WO2022025967A1

WO2022025967A1 - Ultra sensitive methods

Info

Publication number: WO2022025967A1
Application number: PCT/US2020/067518
Authority: WO
Inventors: Jon J. SCHMUKE; Christopher C. Striemer; Bryan WITHERBEE; Ernest J. Mueller; Benjamin L. Miller
Original assignee: Adarza Biosystems, Inc.
Priority date: 2020-07-28
Filing date: 2020-12-30
Publication date: 2022-02-03

Abstract

The present invention provides methods for increasing the sensitivity of arrayed imaging reflectometry (AIR) sensor chip.

Description

ULTRA SENSITIVE METHODS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of U.S. Provisional Patent Application No. 63/057,735, which was filed in the U.S. Patent and Trademark Office on July 28, 2020, the entire contents of which are incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

[0002] The invention describes methods for increasing the sensitivity of arrayed imaging reflectometry (AIR).

BACKGROUND OF THE INVENTION

[0003] Detection of extremely low levels of a broad range of nanoscale targets (small molecules, large molecules, and antibodies) has been a challenge. Of particular interest are blood serum proteins known or suspected to have a critical role in human disease. While individual tests are available for some of these, the dynamic range, sensitivity, and multiplex capability of these tests can be lacking. What is needed is a system for detecting many proteins simultaneously with high sensitivity and dynamic range.

SUMMARY OF THE INVENTION

[0004] In one aspect, provided herein, are methods for increasing the sensitivity of arrayed imaging reflectometry (AIR) with a direct mass enhancement. The method comprising: (a) providing an arrayed imaging reflectometry (AIR) sensor chip comprising at least one surface bound capture antibody or antibody fragment specific to a target antigen (b) treating the sensor chip with a sample solution comprising said target antigen and a mass-enhanced detection antibody or antibody fragment specific to the target antigen; (c) rinsing the sensor chip; (d) drying the sensor chip; (e) measuring the signal of the sensor chip using arrayed imaging reflectometry for the bound target antigen and mass-enhanced detection antibody complex and comparing the signal to an AIR response plot of a known series of target antigen concentrations with the mass- enhanced detection antibody; and (f) determining the concentration of the target antigen in the sample; wherein the mass-enhanced detection antibody 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, high optical density molecules, high optical density molecular complexes, or high optical density particles.

[0005] In another aspect, provided herein, are methods for increasing the sensitivity of arrayed imaging reflectometry (AIR) with a secondary mass enhancement. The method comprising: (a) providing an arrayed imaging reflectometry (AIR) sensor chip comprising at least one surface bound capture antibody or antibody fragment specific to a target antigen; (b) treating the sensor chip with a sample solution comprising said target antigen and a linker-functionalized detection antibody or antibody fragment specific to the target antigen; (c) treating the sensor chip a second time with a mass enhancer functionalized to bind to the linker on the detection antibody or antibody fragment; (d) rinsing the sensor chip; (e) drying the sensor chip; (f) measuring the signal of the sensor chip using arrayed imaging reflectometry for the bound target antigen with the linker-functionalized antibody complexed with the mass enhancer and comparing the signal to an AIR response plot of a known series of target antigen concentrations with the linker functionalized detection antibody complexed with the mass enhancer; and (g) determining the concentration of the target antigen in the sample; wherein the linker functionalized antibody is modified with biotin, a peptide, or other linker bridging chemistry, and the functionalized mass enhancer is modified with the complimentary linker bridge and consists of 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, high optical density molecules, high optical density molecular complexes, or high optical density particles.

[0006] In yet another aspect, provided herein, are methods for increasing the sensitivity of arrayed imaging reflectometry (AIR) with chemical amplification of a direct or a secondary reactive mass enhancer. The method comprising: (a) providing an arrayed imaging reflectometry (AIR) sensor chip comprising at least one surface bound capture antibody or antibody fragment specific to a target antigen; (b) treating the sensor chip with a sample solution comprising said target antigen and a reactive mass- enhanced or linker-functionalized detection antibody or antibody fragment specific to the target antigen; (c) optionally treating the sensor chip with a reactive mass enhancer functionalized to bind to the linker on the detection antibody or antibody fragment; (d) rinsing the sensor chip; (e) exposing the clean sensor to a reactive solution that further increases the detection mass through polymerization, metal deposition, nucleotide replication or other known growth method of a reactive mass tag; (f) rinsing the chip; (g) drying the sensor chip; (h) measuring the signal of the sensor chip using arrayed imaging reflectometry for the bound target antigen and mass-enhanced or linker- functionalized antibody complex with chemical amplification and comparing the signal to an AIR response plot of a known series of target antigen concentrations with the mass- enhanced or linker functionalized antibody with chemical amplification; and (i) determining the concentration of the target antigen in the sample; wherein the linker- functionalized antibody is modified with biotin, a peptide, or other linker bridging chemistry, and the reactive mass-enhancer is optionally modified with the complimentary linker bridge and consist of an enzyme or enzyme complex, a nucleotide tag, a nanoparticle, or high optical density particles.

[0007] Other features and iterations of the invention are described in more detail below.

BRIEF DESCRIPTIONS OF THE FIGURES

[0008] Fig. 1 shows secondary mass enhancement with a streptavidin-protein complex formed from a biotinylated detection antibody, streptavidin, and a protein of known mass on the surface of the array, specific to a site of a surface bound antibody (antibody fragment) with a captured antigen.

[0009] Fig. 2 shows secondary mass enhancement with a dextran complex formed from a biotinylated detection antibody: Fig. 2a shows streptavidin and biotin- dextran; and Fig. 2b shows streptavidin-dextran of known mass specific to a site of a surface bound antibody (antibody fragment) with a captured antigen. [0010] Fig. 3 shows secondary mass enhancement with a protein complex formed from a detection antibody labelled with a peptide tag, an antibody that recognizes the peptide tag and a metal of high atomic number used to build mass on the surface of the array, specific to a site of a surface bound antibody (antibody fragment) with a captured antigen.

[0011] Fig. 4 shows direct mass enhancement with chemical amplification with a nucleotide complex formed from a detection antibody labelled with a nucleotide tag replicated using phi29 DNA polymerase to perform rolling circle amplification, building mass on the surface of the array specific to a site of a surface bound antibody (antibody fragment) with a captured antigen.

[0012] Fig 5a and Fig. 5b shows secondary mass enhancement with chemical amplification (Fig. 5a) and direct mass enhancement with chemical amplification (Fig. 5b) with a protein complex enhanced by reactive precipitation of a polymerized soluble chromogenic substrate when diaminobenzamidine (DAB) is oxidized by hydrogen peroxide in a reaction catalyzed by horseradish peroxidase (HRP) specific to a site of a surface bound antibody (antibody fragment) with a captured antigen.

[0013] Fig 6 shows direct mass enhancement with optional chemical amplification with a small nanoparticle bound to an unfunctionalized detection antibody on the surface of the array specific to a site of a surface bound antibody (antibody fragment) with a captured antigen. A subsequent chemical step allows substantial growth of the small nanoparticle into a significant mass of high refractive index at the binding site.

[0014] Fig. 7 shows direct mass enhancement with a complex formed by a large central particle or molecule covered with functionalized detection antibodies that bind directly to the surface of the array specific to a site of a surface bound antibody (antibody fragment) with a captured antigen.

[0015] Fig. 8 shows direct mass enhancement with a large complex, formed by cross-linked secondary antibody that directly binds to the surface of the array specific to a site of a surface bound antibody (antibody fragment) with a captured antigen. [0016] Fig. 9 shows direct mass enhancement with a functionalized antibody decorated with molecules, molecular complexes, or particles with high optical density that binds to the surface of the array specific to a site of a surface bound antibody (antibody fragment) with a captured antigen.

[0017] Fig. 10 shows an example of the response improvement for 4 assays observed as the size of a secondary mass enhancer is increased. Larger size adds more mass to the sensor surface, leading to a greater optical reflectance seen in the array images to the right and the titration curves on the left.

