Methods and related aspects for analyte detection

Abstract

Provided herein are methods of detecting a target analyte in a sample. The methods include contacting the sample with a set of functionalized plasmonic metal nanoparticles (MNPs) that comprise a target analyte binding agent that selectively binds to the target analyte to produce target analyte bound MNPs, and detecting binding of the resuspended target analyte bound MNPs. Related kits and systems are also provided.

Description

Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f METHODS AND RELATED ASPECTS FOR ANALYTE DETECTION CROSS-REFERENCE TO RELATED APPLICATONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application Ser. Nos.63 / 253,410, filed May 30, 2024, and 63 / 686,499, filed August 23, 2024, the entirety of each of which are incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R21 AI169098, and GM149552 awarded by the National Institutes of Health, 2022-67021-37013 awarded by the National Institute of Food and Agriculture, and 1838443 and 1847324 awarded by the National Science Foundation. The government has certain rights in the invention. BACKGROUND

[0003] Analyte detection is used in a variety of contexts, including for infectious disease diagnostics and for detecting various cancer types. These include, as an example, coronavirus disease (COVID-19), caused by the RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As a further example, thrombospondin-2 (THBS2) is a prevailing prognostic biomarker implicated in different cancer types, such as deadly colorectal, pancreas, and triple-negative breast cancers.

[0004] Currently, most COVID-19 diagnostics are based on reverse-transcription polymerase chain reaction (RT-PCR) assay, enzyme-linked immunosorbent assay (ELISA) and colloidal gold or fluorescence based rapid immunoassay. RT-PCR and ELISA generally require skilled medical practitioners operating bulky equipment and following elaborate diagnostics protocols that can takes up to 5 hours to complete. In addition, centralized laboratories with well-established COVID-19 diagnostics capacities are generally greatly limited due to the strict biosafety regulations (BSL-3), which significantly reduce the diagnostics throughput and increase diagnostic cost taking into account the biological sample shipment charges from the epidemic region to the laboratories. These methods, although widely used in clinical diagnostics, generally lack the stringent requirements of quick, user-friendly, electricity-free and low cost in fieldAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f point-of-care (POC) solutions to COVID-19 diagnostics in medical resource limited regions.

[0005] Rapid lateral flow immunochromatographic technology has been widely adopted in POC applications. It generally meets POC requirements such as fast screening speed, low cost and zero electricity consumption. However, its detection sensitivity is relatively low (up to hundreds of ng / ml in antigen protein detection) compared to RT-PCR and ELISA and entails qualitative analysis unless coupled to readout devices. In addition, there have been confirmed false positive cases (as high as 20%) from field validation, which can leave patients exposed to nosocomial SARS-CoV-2 transmission, potentially limiting its application in field-tests.

[0006] Researchers have reported improved sensitivity using Fe3O4 magnetic nanoparticles and multifunctional nanospheres in certain applications. However, these approaches typically utilize complicated detecting agent preparations and relatively high associated diagnostic costs. Besides these well adopted clinical diagnostics methods, several bioassays have been reported in current literature, with most of the works focusing on antigen-antibody conjugation or oligonucleotide hybridization and sensing signal transduced through florescence resonance energy transfer, single-particle interferometric reflectance imaging, opto-fluidic nanoplasmonic biosensors, nanoantenna array, memristor and field-effect transistors. Some of these methods demonstrate very sensitive detection (limit of detection reaching femtomolar) attributed to delicate signal transduction mechanism. On the other hand, such high sensitivity is often at the cost of elaborate sample preparation and complicated characterization, creating challenges for low cost miniaturized POC applications.

[0007] Moreover, while current methods for cancer-relevant protein detection, such as enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and immunohistochemistry, are feasible at advanced stages, they have shortcomings in sensitivity, specificity, and accessibility, particularly at low concentrations in complex biological fluids towards early detection.

[0008] Accordingly, there is a need for additional methods, and related aspects, of detecting target analytes, such as SARS-CoV-2 proteins and cancer biomarkers, suchAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f as THBS2 that are low cost, sensitive, easy-to-use, and which yield rapid POC results, particularly under low resource conditions. SUMMARY

[0009] In one aspect, the present disclosure provides a method of detecting a target analyte in a sample. The method includes contacting the sample with a set of functionalized plasmonic metal nanoparticles (MNPs) that comprise a target analyte binding agent that selectively binds to the target analyte to produce target analyte bound MNPs. The method also includes precipitating the target analyte bound MNPs to produce precipitated target analyte bound MNPs, and resuspending the precipitated target analyte bound MNPs to produce resuspended target analyte bound MNPs. In addition, the method also includes detecting binding of the resuspended target analyte bound MNPs.

[0010] In another aspect, the present disclosure provides a system for detecting a target analyte in a sample. The system includes a sample container receiving structure configured to receive a sample container that contains resuspended target analyte bound plasmonic metal nanoparticles (MNPs), wherein the sample container receiving structure is configured to substantially prevent ambient light from entering a sample container receiving area of the sample container receiving structure and wherein the sample container receiving structure comprises a light path aligned to measure at least supernatant in the sample container. The system also includes a light source operably connected to the sample container receiving structure, wherein the light source is configured to transmit light through the sample container, and 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. The system also includes 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 that contains the resuspended target analyte bound MNPs using the photodetector. In addition, the system also includes 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 operablyAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f 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

[0011] 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.

[0012] FIG.1A is a flow chart that schematically shows exemplary method steps of detecting a target analyte in a sample according to some aspects disclosed herein.

[0013] FIG.1B. Schematically shows an exemplary system for detecting a target analyte in a sample according to some aspects disclosed herein.

[0014] FIGS.2A-2G. Illustrate nanoparticle-supported, rapid electronic detection (NasRED) of antigens and antibodies for infectious diseases diagnostics. (a) Nanosensor preparation by biotinylating antigens or antibodies and attaching such proteins to streptavidin-coated AuNPs. (b-d) Schematics and optical images illustrating the detection process, using antibody testing as an example. (b) The AuNP sensing solution (18 µL) was mixed with the medium solution (6 µL). Negative controls (NCs) did not contain any target molecules but otherwise contained the same biological fluids and AuNP sensors. (c) The AuNPs were precipitated by centrifugation, incubated, and resuspended after stirring vortex. (d) The readout was collected directly from the tube, which was inserted in a tube holder as part of the NasRED readout system. The amount of AuNP precipitation at the tube bottom correlated with the number of test molecules, while no AuNP precipitates formed in the NC sample. (e) Schematic of a typical sensing curve showingAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f the normalized PED readout signal SPED. (f) Exemplary optical images and bar plots showing sensitivity and specificity in SARS-CoV-2 antigen detection in PBS. Tubes containing N-proteins of two other coronaviruses (HCoV-NL63 and HCoV-229E at 400 nM in PBS buffer) displayed very similar colors and electronic signals comparable to that of the NC sample. In contrast, tubes containing SARS-CoV-2 N-proteins showed much higher transparency and differentiable electronic signals at 4 fM and 400 nM. (g) Exemplary optical images and sensing curve plots showing antibody detection at high sensitivity in diluted whole blood.

[0015] FIGS. 3A-3F. Illustrate electronic circuit design for stabilized NasRED readout. (a) Schematic showing an integrated printed circuit board (PCB), which includes: (i) a built-in voltage regulator, (ii) a constant current LED driver circuit, and (iii) a microcontroller, which processes the signals and communicates with computers through USB, Wi-Fi, or Bluetooth modules. (b) Optical image showing the PCB board for signal processing. (c) The stability of LED currents over time for a circuit without the stabilization circuit (black line) and an integrated PCB board with the stabilization circuit (lighter grayscale line). (d) PED readout signal SPED plotted against antibody AS35 concentrations spiked in PBS, with (lighter grayscale) and without (black) the stabilization circuit. (e) LED irradiance recorded for 60 minutes using four different devices #1 (black line), #2 (grayscale line), #3 (grayscale line), and #4 (grayscale line). (f) Three, three- hour-long, replicate measurements of LED irradiance from one PED reader.

[0016] FIGS.4A-4K. Illustrate optimization of NasRED sensing protocol under different centrifugation and vortex conditions. (a-d) The PED readout signal ^^^^plotted as a function of the antibody concentration, with: (a) the centrifugate force from 0 to 1,500 gravity (g), (b) the centrifugation time from 0 to 5 minutes, (c) the vortex agitation speed from 32 to 40 round per second (rps), and (d) the vortex time from 3 to 15 seconds. (e-f) Schematic representations of the AuNP cluster formation and precipitation mechanism during the sensing process: (1) uniform mixing of AuNPs with target proteins, (2) AuNP sensors react to and bind to target proteins, thus forming clusters, during centrifugation, (3) after centrifugation and during incubation, the AuNP clusters form larger precipitates at the tube bottom, and (4) vortex agitation releasing loose AuNPs back into the solution. (g) Representative cryo-TEM images of 80 nm AuNP clusters at AS35 concentrations ofAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f 40 pM, 4 pM, 400 fM, 40 fM, 40 aM, and NC. (h) Statistical analysis of AuNP cluster sizes formed at different AS35 concentrations. NC: negative control using blank sample. (i–k) Modeling and analysis of vertical AuNP concentration distribution. (i) Schematic of the optical detection setup with dual-position PED readout, showing upper (5.5 mm) and lower (3.5 mm) sensing locations. (j) Reconstructed AuNP concentration profiles using an exponential decay model, fitted to the measured concentrations at both positions. The profiles were generated using Python with a mass conservation constraint. (k) Extracted model parameters ∅(^^) and ^^(^^) as a function of analyte concentration.

[0017] FIGS. 5A-5F.ELISA and spectrometry confirmation of mAb AS35 detection in PBS and biological fluids. (a) Schematics of ELISA assay, with antibody concentrations ranging from 400 nM to 40 aM. NC: PBS only. (b) The PED readout signal ^^^^(black line) plotted the average of the 6 replicate experiments in PBS, with a LoD of 49 aM (0.007 pg / mL). The ELISA absorbance (grayscale line), averaged from the 3 replicate experiments in PBS, were plotted against antibody concentration, showing a LoD of ~155 fM (23.3 pg / mL). ELISA values were normalized between 0 and 1 for direct comparison with ^^^^. Error bars indicate the standard deviation (SD) across replicates for both experiments. (c) Comparison of the ELISA and NasRED sensing performance of AS35 in PBS. The CV analysis showing a larger variation (as large as 52%) in ELISA absorbance but a smaller variation (<10%) in PED signals across the entire tested concentration range. The recovery of ELISA was out of the desired range (100±30%) at moderate and low concentrations (4 pM - 40 aM), while PED showed stable recovery within the range, showing higher reliability. (d-e) Optical images showing samples ready for NasRED readout: (d) in human pooled serum and (e) in 1% diluted human whole blood. Upon mixing with the AuNP sensing solution, the HPS sample was 25% diluted, and the WB was 0.25% diluted. (h) Extracted PED signals plotted against antibody concentration in serum (grayscale line, LoD=76 aM, or 0.011 pg / mL), and whole blood (grayscale line, LoD=360 aM, or 0.054 pg / mL). The samples were analyzed using a standard testing condition, i.e., centrifugation at 1,200 g for 5 minutes, incubation for 5 minutes, and vortex agitation at 34.5 rps for 5 seconds.Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f

[0018] FIGS.6A-6N. Illustrate N-protein antigen detection in biological fluids. (a- c) Optical images showing antigen samples using AuNPs functionalized with different mAbs in PBS buffer: (a) only AS47, (b) only AM223, (c) co-binder (AS47 and AM223) on two sets of AuNPs. The antigen concentrations were 400 nM, 400 pM, 400 fM, and 40 aM. NC: buffer only. (d) Extracted PED signals in PBS buffer using different AuNP sensors, i.e. monobander AS47 / AuNP (black dashed line, LoD=4 nM, or 2×105pg / mL),monobander AM223 / AuNP (black dotted line, LoD=820 pM, or 4 × 104 pg / mL), andcobinders (AS47 / AuNP + AM223 / AuNP, black solid line, LoD=180 aM, or 0.01 pg / mL). (e) Optical images showing samples for specificity test in PBS against human coronavirus (HCoV) (HCoV-NL63 and 229E) and SARS-CoV-2 N-proteins. All N-proteins were at 400 nM. (f) Extracted PED signals plotted against HCoV and SARS-CoV-2 antigens, as shown in figure e. (g) Extracted PED signal plotted antigen testing with the cobinders in saliva (red solid line, LoD=190 aM, or 0.01 pg / mL) and nasal fluid (grayscale solid line, LoD=2 fM, or 0.115 pg / mL). (h-k) The PED readout signal ^^^^plotted as a function of the N- protien concentration, with: (h) the centrifugate force from 0 to 1,500 gravity (g), (i) the centrifugation time from 0 to 5 minutes, (j) the vortex agitation speed from 30 to 36 round per second (rps), and (k) the vortex time from 0 to 5 seconds. (l) Schematics of preparing inactive virus sample in saliva: lysis using triton x-100 to produce product comprising viral RNA and proteins, and mixing the lysis product with NasRED antigen sensor solution for viral N-protein detection. (m) Optical image showing a titration of the inactive virus samples. (n) PED signals plotted against inactivated virus N-protein concentration determined by ELISA (bottom x-axis) and the viral particle RNA count determined by qPCR (top x-axis) in saliva (LoD=4.1 pM, 3×105copies / mL or 1.7×103TCID50 / mL).

[0019] FIGS. 7A-7D. Illustrate a design scheme for NasRED-based THBS2 detection. (a) Streptavidin-coated gold nanoparticles (AuNPs) are functionalized with anti- THBS2 antibodies to create specific probes targeting THBS2. (b) THBS2 is spiked into PBS and body fluids such as whole blood, serum, synovial fluid, and saliva. These samples (red tubes) are prepared through serial dilutions ranging from 10 nM to 100 fM, alongside a negative control containing the fluid matrix without THBS2 (yellow tube). When mixed, a biochemical binding reaction occurs between THBS2 and anti-THBS2 antibodies on the AuNP surface, leading to clustering. (c) Clustering is enhanced usingAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f active fluidic forces generated by centrifugation, which precipitates nanoparticles to the bottom of the tube, thereby locally enhancing reagent concentration. During incubation, clusters grow, while non-reacted nanoparticles are later resuspended using vortex agitation. (d) The colorimetric signals from the tubes are measured using a portable electronic device (PED). This device comprises a 3D-printed tube chamber, an LED, a photodiode, and a custom circuit board to digitize the analog signals from transmitted light through the tube.

[0020] FIGS. 8A-8D. Illustrate sensing protocol optimization by analyzing the impact of active fluidic force. (a-b) Vortex agitation impact: (a) Optical images of the tubes immediately after the NasRED protocol with vortex speeds ranging from 1750 to 2250 rpm. (b) Sensing curves showing collected signals (SPED) plotted against different THBS2 concentrations on a logarithmic scale for different vortexing speeds. Here SPED is normalized between 1 (negative control, NC) and 0 (indicating positive sample). (c-d) Centrifugation impact: (c) Optical images of the tubes immediately after the readout with centrifugation speeds adjusted from 385 to 2400 g. (d) Sensing curves showing collected signals SPED plotted against different THBS2 concentrations on a logarithmic scale for different centrifugation speeds. Error bars represent the standard deviation across five independent readouts of the same tube in different orientations to account for tube variability.

