface bound capture molecule is an antigen that specifically binds the target high abundance protein. Embodiment 27 is the method of any of embodiments 1-26, wherein the target low abundance protein is a cytokine, chemokine, growth factor, T-cell exhaustion marker, a bacterial antigen, a viral antigen, or a cancer antigen.

[0176] Embodiment 28 is the method of embodiment 27, wherein the target low abundance protein is a cytokine selected from the group consisting of IL-6, IL-10, IL-17A, IL-17F, IL-27, and TNFa.

[0177] Embodiment 29 is the method of any of embodiments 1-28, wherein the target high abundance protein is an antibody.

[0178] Embodiment 30 is the method of embodiment 29, wherein the target high abundance protein is an antibody against a bacterial antigen, a viral antigen, or a cancer antigen, or is an autoantibody.

[0179] Embodiment 31 is the method of any of embodiments 1-28, wherein the target high abundance protein is C-Reactive Protein (CRP).

[0180] Embodiment 32 is a system comprising a device for simultaneous detection of low abundance and high abundance proteins in a sample using arrayed imaging reflectometry (AIR), comprising: an AIR sensor chip comprising a first surface bound capture molecule that specifically binds a target low abundance protein, and a second surface bound capture molecule that specifically binds a target high abundance protein; a detection molecule that specifically binds the target low abundance protein; an enzyme-conjugated molecule that specifically binds the detection molecule; and an enzymatic substrate, wherein the enzyme-conjugated molecule generates a polymer from the substrate.

[0181] Embodiment 33 is the system of embodiment 32, further comprising at least one component selected from the group consisting of a light source, a spatial filter, a collimating lens, and a camera.

[0182] Embodiment 34 is the system of embodiment 33, wherein the light source is a HeNe laser.

[0183] Embodiment 35 is the system of embodiment 33 or 34, wherein the light source is polarized. Embodiment 36 is the system of any of embodiments 32-35, wherein the camera is a charge-coupled device (CCD) camera.

[0184] Embodiment 37 is the system of any of embodiments 32-36, wherein the detection molecule comprises at least one molecule selected from the group consisting of a protein, a protein complex, a polymer complex, a dextran complex, a peptide tag, a lanthanide element, a nucleotide tag, a nanoparticle, a polymer nanoparticle, a dendrimer, a large molecular complex, high optical density molecules, high optical density molecular complexes, and high optical density particles.

[0185] Embodiment 38 is the system of embodiment 37, wherein the detection molecule comprises a biotin-conjugated antibody that specifically binds the target low abundance protein.

[0186] Embodiment 39 is the system of any of embodiments 32-38, wherein the enzyme-conjugated molecule comprises at least one molecule selected from the group consisting of streptavidin, horseradish peroxidase (HRP), alkaline phosphatase (AP), a metal nanoparticle, a polymer nanoparticle, and a dendrimer.

[0187] Embodiment 40 is the system of embodiments 39, wherein the enzyme- conjugated molecule comprises streptavidin conjugated to polymerized HRP.

[0188] Embodiment 41 is the system of any of embodiments 32-40, wherein the enzymatic substrate comprises 3,3 ’-diaminobenzidine (DAB), 4-chloro-l-napthol (4CN), 3,3’,5,5’-tetramethylbenzidine (TMB), nitro blue tetrazolium (NBT), or 5-bromo-4-chloro-3- indolyl phosphate (BCIP).

[0189] Embodiment 42 is the system of embodiment 41, wherein the enzymatic substrate comprises DAB.

[0190] Embodiment 43 is the system of any of embodiments 32-42, wherein the sensor chip is incubated with the sample solution comprising the low abundance target protein and the high abundance target protein for 17-20 hours at about 4°C.

[0191] Embodiment 44 is the system of any of embodiments 32-42, wherein the sensor chip is incubated with the sample solution comprising the low abundance target protein and the high abundance target protein for 1 hour at about 22°C. Embodiment 45 is the system of any of embodiments 32-44, wherein the solution comprising the detection molecule comprises the detection molecule at a concentration between 0.1 pg / mL and 1 pg / mL.

