Methods and related aspects for rapid cytokine detection for inflammatory disease diagnosis

Abstract

Provided herein are methods of monitoring Tumor Necrosis Factor-alpha (TNF-a) in a sample. The methods include contacting the sample with a set of Tumor Necrosis Factor Receptor 2 (TNFR2) functionalized plasmonic metal nanoparticles (MNPs), wherein the sample comprises the TNF-a, and detecting a binding level of the TNFR2 functionalized MNPs with the TNF-a. Related kits and systems are also provided.

Description

METHODS AND RELATED ASPECTS FOR RAPID CYTOKINE DETECTION FOR INFLAMMATORY DISEASE DIAGNOSISCROSS-REFERENCE TO RELATED APPLICATONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application Ser. No. 63 / 653,383, filed May 30, 2024, the disclosure of which is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R21 AH 69098 awarded by the National Institutes of Health, and 1847324 awarded by the National Science Foundation. The government has certain rights in the invention.BACKGROUND

[0003] The immune system operates as the body’s defense mechanism, safeguarding living organisms from invasion by viruses, bacteria, and parasites. Tumor Necrosis Factor-alpha (TNF-a), a pro-inflammatory cytokine produced by macrophages / monocytes, participates in the body's immune response. It plays a role in various physiological events in the human body, especially when released during an inflammatory response, and is directly associated with processes such as fever, apoptosis, sepsis, and myocardial suppression. However, overexcretion of TNF-a can also contribute to acute inflammation and chronic autoimmune disorders such as Crohn’s disease, ulcerative colitis, and Rheumatoid Arthritis. Measuring the level of TNF-a in biological fluids such as serum, blood, and saliva provides valuable information about the diagnosis, seriousness, and prognosis of these diseases. Currently, treatment using Anti- TNF-a antibodies (e.g., Adalimumab) is an efficacious method for managing disease progression; however, its efficacy is challenged by the elicitation of immunogenic responses, including anti-drug antibodies (ADA). Therefore, effective patient treatment and disease management require a thorough consideration of crucial factors, including immunogenicity potential, treatment safety profiles, and optimal therapy duration.

[0004] Numerous detection methods have been developed to measure amounts of TNF-a in biofluids. Common methods of TNF-a assays, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and bioassays, are sensitive but costly, time-consuming, and instrument-heavy. Additionally, a large amount of sample is needed to achieve a notable signal-to-noise ratio for detection. Therefore, a persistent need remains for a simple, cost-effective approach for sensitive TNF-a detection, particularly for point-of-care applications.SUMMARY

[0005] In one aspect, the present disclosure provides a method of monitoring Tumor Necrosis Factor-alpha (TNF-a) in a sample. The method includes contacting the sample with a set of T umor Necrosis Factor Receptor 2 (TNFR2) functionalized plasmonic metal nanoparticles (MNPs), wherein the sample comprises the TNF-a. The method also includes detecting a binding level of the TNFR2 functionalized MNPs with the TNF-a, thereby monitoring the TNF-a in the sample..

[0006] In another aspect, the present disclosure provides a kit for monitoring Tumor Necrosis Factor-alpha (TNF-a) in a sample, comprising a container that contains a set of Tumor Necrosis Factor Receptor 2 (TNFR2) functionalized plasmonic metal nanoparticles (MNPs).

[0007] In another aspect, the present disclosure provides a system for monitoring Tumor Necrosis Factor-alpha (TNF-a) in a sample. The system includes a sample container receiving structure configured to receive a sample container that contains the sample and a set of T umor Necrosis Factor Receptor 2 (TNFR2) functionalized plasmonic metal nanoparticles (MNPs), wherein the sample comprises the TNF-a; a light source operably connected to the sample container receiving structure, wherein the light source is configured to transmit light through the sample container; a photodetector operably connected to the sample container receiving structure, wherein the photodetector is configured to measure light intensity transmitted through the sample container from the light source; a controller operably connected to the light source and to the photodetector, wherein the controller is configured to effect transmission of the light through the sample container using the light source and measurement of the light intensity transmittedthrough the sample container to detect a binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample using the photodetector; a power source operably connected to the controller, wherein the power source is configured to selectively supply power to the controller; and, a housing structure operably connected to the sample container receiving structure, wherein the controller and power source are substantially housed in one or more cavities disposed within the housing structure.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the methods, reaction mixtures, devices, kits, and related systems disclosed herein. The description provided herein is better understood when read in conjunction with the accompanying drawings which are included by way of example and not by way of limitation. It will be understood that like reference numerals identify like components throughout the drawings, unless the context indicates otherwise. It will also be understood that some or all of the figures may be schematic representations for purposes of illustration and do not necessarily depict the actual relative sizes or locations of the elements shown.

[0009] FIGS. 1 A-1 E: Schematic of assay design for therapeutic monitoring of TNF-a. (a) TNFR2-AuNP probes are produced by functionalizing 80 nm gold nanoparticles with TNFR2. (b) A hypothetical scenario for TNF-a antigen detection (binding test) for disease diagnosis, (c) Detection in a hypothetical on-treatment scenario where anti-TNF-a antibody is present (Drug competition test), (d) Detection in a hypothetical scenario where both anti-TNF-a and induced ADA are present in the testing sample, (e) Schematics of assay process and readout using PED device.

[0010] FIGS. 2A-2D: Engineering process optimization to enhance TNF-a antigen sensing, (a) different vortexing speed conditions (29 rps to 45 rps) have been tested to find the optimized number for the detection (b) vortexing time conditions (2 seconds to 10 seconds) (c) Centrifugation speed conditions (2.5 krpm (600g) to 4.5 krpm (1950g)) (d) Centrifugation time conditions (1 minute to 8 minutes).