[0021] FIGS.9A-9L. Illustrate NasRED detection of THBS2 spiked in different biological matrixes. (a-b) Detection in PBS: (a) optical images of sensing tubes before (top row) and after (bottom) the NasRED protocol, and (b) normalized sensing curve. (c- d) Detection in HPS: (c) optical images of sensing tubes, and (d) normalized sensing curve. (e-f) Detection in Saliva. (g-h) Detection in 20% WB. (i-j) Detection in CSF. (k-l) Detection in 20% synovial fluid. All SPED sensing curves are normalized between 1 (negative control, NC) and 0 (from positive sample, clear tube) and plotted against THBS2 concentrations on a logarithmic scale. Error bars represent the standard deviation across five independent readouts of the tube in different orientations to account for tube variability.Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f

[0022] FIGS.10A-10D. Illustrate THBS2 specificity analysis in body fluids. (a-b) Specificity analysis in HPS: (a) Optical images of the tubes before (top row) and after (bottom row) the NasRED protocol, with THBS2 spiked at 10 nM concentration. (b) Bar charts representing normalized sensor signals (SPED) for THBS2 compared with CA19-9, BSA, and the negative control (NC). SPED represents changes in optical extinction (see methods). (c-d) Specificity analysis in saliva.

[0023] Those of ordinary skill in the art will understand that the compositions, methods, devices, and kits specifically described herein and illustrated in the accompanying drawings are non-limiting exemplary embodiments and that the scope of the various embodiments of the present disclosure is defined solely by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present disclosure. DEFINITIONS

[0024] 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.

[0025] 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.

[0026] 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. TheAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f 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.

[0027] 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.

[0028] 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.

[0029] 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%, 11%, 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).

[0030] 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.

[0031] Antibody: As used herein, the term “antibody” refers to an immunoglobulin or an antigen-binding domain thereof. The term includes but is not limitedAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f 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: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, 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.

[0032] Antigen Binding Portion: As used herein, the term “antigen binding portion” refers to a portion of an antibody that specifically binds to a target analyte (e.g., a SARS-CoV-2 protein, such as a receptor-binding domain (RBD) protein, Thrombospondin-2 (THBS2), etc.), e.g., a molecule in which one or more immunoglobulin chains is not full length, but which specifically binds to a target analyte. Examples of binding portions encompassed within the term “antigen-binding portion of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VLC, VHC, CL and CH1 domains: (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHC and CH1 domains; (iv) a Fv fragment consisting of the VLC and VHC domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VHC domain; and (vi) an isolated complementarity determining region (CDR) having sufficient framework to specifically bind, e.g., an antigen binding portion of a variable region. An antigen binding portion of a light chain variable region and an antigen binding portion of a heavy chain variable region, e.g., the two domains of the Fv fragment, VLC and VHC, can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLC and VHC regions pair to form monovalent molecules (known as single chain Fv (scFV)). Such single chain antibodies are also encompassed within the term “antigen binding portion” of an antibody. The term “antigen binding portion” encompasses a single-domain antibody (sdAb), also known as a “nanobody” or “VHH antibody,” which is an antibody fragment consisting of a single monomeric variable antibody domain. These antibody portions are obtained using conventional techniquesAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f known to those with skill in the art, and the portions are screened for utility in the same manner as are intact antibodies.

[0033] Bind: As used herein, “bind,” in the context of target analyte detection, refers to a state in which a first chemical structure (e.g., a pathogenic particle or other biomarker) is sufficiently associated a second chemical structure such that the association between the first and second chemical structures can be detected.

[0034] 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.

[0035] 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., a SARS- CoV-2 protein, Thrombospondin-2 (THBS2), etc.) and / or a pathogen (e.g., a SARS-CoV- 2) in a sample.

[0036] Epitope: As used herein, “epitope” refers to the part of an antigen (e.g., a SARS-CoV-2 protein) to which an antibody and / or an antigen binding portion binds.

[0037] Reaction Mixture: As used herein, "reaction mixture" refers a mixture that comprises molecules and / or reagents that can participate in and / or facilitate a given reaction or assay. A reaction mixture is referred to as complete if it contains all reagents necessary to carry out the reaction, and incomplete if it contains only a subset of the necessary reagents. It will be understood by one of skill in the art that reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for application- dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, it will be understood by one of skill in the art that reaction components are packagedAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f separately for commercialization and that useful commercial kits may contain any subset of the reaction or assay components.

[0038] Sample: As used herein, “sample” means anything capable of being analyzed by the methods, devices, and / or systems disclosed herein.

[0039] Severe Acute Respiratory Syndrome Coronavirus-2: As used herein, “severe acute respiratory syndrome coronavirus-2” or “SARS-CoV-2” refers to the coronavirus that emerged in 2019 to cause a human pandemic of an acute respiratory disease, now known as coronavirus disease 2019 (COVID-19).

[0040] Specifically Bind: As used herein, "specifically bind,” in the context of pathogen detection, refers to a state in which substantially only target chemical structures (e.g., target SARS-CoV-2 proteins, Thrombospondin-2 (THBS2), etc.) are sufficiently associated with a corresponding or cognate binding agent (e.g., an antibody, or antigen binding portion thereof), to the exclusion of non-target chemical structures, such that the association between the target chemical structures and the binding agent can be detected.

[0041] 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.”

[0042] 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.Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f

[0043] Thrombospondin-2: As used herein, "Thrombospondin-2" or "THBS2" is a glycoprotein that belongs to the thrombospondin family. THBS2 plays a role in extracellular matrix assembly, angiogenesis, and chondrogenic differentiation. The protein is involved in various biological processes and is also implicated in cancer progression and metastasis, serving as a potential diagnostic and prognostic biomarker for several cancers.

[0044] 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

[0045] 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 disclosure 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 disclosure. The following description is, therefore, merely exemplary.

[0046] I. Introduction

[0047] The present disclosure provides an inexpensive, miniaturized sensing system (using SARS-CoV-2 and Thrombospondin-2 as examples) to detect the antibodies and antigens directly from sera, blood, saliva, and nasal fluids within a short time (10 to 30 min, comparable to LFA) and achieve high sensitivity (atto molar, about 5 orders better than ELISA20 and 6 orders of magnitude better than LFA), enabling rapidAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f and accurate diagnostics of infectious diseases that can be accessed in different clinical settings. Specifically, a digital sensing platform is designed to detect the target proteins in-solution, by simply mixing the bodily fluids with a 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 a 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. While this work leverages a past proof-concept demonstration of Ebola antigen and small molecule sensing using AuNP-based digital platform3, it fundamentally differs in a number of important innovations. First, unlike a prior work that relied on synthetic nanobodies for target antigen or small molecule detection, the present disclosure proves the feasibility of high-sensitivity and high-specificity detection using commercially available monoclonal antibodies (mAbs), and therefore demonstrate the broad and general applicability of the sensing technology of the present disclosure. Second, the present disclosure provides a new circuit system that stabilizes the LED and PD signals for minimized readout errors, which is favorable for detection at ultralow concentrations. Lastly, the inventors comprehensively optimized the sensing protocol, which, together with the new circuit design, helps achieve atto-molar detection in a wide range of bodily fluids, i.e.5 to 6 logs in LODs better than previously demonstrated Ebola secreted glycoprotein and SARS-CoV-2 receptor binding domain (RBD) detection in diluted sera (previously ~10 pg / mL (0.13 pM) and ~40 pg / mL (~1.3 pM), respectively). Notably, the detection time or specificity were not compromised while achieving this significant improvement in sensitivity. Therefore, this new sensing platform and as its design and optimization strategy have a significantly broadened use in applications for highly sensitive, rapid and accessible detection of not only specific infectious diseases but also a wide range of infectious and chronic diseases such as cancer, Alzheimer’s, and the like.

[0048] In some embodiments, the present disclosure provides NasRED as aAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f novel, semiconductor-based, digital sensing platform for rapid, modular, sensitive, specific, portable, and low-cost infectious disease detection. Using AuNPs as the reagent carrier and signal transducing probes, the inventors have engineered both the sensing protocol and designed the electronic readout circuitry with signal stabilization functions to improve the antigen and antibody sensing performance. The sensitivities of SARS-CoV- 2 antibodies and antigens were demonstrated down to attomolar or as small as < 10 fg / mL when tested in PBS, human serum, blood, saliva, and nasal fluids, attributed to the centrifugation-induced concentration enhancement effect and minimized electronic readout errors. The assay specificity was high enough to differentiate SARS-CoV-2 N- proteins across 11 logs (100 nM to 10 aM) from HCoV N-proteins at a high concentration (100 nM), showing minimal non-specific interactions. In addition, the assay time from sample mixing to signal readout was as short as 10 to 30 minutes, thus meeting the needs of rapid diagnostics of infectious diseases. The NasRED assay also requires very small amount of sample (< 10 μL) and reagents (< 20 μL), and therefore minimize the diagnostic costs to a few dollars. In addition, the digitized readout allows on-chip signal processing and automated direct data output though WiFi, Bluetooth and USB, further minimizing data processing time and human errors. With a sensitivity at least two orders of magnitude better than ELISA and a fast turnaround time comparable to LFA, NasRED is a versatile platform with extensive applications in the rapid and accessible detection of a wide range of infectious diseases as well as chronic diseases such as cancer.

[0049] Thrombospondin 2 (THBS2) is a prevailing prognostic biomarker implicated in different cancer types, such as deadly colorectal, pancreas, and triple- negative breast cancers. While current methods for cancer-relevant protein detection, such as enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and immunohistochemistry, are feasible at advanced stages, they have shortcomings in sensitivity, specificity, and accessibility, particularly at low concentrations in complex biological fluids towards early detection. Accordingly, in some aspects, the present disclosure demonstrates a modular, in-solution assay design concept, Nanoparticle- Supported Rapid Electronic Detection (NasRED), as a versatile cancer screening and diagnostic platform. NasRED utilizes antibody-functionalized gold nanoparticles (AuNPs) to capture target proteins from a minute amount of sample (< 10 μL), and achieve optimalAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f performance with a short assay time by introducing active fluidic forces that act to promote biochemical reaction and accelerate signal transduction. This rapid (15-minute) process serves to form AuNP clusters upon THBS2 binding and subsequently precipitate such clusters, resulting in color modulation of the test tubes that are dependent on the THBS2 concentration. Finally, a semiconductor-based, portable, electronic device is used to digitize the optical signals for sensitive detection of THBS2. High sensitivity (femtomolar level) and a large dynamic range (5 orders of magnitude) are obtained to analyze THBS2 spiked in PBS, serum, whole blood, saliva, cerebrospinal fluids, and synovial fluids. High specificity is also preserved in differentiating THBS2 from other markers such as cancer antigen (CA) 19-9 and bovine serum albumin (BSA). This disclosure highlights NasRED's potential to enhance cancer prognosis and screenings by offering a cost-effective, accessible and minimally invasive solution. These and other attributes will be apparent upon a complete review of the present disclosure, including the accompanying figures.

[0050] II. Description of Example Embodiments

[0051] To illustrate aspects of the present disclosure, FIG. 1A provides a flow chart that schematically shows exemplary method steps of detecting a target analyte in a sample according to some embodiments disclosed herein. As shown, method 100 includes contacting the sample with a set of functionalized plasmonic metal nanoparticles (MNPs) that comprise a target analyte binding agent that selectively binds to the target analyte to produce target analyte bound MNPs (step 102). In some embodiments, the sample comprises a volume of about 10 μL or less. Method 100 also includes precipitating the target analyte bound MNPs to produce precipitated target analyte bound MNPs (step 104) and resuspending the precipitated target analyte bound MNPs to produce resuspended target analyte bound MNPs (step 106). In addition, method 100 also includes detecting binding of the resuspended target analyte bound MNPs (step 108). In some embodiments, method 100 comprises quantifying an amount of the target analyte in the sample. In some embodiments, the detecting step comprises detecting one or more aggregations of the resuspended target analyte bound MNPs. In some embodiments, method 100 comprises obtaining the sample from a subject. In some embodiments, method 100 comprises administering one or more therapies to the subject when the target analyte is detected in the sample.Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f

[0052] In some embodiments, method 100 comprises a limit of detection of between about 10 femtogram per milliliter (fg / mL) and about 225 fg / mL. In some embodiments, method 100 comprises a sensitivity at a femtomolar or lower level. In some embodiments, method 100 comprises a dynamic range of at least about five orders of magnitude. In some embodiments, a duration of the method 100 is about 15 minutes or less.

[0053] Method 100 can be adapted to accommodate essentially any sample type. In some embodiments, for example, the sample comprises a sample type, such as whole blood, serum, plasma, saliva, sputum, nasal fluid, cerebrospinal fluid, vaginal fluid, semen, feces, and urine, among others.

[0054] In some embodiments, the target analyte binding agent comprises an antibody or antigen binding portion thereof, and wherein the target analyte comprises an antigen. In some embodiments, the antibody or antigen binding portion thereof comprises a monoclonal antibody. In some embodiments, the antibody or antigen binding portion thereof comprises a nanobody. In some embodiments, the antigen comprises a protein, or a portion thereof, from an infectious agent. In some embodiments, the infectious agent comprises a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the antigen, or a portion thereof, comprises a SARS-CoV-2 protein, or a portion thereof, selected from the group consisting of: an envelope (E) protein, a membrane (M) protein, a nucleocapsid (N) protein, and a spike (S) protein. In some embodiments, the antigen comprises a cancer biomarker. In some embodiments, the cancer biomarker comprises thrombospondin-2 (THBS2).

[0055] In some embodiments, the target analyte binding agent comprises an antigen, and wherein the target analyte comprises an antibody. In some embodiments, the antigen comprises a protein, or a portion thereof, from an infectious agent. In some embodiments, the infectious agent comprises a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the antigen, or a portion thereof, comprises a SARS- CoV-2 protein, or a portion thereof, selected from the group consisting of: an envelope (E) protein, a membrane (M) protein, a nucleocapsid (N) protein, and a spike (S) protein. In some embodiments, the antigen comprises a cancer biomarker or a portion thereof. InAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f some embodiments, the cancer biomarker or a portion thereof comprises thrombospondin-2 (THBS2) or a portion thereof.

[0056] In some embodiments, the present disclosure provides a system for detecting a target analyte in a sample. The system includes a sample container receiving structure (e.g., a tube holder) configured to receive a sample container (e.g., a microcentrifuge tube) that contains resuspended target analyte bound plasmonic metal nanoparticles (MNPs) in which the sample container receiving structure is configured to substantially prevent ambient light from entering a sample container receiving area of the sample container receiving structure and in which the sample container receiving structure comprises a light path aligned to measure at least supernatant in the sample container. The system also includes a light source (e.g., a light-emitting diode (LED) or the like) operably connected to the sample container receiving structure in which the light source is configured to transmit light through the sample container. The system also includes 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 (e.g., configured on a printed circuit board (PCB)) 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 that contains the resuspended target analyte bound MNPs using the photodetector. In some embodiments, the controller is configured to quantify an amount of the target analyte in the sample. The system also includes a power source (e.g., a battery or the like) operably connected to the controller in which the power source is configured to selectively supply power to the controller. The system also includes a housing structure or enclosure 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. To illustrate, FIG. 1B schematically shows an exemplary system for detecting a target analyte in a sample according to some aspects disclosed herein. In some embodiments, systems are packaged as part of kits.

[0057] In some embodiments, the resuspended target analyte bound MNPs areAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f formed by: contacting the sample with a set of functionalized MNPs that comprise a target analyte binding agent that selectively binds to the target analyte to produce target analyte bound MNPs; precipitating the target analyte bound MNPs to produce precipitated target analyte bound MNPs; and resuspending the precipitated target analyte bound MNPs to produce the resuspended target analyte bound MNPs.

[0058] In some embodiments, the target analyte binding agent comprises an antibody or antigen binding portion thereof, and the target analyte comprises an antigen. In some embodiments, the antibody or antigen binding portion thereof comprises a monoclonal antibody. In some embodiments, the antibody or antigen binding portion thereof comprises a nanobody. In some embodiments, the antigen comprises a protein, or a portion thereof, from an infectious agent. In some embodiments, the infectious agent comprises a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the antigen, or a portion thereof, comprises a SARS-CoV-2 protein, or a portion thereof, selected from the group consisting of: an envelope (E) protein, a membrane (M) protein, a nucleocapsid (N) protein, and a spike (S) protein. In some embodiments, the antigen comprises a cancer biomarker. In some embodiments, the cancer biomarker comprises thrombospondin-2 (THBS2).

