CN117136307A - Methods, assays and systems for detecting target analytes - Google Patents

Abstract

The strip systems, methods, devices and related kits disclosed herein are useful for determining the presence and/or level of a target analyte in a sample (e.g., a biological sample, such as saliva or a nasal swab), wherein the target analyte may be a microorganism (e.g., whole virus) or molecule (e.g., a viral antigen) associated with a health state, disease or damaged or otherwise altered physiological condition. In certain embodiments, the systems, methods, devices, and kits provide one or more improved characteristics over lateral flow proteins and other assays known in the art for detection, including but not limited to assay time, ease of use, risk of infection, accuracy, specificity, selectivity, detection limits of assays, quantitative detection, and impact of common interferents on sensor output, cost, simplicity, or a combination thereof. In certain embodiments, the systems, assays, methods, and reagents are multiplexed, i.e., allow for the detection or monitoring of more than one target analyte (e.g., two different viruses or viruses and bacteria).

Description

Methods, assays and systems for detecting target analytes

Cross Reference to Related Applications

The present application requires the following priorities: U.S. provisional application No. 63/137,085 filed on day 13 of month 1 of 2021; U.S. provisional application No. 63/150,990, filed at 18 of 2 of 2021; U.S. provisional application No. 63/156,663, filed on 3/4 at 2021; U.S. provisional application No. 63/156,666 filed on 3/4 of 2021; U.S. provisional application No. 63/170,426 filed on 2/4/2021; U.S. provisional application No. 63/208,694, filed on 6/9 at 2021; U.S. provisional application No. 63/221,375 filed on day 137 of 2021; U.S. provisional application No. 63/271,544 filed on 25/10/2021; and U.S. provisional application No. 63/272,065 filed on day 26 of 10 in 2021. The contents of the above-mentioned documents are incorporated by reference in their entirety.

Technical Field

Disclosed herein are systems, assays, and methods for detecting and monitoring the presence of one or more target analytes in a sample (e.g., a biological sample or an environmental sample). Methods disclosed herein also include detection methods, sample preparation methods, therapeutic methods, and telemedicine services. Kits and reagents for carrying out the methods are also provided.

Background

Assays for quantitatively detecting analytes (e.g., infectious agents) in biological samples are routinely used in medicine to diagnose or track the progression of disease or injury or to monitor health. Historically, these assays have been performed in a healthcare or laboratory setting. Similarly, assays to detect analytes in environmental samples are of importance to industries such as transportation and agriculture, and are typically performed in laboratory environments. In both cases, such assays typically require multiple steps and expert analysis.

There remains a need to improve the number and value of such tests. In particular, there remains a need for simple and economical performance of such assays by laypersons in non-clinical environments (e.g., home, office, or field).

Disclosure of Invention

Disclosed herein are systems, assays (biosensors) and methods for detecting at least one target analyte in a sample. Advantageously, the systems, assays, and methods disclosed herein are fast and allow reliable results even when performed by relatively untrained users, such as laypersons. In certain embodiments, the systems, assays, and methods disclosed herein can be used to detect low concentrations of at least one target analyte using small sample volumes.

In a first aspect, a system for detecting at least one target analyte in a sample (e.g., a biological sample or an environmental sample) added to the system is provided, comprising: (i) An assay comprising at least one capture reagent and at least one detection reagent capable of producing a detectable complex with at least one target analyte (if present) in the presence of a substrate (e.g., a substrate added by a user); and (ii) a detection device for detecting a detectable complex, wherein the detection device comprises an enzyme-based amperometric sensor comprising at least one electrode, wherein the detectable complex forms over the at least one electrode or migrates within the system to become positioned over the at least one electrode.

The time at which the results are provided by the system varies. In one embodiment, the detectable complex is detected in about 30 minutes or less, about 10 minutes or less, about 5 minutes or less, about 2 minutes or less, or about 1 minute or less. In a particular embodiment, the time to produce a result is less than 1 minute or more particularly, about 30 seconds, about 20 seconds, about 10 seconds, or about 1 second.

Target analytes detected by the system may be different. In one embodiment, the target analyte is selected from microorganisms (e.g., viruses, bacteria, protozoa, fungi, or prions), proteins, peptides, cytokines, hormones, steroids, cofactors, small molecules (e.g., therapeutic or abusive drugs), vitamins, and the like.

In one embodiment, the target analyte detected by the system is a viral protein.

In a particular embodiment, the target analyte detected by the system is a nucleocapsid (N) protein of a coronavirus, such as SARS-CoV-2 or a variant thereof.

In a particular embodiment, the target analyte is a spike (S) protein of a coronavirus, such as SARS-CoV-2 or a variant thereof.

In certain embodiments, the system detects more than one target analyte, e.g., two or more viral species, in a biological sample. In some embodiments, one or more viral species are closely related. In certain embodiments, the system detects SARS-CoV-d and influenza.

The detection Limit (LOD) of the system may be different. In one embodiment, the system has an LOD of about 1mg/mL or less, about 1ng/mL or less, about 1pg/mL or less, or about 1fg/mL or less.

In a particular embodiment, the target analyte is a protein or peptide, and the system has an LOD of about 500pg/mL or less, more particularly about 200, about 150, about 100, about 75, about 50, about 25, about 10, about 5, or about 1pg/mL or less.

In one embodiment, the target analyte is a whole virus, such as a whole coronavirus. In a particular embodiment, the whole virus is SARS-CoV-2 or a variant thereof.

In one embodiment, the target analyte is a whole virus having a LOD of about 10000TCID50/mL or less, about 5000TCID50/mL or less, about 1000TCID50/mL or less, about 100TCID50/mL or less, about 50TCID50/mL or less, about 25TCID50/mL or less, about 10TCID50/mL or less, or about 5TCID50/mL or less.

The capture reagent and the detection reagent may be different. In certain embodiments, the capture reagent and the detection reagent are binding reagents selected from the group consisting of aptamers, antibodies, and proteins, or combinations thereof.

The detection reagent may be labeled with an enzyme. Any suitable enzyme label may be used, such as an oxidoreductase. In one embodiment, the enzyme is selected from oxidase, peroxidase, hydrogenase, catalase, dehydrogenase or phosphatase.

In a specific embodiment, the enzyme label is alkaline phosphatase and the added substrate is selected from pyridoxal 5' -phosphate (PLP), 5-bromo-4-chloro-3-indolyl-phosphate, L-ascorbic acid-2-phosphate, acetaminophen phosphate, 4-acetamidophenyl phosphate, 4-aminophenyl phosphate in Diethanolamine (DEA), 1-amino-2-propanol, N-methyl-D-glucosamine or tris buffer.

In another particular embodiment, the enzyme label is glucose phosphatase and the added substrate is glucose.

In certain embodiments, the system further comprises a reporter reagent. The reporter reagent may be different, e.g. selected from an aptamer or an antibody. According to this embodiment, the reporter reagent is conjugated to the detection reagent. Both the detection reagent and the reporter reagent may be labeled, for example, with different enzyme labels, to provide a dual detection system.

In certain embodiments, the capture reagent is present in solution and is optionally added by the user prior to binding to the at least one target analyte.

In alternative embodiments, the capture reagent is immobilized to a solid or porous support, either directly or by means of a first binding reagent, to provide a test site. According to the latter embodiment, the capture reagent is conjugated to a second binding reagent, wherein the second binding reagent binds to the first binding reagent.

In one embodiment, the first binding reagent is selected from the group consisting of streptavidin, gold, silver, malamides, acrylates, amines, carboxylic acids, vinyl sulfones, thiols, silanes, and epoxides.

In a particular embodiment, the first binding agent is streptavidin and the second binding agent is biotin.

The binding reagent within the assay may be crosslinked with one or more additional binding reagents. In one embodiment, the first binding reagent is crosslinked with one or more additional first binding reagents. In another embodiment, the capture reagent is crosslinked with one or more additional capture reagents.

In one embodiment, the detection reagent is added to the system by the user, optionally together with the capture reagent.

The solid or porous support may be any suitable such support, for example a bead, a membrane (e.g. nitrocellulose) or a bead immobilised on a membrane.

In certain embodiments, the assay is contained within a cartridge, such as a disposable cartridge.

In embodiments in which the target analyte is a protein, the affinity of the capture reagent and the detection reagent for the protein may be different. In one embodiment, the capture reagent and the detection reagent have about 10 for the protein ^-10 Or greater, about 10 ^-8 Kd or greater, or about 10 ^-6 Kd or greater.

The accuracy of the system may vary. The system of claims 1-42, wherein the system allows for at least about 90% accuracy or greater, about 93% accuracy or greater, about 95% accuracy or greater, about 98% accuracy or greater, or about 99% accuracy or greater.

In certain embodiments, the system continuously generates an electrochemical signal. In one embodiment, the electrochemical signal is collected discontinuously, e.g., the electrochemical signal is collected at intervals separated by waiting periods.

Any suitable assay format may be utilized in the system, including competitive or non-competitive assay formats. In one embodiment, the system includes a lateral flow assay. In other embodiments, the system comprises a vertical flow assay. The vertical flow assay may have more than one layer, for example a multi-layer vertical flow assay.

In some embodiments, the systems described herein are intended for use outside of a clinical setting, such as for home or workplace use. In some embodiments, the system is intended for self-monitoring by an individual, including over time.

The system may allow qualitative, semi-quantitative or quantitative detection.

In an alternative embodiment, the system is an optical system, wherein the detection means is an optical reader.

In a particular embodiment, the sample is obtained from two or more subjects.

In a second aspect, an assay is disclosed comprising at least one capture reagent and at least one detection reagent, wherein the detection reagent is labeled with an enzyme label and the capture reagent and detection reagent form a detectable complex in the presence of at least one target analyte and added substrate.

The assay may be an electrochemical or optical (e.g., fluorescent or colorimetric) assay.

In one embodiment, the detection does not require a detection device. In another embodiment, the detectable complex is detected by a detection device such as a blood glucose meter, a chronoamperometer (chronoamperometer), or a mobile phone.

The time at which the results are provided by the assay may be different. In one embodiment, the detection occurs in about 30 minutes or less, about 10 minutes or less, about 5 minutes or less, about 2 minutes or less, or about 1 minute or less. In a particular embodiment, the time to produce a result is less than 1 minute or more particularly, about 30 seconds, about 20 seconds, about 10 seconds, or about 1 second.

The target analytes detected by the assay may be different. In one embodiment, the target analyte is a microorganism (e.g., a virus or bacterium), a protein, a peptide, a hormone, a steroid, a cytokine, a small molecule, a cofactor, a vitamin, or the like.

In one embodiment, the target analyte detected by the assay is a protein or peptide, such as a viral protein or peptide.

In a particular embodiment, the target analyte detected by the assay is the spike (S) protein or the nucleocapsid (N) protein of a coronavirus. The coronavirus may be, for example, SARS-CoV-2 or a variant thereof.

In certain embodiments, the assay may detect more than one target analyte sequentially or simultaneously. For example, an assay may detect two different viral species.

The detection Limit (LOD) of the assay may be different. In one embodiment, the assay has an LOD of about 1mg/mL or less, about 1ng/mL or less, about 1pg/mL or less, or about 1fg/mL or less.

In a particular embodiment, the target analyte is a protein and the assay has a LOD of about 1ng/mL or less, about 500pg/mL or less, more particularly about 200, about 150, about 100, about 75, about 50, about 25, about 10, about 5, or about 1pg/mL or less.

The number of target analytes that can be detected by the assay may vary. In one embodiment, the assay has about 100 target analytes/mL or less, about 50, about 20, about 10, or about 5 target analytes/mL or less LOD.

In certain embodiments, the target analyte is a whole virus, such as a whole coronavirus or more particularly SARS-CoV-2 or a variant thereof.

In one embodiment, the target analyte is a whole virus and the assay has an LOD of about 10000TCID50/mL or less, about 100TCID50/mL or less, about 50TCID50/mL or less, about 10TCID50/mL or less, or about 5TCID50/mL or less.

The capture reagent and the detection reagent used in the assay may be different. In one embodiment, the capture reagent and the detection reagent are binding reagents selected from the group consisting of aptamers, antibodies, and proteins.

The detection reagent may be labeled, for example, with an enzyme label. Enzymes may be different. In one embodiment, the enzyme is an oxidoreductase. In a particular embodiment, the enzyme is selected from oxidase, peroxidase, hydrogenase, catalase, dehydrogenase or phosphatase.

In one embodiment, the enzyme is alkaline phosphatase and the added substrate is selected from pyridoxal 5' -phosphate (PLP), 5-bromo-4-chloro-3-indolyl-phosphate, L-ascorbic acid-2-phosphate, acetaminophen phosphate, 4-acetamidophenyl phosphate, 4-aminophenyl phosphate in Diethanolamine (DEA), 1-amino-2-propanol, N-methyl-D-glucosamine or tris buffer.

In another embodiment, the enzyme is glucose phosphatase and the added substrate is glucose.

In certain embodiments, the assay further comprises a reporter reagent. The reporter reagent may be different, e.g. selected from an aptamer or an antibody. According to this embodiment, the reporter reagent is conjugated to the detection reagent. Both the detection reagent and the reporter reagent may be labeled, for example, with different enzyme labels, to provide a dual detection system.

In one embodiment, the capture reagent is immobilized to a solid or porous support to provide a test site.

In a particular embodiment, the capture reagent is immobilized to the solid support by means of a first binding reagent. According to this embodiment, the capture reagent is conjugated to a second binding reagent, wherein the second binding reagent binds to the first binding reagent. The first binding reagent may be different.

In one embodiment, the first binding reagent is selected from the group consisting of streptavidin, gold, silver, malamides, acrylates, amines, carboxylic acids, vinyl sulfones, thiols, silanes, and epoxides.

In a particular embodiment, the first binding agent is streptavidin and the second binding agent is biotin.

The binding reagent associated with the assay may be cross-linked. In one embodiment, the first binding reagent is crosslinked with one or more additional first binding reagents. In another embodiment, the capture reagent is crosslinked with one or more additional capture reagents.

The solid or porous support may be any suitable such support, for example a bead, a membrane (e.g. nitrocellulose) or a bead immobilised on a membrane.

The solid or porous support may optionally include a control site.

The assay may optionally be contained within a cartridge, such as a disposable cartridge.

In embodiments in which the target analyte is a protein or peptide, the affinity of the capture reagent and the detection reagent for the protein or peptide may be different. In certain embodiments, capture reagents and assaysThe test agent has a protein or peptide concentration of about 10 ^-10 Kd or less, about 10 ^-8 Kd or less, or about 10 ^-6 Kd or less.

The accuracy of the assay may vary. In one embodiment, the assay is at least about 90%, at least about 93%, at least about 95%, at least about 98%, or at least about 99% or more.

The form of the assay may vary. The assay may be a competitive or non-competitive assay. The assay may be a lateral flow or a vertical flow assay. In embodiments where the assay is a vertical flow assay, the assay may comprise one or more layers.

In a third aspect, a method for detecting at least one target analyte in a sample (e.g., a biological sample or an environmental sample) is provided, comprising (i) providing a sample, (ii) optionally, treating the sample; (iii) Adding a sample to the system or assay disclosed herein, and (iv) and if at least one target analyte is present, detecting the target analyte.

The method optionally includes transmitting the results to a third party for review and optional further action.

In one embodiment, the further action includes diagnosing the presence of a disease state or a health state. In a particular embodiment, the results may be calibrated for a disease state (e.g., infection) or health state. The disease may be a viral infection or a bacterial infection. The clinical manifestations may be, for example, upper respiratory tract infections, lower respiratory tract infections, hepatitis, meningitis, encephalitis and/or meningoepithymitis, conjunctivitis, keratitis, keratoconjunctivitis, rash or genital lesions.

In one embodiment, the further action includes monitoring the outcome of administering a therapeutic agent, such as a drug, to the user. According to this embodiment, the user is a patient or a clinical trial subject.

In one embodiment, the action includes administering or ceasing administration of the therapeutic agent to the user.

In a particular embodiment, further actions may include adjusting the dose (up, down) of the therapeutic agent previously administered to the user to provide a new dose for administration.

In another particular embodiment, the further action may involve administering an additional (e.g., second) therapeutic agent to the user.

In one embodiment, the approved therapeutic agent is a small molecule drug or biologic (e.g., monoclonal antibody, therapeutic vaccine, or anti-cancer agent).

In a particular embodiment, the approved therapeutic agent is a small molecule antiviral agent. Representative non-limiting antiviral agents include adhesion inhibitors, entry inhibitors, uncoating inhibitors, protease inhibitors, polymerase inhibitors, nucleoside and nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and integrase inhibitors.

In a particular embodiment, the antiviral agent is selected from acyclovir, ganciclovir, foscarnet; ribavirin; amantadine, azidodeoxycthymidine/zidovudine), nevirapine, tetrahydroimidazobenzodiazepinone (TIBO) compounds; efavirenz; adefovir, delavirdine, mo Nupi, nemortevir and ritonavir-enhanced nemortevir.

In one embodiment, the small molecule antiviral agent is adefovir (as a 200-mg loading dose at day one followed by a 100-mg maintenance dose administered once a day for up to ten days).

In one embodiment, the approved therapeutic agent is a biological antiviral agent. Representative non-limiting biological antiviral agents include monoclonal antibodies (mabs), nucleic acid therapies (e.g., RNAi, antisense, DNA vaccines, micrornas, shrnas, or aptamers).

In one embodiment, the therapeutic agent is a viral particle blocker.

In one embodiment, the monoclonal antibody antiviral agent is selected from the group consisting of sotovimab (e.g., administered intravenously as a 500 mg single dose), ba Ma Nishan antibody (bamlanivimab) (700 mg as a single IV infusion), tetomimab Wei Shankang (etesevelab) and ba Ma Nishan antibody (1400 mg tetomimab Wei Shankang plus ba Ma Nishan antibody 700mg as a single IV infusion), or casirimumab (casirivimab) and idemumab (imdevimab) (1, 200mg casirimumab and 1,200mg idemumab in a single IV infusion).

In a particular embodiment, the therapeutic agent is an antibacterial agent. Representative non-limiting antibacterial agents include penicillins, cephalosporins, fluoroquinolones, aminoglycosides, monocyclolactams and carbapenems and macrolides.

In one embodiment, the therapeutic agent is selected from the group consisting of oxacillin (oxacillin), doxycycline, demeclocycline; epothilone, minocycline, omacycline, tetracycline, cefalexin, cefotaxime, ceftazidime, cefuroxime, and ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin, clindamycin, lincomycin, metronidazole, azithromycin; clarithromycin, erythromycin, sulfamethoxazole and trimethoprim; sulfasalazine, amoxicillin and clavulanic acid; vancomycin, daptomycin, orivancin, telavancin, gentamicin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem and ertapenem.

In a particular embodiment, the therapeutic agent is an antifungal agent. Representative non-limiting antifungal agents include azoles, polyenes, and 5-fluorocytosine.

In a particular embodiment, the therapeutic agent is an anti-inflammatory agent.

In one embodiment, the anti-inflammatory agent is selected from the group consisting of aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, nilanib, oxaprozin, pirfenidone, piroxicam, bissalicylester, sulindac, tolmetin, and combinations thereof.

In a particular embodiment, the therapeutic agent is an anticancer agent.

In one embodiment, the anticancer agent is selected from alkylating agents (or alkylating-like agents), antimetabolites, antitumor antibiotics, plant alkaloids, hormonal agents, topoisomerase inhibitors and the like.

(ii) May be different and include, for example, diluting the sample or adding one or more components, such as one or more binding reagents, to the assay. In certain embodiments, one or more of a capture reagent, a detection reagent, and a reporter reagent is added to the system or assay.

In a fourth aspect, a kit is disclosed containing one or more components of the systems or assays disclosed herein and optionally instructions for use.

In a fifth aspect, a blood glucose meter or chronoamperometer configured to read the results of an immunoassay is disclosed.

In certain embodiments, the chronoamperometer utilizes a modified chronoamperometric method (e.g., a technique that alters the length and period of voltage application), optionally in combination with titration of a compound or counter ion (e.g., mgCl 2) critical to the function of the enzyme. According to this embodiment, the modified chronoamperometry allows one or more of the following: collecting signals (current, charge), increasing signals, improving signal-to-noise ratio, improving sensitivity (e.g., limit of detection), reducing signal time, multiplexing on multiple working electrodes, and/or reducing background. Variables include, but are not limited to, forcing potential, pre-measurement delay, measurement time, open circuit time, number of cycles, measurement sampling rate, and the like.

In certain embodiments, the method does not utilize constant chronoamperometry. In another embodiment, the method does not utilize a delay-timed amperometric method.

The systems, assays, and methods disclosed herein advantageously allow for the detection of target analytes present in a sample at low concentrations.

In one particular embodiment, the systems, assays, and methods disclosed herein allow for the detection of a target protein or peptide (e.g., a viral protein or peptide, such as a coronavirus, e.g., SARS-CoV-2, or a variant thereof, e.g., the N protein of the "omacron" variant) at low concentrations in a sample.

In a particular embodiment, the systems, assays and/or methods disclosed herein allow detection of a target virus (e.g., coronavirus, such as SARS-CoV-2 or variants thereof) at a LOD of about 100fg/mL or less, more particularly, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 10, about 5, or about 1fg/mL or less.

In a particular embodiment, the systems, assays, and/or methods disclosed herein allow detection of a target virus (e.g., coronavirus, such as SARS-CoV-2 or variants thereof) at a LOD of about 25fg/mL or less.

In a particular embodiment, the systems, assays, and/or methods disclosed herein allow for a range of about 10 ¹² TCID50/mL or less, more particularly, about 10 ¹¹ About 10 ¹⁰ About 10 ⁹ About 10 ⁸ About 10 ⁷ About 10 ⁶ About 10 ⁵ About 10 ⁴ LOD detection target virus of about 5000, about 2000, about 200, about 100, about 50 or about 25TCID50/mL or less.

In another particular embodiment, the systems, assays, and/or methods disclosed herein allow for a range of about 10 ¹² pfu/mL or less, more particularly, about 10 ¹¹ About 10 ¹⁰ About 10 ⁹ About 10 ⁸ About 10 ⁷ About 10 ⁶ About 10 ⁵ About 10 ⁴ LOD detection target virus of about 5000, about 2000, about 200, about 100, about 50, or about 25pfu/mL or less.

In another particular embodiment, the systems, assays, and/or methods disclosed herein allow for LOD of less than about 100 target analytes/millimeter, about 80 target analytes/mL or less, about 60 target analytes/mL or less, about 40 target analytes/mL or less, about 20 target analytes/mL or less, about 10 target analytes/mL or less, about 5 target analytes/mL or less, or about 1 target analyte/mL.

In certain embodiments, the systems, assays, and/or methods disclosed herein allow for LOD of about 1 analyte/millimeter to about 100,000 target analytes/mL or more.

In certain embodiments, the system allows for LOD of about 1 to about 5, about 5 to about 10, about 10 to about 20, or about 20 to about 30 analytes/mL.

In certain embodiments, the systems and methods disclosed herein utilize pulse detection.

In a particular embodiment, the treatment in (ii) comprises diluting the sample in a liquid medium.

In certain embodiments, the systems, assays, and methods disclosed herein allow for a high degree of specificity and sensitivity. In some embodiments, the systems, assays, and methods have a specificity of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some, the systems, assays, and methods allow a sensitivity of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

In certain embodiments, the systems, assays, and methods disclosed herein allow for qualitative, semi-quantitative, or quantitative detection of at least one target analyte.

In certain embodiments, the systems, assays, and/or methods disclosed herein allow for simultaneous or sequential detection of multiple targets. In one embodiment, the system, assay and/or method allows for the simultaneous or sequential detection of two or more viral species or two or more strains of the same viral species.

In one embodiment, the system, assay and/or method allows for simultaneous or sequential detection of respiratory viruses and adenoviruses selected from coronaviruses (e.g., SARs-CoV-2), respiratory Syncytial Viruses (RSV), influenza viruses, and parainfluenza viruses (PIVs).

In certain embodiments, the systems, assays, and/or methods allow for simultaneous or sequential detection of viruses and bacteria. In one embodiment, the virus is a respiratory virus and the bacteria are selected from the group consisting of streptococcus pneumoniae (s.pneumoniae), haemophilus influenzae (h.influenzae), moraxella catarrhalis (Mcatarrhalis) and staphylococcus aureus (s.aureus).

Multiplexing systems, assays or methods may involve electrochemical or optical detection.

In certain embodiments, simultaneous or sequential detection is qualitative, semi-quantitative, or quantitative.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and the example section, serve to explain the principles and implementations of the disclosure.