[0011] FIGS. 3A-3C: Demonstrated binding assay in TNF-a antigen sensing in PBS using TNFR2-AuNPs. (a-b) Optical images of the test tubes holding AuNP sensing buffer mixed with target TNF-alpha molecules: (a) right after mixing and (b) after the sensing procedure (centrifugation, incubation, and vortex agitation) was completed, (c) Experimental data of normalized electronic signals, showing a high concentration of TNF- a can aggregate AuNP probes.

[0012] FIGS. 4A-4F: Demonstrated ADA affinity towards Adalimumab and TNF- a. (a) schematic of Au / ADA interaction with adalimumab. (b) Optical images of tube arrays to detect Adalimumab in PBS using AuNPs / ADA, showing a high concentration of Adalimumab can aggregate AuNP probes, (c) Measured Adalimumab affinity towards ADA in PBS. (d-f) Schematic, optical images and sensing curves for TNFalpha detection using AuNP / ADA sensors.

[0013] FIGS. 5A-5F: Demonstrated assays in TNF-a antigen sensing at the presence of antibody drug and ADA. (a-c) Schematics, optical images and sensing curves to detect TNF-a using AuNPs / TNFR2 probes at the presence of Adalimumab. (d-f) Schematics, optical images and sensing curves to detect TNF-a using AuNPs / TNFR2 probes at the presence of Adalimumab and ADA.

[0014] FIGS. 6A-6G: Demonstrated assays in TNF-a antigen sensing in artificial patient serum samples, (a-e) Visual images of tube arrays for TNF-a detection in HPS using AuNPs / TNFR2 with different combinations of TNF-a, adalimumab (antibody), and ADA. (a-c) ADA fixed at 10 nM, but TNF-alpha at (a) 50 nM, (b) 5 nM, and (c) 500 pM. (d- e) ADA fixed at 100 nM, but adalimumab at (d) 10 nM, (e) 100 nM. (f-g) Impact of ADA and Adalimumab concentration in HPS as a function of TNF-a concentration (50 nM, 5 nM and 500 pM): (f) plotted in individual lines for each cases that have different variables; (g) plotted in aggregate signals using TNF-alpha as the only variable.DEFINITIONS

[0015] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth throughout the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference,the definition set forth in this application should be used to understand the meaning of the term.

[0016] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0017] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0018] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0019] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Further, 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 disclosure pertains. In describing and claiming the methods, reaction mixtures, devices, kits, and systems, the following terminology, and grammatical variants thereof, will be used in accordance with the definitions set forth below.

[0020] About. As used herein, “about” or “approximately” or “substantially” as applied to one or more values or elements of interest, refers to a value or element that is similar to a stated reference value or element. In certain embodiments, the term “about” or “approximately” or “substantially” refers to a range of values or elements that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value or element unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value or element).

[0021] Administer: As used herein, “administer” or “administering” a therapeutic agent (e.g., an immunological therapeutic agent) to a subject means to give, apply or bring the composition into contact with the subject. Administration can be accomplished by any of a number of routes, including, for example, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal and intradermal.

[0022] Antibody: As used herein, the term “antibody” refers to an immunoglobulin or an antigen-binding domain thereof. The term includes but is not limited to polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, human, canonized, canine, felinized, feline, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. The antibody can include a constant region, or a portion thereof, such as the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes. For example, heavy chain constant regions of the various isotypes can be used, including: IgGi, lgG2, IgGs, lgG4, IgM, IgAi, lgA2, IgD, and IgE. By way of example, the light chain constant region can be kappa or lambda. The term “monoclonal antibody” refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope.

[0023] Bind. As used herein, “bind,” in the context of target analyte detection, refers to a state in which a first chemical structure is sufficiently associated a second chemical structure such that the association between the first and second chemical structures can be detected.

[0024] Conjugate-. As used herein, “conjugate” refers to a reversible or irreversible connection between two or more substances or components. In some embodiments, for example, gold nanoparticles (AuNPs) and / or other plasmonic metal nanoparticles (MNPs) are connected to antibodies and / or to antigen binding portions thereof. In some embodiments, AuNPs and / or other plasmonic metal nanoparticles (MNPs) are conjugated with antibodies and / or to antigen binding portions thereof via one or more linker compounds.

[0025] Detect As used herein, “detect,” “detecting,” or “detection” refers to an act of determining the existence or presence of one or more target analytes (e.g., Tumor Necrosis Factor-alpha (TNF-a), etc.) in a sample.

[0026] Sample: As used herein, “sample” means anything capable of being analyzed by the methods, devices, and / or systems disclosed herein.

[0027] Subject: As used herein, “subject” refers to an animal, such as a mammalian species (e.g., human, dog, cat) or avian (e.g., bird) species. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. Animals include farm animals (e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like), sport animals, and companion animals (e.g., pets or support animals). In certain embodiments, the subject is a human. In certain embodiments, the subject is a companion animal, including, but not limited to, a dog or a cat. A subject can be a healthy individual, an individual that has or is suspected of having a disease or a predisposition to the disease, or an individual that is in need of therapy or suspected of needing therapy. The terms “individual” or “patient” are intended to be interchangeable with “subject.”

[0028] System: As used herein, "system" in the context of analytical instrumentation refers a group of objects and / or devices that form a network for performing a desired objective.