[0192] Embodiment 46 is the system of any of embodiments 32-45, wherein the sensor chip is incubated with the solution comprising the detection molecule for between 10 and 60 minutes.

[0193] Embodiment 47 is the system of any of embodiments 32-46, wherein the solution comprising the detection molecule comprises the detection molecule at a concentration of 0.5 pg / mL and the sensor chip is incubated with the solution comprising the detection molecule for 30 minutes.

[0194] Embodiment 48 is the system of any of embodiments 32-47, wherein the solution comprising the enzyme-conjugated molecule comprising the enzyme-conjugated molecule at a concentration between 1 pg / mL and 5pg / mL.

[0195] Embodiment 49 is the system of any of embodiments 32-48, wherein the sensor chip is incubated with the solution comprising the enzyme-conjugated molecule for between 5 and 15 minutes.

[0196] Embodiment 50 is the system of any of embodiments 32-49, wherein the solution comprising the enzyme-conjugated molecule comprises the enzyme-conjugated molecule at a concentration of 1 pg / mL and the sensor chip is incubated with the solution comprising the enzyme-conjugated molecule for 10 minutes.

[0197] Embodiment 51 is the system of any of embodiments 32-50, wherein the solution comprising the enzymatic substrate comprises the enzymatic substrate at a concentration between 0.1 to 1 mg / mL.

[0198] Embodiment 52 is the system of any of embodiments 32-51, wherein the sensor chip is incubated with the solution comprising the enzymatic substrate for between 5 and 20 minutes.

[0199] Embodiment 53 is the system of any of embodiments 32-52, wherein the solution comprising the enzymatic substrate comprises the enzymatic substrate at a concentration of 0.5 mg / mL and the sensor chip is incubated with the solution comprising the enzymatic substrate for 15 minutes. Embodiment 54 is the system of any of embodiments 32-53, wherein the sample solution comprises the target low abundance protein at a concentration between 1 fg / mL 100 ng / mL.

[0200] Embodiment 55 is the system of any of embodiments 32-54, wherein the sample solution comprises the target high abundance protein at a concentration of at least Ipg / mL.

[0201] Embodiment 56 is the system of any of embodiments 32-55, wherein the sample comprises serum, plasma, whole-blood, saliva, urine, cerebral spinal fluid, tumor aspirate, infection site aspirate, or Medium Enriched for Secreted Antibodies.

[0202] Embodiment 57 is the system of any of embodiments 32-56, wherein the sample comprises serum.

[0203] Embodiment 58 is the system of any of embodiments 32-57, wherein the target low abundance protein is a cytokine, a bacterial antigen, a viral antigen, or a cancer antigen.

[0204] Embodiment 59 is the system of embodiment 58, wherein the target low abundance protein is a cytokine selected from the group consisting of IL-6, IL-10, IL-17A, IL-17F, IL-27, and TNFa.

[0205] Embodiment 60 is the system of any of embodiments 32-59, wherein the target high abundance protein is an antibody.

[0206] Embodiment 61 is the system of embodiment 60, wherein the target high abundance protein is an antibody against a bacterial antigen, a viral antigen, or a cancer antigen, or is an autoantibody.

[0207] Embodiment 62 is the system of any of embodiments 32-59, wherein the target high abundance protein is C-reactive protein. (CRP).

[0208] Embodiment 63 is the system of any of embodiments 32-62, wherein the first surface bound capture molecule is an antibody that specifically binds the target low abundance protein.

[0209] Embodiment 64 is the system of any of embodiments 32-63, wherein the second surface bound capture molecule is an antigen that specifically binds the target high abundance protein. EXPERIMENTAL EXAMPLES

[0210] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0211] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples, therefore, specifically point out exemplary embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0212] Example 1 : Dual-scale multiplex array for simultaneous detection of small proteins, inflammatory cytokines, and pathogen-specific antibodies in human serum.

[0213] Described herein is a multiplex assay technique for simultaneous detection of small proteins, cytokines, and antibodies using Arrayed Imaging Refl ectom etry. Until now, arrayed imaging reflectometry (AIR) has been used for label-free detection of cytokines, antibodies, and small molecules in separate assays. Here, a specific mass-based amplification technique was developed to enable dual scale detection of low abundance cytokines (pg / mL) and high abundance antibodies (>0.1 pg / mL) in human serum using a single AIR assay (Figure 1).