[0059] In some embodiments, the target analyte binding agent comprises an antigen, and the target analyte comprises an antibody. In some embodiments, the antigen comprises a protein, or a portion thereof, from an infectious agent. In some embodiments, the infectious agent comprises a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the antigen, or a portion thereof, comprises a SARS-CoV-2 protein, or a portion thereof, selected from the group consisting of: an envelope (E) protein, a membrane (M) protein, a nucleocapsid (N) protein, and a spike (S) protein. In some embodiments, the antigen comprises a cancer biomarker or a portion thereof. In some embodiments, the cancer biomarker or a portion thereof comprises thrombospondin-2 (THBS2) or a portion thereof.

[0060] III. Examples

[0061] Example 1: Nanoparticle-Supported, Rapid, and Electronic Detection of SARS-CoV-2 Antibodies and Antigens at Sub-Femtomolar LevelAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f

[0062] I. Introduction

[0063] Infectious diseases pose a serious threat to human health and the world economy. For example, as of March 1st, 2025, COVID-19 alone has resulted in more than 777 million infections and 7 million deaths, while causing an estimated $14 trillion in economic losses in the U.S. by the end of 2023. In addition, the pandemic also seriously strained the global healthcare system and caused delayed or missed diagnosis or treatment for many other patients. To better prepare for future pandemics, which could be caused by a currently unknown disease ‘X’, it is of paramount importance to continue investing in innovative diagnostic technologies. These technologies should enable early and accurate disease detection and quantification in a portable, cost-effective, and user- friendly manner, ensuring accessibility for all patients in need, including those in rural areas and low- to middle-income countries where medical resources are scarce. To complement this, various new approaches, including label-free based high-sensitivity diagnostic technologies, have been proposed recently.

[0064] Conventionally, molecular and immunochemical tests are widely used to diagnose infectious diseases, including COVID-19. These tests look for molecules of different genetic information to diagnose diseases and predict the risk of unknown diseases. Nucleic Acid Amplification Tests (NAATs), including Polymerase Chain Reaction (PCR) and Loop-mediated Isothermal Amplification (LAMP), leverage the feasibility of DNA or RNA amplification to maximize the sensitivity. However, as the gold standard for laboratory diagnostics, PCR requires several hours to days to amplify and confirm the results due to the multiple steps of sample handling and the thermal cycling process. Additionally, it requires expensive, bulky equipment, making it suitable for centralized testing but inaccessible in resource-limited settings. Isothermal amplification methods such as LAMP, as an alternative to PCR, require much cheaper instruments; however, the sensitivity heavily depends on the primer design, which constrains target site selection and assay specificity.

[0065] Immunochemical tests diagnose diseases by confirming the presence or absence of a specific protein based on antigen-antibody reactions. The most commonly used methods include Enzyme-Linked Immunosorbent Assay (ELISA) and Lateral FlowAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f Assay (LFA). ELISA typically achieves picomolar-level sensitivity in biological fluids. However, like PCR, ELISA is most suited for high-sensitivity analysis of large amounts of samples in laboratory settings since it requires a complex, multistep workflow, making it highly labor-intensive and expensive. In addition, Rapid Antigen Tests (RATs), such as LFA, are portable and suitable for field applications but generally have lower sensitivity (e.g., 65.3% for COVID-19). This limitation makes them less reliable for accurate detection, particularly at early-stage infection when the viral load is low.

[0066] Here, using SARS-CoV-2 as an example, the inventors report a prototyped, inexpensive, miniaturized sensing system, i.e. nanoparticle-supported rapid electronic detection (NasRED), to detect the antibodies and antigens directly from serum, whole blood, saliva, and nasal fluid within a short time (15 to 30 min, comparable to LFA). NasRED achieves high sensitivity (sub-femtomolar, or fg / mL, about 2 to 4 orders of magnitude better than ELISA and about 5 to 6 orders better than LFA), enabling rapid and accurate diagnostics of infectious diseases that can be accessed in different clinical settings. Specifically, a digital sensing platform was designed to detect the target proteins by simply mixing bodily fluids with a prepared sensing solution comprising functionalized gold nanoparticles (AuNPs) in a testing tube. The entire process, including mixing, reaction, and signal readout, occurs within the same tube without washing, labeling, or fluorescent imaging and significantly reducing operation complexity. The sensing signals were collected, digitized, and transmitted through a customized semiconductor devices and circuits, which minimized signal fluctuations and enable the accurate detection of antigens and antibodies at ultralow concentrations. Further, the inventors comprehensively engineered the sensing protocol, i.e. the centrifugation and vortex agitation speed and time, to evaluate their impact on the limits of detection (LoDs) of SARS-CoV-2 antibody (AS35) and antigen (N-protein). The LoDs were found ~<0.1 pg / mL from a wide range of bodily fluids, including in 20% diluted whole blood without plasma separation. This high sensitivity is attributed to the application of active fluidic forces during centrifugation and vortex agitation, which enhance protein collisions, biochemical binding, and signal transduction, even at low concentrations. Additionally, vortex-induced exponential decay in AuNP concentration along the liquid column enhances optical signal amplification, enabling detection of trace protein levels. Lastly,Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f using inactivated SARS-CoV-2 virion particles, NasRED successfully detected N-protein with high sensitivity (190 aM or 10 fg / mL) in saliva, outperforming ELISA. Based on parallel qRT-PCR measurements, this protein detection level corresponds to an estimated RNA sensitivity of 3×105copies / mL, demonstrating performance comparable to portable nucleic acid amplification tests. Together, the preliminary data demonstrates the strong promise of NasRED as a new and broadly applicable platform for highly sensitive, rapid, and accessible detection of infectious diseases as well as chronic diseases.

[0067] II. Results

[0068] NasRED sensing platform

[0069] The NasRED protein sensing platform can be applied to the detection of both the antigens and the antibodies (Figure 2). The sensors are prepared by attaching biotinylated proteins to streptavidin-coated AuNPs through a biotin-streptavidin reaction, where the protein binders can be antigens (e.g. SARS-CoV-2 spike (S) or receptor binding domain (RBD) proteins) for antibody (neutralizing antibody AS35) sensing or antibodies (mouse IgG1 AS47 and chimeric IgG1 AM223) for antigen (nucleocapsid (N) protein) sensing (Figure 2a). Using antibody sensing as an example (Figure 2b), 18 µL of the RBD-functionalized AuNPs were mixed with 6 µL of medium (e.g., PBS buffer, human pooled serum (HPS), or human whole blood (WB)) containing target SARS-CoV-2 antibodies diluted to desired concentrations. A sample of blank medium without antibodies was used as a negative control (NC). For rapid detection, the 24 µL mixture was centrifuged at 1,200 g for 5 minutes and incubated for 5 minutes to precipitate the functionalized AuNPs to the bottom of the microcentrifuge tube, where their concentration was greatly enhanced. Such a localized and boosted reactant concentration significantly promoted the localized reaction with target proteins, thus accelerating the antibody- antigen reaction at low concentrations to achieve an ideal LoD and reduce assay time. To preserve the detection specificity, a vortex agitation was introduced to disperse unbound AuNPs without disturbing the precipitated AuNP clusters, verified by fully dispersed AuNPs in the NC reference. The optical absorption signal from the supernatant, attributed to free-floating AuNPs after vortexing, was digitized using a portable electronicAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f detector (PED) for quantification (Figure 2d). The electronic signals of the antigen or antibody samples collected from the PED represented the optical transmission of light- emitting diode (LED) light through the supernatant in the microcentrifuge tube and were normalized (Methods). The NasRED system has proven to be feasible to detect antigens and antibodies in complex biological fluids with high sensitivity and specificity (Figure 2f- g).

[0070] Signal-stabilizing circuit for low-noise detection

[0071] The NasRED platform consists of a portable electronic detector (PED) for signal readout, which is composed of two customized components. The first component is a semiconductor-based, digital, signal-collecting device, which includes an LED, a photodetector (PD), and a microcentrifuge tube chamber designed to block ambient light. This device functions to probe the free-floating AuNP concentration in the supernatant of the testing tube and to digitize the optical signals of the AuNPs to electronic current signals. The second component is a circuit board designed to stabilize the LED and PD signals for reliable antibody or antigen detection. Electronically, the stabilizing circuit board (Figure 3a-b) had three key components: a voltage regulator that adjusts the power supply to the desired value for the LED circuit and microprocessor, a constant current LED driver circuit that stabilizes the light intensity, and a microprocessor for signal processing. The regulator circuit includes two electrolytic capacitors, two ceramic capacitors, and one protective Zener diode (Figure 3a (i)). The constant current LED driver circuit utilizes a feedback loop that coordinates double bipolar junction transistors (BJTs, T1 and T2 in Figure 3a (ii)) to stabilize the current supplied to the LED. In addition, a customized microprocessor (Figure 3a (iii)) processes the signals and enables communication with a laptop through USB, Wi-Fi, or Bluetooth modules. Without the stabilization circuit, the LED displayed a large deviation in current (average 0.14 mA over 10 minutes but significantly increased after the 40-min operation, possibly due to heating, Figure 3c), and the PED produced random spike noises when used to test AS35 antibodies in PBS (Figure 3d). This instability can be attributed to the fact the LED light emission is proportional to its electrical current and exponentially depends on the bias voltage, and therefore, a slight voltage fluctuation from a power glitch or device heating can introduce a systematic error, ultimately seriously limiting the sensing performance atAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f low analyte concentrations. In comparison, the improved readout system demonstrated significantly reduced current fluctuation (0.12 mA within the first two minutes during circuit initiation and decreased to 0.02 mA during the rest 60-min testing period, Figure 3c) and greatly minimized system error in antibody detection (Figure 3d), indicating a substantial improvement in stability.

[0072] To further evaluate the reproducibility and reliability of the PED circuit design, four different PED devices were constructed. The LED output power from each PED circuit was measured by a PD and found stable at ~6 μW / cm2over a 60-minute test period (Figure 3e) with a very small (~2.5%) coefficient of variation (CV). Then, one PED circuit was randomly selected, and its LED power was measured three times, 3 hours each (Figure 3f). The measured irradiance was again very stable at ~6 μW / cm2with a CV of 2.4%. These inter- and intra-device tests proved the high reproducibility and suitability of the NasRED reading system for continuous use or repeated operation.

[0073] Impacts of centrifugation and vortex agitation on sensing performance

[0074] The centrifugation and vortex agitation actively modulate the reagent and AuNP distribution in the buffer solutions, providing additional flexibility to optimize the sensitivity and specificity. To examine their impact on the NasRED assay performance, the inventors chose mAb AS35 (6 µL, from 4 µM to 40 fM in PBS) as the target antibody, using AuNPs functionalized with WT-RBD (Wuhan-Hu-1, 18 µL in1×PBS dilution buffer). Electronic signals were collected using the stabilized readout system. Here, a series of tests (Figure 4a-h) were performed by modifying four variables: the centrifugate force gravity (g) (Figure 4a), centrifugation time (Figure 4b), vortex agitation speed (Figure 4c), and the vortex time (Figure 4d). The normalized assay signals, ^^^^(^^), were plotted as a function of AS35 concentration ^^(Figure 4a-d). The default parameters were centrifugation at 1,200 g for 5 minutes, incubation for 5 minutes, and vortex agitation at 34.5 rps for 5 seconds unless otherwise varied. It was clear that the AS35 antibodies could be detected with a large contrast in signal ^^^^, i.e. a high value at lowconcentrations (^^ = ^^^^(^^) ≅1) and a small signal at high concentrations (^^ =^^^^(^^) ≅ 0.1, ^^ = 4µM ), or ∆^^^^ = ^^ − ^^ ≅ 0.9. A high signal contrast ∆^^^^Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f indicated that the AuNP sensors effectively participated in the biochemical reaction at high protein concentrations and subsequently precipitated, which was ideal for maximizing the sensitivity and dynamic range (about 9 logs in this test). Clearly, the sensing performance of NasRED could be optimized within a range of centrifugation conditions, i.e., from 900 g to 1500 g and from 3 to 5 minutes, and at moderate vortex conditions.

[0075] NasRED sensing mechanism

[0076] The dependence of NasRED’s performance on the centrifugation and vortex parameters can be understood from the unique sensing mechanism. First of all, fundamentally, NasRED is an in-solution assay, where diffusion of AuNP sensors and reagents drives signal transduction, leading to AuNP precipitation and ultimately determining the assay performance. In NasRED, the sedimentation time of AuNPs linearly scales with the precipitation length. Unlike conventional assays such as ELISA, which rely on the passive diffusion of target molecules to a stationary reaction surface and require long incubation time to maximize the signal-to-noise ratio, NasRED supports external controls, such as centrifugation, incubation, and vortex agitation, to actively accelerate diffusion, reaction, and readout (Figures 4). Centrifugation pellets AuNPs dispersed across a few millimeters to the tube bottom, with a thickness estimated as <0.5 mm, thus eliminating slow sedimentation and accordingly reducing the readout time to less than 30 minutes (Figure 4a-b). In comparison, passive precipitation of AuNP clusters was previously found to require 3 to 24 hours of assay time from mixing to readout. Even after physical external control, the PED signal was found to remain constant for at least 1 hour. This indicates the samples after vortex agitation reached a quasi-equilibrium state with minimal signal fluctuation, thus desirable for reliable readout.

[0077] Further, the introduction of active fluidic forces is an integral part of NasRED’s unique signal transduction process, which begins with biochemical protein reaction and is dynamically modulated by fluidic interactions (Figure 4e-f). Centrifugation significantly increases the kinetic velocity of AuNPs, enhancing their collision with and effective capture of the target proteins, which in turn promotes the growth of AuNP clusters. Protein binding and AuNP cluster growth are expected to occur throughout theAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f centrifugation process, ultimately leading to precipitation of the majority of the AuNPs (Figure 4e). The dimensionless Peclet number (Pe), which compares nanoparticle centrifugal sedimentation to Brownian motion, scales linearly with the relative centrifugal force (RCF) and quantically with the AuNP diameter. For example, the Pe number was found ~1.92 for 80 nm AuNPs at an RCF of 1200×g, while significantly lower values were obtained for smaller nanoparticles (~0.01, 0.12, and 0.61 for 20 nm, 40 nm, and 60 nm AuNPs, respectively). The estimates suggest that the centrifugal forces dominate over Brownian motion during the sedimentation process of 80 nm and larger AuNPs, aligning with a prior observation that 80 nm AuNPs precipitated faster than smaller ones. On the other hand, the particle Reynolds number (^^^), which quantifies the ratio of inertial to viscous forces, increases proportionally AuNP diameter. ^^^was estimated~0.04 for 80 nm AuNPs, suggesting that fluidic drag forces remain a significant factor in governing the particle motion and dispersion within biological fluids. Consequently, the NasRED sensing protocol should be individually optimized for fluid media with different viscosities.