[0029] Treat: As used herein, the term “treat,” “treating” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease and / or injury, or one or more symptoms of such disease, to which such term applies. Depending on the condition of the subject, the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease (e.g., viral infection). A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Such prevention or reduction of the severity of a disease prior to affliction refers to administration of a treatment to a subject that is not at the time of administration afflicted with the disease. “Preventing” also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease.DETAILED DESCRIPTION

[0030] Reference will now be made in detail to example implementations. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention and it is to be understood that other embodiments may be utilized and that changes may be made without departing from the scope of the invention. The following description is, therefore, merely exemplary.

[0031] I. Description of Example Embodiments

[0032] In some aspects, the present disclosure provides an approach to making use of plasmonic gold nanoparticles (AuNPs) to quantify TNF-o rapidly and accurately in the presence of anti-TNF-o Abs and ADA to improve disease treatment efficacy. In some embodiments, an in-solution assay employs Tumor Necrosis Factor Receptor 2 (TNFR2) functionalized gold nanoparticles (TNFR2-AuNPs) as signaling beacon receptors, TNF-a as the antigen, Adalimumab as the therapeutic agent, and ADA as the immune system indicator. The competition between TNFR2-AuNPs and Adalimumab for TNF-a binding directly influences the AuNPs clustering and, therefore, the amount of free-floating AuNP beacons. With an ample amount of Adalimumab binding to TNF-a, AuNPs are shielded from clustering, generating colorimetric signals due to plasmonic extinction (Fig. 1 ). Conversely, the presence of ADA binds to Adalimumab and blocks Adalimumab to TNF- a binding, allowing free TNF-a to react with TNFR2-AuNPs and form AuNP aggregates. These AuNP extinction signals can be accurately quantified using our portable electronic detector (PED), which includes a light-emitting diode (LED), photodiode, battery, and signal processing circuitry, similar to our prior works . With an optimized centrifugation and vortex process, the interaction time will reduce to less than 30 minutes.

[0033] In some embodiments, the present disclosure provides a nanoparticle- supported rapid electronic detection (NasRED) platform is devised as a novel, digital sensing platform for rapid detection of inflammatory marker Tumor Necrosis Factor (TNF)-alpha. We demonstrate the feasibility of highly sensitive detection of TNF-alpha at femtomolar by functionalizing gold nanoparticles (AuNPs) with TNF Receptor 2 (TNFR2), proving the feasibility of early disease detection. Further, we design the NasRED assay with three components, i.e. TNFR2-AuNPs, TNF-alpha, and treatment antibodyAdalimumab, demonstrating the feasibility of the assay in analyzing the concentrations of TNF-alpha during treatment, proving its use in prognosis and treatment efficacy evaluation. In addition, we also design the assay with four components, including TNFR2- AuNPs, TNF-alpha, Adalimumab, and anti-drug antibody (ADA), to simulate scenarios that drug resistance develop after treatment. Lastly, we explore the assay’s feasibility to detect TNF-alpha in both PBS buffer and human serum and under different clinical conditions with Adalimumab and ADA at different levels. This rapid and digital assay can have broad use in understanding of inflammatory diseases, cancer, and the like.

[0034] In some embodiments, the present disclosure provides a method of monitoring Tumor Necrosis Factor-alpha (TNF-o) in a sample. The method includes contacting the sample with a set of Tumor Necrosis Factor Receptor 2 (TNFR2) functionalized plasmonic metal nanoparticles (MNPs) in which the sample comprises the TNF-a. The method also includes detecting a binding level of the TNFR2 functionalized MNPs with the TNF-a. In some embodiments, the method includes quantifying an amount of the TNF-a in the sample.

[0035] In some embodiments, the sample further comprises an anti-TNF-a antibody and in which the method further comprises detecting the binding level or a change in the binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample. In some embodiments, the sample is obtained from a subject having been administered the anti-TNF-a antibody as a therapy for a disease, condition, or disorder and in which the binding level or the change in the binding level provides an efficacy measure of the therapy. In some embodiments, the anti-TNF-a antibody comprises Adalimumab.

[0036] In some embodiments, the sample further comprises anti-drug antibody (ADA) that binds to the anti-TNF-a antibody and in which the method further comprises detecting the binding level or a change in the binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample. In some embodiments, the sample is obtained from a subject having been administered the anti-TNF-a antibody as a therapy for a disease, condition, or disorder and in which the binding level or the change in the binding level provides a measure of an immunogenic response in the subject.

[0037] In some embodiments, the sample comprises a sample type selected from the group consisting of: whole blood, serum, plasma, saliva, sputum, nasal fluid, cerebrospinal fluid, vaginal fluid, semen, and urine. In some embodiments, the sample is obtained from a subject and in which the method comprises administering a therapy to the subject when the binding level of the TNFR2 functionalized MNPs with the TNF-a exceeds a predetermined threshold.

[0038] In another aspect, the present disclosure provides a kit for monitoring Tumor Necrosis Factor-alpha (TNF-a) in a sample, comprising a container that contains a set of Tumor Necrosis Factor Receptor 2 (TNFR2) functionalized plasmonic metal nanoparticles (MNPs). In some embodiments, the kit further includes a system for monitoring the TNF-a in the sample using the set of TNFR2 functionalized MNPs.