FIG. 1 shows a schematic top view program flow chart method for adding target analytes in a sample applied to a strip with embedded chemicals that provides binding in a region of interest. As indicated by the numbering: 5 is a target; 10 is a sample pad; 20 is the conjugate pad area; 30 is a sample membrane; 40 is the test pad holding area; 50 is an absorbent pad/wicking pad; and 46 is a detectable complex or sandwich (sandwich)

FIG. 2 shows a schematic top view program flow chart method for qualitative or quantitative detection of a target analyte via electrochemical means. PB is Prussian blue.

Fig. 3 shows a picture of a prototype strip cartridge and a schematic of a prototype strip cartridge design that may be used in conjunction with an electrochemical reader.

Fig. 4 includes materials and features that may be used to modify residence time.

Fig. 5 includes materials and mixing options that may be used to prepare the sample.

FIG. 6 shows a schematic top view program flow chart method for adding target analytes in a sample applied to a strip with embedded chemicals that provides binding in a region of interest.

FIG. 7 shows a schematic top view program flow chart method for qualitative or quantitative detection of a target analyte via optical means.

Fig. 8 shows a schematic top view of an alternative physical design incorporating positive and negative controls in combination with optical detection.

Figure 9 shows the use of gel instead of flow to provide glucose addition and possible optical detection.

FIG. 10 shows a schematic top view flow chart method for qualitative or quantitative detection of target analytes via optical means and Ab-GOx-bound HRP.

Figure 11 shows the components typically used in conjunction with a cross flow device.

Fig. 12 shows a schematic of a sample integrator.

Fig. 13 shows a top view of a microfluidic strip comprising 3 electrodes and reagent addition and mixing channels for adding samples.

FIG. 14 shows a scheme and an example of electrochemical detection of H1N 1.

FIG. 15 shows electrochemical detection plots of two different SARS-CoV-2 concentrations. The three bars indicate the virus concentration [ virus ] as the number of pfu/mL, 0, 10E3 and 10E 5.

FIG. 16 shows initial selectivity data for CoV-2 for other viruses.

FIG. 17 shows a scheme and examples of colorimetric detection of H1N1

FIG. 18 shows further examples of colorimetric detection of H1N 1.

Fig. 19 shows an example of colorimetric detection of osteopontin.

Fig. 20 shows further examples of colorimetric detection of osteopontin.

Fig. 21 shows a schematic diagram of a top view of an electrochemical disposable test prototype.

Fig. 22 shows a schematic diagram of a top view of an optical disposable test prototype.

Fig. 23 shows a schematic diagram of a top view of another optical disposable test prototype.

Fig. 24 shows a schematic diagram of a top view of the fixation area.

Fig. 25 shows a schematic diagram of a top view of another fastening region.

FIG. 26 shows the final (stop) current at 10 seconds for 0 or 0.1ng/ml ALP activity in the APP/DEA buffer system relative to total incubation time.

Fig. 27 shows the cumulative current in nA according to the total measurement time and measurement time interval.

FIG. 28 shows a sandwich assay configuration for detecting IL-6.

Figure 29 shows a standard chronoamperometric detection and modified chronoamperometric detection scheme that relies on an incubation period of 5 minutes or 30 seconds intervals prior to a 10 second acquisition time.

FIG. 30 shows the cumulative current difference in the modified chronoamperometric detection according to IL-6 concentration.

FIG. 31 shows the production of MgCl ₂ With respect to the change in measured charge of 10ng/ml ALP in APP/DEA. The concentrations of 0.0, 0.1 and 2ng/ML IL-6 are shown in the current versus time plot.

FIG. 32 shows the results with respect to the presence or absence of 5mM MgCl ₂ 10mM APP in DEA or EAE versus time

Fig. 33 shows a data processing method concerning repeated timing current detection, the result of which uses a termination current at 10 seconds.

Fig. 34 shows a data processing method for repeating the timer current detection, and the result thereof uses the charge at 10 seconds.

Fig. 35 shows the data processing method for repeated timed current detection, the result of which utilizes the slope of the termination current at 10 seconds (integrated current within 10 seconds) relative to the total incubation time (300 seconds).

FIG. 36 shows a sandwich assay configuration for detecting IL-6. Complexes are formed around the IL-6 target via two antibodies, one labeled with alkaline phosphatase and the other labeled with biotin. In this example, the biotin moiety is tightly bound to streptavidin-labeled beads embedded in the membrane, which may be blocked.

FIG. 37 shows a standard chronoamperometric detection of IL-6 within 10 minutes using alkaline phosphatase as the detection enzyme.

FIG. 38 shows the timed current detection of IL-6 10 minutes after the data in FIG. 37 was collected (same electrode, suspended for 10 minutes, then over-potential reapplied within 10 minutes).

Fig. 39 shows a schematic view of a lateral flow device cartridge (top) that may be used in conjunction with an electrochemical reader (bottom).

FIG. 40 shows a method of detecting a target molecule via: 1) adding the sample to a vessel having a reagent capable of binding to the target molecule of interest in a buffer, 2) transferring the vessel contents onto a membrane (not shown washing), and 3) coupling the membrane to an electrode. The substrate is added to the membrane for subsequent electrochemical detection to provide results. This scheme facilitates electrochemical detection of the target molecule.

Figure 41 shows 2 second chronoamperometric detection of extended time intervals of nucleocapsid proteins captured on a membrane positioned on top of an electrode. Error bars depict standard deviations of three samples. Data points and error bars represent current mean and standard deviation.

FIG. 42 shows a method of detecting target molecules via first running a lateral flow immunoassay and then electrochemically detecting targets that bind to designated areas on a membrane atop an electrode covered with a substrate solution. In another embodiment, the membrane may be initially placed on the electrode. The method facilitates electrochemical detection of the target molecule.

Fig. 43 shows the results from a modified chronoamperometric detection scheme that relies on 30 second intervals and 2 second acquisition times. Error bars depict standard deviations of three samples. The line labeled "LoD" represents current values above the 0ng/mL baseline of three standard deviations.

FIG. 44 shows the results from a modified chronoamperometry for detection of IL-6 as low as 20 pg/ml. For clarity, the top plot extends the lowest concentration of 20pg/ml relative to 0; the bottom plot includes a concentration range from 500pg/ml down to 0. Error bars depict standard deviations of three samples. The line labeled "LoD" represents current values above the 0ng/mL baseline of three standard deviations.

FIG. 45 shows the slope of the chronoamperometric termination current from FIG. 44 according to the concentration of IL-6. The top plot includes a concentration range from 500pg/ml down to 0; for clarity, the bottom plot extends the lower concentration of 20pg/ml relative to 0. Error bars depict standard deviations of three samples. The line labeled "LoD" represents current values above the 0ng/mL baseline of three standard deviations.

Fig. 46 shows the results from the modified chronoamperometry for prolactin protein. Error bars depict standard deviations of three samples. The upper left panel shows the current response with respect to prolactin concentrations from 20 to 0 ng/mL. The upper right plot depicts the same enlarged data as the left plot to more closely display the 0.2, 0.1 and 0ng/mL data. The lower left and right panels show the results obtained when the slope of the termination current in nA/s over the time of detection is obtained for the range of prolactin concentrations measured (20 to 0 ng/mL). The bottom right plot is an enlarged version showing the lowest four levels. Error bars depict standard deviations of three samples. The line labeled "LoD" represents current values above the 0ng/mL baseline of three standard deviations.

FIG. 47 shows a method of detecting target molecules by running a competitive immunoassay via lateral flow, and then electrochemically detecting targets that bind to designated areas on a membrane. The method facilitates electrochemical detection of target molecules by measuring a decrease in current based on the presence of target.

FIG. 48 shows the results from a modified chronoamperometric method for detecting biotin via a competitive assay with biotin-conjugated alkaline phosphatase binding. Error bars depict standard deviations of three samples. The line labeled "LoD" represents current values above the 0ng/mL baseline of three standard deviations.

FIG. 49 shows the results from a modified chronoamperometry for detection of biotin-conjugated alkaline phosphatase as low as 80 pg/ml. Error bars depict standard deviations of three samples. The line labeled "LoD" represents current values above the 0ng/mL baseline of three standard deviations.

FIG. 50 shows the results from a modified chronoamperometry for detecting osteopontin as low as 200pg/ml using an electrode with a carbon working electrode, a carbon counter electrode, and an Ag/AgCl reference electrode. Error bars depict standard deviations of three samples. The point labeled "LoD" represents current values above the 0ng/mL baseline of three standard deviations.

FIG. 51 shows the results from an improved chronoamperometry for detecting osteopontin at 100ng/mL, 10ng/mL, 1ng/mL, 0.5ng/mL, and 200pg/mL using an electrode with a carbon working electrode, a carbon counter electrode, and an Ag/AgCl reference electrode. The measurements are shown in terms of a detection time of 300 seconds. Error bars depict standard deviations of three samples. The point labeled "LoD" represents current values above the 0ng/mL baseline of three standard deviations.

FIG. 52 shows the results from a modified chronoamperometry for detecting the theoretical maximum amount of osteopontin applied to a membrane at 10ng, 1ng, 100pg, 50pg, and 20pg using an electrode with a carbon working electrode, a carbon counter electrode, and an Ag/AgCl reference electrode. The measurements are shown in terms of a detection time of 300 seconds. Error bars depict standard deviations of three samples. The point labeled "LoD" represents current values above three standard deviations from the 0ng baseline.

FIG. 53 shows the results from an improved chronoamperometric method for detecting osteopontin at 1ng/mL, 100pg/mL using an electrode with a platinum working electrode, a platinum counter electrode, and an Ag/AgCl reference electrode, and a theoretical maximum amount of osteopontin applied to the membrane at 100pg and 10 pg. The measurements are shown in terms of a detection time of 300 seconds. Error bars depict standard deviations of three samples. The point labeled "LoD" represents current values above the 0ng/mL baseline of three standard deviations.

FIG. 54 shows the results from a chronoamperometric method for detecting SARS-CoV-2 as low as 12.9TCID50/mL using an electrode having a carbon/Prussian blue working electrode, a carbon counter electrode and an Ag/AgCl reference electrode.

FIG. 55 shows the total charge from a chronoamperometric method for detecting SARS-CoV-2 as low as 12.9TCID50/ml using an electrode having a carbon/Prussian blue working electrode, a carbon counter electrode and an Ag/AgCl reference electrode. The total charge (μcoulomb) was obtained from 30 to 300 seconds.

FIG. 56 shows the absolute value of current (|μA|) at 120 seconds from the chronoamperometry used to detect SARS-CoV-2 down to 12.9TCID50/ml using an electrode having a carbon/Prussian blue working electrode, a carbon counter electrode and an Ag/AgCl reference electrode.

FIG. 57 shows the results from a chronoamperometric method for detecting SARS-CoV-2 as low as 9pfu/ml using an electrode having a carbon/Prussian blue working electrode, a carbon counter electrode and an Ag/AgCl reference electrode. The data from FIG. 54 have been converted to pfu/mL.

FIG. 58 shows the total charge from a chronoamperometric method for detecting SARS-CoV-2 as low as 9pfu/ml using an electrode having a carbon/Prussian blue working electrode, a carbon counter electrode and an Ag/AgCl reference electrode. The total charge (μcoulomb) was obtained from 30 to 300 seconds. The data from FIG. 55 have been converted to pfu/mL.

FIG. 59 shows the results from a chronoamperometric method for detecting SARS-CoV-2 as low as 1 plaque forming unit (pfu) using an electrode having a carbon/Prussian blue working electrode, a carbon counter electrode, and an Ag/AgCl reference electrode. The theoretical maximum number of plaque forming units is applied to the membrane. The data from FIG. 57 has been converted to pfu.

FIG. 60 shows the absolute value of current at 120 seconds (|μA|) from a chronoamperometry for detecting SARS-CoV-2 as low as 1 plaque forming unit (pfu) using an electrode having a carbon/Prussian blue working electrode, a carbon counter electrode, and an Ag/AgCl reference electrode. The theoretical maximum number of plaque forming units is applied to the membrane. The data from FIG. 56 has been converted to pfu.

FIG. 61 shows the total charge from a chronoamperometric method for detecting SARS-CoV-2 as low as 1 plaque forming unit (pfu) using an electrode having a carbon/Prussian blue working electrode, a carbon counter electrode, and an Ag/AgCl reference electrode. The theoretical maximum number of plaque forming units is applied to the membrane. The total charge (μcoulomb) was obtained from 30 to 300 seconds. The data from FIG. 58 has been converted to pfu.

Detailed Description

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies illustrative embodiments. Guidance is provided through a list of examples, which may be used in various combinations, in several places throughout the application. In each case, the list serves as a representative group only and should not be construed as an exclusive list.

Disclosed herein are systems, assays, kits, and methods for determining the presence of at least one target analyte in a sample using the same. Advantageously, the disclosed systems, assays, kits and methods allow for rapid, cost-effective detection of analytes, and in certain embodiments,

I. definition of the definition

As used herein, the singular forms "a", "an", "or" the "include plural referents unless the context clearly dictates otherwise.

The term "about" as used herein in connection with any and all values (including the lower and upper ends of a range of values) refers to a range of values that fall 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 or less) of the recited reference value, unless otherwise stated or otherwise apparent from the context (unless such numbers would exceed 100% of the possible values).

As used herein, the term "administering" refers to physically introducing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration include oral, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. Administration may also be performed, for example, one time, multiple times, and/or over one or more extended periods of time.

As used herein, the term "affinity" refers to a measure of the strength of binding between a target molecule and a binding agent. Affinity is generally expressed by dissociation constant (Kd). Greater than about 10 ^-6 Any Kd of M is generally considered to be indicative of non-specific binding.

As used herein, the term "amperometric" refers to chemical titration in which a measure of the current flowing under a potential difference applied between two electrodes in a solution is used to detect an endpoint.

As used herein, the term "antibiotic" refers to a substance that inhibits bacterial growth and replication. Antibiotic compounds are generally classified as aminoglycosides, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonamides and tetracyclines.

As used interchangeably herein, the term "antibody" or "immunoglobulin" includes whole antibodies and any antigen-binding fragment (antigen-binding portion) or single-chain homolog thereof. An "antibody" comprises at least one heavy (H) chain and one light (L) chain. For example, in naturally occurring IgG, these heavy and light chains are interconnected by disulfide bonds, and there are two pairs of heavy and light chains, which are also interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain CL. VH and VL regions can be further subdivided into regions of hypervariability termed Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved, termed Framework (FR) or junction (J) regions (JH or JL, respectively, in the heavy and light chains). Each VH and VL is composed of three CDRs, three FR and one J domain, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, J. The variable regions of the heavy and light chains bind to the antigen. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) or humoral factors such as the first component of the classical complement system (C1 q). The term "antibody" is used herein in its broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

As used herein, the term "antigen" refers to an entity (e.g., a protein entity or peptide) to which an antibody binds. In certain embodiments, the antigen is a coronavirus protein (e.g., a spike protein), or a derivative, fragment, analog, homolog, or ortholog thereof, that serves as an antigen in the systems and methods disclosed herein.

The term "antigen binding region" refers to a portion of a binding agent (e.g., antibody, aptamer) that interacts with a target molecule (e.g., antigen) and imparts specificity and affinity to the binding agent for the target molecule. In embodiments herein, the capture reagent and optional detection reagent bind to an antigen binding region of at least one target analyte. In certain embodiments, the capture reagent and the detection reagent bind to a first antigen binding region and a second antigen binding region, respectively, of the target analyte.

As used herein, the term "antiviral drug" broadly refers to any anti-infective drug or therapy used to treat or ameliorate a viral infection in a subject.

As used herein, the term "aptamer" refers to an oligonucleotide (DNA or RNA) that is capable of adapting in three dimensions to bind another molecule with high affinity in the nanomolar and subnanomolar ranges. Exemplary nucleic acid molecules or polynucleotides comprising such aptamers include, but are not limited to, D-or L-nucleic acids, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose Nucleic Acids (TNAs), ethylene Glycol Nucleic Acids (GNAs), peptide Nucleic Acids (PNAs), locked nucleic acids (LNAs, including LNAs having β -D-ribose configuration, α -LNAs having α -L-ribose configuration (diastereomers of LNAs), 2 '-amino-LNAs having 2' -amino functionalization, and 2 '-amino- α -LNAs having 2' -amino functionalization, or hybrids thereof. Aptamers may be other molecules, including small molecules, proteins, nucleic acids, and even cells, tissues, and organisms (e.g., whole viruses), and may be monovalent or multivalent. The aptamers used in the disclosed embodiments may be obtained by selection from a large random sequence library using methods well known in the art, such as synthetic evolution of exponentially enriched ligands (Synthetic Evolution of Ligands by Exponential Enrichment) (SELEX).

The term "assay" as used herein refers to an analytical procedure for qualitatively evaluating or quantitatively measuring the presence, amount or functional activity of a target analyte. In certain embodiments, the assays disclosed herein are not or are not expected to be foldable. In certain embodiments, the assays disclosed herein do not include a mixing chamber. In certain embodiments, the assays disclosed herein do not comprise a conductive polymer.

As used herein, the term "array" means a plurality of different sites carrying different capture reagents. In certain embodiments, the assay components of the assays, systems, and methods described herein comprise an array.

As used herein, the term "binding agent" refers to a molecule that binds (including hybridizes) with high affinity and high specificity to cognate ligands. Binding reagents are commonly used to identify the presence of their cognate ligands and may be detectably labeled to allow identification. Binding reagents bind with high affinity and high specificity to their target analytes. Examples of binding reagents include, for example, aptamers, antibodies, antibody fragments, antibody mimics, aptamers, affimer, quenchbody, receptor ligands, or molecularly imprinted polymers. In certain embodiments, the binding reagent may be associated, i.e., coupled, linked or linked, to a solid support, e.g., a test strip or bead.

As used herein, the term "binding pair" refers to a pair of molecules that bind to each other with high affinity and specificity. "binding pair member" refers to a molecule of a binding pair. For example, streptavidin and biotin (or biotin analogues) are binding pair members that are non-covalently bound to each other.

As used herein, the term "binding affinity" refers to the propensity of a binding agent to bind or not bind to a target, and describes a measure of the binding strength or affinity of the binding agent to bind to the target molecule.

As used herein, the term "biomarker" generally refers to a molecule associated with a biological change or quantification or characterization. Examples of biomarkers include polypeptides, proteins, or fragments of polypeptides or proteins; and polynucleotides, such as gene products, RNAs or RNA fragments, or encoding polynucleotides, hormones, small molecules; as well as other bodily metabolites. In certain embodiments, a "biomarker" means a small molecule compound that is differentially present (i.e., increased or decreased) in a biological sample from a subject or group of subjects having a first phenotype (e.g., having a disease or condition) compared to a biological sample from a subject or group of subjects having a second phenotype (e.g., not having a disease or condition or having a less severe form of a disease or condition).

As used herein, the term "biosensor" refers to an analytical device that includes a biological detection element and a transducer. Various types of biosensors are known in the art. Electrochemical biosensors are based on enzymatic catalytic reactions that consume or generate electrons and include, for example, amperometric biosensors, potentiometric biosensors, impedance biosensors, and voltammetric biosensors.

The term "buffer" refers to a liquid suitable for supporting a binding reaction between a binding reagent and a target analyte. During the incubation, the sample suspected of containing the target analyte or analytes, the buffer and possibly other liquids form a liquid phase.

The term "calibrated" or "correlated" refers to the level of a target analyte or fragment thereof in a biological sample of a subject that has a statistically significant correlation with a physiological state, such as a disease state or extent of disease, response to treatment, and survival. The strength of the correlation between the level of the target analyte or fragment thereof and the presence or absence of a particular physiological state can be determined by statistical testing for significance.

As used herein, the terms "camera," "photodetector," and the like refer to a component that is capable of detecting light intensity or composition to cause data, such as an image, of the detected light. The terms "camera" and "photodetector" may also refer to any type of detector, including RGB detectors or spectrophotometers.

As used herein, the term "capture reagent" refers to a reagent that is capable of binding to and capturing a target analyte in a sample. Typically, the capture reagent is immobilized or fixable (e.g., not immobilized at the time of capture, but immobilized thereafter). In a sandwich immunoassay, the capture reagent may be any binding reagent, such as an aptamer or an antibody.

As used herein, the term "colorimetric" refers to the physical description and quantification of a colored spectrum, including a human color perception spectrum (e.g., the visible spectrum). In some embodiments, colorimetric assays are particularly useful when quantification is not necessary. In certain embodiments, detection of the color change may be performed by visual inspection by a user (e.g., the person performing the assay), while in other embodiments, a detection device is desired. In some embodiments, calibrated colorimetric measurements may be used to quantitatively determine target amounts.

As used herein, the term "colorimetric material" refers to a material that can produce a detectable change based on one or more substances in contact with the material. The detectable change may include a change in visibility, such as a change in color, optical transmittance, or a change in the intensity or wavelength of emitted fluorescence or chemiluminescence.

As used herein, the term "chronoamperometry" refers to electrochemical measurement techniques for electrochemical analysis or for determining the kinetics and mechanism of electrode reactions. A rapidly rising potential pulse is applied to an active (or reference) electrode of an electrochemical cell and the current flowing through the electrode is measured by a chronoamperometer over time. The chronoamperometric methods disclosed herein may be standard or modified in some manner (e.g., long pulses, short intervals, or repeated pulses).

As used herein, the term "cross-link" refers to a bond that connects one polymer chain to another polymer chain. These linkages may take the form of covalent or ionic bonds, and the polymer may be a synthetic polymer (e.g. polyethylene terephthalate) or a natural polymer (e.g. protein).

As used herein with respect to an assay, the term "competitive" refers to an assay in which the number of binding sites is limited, resulting in competition for binding between the endogenous analyte and the detectable labeled analog. Thus, the amount of bound labeled analog is inversely proportional to the amount of analyte in the sample. As the amount of analyte in the sample increases, the detectable signal decreases. Competitive assays can be categorized as simultaneous (where all components are added at once) or sequential (where the sample is incubated with the antibody prior to the addition of the labeled analog). In contrast, non-competitive immunoassays are designed to have an excess of binding sites and produce a signal that is proportional to the amount of analyte in the sample. In one embodiment, the assays disclosed herein are sequential competitive assays. In another embodiment, the assays disclosed herein are simultaneous addition of a competitive assay.

As used herein, the term "complex" refers to an entity comprising more than one molecule that is bound or related to at least one other molecule, e.g., by chemical correlation. Thus, the term "matrix-aptamer-target molecule complex" relates to the association between matrix, aptamer and target molecule. The term "biotinylated second binding reagent streptavidin (or" b-binding reagent-SA complex ") relates to the correlation between biotin, the second binding reagent, and streptavidin.

As used herein, the term "control element" refers to an element that is used to provide information about the function of an assay, such as binding specificity, the level of non-specific background binding, the degree of binding cross-reactivity, and the performance of assay reagents and detection systems. Preferred controls useful herein include at least one negative control that monitors background signals, at least one negative control that monitors assay specificity, at least one positive colorimetric control, and at least one positive control that monitors assay performance.

As used herein, the term "cross-reactive" refers to the ability of a binding agent (e.g., aptamer, antibody) to a target analyte to successfully bind to a different molecule (i.e., a non-target molecule). The degree of cross-reactivity may vary. In certain embodiments, the target analyte and the non-target analyte share a common epitope, i.e., a feature that is highly conserved across species.

As used herein, the term "cut point" refers to a threshold value used to distinguish between a negative response and a positive response in an assay. It is a statistically determined constant value by analyzing the measured response of a group of diseased human samples that have not been exposed to the drug.

As used herein, the term "cytokine" refers to a class of immunomodulatory proteins, peptides, or glycoproteins. In certain embodiments, the systems, assays, and methods described herein can be used to detect cytokines, such as pro-inflammatory cytokines, e.g., interleukin-8 (IL-8), interleukin-6 (IL-6), interleukin-1 (IL-1), interleukin-11 (IL-11), interleukin-17 (IL-17), interleukin-18 (IL-18), interferon-alpha (IFN-alpha), interferon-beta (IFN-beta), interferon-gamma (IFN-gamma), G-CSF, tumor necrosis factor alpha (TNF-alpha), or tumor necrosis factor beta (TNF-beta).