[0214] The crux of this strategy is combining label -free detection of high abundance proteins with an amplification strategy for low abundance proteins. This consists of three components: 1) a matched pair comprising a surface bound capture molecule that specifically binds a target low abundance protein (i.e., a capture antibody that is covalently attached to the assay chip for capturing the target low abundance protein), and a detection molecule that specifically binds the target low abundance protein (i.e., a biotin-conjugated sandwich antibody that recognizes a distinct epitope on the target low abundance protein), 2) an enzyme conjugated molecule (i.e., a poly-HRP streptavidin protein), and 3) an enzymatic substrate (i.e., 3,3 ’-diaminobenzidine (DAB) substrate) (Figures 2-4). The detection of the target high abundance protein (i.e., antibody) is untouched by the amplification strategy, and the label-free nature of the AIR technology enables direct detection of these high abundance proteins bound to the capture molecules on the array. This ensures that the sensitivity of high abundance proteins is not lost due to overexposure of the array during the data collection process. Meanwhile, the mass amplification of low abundance proteins achieves limits of detection in the pg / mL range (Figures 6-8) and ensures that the AIR reflectivity is on par with that measured with direct detection of high abundance probes. Thus, the dual scale array enables sensitive measurements of proteins in human serum with a dynamic concentration range from pg / mL to mg / mL without the need for multiple sample dilutions or multiple assay substrates. This strategy was shown to successfully to detect cytokines and small proteins as well as antibodies in a diabetic foot infection serum sample (Figure 9).

[0215] While the strategy includes a method for simultaneously detecting small, low abundance inflammatory cytokines and large, high abundance human antibodies in human serum, the technique can be applied to the detection of any protein, including, but not limited to cancer antigens, viral antigens, bacterial antigens, autoantibodies for various disease conditions, protein biomarkers, and proteins from mice or other animals.

[0216] The strategy presented herein uses Horse Radish Peroxidase (HRP) enzyme with 3,3’-diaminobenzidine (DAB) substrate in the presence of Hydrogen Peroxide (H2O2). This is a well-established chromogenic system used for a variety of applications in the life sciences, but in this application it is used to measure a localized mass change due to the insoluble precipitated product rather than a color change. Detection antibodies preconjugated to poly-HRP streptavidin show no significant cross-binding to other cytokines or probes on the assay (Figure 5). Other molecule / materials that could be used for the massbuilding signal amplification purpose include, but are not limited to HRP and 4-chloro-l- napthol (4CN), HRP and 3,3’,5,5’-tetramethylbenzidine (TMB), Alkaline Phosphatase (AP) and Nito blue tetrazolium (NBT) and / or 5-bromo-4-chloro-3-indolyl phosphate (BCIP), metal nanoparticles or polymer nanoparticles, and dendrimers.

[0217] The DAB polymerization by HRP in the presence of H2O2 as used with the Arrayed Imaging Reflectometry chip was tested at four different incubation times: 10, 20, 30, and 60 minutes and at 3 different target concentrations (1000 pg / mL, 100 pg / mL, and 10 pg / mL). The goal was to determine if longer DAB incubation times would increase the AIR response in a way that would improve the lower limit of detection (LLOD) of the assay. A significant increase in AIR response at the 10 pg / mL and 100 pg / mL incubation times would be valuable for improving the LLOD, but this is not what was observed. When all capture proteins are considered in aggregate, there is a significant increase in AIR response with increased DAB incubation time at the 1000 pg / mL target concentration, which is limited by the upper limit of detection of the assay. However, DAB incubation time did not have any effect on the average AIR response at the 100 pg / mL or 10 pg / mL target concentrations (Figure 10). Therefore, it seems that DAB incubation times greater than 10 minutes do not significantly improve performance of the assay at low target concentrations.