[0039] In another aspect, the present disclosure provides a system for monitoring Tumor Necrosis Factor-alpha (TNF-a) in a sample. The system includes a sample container receiving structure configured to receive a sample container that contains the sample and a set of T umor Necrosis Factor Receptor 2 (TNFR2) functionalized plasmonic metal nanoparticles (MNPs), in which the sample comprises the TNF-a. The system also includes a light source operably connected to the sample container receiving structure, in which the light source is configured to transmit light through the sample container, and a photodetector operably connected to the sample container receiving structure, in which the photodetector is configured to measure light intensity transmitted through the sample container from the light source. The system also includes a controller operably connected to the light source and to the photodetector, in which the controller is configured to effect transmission of the light through the sample container using the light source and measurement of the light intensity transmitted through the sample container to detect a binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample using the photodetector. In addition, the system also includes a power source operably connected to the controller, in which the power source is configured to selectively supply power to the controller, and a housing structure operably connected to the sample container receiving structure, in which the controller and power source are substantially housed in one or more cavities disposed within the housing structure.

[0040] In some embodiments, the controller is configured to quantify an amount of the TNF-a in the sample. In some embodiments, the sample further comprises an anti- TNF-a antibody and in which the controller is configured to detect the binding level or a change in the binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample. In some embodiments, the sample is obtained from a subject having been administered the anti-TNF-a antibody (e.g., Adalimumab or the like) as a therapy for a disease, condition, or disorder and in which the binding level or the change in the binding level provides an efficacy measure of the therapy.

[0041] In some embodiments, the sample further comprises anti-drug antibody (ADA) that binds to the anti-TNF-a antibody and in which the controller is configured to detect the binding level or a change in the binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample. In some embodiments, the sample is obtained from a subject having been administered the anti-TNF-a antibody as a therapy for a disease, condition, or disorder and in which the binding level or the change in the binding level provides a measure of an immunogenic response in the subject.

[0042] II. Example

[0043] Example 1 : Rapid TNF-Alpha Quantification Using Gold Nanoparticles Towards Cytokine Monitoring in Inflammatory Diseases

[0044] I. Introduction

[0045] In this example, we present an innovative approach making use of plasmonic gold nanoparticles (AuNPs) to quantify TNF-a rapidly and accurately in the presence of anti-TNF-a Abs and ADA to improve disease treatment efficacy. Our insolution assay employs Tumor Necrosis Factor Receptor 2 (TNFR2) functionalized gold nanoparticles (TNFR2-AuNPs) as signaling beacon receptors, TNF-a as the antigen, Adalimumab as the therapeutic agent, and ADA as the immune system indicator. The competition between TNFR2-AuNPs and Adalimumab for TNF-a binding directly influences the AuNPs clustering and, therefore, the amount of free-floating AuNP beacons. With an ample amount of Adalimumab binding to TNF-a, AuNPs are shielded from clustering, generating colorimetric signals due to plasmonic extinction (FIG. 1 ). Conversely, the presence of ADA binds to Adalimumab and blocks Adalimumab to TNF-a binding, allowing free TNF-a to react with TNFR2-AuNPs and form AuNP aggregates. These AuNP extinction signals can be accurately quantified using our portable electronic detector (PED), which includes a light-emitting diode (LED), photodiode, battery, and signal processing circuitry, similar to our prior works3. With an optimized centrifugation and vortex process, the interaction time will reduce to less than 30 minutes.

[0046] 1. Sensing process overview

[0047] Our digital sensing platform is designed to detect the target protein interactions in-solution, by simply mixing the bodily fluids with our prepared sensing buffer comprising functionalized gold nanoparticles (AuNPs) in a testing tube. The mixing, reaction, and readout are all completed in the same tube without any washing, labeling, or fluorescent imaging, thus greatly reducing complexity of operation. The output signals are digitized using our customized a AuNP-supported rapid, electronic detection (NasRED), comprising semiconductor devices such as light-emitting diodes (LEDs) and photodetectors (PDs) for signal digitation, semiconductor circuits integrated on Printed circuit board (PCB) for signal processing, as well as mechanical components for device mounting and system integration.

[0048] Sensing solution preparation

[0049] Here, the AuNPs in the sensing buffer solutions were functionalized using biotin-streptavidin reaction to coat with TNFR2. In particular, the Streptavidin-coated AuNPs (0.13 nM, 80nm AuNPs, 50 pL) were first mixed with an excessive amount of biotinylated TNFR2 (2 pM, 20 pL). The mixture was then incubated for at least 2 hours to ensure complete streptavidin-biotin conjugation. Next, the mixture was purified by centrifuge (accuSpin Micro 17, Thermo Fisher) at 10,000 rpm for 10 mins and repeated twice to remove unbounded TNFR2. The purified AuNP sensing buffer was diluted by adding extra assay buffer for a tube readout on our PED device, and the AuNP concentration was set to 16.25 pM in the tube array. The assay buffer was prepared by diluting 10x PBS buffer and mixing it with glycerol and BSA to reach a final concentration of 1 xPBS, 20% v / v glycerol, and 1 wt% BSA. The AuNP sensing buffer was aliquoted into 18 pL in 500 pL Labcon microcentrifuge tubes.

[0050] This prepared AuNP-TNFR2 sensing buffer was then mixed with targetmolecules, i.e. TNF-alpha in binding test (Figure 1 b), TNF-alpha and anti-TNF-a Abs in drug efficacy test (Figure 1 c), and TNF-alpha, anti-TNF-a Abs and ADAs in the ADA test (Figure 1 c). The molecules to be tested were diluted from stock solutions to target concentrations in the selected detection medium (e.g., PBS buffer, human pooled serum (HPS), and human whole blood (WB)). The blank medium without antibodies was used as a negative control (NC).