As used herein, the term "label" or "detectable label" refers to any molecule that produces or can be induced to produce a detectable signal. Non-limiting examples of labels include radioisotopes, enzymes, enzyme fragments, enzyme substrates, enzyme inhibitors, colorimetric labels, coenzymes, catalysts, fluorophores, dyes, chemiluminescent agents (chemiluminescers), luminescent agents (luminescers), or sensitizers; non-magnetic or magnetic particles, solid supports, liposomes, ligands or receptors.

As used herein, the term "detection device" refers to any device suitable for detecting a signal generated in the presence of a target analyte. Representative non-limiting detection devices include current devices, coulomb devices, potential devices, and voltammetric devices. In certain embodiments described herein, the detection device is a portable or hand-held device, and in certain embodiments is a blood glucose meter.

As used herein, the term "diagnosis" refers to the identification and (early) detection of a disease or clinical condition in a subject, and may also include differential diagnosis. In addition, in certain embodiments, the assessment of the severity of a disease or clinical condition may be encompassed by the term "diagnostic μ. In some embodiments, the term "diagnosis" encompasses the assessment of the severity of a disease or condition. Certain systems, assays, and methods used herein provide a user with a diagnosis or information about the result that is transmitted to a third party, allowing the third party to provide or confirm the diagnosis.

As used herein, the term "drug" refers to a substance (e.g., a small molecule) that is used to treat or prevent a disease, or to improve the manifestation of a disease, including but not limited to side effects and associated risk factors and co-diseases. Also included in this definition are substances that are being developed for treating or preventing a disease, or improving the manifestation of a disease.

As used herein, the term "drop casting" refers to a process in which a solid film is formed by dropping a solution onto a flat surface, followed by evaporation of the solution.

As used herein, the term "electrode" refers to any medium capable of transporting charge (e.g., electrons) to and/or from a storage molecule. Representative electrodes are metal or conductive organic molecules. In certain embodiments, the electrode comprises gold, silver, copper, platinum, aluminum, stainless steel, tungsten, indium tin oxide, titanium, lead, nickel, silicon, polyimide, parylene, benzocyclobutene, carbon, graphite, or any combination thereof. The electrodes can be fabricated in virtually any 2-dimensional or 3-dimensional shape (e.g., discrete lines, pads, planes, spheres, cylinders, etc.). In certain embodiments, the electrodes may be screen printed. The electrodes may be analyte-specific electrodes, positive control electrodes, negative control electrodes, counter electrodes, reference electrodes, and the like. The term "analyte-specific electrode" refers to an electrode that is coated or otherwise functionalized with a binding reagent. In certain embodiments, the electrode utilized in the assays and/or systems disclosed herein is not an oxide electrode. In certain embodiments, the systems and assays disclosed herein include a "wake-up" electrode to facilitate electronic engagement or measurement.

As used herein, the term "electrochemical system" refers to a system that determines the presence and/or amount of a redox analyte by measuring an electrical signal (e.g., an electrical potential induced by a redox reaction or from ion release or absorption) in a solution between a working electrode and a counter electrode. Redox reactions refer to the loss of electrons (oxidation) or the acquisition of electrons (reduction) that a material undergoes during an electrical stimulus, such as the application of an electrical potential. The redox reaction occurs at the working electrode and for chemical detection the working electrode is typically composed of an inert material such as platinum or carbon. The potential of the working electrode is measured against a reference electrode, which is typically a stable, well performing electrochemical half-cell, such as silver/silver chloride. Electrochemical systems can be used to support many different techniques for determining the presence and concentration of a target biomolecule, including but not limited to various types of voltammetry, amperometry, potentiometry, coulometry, conductometry, and conductivity analysis such as AC voltammetry, differential pulse voltammetry, square wave voltammetry, electrochemical impedance spectroscopy, anodic stripping voltammetry, cyclic voltammetry, and fast scanning cyclic voltammetry. The electrochemical system may further comprise one or more negative control electrodes and a positive control electrode. In the context of the present invention, a single electrochemical system may be used to quantify more than one type of analyte.

As used herein, the term "environmental sample" encompasses a wide variety of sample types, wherein

The term "epitope" or "antigenic determinant" is used interchangeably herein and refers to a portion of a molecule, such as an antigen, that is capable of being recognized and specifically bound by a particular binding agent (e.g., an antibody or aptamer). When the antigen is a polypeptide, the epitope may be formed by both contiguous amino acids as well as non-contiguous amino acids that are abutted by tertiary folding of the protein. Epitopes formed by contiguous amino acids are typically retained after protein denaturation, whereas epitopes formed by tertiary folding are typically lost after protein denaturation. Epitopes typically comprise at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation. An epitope may compete with the intact antigen (i.e., the "immunogen" used to elicit an immune response) for binding to an antibody. The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site (referred to as a paratope) in the variable region of an antibody molecule. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects.

As used herein, the term "false negative" refers to a sample that is erroneously identified as being free of one or more analytes, such as a virus.

As used herein, the term "false positive" refers to a sample that is erroneously identified as containing one or more analytes, such as a virus.

As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to a full-length polypeptide or polynucleotide (length n). The length of the fragment may be appropriately changed according to the purpose thereof. In the case of polypeptides, examples of the lower limit of the length thereof include 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more amino acids, and a length represented by an integer (e.g., 11) not specifically listed herein may also be suitable as the lower limit. In addition, in the case of polynucleotides, examples of the lower limit of the length thereof include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 200, 300, x400, 500, 600, 700, 800, 900, 1000 and more nucleotides, and a length represented by an integer (e.g., 11) not specifically listed herein may also be appropriate as the lower limit. In certain embodiments, in the systems, assays, and methods described herein, the target analyte is a fragment, e.g., a protein or nucleic acid fragment.

The term "blood glucose meter" has been used herein to refer to a medical device that is typically used by diabetics to self-monitor blood glucose levels. Many blood glucose meters use electrochemical methods based on a test medium, such as a test strip. The test strip is a consumable element containing chemicals that react with glucose in one drop of blood used for each measurement in the context of diabetes monitoring. Specifically, a chemical reaction is produced, and the meter reads the glucose level in mg/dl or mmol/l. Although specialized blood glucose meters are known, blood glucose meters are typically portable and used at home.

As used herein, the term "glucose" refers to monosaccharides, commonly hexoses.

As used herein, the term "high affinity" refers to at least 10 ^-8 M, about 10 ^-8 M to about 10 ^-12 M, or more particularly about 10 ^{- 8} M, about 10 ^-9 M; about 10 ^-10 M, about 10 ^-11 M, or about 10 ^-12 Binding affinity of M.

As used herein, the term "hormone" refers to a chemical substance that controls and regulates the activity of certain cells or organs. Hormones can be classified as lipid-derived, amino acid-derived, and peptide-derived. In certain embodiments, the assays, systems, and methods disclosed herein are suitable for detecting lipid-derived (i.e., steroid) hormones including testosterone, estrogen, and progesterone. In certain embodiments, the assays, systems and methods disclosed herein are suitable for detecting prolactin, a protein hormone.

As used herein, the term "immobilized" refers to a molecule (e.g., a binding reagent or analyte) that is reversibly and irreversibly immobilized.

As used herein, the term "instructional material" includes publications, records, illustrations, or any other expression medium that can be used in kits to convey the usefulness of the compositions and/or compounds of the invention. The instructional materials of the kit may, for example, be attached to or carried along with a container containing the compounds and/or compositions of the invention. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient cooperatively use the instructional material and the compound. The delivery of the instructional material may be, for example, physical delivery through publications or other expression media that convey the usefulness of the kit, or alternatively may be accomplished by electronic transmission, for example, by means of a computer, for example, through email, or download from a website.

As used herein, the term "isolated", "purified" or "biologically pure" refers to a material that is substantially or essentially free of components that normally accompany it as found in its natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. It is a protein of the predominant species present in the formulation that is substantially purified. In certain embodiments, the enzyme-labeled capture reagent and/or detection reagent has a purity of about 10% to about 90% or more, more specifically about 10% or more, about 30% or more, about 50% or more, about 70% or more, about 85% or more, about 90% or more, or more specifically about 92%, about 95%, about 97% or about 99% or more.

As used herein, the term "Kd" refers to the equilibrium dissociation constant of a particular binding agent-target molecule interaction. In certain embodiments herein, the Kd of the capture reagent, the detection reagent, or both is about 10 ^-10 Kd. About 10 ^-8 Kd or about 10 ^-6 。

As used herein, the term "kit" refers to a collection of items intended for use together. The items in the kit may or may not be operably connected to each other. The kit may comprise, for example, an antibody or antigen binding fragment as disclosed herein, optionally attached to a solid support, as well as reagents for performing the assay and control reagents. Typically, the items in the kit are contained in a primary container, such as a vial, tube, bottle, box or bag. The separate items may be contained in their own, separate containers or in the same container. The articles in the kit or the primary container of the kit may be assembled into a secondary container, such as a box or bag, which is optionally suitable for commercial sale, such as for shelving, or for transport by public transport means, such as mail or delivery service.

As used herein, the term "labeled" refers to a molecule that produces or can be induced to produce a detectable signal. The label may be conjugated to an analyte, an immunogen, an antibody or another molecule. Non-limiting examples of labels include radioisotopes, enzymes, enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes, catalysts, fluorophores, dyes, chemiluminescent agents, luminescent agents, or sensitizers; non-magnetic or magnetic particles, solid supports, liposomes, ligands or receptors.

As used herein, the term "lateral flow assay" or "LFA" refers to an assay that can be used to identify at least one target analyte in a sample. The general format of LFA is similar to ELISA. Lateral flow technology is well suited for point-of-care (POC) disease diagnosis because it is robust and inexpensive, requiring no electrical power, cold chains for storage and transportation, or specialized reagents. The LFA device may comprise a solid matrix capable of supporting a test and made of a material, such as nitrocellulose, that can absorb a liquid sample and promote capillary action of the liquid sample along the solid support. The solid support may be of any shape or size, one common size being a strip that can be held in the hand. The lateral flow assay may have more than one test line for multiplexed testing of multiple target reagents and is one embodiment of a "multiplexed" assay or system. As used herein, the term "lateral flow" refers to capillary flow through a material in a horizontal direction, but is understood to apply to flow of liquid from a liquid application point to another lateral location, even if, for example, the device is vertical or inclined. Lateral flow depends on the nature of the liquid/matrix interaction (surface wetting or wicking) and does not require or involve the application of external forces, such as by a user's vacuum or pressure. By "capillary flow" is meant a flow of a liquid in which all dissolved or dispersed components of the liquid flow through the membrane at substantially equal rates and in a relatively undamaged lateral direction, as opposed to preferential retention of one or more components, for example, as occurs in materials capable of adsorbing or absorbing one or more components.

As used herein, the term "layman" means a subject lacking significant or any clinical training.

As used herein, the term "limit of detection" or "LOD" refers to the lowest analyte concentration under which detection is feasible. LOD is determined by repeating both the test with measured LOD and a sample known to contain a low concentration of analyte. In some examples, LOD is determined by testing serial dilutions of a sample known to contain the analyte and determining the lowest dilution at which detection occurs.

The term "limit of quantitation" or "LOQ" refers to the lowest concentration under which not only the analyte can be reliably detected, but also some predetermined objective regarding bias and inaccuracy is met.

The terms "measuring" and "determining" are used interchangeably throughout and refer to a method comprising obtaining a patient sample and/or detecting the level of a biomarker in a biological sample. In one embodiment, these terms refer to obtaining a patient sample and detecting the level of one or more biomarkers in the sample. In another embodiment, the terms "measure" and "determine" mean detecting the level of one or more biomarkers in a biological sample. The term "measurement" is also used interchangeably throughout with the term "detection".

As used herein, the term "molecule" is used broadly to refer to a natural, synthetic, or semi-synthetic molecule or compound.

The term "monitoring" as used herein with respect to a disease or disorder refers to keeping track of the disease, disorder, complication, or risk that has been diagnosed, e.g., to analyze the progression of the disease or the effect of a particular treatment on the progression of the disease or disorder.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

The term "multiplexing" as used herein with respect to an assay refers to the simultaneous or sequential use and/or testing of multiple target analytes in a single assay. In certain embodiments, the systems, assays, and methods disclosed herein allow a user to detect more than one virus species or more than one strain of the same virus species. In certain embodiments, the systems, assays, and methods disclosed herein allow a user to detect more than one bacterial species or more than one strain of the same bacterial species. In certain embodiments, the systems, assays, and methods disclosed herein allow a user to detect viruses or bacteria, i.e., to distinguish between viral or bacterial causes of an infection, such as an upper respiratory tract infection. For example, in order to distinguish infection by SARS-CoV-2, parainfluenza, rhinovirus, influenza A virus or influenza B virus. In one example, to distinguish an infection caused by a particular subtype of influenza a from another particular subtype of influenza a, for example, influenza a H1 subtype and influenza a H3 subtype. In a further example, to detect a coronavirus of significant pathogenicity from a less pathogenic coronavirus. In other embodiments, the multiplexing system, assay or method allows for the detection of different immunoglobulins.

As used herein, the term "mutation" refers to an alteration in the amino acid sequence of a native protein. Mutations can be described by using the natural sequence and then identifying the specific acid that has been altered. "mutant" or "variant" refers to a protein that contains a mutation. Full length mutant sequences refer to the complete amino acid sequence of a mutant protein, rather than describing the mutant as being amino acids that differ from the native protein. In certain embodiments, the systems, assays, and methods used herein can be used to detect two or more viruses, wherein the viruses are closely related variants.

As used herein, the term "native protein" refers to a protein in its natural or native state that has not been altered by any denaturing agent, such as heat, chemical mutation, or enzymatic reaction.

As used herein, the term "non-target molecule" refers to a molecule that is not a biomarker of interest. In particular, the non-target molecule may be a molecule that is structurally similar to the biomarker of interest. In certain embodiments, the systems, assays, and methods used herein can be used to distinguish between target analytes and non-target molecules, i.e., to detect the presence of a target analyte and not the presence of a non-target molecule, even when both are present in the same sample.

As used herein, the term "nucleic acid" refers to single-or double-stranded deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and any chemical modifications thereof. The nucleic acid detected according to the systems, assays and/or methods disclosed herein can be a full-length nucleic acid or a fragment thereof.

As used herein, the term "oxidase" refers to an enzyme that belongs to the class of oxidoreductases and that catalyzes a redox reaction using molecular oxygen as an electron acceptor, resulting in the formation of water (H2O) or hydrogen peroxide (H2O 2) as a byproduct. This is in contrast to dehydrogenases which transfer hydrogen to NAD, NADP or flavins in order to oxidize a substrate. The reductase may be an oxidase, as most redox reactions are reversible.

As used herein, the term "oxidoreductase" refers to an enzyme that catalyzes the transfer of electrons from one molecule (reducing agent, also referred to as an electron donor) to another molecule (oxidizing agent, also referred to as an electron acceptor). Oxidoreductases can be classified into different subtypes, including oxidases, dehydrogenases, reductases, peroxidases, hydroxylases and oxidases.

As used herein, the term "pathogen" means any pathogenic agent including, but not limited to, viruses or bacteria, fungi, protozoa, or other microorganisms. Replication pathogens (e.g., viruses, parasites and bacteria) are organisms that replicate by using the resources of the organism while largely avoiding the immune response of the organism causing the disease.

As used herein, the term "pesticide μmeans a chemical for killing pests. Pesticides are generally classified as fungicides, herbicides, insecticides, and rodenticides.

As used herein, the term "point-of-care test" or "POCT" refers to the measurement of a biological sample at or near a patient, assuming that test results will be obtained immediately or within a very short time frame to aid in immediate diagnosis and/or clinical intervention by a caregiver. (Ehrmeyer SS et al (2007) Clin Chem Lab Med 45:766-773). The term is not intended to be limited to patient and home use, but includes various environments (e.g., communities, clinics, peripheral laboratories, and hospitals) and users (e.g., technicians and caregivers). Depending on the circumstances and the user, the purpose of POCT may vary-from triage and triage to diagnosis, treatment and monitoring. In the context of environmental samples, a similar concept is in-situ testing, i.e. testing at the sample collection site.

As used herein, the term "potentiostat" is a broad term and is used in its ordinary sense, including but not limited to an electrical system that controls the potential between the working electrode and the reference electrode of a three-electrode battery to a preset value. As long as the required cell voltage and current do not exceed the compliance limits of the potentiostat, it forces any necessary current to flow between the working and counter electrodes to maintain the required potential. The double potentiostat and the multi-potentiostat are potentiostats capable of controlling two working electrodes and more than two working electrodes, respectively

As used herein, the term "predetermined threshold value" refers to a threshold value below which the classifier gives a desired balance between (costs of) false negatives and false positives. In some embodiments, the "predetermined threshold" is determined, refined, adjusted, and/or validated statistically (and clinically) by, with respect to, or based on: clinical studies and their analysis of results (collectively, "clinical data") and/or preclinical or non-clinical studies (collectively, "non-clinical data") in order to minimize adverse effects of false positives and false negatives.

As used herein, the terms "prevent", "prevention" or "prevention" refer to inhibiting the manifestation of a pathological condition, such as a symptom or indication of a pathology, such as a symptom or indication of a viral infection.

As used herein, the term "processor" is used broadly to refer to a programmable or non-programmable processing device, such as a microprocessor, microcontroller, application Specific Integrated Circuit (ASICS), programmable Logic Devices (PLDs), field Programmable Gate Arrays (FPGAs), or the like. The term "processor" may also include multiple processing devices that work in concert with each other.

As used herein, the term "point mutation" refers to the modification of a polynucleotide that results in the expression of an amino acid sequence that differs from an unmodified amino acid sequence in terms of substitution or exchange, deletion or insertion of one or more single (discontinuous) amino acids or amino acid diads with respect to different amino acids.

The terms "protein," "peptide," and "polypeptide" are used interchangeably herein to refer to an amino acid polymer or a set of two or more interacting or binding amino acid polymers. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers. The protein detected according to the assays, systems and/or methods disclosed herein may be a full-length protein or a protein fragment. In a particular embodiment, the target analyte detected according to the systems, assays and methods disclosed herein is a nucleocapsid (N) protein of a coronavirus and more particularly SARS-CoV-2 or variants thereof. In another particular embodiment, the target analyte detected according to the systems, assays and methods disclosed herein is osteopontin, an integrin binding glycoprotein.

The term "pulse" refers to a burst of current, voltage or electromagnetic field energy. The pulses may last from fractions of nanoseconds up to seconds or even minutes.

The term "quantitative" as used herein with respect to the methods and systems described herein refers to information about the concentration of an analyte relative to a reference (control), which may be reported digitally, wherein a "zero" value may be specified in which the analyte is below the detection limit. "semi-quantitative" methods and systems involve the presentation of a digital representation of the amount of analyte in a sample relative to a reference (e.g., a threshold, such as a normal threshold or an abnormal threshold), where a "zero" value may be specified if the analyte is below a detection limit. In general, semi-quantitative results are compared against accompanying references to provide a qualitative interpretation of the results. In certain embodiments, the systems, assays, and methods disclosed herein allow for quantitative or semi-quantitative results.

As used herein, the term "rapid diagnostic test" refers to a system or assay for testing a sample that may be performed at the point of care or at the user's location (e.g., home, office, site) to obtain rapid diagnosis. Rapid diagnostic tests are quick and easy to perform and may even be performed in the absence of laboratory techniques such as microscopy, enzyme-linked immunosorbent assay (ELISA) or Polymerase Chain Reaction (PCR). As a non-limiting example, rapid diagnostic tests generally require about 30 minutes or less (e.g., about 10 minutes or less, about 2 minutes or less, about 1 minute or less) from the time of sample collection to the time of obtaining a result. It should be noted that the time required for a rapid diagnostic test depends on variables such as the type of sample, the amount of sample, the nature of the analyte, etc.

As used herein, the term "reference value" may be a "threshold" or a "cutoff value. In general, the "threshold" or "cutoff value" may be determined experimentally, empirically, or theoretically.

As used herein, the term "reporter reagent" refers to a reagent that is a component of a dual detection strategy, wherein the reporter reagent may be a labeled detection reagent or reagent (e.g., an enzyme) in solution.

As used herein, the term "risk" refers to the probability of an event occurring within a particular period of time, and may refer to the subject's "absolute" risk or "relative" risk. The absolute risk may be measured with reference to actual observations after measurements on the time-series, or with reference to index values developed from a statistically valid historical series of tracked time-series. Relative risk refers to the ratio of the absolute risk of a subject compared to the absolute risk of a low risk cohort or average population risk, which may vary depending on the manner in which the clinical risk factor is evaluated. Ratio (ratio of positive to negative events for a given test result) is also common (ratio is calculated according to the formula p/(1-p), where p is the probability of an event and (1-p) is the probability of no event) to no transition. Alternative continuous measurements may be evaluated in the context of the present invention.

As used herein, the term "selectivity" refers to the ability of a system, assay, or method to distinguish between specific analytes in a complex mixture without interference from other components.

As used herein, the term "sensor" refers to a device for detecting at least one target analyte. A "sensor system" includes, for example, elements, structures, and architectures that are intended to facilitate sensor use and function. The sensor system may include, for example, compositions such as those having selected material properties, as well as electronic components such as components and devices (e.g., current detectors, monitors, processors, etc.) for signal detection and analysis.

As used herein, the term "small molecule" refers to a low molecular mass (or molecular weight) (e.g., 2000 g/mole). The small molecules may be organic or inorganic, or metal organic. Examples of small molecules include drugs (e.g., therapeutic drugs, drugs of abuse), heavy metals, hormones and growth promoters, molecular markers, pesticides and toxins. The term "small molecule" refers to a molecule, for example, having a molecular weight of about 150 to about 2,000, or about 150 to about 1,500, or about 150 to about 1,000, or about 150 to about 500, or about 300 to about 2,000, or about 300 to about 1,500, or about 300 to about 1,000, or about 500 to about 2,000, or about 500 to about 1,500, or about 500 to about 1,000.

As used herein, the term "solid support" refers to a solid material to which a binding agent can attach. Exemplary solid supports include, but are not limited to, beads or particles (e.g., made of agarose gel), microtiter plates, microchips, filters, membranes, or fibers such as microfibers.

The terms "specifically bind," "selectively bind," and "selectively bind" mean that the binding agent (e.g., antibody, aptamer) exhibits significant affinity for the target molecule and generally does not exhibit significant cross-reactivity with the non-target molecule, which in certain embodiments means having a molecular weight of at least about 1x10 ^-8 Equilibrium dissociation constant of M or lowerA number (e.g., a smaller Kd indicates a tighter bond). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis or surface plasmon resonance. In certain embodiments described herein, the capture reagent and optional detection reagent specifically bind to at least one target analyte.

As used herein, the term "sensitivity" refers to the proportion of positive that is positively identified (e.g., the percentage of positive personnel identified by the system or method). In highly sensitive systems or methods, false negatives are limited.

As used herein, the term "specificity" refers to the proportion of positive identified negatives. In highly specific systems or methods, false positives are limited.

As used herein, the term "screen printing" refers to a technique that includes printing different types of inks on a substrate. The ink composition may vary and include, for example, carbon, silver, gold, and platinum. Screen printing allows high quality disposable electrodes to be produced repeatedly at low cost. Other printing methods or other methods of forming electrodes are known in the art.

The term "subject" refers to a mammal, such as a human. In certain embodiments, the subject is suspected of having, is currently having, is recently recovering from, or is at risk of contracting a disease or disorder (e.g., a viral infection).

As used herein, the term "system" refers to a set of objects and/or devices that form a network for performing a desired objective

As used herein, the term "system noise" refers to, but is not limited to, unwanted electronic noise or diffusion-related noise, which may include, for example, gaussian noise, motion-related noise, flicker noise, kinetic noise, or other white noise. In certain embodiments described herein, the system has reduced noise as compared to detection systems known in the art. In certain embodiments, the noise is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 45%, about 50% or more.

As used herein, the term "target" is a broad term that is used to refer to a substance or chemical constituent in a fluid (e.g., biological fluid) or an environmental sample (e.g., water, oil, fuel, mud, sediment, mold, combinations thereof, etc.). The target may be naturally occurring or may be a foreign substance. The target may be a toxin, may be a catalyst, an additive, or the like.

As used herein, the term "target analyte" refers to a molecule or other analyte that can be found in a test sample and that is capable of binding to a binding reagent. In certain embodiments, the target analyte is a pathogenic organism (e.g., a virus or bacterium), a protein, a peptide, a hormone, a steroid, a vitamin (e.g., biotin), a small molecule (e.g., a drug intermediate), an organic compound, or a toxin. In certain embodiments, the target analyte detected by the assays, systems, and methods disclosed herein is not nicotine. In certain embodiments, the target analyte detected by the assays, systems, and methods disclosed herein is not RNA. In certain embodiments, the target analyte detected by the assays, systems, and methods disclosed herein is not an oxidase microbial oxidoreductase (MRE).