[0218] The incubation conditions for each step of the protocol are essential for achieving the lower limits of detection (LLOD) for cytokines. The following conditions are undesirable due to increased noise and / or insufficient amplification to achieve the required LLOD: HRP-conjugated detection antibodies (Figure 11), pre-incubation of biotin- conjugated detection antibodies with the Streptavidin poly-HRP (SA-poly-HRP, Figure 12), SA-poly-HRP incubation times and concentration greater than 1 pg / mL for 5 or 10 minutes (Figures 13 and 14), and biotinylated detection antibody concentrations of 0.1 pg / mL perform poorly compared to 0.5 pg / mL (Figure 15).

[0219] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

[0220] While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. CLAIMS1. A method for simultaneous detection of low abundance (pg / mL) and high abundance (>0.1 pg / mL) proteins using arrayed imaging reflectometry (AIR), the method comprising: a. providing an AIR sensor chip comprising a first surface bound capture molecule that specifically binds a target low abundance protein, and a second surface bound capture molecule that specifically binds a target high abundance protein; b. incubating the sensor chip with a sample solution comprising the target low abundance protein and the target high abundance protein; c. incubating the sensor chip with a solution comprising a detection molecule that specifically binds the target low abundance protein; d. incubating the sensor chip with a solution comprising an enzyme-conjugated molecule that binds the detection molecule, thereby creating a mass-enhanced detection molecule complex comprising the first surface bound capture molecule, the target low abundance protein, the detection molecule, and the enzyme- conjugated molecule; e. incubating the sensor chip with a solution comprising an enzymatic substrate, wherein the enzyme-conjugated molecule generates a polymer from the substrate at the site of the mass-enhanced detection complex; and f. measuring the signal of the sensor chip using AIR for (1) the bound mass- enhanced detection molecule complex and polymer, and (2) the bound target high abundance protein.

2. The method of claim 1, further comprising: a. comparing the measured signal to an AIR response calibration of a known series of target low abundance protein concentrations after formation of a mass- enhanced detection molecule complex and incubation with a solution comprising an enzymatic substrate, and a known series of target high abundance protein concentrations; and b. determining the concentration of target low abundance protein and target high abundance protein in the sample.

3. The method of claim 1 or 2, wherein the detection molecule comprises at least one molecule selected from the group consisting of a protein, a protein complex, a polymer complex, a dextran complex, a peptide tag, a lanthanide element, a nucleotide tag, a nanoparticle, a polymer nanoparticle, a dendrimer, a large molecular complex, high optical density molecules, high optical density molecular complexes, and high optical density particles.

4. The method of claim 3, wherein the detection molecule comprises a biotin-conjugated antibody that specifically binds the target low abundance protein.

5. The method of any of claims 1-4, wherein the enzyme-conjugated molecule comprises at least one molecule selected from the group consisting of streptavidin, horseradish peroxidase (HRP), alkaline phosphatase (AP), a metal nanoparticle, a polymer nanoparticle, and a dendrimer.

6. The method of claim 5, wherein the enzyme-conjugated molecule comprises streptavidin conjugated to polymerized HRP.

7. The method of any of claims 1-6, wherein the enzymatic substrate comprises 3,3’- diaminobenzidine (DAB), 4-chloro-l-napthol (4CN), 3, 3 ’,5, 5 ’-tetramethylbenzidine (TMB), nitro blue tetrazolium (NBT), or 5-bromo-4-chloro-3-indolyl phosphate (BCIP).

8. The method of claim 7, wherein the enzymatic substrate comprises DAB.

9. The method of any of claims 1-8, wherein the sensor chip is incubated with the sample solution for 17-20 hours at about 4°C.

10. The method of any of claims 1-8, wherein the sensor chip is incubated with the sample solution for 1 hour at about 22°C.

11. The method of any of claims 1-10, wherein the solution comprising the detection molecule comprises the detection molecule at a concentration between 0.1 pg / mL and 1 pg / mL.

12. The method of any of claims 1-11, wherein the sensor chip is incubated with the solution comprising the detection molecule for between 10 and 60 minutes.

13. The method of any of claims 1-12, wherein the solution comprising the detection molecule comprises the detection molecule at a concentration of 0.5 pg / mL and the sensor chip is incubated with the solution comprising the detection molecule for 30 minutes.

14. The method of any of claims 1-13, wherein the solution comprising the enzyme- conjugated molecule comprises the enzyme-conjugated molecule at a concentration between Ipg / mL and 5pg / mL.