[0051] 2. Impact of sensing protocol on performance

[0052] The centrifugation and vortex agitation can significantly modulate the reagent and AuNP distribution in the buffer solutions, and thus strongly affect the optimal conditions to obtain the best sensitivity and specificity in a given biological medium. On one hand, sufficiently strong and long centrifugation plays several important roles to promote rapid and highly sensitive antibody-antigen reactions. First, it serves to spin the AuNPs and reagents at a high speed for active mixing. Second, centrifugation also acts to shorten the assay time, which is mainly limited by the sedimentation speed of AuNPs. With centrifugation, the precipitation length of AuNPs significantly drops from a few millimeter (here the height of colloid liquid, dependent on the buffer volume and the tube size) to micrometer scale, therefore eliminating hours-long precipitation time that is otherwise required and significantly speeding up signal transduction and readout. Third, centrifugation also localizes AuNPs and their clusters with a greatly boosted concentration at the bottom of the reaction tube, thus moving the dynamic equilibrium of antigen-antibody reaction to favor AuNP cluster formation even at a low reagent concentration in the tube.

[0053] On the other hand, sufficient vortex agitation is needed to resuspend monomer AuNPs not incorporated into clusters back to free-floating in order to ensure high specificity, i.e. the buffer tube of negative control should return to their initial states, but the vortex force cannot be excessively high to break the AuNP clusters “glued” together that would negatively affect the sensitivity. Therefore, the speed and time of vortex agitation need to be adjusted together with the centrifugation conditions. For example, excessively high centrifugal speed and time could form tight AuNPs aggregates that become harder to break under vortex agitation, and were therefore avoided in ourprotocols.

[0054] To find out the impact of centrifugation and vortex agitation on the assay performance, TNF-a stock solutions (4 pM, in 1 xPBS) were serially diluted, and a 6 pL TNF-a solution of each concentration (4 fM to 4 pM) was mixed with 18 pL AuNP assay colloid, obtaining a final concentration of 1 pM to 1 fM in the mixture. The mixture was briefly vortexed (mini vortexer, Thermo Fisher) at 2000 rpm for 5 seconds to produce a homogenous colloid solution. Then the TNF-a in PBS buffer at different concentrations was tested under different centrifugation speed and time as well as vortexing speed and time (Fig. 3). All the other parameters were constant for each stage, and only one variable was changed at a time.

[0055] The tubes were then measured on our PED reader. For data analysis, a background signal calibration was performed. Each tube was filled with buffer, and 12 measurements were collected, and averaged to establish the background electronic readoutref~12( / =1 to 12) as a reference. The same tubes were then used to hold the AuNP sensing buffer, during which 12 measurements were performed, and the electronic signals were averaged as( / =1 to 12) for each TNF-alpha concentrationC. Therefore, the antibody signal was normalized as. The error of the measurement is the standard deviation (SD) of the 12 normalized signals, or( / =1 to 12). Furthermore, the limit of detection (LoD) was calculated such that the measured signal distinguishes from the reference signal by 1 .645 times the standard deviation of the reference and the lowest concentration sample, following S(LoD)=S(NC) -1 ,645*(SD(NC) + SDfCmin)), where Cmin is the lowest TNF-alpha concentration in the test.

[0056] Ideally, a large contrast in signal S was desired for maximizing the sensitivity, and a large dynamic range, i.e. from <10 fM to 1 pM, was useful for detection of heterogenous patient samples. Interestingly, these requirements could be achieved within a relatively large range of centrifugation conditions, i.e. from 900 g to 1500 g and from 3 to 5 minutes (Figure 2). In addition, the sensing performance was found moresensitive to the vortex conditions, because too long or too strong agitation would break the AuNP clusters into AuNPs in the sensing buffer. Considering all the conditions, we chose a standard processing condition for all the tests in this work, i.e. centrifugation for 5 minutes at 1 ,200g (3.5 krpm), incubation for 10 minutes, and vortexing for 5 seconds at 32.5 rps.

[0057] 3. TNF-alpha Binding test

[0058] To prove the concept of TNF-alpha detection for diagnosis purpose, different concentrations of TNF-a (1 pM to 1 fM after mixing with sensing buffer) and NC samples were prepared in PBS buffer, incubated with AuNPs / TNFR2 for 10 minutes, and centrifuged at 1 ,200g for 5 mins (Figure 3a). This centrifugation step helps accelerate the reaction by creating a highly concentrated temporary reaction zone at the bottom of the tubes. After 15 minutes of incubation, a brief (5 seconds) vortex agitation (32.5 rps) was introduced to redisperse monomer AuNPs, mostly in low concentrations (Fig. 3b). The tubes were then measured on our PED reader and analyzed as discussed above. Our analysis showed that TNF-alpha could be detected at a low concentration of ?1 fM, much lower than prior work (cite and provide what sensitivities others could achieve), demonstrating the feasibility of our NasRED platform to detect cytokines at a high sensitivity.

[0059] 4. Protein interaction evaluation

[0060] In a solution with diverse biomolecules, finding the affinity of all parameters towards each other is crucial to rule out nonspecific reactions. In our case where multiple proteins can interact with each other (Figure 1 ), it is essential to inspect ADA interaction with TNF-a and Adalimumab to determine whether the platform design is valid. Similar to the approach to prepare AuNPs / TNFR2 sensors, mono-biotinylated Anti-Adalimumab Antibody (ADA) (AY19, Acrobiosystems) stock solution (6.9 pM, in 1 xPBS) was serially diluted, conjugated with streptavidin-coated AuNPs, and purified.

[0061] First to examine the interactions between ADA and Adalimumab, a dilution series of Adalimumab (1 pM to 1 fM and negative sample) were mixed with AuNP / ADA probes in PBS buffer (Fig. 4a-c). As shown in the tube images, Adalimumab proved to bind strongly to AuNP / ADA over a large concentration range (7 log), with a detection limitof ~1 pM. In another test, AuNP / ADA probes were mixed with a dilution series of TNF-a (1 pM to 1 fM and negative sample) (Fig. 4d-f). As expected, TNF-a and ADA did not show clear affinity, evidenced by the full dispersion of AuNP / ADA probes in all concentrations of TNF-a.