As used herein, the terms "treatment" and "treatment" refer to preventing, inhibiting, and alleviating conditions and symptoms associated with a disorder or disease.

As used herein, the term "therapeutically effective amount" refers to that amount of an active compound or pharmaceutical agent (e.g., antiviral drug) that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes preventing, ameliorating or reducing the symptoms of the disease or disorder being treated. Methods for determining a therapeutically effective dose for the pharmaceutical compositions of the present invention are known in the art. In certain methods described herein, the method comprises administering to the subject a therapeutically effective amount of at least one approved therapeutic agent.

The term "dual binding reagent assay" refers to a further incubation of a target analyte attached to a first binding reagent that binds to a substrate in the presence of a second binding reagent that is associated with a chemically reactive group. Incubation of the two binding reagents may be simultaneous.

As used herein, the term "variant" is a relative term that describes the relationship between a polypeptide of particular interest and a "parent" or "reference" polypeptide to which its sequence is compared. A polypeptide of interest is considered to be a "variant" of a parent polypeptide or reference polypeptide if it has an amino acid sequence equivalent to the parent, except for a small number of sequence changes at a particular position. Variants include, for example, substitution variants, insertion variants, or deletion variants. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted compared to the parent. In some embodiments, the variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residues compared to the parent. Often, variants have a very small number (e.g., less than 5, 4, 3, 2, or 1) of substituted functional residues (i.e., residues involved in a particular biological activity). Furthermore, variants typically have no more than 5, 4, 3, 2, or 1 additions or deletions compared to the parent, and often do not have additions or deletions. Further, any addition or deletion is typically less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6 residues, and typically less than about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent polypeptide or reference polypeptide is a polypeptide found in nature. As will be appreciated by one of ordinary skill in the art, multiple variants of a particular polypeptide of interest may generally be found in nature, particularly when the polypeptide of interest is an infectious agent polypeptide. In a particular embodiment, the variant is a viral protein (e.g., spike protein) which is similar in particular in its function to a reference viral protein, but has a mutation in its amino acid sequence which makes it different in sequence from the wild-type viral protein at one or more positions. In the context of SARS-CoV-2, the "wild-type" genome has been sequenced and is known in the art (see, e.g., wu et al (2020) cell Host & Microbe 27 (3): 325-328; wang, H et al (2020): eur J Clin Microbiol Infect Dis, 1629-1635). Thus, "SARS-CoV-2 variant" includes variants that are currently present, as well as variants that may occur or be discovered in the future.

The term "vertical flow assay" (also referred to as flow-through assay) refers to an assay in which a liquid sample flows vertically in the assay, in contrast to the lateral flow in the LFA. One embodiment replaces the conventional cross-flow section in a stacked manner (e.g., stacked membranes) that allows liquid to diffuse from the bottom layer to the top layer. See, e.g., E.Eltzov, biosens.Bioelectron.,87 (2017), pages 572-578. Another embodiment pushes the reagents stepwise across a single membrane and allows the target to react with the reagents on the membrane. Multiplexing is achieved in a vertical flow assay by providing captured antigens for different antigens at predetermined locations (spatial multiplexing) and/or patterns on a solid support, such as a polymer membrane. Advantageously, the vertical flow assay described herein does not require a syringe pump for fluid handling or a bench-top readout for assay analysis. In certain embodiments, the vertical flow assays described herein avoid diffusion limiting kinetics and exhibit significantly reduced assay times compared to conventional assays.

As used herein, the term "viral species" refers to a single family of viruses whose characteristics can be distinguished from those of other species by multiple criteria.

As used herein, the term "wild-type" refers to a protein or nucleic acid in its native full-length form, as found in nature. As used herein, the term full-length native protein sequence refers to the amino acid sequence found in a full-length native protein. Wild-type proteins may be obtained, for example, from biological samples.

As used herein, the term "whole virus" refers to a virus agent that is intact or largely intact. In certain embodiments, the methods and systems disclosed herein are not used to detect or quantify whole virus particles, but rather to detect or quantify specific viral proteins. In one embodiment, the methods and systems disclosed herein are used to detect or quantify soluble viral proteins.

For any of the methods disclosed herein including discrete steps, the steps may be performed in any order possible. Also, any combination of two or more steps may be performed simultaneously, as appropriate.

I. System and assay

Disclosed herein are systems and assays (sensors) for detecting at least one target analyte in a sample, such as a fluid sample. In certain embodiments, the sample is treated but not extracted. The sample may contain one or more target analytes (e.g., one, two, three, four, five, six, seven, eight, nine, or ten target analytes, or more).

The system and assay are suitable for rapid diagnostic testing, which is relatively less time consuming and labor intensive than conventional methods, and in certain embodiments, the ease of processing and interpretation of results enables testing outside of conventional environments (e.g., in the home or in the field as opposed to laboratory or clinical environments) by relatively untrained users (e.g., laypersons). In certain embodiments, the systems and assays disclosed herein are intended for a single user. Advantageously, the system and assay allow for relatively low limit of detection (LOD) and high accuracy, as further described herein. In certain embodiments, the systems described herein utilize a modified chronoamperometric method to allow for faster and/or more sensitive measurements (e.g., provide lower LOD).

In one embodiment, a system for detecting at least one target analyte (e.g., whole virus or viral protein) in a sample (e.g., a biological sample, such as blood, nasal mucus, sputum, saliva, or urine) is provided, wherein the system comprises (i) an assay comprising at least one capture reagent (e.g., an aptamer or antibody) capable of directly or indirectly generating an enzyme-mediated signal (e.g., an oxidase-mediated signal) in the presence of the at least one target analyte and an added substrate (e.g., a glucose solution), and (ii) a detection device (e.g., a blood glucose meter) for detecting the signal. In certain embodiments, the detection device comprises a sensor selected from the group consisting of an electrochemical sensor, an optical sensor, or a combination thereof. In certain embodiments, the detection device produces results in about thirty minutes or less. In certain embodiments, the signal is calibrated for the concentration of the enzyme.

In certain embodiments, the system further comprises a detection reagent (e.g., an aptamer or an antibody). In one embodiment, the detection reagent is added to the system by the user, i.e., the added detection reagent. In a particular embodiment, the detection reagent (e.g., labeled with an enzyme) is labeled and binds to the target analyte, producing a detectable complex.

In a particular embodiment, the capture reagent is immobilized to a solid support, such as a test strip, to provide a test site.

In certain embodiments, the capture reagent is immobilized, but not initially, i.e., upon binding of the target analyte or prior to detectable complex formation. According to this embodiment, the capture reagent and the detection reagent are present in the system upstream of the electrode and form a detectable complex upon addition of the analyte solution to the system. The detectable complex is then captured on a solid support near the electrode.

In certain embodiments, the substrate (e.g., a sugar such as glucose or diethanolamine) is an added substrate, i.e., added to the system by the user.

In certain embodiments, the system further comprises a first binding reagent. According to this embodiment, the first binding reagent is immobilized to a solid support.

In certain embodiments, the first binding reagent comprises a first binding site that binds to a second binding reagent (e.g., biotin) conjugated to a capture reagent.

In certain embodiments, the first binding reagent contains a second binding site and the assay further comprises a polymer (e.g., PEG), wherein the polymer binds to the first binding reagent at the second binding site. According to this embodiment, the first binding agent is cross-linked.

In certain embodiments, the second binding reagent comprises a third binding reagent, wherein the third binding reagent (e.g., biotin) is conjugated to the capture reagent to allow binding to one or more additional capture reagents. According to this embodiment, the capture reagent is crosslinked.

In certain embodiments, the solid support or matrix is a bead, a membrane, or a bead immobilized on a membrane. In certain embodiments, the solid matrix is not a metal particle.

In a particular embodiment, the capture reagent, the detection reagent, or both are added reagents, i.e., added to the system by the user.

In certain embodiments, the substrate (e.g., a sugar such as glucose or diethanolamine) is an added substrate, i.e., added to the system or assay by the user. In one embodiment, an excess of substrate is added.

In one embodiment, the enzyme label is an oxidoreductase. The oxidoreductase may be selected from the group consisting of oxidases, dehydrogenases, hydrogenases, peroxidases, phosphatases, hydroxylases, oxygenases, catalases and reductases.

Representative non-limiting oxidases include glucose oxidase, galactose oxidase, D-glucose: d-fructose oxidoreductase and cellobiose oxidase.

In a particular embodiment, the enzyme label is selected from horseradish peroxidase (HRP), alkaline Phosphatase (AP), glucose Oxidase (GO), and β -galactosidase.

In one embodiment, the enzyme is glucose oxidase and the substrate is glucose.

In one embodiment, the enzyme is alkaline phosphatase and the substrate is pyridoxal 5' -phosphate (PLP), or 5-bromo-4-chloro-3-indolyl-phosphate, or L-ascorbic acid-2-phosphate, acetaminophen phosphate, 4-acetamidophenyl phosphate, or 4-aminophenyl phosphate in Diethanolamine (DEA), 1-amino-2-propanol, N-methyl-D-glucosamine, or tris buffer.

In one embodiment, the enzyme is β -galactosidase and the substrate is galactose.

In one embodiment, the enzyme is horseradish peroxidase and the substrate is a chromogenic HRP substrate, such as 3,3', 5' -Tetramethylbenzidine (TMB) and 2,2' -azino-bis- [ 3-ethylbenzothiazoline-6-sulfonic acid ] (ABTS).

In one embodiment, the enzyme-mediated signal comprises a dual detection system, wherein the dual detection system comprises a first enzyme label and a second enzyme label, such as an oxidase label (e.g., oxidase label) and a peroxidase label (e.g., hydrogen peroxide).

In another embodiment, the Vmax of the enzyme linked to the detection reagent is greater than 0.0001 mM/min, greater than 0.01 mM/min, greater than 0.1 mM/min, or greater than > 10 mM/min.

In another embodiment, the kcat of the enzyme linked to the detection reagent is greater than 1s ^-1 More than 10s ^-1 More than 50s ^-1 Or greater than 100s ^-1 。

In another embodiment, the kcat/Km value of the enzyme linked to the detection reagent is greater than 0.00001mM s ^-1 Greater than 0.01mM s ^-1 Greater than 1mM s ^-1 Or greater than 10mM s ^-1 。

In a particular embodiment, the capture reagent is provided as a hydrogel positioned on or within the solid support. Hydrogels may be saturated with a substrate such as glucose.

In one embodiment, the assay is a Lateral Flow Assay (LFA). In a particular embodiment, the lateral flow assay comprises at least one test site comprising at least one capture binding reagent. Optionally, the lateral flow assay further comprises at least one control site comprising at least one control element in order to monitor the performance of the system.

In one embodiment, the system is an electrochemical system or an optical system. In particular, the sensor produces an output that is calibrated for the presence or concentration of the target analyte. In a particular embodiment, electrochemical detection is performed only upon insertion of the strip into the electrochemical device, providing a differential voltage and detecting the current output provided by the strip and accompanying electrodes.

In a particular embodiment, the system is an electrochemical system comprising at least one electrode located at, above or below the target site. Optionally, at least one binding reagent may be bound to the electrode.

In one embodiment, the system is a system for self-monitoring. In a particular embodiment, the detection device is a blood glucose meter or a mobile phone.

In embodiments, information about the signal is transmitted to a third party for diagnosis and optional treatment.

In one embodiment, the system allows for detection of at least one target analyte in about 10 minutes or less, about 5 minutes or less, about 2 minutes or less, or about 1 minute or less.

In one embodiment, the systems disclosed herein allow for improved disease diagnosis, monitoring, management, or a combination thereof.

In certain embodiments, the system stores multiple test results obtained at different times with respect to the same user and compares the results to monitor or predict the likely development of a disease or condition. In one embodiment, the system allows two or more results, three or more results, or five or more results to be obtained for the same user at different times with respect to the amount of target analyte to allow monitoring of trends in analyte levels over time.

In another embodiment, an assay (e.g., a hand-held assay) for detecting at least one target analyte (e.g., a whole virus) is provided that comprises a first binding reagent (e.g., streptavidin) and a capture reagent (e.g., an aptamer or antibody), wherein the capture reagent is capable of directly or indirectly generating an enzyme-mediated signal (e.g., an oxidase-mediated signal) in the presence of the at least one target analyte and a substrate (e.g., a glucose solution). In certain embodiments, the signal is proportional to the concentration of the enzyme. In certain embodiments, the signal may be detected in 30 minutes or less.

In certain embodiments, the system further comprises a detection reagent (e.g., an aptamer or an antibody), wherein the capture reagent and the detection reagent form a detectable complex when the target analyte is present.

In one embodiment, the detection reagent is an added detection reagent, i.e., added to the system by the user.

In a particular embodiment, the substrate is an added substrate, i.e., added to the system by the user.

In certain embodiments, the first binding reagent comprises a first binding site that binds to a second binding reagent (e.g., biotin) conjugated to a capture reagent.

In certain embodiments, the first binding reagent contains a second binding site and the assay further comprises a polymer (e.g., PEG), wherein the polymer binds to the first binding reagent at the second binding site. According to this embodiment, the first binding agent is cross-linked.

In certain embodiments, the second binding reagent comprises a third binding reagent, wherein the third binding reagent (e.g., biotin) is conjugated to the capture reagent to allow binding to one or more additional capture reagents. According to this embodiment, the capture reagent is crosslinked.

In certain embodiments, the solid substrate is a bead, a membrane, or a bead immobilized on a membrane.

In a particular embodiment, the capture reagent, the detection reagent, or both are added reagents, i.e., added to the system by the user.

In certain embodiments, the substrate is an added substrate, i.e., added to the system by the user.

In a particular embodiment, the enzyme label is an oxidase (e.g., glucose oxidase) or dehydrogenase.

In one embodiment, the assay is a lateral flow assay. In a particular embodiment, the assay is a multiplexed lateral flow assay.

In another embodiment, the assay is a vertical flow assay. In one embodiment, the vertical flow assay consists of one, two, or three or more layers (e.g., one, two, or three film layers or more). In one embodiment, the assay is a multiplexed sandwich vertical flow assay.

In a particular embodiment, the vertical flow assay comprises a first membrane layer comprising an immobilized capture reagent, a second membrane layer comprising a target analyte, and a third membrane layer comprising a labeled detection reagent.

In one embodiment, the detectable complex is detected without the aid of a detection device.

In another embodiment, the detectable complex is detected by a detection device. The detection means may be, for example, a blood glucose meter or a mobile phone.

In one embodiment, the enzyme-mediated signal comprises a dual detection system, wherein the dual detection system comprises an enzyme label (e.g., oxidase) and a reporter label (e.g., horseradish peroxidase). In certain embodiments, the signal is colorimetric.

In one embodiment, the target analyte is a protein or peptide (e.g., a viral protein such as a nucleocapsid protein or protein-derived hormone), and the system allows for a detection Level (LOD) of about 1.0ng/mL or less, about 0.8ng/mL or less, about 0.6ng/mL or less, about 0.4ng/mL or less, about 0.2ng/mL or less, about 0.1ng/mL or less. In certain embodiments, the systems, assays, and/or methods disclosed herein allow for the LOD in 30 minutes or less, in 15 minutes or less, in 10 minutes or less, in 5 minutes or less, or in 1 minute or less.

In certain embodiments, the target analyte is a small molecule and the system allows LOD ranging from about 0.01 to about 100ng/mL, about 0.1 to 10ng/mL, 0.2 to 5ng/mL, or about 0.2 to about 1.0 ng/mL. In certain embodiments, the systems, assays, and/or methods disclosed herein allow for the LOD in 30 minutes or less, in 15 minutes or less, in 10 minutes or less, in 5 minutes or less, or in 1 minute or less.

In certain embodiments, the target analyte is a whole virus, and the system allows for about 10 ¹² TCID50/mL or less, more particularly, about 10 ¹¹ About 10 ¹⁰ About 10 ⁹ About 10 ⁸ About 10 ⁷ About 10 ⁶ About 10 ⁵ About 10 ⁴ About 5000TCID50/mL or less, about 20000TCID50/mL or less, about 10000TCID50/mL or less, about 5000TCID50/mL or less, about 1000TCID50/mL or less, about 500TCID50/mL, about 300TCID50/mL, about 100TCID50/mL, about 50TCID50/mL or less, about 20TCID50/mL, or LOD of about 15TCID50/mL or less. In certain embodiments, the systems, assays, and/or methods of the present disclosure allow for the LOD in 30 minutes or less, in 15 minutes or less, in 10 minutes or less, in 5 minutes or less, or in 1 minute or less.

In another embodiment, the target analyte is a whole virus and the system allows for a range of about 13 to about 50000TCID50/mL, more particularly about 13 to about 20000TCID50/mL, more particularly about 50 to about 10,000TCID50/mL, 50 to about 10 ⁴ TCID50/mL, 50 to about 10 ⁵ TCID50/mL, 50 to about 10 ⁶ TCID50/mL, 50 to about 10 ⁷ TCID50/mL, 50 to about 10 ⁸ TCID50/mL, 50 to about 10 ⁹ TCID50/mL, 50 to about 10 ¹⁰ TCID50/mL, 50 to about 10 ¹¹ TCID50/mL, 50 to about 10 ¹² LOD of TCID 50/mL. In certain embodiments, the systems, assays, and/or methods disclosed herein allow for the LOD in 30 minutes or less, in 15 minutes or less, in 10 minutes, in 5 minutes or less, or in 1 minute or less.

In one embodiment, the systems, assays, and methods disclosed herein allow for about 10 per mL ¹ To about 10 ¹¹ LOD in the range of individual protein copies.

In one embodiment, the systems and assays disclosed herein allow for the detection of about 10 to 1,000 small molecules in solution, well below the current range of clinical interest. In one embodiment, the system allows for the detection of about 10 to about 100 small molecules in solution, or about 10 to about 50, and more particularly about 10 to about 20 small molecules in solution.

In one embodiment, the systems disclosed herein have a detection limit of about 10 small molecules/mL or 10 analytes/mL or similar concentrations.

In one embodiment, the systems, assays and/or methods disclosed herein allow detection of about 1000, about 900, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100 or less viral particles per mL or similar concentrations.

In a particular embodiment, the systems, assays, and/or methods disclosed herein allow detection of about 100 or less viral particles per mL, or more particularly, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, or about 50 viral particles per mL.

In one embodiment, the time to produce the result is from 1 second to 30 minutes, preferably from 10 seconds to 15 minutes, more preferably from about 20 seconds to 8 minutes, still more preferably from 30 seconds to 5 minutes.

In one embodiment, the assay allows for detection of at least one target analyte in 10 minutes or less, 5 minutes or less, 2 minutes or less, or 1 minute or less.

The systems and assays disclosed herein exhibit desirable characteristics for the user and, in some cases, improved characteristics over prior art assays and systems. These characteristics may include, but are not limited to, speed and duration of sensing (< about 1 minute), specificity (> about 90%), selectivity (> about 90%), limit of detection of the assay (1 target analyte per milliliter to > 100,000 target analytes per milliliter), quantitative detection (> about 90% accuracy and > about 90% accuracy), effects of common disturbances on sensor output, cross-reactivity (> about 90% selectivity with respect to target analytes) (e.g., between related proteins such as phosphorylated or unphosphorylated), dynamic range, coefficient of variation of repeated measurements (< about 0% variance), operational stability, or combinations thereof. In one embodiment, an analysis of variance with five (5) variables may use five (5) measurements to achieve convergence greater than 0.95, depending on the statistical method used. By reducing the variables to one or two by normalization of the manufacturing, two (2) measurements can be used to obtain a confidence level. In certain embodiments, the systems and assays disclosed herein utilize improved chronoamperometry to achieve desired characteristics, i.e., characteristics that are superior to those achievable with constant or delayed chronoamperometry.

In one embodiment, the system or assay allows a sensitivity of about 90% or greater, about 91% or greater, about 92% or greater, about 93% or greater, about 94% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, or about 99% or greater.

In a particular embodiment, the system allows 9 true positive tests to accompany 1 false negative test out of 10 tests.

In one embodiment, the system or assay allows a sensitivity of about 90% or greater, about 91% or greater, about 92% or greater, about 93% or greater, about 94% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, or about 99% or greater. The advantage of such high sensitivity is that very early detection can be performed, for example, before any symptoms are apparent. This is particularly useful for detecting disease states in subjects who have been in contact with other individuals who are infected.

In one embodiment, the redox analyte solution may become more or less sensitive to the influence of the compound and counter ion, which may affect the system sensitivity. In certain embodiments, titration of compounds or counterions critical to enzyme function may allow for more sensitive detection of target analytes. See, e.g., example 19, which shows the preparation of MgCl ₂ Titration into DEA buffer can increase the sensitivity of the assay, especially depending on the enzyme source.

In one particular embodiment, the system or assay allows 9 true negative tests to accompany 1 false negative in ten tests.

Other such characteristics may include test scale, assay time, ease of use, and incidental (healthcare) infections. In particular, accuracy is of paramount importance, as false negative results may lead to erroneous diagnosis or treatment when used for detecting target analytes in biological samples.

In one embodiment, the systems and assays disclosed herein provide results to a user about 10 minutes or less, and more particularly about 5 minutes or less, about 2 minutes or less, or about 1 minute or less, after the addition of a biological (e.g., saliva or blood) sample. In a particular embodiment, the system allows results to be provided to the user in about 1 to about 2 minutes.

In one embodiment, the systems and assays disclosed herein have a false positive rate of less than about 33%. In a particular embodiment, the false positive rate is about 32%, about 30%, about 28%, about 26%, about 24%, about 22%, about 20%, about 18%, about 16%, about 14%, about 12%, about 10%, about 8%, about 6%, about 4%, or about 2% or less.

In another embodiment, the systems and assays disclosed herein have a false negative rate of less than about 20%, about 18%, about 16%, about 14%, about 12% about 10%, about 8%, about 6%, about 4%, or about 2% or less.

In one embodiment, the system disclosed herein allows (with 95% confidence interval) a sensitivity of 95% and a specificity of 95%.

In another embodiment, the system disclosed herein allows for a minimum target clinical sensitivity of about 90% and an optimal target sensitivity of about 98%; about 90% minimum target specificity and > 98% optimal target sensitivity.

In one embodiment, the systems disclosed herein allow for improved disease diagnosis, monitoring, management, or a combination thereof.

The samples and assays described herein may differ in form and detection strategy, but share certain common elements as discussed below.

A.Sample of

The samples utilized in the systems, assays, and methods disclosed herein may vary.

In one embodiment, the sample is a biological sample. Biological samples various sample types obtained from an individual, including clinical samples or non-clinical samples. Biological samples may vary and include, for example, sweat, saliva, tears, blood, serum, milk, urine, mucus, fecal matter, sebum, ocular fluids such as aqueous humor, respiratory tract droplets, pleural effusion, cerebral spinal fluid, semen, ejaculation, vaginal mucus, lymph, ascites, peritoneal fluid, pericardial fluid, amniotic fluid, synovial fluid, intestinal fluid, cerumen, epidermal cells, white blood cells, nasal or nasopharyngeal samples, blood, or combinations thereof.

In a particular embodiment, the biological sample is saliva. Advantageously, the use of saliva as a biological sample eliminates the use of uncomfortable sample collection techniques and allows for simple sample collection. Saliva is a viscous, dense viscous fluid that inherently contains microorganisms such as bacteria and fungi, intact human cells, cell debris, and many soluble substances such as enzymes, hormones, antibodies, and other molecules.

Saliva samples can be easily collected from the subject in any suitable manner, and in certain embodiments, without the use of specialized equipment, such as by splitting the subject into vessels whose contents are then diluted and applied to a system or assay, or alternatively spitting directly onto a cartridge or test strip. See, e.g., navazesn M (1993), methods for collecting saliva, ann N Y Acad Sci 694:72-77. In other embodiments, saliva may be treated (e.g., by centrifugation) to provide a cell-free fluid phase.

In certain embodiments, the blood sample may be readily collected from the subject in any suitable manner without the use of specialized equipment.

In another particular embodiment, the biological sample is not blood.

In another particular embodiment, the biological sample is not urine.

Biological samples may be derived from a subject (e.g., a human) using well known techniques such as venipuncture, lumbar puncture, fluid samples such as saliva or urine, or tissue biopsies, etc.