[0018] Fig. 11 shows an example of the signal amplification observed when secondary mass enhancement is followed by chemical amplification. In this example, diaminobenzamidine (DAB) is oxidized by hydrogen peroxide in a reaction catalyzed by horseradish peroxidase (HRP) specific to a site of a surface bound antibody (antibody fragment) with a captured antigen

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present disclosure provides methods for increasing the sensitivity of arrayed imaging reflectometry (AIR) sensor chip. The methods comprising utilizing a variety of chemistry based techniques to increase the mass (molecular weight) of the bound antibody-capture antigen-antibody complex. By increasing, the mass of the bound antibody-capture antigen-antibody complex wherein the capture antibody is further functionalized, increased sensitivity and improved signal to noise ratio can be achieved in extremely low concentrations of the target antigen.

I. Methods of Increasing the Sensitivity of Array Imaging Reflectometry (AIR) by Direct Mass Enhancement

[0020] One aspect of the present disclosure encompasses methods for increasing the sensitivity of arrayed imaging reflectometry (AIR) with a direct mass enhancement, the method comprising: (a) providing an arrayed imaging reflectometry (AIR) sensor chip comprising at least one surface bound capture antibody or antibody fragment specific to a target antigen; (b) treating the sensor chip with a sample solution comprising said target antigen and a mass-enhanced detection antibody or antibody fragment specific to the target antigen; (c) rinsing the sensor chip; (d) drying the sensor chip; (e) measuring the signal of the sensor chip using arrayed imaging reflectometry for the bound target antigen and mass-enhanced detection antibody complex and comparing the signal to an AIR response plot of a known series of target antigen concentrations with the mass-enhanced detection antibody; and (f) determining the concentration of the target antigen in the sample; wherein the mass-enhanced detection antibody 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, high optical density molecules, high optical density molecular complexes, or high optical density particles.

(a) providing an arrayed imaging reflectometry (AIR) sensor chip comprising at least one surface bound capture antibody or antibody fragment specific to a target antigen

[0021] The arrayed imaging reflectometry (AIR) sensor chip has previously been shown to accurately measure low concentration of large molecules, small molecules, and various antibodies. The AIR detection system is described in U.S. Pat. No. 7,292,349 to Miller et al. , U.S. Pat. No. 7,692,798 to Striemer et al. , and U.S. Pat. No. 10,209,254 to Miller et al., the disclosures of which are incorporated herein by reference in its entirety.

[0022] A common trait inherent to all biosensors, regardless of labeling status or means of signal transduction, is probe immobilization. The role of the terminal hydroxyl of a silicon dioxide surface is highly flexible as it may act as a nucleophile (Bikiaris et al., “Compatibilisation Effect of PP-g-MA Copolymer on iPP/Si02 Nanocomposites Prepared by Melt Mixing,” Eur. Polym. J. 41:1965-1978 (2005); Tripp et al. , “Chemical Attachment of Chlorosilanes to Silica: A Two-step Amine-promoted Reaction,” J. Phys. Chem. 97:5693-5698 (1993), the disclosures of which are incorporated herein by reference in their entirety) or support adsorption. For this reason, silicon dioxide is readily derivitized through a variety of chemical methods. These chemical reactions result in the effective transformation of the hydroxyl group to any of a number of chemical functionalities including, but certainly not limited to, amines (Huang et al. , “Directed Assembly of One dimensional Nanostructures Into Functional Networks,” Science 291:630-633 (2001), the disclosure of which is incorporated herein by reference in its entirety) or halides (Hergenrother et al., “Small-molecule Microarrays: Covalent Attachment and Screening of Alcohol-containing Small Molecules on Glass Slides,” J. Am. Chem. Soc. 122:7849- 7850 (2001), the disclosure of which is incorporated herein by reference in its entirety). From each initial reaction, a secondary chemical can be added to further alter the surface reactivity or probes may be directly coupled. Moreover, a multitude of functionalized silanes, molecules that couple to and self-assemble on silicon dioxide (Onclin et al., “Engineering Silicon Oxide Surfaces Using Self-assembled Monolayers,” Angew Chemie Int. Ed. 44:2-24 (2005), the disclosure of which is incorporated herein by reference in its entirety), are commercially available, and may confer a diverse chemical landscape to the surface of the substrate (amines, epoxides, alkenes, etc.). A number of these approaches are generally described in U.S. Pat. No. 7,226,733 to Chan et al. and U.S. Pat. No. 7,292,349 to Miller et al., the disclosures of which are incorporated herein by reference in their entirety.

[0023] PCT Publication No. WO 2010/039808 to Mace et al., the disclosure of which is incorporated herein by reference in its entirety, teaches the use of a non- nucleophilic additive in a formulation containing a probe molecule to be bound to an array surface (sensor chip). The non-nucleophilic additive is used in an amount effective to avoid or reduce the severity of surface morphological anomalies caused by non- homogeneous distribution of the reactant across a spot on the array where the reactant is bound. These surface morphological anomalies include bright center spots and “coffee stain” rings (or halos) that can interfere with accurate detection of target molecule binding at a particular spot. In other words, the use of effective amounts of the non-nucleophilic additive promotes substantially homogeneous distribution of the reactant across each of the spots on the array where the probe is located. By homogeneous distribution, it is intended that the variance of reactant concentration across the surface of a spot is minimized (relative to spots prepared in the absence of the non-nucleophilic additives). Stated another way, there is e.g., less than about 10 percent pixel variation across the array spot, or less than about 5 percent variation, or less than about 3 percent variation, 2 percent variation, or even less than about 1 percent variation.

[0024] As described above, antibodies or antibody fragments can be bound directly to the sensor chip at various locations on the sensor chip surface. These covalently or non-covalently bound antibodies are capture molecules designed to specifically bind to antigen targets in a biological sample to which the sensor chip is subsequently exposed. Non-limiting examples of these antibodies and antibody fragments may be monoclonal or polyclonal IgG serotypes, single-chain antibodies, other immunoglobulins, proteins, or engineered molecules such as nucleotide aptamers, etc.

(b) treating the sensor chip with a sample solution comprising target antigen and a mass-enhanced detection antibody or antibody fragment specific to the target antigen [0025] The next step in the method comprises treating the sensor chip with a sample solution comprising the target antigen of interest and the mass-enhanced detection antibody specific to the capture antigen. During this treatment, a site specific antibody-antigen sandwich structure forms on the sensor surface, comprising the surface-bound capture antibody, the captured target antigen, and the mass-enhanced detection antibody. Non-limiting examples of these target antigens can and will vary.

As appreciated by the skilled artisan, the target antigen is specifically complexed to the bound antibody or antibody fragment. Non-limiting examples of these target antigens may be cytokines, chemokines, inflammation marker proteins, metabolic marker proteins, cancer marker proteins, antibodies, membrane proteins, virus proteins, bacterial proteins, toxins, pollutants, nucleic acids, drug molecules, etc. The mass- enhanced capture antibody is complexed with 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, high optical density molecules, high optical density molecular complexes, or high optical density particles through a covalent or non-covalent bond.

[0026] It should be noted that someone of skill in the art would recognize that this step could also be separated into two separate capture steps where the sensor chip is first exposed to a sample containing the target antigen followed by exposure to a second solution containing the mass-enhanced detection antibody. In the first step, the antigen would bind to the surface bound capture antibody and in the second step the mass-enhanced detection antibody would complete the sandwich structure by binding to the bound antigen. A rinse step may or may not be incorporated between these steps.

[0027] In various embodiments, the antibodies being used in the assay can be derived from various serological fluids, where the serum comprises antibodies formed in response to an infection (e.g. microorganism, bacteria, and virus), other foreign proteins, or proteins or antibodies derived from an autoimmune disease. Antibodies are also available commercially. The mass-enhanced antibody can be prepared from the unfunctionalized antibody using methods known in the art.

[0028] In one embodiment, the mass-enhanced antibody may comprise a protein complex. As an example, the antibody may be formed using a biotinylated detection antibody, streptavidin, and a protein of known mass complex as shown in Fig. 1. Non limiting examples of these proteins of known mass may be horseradish peroxidase, beta-galactosidase, bovine serum albumin, and similar proteins which complex with the antibody-biotin-streptavidin-protein complex. As a result, the addition of the protein complex increases the molecular weight of the antibody.

[0029] In another embodiment, the mass-enhanced antibody may be bound to a large complex molecule or nanoparticle, a protein or protein complex, a polymer complex, or a dextran complex, and may comprise a single or a plurality of antibodies that will specifically bind to the capture antigen as shown in Fig. 7 where a particle is shown as an example. At least one antibody complexes with the bound antibody- capture antigen complex. This large additional mass will enhance the AIR optical reflectance response for this target antigen and in practice would cause an array spot comprising this surface structure to show a higher signal in the array image. The higher the refractive index of this added mass, the stronger its enhancement will be realized.