[0062] 5.Drug evaluation test

[0063] A 6 pL TNF-a solution (40nM) was mixed with 18 pL AuNP assay colloid solution, and then vortexed at 2000 rpm for 5 seconds to produce a homogeneous mixture solution. After incubation for 12 hours, 2 pL solutions of Adalimumab (at 8pl / ml and 1 pl / ml) were added to the mixture solution tubes, and vortexed at 2000 rpm for 5 seconds. Finally, after another 24 hours incubation, another sets of ADA solutions (2 pL, at different concentrations of 1 .4 pM, 140nM, and 1 .4nM) were added, followed by vortex agitation.

[0064] To demonstrate the feasibility of TNF-a detection in a hypothetical anti- TNF-a therapy situation treated by Adalimumab (16H5, Acrobiosystems) (Fig. 5a), we performed analysis by mixing three components in PBS buffer: a dilution series of Adalimumab (4 fM to 4 pM and negative sample) as the treatment antibody, a solution of AuNPs / TNFR2 probes (12 pL, as described above) as signal reporters, and free-floating TNF-a as the cytokine antigen (6 pL, at two different concentrations of 10 nM and 1 nM) (Fig. 5a). These concentrations of TNF-a were used to evaluate different disease severity, and determined experimentally based on their ability to produce visually recognizable solution color difference at the absence of Adalimumab. The assay fundamentally works to evaluate the competition effects between Adalimumab and TNFR2 to bind to TNF-a. At a high Adalimumab concentration, such antibodies can neutralize TNF-a, and accordingly prevent the chemical interaction between TNFR2 and TNF-a, resulting in complete suspension of the AuNPs / TNFR2 probes and a high electronic signal (close to 0.5, Fig. 5b). However, when Adalimumab was lower than 1 nM, it could not fully inhibit the TNF-a binding to TNFR2, therefore the amount of AuNP aggregation and the collected electronic signals were mainly determined by the TNF-a concentration (Fig. 5c).

[0065] Furthermore, to evaluate the impact of ADA on the effectiveness of anti- TNF-a in some patients, we further added ADA at different concentrations to the above three-component mixture of AuNPs / TNFR2, TNF-a, and Adalimumab (Fig. 5d).Therefore, the amount of AuNP aggregation depend on the binding of AuNPs / TNFR2 probes to the available TNF-a, which is affected by the amount of Adalimumab that is not neutralized by ADA. In clinical practice, the adalimumab dose concentration in on- treatment patients is usually greater than 4.5 pg / mL, or typically between 8-12 pg / mL. Here we chose to use 50 nM (approximately 8 pg / mL) adalimumab. This concentration is clinically relevant, and on the other hand sufficient in our assay design to produce noticeable signals for two exemplary TNF-a concentrations that represent a more severe case (10 nM) and a moderate case (1 nM) (Fig. 5e). Clearly shown in the tube images and the electronic readout signals, Adalimumab was not able to neutralize all TNF-a at the presence of ADA. Rather, Adalimumab was more available at low ADA concentrations, and thus more effective to neutralize TNF-alpha. In turn, under these conditions Adalimumab would be more effective to protect the AuNPs / TNFR2 probes, thus producing a more red color in the tube images and higher electronic signals. In the contrary, at higher ADA concentrations, the amount of Adalimumab would be lower, and the AuNPs would aggregate more due to more available TNF-a. Overall, these data prove the feasibility of a simple in-solution test that can effectively analyze the impact of both anti-TNF-a and ADA on TNF-a detection.

[0066] 6. Artificial serum test

[0067] We further evaluated the use of the tests in artificial patient serum samples made by mixing TNFR2-functionalized AuNPs with varying concentrations of TNF-a, Adalimumab, and ADA in 100% human pooled serum (HPS). For example, a 4 pL TNF- a solution (600 nM, 60 nM and 6 nM), a 4 pL Adalimumab solution (300 nM, 30 nM and 3 nM, 30 pM), and a 4 pL Adalimumab (600 nM, 60 nM) were mixed in different combination with 12 pL AuNP assay, and then briefly vortexed at 2000 rpm for 5 seconds. For proof-of-concept demonstration, the concentration of ADA was fixed (10 nM) while the concentrations of TNF-a and Adalimumab varied (Fig. 6a). In another scenario, the concentration of ADA was fixed at a high amount (100nM) while the concentrations of TNF-a and Adalimumab varied (Fig. 6b). At high TNF-a concentrations (50 nM), with varying concentrations of Adalimumab (1 pM to 10 pM), the functionalized AuNPs were fully aggregated, showing complete Adalimumab inhibition and resulting in transparent tubes. Furthermore, at 5nM TNF-a concentration, the interaction between Adalimumaband TNF-a increased regardless of ADA presence. The color of tubes gradually faded as Adalimumab concentrations decreased, indicating insufficient capture of TNF-a. However, at low TNF-a concentrations (500 pM), no cluster was formed (Fig. 6c). Considering the fact that detecting TNF-a despite the presence of ADA and adalimumab is essential, without knowing all the parameters, detection of active TNF-a is possible (Fig. 6d). Our methodology proved to be highly effective in rapid, and sensitive detection of TNF-a using a portable optoelectronic system with a detection time of 30 minutes and a low LoD in the picomolar range across different stages of clinical treatment.