The volumes of biological samples may vary. In one embodiment, the biological sample has a volume of about 1 μl, 10 μl, 20 μl, 50 μl, or 100 μl to about 2000 μl, more particularly about 100 μl, about 150 μl, about 200 μl, about 250 μl, about 300 μl, about 350 μl, about 400 μl, about 450 μl, about 500 μl, about 550 μl, about 600 μl, about 650 μl, about 700 μl, about 750 μl, about 800 μl, about 850 μl, about 900 μl, about 950 μl, about 1000 μl, about 1250 μl, about 1500 μl, about 1750 μl, or about 2000 μl.

In another embodiment, the sample is an environmental sample. Environmental samples may vary and include water, soil, waste (liquid, solid or sludge, including, for example, sewage), fuel, sediment, mud, and the like. In certain embodiments, the environmental sample is an industrial product, such as a food or beverage or a starting material for producing the same.

In some embodiments, the system incorporates sample preparation (e.g., solvation, dilution, and mixing) via components such as a collection chamber and/or fluidic design. The matrix on the strip may comprise any solvating material. Alternatively, sample preparation may be processed independent of the system and then added to the system.

In certain embodiments, the sample may be manipulated or processed in some manner after being taken but prior to testing for the analyte of interest. In particular, the sample may be diluted in a liquid medium to provide a diluted sample. The liquid medium used to dilute the sample may be, for example, water, saline, cell culture medium, or any solution, and may contain any number of salts, surfactants, buffers, reducing agents, denaturants, preservatives, and the like. The sample may be diluted, for example 2X, 4X or 6X or more.

The pH of the samples may vary, but in certain embodiments is between about 6.0 and about 8.0.

The solid sample may be dissolved in a liquid medium or otherwise prepared as a liquid sample to facilitate flow. In the case where biological cells or particles are used, the biological cells or particles may be lysed or otherwise disrupted such that the contents of the cells or particles are released into the liquid medium. In this case, the molecules contained in the cell membrane and/or the cell wall may also be released into the liquid medium.

In other embodiments, the sample may be mixed with at least one reagent, such as a capture reagent, a binding reagent, a secondary binding reagent, and/or a substrate, prior to addition to the assay.

In certain embodiments, the target analyte is not enriched or incubated prior to performing the diagnostic assay itself.

In certain embodiments, the sample is a raw sample, i.e., taken directly from a source and not subjected to other treatments prior to testing.

B. Target analytes

The at least one target analyte detected by the systems, assays, and methods disclosed herein may be different. In certain embodiments, the systems, assays, and methods disclosed herein allow for the simultaneous or sequential detection of two or more target analytes, i.e., are multiplexed systems, assays, or methods.

In certain embodiments, the target analyte may be associated with a normal health condition or a non-pathogenic condition or a physiological condition that is otherwise altered due to disease or injury. In other embodiments, the target analyte is associated with an altered physiological condition, and the test allows monitoring of the progression of the condition or response to treatment.

In one embodiment, the target analyte is associated with an allergic disease, an infectious disease, an autoimmune disease, a heart disease, a cancer, or a graft versus host disease.

In a particular embodiment, the target analyte is not glucose.

In one embodiment, the sample is a biological sample and the target analyte is an analyte selected from the group consisting of: microorganisms such as pathogenic microorganisms (e.g., viruses, bacteria, fungi, parasites, or fungal spores), allergens, proteins, peptides, nucleic acids, small molecules, hormones, steroids, cofactors, vitamins, metabolites, and the like. In certain embodiments, the target analyte is an intact microorganism, such as a whole virus.

In other embodiments, the target analyte is an antigen associated with a microorganism, such as a protein, peptide, polysaccharide, toxin, cell wall, cell capsule, viral coat, flagella, pili or pili, microorganism, nucleic acid complexed with protein or polysaccharide, lipid complexed with protein or polysaccharide, polynucleotide, polypeptide, carbohydrate, chemical moiety, or combination thereof (e.g., phosphorylated or glycosylated polypeptide, etc.)

In particular, the target analyte may be a virus or a part of a virus, wherein at least one polyclonal or monoclonal antibody or aptamer or protein with respect to the virus or part of a virus is currently known or becomes known.

In one embodiment, the virus is a DNA virus. For example, single-or double-stranded DNA viruses.

In another embodiment, the virus is an RNA virus. For example, single-stranded or double-stranded RNA viruses.

Representative non-limiting viruses that can be detected according to the systems, assays and methods disclosed herein include adenovirus, adeno-associated virus, influenza virus, parainfluenza virus, cytomegalovirus, coronavirus, hepatitis virus (e.g., hepatitis a, hepatitis B, hepatitis c, hepatitis d), human immunodeficiency virus, avian influenza virus, respiratory Syncytial Virus (RSV), herpes simplex virus, ebola virus, herpes simplex virus 1, herpes simplex virus 2, human papilloma virus, marburg virus, lassa virus, pestivirus, porcine parvovirus, pseudorabies virus, rotavirus, calicivirus, epstein barr virus, human cytomegalovirus, human bocavirus, parvovirus B19, human astrovirus, norwalk virus, coxsackie virus, measles virus, mumps virus, rubella virus or rotavirus or canine distemper virus.

In a particular embodiment, the target analyte is a coronavirus, such as a whole coronavirus or a coronavirus protein or peptide. Coronaviruses consist of a large and diverse family of enveloped, sense, single-stranded RNA viruses. Each coronavirus contains four structural proteins, such as spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N). Among them, the S protein plays the most important role in viral attachment, fusion and entry.

In one embodiment, the target analyte is a coronavirus S protein or fragment or epitope thereof. The S protein is a trimeric type I transmembrane glycoprotein that forms a large, prominent, characteristic corona of spikes on the surface of the virion and mediates binding to host cell receptors and fusion with host cell membranes. In many coronaviruses, S is posttranslationally cleaved into two subunits, designated S1 and S2, which are triad and folded into a metastable pre-fusion conformation. The S1 subunit forms the "head" of the spike and contains two domains: amino (N) terminal domain (NTD) and carboxy (C) terminal domain (CTD), wherein the latter generally contains a Receptor Binding Domain (RBD). The S2 subunit contains two Heptad Repeat (HR) regions. When S1 recognizes and binds to the corresponding host receptor, S2 undergoes a conformational change, straightening itself from a compressed form to a nail-like shape, referred to as a post-fusion state. This enables the viral envelope to fuse with the outer membrane and deposit viral genetic material inside the cell. The life cycle of the virus then progresses to include biosynthesis, assembly and release.

In one embodiment, the target analyte is S1 or S2, and more particularly, NTD, RBD, CTD1, CTD2, S1/S2 cleavage site, S2' cleavage site, fusion Peptide Proximal Region (FPPR), heptad repeat 1 (HR 1), heptad repeat 1, central helical region (CHD), linking domain (CD, heptad repeat 2 (heptad repeat 2), transmembrane anchor (TM), cytoplasmic tail (CT, or a combination thereof).

The diversity of coronaviruses is reflected in the variable S protein, which has evolved into different forms in terms of its receptor interactions and its responses to various environmental triggers of virus-cell membrane fusion. In particular, the RBD of the S protein is the most variable genomic part of the β coronavirus group.

In another embodiment, the target analyte is a coronavirus nucleocapsid (N) protein or a fragment of an epitope thereof. N proteins are characterized by three distinct and highly conserved domains: two structural and independently folded domains, the N-terminal domain (NTD/domain 1) and the C-terminal domain (CTD/domain 3), separated by an essentially disordered central region (RNA binding domain/domain 2). In a particular embodiment, the target analyte is an RNA binding domain of an NTD, CTD or N protein.

Four serologically distinct groups of coronaviruses have been described, namely α, β (previously referred to as group 2), δ and γ. Within each group, the virus is characterized by its host range and genomic sequence. The alpha and beta coronaviruses only infect mammals, while the gamma and delta coronaviruses primarily infect birds, although some of these may also infect mammals. Novel mammalian coronaviruses are now regularly identified. (Su et al, trends Microbiol.2016; 24:490-502). Beta coronaviruses (Beta-CoV) known to be clinically important for humans include viruses of the A, B and C lineages, and more particularly, the a lineages: OC43 (which may cause the common cold) and HKU1; b lineage: LPH-COV, SARS-COV-2 (which causes the disease COVID-19) and SARS-COV-n (where n is any integer); c: MERS-CoV.

In one embodiment, the systems, assays, and methods disclosed herein relate to the detection of a β -coronavirus infection, and more particularly, a-, B-, or C-lineage coronavirus infection. These are viruses with a plus-sense single-stranded RNA of about 32Kb, which encodes multiple structural and non-structural proteins. Viral particles contain four major structural proteins: spikes, membranes, envelope proteins, and nucleocapsids. Spike proteins protrude from the envelope of the virion and play a critical role in receptor host selectivity and cell attachment. Beta coronaviruses share many similarities within the ORF1ab polyprotein and most structural proteins; however, spike proteins and helper proteins show significant diversity. Mutations in spike proteins can alter viral tropism, including new hosts or increase pathogenesis

In a particular embodiment, the at least one target analyte is a virus, and more specifically, a coronavirus such as a β coronavirus, and even more specifically, SARS-CoV-1 or SARS-CoV-2.

In another particular embodiment, the systems, assays and methods disclosed herein relate to the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. SARS-CoV-2 (also known as 2019-nCoV) was identified as a causative agent of severe acute respiratory syndrome 2 (also known as Covid-19) in month 1 of 2020. The new coronavirus infection spread rapidly and the world health organization (World Health Organization) (WHO) announced Covid-19 as a pandemic in month 3 of 2020. To date, the virus has infected more than 700 tens of thousands of people and killed more than 400,000 individuals. Individuals living or working at high density and in close contact (e.g., military personnel) are particularly at risk.

Clinical signs associated with SARS-CoV-2 include pneumonia, fever, dry cough, headache, and dyspnea, which may progress to respiratory failure and death. The latency period for SARS-CoV-2 appears to be longer than for SARS-CoV and MERS-CoV, which have an average latency period of 5 to 7 days.

SARS-CoV-2 was sequenced and isolated at month 1 of 2020 (e.g., zhou N.N Engl J Med.,382 (2020), pages 727-733). Several sequences of SARS-CoV-2 have been published hereafter. Similar to other coronaviruses, spike (S) protein is the major glycoprotein on the surface of SARS-CoV-2 virus. SARS-CoV-2 appears to have a Receptor Binding Domain (RBD) that binds with high affinity to ACE2 from humans, ferrets, cats and other species with high receptor homology (Wan et al, (2020) J.Virol.https:// doi.org/10.1128/JVi.00127-20).

SARS-CoV-2S1 RBD is 193 amino acids in length (N318-V510).

The SARS-CoV-2S protein has been reported to share 76% amino acid sequence identity with SARS-CoV S Urbani and 80% identity with bat SARSr-CoV ZXC21S and ZC 45S glycoproteins. With respect to sequence alignment of the interaction domains of SARS-CoV-2 (MN 938384), bat-CoV (MN 996532 and MG 772933) and SARS-CoV (NC 004718). The RBD of SARS-CoV-2 differs greatly from SARS-CoV at the C-terminal residue.

The S1 subunit of SARS-CoV-2 contains the Receptor Binding Domain (RBD), while the S2 subunit contains a hydrophobic fusion peptide and two heptad repeat regions. S1 contains two structurally independent domains, an N-terminal domain (NTD) and a C-terminal domain (C-domain). Depending on the virus, either the NTD or C domain may act as a Receptor Binding Domain (RBD).

In one embodiment, the systems, assays and methods disclosed herein allow for the detection of the S protein of SARS-CoV-2 or subunits or fragments thereof, and more specifically, one or more epitopes of the S protein of SARS-Co-V-2, including, but not limited to, RBD, S1 amino terminal domain (S1-NTD), ORF3 (3 a and 3 b), and the accessory gene ORF8.

In one embodiment, the capture reagent and the binding reagent bind to SARS-CoV-2 spike (S) protein using human Angiotensin Converting Enzyme (ACE) protein. In a particular embodiment, the ACE protein binds to the Receptor Binding Domain (RBD) of the S protein.

In one embodiment, the capture reagent and the detection reagent bind to different epitopes on SARS-CoV-1 spike (S) protein. In a particular embodiment, at least one epitope is within the Receptor Binding Domain (RBD) of the S1 protein.

In one embodiment, one of the binding agents binds SARS-CoV-1 spike (S) protein using human Angiotensin Converting Enzyme (ACE). In a particular embodiment, the ACE protein binds to the Receptor Binding Domain (RBD) of the S protein.

In one embodiment, the systems, assays and methods herein allow for detection of whole viruses, i.e., SARS-CoV-2 particles.

In one embodiment, the systems, assays and methods herein allow for the detection of one or more epitopes of the N-terminal domain (NTD) and the C-terminal domain (C-domain) of SARS-CoV-2.

In one embodiment, the systems, assays, and methods herein allow for the detection of one or more epitopes in the RBD of SARS-CoV-2, and more specifically, one or more epitope residues within residues 319 and 510 of the RBD.

In a particular embodiment, the systems, assays and methods disclosed herein relate to the detection of SARS-CoV infection. SARS-CoV was identified in month 4 of 2003 as the causative agent responsible for Severe Acute Respiratory Syndrome (SARS) (Drosten et al, new Engl. J. Med.2003; 348:1967-1976). Clinically, SARS-CoV shows a biphasic course, namely, first high fever, parainfluenza syndrome, followed by increased respiratory distress. Spray plays a key role in the propagation. Diagnosis is based on clinical manifestations, epidemiological data supported by positive serology, PCR or the presence of viruses in cell culture. Shortly thereafter, the consensus genomic sequence of SARS-CoV is disclosed, most closely resembling group B. Beta. Coronavirus (Marra et al, science.2003;300:1399-14040; ruan et al, lancet.2003; 361:1779-1785).

SARS-CoV spike protein has been shown to consist of two functional domains: s1 (amino acids 12-680) and S2 (amino acids 681-1255) (Li et al science 2005; 309:1864-1868). RBD is located within the S1 subunit and has been mapped to a fragment consisting of amino acids (aa) 318-510 in the S1 domain. (Wong et al, J Biol chem.2004; 279:3197-3201).

In one embodiment, the systems, assays, and methods disclosed herein allow for the detection of the S protein of SARS-CoV-2, or a fragment or epitope thereof, and more specifically, one or more epitopes of the S protein of SARS-CoV-2, including but not limited to RBD.

In one embodiment, the systems, assays, and methods herein allow for the detection of one or more epitopes in the RBD of SARS-CoV-2, and more specifically, one or more epitope residues within residues 318 and 510 of the RBD.

In one embodiment, the systems, assays, and methods described herein allow for the detection of the N protein of SARS-CoV-2 or fragments or epitopes thereof.

In a particular embodiment, the systems and methods disclosed herein relate to the detection of middle east respiratory syndrome coronavirus (MERS-CoV) infection. MERS-CoV is a recently emerging beta coronavirus that causes severe acute respiratory illness. It was first isolated in sauter arabia in 2012 (Zaki et al 2012,NEJM 367:1814-1820) and has been spread to about 18 countries thereafter, with most cases being combined in sauter arabia He Ala in the united states. The clinical profile of MERS-CoV infection in humans ranges from asymptomatic infection to very severe pneumonia, with the potential development of acute respiratory distress syndrome leading to death, septic shock, and multiple organ failure. The virus uses its spike protein to interact with a cellular receptor for entry into a target cell. Viruses have been shown to bind dipeptidyl peptidase 4 (DPP 4) on human epithelial cells and endothelial cells via the receptor binding domain of their spike proteins (Raj et al 2013,Nature 495:251-256). Lu et al 2013 have shown that MERS-CoV receptor binding domain consists of a core and receptor binding subdomain that interacts with DPP4 (Lu et al 2013,Nature 500:227-231).

MERS-CoV spike protein is a 1353 amino acid type I membrane glycoprotein that is assembled into trimers that constitute spikes or envelope protrusions on the surface of enveloped MERS coronavirus particles. The protein has two basic functions, host receptor binding and membrane fusion, which are attributed to the N-terminal (S1, amino acid residues 1-751) and C-terminal (S2, amino acid residues 752-1353) halves of the S protein. MERS-CoV-S binds to its cognate receptor dipeptidyl peptidase 4 (DPP 4) via a Receptor Binding Domain (RBD) of about 230 amino acids in length that is present in the S1 subunit. Mou et al (2013) have shown in J.virology (volume 87, pages 9379-9383) that MERS-CoV RBD is located within residues 358-588 of spike protein. The amino acid sequence of the full length MERS-CoV spike protein is exemplified by the amino acid sequence of the spike protein of MERS-CoV isolate EMC/2012 provided as accession number AFS88936.1 in GenBank. The term "MERS-CoV-S" also includes protein variants of MERS-CoV spike proteins isolated from different MERS-CoV isolates, such as Jordan-N3/2012, england-Qatar/2012, A1-Hasa_1_2013, al-Hasa_2_2013, al-Hasa_3_2013, al-Hasa_4_2013, al-Hasa_12, A1-Hasa_15, A1-Hasa_16, A1-Hasa_17, al-Hasa_18, A1-Hasa_19, al-Hasa_21, al-Hasa_25, bisha_1, buralidah_1, england 1, hafr-Al-Batin_1, hafr-Al-Batin_2, hafr-Al-Batin_6, jeddah_1, KFU-U1, KFU HK_ataU 13, munich, qr 3, qr 4, ridh_1, ridha_2, ridha_18, ridha_1, ridha_9, ridha_Ridha_14, ridha_9, ridha_Ridha_4, ridha_14, ridha_9 and Ridha_4. The term "MERS-CoV-S" includes recombinant MERS-CoV spike proteins or fragments thereof.

In one embodiment, the capture reagent and detection reagent bind to common human coronaviruses including spike, membrane, hemagglutinin, envelope, or envelope proteins of the 229E, NL, OC43, and HKU1 types.

In a particular embodiment, the at least one target analyte is a virus, and more particularly, a rhinovirus. In one embodiment, the first binding agent and the second binding agent bind to one of 4 possible capsid proteins of the rhinovirus.

In a particular embodiment, the at least one target analyte is a virus, and more specifically, respiratory Syncytial Virus (RSV), parainfluenza (PIV), or H1N1.

In one embodiment, the first binding agent and the second binding agent bind to a fusion protein, membrane protein, hemagglutinin protein, neuraminidase protein, envelope or envelope protein of Respiratory Syncytial Virus (RSV), parainfluenza (PIV) or H1N1.

In a particular embodiment, the at least one target analyte is a virus, and more particularly, a human metapneumovirus.

In one embodiment, the capture reagent and the detection reagent bind to a fusion protein, SH protein, matrix protein, glycoprotein, envelope or envelope protein of a human metapneumovirus.

In a particular embodiment, the at least one target analyte is a virus, and more particularly, a Human Immunodeficiency Virus (HIV).

In one embodiment, the capture reagent and the detection reagent bind to an MHC protein, a p17 matrix protein, a gp120 docking glycoprotein, a gp41 transmembrane glycoprotein, an envelope or envelope protein of Human Immunodeficiency Virus (HIV).

In a particular embodiment, the at least one target analyte is a virus, and more particularly, ebola virus.

In one embodiment, the capture reagent and the detection reagent bind to a glycoprotein, matrix protein, nucleoprotein, envelope or envelope protein of an ebola virus.

In a particular embodiment, the at least one target analyte is a virus, and more particularly, a marburg virus.

In one embodiment, the capture reagent and the detection reagent bind to a glycoprotein, VP40 matrix protein, nucleoprotein, envelope, or envelope protein of a marburg virus.

In a particular embodiment, the at least one target analyte is a virus, and more particularly, a lassa virus.

In one embodiment, the capture reagent and the detection reagent bind to glycoprotein 1, glycoprotein 2, large protein, zinc protein, stable Signal Peptide (SSP), nucleoprotein, envelope, or envelope protein of the lassa virus.

In one embodiment, the capture and detection reagents bind to TRAP, SPECT, MAEBL, PPLP, LSA, STARP, CS, SALSA, SPATR, pxSR, or PfEMP3 proteins of malaria plasmodium.

In certain embodiments, one or more target analytes or pathogens are found in biological samples from animals other than humans, such as west nile virus and zoonotic pathogens in bats.

The target analyte is any protein or peptide, wherein at least one polyclonal or monoclonal antibody or aptamer is currently known or becomes known.

Representative non-limiting targets include proteins such as: n-terminal pro-B-type natriuretic peptide (NTproBNP) [ congestive heart failure ]; insulin [ diabetes ]; glucagon [ diabetes ]; autoantibodies (anti-dsDNA, anti-dsg 1, ANA, etc.); prostate Specific Antigen (PSA) [ cancer ]; osteopontin (OPN) [ arthritis and cancer ]; carcinoembryonic antigen (CEA) [ cancer ]; luteinizing hormone [ gestation ]; follicle stimulating hormone [ development ]; prolactin [ cancer ]; human chorionic gonadotrophin (hCG) [ pregnancy and development ]; gluten [ food allergy ]; wheat [ food allergy ]; peanut [ food allergy ]; almonds [ food allergy ]; casein and whey [ food allergy ]; sesame [ food allergy ]; egg [ food allergy ]; tissue transglutaminase antibody [ celiac disease ]; liver type arginase [ liver disease ]; soluble liver antigen [ liver disease ]; mitochondrial 2 (M2) antigen [ liver disease ]; t3 triiodothyronine, ASA anti-sperm antibody, troponin I, T thyronine, ACA anti-cardiolipin antibody, CKMB, TSH, AEA anti-endometrial antibody, FT3 free triiodothyronine, AOA anti-ovarian antibody, FT4 free thyronine, ATB anti-trophoblast antibody, anti-TM thyromicrosomal antibody, ZP anti-zona pellucida antibody, anti-TG anti-thyroglobulin (thiogloblin) antibody, anti-HCG antibody, human placental lactogen, anti-TPO thyroperoxidase antibody, HCG, TOX antibody, FSH, AFP alpha fetoprotein, CEA carcinoembryonic antigen, FPSA free prostate specific antigen, CMV cytomegalovirus antibody PRO progesterone, ferritin, TOX-Ag toxoplasma circulating antigen, E2 estradiol, C A125, E3 estriol, CA153, CA199, HBsAg, NSE neuron-specific enolase, HBsAb, CA50, HBeAg, β2 microglobulin, HBeAb, coxsackievirus antibody, HBcAb, BGP bone Gla protein, D-Pyr deoxypyridinin, vitamin D, insulin, PCIII type procollagen, C peptide, type IV collagen, insulin antibody, LN laminin, glucagon, HA hyaluronic acid, GAD-AB glutamate decarboxylase antibody, fn fibronectin, alpha Fetoprotein (AFP) human chorionic gonadotrophin-like hormone such as HCG, LH, FSH, TSH; troponin I [ myocardial infarction ]; troponin T [ myocardial infarction ]; creatinine Phosphokinase (CPK) [ myocardial infarction ]; MB isozymes (CPKMB) [ myocardial infarction ], myoglobin [ myocardial infarction ]; s100 proteins such as S100B and enolase [ cerebral ischemia ]; beta-amyloid [ Alzheimer's disease ]; alpha-synuclein [ parkinson's disease ]; beta-amyloid and myelin basic protein [ multiple sclerosis ]; albumin and liver enzymes [ hepatitis c ]; avidin; streptavidin; alpha 1-antitrypsin and surfactant proteins [ chronic obstructive pulmonary disease ]; alpha 1-antitrypsin and surface active protein [ asthma ]; surfactant proteins and elastase [ adult respiratory distress syndrome ]; rheumatoid factors, collagen and elastase [ autoimmune disease ]; albumin and elastase [ organ failure ]; lipopolysaccharide binding protein [ sepsis ]; angiotensin and erythropoietin [ eclampsia and preeclampsia ]; calprotectin is synonymous expression of "L1 albumin", "MRP 8/14", "cystic fibrosis (related) antigen (CFA)" and "calgranulin" [ heart disease and others ]; oxidoreductases, transferases; a kinase; a hydrolase; a lyase; an isomerase; a ligase; a polymerase; a cathepsin; calpain; aminotransferases such as AST and ALT, proteases such as caspases, nucleotide cyclases, transferases, lipases, heart attack-related enzymes, spike proteins from SARS-CoV-2, and the like.

In certain embodiments, the analyte may be an antibody, such as nAb, igA, igE, igG or IgM. For example, specific nAb, igA, igE, igG or IgM is present in vivo due to a covd infection. Antibodies originating from a disease or infection can be captured and detected.