15. The method of any of claims 1-14, wherein the sensor chip is incubated with the solution comprising the enzyme-conjugated molecule for between 5 and 15 minutes.

16. The method of any of claims 1-15, wherein the solution comprising the enzyme- conjugated molecule comprises the enzyme-conjugated molecule at a concentration of Ipg / mL and the sensor chip is incubated with the solution comprising the enzyme- conjugated molecule for 10 minutes.

17. The method of any of claims 1-16, wherein the solution comprising the enzymatic substrate comprises the enzymatic substrate at a concentration between 0.1 and 1 mg / mL.

18. The method of any of claims 1-17, wherein the sensor chip is incubated with the solution comprising the enzymatic substrate for between 5 and 20 minutes.

19. The method of any of claims 1-18, wherein the solution comprising the enzymatic substrate comprises the enzymatic substrate at a concentration of 0.5 mg / mL and the sensor chip is incubated with the solution comprising the enzymatic substrate for 15 minutes.

20. The method of any of claims 1-19, wherein the sample solution comprises the target low abundance protein at a concentration between 1 fg / mL and 100 ng / mL.

21. The method of any of claims 1-20, wherein the sample solution comprises the target high abundance protein at a concentration of at least 1 pg / mL.

22. The method of any one of claims 1-21, wherein the signal is a differential reflectance signal.

23. The method of any of claims 1-22, wherein the sample solution comprises serum, plasma, whole-blood, saliva, urine, cerebral spinal fluid, tumor aspirate, infection site aspirate, Medium Enriched for Secreted Antibodies.

24. The method of any of claims 1-23, wherein the sample solution comprises serum.

25. The method of any of claims 1-24, wherein the first surface bound capture molecule is an antibody that specifically binds the target low abundance protein.

26. The method of any of claims 1-25, wherein the second surface bound capture molecule is an antigen that specifically binds the target high abundance protein.

27. The method of any of claims 1-26, wherein the target low abundance protein is a cytokine, chemokine, growth factor, T-cell exhaustion marker, a bacterial antigen, a viral antigen, or a cancer antigen.

28. The method of claim 27, wherein the target low abundance protein is a cytokine selected from the group consisting of IL-6, IL-10, IL-I7A, IL-17F, IL-27, and TNFa.

29. The method of any of claims 1-28, wherein the target high abundance protein is an antibody.

30. The method of claim 29, wherein the target high abundance protein is an antibody against a bacterial antigen, a viral antigen, or a cancer antigen, or is an autoantibody.

31. The method of any of claims 1-28, wherein the target high abundance protein is C- Reactive Protein (CRP).

32. A system comprising a device for simultaneous detection of low abundance and high abundance proteins in a sample using arrayed imaging reflectometry (AIR), comprising: an AIR sensor chip comprising a first surface bound capture molecule that specifically binds a target low abundance protein, and a second surface bound capture molecule that specifically binds a target high abundance protein; a detection molecule that specifically binds the target low abundance protein; an enzyme-conjugated molecule that specifically binds the detection molecule; and an enzymatic substrate, wherein the enzyme-conjugated molecule generates a polymer from the substrate.

33. The system of claim 32, further comprising at least one component selected from the group consisting of a light source, a spatial filter, a collimating lens, and a camera.

34. The system of claim 33, wherein the light source is a HeNe laser.

35. The system of claim 33 or 34, wherein the light source is polarized.

36. The system of any of claims 32-35, wherein the camera is a charge-coupled device (CCD) camera.

37. The system of any of claims 32-36, wherein the detection molecule comprises at least one molecule selected from the group consisting of a protein, a protein complex, a polymer complex, a dextran complex, a peptide tag, a lanthanide element, a nucleotide tag, a nanoparticle, a polymer nanoparticle, a dendrimer, a large molecular complex, highoptical density molecules, high optical density molecular complexes, and high optical density particles.

38. The system of claim 37, wherein the detection molecule comprises a biotin-conjugated antibody that specifically binds the target low abundance protein.

39. The system of any of claims 32-37, wherein the enzyme-conjugated molecule comprises at least one molecule selected from the group consisting of streptavidin, horseradish peroxidase (HRP), alkaline phosphatase (AP), a metal nanoparticle, a polymer nanoparticle, and a dendrimer.