[0030] In another embodiment, the mass-enhanced antibody is part of a large complexed molecule comprising a plurality of antibodies. The antibodies in the complex are specific to the target antigen and are cross-linked as shown in Fig. 8. The additional mass of the unbound antibodies in the complex would produce a much larger AIR optical reflectance signal.

[0031] In still another embodiment, the functionalized antibody is part of a high optical density molecular complexes, or bound to high optical density particles as shown in Fig. 9. These particles could be metal atoms, metal particles, dyes, fluorescent particles, semiconductor quantum dots or other materials know in the art that strongly interact with light, and would increase the AIR optical reflectance signal.

[0032] In all embodiments, the mass-enhanced antibody with a protein or protein complex, a polymer complex, a dextran complex, a nanoparticle, a large molecular complex, high optical density molecules, high optical density molecular complexes, or high optical density particles is used in excess to ensure all the capture antigens are complexed. Non-limiting examples of treating the sensor include but are not limited to a spray or a dropper or flow nozzle. Manual methods or fully automated instruments may be used for this treatment step.

[0033] Generally, the concentration of the functionalized antibody with a protein, a protein complex, a polymer complex, a dextran complex, a nanoparticle, a large molecular complex, high optical density molecules, high optical density molecular complexes, or high optical density particles may range from about 1 pg/mL to about 10 mg/ml_. In various embodiments, the concentration may range from about 1 pg/mL to about 1 pg/mL, from about 1 pg/mL to about 100 pg/mL, from about 100 pg/mL to about 1 mg/mL, or from about 1 mg/mL to about 10 mg/mL.

[0034] In general, the time for treating the sensor may range from about 1 minute to about 15 hours. In various embodiments, the time for treating the sensor may range from about 1 minute to about 15 minutes, from about 15 minutes to about 1 hour, from about 1 hour to about 4 hours, or from about 4 hours to about 15 hours. [0035] Generally, the temperature of treating the sensor chip with the solution of target antigen and the mass-enhanced antibody with a protein or protein complex, a polymer complex, a dextran complex, a nanoparticle, a large molecular complex, high optical density molecules, high optical density molecular complexes, or high optical density particles may range from 0°C to about 40°C. In various embodiments, the temperature of treating the sensor chip with the solution of the target antigen and complexed antibody may range from about 0°C to about 40°C, from about 10°C to about 30°C, or from about 20°C to about 25°C. In one preferred embodiment, the temperature of treating the sensor chip with the solution of the functionalized antibody is about room temperature or 23°C.

(c) rinsing the sensor chip

[0036] The next step in the processes is rinsing the sensor chip. This step is necessary to remove contaminants and impurities from the sample solution that are not bound to the sensor chip. By removing these other contaminants and impurities, an accurate measurement of the target antigen in the solution can be determined.

[0037] Generally, the sensor chip is rinsed with a solution comprising an aqueous buffer. This rinsing step maintains the bound antibody-capture antigen-mass-enhanced antibody sandwich but removes other contaminants and impurities. Non-limiting examples of buffers may be a phosphate buffer, and acetate buffer, a citrate buffer,

PBS, PBS-ET, or other buffers or combinations of buffers known in the art.

[0038] The buffer may be at various concentrations and various pHs. Generally, the pH of the buffer may range from about 5.0 to about 10.0. In various embodiment, the pH may range from about 5.0 to about 10.0, from about 6.0 to about 9.0, or from about 7.0 to about 8.0. As appreciated by the skilled artisan, the pH of the buffer is adjusted to the target pH of interest using either an aqueous acid or a base.

[0039] The concentration of the buffer can and will vary depending on the antigen target, capture antibody, mass-enhanced antibody, contaminants, and impurities. Generally, the concentration of the buffer may range from 0.1 wt% to about 10 wt%. In various embodiments, the concentration of the buffer may range from 0.1 wt% to 10 wt%, from 0.5 wt% to 7.5 wt%, from 1 wt% to about 5 wt%, or from 2 wt% to about 4 wt%.

[0040] The volume of the rinse can and will vary depending on the nature of the mass-enhanced antibody, what buffer is used, size of the chip and container, and the temperature of the treatment. Generally, the volume of the rinse may range from 0.1 ml_ to about 10 ml_ per chip. In various embodiments, the volume of the rinse may range from 0.1 ml_ to about 10 ml_, from about 0.5 ml_ to about 5.0 ml_, or from about 1.0 ml_ to about 2.0 ml_. The rinse may be applied in a number of ways. More than one rinse of the buffer may be used, followed by a final rinse with purified Dl water may be used to displace buffer components and prevent dissolved solids from drying onto the sensor surface and interfering with the optical signal. Non-limiting examples of applying a rinse include but are not limited to a spray or a dropper or flow nozzle. Manual methods or fully automated instruments may be used for this rinsing step.

[0041] The temperature of the rinsing step can and will vary depending on antigen target, capture antibody, mass-enhanced antibody, contaminants, and impurities in the sample solution. In general, the temperature of the rinsing step may range from 0°C to about 40°C. In various embodiments, the rinsing temperature may range from about 0°C to about 40°C, from about 10°C to about 30°C, or from about 20°C to about 25°C. In one preferred embodiment, the rinsing temperature is about room temperature or23°C.

(d) drying the sensor chip

[0042] The next step in the process is drying the chip. The sensor chip is dried to provide an accurate reading of the signal of reflectance corresponding to the optical thickness change associated with antigen binding and mass-enhancement.

[0043] Generally, the drying of the sensor chip may be conducted using air, nitrogen, an argon atmosphere, or a combination thereof. The drying may further be conducted under reduced pressure. In various embodiments, this drying step may be conducted in an instrument. [0044] The temperature of the drying may range from 0°C to about 40°C. In various embodiments, the temperature of drying may range from about 0°C to about 40°C, from about 10°C to about 30°C, or from about 20°C to about 25°C. In one preferred embodiment, the temperature of drying is about room temperature or 23°C.

[0045] The duration of drying of the sensor chip may range from about 0.1 seconds to about 2 minutes. In various embodiments, the duration of drying the sensor chip may range from about 0.1 seconds to about 1 second, from about 1 second to 5 seconds, from about 5 seconds to about 1 minute, or from about 1 minute to about 2 minutes.

(e) measuring the signal of the sensor chip using arrayed imaging reflectometry for the bound target antigen and mass-enhanced detection antibody complex and comparing the signal to an AIR standard response plot of a known series of target antigen concentrations with the mass-enhanced detection antibody

[0046] The next step in the method is to measure the signal from the sensor chip using the arrayed imaging reflectometry. As described above, the signal from arrayed imaging reflectometry measures the optical thickness and refractive index of the bound antibody-capture antigen-mass-enhanced antibody complex to a standard response plot of a known series of target antigen concentrations in the sample solution with the mass- enhanced antibody wherein the mass-enhanced antibody comprises a protein, a protein complex, a polymer complex, a dextran complex, a nanoparticle, a large molecular complex, high optical density molecules, high optical density molecular complexes, or high optical density particles.

(f) determining the concentration of the target antigen in the sample

[0047] A comparison of the signal from the sensor chip using the arrayed imaging reflectometry from the antibody-target antigen-mass-enhanced antibody complex comprising a protein, a protein complex, a polymer complex, a dextran complex, a nanoparticle, a large molecular complex, high optical density molecules, high optical density molecular complexes, or high optical density particles to a response plot of a known target antigen concentrations in sample solution with the mass-enhanced detection antibody provides a concentration of the target antigen in the sample. This comparison provides an accurate determination of the concentration of the target antigen in the biological sample. As a comparison, the antigen specific binding of the mass-enhanced detection antibody increases the thickness and/or refractive index (both real and imaginary parts of the dielectric function) of the optical interference layer, causing large changes in the magnitude of reflected light. This strategy described above is designed to increase the signal to noise ratio of a binding event in the AIR platform by up to several orders of magnitude, resulting in increased sensitivity, reduced background variability, increased utility across sample types, and a more accurate determination of the target antigen concentration at extremely low levels.

[0048] In general, the signal has increased at least 2 times in magnitude. In various embodiments, the signal has increased at least 2 times, at least 3 times, at least 10 times, at least 50 times, or more than 50 times.