In certain embodiments, the analyte may be a post-translationally modified protein (e.g., phosphorylated, methylated, glycosylated), and the capture component may be an antibody specific for the post-translational modification. The modified proteins can be captured with a multi-specific antibody and then detected using a specific secondary antibody directed against the post-translational modification. Alternatively, the modified proteins may be captured with antibodies specific for post-translational modifications and then detected with antibodies specific for each modified protein.

In certain embodiments, the target analyte is a hormone. The hormone may be a peptide hormone, an amino acid hormone, a steroid hormone or a eicosanoid hormone. Representative non-limiting hormones that may be detected by the systems, assays and methods herein include estrogen, progesterone, follicle Stimulating Hormone (FSH), testosterone/DHEA, thyroid hormone, testosterone, cortisol, androstenedione or aldosterone.

In certain embodiments, the target analyte is a small molecule, wherein at least one polyclonal or monoclonal antibody or aptamer with respect to the small molecule is or becomes known.

Small molecule target analytes may be different. In one embodiment, the target analyte is a biomarker, a drug (e.g., a therapeutic drug and/or an abused drug), a heavy metal, a hormone, a growth promoter, a nutrient, an insecticide, a food additive, or a toxin.

Any suitable therapeutic agent may be tested, for example, for any condition in a human or other mammal. In one embodiment, the drug to be detected is selected from antibiotics, antifungals, antiparasitics, antiviral or anticancer drugs.

In a particular embodiment, the drug to be detected is an antibiotic selected from the group consisting of penicillins, cephalosporins, macrolides, fluoroquinolones, sulfonamides, tetracyclines and aminoglycosides. In some embodiments of the present invention, in some embodiments, the antibiotic is selected from amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, chlorocarbon cephalosporin, ertapenem, doripenem, imipenem/cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, teicoplanin, vancomycin, telavancin, dapaglycone, olanzapine, clindamycin, lincomycin, daptomycin Azithromycin, avermectin, clarithromycin, dirithromycin, erythromycin, roxithromycin, valcomycin, telithromycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, linezolid, prednisolide, radzolid, tedizolamine (torezolid), amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin g, penicillin v, piperacillin, penicillin g, temoxicillin, ticarcillin, amoxicillin, oxacillin/sulbactam, piperacillin/tazobactam, ticarcillin/clavulanate, bacitracin, colicin, polymyxin b, ciprofloxacin, enoxacin, enrofloxacin, gatifloxacin, gemifloxacin, ciprofloxacin, levofloxacin, lomefloxacin, moxifloxacin, monensin, nalidixic acid, norfloxacin, ofloxacin, sarafloxacin, spectinomycin, streptomycin, trovafloxacin, glapafloxacin, salinomycin, sparfloxacin, temafloxacin, bambemycin (bamemyin), sulfamilbemycin, sulfacetamide, sulfachloropyridazine, sulfadiazine silver, sulfadimidine, sulfamethylthiadiazole, sulfamethoxazole, sulfa (sulfafanilimide), sulfasalazine, sulfamisoxazole, trimethoprim-sulfamethoxazole (tmp-smx), azo sulfanilamide (sulfonamimdochromyside), dimidine, doxycycline minocycline, terramycin, tetracycline, clofazimine, dapsone, frizzled, cycloserine, ethambutol (bs), ethionamide, isoniazid, pyrazinamide, erythromycin, rifampin, rifabutin, rifapentine, streptomycin, arsenicum, chloramphenicol, fosfomycin, fusidic acid, florfenicol, metronidazole, mupirocin, cefquinome, quinolones (e.g., fluoroquinolones), plamycin (platensimycin), quinidine/daptomycin, neomycin, pirlimycin, ofloxacin (oflacaxin), thiamphenicol, tigecycline, tilmicosin, tinidazole, and trimethoprim.

In a particular embodiment, the drug to be tested is an antifungal agent selected from the group consisting of allylamines, azoles, echinocandins, and polyenes. In certain embodiments, the antifungal agent is selected from amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, spinosad, bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, metronidazole, miconazole, omuconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, triazoles, tylosin, abaconazole, alfluconazole, epoxiconazole, fluconazole, isaconazole, itraconazole, posaconazole, propiconazole, rafconazol, terconazole, voriconazole, thiazoles, abafungin, amorolfine, butenafine, naftifine, terbinafine, anidulafungin, caspofungin, ciclopirox, flupyrimidine, griseofulvin, tolnaftate, vancomycin, and undecylenic acid.

In a particular embodiment, the drug to be detected by the systems and methods disclosed herein is an antiparasitic drug. In certain embodiments, the drug is albendazole, abamectin, or Sulfaquinoxaline (SQX),

In a particular embodiment, the drug to be detected by the systems, assays and methods disclosed herein is an antiviral agent. In certain embodiments, the antiviral drug is selected from the group consisting of adhesion inhibitors, entry inhibitors, uncoating inhibitors, protease inhibitors, polymerase inhibitors, nucleoside and nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and integrase inhibitors. In a particular embodiment, the antiviral drug is ribavirin.

In a particular embodiment, the drug to be detected by the systems and methods disclosed herein is an anticancer drug. In certain embodiments, the anticancer agent is methotrexate or paclitaxel, cisplatin, lenalidomide, ibrutinib, palbociclib, and enzalutamide. Other therapeutic agents that may be detected according to the systems and methods disclosed herein include antipyretics, anesthetics, anthelmintics (antihelmintics), analgesics (e.g., acetaminophen, mycophenolic acid), anti-inflammatory agents (e.g., analgin), anticoagulants, antihistamines, anticonvulsants, mood stabilizers, hormone substitutes, oral contraceptives, stimulants, tranquilizers, and statins.

Representative non-limiting therapeutic agents that can be detected by the systems and methods disclosed herein include 3-methylquinoxaline-2-carboxylic acid, LMG, and olaquindox.

Representative non-limiting nutrients that can be detected by the systems and methods disclosed herein include biotin (vitamin B7), ferulic acid, folic acid, and vitamin B12.

Representative non-limiting drugs of abuse that can be detected by the systems and methods disclosed herein include cannabinoids, clonazepam, cocaine, diazepam, hydrochlorothiazide, diphenhydramine, ethylglucuronide, LSD, heroin, harijuana, MDPV, nitrazepam, salicylic acid, tramadol, and venlafaxine.

In one embodiment, the target analyte is a biomarker. In one embodiment, the biomarker is associated with an infection, such as a viral infection. In one embodiment, the biomarker is selected from the group consisting of GM-CSF, granzyme A, granzyme B, IFN- α2α, IFN- β, IFN- γ, IL-1β, IL-1RA, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12p70, IP-10, I-TAC, MCP-1, MCP-2, MCP-4, MDC, MIP-1αMIP-1β, TNF- α, or VEGF-A.

Representative non-limiting biomarkers that can be detected according to the systems, assays and methods disclosed herein include, but are not limited to, erythropoietin (EPO), ferritin, folic acid, hemoglobin, alkaline phosphatase, transferrin, apolipoprotein E, CK, CKMB, parathyroid hormone, cholesteryl Ester Transfer Protein (CETP), cytokines, cytochrome C, apolipoprotein AI, apolipoprotein AII, apolipoprotein BI, apolipoprotein B-100, apolipoprotein B48, apolipoprotein CII, apolipoprotein CIII, apolipoprotein E, triglycerides, HD cholesterol, LDL cholesterol, lecithin cholesterol lipid transferase, paraoxonase (Paraonase), alanine Aminotransferase (ALT), aspartate transferase (AST), CEA HER-2, bladder tumor antigen, thyroglobulin, alpha fetoprotein, PSA, CA 125, CA 19.9, CA 15.3, leptin, prolactin, osteopontin, CD 98, fascin (fascin), troponin I, CD, HER2, CD33, EGFR, VEGFA, etc.), drugs (e.g., tetrahydrocannabinol (THC), cannabidiol (CBD), and Cannabinol (CBN), etc.), opioids (e.g., heroin, opium, fentanyl, etc.), agonists (e.g., cocaine, amphetamine, methamphetamine, etc.), club drugs (e.g., MDMA, flunidazole, gamma-hydroxybutyrate, etc.), dissociative drugs (e.g., ketamine, phencyclidine, sage, dextromethorphan, etc.), hallucinogens (e.g., LSD, mescaline, sal, etc.), drugs (e.g., galban, etc.), siroccin, etc.), explosives (e.g., 2,4, 6-trinitrotoluene (TNT) and hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX), pentaerythritol tetranitrate (PETN), etc.), toxic chemicals (e.g., metronidazole (GA), sarin (GB), soman (GD), cyclosarin (GF), 2- (dimethylamino) ethyl N, N-dimethylphosphamide fluoride (GV), VE, VG, VM, VP, VR, VS, or VX neurotoxic), etc

The biomarker may be differentially present at any level, but is generally present at a level that is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% (i.e., is not present); or at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more. Alternatively, the differential presence of a biomarker may be characterized by a fold change in level, including, for example, the following levels: reduced to 1/1.1, up to 1/1.2, up to 1/1.3, up to 1/1.4, up to 1/1.5, up to 1/2.0, up to 1/2.5, up to 1/3.0, up to 1/3.5, up to 1/4.0, up to 1/5, up to 1/5.5, up to 1/6, up to 1/6.5, up to 1/7.0, up to 1/7.5, up to 1/8.0, up to 1/9, up to 1/10, up to 1/11, up to 1/12, up to 1/13, up to 1/14, up to 1/15, up to 1/16, up to 1/17, up to 1/18, up to 1/19, up to 1/20, up to 1/25, up to 1/30, up to 1/40, or up to 1/50; or at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7.0-fold, at least 7.5-fold, at least 8.0-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, or at least 50-fold. Biomarkers are preferably present differentially at statistically significant levels (e.g., p-values less than 0.05 and/or q-values less than 0.10 as determined using, for example, the Welch T-test or the Wilcoxon rank sum test).

In one embodiment, the sample is an environmental sample and the target analyte is an analyte selected from the group consisting of: toxins, pesticides, asbestos, pollutants, organic compounds (e.g. petroleum hydrocarbons, polyaromatics) or residues, perfluorinated compounds, organochlorine species, endocrine disruptors, drugs, growth factors, detergents, triclosan, sweeteners, N-Nitrosodimethylamine (NDMA), heavy metals (e.g. lead, cadmium), microorganisms, algal toxins, illegal drugs, flame retardants, antibacterial agents, hormones, moulds, prions or nanomaterials.

Representative non-limiting pesticides that can be detected by the systems and methods disclosed herein include acetamiprid, acetochlor, carbaryl, carbendazim/benomyl, chlorothalonil, chlorpyrifos, fenpropathrin, imidacloprid, parathion, and pentachlorophenol. Representative non-limiting hormones that can be detected by the systems and methods disclosed herein include steroid hormones (natural and synthetic), such as estrogens (estron, estradiol, estriol and derivatives thereof), androgens (testosterone, dihydrotestosterone, androstenediol, androstenedione, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S) and derivatives thereof), progesterone (such as progesterone, 17-hydroxyprogesterone, pregnenolone, 17-hydroxy pregnenolone and derivatives thereof), testosterone, corticosteroids (such as glucocorticoids, mineralocorticoids, cortisol, 11-deoxycortisol, corticosterone, 1-deoxycorticosterone, 18-hydroxy corticosterone, aldosterone and derivatives thereof), and melatonin.

Representative non-limiting food additives that can be detected by the systems and methods disclosed herein include acrylamide; ALP; b acid; benzophenone, benzothiazine, BHT, BTZ, coryza, DBP, dimethyl phthalate, econazole, erythrosine, optical brighteners KSN, MBT, melamine, rhodamine, sudan I, sudan red, lemon yellow, and beta-lactamase.

Representative non-limiting fuel additives that can be detected by the systems and methods disclosed herein include antiknock agents such as ferrocene or toluene, or detergents such as polybutyleneamine, or antioxidants such as p-phenylenediamine. These can be more easily sensed by aptamer combinations that do not involve any antibodies.

In certain embodiments, the target analyte is important in industries selected from the agricultural, transportation, or food and beverage sectors.

In some embodiments, the analyte of interest is a chemical warfare agent or a biological warfare agent. The target analytes may also be analogues, metabolites and derivatives of such chemical or biological warfare agents.

In certain embodiments, the assay is a multiplexed assay, i.e., allowing detection of more than one target analyte. For example, the assay may detect two or more different target analytes, three or more different target analytes, four or more different target analytes, or five or more different target analytes. In certain embodiments, the assay is an array suitable for detecting multiple different target analytes.

In one embodiment, the systems, methods, and assays disclosed herein allow for the simultaneous or sequential detection of one or more of the following: SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1, influenza A, influenza B and RSV. In certain embodiments, multiplexed assays detect two or more target analytes, such as viral variants, that are closely related.

C.Measurement

(i) Form of the invention

The format of the assay may vary, whether it is a component of the system described herein or a separate assay.

In one embodiment, the assay is a lateral flow assay. Typically, a Lateral Flow Assay (LFA) runs a liquid sample along the surface of a solid support with capture reagents (e.g., antibodies) that bind to target analytes, if present, to generate a signal directly or indirectly (e.g., by means of labeled detection reagents, e.g., antibodies, that are localized within the assay, e.g., in conjugate regions). Conventional lateral flow assays include strips (e.g., nitrocellulose strips), but other substrates may also be suitable, such as dipsticks, flow devices, or microfluidic devices.

In one embodiment, the assay is a lateral flow assay. In a test strip format, a fluid sample containing or suspected of containing at least one target analyte is placed on a sample receiving zone. The target analyte becomes labeled upon contact with the test strip. The now labeled target analyte of interest then flows (e.g., by capillary action) through the strip.

In one particular embodiment, the Lateral Flow Assay (LFA) consists of four parts: a sample pad, which is an area on which a sample drops; a test area on a solid support (e.g., a polymer membrane) in which the reaction occurs and an absorbent pad that retains waste. The sample pad may be present on a fiberglass, quartz or cellulose substrate for receiving the sample. The absorbent pad (50) may comprise absorbent material that facilitates acquisition, such as cotton, polymer, porex, paper, or may be empty.

In certain embodiments, the sample pad is the same pad as the test pad.

In certain embodiments, at least one first binding reagent (e.g., a capture reagent, or in certain embodiments, a first binding reagent such as streptavidin) is bound to the solid support, e.g., in the form of a test line. The region in which at least one first binding agent binds is referred to as the immobilization region or the test region.

In certain embodiments, the lateral flow assay further comprises a conjugate pad comprising one or more labeled detection reagents.

In certain embodiments, the lateral flow assay also contains one or more control zones that contain control elements to monitor the performance of the assay or system.

In a particular embodiment, the sample is a liquid sample or has been diluted to provide a liquid sample. The liquid fluid flow may be used to mix solutions, split solution directions, provide control sample detection, provide directions for analytical detection, or provide sample control detection.

Vertical flow assays are also provided, wherein the sample flows vertically through the assay. The vertical flow sample may include one or more test zones and optionally one or more control zones.

In one embodiment, the sample is added to the sample pad area and the components are separated vertically from the biological sample using a separation membrane, leaving a filtered sample, wherein the filtered sample then flows into the test area.

The assay comprises at least one capture reagent and a detection reagent, but in certain embodiments may comprise a first binding reagent (e.g., streptavidin) to facilitate the production of a test strip or other substrate that is not known to the target analyte. In certain embodiments, the first binding reagent is crosslinked to reduce or prevent its dissociation from the test strip or other solid support.

In one embodiment, the assay is a competitive assay in which the capture reagent binds to the labeled target analyte. Unlabeled target analyte and labeled target compete for binding to capture reagent, and the amount of target analyte bound can be determined by the proportion of labeled target analyte detected. In other embodiments, the assay is a non-competitive assay.

In another embodiment, the assay is a dual binding reagent assay. The dual binding reagent assay includes a capture reagent that binds to the target analyte and a detection reagent that also binds to the target analyte. According to this embodiment, the capture reagent and the detection reagent must recognize two non-overlapping epitopes of the target antigen such that when the first binding reagent binds to the target analyte, the epitope recognized by the second binding reagent is not masked or altered. In one embodiment, the capture reagent binds to a first epitope on the target analyte and the detection reagent binds to a second epitope on the target analyte. Both capture and detection reagents can bind to the target due to the presence of an excessive copy of the site on the target.

In another embodiment, the assay is a three-binding reagent assay. The three-binding reagent assay includes a capture reagent, a detection reagent, and a reporter reagent that bind to the target analyte, wherein the three binding reagents form a detectable complex with the target analyte. Horseradish peroxidase conjugate (e.g., antibody-HRP conjugate).

In a further embodiment, the assay is a four-binding reagent assay. The four-binding reagent assay includes a capture reagent that binds to the target analyte and a detection reagent that also binds to the target analyte, and a first binding reagent that binds to a second binding reagent, wherein the second binding reagent is conjugated to the capture reagent (e.g., a biotinylated antibody). The first binding agent and the second binding agent are a universal binding pair in the sense that neither specifically binds to the target agent. Instead, the first binding reagent is immobilized to a solid support, and the second binding reagent, capture reagent, and detection reagent are added to the system or assay by the user. According to this embodiment, the assay or test strip is universal with respect to the target analyte, i.e., the analyte is agnostic.

In a particular embodiment, the first binding agent comprises multiple binding sites. The first binding site allows binding of the second binding agent, while the additional binding site allows binding to a polymer (e.g., PEG) such that the first binding agent crosslinks with one or more additional first binding agents to reduce or prevent dissociation of the first binding agent from the solid support.

In yet a further embodiment, the assay is a five-binding reagent assay in which a capture reagent is conjugated to a third binding reagent to allow cross-linking with other capture reagents to reduce or prevent dissociation.

(ii) Binding reagent

The systems and assays described herein include at least one binding reagent, and in certain embodiments, multiple binding reagents. In certain embodiments, the binding reagent is specific for the target analyte (e.g., capture reagent, detection reagent), while in other embodiments, the binding reagent is universal with respect to the target analyte (e.g., first binding reagent and second binding reagent).

The at least one binding agent may be different. In one embodiment, the binding reagent (e.g., capture reagent, detection reagent, or universal binding pair component) is selected from an aptamer, an antibody, a nanobody, a protein, a peptide, a nucleic acid, or a combination thereof.

In certain embodiments, the binding reagent (e.g., capture reagent, detection reagent) is specific for an antigenic site on the target analyte.

In one embodiment, the capture reagent and/or detection reagent is an aptamer having a size of about 10-15kDa (20-45 nucleotides), binds its target analyte with at least micromolar affinity, and distinguishes closely related target analytes.

In one embodiment, the capture reagent and/or detection reagent is an aptamer having a size of about 10-15kDa (20-45 nucleotides), binds its target analyte with at least nanomolar affinity, and distinguishes closely related target analytes.

In a particular embodiment, the capture reagent and/or the detection reagent is an aptamer, wherein the Kd of the aptamer to the target molecule is 10nM or less, more preferably 5nM or less, and can be as low as 100pM.

Antibodies suitable for use in the present invention include antisera, polyclonal antibodies, omniclon antibodies, monoclonal antibodies, bispecific antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, fab fragments, F (ab') 2 fragments, fragments generated from Fab expression libraries, epitope-binding fragments and Complementarity Determining Regions (CDRs) of any of these.

In one embodiment, the antibody is a monoclonal antibody. In some other embodiments, the antibody is a polyclonal antibody. In some examples, the polyclonal antibody is an affinity purified polyclonal antibody.

In certain embodiments, the nanobody is a particle, such as a magnetic particle. The size of the particles may vary, but in one embodiment is about 100nm to 100nm and about 50,000nm.

In another embodiment, the system or assay comprises micro magnetic particles.

In a particular embodiment, the binding reagent is produced via a fermentation process.

In one embodiment, the concentration range of the detection reagent and/or capture reagent may be millimolar, submillimolar, micromolar, nanomolar, picomolar, or femtomolar.

In certain embodiments, the detection reagent is labeled, i.e., coupled to an enzyme or substrate. The efficiency or yield of the enzyme-labeled detection reagent may be greater than about 10%, greater than about 50%, greater than about 75%, or greater than about 90%. The enzyme-labeled detection reagent and/or capture reagent may have a purity of greater than about 10%, greater than about 50%, greater than about 75%, or greater than about 90%.

In some examples, the enzyme is capable of changing color upon exposure to a substrate. In some examples, the substrate is capable of changing color upon exposure to a reagent (e.g., an enzyme), respectively. As such, the detection reagent may be labeled with a dye, a metal particle (e.g., gold), a compound capable of producing chemiluminescence or fluorescence. In alternative embodiments, the detection reagent may be attached to magnetic beads, cellulose beads, polymer beads labeled with a dye, affinity probes, or the like.

The affinity of the capture reagent and/or the detection reagent for the target analyte may be different. In one embodiment, the capture reagent and/or detection reagent has about 10 for the target analyte ^-3 To about 10 ^-15 Kd of M.

In another embodiment, the capture reagent and/or detection reagent has a protein to protein ratio of greater than about 10 ^-10 Greater than about 10 ^-8 Or greater than about 10 ^-6 Kd of M.

In one embodiment, the capture reagent and/or detection reagent has a Kd of about 10nM or less, or about 5nM or less, for the target analyte.

In a particular embodiment, the capture reagent and/or the detection reagent has a sub-nanomolar Kd for the target analyte. In certain embodiments, kd is about 100pM.

The affinity of the capture reagent and the detection reagent for the target analyte may be different. In a particular embodiment, the capture reagent has a weaker affinity for the target analyte than the detection reagent. In another particular embodiment, the capture reagent has a stronger affinity for the target analyte than the detection reagent.

In a particular embodiment, the affinity of the capture reagent for the first epitope is greater than the affinity of the detection reagent for the second epitope. The ratio of Kd of the first epitope to Kd of the second epitope may range from 1:10,000 to 10,000:1.

In a particular embodiment, the affinity of the capture reagent for the first epitope is greater than the affinity of the detection reagent for the second epitope. The ratio of Kd of the first epitope to Kd of the second epitope may range from 1:10,000 to 10,000:1.

In a particular embodiment, the capture reagent is an aptamer and the detection reagent is an antibody, and more particularly, a detectably labeled antibody.

In one embodiment, the capture reagent is an aptamer and the detection reagent is an antibody (e.g., a monoclonal antibody), and a third binding reagent, and in particular, an antibody (e.g., a monoclonal antibody), is present.

In one embodiment, the first binding reagent is an antibody and the second binding reagent is an antibody (e.g., a monoclonal antibody), wherein the antibodies are the same or different, or wherein the targets for the antibodies are the same or different, wherein the third binding reagent is an antibody for glucose oxidase.

In certain embodiments, the capture reagent binds to a first site on the target analyte and the detection reagent binds to a second (different) site of the target analyte or molecule, wherein the third binding reagent is an antibody to glucose oxidase.

In certain embodiments, the capture reagent binds to a first site on the target analyte and the detection reagent binds to the same site of the target analyte. Both the capture reagent and the detection reagent can bind to the target due to the presence of an excess of site copies on the target, wherein the third binding reagent is an antibody to glucose oxidase.

In certain embodiments, the binding reagent (e.g., the first binding reagent or capture reagent) is immobilized in or on a solid support such as a bead or membrane using any suitable method, including, for example, depositing, spraying, immersing, pouring, or pouring the capture reagent onto or into the assay membrane.

In one embodiment, the assay comprises a first binding reagent immobilized on a solid support that does not bind to the target analyte, but provides one component of a universal binding pair (e.g., streptavidin or avidin and biotin or a biotin analog, or neutravidin and biotin or a biotin analog) that allows the strip to be manufactured in a manner that is not limited to a particular target analyte. According to this embodiment, the system or assay comprises a first binding reagent, a second binding reagent (collectively, a universal binding pair) that binds to the first binding reagent, a capture reagent conjugated to the second binding reagent, and a fourth labeled detection reagent, wherein the capture reagent and detection reagent bind to a target analyte to produce a detectable complex.

Universal binding pairs (e.g., first binding reagent, second binding reagent, third binding reagent) include, for example, streptavidin and biotin or biotin analogs; avidin and biotin or biotin analogues; gold, silver, malamide, vinyl sulfone and thiol. In certain embodiments, the thiol may be obtained from a cysteine amino acid (possibly on a protein or antibody). Epoxide and thiol chemistry can also be used to provide universal binding pairs, and silane and hydroxyl chemistry can also be utilized.

In certain embodiments, the system or method comprises multiple capture reagent-detection reagent binding pairs that bind to different epitopes on the same target analyte. For example, at least two, at least three, at least four, at least five, or more such capture reagent-detection reagent binding pairs.