[0078] The AuNP precipitates at the tube bottom are thought to consist of inhomogeneous clusters, because AuNPs dispersed in the sensing solution are expected to experience different precipitation lengths, and have the opportunity to grow into different sizes while capturing proteins during their sedimentation process, eventually forming a mixture of clusters which statistically are larger in size at higher protein concentrations (Figure 4f left). Importantly, centrifugation greatly localizes AuNPs and their clusters into a much smaller volume (estimated <1 nL, or 3 to 4 orders more condensed) and drastically boost the AuNP concentration at the bottom of the reaction tube. This effectively modulates the dynamic equilibrium of antigen-antibody reaction to favor AuNP cluster formation both during centrifugation and incubation, even at ultralow reagent concentration in the tube. During incubation, AuNPs deposited on the tube sidewalls gradually settle to the tube bottom, thus minimizing the nonspecific particle interference with the LED-PD signal-collecting optical path in the upper solution. Additionally, AuNPs may possibly rearrange during incubation, forming more stable clusters that enhance sensitivity.Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f

[0079] After incubation, sufficient vortex agitation is required to fully resuspend monomer AuNPs that have not incorporated into clusters, ensuring high specificity, i.e., the negative control should return to their initial states. However, excessively high agitation risks breaking the AuNP clusters biochemically “glued” together, even at moderate or high protein concentrations. This would diminish the signal contrast ∆^^^^(Figure 4c-d arrows on the right) and negatively affect the NasRED sensitivity. Therefore, optimizing the agitation force and duration (e.g. 5 sec at 34.5 rps for the chosen centrifugation conditions) is necessary to maximize the signal contrast ∆^^^^and to best differentiate samples at low concentrations, for example from attomolar to picomolar for AS35 (Figure 4e optical images). Interestingly, the 40 aM and 4 pM samples could be clearly differentiated from their precipitate formation in the tubes, indicating feasible attomolar detection. For more precise analysis, the AuNP precipitates were collected from the bottom of the tubes and imaged by Cryogenic Electron Microscopy (cryo-EM) (Figure 4g). Then, the AuNP clusters at each AS35 concentration were measured and analyzed (Figure 4h). Evidently, large clusters (e.g. >50 µm2, up to ~800 µm2) formed at moderate AS35 concentrations (e.g., 40 pM, 4 pM, and 400 fM) consisted of densely packed and stacked AuNPs. In comparison, smaller clusters were found at low antibody concentrations (<~50 µm2at 4 fM and <~25 µm240 aM), consisting of loosely connected AuNPs. Notably, AuNP clusters of >5 µm2were only formed in the presence of AS35, whereas very small (<2 µm2) AuNP clusters or oligomers were detected from the NC (blank) sample, likely resulting from minor nonspecific inter-AuNP interactions during sample preparation (such as pipetting, TEM grid loading, and freezing). These Cryo-EM imaging provided direct physical evidence to confirm that the NasRED sensing protocol effectively dispersed the AuNPs for blank sample while differentiating antibodies at attomolar levels from higher-concentration and blank samples.

[0080] The fluidic dynamic behavior of AuNPs in a turbulent flow during vortex agitation is complex, potentially involving both shear- and spin-related fluidic drag forces, and is strongly dependent on the ^^^value. Both shear- and spin-induced lift forces may diminish at relatively high ^^^values, and are highly dependent on the AuNP size, shape, flow velocity, rotation, and position within the fluid. As such, it is reasonable to expect that AuNP clusters, which exhibit a substantially higher effective ^^^, experience reduced liftAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f forces compared to AuNP monomers with smaller ^^^. Therefore, AuNP clusters are far less likely to move upwards during vortex agitation. Precise physical modeling of these behaviors is challenging due to the complex fluidic dynamics, the protein-concentration- dependent, inhomogeneous mixture of AuNP monomers and clusters in the precipitates, and the three-dimensional conical geometry of the microcentrifuge tubes. Therefore, a detailed quantitative model will require extensive future studies and thus beyond the scope of this work.

[0081] To provide an intuitive understanding of the signal transduction mechanisms, the inventors first established a correlation between the PED signal and AuNP concentration, by cross-validating results using the manufacturer-provided data, NanoSight calibration, and AuNP extinction calculations based on Beer–Lambert law. Then the inventors built a new PED device capable of measuring the AuNP extinction signals at two different fluidic levels, and converted the measured two-channel signals into the AuNP concentrations ^^^^^at the two positions, for each tested AS35 antibody concentration ^^(Figure 4i). The inventors hypothesize that the AuNP concentration profile after vortex agitation follows a simplified exponential decay, consistent with the Mason-Weaver theory and assume mass conservation of AuNPs. To test this hypothesis,we modeled the AuNP concentration distribution (Figure 4j) in Python as: ^^^^^ ^^, ^" =# ^^"exp (− (() ^*") . Here # ^^" and ^^ ^^" are empirical, analyte-concentration-dependent(Figure 4k) that characterize the AuNP concentration at tube bottom (z=0) and characteristic decay length, respectively. The PED-measured and theoretically calculated ^^^^^values showed strong overall agreement (<20% error, Figure 4j). This physics-based model supports a hypothesis that the AuNPs tend to aggregate more at the tube bottom and diffuse back less to the suspension at higher ^^, also in agreement with the inventors’ cryo-EM analyses.

[0082] The optical tube images, PED readout signals, TEM analyses, and AuNP concentration modelling collectively demonstrated the success of attomolar detection from a small sample volume (6 µL). While further investigations are needed to fully elucidate the mechanisms behind the high-sensitivity of NasRED detection, a few factors possibly contribute. First, a strong antigen-antibody binding was achieved throughAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f multiple mechanism: (1) Multivalent binding on the AuNP sensor surfaces promotes high avidity, which has been demonstrated in pentameric IgM and nanobody oligomers to enhance the effective affinity by three to four orders of magnitude, (2) Protein capture is enhanced during centrifugation, and their disassociation from AuNPs is suppressed, and (3) The reagent concentration at the tube bottom was enhanced about 4 orders of magnitude (supplementary section 4.3). Second, unlike fluorescent imaging that suffers from a high background noise, the PED readout has minimized background noise owing to successful strategic assay system design: (i) Nonspecific interparticle interactions and tube wall adsorption are minimized by using inert AuNPs with covalently coated streptavidin as the signal carrier and by introducing glycerol and BSA in the AuNP sensor solution. (ii) A post-centrifugation incubation step allows the AuNP precipitates to settle at the tube bottom, thereby minimizing unintentional interference to signal collection from free-floating AuNP monomers. (iii) The stabilized PED readout device successfully suppresses signal fluctuations. Third, AuNP clusters bound by proteins may recruit non- protein-binding AuNP monomers through physical trapping, thus physically enlarging the AuNP precipitates and amplifying the PED signals. This could occur through disruption of the fluidic flow during centrifugation and / or vortex agitation, or by long-range attractive forces such as electrostatic interactions. TEM images revealed inter-particle distance (e.g., >40 nm) far exceeding typical protein dimensions between AuNPs at the perimeters of clusters, suggesting possible long-range electrostatic interactions between protein analyte and protein-coated AuNPs. Lastly, larger AuNP clusters (Figure 4h) not only resist fluidic lift force than AuNP monomers during vortex agitation, but also influence the resuspension of smaller AuNPs. Consequently, the analyte concentration (^^), which determines the size and amount of AuNP precipitates, plays an important role in determining the redistribution of AuNPs. The significant differences in the fitting parameters # ^^" and ^^^^" (Figure 4j) between sub-femtomolar analyte concentrations and blank samplesin a detectable change in AuNP concentration ^^^^^(about4 × 10,). This change exceeded the minimum PED-resolvable concentration differencein ^^^^^ (3 × 10.), thus enabling sub-femtomolar protein detection on the NasRED assay.

[0083] NasRED comparison to ELISAAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f

[0084] To benchmark the sensitivity of NasRED, the AS35 antibody was spiked in PBS at concentration range from 40 aM to 400 nM and analyzed using identical reagents on both standard ELISA assay (Figure 5a-b) and NasRED (Figures 5c) platforms. The LoDs, calculated using two slightly different definitions, and LoQs were determined as described in the Method section and summarized in Table 1. For consistency, the LoDs reported in this work refer to the protein concentration ^^at which the PED signal on the fitted sensing curve is distinguishable from the blank (NC) sampleby three standard deviations ( 3 / ) of the NC measurements, i.e. ^^^^,^0(123) =^^^^(^^) − 3 × ^3(^^) . Here intra-assay imprecision (or within-test error, ^3^ )represents the systematic readout errors from the PED device at each ^^. In contrast, inter-assay error (or between-test variation, ^34) captures the variability across replicate experiments and is used to assess assay reproducibility. For NasRED assay LoDreporting, / = ^34(^^) was determined from replicate blank samples (20 in PBS).

[0085] Remarkably, NasRED (LoD ~49 aM, or 7 fg / mL, from six replicates) outperformed ELISA (LoD 155 fM, or 23.3 pg / mL, from three replicates) in sensitivity by about 3,000 times while requiring only 16 times less sample volume (6 µL compared to 100 µL) and 30 time shorter assay time (10 min compared to 5 hours). Additionally, the coefficient of variation (CV), an indicator of the variability of test results, and recovery analysis, measurement of assay precision in quantification, were also analyzed using for both NasRED and ELISA replicate tests. The inventors’ analyses showed repeated NasRED tests produced comparable LoDs (90 aM, 45 aM, 93 aM, 58 aM, 71 aM, and 60 aM, respectively), determined by using the measurement error of the same NC samplealong different tube orientations, or / = ^3^(^^). In addition, NasRED exhibited <10%CV values and moderate recovery rates mostly within a range of 100±30%, suggesting consistent and reproducible detection with acceptable assay precision across the 10 logs of concentrations. In comparison, ELISA analyses exhibited significantly higher variation in absorbance intensity, CV, and recovery values, particularly at low to moderate antibody concentrations (attomolar to picomolar range) (Figure 5b-c). The comparison demonstrated that NasRED outperformed ELISA in both sensitivity and accuracy, particularly at low protein concentrations.Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f

[0086] Additionally, unlike ELISA or other assays that usually are characterized by a sigmoidal dose response (Figure 4b), NasRED data fitting was best modeled using a biphasic dose response. The biphasic response yielded better fitting (adjusted R2= 0.999 versus 0.991) and better recovery values compared to sigmoidal fitting. While further theoretical and experimental investigations in the future are needed to completely decipher the underlying mechanisms, it is hypothesized that the affinity-driven, biochemical protein binding and the centrifugation / vortex-driven fluidic dynamic interactions work as two mechanisms that affect the AuNP size distribution in the precipitates, concentration gradient in the supernatant, and the subsequent signal transduction in the NasRED assay. These two mechanisms are thought to contribute to the two-phase transitions observed in the response curves. Furthermore, 20 replicates of NC samples in PBS were analyzed after mixing and the completion of the sensing protocol, displaying comparable signals within a 6% error. These results confirm that the NasRED assay exhibits minimal non-specific background signals.

[0087] Antibody detection in biological fluids

[0088] SARS-CoV-2 antibody AS35 was then spiked into undiluted human serum in three replicate tests and 1% diluted whole blood (WB) in two replicate tests, and analyzed following the above-established sensing protocol (Figure 5d-f). Unlike PBS buffer, human serum contains proteins, amino acids, and antibodies and is more viscous. WB presents additional challenges due to the presence of various blood cells and platelets, which may hinder protein binding. In addition, hemoglobin in the WB strongly absorbs blue and green light, producing a red color that could interfere with the solution color and electronic signals. To mitigate these physical and chemical impacts, WB was diluted to 1% or 20% to ensure sufficient light transmission. Further, the sensing protocols (Table 1) were adjusted to increase the centrifugation speed to 1,500 g, the incubation time to 20 minutes, and the vortex force to 37.2 rps. This modified protocol facilitated the formation of AuNP precipitates at the tube bottoms for the antibody samples in HPS, 1% WB, and 20% WB, similar to those formed in PBS (Figure 5d-e).

[0089] The NasRED LoD for antibody detection in undiluted serum (Figure 5f) was found 76 aM (11 fg / mL), comparable to the estimated LoDs from each individual testAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f (70 aM, 87 aM and 200 aM, respectively). In comparison, the NasRED LoD was found360 aM or 54 fg / mL in 1% WB (corresponding to 36 fM in undiluted blood), where / =^34(^^) was obtained from 10 replicates of NC samples in 1% WB. Additionally, NasRED LoD was found 154 aM or 23 fg / mL in 20% WB (corresponding to 770 aM in undiluted blood) using a triplicate test. Further, 5 µL of the WB supernatants were collected and loaded in a PDMS plate to quantify the optical extinction on a laboratory spectrometer (Horiba). Clearly, high-sensitivity (femtomolar) antibody detection was evidently feasible without blood separation, as shown from the spectral intensity modulation. This further simplifies sample preparation for near-patient use. Noticeably, the signal contrast Δ^^^^was close to 1 in 1% WB (similar to tests in PBS), which is ideal for a steeper sensing curve slope, broad dynamic range, and potentially more accurate quantification. However, Δ^^^^dropped to only ~ 0.23 in 20% WB, possibly attributed to biological matrix effect in thicker blood due to the presence of diverse proteins and biomolecules. In case of antibody detection in 20% WB, blood cells also precipitated to the tube bottom after centrifugation. They might also contribute to fluidic and electrostatic interactions with AuNP clusters and monomers that could modulate the AuNP concentration in the supernatant to affect the sensitivity. Therefore, future closer examination of the dilution effect and protocol engineering may be necessary in applications where optimal LoDs and precise quantification are both critical. These analyses proved the feasibility of NasRED in high-sensitivity antibody detection in complex biological fluids for disease diagnostics and immunology studies.

[0090] N-protein antigen detection in biological fluids

[0091] NasRED was also demonstrated for SARS-CoV-2 antigen detection by functionalizing the AuNPs, using commercially available monoclonal antibodies (AS47 and AM223, both from Acrobiosystems) targeting the N-protein in PBS (Figure 6a-c). Interestingly, sensing using AS47-functionalized AuNP sensors displayed a poor colorcontrast (∆^^^^ = ^^^^(^^) − ^^^^(40067) ≅ 0.2) and worst sensitivity (LoD 4 nM, or200 ng / mL), AM223-AuNP sensors produced more differentiable optical contrast (∆^^^^~ 0.9) and better LoD (820 pM, or 41 ng / mL), and finally the use of two sets of AuNPs, functionalized with AS47 and AM223, respectively, produced the best signalAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f contrast (∆^^^^~ 0.99) and best sensitivity (LoD 180 aM, or 9 fg / mL). The co-binding AuNP sensors also produced LoDs of 190 aM (10 fg / mL) and 2 fM (115 fg / mL) for N- proteins spiked in saliva and nasal fluid (Figure 6g). This greatly enhanced sensitivity is attributed to the improved binding avidity because the two mAbs cooperatively target distant, non-overlapping epitopes of the N-protein (AS47 on the C terminus side and AM223 on the N-terminus side), which favors much more stable protein complex formation and accordingly larger AuNP clusters upon antibody-antigen binding. On the other hand, the cobinder-based NasRED sensors easily distinguished SARS-CoV-2 from HCoV-NL63 and HCoV-229E, which cause the common cold and result in similar symptoms to SARS-CoV-2 infection (Figure 6e-f). In fact, even 4 fM SARS-CoV-2 N- protein produces a signal (^^^^~0.63, Figure 1f) differentiable from 400 nM HCoV-NL63 and HCoV-229E (^^^^~0.95, Figure 2f), indicating a very high specificity of the NasRED system. To validate the performance of NasRED in real physiological samples, the inventors systematically analyzed key physical parameters affecting detection sensitivity after spiking SARS-CoV-2 N-protein into saliva. In particular, the inventors quantified the change in PED signal according to the N-protein concentration by controlling the centrifugation conditions (gravitational force, time) and vortex agitation conditions (speed, time), respectively (Figure 6h-k), and visualized the signal intensity difference for eachcondition ( ∆^^^^ = ^^ − ^^ ). In saliva, N-protein showed a high PED signal at lowconcentration (^^≅ 1) and a low signal at high concentration (^^ ≅ 0.2, ^^ = 4006M),showing a clear signal contrast of approximately ∆^^^^ ≅ 0.8. This indicates that thebiochemical reaction and precipitation between the high-concentration target protein and the AuNPs proceeded efficiently even in the saliva environment. In addition, under centrifugation conditions of 900 g to 1,500 g and a vortex speed in the range of from 30 to 36 rps, and a short agitation time of several seconds was confirmed to be an effective condition for maintaining sensitivity and signal reproducibility even in complex biological samples. These results again demonstrate that NasRED has the potential to maintain stable performance even in complex biological matrices.