II. Methods of Increasing the Sensitivity of Array Imaging Reflectometry (AIR) by Secondary Mass Enhancement

[0049] Another aspect of the present disclosure encompasses methods for increasing the sensitivity of arrayed imaging reflectometry (AIR) with a secondary mass enhancement, the method comprising: (a) providing an arrayed imaging reflectometry (AIR) sensor chip comprising at least one surface bound capture antibody or antibody fragment specific to a target antigen; (b) treating the sensor chip with a sample solution comprising said target antigen and a linker-functionalized detection antibody or antibody fragment specific to the target antigen; (c) treating the sensor chip a second time with a mass enhancer functionalized to bind to the linker on the detection antibody or antibody fragment; (d) rinsing the sensor chip; (e) drying the sensor chip; (f) measuring the signal of the sensor chip using arrayed imaging reflectometry for the bound target antigen with the linker-functionalized antibody complexed with the mass enhancer and comparing the signal to an AIR response plot of a known series of target antigen concentrations with the linker functionalized detection antibody complexed with the mass enhancer; and (g) determining the concentration of the target antigen in the sample; wherein the linker functionalized antibody is modified with biotin, a peptide, or other linker bridging chemistry, and the functionalized mass enhancer is modified with the complimentary linker bridge and consists of 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, high optical density molecules, high optical density molecular complexes, or high optical density particles.

[0050] The arrayed imaging reflectometry (AIR) sensor chip is described in more detail above.

(b) treating the sensor chip with a sample solution comprising said target antigen and a linker-functionalized detection antibody or antibody fragment specific to the target antigen

[0051] The next step in the method comprises treating the sensor chip with a sample solution comprising the target antigen of interest and the linker functionalized detection antibody specific to the capture antigen. The linker on the detection antibody allows for binding of a secondary mass enhancing structure in a subsequent treatment step to allow for process and optimization flexibility. During this treatment, a site specific antibody-antigen sandwich structure forms on the sensor surface, comprising the surface-bound capture antibody, the captured target antigen, and the linker- functionalized detection antibody. Non-limiting examples of these capture antigens can and will vary. As appreciated by the skilled artisan, the target antigen is specifically complexed to the bound antibody or antibody fragment. Non-limiting examples of these target antigens are listed above. The linker-functionalized antibody is modified with biotin, a peptide, or other linker bridging chemistry. [0052] It should be noted that someone of skill in the art would recognize that this step could also be separated into two separate capture steps where the sensor chip is first exposed to a sample containing the target antigen followed by exposure to a second solution containing the linker functionalized detection antibody. In the first step, the antigen would bind to the surface bound capture antibody and in the second step the linker functionalized detection antibody would complete the sandwich structure by binding to the bound antigen. A rinse step may or may not be incorporated between these steps.

[0053] In various embodiments, the antibodies being used in the assay can be derived from various serological fluids, where the serum comprises antibodies formed in response to an infection (e.g. microorganism, bacteria, and virus), other foreign proteins, or proteins or antibodies derived from an autoimmune disease. Antibodies are also available commercially. The linker-functionalized antibody can be prepared from the unfunctionalized antibody using methods known in the art.

[0054] Non-limiting examples of treating the sensor include but are not limited to a spray or a dropper or flow nozzle. Manual methods or fully automated instruments may be used for this treatment step.

(c) treating the sensor chip with a mass enhancer functionalized to bind to the linker on the detection antibody or antibody fragment

[0055] The next step in the method comprises treating the sensor chip with a sample solution comprising a functionalized mass enhancer modified with the complimentary linker bridge and consists of 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, high optical density molecules, high optical density molecular complexes, or high optical density particles. The complimentary linker may consist of streptavidin molecule to bind a biotin tag, an antibody specific to a peptide tag or other linker chemistry know in the art.

[0056] In one embodiment, the mass enhancer may comprise a protein or a protein complex. As an example, the detection antibody may be formed using a biotinylated detection antibody, which then binds a streptavidin-functionalized protein or protein complex of known mass as shown in Fig. 1. Non-limiting examples of these proteins or protein complexes of known mass may be horseradish peroxidase, alkaline phosphatase, beta-galactosidase, bovine serum albumin, and similar proteins which complex with the antibody-biotin-streptavidin-protein complex. As a result, the addition of the protein or the protein complex increases the molecular weight of the of the signal enhancing complex.

[0057] In another embodiment, the mass enhancer may comprise a dextran complex. As an example, the dextran complex may be formed through a biotinylated detection antibody and streptavidin-dextran or a biotinylated dextran complex of known mass as shown in Fig. 2a and 2b.

[0058] In still another embodiment, a peptide linker functionalized detection antibody is bound by an anti-peptide antibody enhancer element functionalized with a lanthanide series element as shown in Fig. 3. The peptide tag may be covalently or non-covalently bound to the functionalized antibody and may comprise a peptide such as FLAG (FLAG octapeptide, FLAG-tag, or FLAG epitope), HIS (polyhistidine-tag), HA (hemagglutinin-tag), or similar compounds. The lanthanide series element may be covalently or non-covalently bound to the anti-peptide mass enhancer complex. Non limiting examples of these elements may be Er, Tm, Yb, and Lu.

[0059] Non-limiting examples of treating the sensor include but are not limited to a spray or a dropper or flow nozzle. Manual methods or fully automated instruments may be used for this treatment step.

[0060] Generally, the concentration of the functionalized mass enhancer modified with the complimentary linker bridge and consists of 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, high optical density molecules, high optical density molecular complexes, or high optical density particles may range from about 1 pg/mL to about 10 mg/mL. In various embodiments, the concentration may range from about 1 pg/mL to about 1 pg/mL, from about 1 pg/mL to about 100 pg/mL, from about 100 pg/mL to about 1 mg/mL, or from about 1 mg/mL to about 10 mg/mL. [0061] In general, the time for treating the sensor may range from about 1 minute to about 15 hours. In various embodiments, the time for treating the sensor may range from about 1 minute to about 15 minutes, from about 15 minutes to about 1 hour, from about 1 hour to about 4 hours, or from about 4 hours to about 15 hours.

[0062] Generally, the temperature of treating the sensor chip with the solution of target antigen and the mass-enhanced antibody with a protein or protein complex, a polymer complex, a dextran complex, a nanoparticle, a large molecular complex, high optical density molecules, high optical density molecular complexes, or high optical density particles may range from 0°C to about 40°C. In various embodiments, the temperature of treating the sensor chip with the solution of the target antigen and complexed antibody may range from about 0°C to about 40°C, from about 10°C to about 30°C, or from about 20°C to about 25°C. In one preferred embodiment, the temperature of treating the sensor chip with the solution of the functionalized antibody is about room temperature or 23°C.

(d) rinsing the sensor chip

[0063] The next step in the processes is rinsing the sensor chip. This step is necessary to remove contaminants and impurities from the sample solution that are not bound to the sensor chip. By removing these other contaminants and impurities, an accurate measurement of the target antigen in the solution can be determined.

[0064] Generally, the sensor chip is rinsed with a solution comprising an aqueous buffer. This rinsing step maintains the bound antibody-capture antigen-antibody-mass enhancer structure but removes other contaminants and impurities. Non-limiting examples of buffers may be a phosphate buffer, and acetate buffer, a citrate buffer,

PBS, PBS-ET, or other buffers or combinations of buffers known in the art.

[0065] The buffer may be at various concentrations and various pHs. Generally, the pH of the buffer may range from about 5.0 to about 10.0. In various embodiment, the pH may range from about 5.0 to about 10.0, from about 6.0 to about 9.0, or from about 7.0 to about 8.0. As appreciated by the skilled artisan, the pH of the buffer is adjusted to the target pH of interest using either an aqueous acid or a base. [0066] The concentration of the buffer can and will vary depending on the antigen target, capture antibody, detection antibody, mass enhancer, contaminants, and impurities. Generally, the concentration of the buffer may range from 0.1 wt% to about 10 wt%. In various embodiments, the concentration of the buffer may range from 0.1 wt% to 10 wt%, from 0.5 wt% to 7.5 wt%, from 1 wt% to about 5 wt%, or from 2 wt% to about 4 wt%.