The liquid fluid flow through the assay may be used to mix solutions, split solution directions, dilute samples, provide control sample detection, provide directions for analytical detection, or provide sample control detection

(iii) Solid support

The solid support may vary. In certain embodiments, the solid support comprises a strip or other substrate upon which the reaction occurs. The term "strip" or "test strip" also includes kits or devices in which the "strip" has different dimensions and may be referred to as a "card". In certain embodiments, the strap has the dimensions of a bow tie (bow-tie).

The test strip may include an insertion portion and an exposed portion. The exposed portion of the test strip may be arranged to receive a biological sample (e.g., saliva, blood) from a subject.

The strip or substrate may be made of any suitable material. In certain embodiments, the membrane is selected from a polymer (e.g., hydrogel), a metal, a glass fiber or ceramic membrane, cellulose, nylon, cross-linked dextran, or various chromatographic papers.

In one embodiment, the substrate is selected from nitrocellulose (e.g., in the form of a membrane or microtiter well), polyvinyl chloride (e.g., a sheet or microtiter well), polystyrene latex (e.g., a bead or microtiter plate), polyvinylidene fluoride, diazotized paper, fiberglass membrane, nylon membrane, activated beads, or magnetically responsive beads.

In one embodiment, the substrate is an anionic polymer, such as a nitrocellulose membrane. In other embodiments, the substrate is sulfonated tetrafluoroethylene, poly (acrylic acid), or poly (2-acrylamido-2-methyl-1-propanesulfonic acid) (poly AMPS).

The strips or other substrates may be patterned areas comprising, for example, different materials, textures, hydrophobicity, or hydrophilicity.

In a particular embodiment, the assay comprises a crosslinked hydrogel containing a capture reagent (e.g., an aptamer or antibody) or a first binding reagent of a universal binding pair (e.g., streptavidin). The crosslinked hydrogel may be present on or in the test strip.

The assay may comprise one of a plurality of functional zones, which are preferably different and non-overlapping. These may include one or more test zones or one or more control zones.

In certain embodiments, the strip comprises one or more channels selected from a split channel, a parallel channel, or an adjacent channel.

In some examples, the channels may have different lengths, various materials or textures, geometric features, recessed features, patterned features, or recessed chambers.

In a particular embodiment, the membrane also collects a biological sample and provides a sink region to allow the sample to flow from one location to another on the membrane.

The assay may be a multiplexed assay, i.e. allowing detection of two or more target analytes. In particular, the strip may have one or more test zones each comprising at least one binding reagent (e.g., capture reagent, first binding reagent of a universal binding pair) capable of directly or indirectly generating a signal in response to the presence of a particular target analyte.

In other embodiments, multiple binding reagents (e.g., capture reagents) may be present within the same test zone to allow detection of multiple target analytes within a common test zone.

In some areas, the test zone may include a test area or test line "T". The detection zone may further comprise a control zone or control line "C.

In a particular embodiment, the system comprises a second strip, wherein the first and second strips allow detection of different target analytes.

In one embodiment, the color change is used to determine the concentration of at least one target analyte.

In one particular embodiment, an intermediate semi-quantitative colorimetric assay is provided that includes a gradient (deposition density) of a first binding reagent (e.g., streptavidin) on a solid support (e.g., a polymer membrane) so as to provide a range or concentration of bound target analytes.

In some embodiments, the strip may be adapted or manufactured for use in a colorimetric method. In these examples, the electrode may not be utilized (e.g., the working electrode region may not be provided).

In a particular embodiment of the strip, the test area is located over at least one electrode that provides a chamber for determining the current and analyte concentration.

In another embodiment of the strip, the electrodes are positioned at, above or below at least one electrode.

In one embodiment, the test strip includes at least one test site and two or more electrodes (e.g., a working electrode and a reference electrode), and means for establishing a connection between the electrodes and the meter.

In a particular embodiment, the test site is positioned between two electrodes.

In certain embodiments, at least one binding reagent (e.g., a capture reagent, a first binding reagent of a universal binding pair) is bound to the electrode. Direct electrode functionalization may be achieved by any suitable method, for example, thin-film dry-phase-inversion methods using nitrocellulose, methylcellulose, ethylcellulose, hydroxypropylethylcellulose, or any solid fixed structure dissolved or suspended in a solvent and/or water. Deposition on the electrode may be accompanied by prior to further modification with at least one binding reagent, or may be accompanied by co-deposition of at least one binding reagent.

In certain embodiments, the strip comprises two or more electrodes or three or more electrodes. In a particular embodiment, the strip comprises a first set of two or more electrodes and a second set of two or more electrodes, so as to allow a positive control and a negative control.

Any suitable electrode may be utilized. In one embodiment, the electrode is a carbon, iron, palladium, platinum or gold electrode. In another embodiment, the electrode is a (semi) conductive solid.

In a certain embodiment, at least one electrode is coated with a medium. Representative non-limiting mediums include Prussian blue, platinum, ferricyanide, hexacyanoferrate III/hexacyanoferrate II, 1, 10-phenanthroline quinone, quinone imine/phenylenediamine, or osmium-based mediums.

In a particular embodiment, the electrode is carbon or carbon coated with Prussian blue or platinum.

In a particular embodiment, the electrode is porous.

In a particular embodiment, the electrodes are interdigitated.

In certain embodiments, at least one electrode is associated with, e.g., coated with, a gel, wherein a sugar (e.g., glucose) is present within the gel.

In a particular embodiment, a binding reagent (e.g., a first binding reagent, a capture reagent) is incorporated into the carbon-based electrode or bound to the gold electrode.

In certain embodiments, a strip or solid support is inserted into the detection device.

When a voltage is applied between the two electrodes, free electrons can move through the circuit. If necessary, each enzyme and mediator molecule may repeat this transfer again and again. The amount of charge that moves through the circuit will represent the glucose level in the system, which reflects the concentration of the analyte in the sample.

In a particular embodiment, glucose oxidase is used as the enzyme and the electrochemical reaction that occurs is shown in the following:

glucose O ₂ D-glucono-1, 5-lactone +H ₂ O ₂

The oxidation reaction produces an electrical current. The magnitude of this current is directly related to the blood glucose concentration. Oxidation of glucose by Gox results in o-glucono-o-lactone. H at Prussian Blue (PB) film ₂ O ₂ The reduction is measured by electrons transferred from the working electrode. In certain embodiments, the current is continuous, rather than gentle.

In one embodiment, the test area is located below the optical chamber for the development of a color change, and the color change is used to determine the analyte concentration.

In a particular embodiment, the strips may be laminated or contained within a cartridge, such as a disposable cartridge.

In some embodiments, the cassette does not include glucose pods (pod) for glucose storage or delivery.

In a particular embodiment, the strip is part of a disposable article containing a waterproof barrier, waterproof base material such as polyethylene terephthalate (PET).

In one embodiment, the test area may include a bow tie structure to adhere the film material under the waterproof barrier.

The cartridge may include one or more of a reagent chamber, a channel for sample collection material, one or more microfluidic channels, and/or a working electrode region. The cartridge may include a waste pod for waste storage. In some examples, the case may include a hinge configured to place the case in an open position and a closed position. The hinge is actuatable between an open position and a closed position. In the open position, the sample may be in fluid communication with the sample collection material. The channel may include a proximal end having saliva sample collection material.

A cartridge comprising a hinge in a closed position may substantially seal a sample in the cartridge. In some embodiments, the hinge that places the cartridge in the closed position is operated to substantially prevent the cartridge from being opened (e.g., the hinge may operate in an irreversible manner).

In one embodiment, the reagents (e.g., binding reagents) utilized in the test strip are storage stable. In certain embodiments, the reagents used with the test strip are freeze-dried to extend shelf life.

The test strip typically includes layers of conductive and non-conductive constituent components disposed on top of each other to create a sensor structure.

In one embodiment, the test strip includes a base substrate, a conductive layer, an insulating layer, a reagent layer, an adhesive layer, a hydrophilic (e.g., nitrocellulose) membrane to which a first binding reagent (e.g., an aptamer) is attached to capture a target analyte (e.g., an antigen), a freeze-dried, detectably labeled second binding reagent (e.g., ab-GOx), and glucose, and a top layer.

In another embodiment, a test strip includes a base substrate, a conductive layer, an insulating layer, a reagent layer, an adhesive layer, a hydrophilic (e.g., nitrocellulose) membrane to which a first binding reagent (e.g., an aptamer) is attached to capture a target analyte (e.g., an antigen), and freeze-dried glucose, and a labeled second binding reagent (e.g., ab-GOx) is added to a biological sample containing the target analyte.

The base substrate serves as a matrix of a plurality of constituent components which are stacked on top of one another and constitute the functional sensor. The base component may be made of a wide variety of materials having desirable qualities such as dielectric properties, water impermeability, gas impermeability, and gas tightness. Some materials include metal and/or ceramic and/or polymeric substrates, and the like.

A conductive layer is disposed over the base substrate, wherein the conductive layer includes at least one electrode (e.g., one, two, or three electrodes) comprising a conductive material for contacting an analyte to be assayed or a byproduct thereof (e.g., oxygen and/or hydrogen peroxide). The one or more electrodes may include one or more working electrodes and one or more counter, reference and/or counter/reference electrodes.

The electrodes may be screen printed electrodes, for example screen printed using conductive carbon ink. The materials used may vary. Conductive ink compositions useful in the glucose sensor system of the present invention include, but are not limited to, silver, carbon, or mixed conductive inks. Examples of inks that may be used to print the working electrode include, but are not limited to, carbon, platinum, carbon/platinum, carbon nanotubes, or other conductive materials suitable for detecting peroxides in a sample.

The electrodes used and the sensitivity required generally determine the enzyme chemistry that can be employed. For example, the second binding reagent, which is linked to glucose oxidase, requires an excess of glucose to detect the analyte in the sample.

A "working electrode" is an electrode at which an analyte undergoes electrooxidation or electroreduction, with or without the aid of a redox mediator. The working electrode may measure an increase or decrease in current in response to exposure to a stimulus, such as a change in concentration of a target analyte or molecule or by-product thereof. The electrodes provide a detectable signal in the presence of various concentrations of molecules such as hydrogen peroxide or oxygen.

In addition to the working electrode, the conductive layer may also include a Reference Electrode (RE) or a combined reference and counter electrode (also referred to as a quasi-reference electrode or counter electrode/reference electrode).

In one embodiment, the electrode provides a minimum sensitivity of at least about 50 micromolar glucose concentration and a noise level of less than about 100nA to 0.5nA per square millimeter.

The insulating layer may be a film of insulating (e.g., electrically insulating or water impermeable) material including poly (vinyl chloride), polyethylene, polypropylene, aromatic and aliphatic polyurethanes, poly (butylene terephthalate), polybutadiene, silicone rubber, thiol-ene copolymers, or poly (ethylene-co-vinyl acetate)

In certain embodiments, the reagent layer contains a medium that facilitates electron exchange. In one embodiment, the reagent layer includes a binder, silica, ferricyanide, 1, 10-phenanthroline quinone, or osmium-based medium.

The adhesive layer may be an acrylic copolymer including poly (ethyl acrylate), poly (cyanoacrylate), poly (butyl acrylate), poly (2-ethylhexyl acrylate), and urethane acrylate copolymers.

The hydrophilic membrane may be composed of an anionic hydrophilic copolymer comprising nitrocellulose, sulfonated tetrafluoroethylene, poly (acrylic acid) or poly (2-acrylamido-2-methyl-1-propanesulfonic acid (poly AMPS). The membrane may be coated with streptavidin-NC and a first binding reagent (e.g., biotinylated aptamer) attached thereto to act as a capture reagent for the target analyte or molecule.

In one particular embodiment, the cartridge or test strip includes a base substrate, typically made of PET; a conductive layer [8] comprising three electrodes; an insulating layer [6] exposing only part of the electrode in which we will drip the sample to be tested; a reagent layer containing a medium that facilitates electron exchange; [6] an adhesive layer; a hydrophilic nitrocellulose membrane, the proximal membrane containing a first binding reagent (e.g., an aptamer) to capture a target analyte (e.g., antigen) (if not directly functionalized, on 6) and freeze-dried glucose, and the distal end being a paper channel (5); (G) freeze-dried Ab-GOx; (4) top layer.

In a particular embodiment, the cartridge or test strip comprises (a) a base substrate; (B) a conductive layer comprising two electrodes; (C) An insulating layer exposing only a portion of the electrode in which a sample to be tested is dropped; (D) a reagent layer containing a medium facilitating electron exchange; (E) an adhesive layer; (F) A hydrophilic nitrocellulose membrane, the proximal membrane containing a first binding reagent (e.g., an aptamer) to capture a target analyte (e.g., an antigen) and freeze-dried glucose, and the distal end being a paper channel (13); (G) freeze-dried Ab-GOx; (H) top layer.

According to this embodiment, the base substrate is polyester and an acrylic coating is applied to improve ink adhesion. The mask was laser cut onto the base substrate using a CAD model of the electrode mask. Then using conductive carbon oil Ink (Ercon Inc) screen-prints the electrodes followed by an insulating layer (Ercon Inc, instrlayer ink). The two working electrodes will have 0.6mm each ² And the reference electrode will have a surface area of 1.2mm ² . The reagent layer is a dielectric layer and is composed of an adhesive, silicon dioxide, and ferricyanide. This layer was screen printed on the working electrode for two cycles. The adhesive layer on top will be an acrylic copolymer, the hydrophilic membrane will be a nitrocellulose membrane with streptavidin-NC, and bind biotinylated aptamer to capture viral antigen. The top layer may be PET with a small transparent portion to view sample movement on the strip. The overall dimensions will be similar to those described in the patent to ensure compatibility with the reader of the Lifescan, or can be modified to be compatible with other commercial blood glucose meters.

In one embodiment, the drop cast GOx is directly drop cast onto the working electrode prior to the addition of the lyophilized biological agent and aptamer fixation.

In certain embodiments, the test strip components may be pre-blocked by any suitable blocking agent in order to reduce or eliminate non-specific binding. Non-limiting examples of coating materials are proteins, acrylamides, synthetic polymers and polysaccharides. In one embodiment, BSA is used as a blocking agent. In one embodiment, denatured BSA is used as a blocking agent. In another embodiment, milk proteins, TWEEN or other surfactants are used as blocking agents. In another embodiment, BSA, milk protein, casein, triton, SDS, TWEEN, IGEPAL or other surfactants are used as blocking agents.

In certain embodiments, the system allows for low signal-to-noise ratios, such as limiting transient non-glucose related signal noise. The composition of the base layer, the method used to deposit the electrodes, the electrode configuration, the electrode materials, the enzyme chemistry used, and other design factors all contribute to the noise of the system.

In one embodiment, the strip has a shelf life of more than 1 year or more than two years, or more than three years.

(iv) Marking and detection system

One or more binding reagents (e.g., detection reagents) may be bound to the label. The label may be directly attached to the binding agent (e.g., via a covalent bond), or the attachment may be indirect (e.g., using a chelator or linker molecule).

In a particular embodiment, the detection binding reagent is conjugated to a label.

In certain embodiments, the label is directly attached to the capture reagent, enabling direct detection.

Examples of detectable labels include, but are not limited to, biotin/streptavidin labels, nucleic acid (e.g., oligonucleotide) labels, chemically reactive labels, fluorescent labels, enzyme labels, radiolabels, quantum dots, polymer dots, mass labels, colloidal gold, and combinations thereof.

In some embodiments, the label is an enzyme, such as an oxidoreductase, and the at least one target analyte is detected by detecting a product produced by the enzyme. Suitable enzymes include, but are not limited to, oxidases, dehydrogenases, amylases, and invertases.

In one embodiment, where the label is an enzyme, the enzyme may react with a substrate of the enzyme such that upon reaction with the enzyme label, a colored, fluorescent or chemiluminescent substance is produced from the substrate. In a particular embodiment, the colored substrate is selected from the group consisting of o-phenylenediamine (OPD), 3', 5' -Tetramethylbenzidine (TMB), 3 '-diaminobenzidine tetrahydrochloride (DAB), 2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS), and the like.

In a particular embodiment, the enzyme is an oxidase. Representative non-limiting oxidases include sugar oxidases (e.g., glucose oxidase, galactose oxidase, lactate oxidase, and glucose 6-phosphate dehydrogenase), cellobiose oxidase, monoamine oxidase, cytochrome P450 oxidase, NADPH oxidase, heterocyclic oxidases (e.g., uricase and xanthine oxidase), L-gulonolactone oxidase, laccase, lysyl oxidase, polyphenol oxidase, thiol oxidase, and ascorbate oxidase.

In certain embodiments, the enzyme is a dehydrogenase or oxidoreductase (e.g., D-glucose: D-fructose oxidoreductase). Representative non-limiting enzymes include acetaldehyde dehydrogenase; aldehyde dehydrogenase, alcohol dehydrogenase, delta 12-fatty acid dehydrogenase, glutamate dehydrogenase (an enzyme that can convert glutamate to alpha-ketoglutarate and vice versa), lactate dehydrogenase, pyruvate dehydrogenase, fructose dehydrogenase, sucrose dehydrogenase, glucose 6-phosphate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, sorbitol dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, yellow enzyme, glutamate dehydrogenase, 1-phosphoglycerate dehydrogenase.

In one embodiment, the enzyme is horseradish peroxidase (HRP) or catalase.

In one embodiment, the enzyme is selected from the group consisting of PQQ-glucose dehydrogenase, NAD-glucose dehydrogenase and FAD-glucose dehydrogenase.

In one embodiment, an alkaline phosphatase/NBT-BCIP (4-nitrotetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate) assay system is utilized.

In one embodiment, the alkaline phosphatase detection system can be used with the chromogenic substrate p-nitrophenyl phosphate, which produces a soluble product that is readily detectable at 405 nm.

In one embodiment, two or more labels are utilized. In certain embodiments, each label (e.g., a first label attached to a capture reagent, a second label attached to a detection reagent) generates a detectable signal, and the signals (e.g., a first signal generated by the first label, a second signal generated by the second label, etc.) are distinguishable. In some embodiments, the two or more labels comprise the same type of reagent (e.g., a first label that is a first fluorescent agent and a second label that is a second fluorescent agent). In some embodiments, two or more labels (e.g., a first label, a second label, etc.) are combined to produce a detectable signal that is not generated in the absence of the one or more labels.

In some embodiments, at least two, at least three, or at least four binding reagents (e.g., capture reagent, detection reagent, first binding reagent and second binding reagent of a universal binding pair) are each labeled with an enzyme (e.g., first binding reagent is labeled with a first enzyme, second binding reagent is labeled with a second enzyme, etc.), and each binding reagent labeled with an enzyme is detected by detecting a product generated by the enzyme. In some embodiments, all binding reagents are labeled with enzymes, and each enzyme-labeled binding reagent is detected by detecting a product produced by the enzyme.

In some embodiments, two or more labels (e.g., a first label, a second label, etc.) are combined to produce a detectable signal that is not generated in the absence of the one or more labels. For example, in some embodiments, the labels are each enzymes, and the activities of the enzymes combine to generate a detectable signal indicative of the presence of the label (and thus of the protein of each label). Examples of enzymes that combine to generate a detectable signal include coupled assays, such as coupled assays using hexokinase and glucose 6-phosphate dehydrogenase; and chemiluminescent assays for NAD (P) H coupled to glucose 6-phosphate dehydrogenase, beta-D-galactosidase or alkaline phosphatase.

Various methods may be used to covalently bind these labels to the binding reagent. For example, coupling agents such as dialdehydes, carbodiimides, dimaleimides, diimidates, dual nitrogen benzidines, and the like may be used to label binding agents with such labels.

In one embodiment, the enzyme label may be conjugated directly to a binding reagent (capture reagent) that detects the target analyte, or introduced through a second binding reagent (detection reagent) that binds to the capture binding reagent. If the capture reagent is biotin-labeled, it may also be conjugated to a protein such as streptavidin.

In a particular embodiment, the assay utilizes a dual labeling strategy that includes a first enzyme label, such as oxidase (e.g., glucose oxidase), and a second enzyme label, such as oxidoreductase (e.g., HRP). According to this embodiment, the enzyme catalyzes the oxidation of a substrate to form hydrogen peroxide, which is then quantified by an enzymatic reaction with horseradish peroxidase and a dye such as 3,3', 5' -tetramethylbenzidine or TMB, by a change in color. Since the amount of glucose in the biological sample is excessive and is added for detection, the quantification is for the target analyte.

In a particular embodiment, the detection reagent is an antibody. In one embodiment, the antibodies are combined or linked to an enzyme (e.g., glucose oxidase) at a fixed integer ratio (e.g., 2 enzymes/1 antibody or 3 enzymes/1 antibody or 4 enzymes/1 antibody).

In a particular embodiment, the antibody is combined with glucose oxidase to provide an antibody-GOx conjugate. In another embodiment, an alternative conjugation strategy is utilized that uses chemical linkers for site-specific conjugation to Gox, such as non-cleavable thioether and peptide bonds. As shown in fig. 2, the aptamer captures the target analyte (protein) and Ab-GOx binds only when the protein is present. When a constant potential is applied, gox oxidizes glucose, transfers electrons to oxygen, generates hydrogen peroxide, and generates a current output via an electrode that reacts with hydrogen peroxide.

In a particular embodiment, the antibody is combined with an oxidase to provide a polypeptide consisting of galactose oxidase, D-glucose: antibody-Ox conjugates prepared with D-fructose oxidoreductase and cellobiose oxidase.

In a particular embodiment, the antibody is combined with a dehydrogenase to provide an antibody-DH conjugate prepared from a glucose dehydrogenase, a glucose 6-phosphate dehydrogenase, a fructose dehydrogenase, a sucrose dehydrogenase, a glucoside dehydrogenase, an alcohol dehydrogenase, a sorbitol dehydrogenase, a lactate dehydrogenase, and a malate dehydrogenase.

In a particular embodiment, the detection reagent antibody is combined with glucose oxidase to provide an antibody-GOx conjugate, and the third binding reagent antibody is combined with horseradish peroxidase to provide an antibody-HRP conjugate. In another embodiment, an alternative conjugation strategy is utilized that uses a chemical linker for site-specific conjugation to Gox or HRP, such as non-cleavable thioether and peptide linkages. In embodiments of the three-binding reagent assay, the first binding reagent and the second binding reagent may be specific for at least one protein, wherein the third binding reagent is an antibody linked to horseradish peroxidase.

Representative non-limiting colorimetric labels that can be used in the systems and assays described herein include colored latex (polystyrene) particles, colored polymer particles, colored cellulose particles, metal (e.g., gold) sols including gold nanoparticles, nonmetallic element (e.g., selenium, carbon) sols, and dye sols.

D. Detection device

Any suitable method of detecting a signal may be utilized. In certain embodiments, the detection device is a portable (e.g., hand-held), battery-powered device.

In certain embodiments, the detection device allows for analysis of the analyte in the sample by, for example, coulometry, amperometry, and/or potentiometry.

In one embodiment, the device is selected from a current device, a coulomb device, a potential device, or a voltammetric device.

The device may have multiple electrodes, for example at least two, at least three, at least four, at least five, at least six or more electrodes.

In one embodiment, the electrode is unmodified. In other embodiments, the electrodes are modified, for example, using metal (oxide) NPs, polymers, and other carbonaceous materials

In one embodiment, the device includes three (3) electrodes, including a working electrode, a counter electrode, and a reference electrode.

In one embodiment, the device measures reactant or product concentrations, such as hydrogen peroxide concentration produced or oxygen concentration consumed.

In another embodiment, the device is based on the use of redox mediators (Mox and MRED). According to this embodiment, the concentration of the analyte involved in the reaction is related to the response to oxidation or reduction of the medium

In yet another embodiment, the device allows for direct electron transfer between the GOx-FADH 2-nanomaterial conjugate and the electrode. According to this embodiment, the analyte concentration is proportional to the redox current generated at the polarized electrode set at a low operating potential (typically close to the reversible redox potential of the enzyme) without the need for a mediator.

In embodiments where the signal is electrochemical, the detection device may be an electrochemical device capable of performing a current measurement or a potentiostat-based measurement tool.