[0092] The inventors further demonstrated direct detection of inactivated SARS- CoV-2 virion particles in saliva using the antigen sensors (Figure 6l-n). This was achieved by mixing the AuNP sensor buffer with PBS-diluted viruses (by 10 log titrations) and lysisAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f reagents (Triton X-100, 0.3% v / v concentration) to extract N-proteins (Methods). By referencing the initial concentrations of N-proteins (2995 ng / mL or ~30 nM by ELISA) andRNA copy numbers ( 4.7 × 10, copies / mL by quantitative PCR), the LoDs weredetermined as 4 pM (185 pg / mL) for the N-protein, corresponding to an estimated RNAsensitivity of 2.9 × 10<copies / ml without amplification, and 1.7 × 10= TCID50 / mL for thevirus particles. In saliva samples, the presence of enzymes such as protease degrade N- protein molecules, thus lowering the sensitivity. Here the LoD for NasRED tests in saliva was found comparable to the reported sensitivity (3.24 Log10 IU / mL, or about 2×103copies / mL) using qPCR. Considering the fundamental limitations of ELISA and qPCR in quantitation and possible impurities in the standard sample (ZeptoMetrix), the results suggest that NasRED offers comparable sensitivity to ELISA and qPCR.

[0093] Discussion

[0094] In the NasRED platform, AuNP sensors play multiple roles in promoting the assay performance. They act as multivalent sensors to enhance protein binding and also effective optoelectronic beacons to convert the optical extinction to electronic signals, thus enabling reliable detection using a simple PED readout device for portable diagnostics. Through controlled fluidic dynamic interactions, AuNPs actively capture proteins during centrifugation-induced sedimentation, generating a concentration- boosting effect to greatly strengthen the protein-antibody binding, and then redistribute upon vortex agitation, establishing a concentration gradient profile that allows sensitive signal differentiation at sub-femtomolar level analyte concentrations. To compare NasRED with existing assay formats, LoDs were calculated to represent the assay’s sensitivity in differentiating the minimum target SARS-CoV-2 antibody or antigen concentration from the background.

[0095] Here the inventors assessed the NasRED analytical sensitivity followingtwo commonly used LoD definitions (Table 1), ^^^^(123) = ^^^^(^^) − 1.645( / + / ′)and ^^^^(123) = ^^^^(^^) − 3 / , where / and / ′ denote the standard deviation inmeasuring the blank and least-concentrated protein samples, respectively. The first definition takes into account of the signal variations from both blank sample and from introducing analyte, and thus include more objective data to determine the minimalAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f concentration of analyte that empirically can be distinguished from its absence. In comparison, the second approach calculates LoD based on only the measurement errors from blank sample without requiring additional consideration from a low concentration of analyte, and therefore is much more widely used for its simplicity. While the 3 / approach was adopted when reporting the LoD values based on reviewer’s feedback, the inventors’ comprehensive analysis showed that the two definitions indeed yielded comparable numbers for both SARS-CoV-2 antibodies and antigens in all the tested biological fluids.Clearly, the 3 / approach produced a slightly more optimistic estimate while the 1.645( / + / ′) approach was more conservative. Additionally, the inventors introduced the limit ofquantitative (LoQ) as another measure, defined as ^^^^(12B) = ^^^^(^^) − 10 / , to helpassess NasRED assay’s ability for reliable quantification, and also calculated the LoQ(10 / ) / LoD(3 / ) ratio as a reference. Evidently, the LoD and LoQ values were both in the sub-femtomolar range for antibody sensing in PBS and HPS, and LoQ / LoD ratios were also moderate (<20), showing NasRED had comparable sensitivity and quantification ability in HPS and PBS. In comparison, the LoD and LoQ values were in the femtomolar range (30 to 50 fM) for antibody tests in 1% WB and antigen tests in saliva, which was accompanied by a larger LoQ / LoD ratio (100 to 250), indicating increased background effect in more complex biological matrices. Lastly, sensing in more complex and viscose fluids, for example antibody detection in 20% WB and antigen detection in nasal fluids, was more challenging and not strategically optimized in this work due to resource constraint, showing sub-femtomolar to femtomolar LoDs, picomolar LoQs and very large LoQ / LoD ratios (~104). These above analyses proved that sensing optimization in each biological fluids would be necessary to achieve optimal NasRED performance.

[0096] The inventors also attempted to compare NasRED with the reported performance from other traditional assay formats. LFAs usually have much poorer sensitivity (e.g., LoD of about 3 ng / mL) compared to ELISA (e.g., literature reported LoD of 8.4 pg / mL). In comparison, NasRED achieved exceptional antigen sensitivity (10 fg / mL and 115 fg / mL in saliva and nasal fluid), which is about 102to 105times more sensitive than LFA and 102to 103times better than ELISA. Importantly, NasRED also directly detected virion particles from saliva with an estimated LoD of ~105RNA copies / mL, which is still higher than the reported qPCR sensitivity in saliva, but comparable to that of someAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f commercially available, isothermal NAAT tests such as Abbott ID NOW (~105RNA copies / mL). It is also worth pointing out that NasRED detection integrates the RNA extraction and readout process in one-pot, one-tube reaction, significantly reducing the sensing complexity for practical applications. The presence of lysis buffer and additional biochemicals probably complicated the reaction and limited the sensitivity, which could be potentially improved in future work.

[0097] Besides the analytical performance, many other factors, such as the assay time, cost, complexity of the readout instrumentation, sample volume, etc., also strongly influence the success of antigen tests in practical use. For example, despite its low sensitivity, LFA became widely used during the COVID-19 pandemic due to its relatively low cost (~$10), rapid turnaround time (~15 min), and simpler sample preparation and detection schemes. In comparison, the use of ELISA and qPCR in POC applications is limited due to their higher test cost, bulky and costly instrument (e.g. ~$10,000 or more for ELISA absorbance reader or a PCR machine), long turnaround time (typically hours), and the need for specially trained personnel. In contrast, NasRED performs tests in microcentrifuge tubes without any labeling, washing, or enzymatic reaction, therefore simplifying the sensing process, reducing turnaround time (15 to 30 minutes from sample mixing to readout), and minimizing both sample volume (only 6 µL, much smaller than a single drop of blood) and reagent usage. Additionally, the overall sensing cost could be under $2 per NasRED test (~$0.1 per µL AuNP sensing solution, or ~$1.8 per test) using commercially available AuNPs (Cytodiagnostics) and can be further decreased to <$1 if the AuNPs are produced at scale by solution synthesis. With performance comparable to ELISA- and PCR but significantly reduced assay time, sample volume, system footprint, and reagent cost, NasRED is an ideal candidate for rapid, precise point-of-care testing, which is currently unavailable. Future development, such as integration and automation of centrifugation, vortex, and data analysis processes, will support the deployment of the NasRED platform not only in clinics, hospitals, and community centers but also in the field next to the users.

[0098] METHODS

[0099] Sources of reagents and instrumentationAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f

[0100] Biotinylated WT-RBD (Wuhan-Hu-1 Catalog No. SPD-C82E9), AS35 anti- SARS-CoV-2 antibody (Catalog No. SAD-S35), biotinylated anti-SARS-CoV-2 Nucleocapsid antibody, Chimeric mAb, Human IgG (AM223 Catalog No. NUN-BM272), mouse IgG (AS47 Catalog No. NUN-S47L8) and SARS-COV-2 Nucleocapsid protein (Catalog No. NUN-C5227) were commercially purchased from ACROBiosystems. TritonTMX-100 was purchased from Millipore Sigma. The microcentrifuge (accuSpin Micro 17) and the vortexer (Analog Vortex Mixer Catalog No. 02-215-365 for antibody detection and Digital Vortex Mixer Catalog No. 02-215-418 for antigen detection) were both purchased from Thermo Fisher.

[0101] Electronic readout system

[0102] The NasRED readout system included a reading device and a circuit board for signal processing. The reading device consisted of three main components: an LED (WP7113PGD, Kingbright), a photodiode-integrated circuit (SEN-12787, SparkFun Electronics, integrated with a digital light-sensor APDS-9960 from Broadcom), and a microcentrifuge tube holder that was 3D printed (Qidi Tech X-Plus 3D Printer) using black carbon fiber polycarbonate filament to fit snugly into a standard 0.5 mL Labcon microcentrifuge tube. An alkaline battery (9 V) was converted to 5V by a voltage regulator circuit as the power supply. The photodiode APDS-9960 was biased at 3.3 V and interfaced with a microcontroller (Atmega328) to convert the output into a digital signal. The regulator circuit was comprised of two electrolytic capacitors (10 µF), two ceramic capacitors (0.1 µF), and one protective Zener diode (Figure 2a (i)). The constant current LED driver circuit utilized a feedback loop to stabilize the current supplied to the LED. For example, a sudden increase in the base voltage of transistor T2 (VB,2) would increase its base-to-emitter bias (VBE,2) and, therefore, conduct a higher amount of current (iLED= iC,2≈iE,2) through the LED and the T2 collector, which would tend to cause the LED current to rise. However, the higher iE,2would also boost the voltage drop across resistor R3, or the base-to-emitter bias voltage of transistor T1 (VBE,1). This, in turn, would lead to enhanced emitter current (iE,1) and collector current (iC,1≈iE,1). This increase in iC,1 would cause larger voltage to drop across resistor R2, and, therefore lower VB,2 to counter the voltage fluctuation and stabilize the LED signals. --All the above electronic components, including microcontroller, capacitors, resistors, transistors, etc., were purchased fromAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f DigiKey unless specified otherwise.

[0103] Optical power calculations

[0104] The output power of a green LED (peak emission wavelength 557 nm, full- width-at-half maximum (FWHM) 30 nm) was measured and digitized using an APDS- 9960 sensor. The APDS-9960 sensor operates at a gain of 16 and an integration time (ATIME) of 175 msec, measures the light intensity in Clear, Blue, Green, and Red channels, and outputs the emission power in the Clear channel with a responsibility of^D = 23.60 ^2E6FG / (IJ / ^KL). The LED output power (irradiance) was calculated fromthe counts obtained via the inter-integrated circuit (I2C) interface. The same LED and APDS-9960 sensor were used throughout the measurements for consistency.

[0105] Polydimethylsiloxane (PDMS) well plate fabrication

[0106] Sylgard 184 silicone elastomer base was thoroughly mixed with the curing agent (mass ratio 10:1) for 30 minutes in a petri dish and placed in a vacuum container for 2 hours to degas. The mixture was then fully cured at room temperature into a PDMS membrane, which was then cut to the desired size and punched to create 2 mm wells. The as-prepared PDMS membrane and a diced fused silica chip (500 μm thick) were rinsed with isopropyl alcohol, dried by nitrogen, and treated with oxygen plasma. Immediately after, the two were bonded to form a PDMS well plate. The PDMS plate was treated with 1 wt% PVA in water solution for 10 minutes to prevent the non-specific binding of proteins, adapted from previously reported methods. Finally, it was dried with nitrogen, heated on a 110oC hotplate for 15 minutes, and cooled to room temperature.

[0107] 1×PBS dilution buffer

[0108] The 10× Phosphate-buffered saline (PBS, Fisher Scientific) stock buffer was mixed with glycerol (Sigma-Aldrich) and BSA (Sigma-Aldrich) and deionized water (Fisher Scientific) to create a 1×PBS dilution buffer with a final concentration of 1×PBS, 20 % v / v glycerol, and 1 wt % BSA. This dilution buffer (pH~7.4) was used to prepare the AuNP sensors, antigen and antibody solutions, and diluted biological media.

[0109] Sensing solution preparation and quantification

[0110] Streptavidin-functionalized AuNPs (∼0.13 nM, 80 nm, OD10) wereAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f purchased from Cytodiagnostics, and quantified by Nanosight (NS3000, Malvern Panalytical). To create AuNP sensing solution (e.g.200 μL, ODPED = 0.5), the streptavidin- coated AuNPs (50 μL, OD10) were mixed with an excessive amount of biotinylated antigens or antibodies (e.g. about 1.2 μM, 20 μL), incubated for 2 hours, diluted by adding 1000 μL of 1×PBS dilution buffer and purified by centrifugation at 9,600 g for 10 minutes, followed by removing 1050 μL supernatant and adding 1050 μL 1×PBS dilution buffer. The above purification was repeated another time to remove unbound biotinylated antigens or antibodies. The purified sensing solutions were measured by Nanodrop 2000 (Thermo Fisher) to determine the AuNP concentration and then adjusted to the desired optical extinction level (e.g., in this case ∼0.019 nM for 80 nm AuNPs) by mixing with 1×PBS dilution buffer. Prior to each sensing experiment, the stock sensing solutions were aliquoted into Eppendorf tubes of 18 μL each.

[0111] Antibody and antigen detection in biological buffer

[0112] Sensing in PBS: The target antigen (N-protein) or antibody was adjusted to a target concentration (e.g., 40 aM to 4 μM) by mixing with 1×PBS dilution buffer. Then 6 μL of such antigen or antibody solutions were mixed with an 18 μL AuNP sensing solution, followed by vortex agitation at 34.5 rps for 5 seconds. The final antigen or antibody concentration in the mixed reaction buffer (24 μL) was therefore diluted four times from the original solutions, e.g.40 aM to 10 aM and 4 μM to 1 μM.

[0113] Biological media preparation: Human Pooled Serum (HPS) was used as purchased. Human whole blood (WB) was diluted with 1×PBS dilution buffer to 20% and 1% to minimize optical and biochemical interference. Saliva and nasal fluid were purified by centrifugation at 9,600 g for 5 minutes to precipitate the mucus, and the supernatant was used. Their corresponding final concentration in the sensing mixture was 25% HPS, 25% nasal fluid, 25% saliva, and 5% WB. Single donor human WB, HPS, human nasal fluid, and saliva were purchased from Innovative Research, Inc.

[0114] Inactivated virus N-protein extraction: PROtrol SARS-CoV-2 (Isolate: USA-WA1 / 2020) was purchased from ZeptoMetrix LLC in standard Vero E6 culturing medium (2% minimum essential medium (MEM)). The N-Protein was extracted by adding 0.6% v / v Triton X-100 (non-ionic surfactant, from Millipore Sigma) at a 1:1 v / v ratio to theAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f SARS-CoV-2 sample, followed by 5 minutes incubation to release the N-protein via viral envelope micellization.

[0115] qPCR assay was performed by ZeptoMetrix, and the protocol and the data were shared with their permission. The SARS-CoV-2 viral RNA was extracted from 200 µL of PROtrol sample (part # PROSARS(COV2)-587) using QIAmp MinElute Virus extraction. Briefly, the sample was lysed using the lysis buffer (NaCl, Tris-HCL, SDS), followed by RNA binding to silica in a spin column, sequentially washed by guanidine hydrochloride and ethanol to remove impurities and salts, and the viral RNA was eluted into 60 µL of aqueous viral elution (AVE) buffer (RNase-free water, 0.04% Sodium Azide (NaN3)) and immediately used to measure genomic copies in real-time quantitative PCR assay. Furthermore, the RNA from working quantitative standard and extraction control were extracted alongside the PROtrol samples. The extracted SARS-CoV-2 RNA was tested in the SAR-CoV-2 qRT-PCR assay that targets the N protein-coding gene. The PCR reaction buffer included 6.25 µL TaqMan Fast Virus 1-Step Multiplex Master Mix for qPCR (carboxy rhodamine dye free), 1 µL of each forward and reverse primers at 500 nM final concentration, 1 µL fluorescent probe at 200 nM final concentration (Table S4), 5 µL of extracted RNA as PCR template, and 11.75 µL RNase-free water (25 µl total mixture volume). PCR was run in a QuantStudio 5 System. A standard curve was generated using 10-fold serial dilutions of the extracted RNA from a working standard with known copies / mL, determined by droplet digital PCR (ddPCR). The efficiency of the PCR reaction was found to be 93.4%, derived from a slope of -3.492 from the standard CT curve. The same 10-fold serial dilutions were made with the extracted RNA from the PROtrol sample (n=2 sets) and tested against the standard curve to determine the quantity of viral RNA in the PROtrol sample. The working standard and the PROtrol qPCR data were analyzed using the QuantStudio Design & Analysis Software (V1.4.3).