[0067] The volume of the rinse can and will vary depending on the nature of the functionalized antibody, what buffer is used, size of the chip and container, and the temperature of the treatment. Generally, the volume of the rinse may range from 0.1 ml_ to about 10 ml_ per chip. In various embodiments, the volume of the rinse may range from 0.1 ml_ to about 10 ml_, from about 0.5 ml_ to about 5.0 ml_, or from about 1.0 ml_ to about 2.0 ml_. The rinse may be applied in a number of ways. More than one rinse of the buffer may be used, followed by a final rinse with purified deionized (Dl) water may be used to displace buffer components and prevent dissolved solids from drying onto the sensor surface and interfering with the optical signal. Non-limiting examples of applying a rinse include but are not limited to a spray, a dropper, or a flow nozzle. Manual methods or fully automated instruments may be used for this rinsing step.

[0068] The temperature of the rinsing step can and will vary depending on antigen target, capture antibody, detection antibody, mass enhancer, contaminants, and impurities in the sample solution. In general, the temperature of the rinsing step may range from 0°C to about 40°C. In various embodiments, the rinsing temperature may range from about 0°C to about 40°C, from about 10°C to about 30°C, or from about 20°C to about 25°C. In one preferred embodiment, the rinsing temperature is about room temperature or23°C.

(e) drying the sensor chip

[0069] The next step in the process is drying the chip. The sensor chip is dried to provide an accurate reading of the signal of reflectance corresponding to the optical thickness change associated with antigen binding and mass-enhancement. [0070] Generally, the drying of the sensor chip may be conducted using air, nitrogen, an argon atmosphere, or a combination thereof. Additionally, the drying may further be conducted under reduced pressure. In various embodiments, this drying step may be conducted within an instrument.

[0071 ] The temperature of the drying may range from 0°C to about 40°C. In various embodiments, the temperature of drying may range from about 0°C to about 40°C, from about 10°C to about 30°C, or from about 20°C to about 25°C. In one preferred embodiment, the temperature of drying is about room temperature or 23°C.

[0072] The duration of drying of the sensor chip may range from about 0.1 seconds to about 2 minutes. In various embodiments, the duration of drying the sensor chip may range from about 0.1 seconds to about 1 second, from about 1 second to 5 seconds, from about 5 seconds to about 1 minute, or from about 1 minute to about 2 minutes.

(f) measuring the signal of the sensor chip using arrayed imaging reflectometry for the bound target antigen with the linker-functionalized antibody complexed with the mass enhancer and comparing the signal to an AIR response plot of a known standard series of target antigen concentrations with the linker functionalized detection antibody complexed with the mass enhancer

[0073] The next step in the method is to measure the signal from the sensor chip using the arrayed imaging reflectometry. As described above, the signal from the arrayed imaging reflectometry sensor measures the optical thickness and refractive index of the bound antibody-capture antigen-detection antibody-mass enhancer complex relative to a standard response plot of a known series of target antigen concentrations in the sample solution with the linker-functionalized antibody and subsequent exposure to a mass enhancer solution wherein the mass enhancer 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, high optical density molecules, high optical density molecular complexes, or high optical density particles. (g) determining the concentration of the target antigen in the sample

[0074] A comparison of the signal from the sensor chip using the arrayed imaging reflectometry from the antibody-target antigen-detection antibody-mass enhancer complex comprising a protein or protein complex, a polymer complex, a dextran complex, a peptide tag, a lanthanide element, a nucleotide tag, a nanoparticle, a large molecular complex, high optical density molecules, high optical density molecular complexes, or high optical density particles to a standard response plot of a known target antigen concentrations in the sample solution with the linker functionalized detection antibody provides a concentration of the target antigen in the sample. This comparison provides an accurate determination of the concentration of the target antigen in the biological sample. As a comparison, the antigen specific binding of the linker-functionalized detection antibody and subsequent bound mass enhancer increased the thickness and/or refractive index (both real and imaginary parts of the dielectric function) of the optical interference layer, causing large changes in the magnitude of reflected light. This strategy described above is designed to increase the signal to noise ratio of a binding event in the AIR platform by up to several orders of magnitude, resulting in increased sensitivity, reduced background variability, increased utility across sample types, and a more accurate determination of the target antigen concentration at extremely low levels.

[0075] In general, the signal has increased at least 2 times in magnitude. In various embodiments, the signal has increased at least 2 times, at least 3 times, at least 10 times, at least 50 times, or more.

III. Methods of Increasing the Sensitivity of Array Imaging Reflectometry (AIR) by Chemical Amplification of a Direct or a Secondary Mass Enhancer

[0076] Another aspect of the present disclosure encompasses methods for increasing the sensitivity of arrayed imaging reflectometry (AIR) with chemical amplification of a direct or a secondary reactive mass enhancer, the method comprising: (a) providing an arrayed imaging reflectometry (AIR) sensor chip comprising at least one surface bound capture antibody or antibody fragment specific to a target antigen;

(b) treating the sensor chip with a sample solution comprising said target antigen and a reactive mass-enhanced or linker-functionalized detection antibody or antibody fragment specific to the target antigen; (c) optionally treating the sensor chip with a reactive mass enhancer functionalized to bind to the linker in the detection antibody or antibody fragment; (d) rinsing the sensor chip; (e) exposing the clean sensor to a reactive solution that further increases the detection mass through polymerization, metal deposition, nucleotide replication or other known growth methods of a reactive mass tag; (f) rinsing the chip; (g) drying the sensor chip; (h) measuring the signal of the sensor chip using arrayed imaging reflectometry for the bound target antigen and reactive mass-enhanced or linker-functionalized antibody complex with chemical amplification and comparing the signal to an AIR response plot of a known series of target antigen concentrations with the reactive mass-enhanced or linker functionalized antibody with chemical amplification; and (i) determining the concentration of the target antigen in the sample; wherein the linker-functionalized antibody is modified with biotin, a peptide, or other linker bridging chemistry, and the reactive mass-enhancer is optionally modified with the complimentary linker bridge and consists of an enzyme or enzyme complex, a nucleotide tag, a nanoparticle, or high optical density particles.

[0077] The arrayed imaging reflectometry (AIR) sensor chip is described in more detail above.

(b) treating the sensor chip with a sample solution comprising said target antigen and a reactive mass-enhanced or linker-functionalized detection antibody or antibody fragment specific to the target antigen

[0078] The next step in the method comprises treating the sensor chip with a sample solution comprising the target antigen of interest and the reactive mass- enhanced or linker-functionalized detection antibody specific to the capture antigen.

The linker on the detection antibody allows for binding of a secondary reactive mass enhancer in a subsequent treatment step to allow for process and optimization flexibility. During this treatment, a site specific antibody-antigen sandwich structure forms on the sensor surface, comprising the surface-bound capture antibody, the captured target antigen, and the reactive mass-enhanced or linker-functionalized detection antibody. Non-limiting examples of these target antigens can and will vary. As appreciated by the skilled artisan, the target antigen is specifically complexed to the bound antibody or antibody fragment. Non-limiting examples of these target antigens are listed above.

The reactive mass-enhanced antibody would be modified with an enzyme or enzyme complex, a nucleotide tag, a nanoparticle, or high optical density particles. The linker- functionalized antibody would be modified with biotin, a peptide, or other linker bridging chemistry.

[0079] It should be noted that someone of skill in the art would recognize that this step could also be separated into two separate capture steps where the sensor chip is first exposed to a sample containing the target antigen followed by exposure to a second solution containing the reactive mass-functionalized or linker-functionalized detection antibody. In the first step, the antigen would bind to the surface bound capture antibody and in the second step the reactive mass-enhanced or linker- functionalized detection antibody would complete the sandwich structure by binding to the bound antigen. A rinse step may or may not be incorporated between these steps.

[0080] In various embodiments, the antibodies being used in the assay can be derived from various serological fluids, where the serum comprises antibodies formed in response to an infection (e.g. microorganism, bacteria, and virus), other foreign proteins, or proteins or antibodies derived from an autoimmune disease. Antibodies are also available commercially. The reactive mass-enhanced or linker-functionalized antibody can be prepared from the unfunctionalized antibody using methods known in the art. [0081] Non-limiting examples of treating the sensor include but are not limited to a spray or a dropper or flow nozzle. Manual methods or fully automated instruments may be used for this treatment step.