[0068] Conclusions:

[0069] In conclusion, our study demonstrates the efficacy of using gold nanoparticles for rapid TNF-a quantification, preparing the way for improved cytokine monitoring in inflammatory diseases utilizing our portable electronic detector (PED). Our low-cost optoelectronic system for cytokine detection shows significant potential across various areas, including therapeutic and personalized drug dosage with a 30-minute detection time that can be valuable in clinical centers.

[0070] Some further aspects are defined in the following clauses:

[0071] Clause 1 : A method of monitoring Tumor Necrosis Factor-alpha (TNF-a) in a sample, the method comprising: contacting the sample with a set of Tumor Necrosis Factor Receptor 2 (TNFR2) functionalized plasmonic metal nanoparticles (MNPs), wherein the sample comprises the TNF-a; and detecting a binding level of the TNFR2 functionalized MNPs with the TNF-a, thereby monitoring the TNF-a in the sample.

[0072] Clause 2: The method of Clause 1 , wherein the sample further comprises an anti-TNF-a antibody and wherein the method further comprises detecting the binding level or a change in the binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample.

[0073] Clause 3: The method of Clause 1 or Clause 2, wherein the sample is obtained from a subject having been administered the anti-TNF-a antibody as a therapy for a disease, condition, or disorder and wherein the binding level or the change in the binding level provides an efficacy measure of the therapy.

[0074] Clause 4: The method of any one of the preceding Clauses 1 -3, wherein the anti-TNF-a antibody comprises Adalimumab.

[0075] Clause 5: The method of any one of the preceding Clauses 1 -4, wherein the sample further comprises anti-drug antibody (ADA) that binds to the anti-TNF-a antibody and wherein the method further comprises detecting the binding level or a change in the binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample.

[0076] Clause 6: The method of any one of the preceding Clauses 1 -5, wherein the sample is obtained from a subject having been administered the anti-TNF-a antibody as a therapy for a disease, condition, or disorder and wherein the binding level or the change in the binding level provides a measure of an immunogenic response in the subject.

[0077] Clause 7: The method of any one of the preceding Clauses 1 -6, wherein the sample comprises a sample type selected from the group consisting of: whole blood, serum, plasma, saliva, sputum, nasal fluid, cerebrospinal fluid, vaginal fluid, semen, and urine.

[0078] Clause 8: The method of any one of the preceding Clauses 1 -7, comprising quantifying an amount of the TNF-a in the sample.

[0079] Clause 9: The method of any one of the preceding Clauses 1 -8, wherein the sample is obtained from a subject and wherein the method comprises administering a therapy to the subject when the binding level of the TNFR2 functionalized MNPs with the TNF-a exceeds a predetermined threshold.

[0080] Clause 10: A kit for monitoring Tumor Necrosis Factor-alpha (TNF-a) in a sample, comprising a container that contains a set of Tumor Necrosis Factor Receptor 2 (TNFR2) functionalized plasmonic metal nanoparticles (MNPs).

[0081] Clause 11 : The kit of Clause 10, further comprising a system for monitoring the TNF-a in the sample using the set of TNFR2 functionalized MNPs.

[0082] Clause 12: A system for monitoring Tumor Necrosis Factor-alpha (TNF-a) in a sample, comprising: a sample container receiving structure configured to receive asample container that contains the sample and a set of Tumor Necrosis Factor Receptor 2 (TNFR2) functionalized plasmonic metal nanoparticles (MNPs), wherein the sample comprises the TNF-a; a light source operably connected to the sample container receiving structure, wherein the light source is configured to transmit light through the sample container; a photodetector operably connected to the sample container receiving structure, wherein the photodetector is configured to measure light intensity transmitted through the sample container from the light source; a controller operably connected to the light source and to the photodetector, wherein the controller is configured to effect transmission of the light through the sample container using the light source and measurement of the light intensity transmitted through the sample container to detect a binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample using the photodetector; a power source operably connected to the controller, wherein the power source is configured to selectively supply power to the controller; and, a housing structure operably connected to the sample container receiving structure, wherein the controller and power source are substantially housed in one or more cavities disposed within the housing structure.

[0083] Clause 13: The system of Clause 12, wherein the controller is configured to quantify an amount of the TNF-a in the sample.

[0084] Clause 14: The system of Clause 12 or Clause 13, wherein the sample further comprises an anti-TNF-a antibody and wherein the controller is configured to detect the binding level or a change in the binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample.

[0085] Clause 15: The system of any one of the preceding Clauses 12-14, wherein the sample is obtained from a subject having been administered the anti-TNF-a antibody as a therapy for a disease, condition, or disorder and wherein the binding level or the change in the binding level provides an efficacy measure of the therapy.

[0086] Clause 16: The system of any one of the preceding Clauses 12-15, wherein the anti-TNF-a antibody comprises Adalimumab.

[0087] Clause 17: The system of any one of the preceding Clauses 12-16, wherein the sample further comprises anti-drug antibody (ADA) that binds to the anti-TNF-a antibody and wherein the controller is configured to detect the binding level or a change in the binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample.

[0088] Clause 18: The system of any one of the preceding Clauses 12-17, wherein the sample is obtained from a subject having been administered the anti-TNF-a antibody as a therapy for a disease, condition, or disorder and wherein the binding level or the change in the binding level provides a measure of an immunogenic response in the subject.

[0089] Clause 19: The system of any one of the preceding Clauses 12-18, wherein the sample comprises a sample type selected from the group consisting of: whole blood, serum, plasma, saliva, sputum, nasal fluid, cerebrospinal fluid, vaginal fluid, semen, and urine.