[0116] NasRED data processing

[0117] The NasRED data analysis involved signal collection, calibration, and normalization. First, the target proteins in biological media were measured on the designed PED device, together with a negative control (NC, 18 µL functionalized AuNPs mixed with 6 µL biological medium without target proteins). Then, each sample tube (18Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f µL AuNP sensing solution mixed with 6 µL target protein solution, in total 24 µL) and the NC tube underwent measurements along five orientations relative to a tube holder position to account for tube variations and inhomogeneous matrix optical impacts such as turbidity and color. The measured digital photodiode sensor signals for each sample tube were averaged, collected to a datasheet (.csv), and saved to a predefined local folder on a computer, all automated by Python scripts. The averaged sample signals NO=∑S (QR,S(^*))<(T=1 to 5 for the five measurements, at protein concentration ^^) represented the supernatant and the microcentrifuge tubeincubation, and vortex agitation), another five measurements were performed for each tube along the same orientations,and the electronic signals were again recorded as N ∑S (QV,S ^*")U(^^) = < ( T =1 to 5).Subsequently, the calibrated electronic signals, representingin opticalextinction caused only by the decrease of free-floating AuNPs due to precipitation, werecalculated as N ^ ∑S (QX,S(^*))^,W ^" = NU,W(^^) − NO,W(^^), and N^(^^) = < . The difference of NCsignalsalso calculated,N^(^^)<6% NO(^^), i.e. most AuNPs redisperse and return to their original state right after mixing, was used as a criteria to validate the sensing protocol. Finally, considering the batch-to-batch difference in AuNP sensor concentrations as well as physical variations of biological fluids on the NasRED signals, the collected PED signals werefurther normalized to a range from 0 to 1 as ^ QX ^*"YQX(^D)^^^ ^^" = 1 − ) . Here, a positivecontrol signal (PC) was chosen for normalizationthe highest possible optical transmission, i.e. AuNPs completely precipitate. For mostly clear biological media, including PBS, saliva, nasal fluid, and serum, a mixture sample of 18 µL 1×PBS dilution buffer (representing sensing solution after AuNPs precipitate) and 6 µL biological media was used as a reference for simplicity, and the transmission signal was collected as the N(Z^). To account for the complex physical and chemical interactions between blood and AuNPs, the PC reference in WB was designed by mixing 18 µL WT RBD-coated AuNP sensing solution with 6 µL 20% WB spiked with high-concentration(^^=800 nM) antibodies. In this case, the PC signals were defined as N(Z^) = NU(^^) −Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-fNO(^^) = N^(^^).

[0118] In addition, the intra-assay imprecision, or within-test error (^3^), of the measurements were calculated as the standard deviation of the 5 normalized signals for esample ^3^(^^) = ∑ (Q ^*"YQ (^ ))\the sam [ S X,S X *<(Q(^D)YQ R(^D))\ ( T =1 to 5, for the 5 measurementorientations). The ^^^^ (^^) ± ^3^(^^) were plotted againstthe antigen or antibody biological media. For thereproducibility experiment performed in this average of each concentrationmeasurement ^^̂^ ^^̂ ^^̂ (^^) was calculated as ^^̂^ ^^̂ ^^̂ (^^) = ∑S (0_`X,a ^*")^ (j=1 to N for the Nreplicate measurements) and the inter-assay test variation) was∑(0^ ^*")\calculated as ^34(^^) = [ a b,a^ . Here ^3^(^^) characterizes the systematicreadout errors from human operations, and ^34(^^) accounts for thevariability of the repeated experiments. ^3^and ^34are found to be quite close in experiments in different biological media, indicating the measurement system errors were the key contributing factors limiting the analytical precision.

[0119] Data fitting, LoD, LoQ, CV and Recovery calculations

[0120] The normalized signals SPEDwere fitted using Origin 2024b software (OriginLab, USA) by orthogonal distance regression algorithm to consider the data and error weight in fitting calculations. Biphasic dose-response model was used for fitting, following where SH and SLwere from NC (or lowest concentrations) and the highest protein concentrations, respectively, EC50 was the half maximal effective concentration, and r describes the steepness of the sensing curve. In general, the EC50 and r values would determine the LoD. A large signal contrast SH- SL, resulting from a small SL (i.e. high level of AuNP precipitation), in combination with a small slope r, would be desired for obtaining a large dynamic range. In addition, a biphasic dose-response model was explored when a simple sigmoidal model failed to produceAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f acceptable fitting, following where two

[0121] Fitting parameters were adjusted so that the goodness of fit parameters such as reduced Chi-square, R-squared coefficient of determination (COD), and reduced sum of squares (RSS) were calculated close to 1, 1, and 0, respectively. The LoD and LoQ values were then calculated such that the PED signal at this concentration on the fitted curve was distinguished from the measured NC sample by 3 / ^D(LoD) and 10 / ^Dtimes (LoQ) the intra-assay or inter-assay errors (^3: ^3^or ^34) of the NC sample, respectively. ^^^^,^0(123) = ^^^^(^^) − 3 × ^3(^^)

[0122] Thesuch that the PED signal at this concentration on the fitted curve was distinguished from the measured NC sample by 3 / ^D(LoD) and 10 / ^Dtimes (LoQ) the intra-assay or inter-assay errors (^3: ^3^or ^34) of the NC sample, respectively.

[0123] The coefficient of variation (CV) was calculated as the ratio of the standard [ \ ∑e0^ ^ "fation to the mean value of each data set, following ^c a a * / ^devi ( × 100%(j=1 to N for the N replicate measurements). The recovery (%)as the ratio of measured PED signals ^^^^(^) to the values from standard biphasic dose-responsefitting ^^^^,^0 at different protein concentrations ( ^^ ), following ^^^2g^hi(%) =( 0_`X(^*)) × 100%.ELISA assay

[0125] The MICROLON® 96-well polystyrene microplate (Cat# 5665-5061, USA Scientific) was coated with 100 µL of 10 µg / mL streptavidin solution (Sigma-Aldrich) and incubated overnight at 4°C. The plate was then washed three times using 200 µL of PBSAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f containing 0.5% (v / v) Tween-20. To block non-specific surface adsorption, 100 µL of a 5% (w / v) BSA solution (Sigma-Aldrich) was added and incubated for 2 hours at room temperature and washed four times with the same washing solution. Biotinylated RBD was added to each well (100 µL) and incubated for 2 hours at room temperature followed by four times washing. Serially diluted samples in three replicates, ranging from 400 nM to 40 aM, including a negative control, were added to the wells (100 µL per well) and incubated for 2 hours at room temperature and washed four times using the washing solution. Subsequently, 100 µL of 1.2 µg / mL secondary antibody (Peroxidase AffiniPure™ Goat Anti-Human IgG (H+L), Jackson ImmunoResearch, Cat# 109-035-088) was added to the wells and incubated for 2 hours at room temperature and washed four times with the washing solution. To develop the reaction, 50 µL of 1-Step Ultra TMB- ELISA substrate solution (Cat# 34028, Thermo Fisher Scientific) was added to the wells and incubated for 15 minutes. The reaction was stopped by adding 100 µL of 2 M H₂SO₄ (Thermo Fisher Scientific) to each well. The absorbance was measured on a BioTek Synergy Neo2 Hybrid Multimode Reader at 450 nm (Agilent Technologies).

[0126] The ELISA absorbance signals at 450 nm were calculated as m1n^o ^^" =∑S (^^40^,a ^*")=(j=1 to 3 for the 3 replicates). To facilitate comparison with ^^^^, the ELISA signalswith 0 representing the minimum absorption at negative controls and 1 representing maximum absorption (approaching device limit at high analyte concentrations such as 40 nM). The error bars, i.e. ^3(m1n^op^^") (j=1 to 3), represented the standard deviations of the ELISA signals between the 3 replicate experiments. The LoD was calculated based on the same principle described in the NasRED data processing, where the assay can differentiate the sample from blanksample following the equation, m1n^o(123) = m1n^o(^^) − 3 × ^3(^^).

[0127] Spectrometry measurement in blood sample

[0128] AS35 was detected in 20% diluted human WB, following the modified protocol, and analyzed by spectroscopic measurements. Here, the samples' supernatant liquids (5 μL) were loaded into a PDMS well plate and measured by the spectrometer (iHR-320, Horiba Instruments Inc.). To achieve the best contrast, the focal plane wasAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f adjusted at the well plate surface, and then three spots were selected from within each well (one at the center and two near the edges) to account for possible inhomogeneity of AuNPs’ distribution. The transmitted light was collected by a 50×objective lens (NA=0.8) from each spot, and the transmission spectra were measured 64 times from 350 to 750 nm range with 0.01 seconds integration time. The signals collected from the three spots were then averaged and normalized against the reflectance from a silver-coated mirror as TPDMS(λ,C), where λ was the wavelength, and C was the protein concentration. The optical extinction spectra were obtained as EPDMS(λ,C) = 1 - TPDMS(λ,C), from which the values at λ = 570 nm were selected as the signal Etest(C). In addition, Etest(PC) = Etest(CH) was similarly collected for the positive control (18 μL WT RBD-coated AuNP sensing solution mixed with high-concentration (CH= 800 nM) antibodies spiked in 6 μL 20% WB), and Etest(NC) was determined for the NC sample (sensing solution mixed with the biological medium at 3:1 ratio). The spectrometer signals were calculated as and the error of the measurement was calculated as the standardsignals (i=1 to 3, for the 3 measurement spots).

[0129] AS35 was detected in 20% diluted human WB, following the modified protocol, and analyzed by spectroscopic measurements. Here, the samples' supernatant liquids (5 μL) were loaded into a PDMS well plate and measured by the spectrometer (iHR-320, Horiba Instruments Inc.). To achieve the best contrast, the focal plane was adjusted at the well plate surface, and then three spots were selected from within each well (one at the center and two near the edges) to account for possible inhomogeneity of AuNPs’ distribution. The transmitted light was collected by a 50×objective lens (NA=0.8) from each spot, and the transmission spectra were measured 64 times from 350 to 750 nm range with 0.01 seconds integration time. The signals collected from the three spots were then averaged and normalized against the reflectance from a silver-coated mirroras N^^q0(r, ^^), where r was the wavelength, and ^^ was the protein concentration. Theoptical extinction spectra were obtained as m^^q0 r, ^^" = 1 − N^^q0(r, ^), from which thevalues at r =570 nm were selected as the signal mUstUm UstU(Z^) =mUstU(^^)was similarly collected for the positive control (18 µL WT RBD-coated AuNPAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f sensing solution mixed with high-concentration (^^=800 nM) antibodies spiked in 6 µL 20% WB), and mUstU(^^) was determined for the NC sample (sensing solution mixed with the biological medium at 3:1 ratio). The spectrometer signals were calculated as ^t^s^(^^) = ^ VuvV(^D)Y^VuvV D*"^VuvV(^D)Y^ VuvV(^D) and the error of the measurement was calculated as the∑S (0 ^*"Y0v*u) ^*")\standard deviation (SD) of the 3 normalized signals ^3t^s^ ^^" = [ v*u),S=(T=1 to 3, for the 3 measurement spots).Table 1. NasRED design and performance in detecting SARS-CoV-2 antigens and antibodies Centrifu LoD Protein ge(1.645( / +LoD (3 / ) ## LoQ (10 / ) ## o o 1 3Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f SARS- 9.4×105RNA 2.9×105RNA 3.9×106RNA CoV-2 copies / mL copies / mL copies / mL AS47 C12, Analog Mixer (at 2,100 rpm) was used for antibody and virion particle detection, and a Digital Mixer (at 1550 rpm) was used for antigen detection (Methods section). *LoD and LoQ were calculated based on intra error within a single experiment. **LoD and LoQ were calculated based on inter error, which reflects the variability between replicate experiments. ## The LoD (3 / ) and LoQ (10 / ) values in the table were calculated in the diluted blood. Therefore, their corresponding values in undiluted blood need to be multiplied with the dilution factor. For example, the LoD was found to be 360 aM in diluted 1% blood and 154 aM in 20% blood, corresponding to 36 fM and 0.77 fM, respectively, in an undiluted 100% blood sample.

[0130] Example 2: Rapid and Sensitive Detection of Thrombospondin-2 Using Nanoparticle Sensors Towards Cancer Screening and Prognosis

[0131] I. Introduction

[0132] Thrombospondin-2 (THBS2) has emerged as a significant prognostic biomarker across a broad spectrum of cancers, including colorectal cancer (CRC), gastric cancer (GC), colitis-associated colorectal cancer (CAC), pancreatic ductal adenocarcinoma (PDAC), ovarian cancer, triple-negative breast cancer (TNBC), non- small cell lung cancer (NSCLC), oral cavity squamous cell carcinoma (OSCC), cervical cancer, bladder cancer, hepatocellular carcinoma, and melanoma. THBS2 plays a critical role in the remodeling of the tumor microenvironment, promotion of tumor angiogenesis, and cancer cell migration and invasion. Therefore, there is a strong association between high levels of THBS2 and poor clinical outcomes. Its relevance to a wide variety of diseases makes THBS2 a potential cornerstone biomarker for not only prognosticAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f evaluation but also early cancer screening. Its detection in various biological fluids, including blood, plasma, serum, urine, and synovial fluid, enables its use as a minimally invasive biomarker for diagnostics and monitoring.

[0133] Conventional diagnostic technologies typically used to measure THBS2 include enzyme-linked immunosorbent assay (ELISA), mass spectrometry, polymerase chain reaction (PCR), and immunohistochemistry (IHC). However, achieving sufficient sensitivity and specificity using point-of-care (POC) platforms remains challenging, particularly at low levels in complex biological fluids. Furthermore, THBS2 detection in clinical settings needs to be differentiated from background biomarkers, for example, CA19-9 for PDAC, to ensure accuracy.

[0134] To meet the urgent need for high-accuracy POC cancer screening, this study introduces an in-solution, single-tube, modular assay platform specifically designed to deliver high sensitivity, specificity, and operational efficiency for biomarker analysis. Leveraging prior success in developing nanosensors, the inventors introduce nanoparticle-supported rapid electronic detection (NasRED) as a versatile and simple-to- use platform and demonstrate its applications in detecting THBS2 in various biological fluids. Briefly, upon the introduction of THBS2 in solution, antibody-functionalized gold nanoparticles (AuNPs) bind to the target and precipitate in clusters, with the cluster sizes and quantities correlating with the concentration of THBS2 in the solution. The physical precipitation process of AuNPs is accelerated by centrifugation, which acts to increase the AuNP and protein collision rate in solution and create a high-concentration region of the reagents at the bottom of the tube to favor detection at enhanced sensitivity. Afterward, vortex agitation is used following incubation to resuspend non-reacting AuNPs, thus preserving the specificity. The above sensing process essentially separates AuNP into clusters and free-floating monomers. Because the AuNP clusters increase with THBS2 concentration, fewer AuNP monomers will be present in the supernatant, thus causing a decrease in the optical extinction. The optical signals are collected using a semiconductor-based portable electronic device (PED), digitalized using customized circuits that stabilize the readout signals, transmitted through Wi-Fi or Bluetooth modules, and then readily stored for analysis. Using THBS2 detection as an example, NasRED demonstrates its feasibility as a novel platform that can make an impact in physician’sAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f offices to transform cancer screening and prognostics.