[0082] Generally, the concentration of the target antigen of interest and the reactive mass-enhanced or linker-functionalized detection antibody specific to the capture antigen may range from about 1 pg/mL to about 10 mg/ml_. In various embodiments, the concentration may range from about 1 pg/mL to about 1 pg/mL, from about 1 pg/mL to about 100 pg/mL, from about 100 pg/mL to about 1 mg/mL, or from about 1 mg/mL to about 10 mg/mL.

[0083] In general, the time for treating the sensor may range from about 1 minute to about 15 hours. In various embodiments, the time for treating the sensor may range from about 1 minute to about 15 minutes, from about 15 minutes to about 1 hour, from about 1 hour to about 4 hours, or from about 4 hours to about 15 hours.

[0084] Generally, the temperature of treating the sensor chip with the solution of target antigen and the mass-enhanced antibody with a protein, a protein complex, a polymer complex, a dextran complex, a nanoparticle, a large molecular complex, high optical density molecules, high optical density molecular complexes, or high optical density particles may range from 0°C to about 40°C. In various embodiments, the temperature of treating the sensor chip with the solution of the target antigen and complexed antibody may range from about 0°C to about 40°C, from about 10°C to about 30°C, or from about 20°C to about 25°C. In one preferred embodiment, the temperature of treating the sensor chip with the solution of the target antigen of interest and the reactive mass-enhanced or linker-functionalized detection antibody specific to the capture antigen is about room temperature or 23°C.

(c) optionally treating the sensor chip with a reactive mass enhancer functionalized to bind to the linker on the detection antibody or antibody fragment

[0085] The next step in the method comprises treating the sensor chip with a sample solution comprising with a reactive mass enhancer functionalized to bind to the linker on the detection antibody or antibody fragment. The complimentary linker may consist of a streptavidin molecule to bind a biotin tag, an antibody specific to a peptide tag or other linker chemistry know in the art.

[0086] Non-limiting examples of treating the sensor include but are not limited to a spray or a dropper or flow nozzle. Manual methods or fully automated instruments may be used for this treatment step.

[0087] Generally, the concentration of the reactive mass enhancer may range from about 1 pg/mL to about 10 mg/ml_. In various embodiments, the concentration may range from about 1 pg/mL to about 1 pg/mL, from about 1 pg/mL to about 100 pg/mL, from about 100 pg/mL to about 1 mg/mL, or from about 1 mg/mL to about 10 mg/mL.

[0088] In general, the time for treating the sensor may range from about 1 minute to about 15 hours. In various embodiments, the time for treating the sensor may range from about 1 minute to about 15 minutes, from about 15 minutes to about 1 hour, from about 1 hour to about 4 hours, or from about 4 hours to about 15 hours.

[0089] Generally, the temperature of treating the sensor chip with the solution of target antigen and the mass-enhanced antibody with a reactive mass enhancer functionalized to bind to the linker on the detection antibody or antibody fragment may range from 0°C to about 40°C. In various embodiments, the temperature of treating the sensor chip with the solution of the with a reactive mass enhancer functionalized to bind to the linker on the detection antibody or antibody fragment may range from about 0°C to about 40°C, from about 10°C to about 30°C, or from about 20°C to about 25°C. In one preferred embodiment, the temperature of treating the sensor chip with the solution of the reactive mass enhancer is about room temperature or 23°C.

(d) rinsing the sensor chip

[0090] The next step in the processes is rinsing the sensor chip. This step is necessary to remove unbound reactive mass enhancer material before the chemical amplification step. This ensures that only antigen-specific bound complexes drive the amplification process and thereby generate much stronger AIR signal response. [0091] Generally, the sensor chip is rinsed with a solution comprising an aqueous buffer. This rinsing step maintains the bound antibody-capture antigen-antibody- reactive mass enhancer structure and removes excess reactive material. Non-limiting examples of buffers may be a phosphate buffer, and acetate buffer, a citrate buffer,

PBS, PBS-ET, or other buffers or combinations of buffers known in the art.

[0092] The buffer may be at various concentrations and various pHs. Generally, the pH of the buffer may range from about 5.0 to about 10.0. In various embodiments, the pH may range from about 5.0 to about 10.0, from about 6.0 to about 9.0, or from about 7.0 to about 8.0. As appreciated by the skilled artisan, the pH of the buffer is adjusted to the target pH of interest using either an aqueous acid or a base.

[0093] The concentration of the buffer can and will vary depending on the antigen target, capture antibody, detection antibody, reactive mass enhancer, contaminants, and impurities. Generally, the concentration of the buffer may range from 0.1 wt% to about 10 wt%. In various embodiments, the concentration of the buffer may range from 0.1 wt% to 10 wt%, from 0.5 wt% to 7.5 wt%, from 1 wt% to about 5 wt%, or from 2 wt% to about 4 wt%.

[0094] The volume of the rinse can and will vary depending on the nature of the reactive mass-enhanced antibody, what buffer is used, size of the chip and container, and the temperature of the treatment. Generally, the volume of the rinse may range from 0.1 ml_ to about 10 ml_ per chip. In various embodiments, the volume of the rinse may range from 0.1 ml_ to about 10 ml_, from about 0.5 ml_ to about 5.0 ml_, or from about 1.0 ml_ to about 2.0 ml_. The rinse may be applied in a number of ways. More than one rinse of the buffer may be used, followed by a final rinse with purified Dl water may be used to displace buffer components and prevent dissolved solids from drying onto the sensor surface and interfering with the optical signal. Non-limiting examples of applying a rinse include but are not limited to a spray, a dropper, or a flow nozzle. Manual methods or fully automated instruments may be used for this rinsing step.

[0095] The temperature of the rinsing step can and will vary depending on antigen target, capture antibody, detection antibody, reactive mass enhancer, contaminants, and impurities in the sample solution. In general, the temperature of the rinsing step may range from 0°C to about 40°C. In various embodiments, the temperature of rinsing may range from about 0°C to about 40°C, from about 10°C to about 30°C, or from about 20°C to about 25°C. In one preferred embodiment, the temperature of rinsing is about room temperature or 23°C.

(e) exposing the sensor to a reactive solution that further increases the detection mass through polymerization, metal deposition, nucleotide replication or other known growth methods of a reactive mass enhancer

[0096] This is the chemical amplification step where the antigen site specific mass can be multiplied by many fold. The reactive mass enhancer seeds or catalyzes the deposition of insoluble material from soluble components in the reactive solution. Common reactive mass enhancers include enzymes that catalyze the polymerization of soluble monomers into an insoluble form that binds to the labeled complex; metal particles that seed the deposition of additional metal ions from solution, growing the particle mass substantially; and template nucleic acid strands that can be replicated repeatedly to build mass.

[0097] In one embodiment, the reactive mass-enhanced antibody may comprise a horseradish peroxidase enzyme complex. As an example with secondary mass enhancement, the detection antibody may be functionalized with biotin linker that subsequently binds to a streptavidin functionalized horseradish peroxidase enzyme complex, as shown in Fig. 1. The horseradish peroxidase enzyme may also be coupled to the detection antibody as a direct mass enhancer. The direct mass enhancer may then be further enhanced through a polymerized soluble chromogenic substrate catalyzed by the direct reactive mass enhancer as shown in Fig. 5b, or by the secondary reactive mass enhancer of Fig 5a. The polymerized soluble chromogenic substrate may be prepared in a variety of methods, such as for example, contacting diaminobenzamidine (DAB) with hydrogen peroxide in the presence of horseradish peroxidase. Other enzymes which may be used are alkaline phosphatase, acid phosphatase and similar enzymes. Other substrates which may be useful, include, but are not limited to (PNPP - p-nitrophenyl phosphate - disodium salt, ABTS - 2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt, OPD - 2,2'-azinobis [3- ethylbenzothiazoline-6-sulfonic acid]-diammonium salt, TMB - 3, 3', 5,5'- tetramethylbenzidine, 4-chloro-1-naphthol, and NBT/BCIP - nitro blue tetrazolium / 5- bromo-4-chloro-3-indolyl phosphate). This method is referred to as the precipitation method.

[0098] In another embodiment, the reactive mass-enhanced antibody may comprise a polymer formation. In this case, the antibody is covalently or non-covalently bound to at least one nucleic acid tag. This nucleic acid tag is replicated through, for example, rolling circler amplification using for example, phi29 DNA polymerase as shown in Fig. 4 to add substantial additional mass to the bound antigen specific complex, amplifying the AIR signal response.