40. The system of claim 39, wherein the enzyme-conjugated molecule comprises streptavidin conjugated to polymerized HRP.

41. The system of any of claims 32-40, wherein the enzymatic substrate comprises 3,3’- diaminobenzidine (DAB), 4-chloro-l-napthol (4CN), 3, 3 ’,5, 5 ’-tetramethylbenzidine (TMB), nitro blue tetrazolium (NBT), or 5-bromo-4-chloro-3-indolyl phosphate (BCIP).

42. The system of claim 41, wherein the enzymatic substrate comprises DAB.

43. The system of any of claims 32-42, wherein the sensor chip is incubated with the sample solution comprising the low abundance target protein and the high abundance target protein for 17-20 hours at about 4°C.

44. The system of any of claims 32-42, wherein the sensor chip is incubated with the sample solution comprising the low abundance target protein and the high abundance target protein for 1 hour at about 22°C.

45. The system of any of claims 32-44, wherein the solution comprising the detection molecule comprises the detection molecule at a concentration between 0.1 pg / mL and 1 pg / mL.

46. The system of any of claims 32-45, wherein the sensor chip is incubated with the solution comprising the detection molecule for between 10 and 60 minutes.

47. The system of any of claims 32-46, wherein the solution comprising the detection molecule comprises the detection molecule at a concentration of 0.5 pg / mL and the sensor chip is incubated with the solution comprising the detection molecule for 30 minutes.

48. The system of any of claims 32-47, wherein the solution comprising the enzyme- conjugated molecule comprising the enzyme-conjugated molecule at a concentration between 1 pg / mL and 5pg / mL.

49. The system of any of claims 32-48, wherein the sensor chip is incubated with the solution comprising the enzyme-conjugated molecule for between 5 and 15 minutes.

50. The system of any of claims 32-49, wherein the solution comprising the enzyme- conjugated molecule comprises the enzyme-conjugated molecule at a concentration of Ipg / mL and the sensor chip is incubated with the solution comprising the enzyme- conjugated molecule for 10 minutes.

51. The system of any of claims 32-50, wherein the solution comprising the enzymatic substrate comprises the enzymatic substrate at a concentration between 0. 1 to 1 mg / mL.

52. The system of any of claims 32-51, wherein the sensor chip is incubated with the solution comprising the enzymatic substrate for between 5 and 20 minutes.

53. The system of any of claims 32-52, wherein the solution comprising the enzymatic substrate comprises the enzymatic substrate at a concentration of 0.5 mg / mL and the sensor chip is incubated with the solution comprising the enzymatic substrate for 15 minutes.

54. The system of any of claims 32-53, wherein the sample solution comprises the target low abundance protein at a concentration between 1 fg / mL 100 ng / mL.

55. The system of any of claims 32-54, wherein the sample solution comprises the target high abundance protein at a concentration of at least I pg / mL.

56. The system of any of claims 32-55, wherein the sample comprises serum, plasma, wholeblood, saliva, urine, cerebral spinal fluid, tumor aspirate, infection site aspirate, or Medium Enriched for Secreted Antibodies.

57. The system of any of claims 32-56, wherein the sample comprises serum.

58. The system of any of claims 32-57, wherein the target low abundance protein is a cytokine, a bacterial antigen, a viral antigen, or a cancer antigen.

59. The system of claim 58, wherein the target low abundance protein is a cytokine selected from the group consisting of IL-6, IL-10, IL-17A, IL-17F, IL-27, and TNFa.

60. The system of any of claims 32-59, wherein the target high abundance protein is an antibody.

61. The system of claim 60, wherein the target high abundance protein is an antibody against a bacterial antigen, a viral antigen, or a cancer antigen, or is an autoantibody.

62. The system of any of claims 32-59, wherein the target high abundance protein is C- reactive protein. (CRP).

63. The system of any of claims 32-62, wherein the first surface bound capture molecule is an antibody that specifically binds the target low abundance protein.

64. The system of any of claims 32-63, wherein the second surface bound capture molecule is an antigen that specifically binds the target high abundance protein.

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