[0135] 2. Materials and Methods

[0136] 2.1 Materials and Reagents

[0137] Recombinant Human Thrombospondin-2 Protein (1635-T2) and Human Thrombospondin-2 Biotinylated Antibody (BAF1635) were purchased from R&D Systems. Human Pooled Serum (HPS) (C817B46), Single donor Human Whole Blood (WB) (IWB1K2E10ML), Single Donor Human Saliva (IRHUSLS5ML), and Pooled Human Cerebrospinal Fluid (CSF) (IRHUCSF1ML) were purchased from Innovative Research Inc. Single Human Synovial Fluid (991-42-S-1) was purchased from Medix Biochemica. CA 19-9 antigen (DAGA-768) was purchased from Creative Diagnostics. Bovine Serum Albumin (BSA) and molecular biology grade glycerol from Sigma-Aldrich. Phosphate- buffered saline (PBS) and DNase / RNase-free distilled water used in the experiments were purchased from Fisher Scientific. The 80nm streptavidin-functionalized AuNPs were purchased from Cytodiagnostics Inc. and dispersed in 20% v / v glycerol and 1 wt % BSA buffer.

[0138] 2.2 Selection of Optimal AuNPs Size for Biosensing Probe Preparation

[0139] The size of AuNPs significantly impacts their absorption wavelength, directly influencing the localized surface plasmon resonance (LSPR) phenomenon. Larger nanoparticles typically exhibit absorption peaks at longer wavelengths due to enhanced oscillations of conduction electrons on the particle surface. Conversely, smaller nanoparticles absorb light at shorter wavelengths. Additionally, the number of binding sites on each particle decreases with the size of the nanoparticles. The AuNP optimization also takes into account the assay time, which is shortened at larger AuNP sizes. Based on previous studies, 80 nm AuNPs achieved a large dynamic range, high sensitivity, and rapid signal transduction well.

[0140] 2.3 Sensing Dilution Buffer Preparation

[0141] The 10× Phosphate-buffered saline (10×PBS, Fisher Scientific) stock buffer was mixed with glycerol (Sigma-Aldrich) and BSA (Sigma-Aldrich) and deionized water (Fisher Scientific) to create a 1×PBS dilution buffer with a final concentration ofAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f 1×PBS, 20 % v / v glycerol, and 1 wt% BSA. This dilution buffer (pH~7.4) was used to prepare AuNP sensors, antigen and antibody solutions, and diluted biological media.

[0142] 2.4 AuNPs Functionalization

[0143] The AuNPs in the sensing dilution buffer solutions were functionalized with anti-THBS2 antibodies using a biotin-streptavidin reaction. In detail, 50 μL of AuNPs (0.13 nM) were mixed with an excessive amount of 20 μL of antibodies (2 μM) in a 1.5 ml Eppendorf tube and incubated for 2 hours at room temperature followed by 12 hours at 4 °C. Then, the tube was gently vortexed, and 630 μL of sensing dilution buffer was added to the tube and centrifuged for 10 minutes at 10 krpm (9600g) to precipitate. After that, the supernatant (650 μL) was removed and replaced with 650 μL sensing dilution buffer. The procedure was repeated 2 times to remove excessive antibodies. Finally, the concentration of coated AuNPs was adjusted to the desired optical density (OD) level (OD~0.5), or around 0.026 nM, using sensing dilution buffer.

[0144] 2.5 THBS2 Detection experiments

[0145] First, 40 μL of WB and Synovial fluid was mixed separately with 160 μL of sensing dilution buffer to make 20% of each matrix to minimize matrix induced interferences. To prepare the target solutions, 5 μL of 4 μM THBS2 were diluted into different matrices individually (PBS, HPS, 20% WB, 20% Synovial fluid, Saliva, and CSF) to achieve final concentrations ranging from 10 nM to 100 fM. For detection, 18 μL of prepared AuNP probes were mixed with 6 μL of THBS2 solutions at each concentration. As a negative control (NC), 6 μL of each biological matrix without THBS2 was mixed with the AuNP probe solutions, and the mixture went through the same sample processing steps to evaluate the matrix effect on the sensing solution. THBS2 sample tubes and the NC control tube were centrifuged at 3500 rpm, or 1180 g, incubated for 10 minutes, and vortexed for 5 seconds at 32.5 rps (1950 rpm).

[0146] 2.6 Specificity in Different Matrices

[0147] To determine the specificity of the sensing probes, two other model proteins, e.g., Carbohydrate Antigen 19-9 (CA19-9) and BSA protein were chosen as a control. For this purpose, CA 19-9 and BSA were spiked and diluted into 100% HPS andAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f saliva to reach the concentration of 10 nM. HPS and saliva solutions without THBS2 were used as negative control, while THBS2 (10 nM) served as positive control.6 μL of each sample was mixed with 18 μL functionalized AuNPs in a microcentrifuge tube and centrifuged at 1200 g, incubated for 10 minutes followed by vortexing at 32.5 rps for 5 seconds.

[0148] 2.7 Electronic Reader Design

[0149] The portable electronic detection (PED) system similar to another recent study consisted of three essential components: (1) a light-emitting diode (LED; WP7113PGD, Kingbright), (2) a photodiode-integrated circuit (SEN-12787, SparkFun Electronics), which incorporates a digital light sensor (APDS-9960, Broadcom), and (3) a custom-fabricated tube holder created using a 3D printer (Qidi Tech X-Plus) with black carbon fiber polycarbonate filament. The tube holder was specifically designed to securely accommodate standard 0.5 mL Labcon microcentrifuge tubes, ensuring precise alignment of the optical components. The photodiode APDS-9960 was operated with a bias voltage of 3.3 V and interfaced with a microcontroller (Atmega328) to process and digitize the signal output. This configuration enabled reliable electronic readouts of light intensity variations with minimal noise. All electronic components, unless otherwise noted, were procured from DigiKey, and their integration was optimized for consistent performance in the detection setup.

[0150] 2.8 Data Analysis

[0151] Our portable electronic reader measured the optical values of the tubes. Each tube was put in the tube holder, and its light transmission value was converted to a digital signal. For each individual tube, two values were recorded: one after mixing the AuNP sensors with target solutions (Tbefore) and another one after completion of the sensing protocol (Tafter). These two sets of values were subtracted to remove backgroundnoises and obtain a delta transmission value (NW = NwxUsy,W − Nzsx{ys,W). Then the tubeswere positioned in 5 different orientations, and such calibrated signals NWwere measured along each orientation, and then averaged to account for tube variability. A baseline value for each matrix was measured by loading a tube with 18 μL of AuNP sensors and 6 μL of the media, representing the highest AuNP concentration and lowest signal intensity N|inAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f the absence of THBS2. In addition, 24 μL of the media were loaded into the tube and measured, representing the highest possible signal intensity Nqin a hypothetical total aggregation of AuNPs. Then, the measured transmission values were normalized as^^^^,W = QSYQ}Q~YQ} from 1 to 0, e.g., 1 representing no aggregation and no target, and 0indicating all AuNPs precipitated at a high target concentration. The signals (SPED,i) were plotted into a graph and fitted using a biphasic dose-response equation in Origin 2024b software (OriginLab, USA) by orthogonal distance regression algorithm to consider the data and error weight in fitting calculations. The fitting equation is: ^1 − ^^^^^(^) = ^^ + (^^ − ^^) ^1 + 10(^{^^R^YD)^^ +1 + 10(^{^^R\YD)^\^which SPEDand SH are the lowest and highest signal, x01 and x02 are half maximal effective concentration (EC50-1), and EC50-2, h1, and h2 are hill slopes for the first and second phases, and p is the steepness. Also, error bars were calculated by the standard deviation of the five values efollowing / = ^∑(Q ^ \of each tub SY^)<Y^ . The limit of detection (LoD) was defined as the lowestconcentration of theusing the modified formula: ^^{^ = ^^D − 1.645 × ( / ^D + / ^D)where / is the error from the sample withzero protein ^^{^was then plotted against the corresponding target concentrations to determine the LoD.

[0152] 3. Results and Discussion

[0153] 3.1 Sensing Mechanism

[0154] Within the context of the sensing assay disclosed herein, streptavidin- coated AuNPs were functionalized with biotinylated THBS2 antibodies, forming multivalent sensing probes (Fig.7a). THBS2 samples were prepared by spiking in PBS and body fluids (Fig.7b) along with negative controls (NC, here blank samples without THBS2). The prepared samples were then mixed with AuNP colloids, triggering antibody- antigen interactions and inducing AuNP aggregation in positive samples. The signalAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f transduction was accelerated by the active fluidic force generated during centrifugation. Subsequently, vortex agitation redispersed unbound AuNPs (Fig 7c). The transparency of the aggregated tubes would increase because of AuNP precipitation, which was quantified by a PED (Fig.7d).

[0155] 3.2 Sensing Protocol Optimization

[0156] Unlike conventional assays, such as ELISA, which rely on passive diffusion and equilibrium conditions over long incubation times (often hours), NasRED operates in a quasi-equilibrium state. External controls, such as centrifugation, actively accelerate diffusion and reaction kinetics, bypassing the need for passive equilibrium. Vortex fluidic force helps ensure the elimination of non-specific interactions. Centrifugation and vortex agitation dynamically adjust the distribution of AuNPs and proteins in the sensing buffer solutions, creating controlled conditions that promote efficient interactions between the nanoparticles and target molecules. This unique approach enables NasRED to achieve rapid signal transduction and significantly shorter assay times while maintaining sufficient biochemical stability for reliable sensing.

[0157] Finding an optimal sensing condition is therefore needed to achieve the best sensitivity and specificity. On one hand, sufficient centrifugation power is needed to accelerate the agglomeration. It drives the AuNPs and bound protein molecules to concentrate at the bottom of the tube, thus reducing the precipitation length from millimeters (solution height) to micrometers (cluster thickness), and in the meanwhile promotes the molecule interactions with the AuNP sensors even at ultralow target concentrations in a concentrated microenvironment at the bottom of the tube. As a result, NasRED assay time is reduced to minutes while achieving high sensitivity. On the other hand, adequate vortex agitation is necessary to redisperse AuNP precipitate caused by the nonspecific binding after centrifugation. Vortex agitation disperses unbound AuNPs back into the supernatant, creating a clear distinction between the precipitated AuNP clusters (representing positive detection) and the free-floating AuNPs (for signal readout). Importantly, excessive vortex agitation can break down the AuNP clusters formed during the antigen-antibody reaction, thus negatively impacting sensitivity and specificity.

[0158] To examine the impacts of centrifugation and vortex forces on theAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f NasRED sensing performance, different concentrations of THBS2 (10 nM to 100 fM, and NC) in PBS media were mixed with functionalized AuNPs and tested under different parameters. For consistency, the sensing protocol was kept the same except vortex agitation speed (Fig.8 a-b) and the centrifugation speed (Fig.8 c-d). Clearly, low vortex speeds (1750 and 1850 rpm) did not create enough force to resuspend NC samples, which represented a blank solution without target proteins. The threshold value to redisperse the NC sample was found to be about 1950 rpm, but higher speeds could cause unintentional AuNP cluster breakdown and negatively impact the dynamic range (Fig. 8b). Similarly, a large range of centrifugation conditions were tested, e.g. 400 to 2400 g (2000 krpm to 4500 krpm). Clearly, AuNP agglomeration was excessive at higher centrifugation speeds (Fig.8c), leading to aggregate formation even for negative control samples that could not be broken up to resuspend after vortex agitation. These results (Fig.8d) indicated that higher centrifugation speeds (e.g.1950 g and 2400 g) limited the maximum achievable PED signal values (to <0.8 and <0.6) and reduced the dynamic range compared to lower speeds. In comparison, lower centrifuge speeds (e.g.385 g and 600 g) limited the minimum PED signals (to >0.6 and >~0.4) and also constrained the dynamic range of detection. These observations were used to empirically determine the optimal condition (1180 g and vortex at 1950 rpm) for the optimum dynamic range and LoDs.

[0159] 3.3 THBS2 Detection

[0160] Our in-solution protein-sensing platform is adaptable to various targets in different media to detect THBS2 (Fig.9). In a previous study, the inventors demonstrated the superior sensitivity of NasRED compared to ELISA by 4 orders of magnitude while providing accessibility, portability, and inexpensiveness. THBS2, with a molecular weight of 129 kDa, has a neutralizing ability towards the anti-THBS2 antibody on the AuNPs, with the median effective dose (ED50) of 0.07-0.7μg / mL and LoD of 0.027μg / mL (213 pM) in PBS (R&D Systems, Inc.). The median reported THBS2 level in healthy patients’ serum was 5.8 ng / mL (45 pM) and 24 ng / mL (186 pM) measured on ELISA, while its cut- off values were found 40.9 ng / ml (317 pM) and 31.88 ng / mL (247 pM) for PDAC and NSCLC patients. The salivary level of THBS2 in healthy patient samples was 0.68±0.73 ng / mL (5.2±5.6 pM) which was significantly lower than that from OSCC patients atAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f 12.90±32.8 ng / mL (100±254 pM). Additionally, THBS2 in the synovium is also an important endogenous regulator of angiogenesis and inflammation. Based on these reports, THBS2 was spiked in PBS and biological media, including serum, WB, saliva, synovial fluid, and CSF, in a concentration range of 10 nM to 1 fM, and tested on the NasRED platform to evaluate the feasibility of clinical cancer detection. Here, WB matrix was diluted to 20% to minimize potential signal interference due to overlapping of optical absorption between hemoglobin and AuNPs. Also, synovial fluid has a high viscosity, which can affect the effectiveness of vortex agitation and thus is diluted to 20%.

[0161] There were several notable observations. First, the microcentrifuge tubes in PBS (Fig.9a) displayed a color transition from transparent at 10 nM to reddish color at 100 fM and negative control, yielding a LoD of ~331 fM (42.7 pg / mL) (Fig.9b). The same was observed for other biological matrices (Fig.9c, e, i, k) except for 20% WB, where the tube images couldn’t properly illustrate this transition (Fig.9g) due to the red colors from both the red blood cells and AuNPs. However, the PED signal measured from the light extinction changes at 532 nm (Fig. 9h) was able to delineate the different THBS2 concentrations, suggesting the feasibility of direct detection from whole blood without blood cell separations to further simplify the clinical use. In terms of sensitivity, the readout signals (Fig.9d, f, h, j, l) proved the capability of THBS2 screening in different media with calculated LoDs as ~0.316 pM (40.8 pg / mL), 0.025 pM (3.2 pg / mL), 12.88 pM (1.6 ng / mL), 7 pM (903 pg / mL), and 0.16 pM (20.64 pg / mL) for HPS, CSF, Saliva, 20% WB, and 20% Synovial Fluid, respectively. These measured values are comparable or better than reported by ELISA assays. Lastly, considering the need for a very small amount of blood (6 µL of 20% whole blood, or only ~1.2 µL), NasRED can be adopted to analyze minute amounts of samples from capillary blood, further minimizing the complexity of sample preparation.