[0099] In yet another embodiment, the reactive mass-enhanced antibody may further comprise at least one seed particle for growth of a nanoparticle as shown in Fig. 6. In this embodiment, the functionalized antibody further comprises a seed particle that is covalently or non-covalently bound to the antibody. Non-limiting examples of the seed particle may be a gold particle, a silver particle, a palladium particle, a platinum particle, a copper particle, or a nickel particle.

[00100] Subsequent chemical modifications by contacting the seed particle with a compound that allows for growth of the small seed crystal into a nanoparticle. As an example, the seed crystal may comprises a gold particle covalently or non-covalently bound to the antibody. Deposition of silver ions through methods known in the art allows for the growth of a silver nanoparticle on the gold seed. Other methods for nanoparticle growth known in the art can also be applied for this class of mass enhancement.

[00101 ] In general, the time for exposing the clean sensor chip to the reactive solution may range from about 1 second to about 5 minutes. In various embodiments, the time for treating the sensor may range from about 1 second to about 15 seconds, from about 15 seconds to about 30 seconds, from about 30 seconds to about 1 minute, or from about 1 minute to about 5 minutes. [00102] Generally, the temperature of treating the sensor chip with the reactive solution may range from 0°C to about 40°C. In various embodiments, the temperature of treating the sensor chip with the solution of the with a reactive mass enhancer functionalized to bind to the linker on the detection antibody or antibody fragment may range from about 0°C to about 40°C, from about 10°C to about 30°C, or from about 20°C to about 25°C. In one preferred embodiment, the temperature of treating the sensor chip with the solution of the reactive mass enhancer is about room temperature or 23°C.

[00103] Non-limiting examples of applying a reactive solution include but are not limited to a spray, a dropper, or a flow nozzle. Manual methods or fully automated instruments may be used for this step.

(f) rinsing the sensor chip

[00104] The next step in the processes is rinsing the sensor chip. This step is necessary to remove contaminants and impurities from the sample solution that are not bound to the sensor chip. By removing these other contaminants and impurities, an accurate measurement of the target antigen in the solution can be determined.

[00105] Generally, the sensor chip is rinsed with a solution comprising an aqueous buffer. This rinsing step maintains the bound antibody-capture antigen- antibody-reactive mass enhancer-chemically amplified precipitate structure but removes other contaminants and impurities. Non-limiting examples of buffers may be a phosphate buffer, and acetate buffer, a citrate buffer, PBS, PBS-ET, or other buffers or combinations of buffers known in the art. Deionized water may also be used as a rinse for the reactive growth solution.

[00106] The buffer may be at various concentrations and various pHs. Generally, the pH of the buffer may range from about 5.0 to about 10.0. In various embodiment, the pH may range from about 5.0 to about 10.0, from about 6.0 to about 9.0, or from about 7.0 to about 8.0. As appreciated by the skilled artisan, the pH of the buffer is adjusted to the target pH of interest using either an aqueous acid or a base. [00107] The concentration of the buffer can and will vary depending on the antigen target, capture antibody, detection antibody, mass enhancer, chemically amplified precipitate contaminants, and impurities. Generally, the concentration of the buffer may range from 0.1 wt% to about 10 wt%. In various embodiments, the concentration of the buffer may range from 0.1 wt% to 10 wt%, from 0.5 wt% to 7.5 wt%, from 1 wt% to about 5 wt%, or from 2 wt% to about 4 wt%.

[00108] The volume of the rinse can and will vary depending on the nature of the reactive mass-enhanced antibody, the composition of the reactive growth solution, what rinse buffer is used, size of the chip and container, and the temperature of the treatment. Generally, the volume of the rinse may range from 0.1 mL to about 10 ml_ per chip. In various embodiments, the volume of the rinse may range from 0.1 mL to about 10 mL, from about 0.5 mL to about 5.0 mL, or from about 1.0 mL to about 2.0 mL. The rinse may be applied in a number of ways. More than one rinse of the buffer may be used, followed by a final rinse with purified Dl water may be used to displace buffer components and prevent dissolved solids from drying onto the sensor surface and interfering with the optical signal. Non-limiting examples of applying a rinse include but are not limited to a spray, a dropper, or a flow nozzle. Manual methods or fully automated instruments may be used for this rinsing step.

[00109] The temperature of the rinsing step can and will vary depending on antigen target, capture antibody, detection antibody, reactive mass enhancer, chemically amplified precipitate, contaminants, and impurities in the sample solution. In general, the temperature of the rinsing step may range from 0°C to about 40°C. In various embodiments, the rinse temperature may range from about 0°C to about 40°C, from about 10°C to about 30°C, or from about 20°C to about 25°C. In one preferred embodiment, the rinse temperature is about room temperature or 23°C.

(g) drying the sensor chip

[00110] The next step in the process is drying the chip. The sensor chip is dried to provide an accurate reading of the reflectance signal corresponding to the optical thickness change associated with antigen binding, mass-enhancement, and chemical amplification.

[00111 ] Generally, the drying of the sensor chip may be conducted using air, nitrogen, an argon atmosphere, or a combination thereof. Additionally, the drying may further be conducted under reduced pressure.

[00112] The temperature of the drying may range from 0°C to about 40°C. In various embodiments, the temperature of drying may range from about 0°C to about 40°C, from about 10°C to about 30°C, or from about 20°C to about 25°C. In one preferred embodiment, the drying temperature is about room temperature or23°C.

[00113] The duration of drying of the sensor chip may range from about 0.1 seconds to about 2 minutes. In various embodiments, the duration of drying the sensor chip may range from about 0.1 seconds to about 1 second, from about 1 second to 5 seconds, from about 5 seconds to about 1 minute, or from about 1 minute to about 2 minutes.

(h) measuring the signal of the sensor chip using arrayed imaging reflectometry for the bound target antigen and reactive mass-enhanced or linker-functionalized antibody complex with chemical amplification and comparing the signal to an AIR standard response plot of a known series of target antigen concentrations with the reactive mass-enhanced or linker functionalized antibody with chemical amplification

[00114] The next step in the method is to measure the signal from the sensor chip using arrayed imaging reflectometry. As described above, the signal from the arrayed imaging reflectometry sensor measures the optical thickness and refractive index of the bound antibody-capture antigen-detection antibody-reactive mass enhancer-chemically amplified precipitate complex relative to a standard response plot of a known series of target antigen concentrations in the sample solution with the reactive mass-enhanced or linker-functionalized antibody and subsequent exposure to the mass enhancer and chemical amplification solutions wherein the reactive mass enhancer comprises an enzyme or enzyme complex, a nucleotide tag, a nanoparticle, or high optical density particles. (i) determining the concentration of the target antigen in the sample

[00115] A comparison of the signal from the sensor chip using the arrayed imaging reflectometry from the antibody-target antigen-detection antibody-reactive mass enhancer-chemically amplified precipitate complex to a standard response plot of a known target antigen concentrations in the sample solution with the reactive mass- enhanced or linker-functionalized detection antibody provides a concentration of the target antigen in the sample. This comparison provides an accurate determination of the concentration of the target antigen in the biological sample. As a comparison, the antigen specific binding of enhancer and chemically amplified precipitate substantially increases the thickness and/or refractive index (both real and imaginary parts of the dielectric function) of the optical interference layer, causing large changes in the magnitude of reflected light. This strategy described above is designed to increase the signal to noise ratio of a binding event in the AIR platform by up to several orders of magnitude, resulting in increased sensitivity, reduced background variability, increased utility across sample types, and a more accurate determination of the target antigen concentration at extremely low levels.

[00116] In general, the signal has increased at least 2 times in magnitude.

In various embodiments, the signal has increased at least 2 times, at least 3 times, at least 10 times, at least 50 times, or more.

DEFINITIONS

[00117] When introducing elements of the embodiments described herein, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[00118] As various changes could be made in the above-described methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.

Example 1

[00119] Fig. 10 illustrates one example of substantially increased response for a protein assay when secondary mass enhancers of different sizes are used in the structural arrangement of shown in Fig. 1. Four different proteins were printed in a microarray on a set of chips and these chips were exposed to 10 concentrations of sample titrated in a dilution series. The response plots show substantial increases in response as the size of the mass enhancer is increased (adding more mass generates increased response), providing greater detection sensitivity. This data illustrates practical and achievable benefit in real world assays. In these plots, the X-axis is concentration in pg/mL and the Y-axis is thickness in arbitrary units. At the right are three raw data images used to generate the values for one mid concentration on the titration curves. In the top “small enhancer” image, the detection spots are dim; they get brighter in the center “medium enhancer” image, and are quite bright in the “large enhancer” bottom image, qualitatively illustrating the signal increase with enhancer size.