[0090] Clause 20: A kit comprising the system of any one of the preceding Clauses 1 -19.

[0091] Although this disclosure contains many specific embodiment details, these should not be construed as limitations on the scope of the subject matter or on the scope of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments. Certain features that are described in this disclosure in the context of separate embodiments can also be implemented, in combination, in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments, separately, or in any suitable sub-combination. Moreover, although previously described features may be described as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can, in some cases, be excised from the combination, and the claimed combination may be directed to a sub-combination or variation of a sub-combination.

[0092] Particular embodiments of the subject matter have been described. Other embodiments, alterations, and permutations of the described embodiments are within the scope of the following claims as will be apparent to those skilled in the art. While operations are depicted in the drawings or claims in a particular order, this should not beunderstood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed (some operations may be considered optional), to achieve desirable results.

[0093] Accordingly, the previously described example embodiments do not define or constrain this disclosure. Other changes, substitutions, and alterations are also possible without departing from the spirit and scope of this disclosure.

Claims

WHAT IS CLAIMED IS:1 . A method of monitoring Tumor Necrosis Factor-alpha (TNF-a) in a sample, the method comprising: contacting the sample with a set of Tumor Necrosis Factor Receptor 2 (TNFR2) functionalized plasmonic metal nanoparticles (MNPs), wherein the sample comprises the TNF-a; and, detecting a binding level of the TNFR2 functionalized MNPs with the TNF-a, thereby monitoring the TNF-a in the sample.

2. The method of claim 1 , wherein the sample further comprises an anti- TNF-a antibody and wherein the method further comprises detecting the binding level or a change in the binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample.

3. The method of claim 2, wherein the sample is obtained from a subject having been administered the anti-TNF-a antibody as a therapy for a disease, condition, or disorder and wherein the binding level or the change in the binding level provides an efficacy measure of the therapy.

4. The method of claim 2, wherein the anti-TNF-a antibody comprises Adalimumab.

5. The method of claim 2, wherein the sample further comprises anti-drug antibody (ADA) that binds to the anti-TNF-a antibody and wherein the method further comprises detecting the binding level or a change in the binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample.

6. The method of claim 5, wherein the sample is obtained from a subject having been administered the anti-TNF-a antibody as a therapy for a disease, condition,or disorder and wherein the binding level or the change in the binding level provides a measure of an immunogenic response in the subject.

7. The method of claim 1 , wherein the sample comprises a sample type selected from the group consisting of: whole blood, serum, plasma, saliva, sputum, nasal fluid, cerebrospinal fluid, vaginal fluid, semen, and urine.

8. The method of claim 1 , comprising quantifying an amount of the TNF-a in the sample.

9. The method of claim 1 , wherein the sample is obtained from a subject and wherein the method comprises administering a therapy to the subject when the binding level of the TNFR2 functionalized MNPs with the TNF-a exceeds a predetermined threshold.

10. A kit for monitoring Tumor Necrosis Factor-alpha (TNF-a) in a sample, comprising a container that contains a set of Tumor Necrosis Factor Receptor 2 (TNFR2) functionalized plasmonic metal nanoparticles (MNPs).1 1. A kit of claim 10, further comprising a system for monitoring the TNF-a in the sample using the set of TNFR2 functionalized MNPs.

12. A system for monitoring Tumor Necrosis Factor-alpha (TNF-a) in a sample, comprising: a sample container receiving structure configured to receive a sample container that contains the sample and a set of Tumor Necrosis Factor Receptor 2 (TNFR2) functionalized plasmonic metal nanoparticles (MNPs), wherein the sample comprises the TNF-a; a light source operably connected to the sample container receiving structure, wherein the light source is configured to transmit light through the sample container;a photodetector operably connected to the sample container receiving structure, wherein the photodetector is configured to measure light intensity transmitted through the sample container from the light source; a controller operably connected to the light source and to the photodetector, wherein the controller is configured to effect transmission of the light through the sample container using the light source and measurement of the light intensity transmitted through the sample container to detect a binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample using the photodetector; a power source operably connected to the controller, wherein the power source is configured to selectively supply power to the controller; and, a housing structure operably connected to the sample container receiving structure, wherein the controller and power source are substantially housed in one or more cavities disposed within the housing structure.

13. The system of claim 12, wherein the controller is configured to quantify an amount of the TNF-a in the sample.

14. The system of claim 12, wherein the sample further comprises an anti- TNF-a antibody and wherein the controller is configured to detect the binding level or a change in the binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample.

15. The system of claim 14, wherein the sample is obtained from a subject having been administered the anti-TNF-a antibody as a therapy for a disease, condition, or disorder and wherein the binding level or the change in the binding level provides an efficacy measure of the therapy.

16. The system of claim 14, wherein the anti-TNF-a antibody comprisesAdalimumab.

17. The system of claim 14, wherein the sample further comprises anti-drug antibody (ADA) that binds to the anti-TNF-a antibody and wherein the controller is configured to detect the binding level or a change in the binding level of the TNFR2 functionalized MNPs with the TNF-a in the sample.

18. The system of claim 17, wherein the sample is obtained from a subject having been administered the anti-TNF-a antibody as a therapy for a disease, condition, or disorder and wherein the binding level or the change in the binding level provides a measure of an immunogenic response in the subject.

19. The system of claim 12, wherein the sample comprises a sample type selected from the group consisting of: whole blood, serum, plasma, saliva, sputum, nasal fluid, cerebrospinal fluid, vaginal fluid, semen, and urine.

20. A kit comprising the system of claim 12.

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