[0162] 3.4 Specificity Test

[0163] To ensure that a diagnostic assay accurately identifies the target analyte and does not produce false positive results due to cross-reactivity with other substances, other proteins should be tested in the same conditions in similar sample media. To validate the specificity of NasRED for THBS2 detection, the inventors included CA19-9Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f and BSA as comparison models in the analysis (Fig 10). These proteins were chosen due to their distinct biological relevance with the THBS2-specific antibody-functionalized AuNPs. CA19-9 serves as a clinically implemented tumor marker, particularly for pancreatic cancer. While CA19-9 and THBS2 can be elevated in certain cancers, they have different characteristics and roles and should be considered separate markers. Protein blockers, such as BSA, are used to block non-specific bindings and stabilizers for the probes when performing the testing and should not impact the signal. Here HPS and saliva were chosen to assess the specificity of THBS2 sensing in biological matrices. Evidently, AuNP aggregation only happened in the tube samples with THBS2 (Fig.10a, c), and the tubes holding CA19-9, BSA, and blank samples (NC) did not produce noticeable color changes. Indeed, no significant PED signal was detected for these proteins (Fig.10b, d), confirming the absence of nonspecific binding or cross-reactivity. These results underscored the high specificity of NasRED, ensuring that the observed signals were not negatively affected by the biological matrixes or the presence of other protein markers.

[0164] 4. Conclusion

[0165] Determining THBS2 concentration in body fluids is a potentially important tool for cancer screening due to the overexpression of THBS2 in various cancers. Current diagnostic methods such as ELISA, PCR and IHC are the main methods; however, they require lengthy assay times, bulky instruments and professional operation, thus limiting their use in clinics and physician’s offices for rapid and convenient screening. Here, as a proof of concept demonstration the inventors studied the application of NasRED as a plug and play, in-solution, POC platform to detect THBS2 for cancer prescreening. The NasRED platform was able to achieve a high sensitivity (LoD 331 fM in PBS) within 15 min, orders of magnitude better compared to ELISA (LoD 213 pM in PBS and typically >6 hours turnaround time). In this study, the inventors optimized the NasRED assay protocol by analyzing the impacts of the active fluidic forces on the assay sensitivity in PBS, and further demonstrated the feasibility of THBS2 detection in various biological matrices with high sensitivity (e.g. LoDs of 25 fM in CSF, 12.9 pM in saliva, and 7 pM in WB) and high specificity. The small amount of sample needed (<6 µL per test) also made NasRED possible to use less invasive capillary blood and a minimal amount of CSF for routineAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f screening, which would otherwise be much more challenging. The modularity of NasRED makes it easily engineered in future work to detect other cancer biomarkers for cancer prescreening, prognostics, and diagnostics for a wide variety of cancer types, from oral cavity cancer and lymphoma to medulloblastoma, cervical cancer, PDAC, NSCLC, and melanoma.

[0166] Some further aspects are defined in the following clauses:

[0167] Clause 1: A method of detecting a target analyte in a sample, the method comprising: contacting the sample with a set of functionalized plasmonic metal nanoparticles (MNPs) that comprise a target analyte binding agent that selectively binds to the target analyte to produce target analyte bound MNPs; precipitating the target analyte bound MNPs to produce precipitated target analyte bound MNPs; resuspending the precipitated target analyte bound MNPs to produce resuspended target analyte bound MNPs; and, detecting binding of the resuspended target analyte bound MNPs, thereby detecting the infectious agent.

[0168] Clause 2: The method of Clause 1, wherein the method comprises a limit of detection of between about 10 femtogram per milliliter (fg / mL) and about 225 fg / mL.

[0169] Clause 3: The method of Clause 1 or Clause 2, wherein the method comprises a sensitivity at a femtomolar or lower level.

[0170] Clause 4: The method of any one of the preceding Clauses 1-3, wherein the method comprises a dynamic range of at least about five orders of magnitude.

[0171] Clause 5: The method of any one of the preceding Clauses 1-4, wherein a duration of the method is about 15 minutes or less.

[0172] Clause 6: The method of any one of the preceding Clauses 1-5, wherein the sample comprises a volume of about 10 μL or less.

[0173] 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.

[0174] Clause 8: The method of any one of the preceding Clauses 1-7, fAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f comprising quantifying an amount of the target analyte in the sample.

[0175] Clause 9: The method of any one of the preceding Clauses 1-8, wherein the detecting step comprises detecting one or more aggregations of the resuspended target analyte bound MNPs.

[0176] Clause 10: The method of any one of the preceding Clauses 1-9, wherein the target analyte binding agent comprises an antibody or antigen binding portion thereof, and wherein the target analyte comprises an antigen.

[0177] Clause 11: The method of any one of the preceding Clauses 1-10, wherein the antibody or antigen binding portion thereof comprises a monoclonal antibody.

[0178] Clause 12: The method of any one of the preceding Clauses 1-11, wherein the antibody or antigen binding portion thereof comprises a nanobody.

[0179] Clause 13: The method of any one of the preceding Clauses 1-12, wherein the antigen comprises a protein, or a portion thereof, from an infectious agent.

[0180] Clause 14: The method of any one of the preceding Clauses 1-13, wherein the infectious agent comprises a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the antigen, or a portion thereof, comprises a SARS-CoV-2 protein, or a portion thereof, selected from the group consisting of: an envelope (E) protein, a membrane (M) protein, a nucleocapsid (N) protein, and a spike (S) protein.

[0181] Clause 15: The method of any one of the preceding Clauses 1-14, wherein the antigen comprises a cancer biomarker.

[0182] Clause 16: The method of any one of the preceding Clauses 1-15, wherein the cancer biomarker comprises thrombospondin-2 (THBS2).

[0183] Clause 17: The method of any one of the preceding Clauses 1-16, wherein the target analyte binding agent comprises an antigen, and wherein the target analyte comprises an antibody.

[0184] Clause 18: The method of any one of the preceding Clauses 1-17, wherein the antigen comprises a protein, or a portion thereof, from an infectious agent.

[0185] Clause 19: The method of any one of the preceding Clauses 1-18, whereinAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f the infectious agent comprises a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the antigen, or a portion thereof, comprises a SARS-CoV-2 protein, or a portion thereof, selected from the group consisting of: an envelope (E) protein, a membrane (M) protein, a nucleocapsid (N) protein, and a spike (S) protein.

[0186] Clause 20: The method of any one of the preceding Clauses 1-19, wherein the antigen comprises a cancer biomarker or a portion thereof.

[0187] Clause 21: The method of any one of the preceding Clauses 1-20, wherein the cancer biomarker or a portion thereof comprises thrombospondin-2 (THBS2) or a portion thereof.

[0188] Clause 22: The method of any one of the preceding Clauses 1-21, comprising obtaining the sample from a subject.

[0189] Clause 23: The method of any one of the preceding Clauses 1-22, comprising administering one or more therapies to the subject when the target analyte is detected in the sample.

[0190] Clause 24: A system for detecting a target analyte in a sample, comprising: a sample container receiving structure configured to receive a sample container that contains resuspended target analyte bound plasmonic metal nanoparticles (MNPs), wherein the sample container receiving structure is configured to substantially prevent ambient light from entering a sample container receiving area of the sample container receiving structure and wherein the sample container receiving structure comprises a light path aligned to measure at least supernatant in the sample container; 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 that contains the resuspended target analyte bound MNPs using the photodetector; a power source operably connected to the controller, whereinAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f 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.

[0191] Clause 25: The system of Clause 24, wherein the controller is configured to quantify an amount of the target analyte in the sample.

[0192] Clause 26: The system of Clause 24 or Clause 25, wherein the resuspended target analyte bound MNPs are formed by: contacting the sample with a set of functionalized MNPs that comprise a target analyte binding agent that selectively binds to the target analyte to produce target analyte bound MNPs; precipitating the target analyte bound MNPs to produce precipitated target analyte bound MNPs; and, resuspending the precipitated target analyte bound MNPs to produce the resuspended target analyte bound MNPs.

[0193] Clause 27: The system of any one of the preceding Clauses 24-26, wherein the target analyte binding agent comprises an antibody or antigen binding portion thereof, and wherein the target analyte comprises an antigen.

[0194] Clause 28: The system of any one of the preceding Clauses 24-27, wherein the antibody or antigen binding portion thereof comprises a monoclonal antibody.

[0195] Clause 29: The system of any one of the preceding Clauses 24-28, wherein the antibody or antigen binding portion thereof comprises a nanobody.

[0196] Clause 30: The system of any one of the preceding Clauses 24-29, wherein the antigen comprises a protein, or a portion thereof, from an infectious agent.

[0197] Clause 31: The system of any one of the preceding Clauses 24-30, wherein the infectious agent comprises a severe acute respiratory syndrome coronavirus- 2 (SARS-CoV-2) and the antigen, or a portion thereof, comprises a SARS-CoV-2 protein, or a portion thereof, selected from the group consisting of: an envelope (E) protein, a membrane (M) protein, a nucleocapsid (N) protein, and a spike (S) protein.

[0198] Clause 32: The system of any one of the preceding Clauses 24-31, wherein the antigen comprises a cancer biomarker.Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f

[0199] Clause 33: The system of any one of the preceding Clauses 24-32, wherein the cancer biomarker comprises thrombospondin-2 (THBS2).

[0200] Clause 34: The system of any one of the preceding Clauses 24-33, wherein the target analyte binding agent comprises an antigen, and wherein the target analyte comprises an antibody.

[0201] Clause 35: The system of any one of the preceding Clauses 24-34, wherein the antigen comprises a protein, or a portion thereof, from an infectious agent.

[0202] Clause 36: The system of any one of the preceding Clauses 24-35, wherein the infectious agent comprises a severe acute respiratory syndrome coronavirus- 2 (SARS-CoV-2) and the antigen, or a portion thereof, comprises a SARS-CoV-2 protein, or a portion thereof, selected from the group consisting of: an envelope (E) protein, a membrane (M) protein, a nucleocapsid (N) protein, and a spike (S) protein.

[0203] Clause 37: The system of any one of the preceding Clauses 24-36, wherein the antigen comprises a cancer biomarker or a portion thereof.

[0204] Clause 38: The system of any one of the preceding Clauses 24-37, wherein the cancer biomarker or a portion thereof comprises thrombospondin-2 (THBS2) or a portion thereof.

[0205] Clause 39: A kit comprising the system of any one of the preceding Clauses 24-38.

[0206] 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 theAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f combination, and the claimed combination may be directed to a sub-combination or variation of a sub-combination.

[0207] 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 be understood 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.

[0208] 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

Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f WHAT IS CLAIMED IS:

1. A method of detecting a target analyte in a sample, the method comprising: contacting the sample with a set of functionalized plasmonic metal nanoparticles (MNPs) that comprise a target analyte binding agent that selectively binds to the target analyte to produce target analyte bound MNPs; precipitating the target analyte bound MNPs to produce precipitated target analyte bound MNPs; resuspending the precipitated target analyte bound MNPs to produce resuspended target analyte bound MNPs; and, detecting binding of the resuspended target analyte bound MNPs, thereby detecting the infectious agent.

2. The method of claim 1, wherein the method comprises a limit of detection of between about 10 femtogram per milliliter (fg / mL) and about 225 fg / mL.

3. The method of claim 1, wherein the method comprises a sensitivity at a femtomolar or lower level.

4. The method of claim 1, wherein the method comprises a dynamic range of at least about five orders of magnitude.

5. The method of claim 1, wherein a duration of the method is about 15 minutes or less.

6. The method of claim 1, wherein the sample comprises a volume of about 10 μL or less.Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f 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 target analyte in the sample.

9. The method of claim 1, wherein the detecting step comprises detecting one or more aggregations of the resuspended target analyte bound MNPs.

10. The method of claim 1, wherein the target analyte binding agent comprises an antibody or antigen binding portion thereof, and wherein the target analyte comprises an antigen.

11. The method of claim 10, wherein the antibody or antigen binding portion thereof comprises a monoclonal antibody.

12. The method of claim 10, wherein the antibody or antigen binding portion thereof comprises a nanobody.

13. The method of claim 10, wherein the antigen comprises a protein, or a portion thereof, from an infectious agent.

14. The method of claim 13, wherein the infectious agent comprises a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and wherein the antigen, or a portion thereof, comprises a SARS-CoV-2 protein, or a portion thereof, selected from the group consisting of: an envelope (E) protein, a membrane (M) protein, a nucleocapsid (N) protein, and a spike (S) protein.

15. The method of claim 10, wherein the antigen comprises a cancer biomarker.Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f 16. The method of claim 15, wherein the cancer biomarker comprises thrombospondin-2 (THBS2).

17. The method of claim 1, wherein the target analyte binding agent comprises an antigen, and wherein the target analyte comprises an antibody.

18. The method of claim 17, wherein the antigen comprises a protein, or a portion thereof, from an infectious agent.

19. The method of claim 18, wherein the infectious agent comprises a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the antigen, or a portion thereof, comprises a SARS-CoV-2 protein, or a portion thereof, selected from the group consisting of: an envelope (E) protein, a membrane (M) protein, a nucleocapsid (N) protein, and a spike (S) protein.

20. The method of claim 17, wherein the antigen comprises a cancer biomarker or a portion thereof.

21. The method of claim 20, wherein the cancer biomarker or a portion thereof comprises thrombospondin-2 (THBS2) or a portion thereof.

22. The method of claim 1, comprising obtaining the sample from a subject.

23. The method of claim 22, comprising administering one or more therapies to the subject when the target analyte is detected in the sample.

24. A system for detecting a target analyte in a sample, comprising: a sample container receiving structure configured to receive a sample container that contains resuspended target analyte bound plasmonic metal nanoparticles (MNPs), wherein the sample container receiving structure is configured to substantially preventAttorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f ambient light from entering a sample container receiving area of the sample container receiving structure and wherein the sample container receiving structure comprises a light path aligned to measure at least supernatant in the sample container; 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 that contains the resuspended target analyte bound MNPs 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.

25. The system of claim 24, wherein the controller is configured to quantify an amount of the target analyte in the sample.

26. The system of claim 24, wherein the resuspended target analyte bound MNPs are formed by: contacting the sample with a set of functionalized MNPs that comprise a target analyte binding agent that selectively binds to the target analyte to produce target analyte bound MNPs; precipitating the target analyte bound MNPs to produce precipitated target analyte bound MNPs; and resuspending the precipitated target analyte bound MNPs to produce the resuspended target analyte bound MNPs.Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f 27. The system of claim 26, wherein the target analyte binding agent comprises an antibody or antigen binding portion thereof, and wherein the target analyte comprises an antigen.

28. The system of claim 27, wherein the antibody or antigen binding portion thereof comprises a monoclonal antibody.

29. The system of claim 27, wherein the antibody or antigen binding portion thereof comprises a nanobody.

30. The system of claim 27, wherein the antigen comprises a protein, or a portion thereof, from an infectious agent.

31. The system of claim 30, wherein the infectious agent comprises a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and wherein the antigen, or a portion thereof, comprises a SARS-CoV-2 protein, or a portion thereof, selected from the group consisting of: an envelope (E) protein, a membrane (M) protein, a nucleocapsid (N) protein, and a spike (S) protein.

32. The system of claim 27, wherein the antigen comprises a cancer biomarker.

33. The system of claim 32, wherein the cancer biomarker comprises thrombospondin-2 (THBS2).

34. The system of claim 26, wherein the target analyte binding agent comprises an antigen, and wherein the target analyte comprises an antibody.

35. The system of claim 34, wherein the antigen comprises a protein, or a portion thereof, from an infectious agent.Attorney Docket Number 0391.0110-PCT / M24-311L^-WO1-f 36. The system of claim 35, wherein the infectious agent comprises a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and wherein the antigen, or a portion thereof, comprises a SARS-CoV-2 protein, or a portion thereof, selected from the group consisting of: an envelope (E) protein, a membrane (M) protein, a nucleocapsid (N) protein, and a spike (S) protein.

37. The system of claim 34, wherein the antigen comprises a cancer biomarker or a portion thereof.

38. The system of claim 37, wherein the cancer biomarker or a portion thereof comprises thrombospondin-2 (THBS2) or a portion thereof.

39. A kit comprising the system of claim 24.

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