Example 2

[00120] Fig. 11 shows titration curves similar to the figure described in Example 1 (Fig. 10). However, in this case, the enhancement is generated with chemical amplification of a reactive secondary mass enhancer, as illustrated in Fig 5a. This data was produced using HRP as the reactive secondary mass enhancer and DAB is the substrate. In all 12 of the curves, signal enhancement is obvious, resulting in greater sensitivity for these assays.

Claims

CLAIMS What is claimed is:

1. A method for increasing the sensitivity of arrayed imaging reflectometry, the method comprising:

(a) providing an arrayed imaging reflectometry (AIR) sensor chip comprising at least one surface bound capture antibody or antibody fragment specific to a target antigen;

(b) treating the sensor chip with a sample solution comprising said target antigen and a mass-enhanced detection antibody or antibody fragment specific to the target antigen;

(c) rinsing the sensor chip;

(d) drying the sensor chip;

(e) measuring the signal of the sensor chip using arrayed imaging reflectometry for the bound target antigen and mass-enhanced detection antibody complex and comparing the signal to an AIR response plot of a known series of target antigen concentrations with the mass-enhanced detection antibody; and

(f) determining the concentration of the target antigen in the sample; wherein the mass-enhanced detection antibody 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, high optical density molecules, high optical density molecular complexes, or high optical density particles.

2. The method of claim 1 , wherein the sensor chip comprises a plurality of bound antibodies or bound antibody fragments and a plurality of bound antigens specific to the bound antibodies or bound antibody fragments.

3. The method of either claims 1 or 2, wherein the solution comprising the functionalized antibody specific to the capture antigen has a concentration from 1 pM to 10 mM.

4. The method of any one of the claims 1 -3, wherein the signal from the sensor chip using arrayed imaging reflectometry for the bound antibody(antibody fragment)-capture antigen-functionalized antibody complex is compared to the signal to an unfunctionalized antibody not comprising 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, high optical density molecules, high optical density molecular complexes, or high optical density particles wherein the signal is at least 2 times greater in magnitude.

5. The method of any one of the claims 1 -4, wherein the signal is a differential reflectance signal.

6. The method of any one of the claims 1 -5, wherein the bound antibody is the same or different from the functionalized antibody in the sample.

7. The method of any one of the claims 1 -6, wherein the bound antibody and capture antigen are covalently or non-covalently bound.

8. The method of any one of the claims 1 -7, wherein the functionalized antibody and capture antigen are covalently or non-covalently bound.

9. The method of any one of the claims 1 -8, wherein the sensor chip is washed with a buffer.

10. The method of any one of the claims 1 -9, wherein the sensor chip is treated for a period of time from about 1 minute to about 15 hours.

11. The method of any one of the claims 1 -10, wherein the optical thickness of the sensor chip is at least 200 nm.

12. A method for increasing the sensitivity of arrayed imaging reflectometry, the method comprising:

(b) treating the sensor chip with a sample solution comprising said target antigen and a linker-functionalized detection antibody or antibody fragment specific to the target antigen;

(c) treating the sensor chip a second time with a mass enhancer functionalized to bind to the linker on the detection antibody or antibody fragment;

(d) rinsing the sensor chip; (e) drying the sensor chip;

(f) measuring the signal of the sensor chip using arrayed imaging reflectometry for the bound target antigen with the linker-functionalized antibody complexed with the mass enhancer and comparing the signal to an AIR response plot of a known series of target antigen concentrations with the linker functionalized detection antibody complexed with the mass enhancer; and

(g) determining the concentration of the target antigen in the sample; wherein the linker functionalized antibody is modified with biotin, a peptide, or other linker bridging chemistry, and the functionalized mass enhancer is modified with the complimentary linker bridge and consists of 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, high optical density molecules, high optical density molecular complexes, or high optical density particles.

13. The method of claim 12, wherein the sensor chip comprises a plurality of bound antibodies or antibody fragments and a plurality of capture antigens specific to the antibodies or antibody fragments.

14. The method of either claims 12 or 13, wherein the solution comprising the unfunctionalized antibody specific to the capture antigen has a concentration from 1 pM to 10 mM.

15. The method of any one of the claims 12-14, wherein the signal of the functionalized antibody in the bound antibody (antibody fragment)-capture antigen- antibody complex using arrayed imaging reflectometry is compared to the signal to the unfunctionalized antibody complex wherein the signal is at least 2 times greater in magnitude.

16. The method of any one of the claims 12-15, wherein the signal is a differential reflectance signal.

17. The method of any one of the claims 12-16, wherein the bound antibody (bound antibody fragment) is the same or different from the unfunctionalized antibody in the sample.

18. The method of any one of the claims 12-17, wherein the bound antibody (antibody fragment) and capture antigen are covalently or non-covalently bound.

19. The method of any one of the claims 12-18, wherein the capture antigen and unfunctionalized antibody are covalently or non-covalently bound.

20. The method of any one of the claims 12-19, wherein the sensor chip is washed with a buffer.

21. The method of any one of the claims 12-20, wherein the sensor chip is treated for a period of time from about 1 minute to about 15 hours.

22. The method of any one of the claims 12-21 , wherein the optical thickness of the sensor chip is at least 200 nm.

23. A method for increasing the sensitivity of arrayed imaging reflectometry (AIR) with chemical amplification of a direct or a secondary reactive mass enhancer; the method comprising:

(b) treating the sensor chip with a sample solution comprising said target antigen and a reactive mass-enhanced or linker-functionalized detection antibody or antibody fragment specific to the target antigen;

(c) optionally treating the sensor chip with a reactive mass enhancer functionalized to bind to the linker on the detection antibody or antibody fragment;

(d) rinsing the sensor chip;

(e) exposing the clean sensor to a reactive solution that further increases the detection mass through polymerization, metal deposition, nucleotide replication or other known growth method of a reactive mass tag;

(f) rinsing the chip;

(g) drying the sensor chip;

(h) measuring the signal of the sensor chip using arrayed imaging reflectometry for the bound target antigen and mass-enhanced or linker-functionalized antibody complex with chemical amplification and comparing the signal to an AIR response plot of a known series of target antigen concentrations with the mass- enhanced or linker functionalized antibody with chemical amplification; and

(i) determining the concentration of the target antigen in the sample; wherein the linker-functionalized antibody is modified with biotin, a peptide, or other linker bridging chemistry, and the reactive mass-enhancer is optionally modified with the complimentary linker bridge and consist of an enzyme or enzyme complex, a nucleotide tag, a nanoparticle, or high optical density particles.

24. The method of claim 23, wherein the sensor chip comprises a plurality of bound antibodies or antibody fragments and a plurality of capture antigens specific to the antibodies or antibody fragments.

25. The method of either claims 23 or 24, wherein the solution comprising target antigen and a reactive mass-enhanced or linker-functionalized detection antibody or antibody fragment specific to the target antigen has a concentration from 1 pM to 10 mM.

26. The method of any one of the claims 23-25, wherein the solution comprising a reactive mass enhancer functionalized to bind to the linker on the detection antibody or antibody fragment has a concentration from 1 pM to 10 mM.

27. The method of any one of the claims 23-26, wherein step (e) is treated from about 1 second to about 5 minutes.

28. The method of any one of the claims 23-27, wherein the signal of the functionalized bound target antigen and mass-enhanced or linker-functionalized antibody complex with chemical amplification using arrayed imaging reflectometry is compared to the signal to the unfunctionalized antibody complex wherein the signal is at least 2 times greater in magnitude.

29. The method of any one of the claims 23-28, wherein the signal is a differential reflectance signal.

30. The method of any one of the claims 23-29, wherein the bound antibody (bound antibody fragment) is the same or different from the unfunctionalized antibody in the sample.

31. The method of any one of the claims 23-30, wherein the bound antibody (antibody fragment) and capture antigen are covalently or non-covalently bound.

32. The method of any one of the claims 23-31 , wherein the capture antigen and unfunctionalized antibody are covalently or non-covalently bound.