In a particular embodiment, the detection device is a blood glucose meter or a personal blood glucose meter (PGM). Conventionally, PGM is a portable hand-held device for measuring blood glucose levels of users suffering from type I or type II diabetes. Typically, the user purchases a small bar (about 20-30mm x about 5-9 mm) connected to the PGM. A user draws a small amount of blood (a few microliters) from a finger or other area using a lancet (separator), applies a drop of blood sample onto the exposed end of the strip, and then inserts the connection end of the strip into the PGM connection port. Chemical reactions occur between the blood sample and the chemicals on the strip, which are measured by PGM to determine blood glucose levels in mg/dL, mmol/L or Kg/L. After repeated measurements of blood glucose levels, the used test strips are removed from the PGM and new test strips are loaded into the connection ports.

In one embodiment, the blood glucose meter in the systems and methods herein is a standard commercially available hand-held blood glucose meter. Non-limiting examples of commercially available blood glucose meters include Accu (Roche Diabetes Care，Inc.，Indianapolis，Indiana)，Van/>(AgaMatrix，Salem，NH)，Wavesense/>(AgaMatrix，Salem，NH)，/>(Assensia, basel, switzerland),(Assensia, basel, switzerland),>(Abbott Diabetes Care Inc.Abbott Park，Ill)，/>(Trividia Health，Fort Lauderdale，Florida)。

in certain embodiments, the glucose meter is a limited use or disposable glucose meter or chronoamperometric device.

Blood glucose meters typically include a base unit that houses the control and test electronics necessary to test the blood glucose level in a blood sample. In other embodiments, the glucose meter has been modified in one or more ways to enhance functionality for detecting analytes in general or analytes from saliva.

In one particular embodiment, the detection device is a blood glucose meter or chronoamperometric device having a base unit with a cartridge or test strip well, and a reader configured to analyze a biological sample (e.g., saliva sample). In one embodiment, the glucose meter measures a glucose signal (e.g., quantitatively). The base units may differ in shape and size. The test strip slot is configured to accept a glucose-type test strip or cartridge, such as those described herein, that can be removably inserted into the test strip slot. The glucose meter may also have means for storing data and transmitting data.

Glucose measurement may be performed by standard amperometric detection of glucose using glucose oxidase. In this embodiment, a sensor is used to convert the glucose concentration in the biological fluid into a voltage or current signal. The sensor uses platinum and silver electrodes to form part of the circuit in which hydrogen peroxide is electrolyzed. Hydrogen peroxide is generated by oxidation of glucose on the glucose oxidation film. The current flowing through the circuit provides a measure of the hydrogen peroxide concentration, giving a glucose concentration.

Glucose measurement may be performed by standard amperometric detection of glucose using glucose oxidase. In this embodiment, a sensor is used to convert the glucose concentration in the biological fluid into a voltage or current signal. The sensor uses carbon electrodes to form part of the circuit in which hydrogen peroxide is electrolyzed. Hydrogen peroxide is generated by oxidation of glucose on the glucose oxidation film. The current flowing through the circuit provides a measure of the hydrogen peroxide concentration, giving a glucose concentration.

In certain embodiments, the system includes one or more signal processing applications or electronic amplifiers in the circuit to amplify the signal.

In another embodiment, signal collection and processing may be achieved via a static or pulsed process, wherein the pulses wait from about 1 second to about 5 minutes, about 1 second to about 2 minutes, about 2 seconds to about 1 minute, or about 2 seconds to about 30 seconds.

In one embodiment, the apparatus includes a display unit for displaying the results. The display may show the most recent test and, optionally, the previous test. In certain embodiments, the blood glucose meter includes voice control functions to facilitate use by visually impaired subjects. The glucose meter may include other features unrelated to glucose measurement, such as measurement of other physiological functions. The glucose reading displayed on the glucose meter will be positively correlated with the enzyme concentration on the sensor surface, which in turn is correlated with the number of analytes (e.g., proteins present in the biological sample).

The glucose meter may have a software element. Various software algorithms for blood glucose meters are known.

In one embodiment, the glucose meter has a wireless transmitter configured to communicate a message to a second device, such as a mobile device, e.g., a cell phone or tablet. In one embodiment, the message is sent to the second device via a short range communication protocol, such as the bluetooth protocol. The message may also be, for example, a text message or an email.

In one embodiment, the blood glucose meter produces results rapidly after the test has begun, for example, less than about 5 minutes, less than about 1 minute 30 seconds, less than about 15 seconds, or less than about 5 seconds.

The accuracy of the glucose meters may vary, but generally does not exceed an error of 20%, and more particularly, does not exceed an error of about 15%, about 10%, about 5%, or less than about 5%, such as an error of about 4%, about 3%, about 2%, or about 1% or less. In certain embodiments, the cross-sensitivity of the glucose meter is reduced or limited based on experimental determination and validation of new correction factors. In one embodiment, the accuracy of the glucose meter ranges from about 85% to about 95%.

In one embodiment, the glucose meter allows the user to save the latest test values and calculate a two (2) week blood glucose average, allowing monitoring over time.

In certain embodiments, improved chronoamperometry methods are disclosed that are different from either a) constant chronoamperometry (applying a potential, e.g., a few minutes, while obtaining a current), or b) delayed chronoamperometry (allowing a substrate to be incubated on an electrode for a period of time, e.g., a few minutes, and then subjected to a constant chronoamperometric investigation, e.g., a few minutes, while obtaining a current), allowing for faster and/or more sensitive measurements (e.g., providing a lower detection limit) associated with the detection of a target analyte.

This improved chronoamperometric method is used to collect signals (current, charge), increase signals, improve signal-to-noise ratio, improve sensitivity (e.g., limit of detection), reduce signal time, multiplex on multiple working electrodes, and/or reduce background. Variables include, but are not limited to, forcing potential, pulse time, pre-measurement delay, measurement time, open circuit time, number of cycles, measurement sampling rate, and the like. Other variables will be known to those skilled in the art.

In certain embodiments, a compound or counterion (e.g., mgCl ₂ ) A modified chronoamperometric method (e.g., pulse detection) of titration combination.

In certain embodiments, the glucose meter is "display-less" in order to minimize the complexity and cost of the meter unit. According to this embodiment, the glucose meter is wirelessly enabled and the result or readout is sent to a second device, such as a cell phone or personal computer.

Optionally, the glucose meter further comprises a transmitter configured to wirelessly transmit data regarding the analysis result encoded within the audio signal, and a controller configured to facilitate encoding.

Also disclosed herein are remote computing devices and remote computing devices that are glucose meters that may be used in the systems and methods herein. In one embodiment, the remote communication device may be, for example, a smart phone or any other suitable device, such as a communication device, and it may constitute an output device.

The glucose meter transmits the measurement to the remote computing device by a transmitter unit, for example, via a wireless audio-based channel.

The remote computing device may further communicate information to a remote device, such as a central repository device to recipient list, over a network, such as an internet-based or mobile-based device. For example, the detection device may transmit medical data through a remote computing device. Thereafter, the data may be transmitted to a remote care provider, for example, via a computer or handheld device, such as a smart phone.

In this embodiment, a software algorithm is disclosed that triggers an electrochemical reaction in the detection system such that one or more detectable chemical species is the reaction product of the cartridge or test strip and the biological sample within the detection device.

In one embodiment, the mathematical operation of the algorithm's limitation calculation is performed on the detection device such that the chemical reaction providing the detectable reaction product is performed between the biological sample, cartridge or test strip component and the detection device.

In one embodiment, the mathematical operation is performed using cloud computing on a server in a physical location external to the location of the detection device such that a chemical reaction providing a detectable reaction product is performed between the biological sample, cartridge or test strip component and the detection device.

In one embodiment, a data card containing additional algorithms not originally programmed on the test device is inserted into a data card slot on the test device such that a chemical reaction that provides a detectable reaction product is performed between the biological sample, cartridge, or test strip component and the test device.

In one embodiment, a non-transitory computer readable storage medium encoded with executable instructions for execution by a processor to detect a target analyte is disclosed.

In embodiments where the signal is optical, e.g. colorimetric, the detection means may be a camera or a mobile phone.

The detection means may detect, for example, hue, intensity, fluorescence, electrons, voltage variations, impedance variations, etc.

In certain embodiments, the signal may be detected without a detection device, i.e., the signal may be visually detected.

In certain embodiments, detection may involve the formation of a precipitate, e.g., binding to the precipitate via an alkaline phosphatase-mediated reaction resulting in NBT-BCIP.

In one embodiment, the glucose oxidase catalyzes the oxidation of glucose to form hydrogen peroxide, which is then quantified by amperometric measurement (e.g., change in current) via one or more electrodes. Since the amount of glucose in the sample is excessive, the amperometric quantification is directed against the target analyte.

In certain embodiments, the results are recorded as a loss of detectable complex. In other embodiments, the results are recorded as the gain of the detectable complex.

The detection device may optionally comprise a collection chamber for receiving the sample and/or a diluter unit.

The detection reader or electrochemical detection reader may include a housing or casing that houses the computing device and a display. The display may include a Liquid Crystal Display (LCD) display, a gas plasma based flat panel display, an Organic Light Emitting Diode (OLED) display, an electrophoretic ink (E-ink) display, an LCD projector, or other type of display device.

The detection reader may be adapted to removably accept a cartridge or strip. In some examples, the detection reader may include a receptacle instrument disposed within the housing and

the detector means may comprise data storage, bluetooth, wireless capability, transmission capability, etc. The system may comprise an electronic device, a database or a cloud server for receiving information about the signal from the detection device.

In certain embodiments, the system includes an algorithm that triggers electrochemical reaction monitoring in the detection system. In certain embodiments, the system includes an algorithm that determines the concentration of at least one analyte. The algorithm may be localized or cloud-based.

According to an example, applications or other functionality may be executed in a network environment. The network environment may include a detection device and one or more client devices that communicate via a network. The network may include the internet, one or more intranets, extranets, wide Area Networks (WANs), local Area Networks (LANs), wired networks, wireless networks, or any combination of two or more such networks. The network may include satellite networks, cable networks, ethernet networks, cellular networks, and telephone networks.

The detection device may execute detection applications for detection (e.g., current detection), as well as other applications, services, processes, systems, engines, or functionalities not discussed in detail herein. Detection devices including detection applications may include, provide, or perform other forms of electrochemical characterization such as impedance spectroscopy, and/or optical characterization (e.g., via spectrophotometry, intensity, fluorescence, chemiluminescence, UV/Vis). One or more measurements may be stored in a data store (e.g., based on a time record). The data transmitted via the network may be qualitative or quantitative. Test results (raw, processed, etc.), control data, and potentially other types of data may be stored or transmitted.

The client device may include a processor-based system, such as a computer system, which may include a desktop computer, a laptop computer, a personal digital assistant, a cellular telephone, a smart phone, a tablet computer system, an IoT device, or any other device with similar capabilities.

In some examples, a system is provided. The system may comprise a detection means comprising a computing means. The detection reader may be adapted to removably receive the cartridge. The system may include program instructions executable in the computing device that, when executed by the computing device, cause the computing device to, among other things, detect insertion of the cartridge. The program instructions may cause actuation of an actuator arm of the test reader or the electrochemical test reader, thereby causing glucose to be released from the glucose pod.

The program instructions may generate output data. The output data may include data calibrated for the presence or concentration of a target analyte within the biological sample, which may not be glucose. The program instructions may cause the output data to be presented on a display of the detection reader or cause a user interface to be generated and sent to the client device to present the output data on the display of the client device.

Many of the software components are stored in a memory of a computing device and are executable by a processor. In this regard, the term "executable" means a program file in a form that may ultimately be run by a processor. An example of an executable program may be a compiled program, which may be translated into machine code in a format that can be loaded into a random access portion of memory and executed by a processor, source code that may be expressed in a suitable format, such as object code that can be loaded into a random access portion of memory and executed by a processor, or source code that can be interpreted by another executable program to generate instructions to be executed by a processor in a random access portion of memory. The executable program may be stored in any portion or component of memory, including Random Access Memory (RAM), read Only Memory (ROM), hard disk drive, solid state drive, USB flash drive, memory card, compact disk such as Compact Disk (CD) or Digital Versatile Disk (DVD), floppy disk, magnetic tape, or other storage component.

The memory may include volatile memory and nonvolatile memory and data storage components. Volatile components are those that do not retain data values when powered down. Nonvolatile components are those that retain data when powered down. Thus, the memory may include Random Access Memory (RAM), read Only Memory (ROM), hard disk drives, solid state drives, USB flash drives, memory cards accessed via a memory card reader, floppy disks accessed via an associated floppy disk drive, optical disks accessed via an optical disk drive, magnetic tape accessed via an appropriate tape drive, and/or other memory components, or a combination of any two or more of these memory components. In addition, RAM may include Static Random Access Memory (SRAM), dynamic Random Access Memory (DRAM), or Magnetic Random Access Memory (MRAM), and other such devices. The ROM may include programmable read-only memory (PROM), erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), or other similar memory devices. In addition, a processor may represent multiple processors and/or multiple processor cores, and a memory may represent multiple memories that operate in parallel processing circuits, respectively.

Although the detection reader, electrochemical detection reader, and any applications or services described herein may be embodied in software or code executed by specially configured or programmed general-purpose hardware as discussed above, it may alternatively be embodied in dedicated hardware or a combination of software/general-purpose hardware and dedicated hardware. If embodied in dedicated hardware, each may be implemented as a circuit or state machine that employs any one or combination of various techniques. These techniques may include, but are not limited to, discrete logic circuits with logic gates for implementing various logic functions upon application of one or more data signals, application Specific Integrated Circuits (ASICs) with appropriate logic gates, field Programmable Gate Arrays (FPGAs), or other components. Such techniques are generally well known to those skilled in the art and thus are not described in detail herein.

In one embodiment, the assay comprises a lateral flow assay and an electrochemical detection system. In a particular embodiment, the lateral flow assay comprises at least one target binding site. Optionally, at least one target binding site on the membrane is placed at, above or below the electrode. Optionally, the lateral flow assay further comprises at least one control site comprising at least one control element in order to monitor the performance of the system. Optionally, a control site may be placed over the other electrode.

In a particular embodiment, electrochemical detection is performed only upon insertion of the strip into the electrochemical device, providing a differential voltage and detecting the current output provided by the strip and accompanying electrodes.

II methods of use

Methods of detecting at least one target analyte using the systems and assays disclosed herein are disclosed. Also disclosed are methods of treating a subject using the systems and assays disclosed herein, as well as methods of making the systems and assays disclosed herein.

In certain embodiments, the methods may be performed in a number of environments, including home, office, job site, or resource limited environments, with the low detection limits and high accuracy required to be truly useful in terms of analyte detection. POC testing may be necessary for rapid detection of disease at an early stage to facilitate better disease diagnosis, monitoring and management. In other embodiments, the method may be performed in a healthcare environment, such as a clinic or emergency room.

Detection of the analyte is performed between 5 and 30 ℃. In a particular embodiment, the detection of the analyte is performed between 17 ℃ and 25 ℃.

In one embodiment, the method comprises (i) obtaining a sample; (ii) optionally, treating the sample; (iii) adding the sample to the system disclosed herein; (iv) Allowing the target analyte (if present) to bind to the capture binding reagent, thereby generating a signal, and (v) detecting the presence of the target analyte in the sample by detecting the signal. In certain embodiments, diagnosis is possible when the concentration of the target analyte is higher than a reference value, as indicated by the signal.

In one embodiment, a sample preparation method is provided that includes (i) obtaining a sample; (ii) treating the sample; (iii) adding the sample to a system or assay disclosed herein; (iv) Allowing the target analyte (if present) to bind to the capture reagent, thereby generating a signal, and (v) detecting the presence of the target analyte in the sample by detecting the signal.

(ii) May be different in the above embodiments. The treatment may be completed prior to adding the sample to the system or to the assay. In one embodiment, the processing includes diluting the sample.

In embodiments, the treatment comprises adding one or more assay components or reagents to the sample. In a particular embodiment, the treatment comprises adding capture reagents and/or detection reagents to the sample prior to adding the sample to the assay.

In a particular embodiment, the treatment comprises adding a biotin-conjugated capture reagent (e.g., an aptamer or antibody) and a labeled detection reagent (e.g., an aptamer or antibody) to the system or assay disclosed herein.

In one embodiment, the treatment comprises adding a substrate (e.g., an enzyme substrate) to the system or assay disclosed herein. The substrate may be, for example, sucrose, fructose, maltose, galactose, cellulose, or any combination comprising an enzyme, oxidase, amylase, or invertase. The concentration of sugar may vary. In one embodiment, the sugar is present at a concentration of 0.01mM to 5M, and more specifically, about 0.3 to about 0.8, and even more specifically, about 0.05M. In other embodiments, glucose is present within the strip, as opposed to being added by the user.

In one embodiment, the target analyte is mixed with a substrate (e.g., glucose) and a dye such as 3,3', 5' -Tetramethylbenzidine (TMB). In a particular embodiment, the target analyte is admixed with glucose at a concentration of about 0.01mM to about 5M and TMB at a concentration of about 0.001mM to 5M.

In other embodiments, the target analyte is mixed with horseradish peroxidase at a concentration of about 0.0000001mM to about 1M.

In other embodiments, the target analyte is mixed with catalase at a concentration of about 0.0000001mM to about 1M.

In a particular embodiment, the allowing in (iii) comprises an incubation period, for example an incubation period of about a few seconds to about 10 minutes.

In a particular embodiment, the method may further comprise one or more washing steps, e.g. after the incubation period and before the detection period, wherein a washing step is required for removing or wicking some portion of the solution or removing any unbound sample. In some embodiments, the method comprises less than five washing steps, less than three washing steps, less than two washing steps, or one washing step.

In certain embodiments, analysis of the chronoamperometric collected data enables distinguishing samples containing the analyte from control samples containing no analyte. For example, the area of the curve, the end point measurement, the initial slope of the curve, the derivative of the curve, the point in time or points selected before the data platform are collected, and the differences between the measured samples are revealed.

In one embodiment, a method for treating a subject is provided, comprising (i) providing a biological sample (e.g., saliva) from the subject, (ii) optionally, treating the sample; (iii) adding the sample to a system or assay disclosed herein; (iv) If a target analyte (e.g., whole virus) is present, detecting the target analyte to provide a result, and (v) if necessary, administering an approved therapeutic agent to the subject, thereby treating the subject.

In a particular embodiment, the results are calibrated for a disease state (e.g., infection) or health state. In a particular embodiment, the result is associated with infection by a virus, bacterium, fungus, or other microorganism. In certain embodiments, the result is correlated with the presence of an allergen. In other embodiments, the result is associated with inflammation, cancer, or heart disease.

In a particular embodiment, the detection in (iv) is via a blood glucose meter or a mobile phone.

Approved therapeutic agents may vary. In one embodiment, the approved therapeutic agent is a small molecule agent (e.g., antiviral agent) or a biological agent (e.g., protein, antibody, therapeutic vaccine).

In a particular embodiment, the therapeutic agent is an antiviral agent.

In one embodiment, the antiviral agent is an adhesion inhibitor, an entry inhibitor, a uncoating inhibitor, a protease inhibitor, an integrase inhibitor, a nucleoside or nucleotide reverse transcriptase inhibitor, or a replication or transcription complex blocker.

In a particular embodiment, the therapeutic agent is an anti-inflammatory agent.

In one embodiment, the anti-inflammatory agent is a non-steroidal anti-inflammatory drug (NSAID). In a particular embodiment, the anti-inflammatory agent is a derivative of acetic acid, anthranilic acid, alkenoic acid, or propionic acid. In one embodiment, the anti-inflammatory agent is selected from celecoxib, naproxen, meloxicam, nabumetone, oxaprozin, and piroxicam.

In a particular embodiment, the therapeutic agent is an anticancer agent.

In one embodiment, the anticancer agent is selected from alkylating agents (or alkylating-like agents), antimetabolites, antitumor antibiotics, mitotic inhibitors, protein kinase inhibitors, plant alkaloids, hormonal agents, topoisomerase inhibitors (e.g. topoisomerase I inhibitors, topoisomerase II inhibitors) and the like.

In a particular embodiment, the anticancer agent is selected from alkylating agents selected from mustard derivatives, ethyleneimine, alkyl sulfonates, hydrazine, triazines, nitrosoureas (nutrosuea) or metal salts.

In a particular embodiment, the anti-cancer agent is an antimetabolite selected from the group consisting of a folic acid antagonist, a pyrimidine antagonist, a purine antagonist, or an adenosine deaminase inhibitor.

In a particular embodiment, the anticancer agent is an antitumor antibiotic selected from the group consisting of anthracyclines, chromomycins, and the like.

In certain embodiments, the anticancer agent is selected from cyclophosphamide, nitrosourea, cisplatin, methotrexate, cytarabine, 5-fluorouracil, doxorubicin, daunorubicin, bleomycin, vincristine, vinblastine, vindesine, or a combination thereof.

In one embodiment, the method further comprises transmitting the results to a third party for diagnosis and optionally prescribing an approved therapeutic agent.

In one embodiment, if the treatment regimen does not produce a detectable improvement in one or more symptoms or clinical measures of the disease (e.g., a decrease in virus count over a defined period of time, e.g., several days), the treatment may be discontinued, an alternative treatment regimen turned to, or in some embodiments, the treatment regimen supplemented with a second treatment regimen.

In one embodiment, a method comprises (i) providing a biological sample from a subject, wherein the biological sample is blood; (ii) Adding a biological sample to a test strip, wherein the strip contains a first binding reagent and a second binding reagent capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) introducing the test strip into a blood glucose meter or similar device; (iv) incubating the biological sample with the test strip; (v) Detecting the level of a detectable complex, if any, in the form of hydrogen peroxide generated from the oxidation of glucose in the presence of excess glucose; and (vi) calibrating the level of detectable complex (if produced) with the amount of target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, a method comprises (i) collecting a biological sample from a subject, wherein the biological sample is urine, saliva, sweat, ocular fluid including aqueous humor, blood, fecal matter, sebum, respiratory tract droplets, semen, vaginal mucus, cerumen, epidermal cells, nasal cavity samples, cerebrospinal fluid, pleural effusion, nasopharyngeal samples, or a combination thereof; (ii) wiping the biological sample; (iii) Adding a biological sample to the strip, wherein the test strip contains a first binding reagent and a second binding reagent capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iv) Introducing the test strip into a blood glucose meter or similar chronoamperometric device; (v) incubating the biological sample with the test cartridge or strip; (vi) Detecting the level of a detectable complex, if any, in the form of hydrogen peroxide generated from the oxidation of glucose in the presence of excess glucose; and (vii) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method comprises (i) collecting a biological sample from a subject in a tube that dilutes the biological sample by 1X to 1,000,000,000X and contains a second binding reagent, wherein the biological sample is blood; (ii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) Introducing the test strip into a blood glucose meter or a chronoamperometric device; (iv) incubating the biological sample with the test strip; (v) Detecting the level of a detectable complex, if any, in the form of hydrogen peroxide generated from the oxidation of glucose in the presence of excess glucose; and (vi) calibrating the level of detectable complex (if produced) with the amount of target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method comprises (i) collecting a biological sample from a subject in a tube diluted 1X to 1,000,000,000X and containing a second binding agent, wherein the biological sample is urine, sweat, ocular fluid including aqueous humor, blood, fecal matter, sebum, respiratory droplets, semen, vaginal mucus, cerumen, epidermal cells, or nasopharyngeal samples; (ii) wiping the biological sample; (iii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iv) Introducing the test strip into a blood glucose meter or a chronoamperometric device; (v) incubating the biological sample with the test strip; (vi) Detecting the level of a detectable complex, if any, in the form of hydrogen peroxide generated from the oxidation of glucose in the presence of excess glucose; and (vii) correlating the level of detectable complex (if produced) with the amount of target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, a method comprises (i) providing a biological sample from a subject, wherein the biological sample is blood; (ii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent that competes with a target analyte conjugated to glucose oxidase, which is capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) introducing the test strip into a blood glucose meter or similar device; (iv) incubating the biological sample with the test strip; (v) Detecting the level of a detectable complex, if any, in the form of hydrogen peroxide generated from the oxidation of glucose in the presence of excess glucose; and (vi) calibrating the level of detectable complex (if produced) with the amount of target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, a method comprises (i) collecting a biological sample from a subject, wherein the biological sample is urine, saliva, sweat, ocular fluid including aqueous humor, blood, fecal matter, sebum, respiratory tract droplets, semen, vaginal mucus, cerumen, epidermal cells, nasal cavity sample, cerebrospinal fluid, pleural effusion, or nasopharyngeal sample; (ii) wiping the biological sample; (iii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent that competes with a target analyte conjugated to glucose oxidase, which is capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iv) Introducing the test strip into a blood glucose meter or a chronoamperometric device; (v) incubating the biological sample with the test strip; (vi) Detecting the level of a detectable complex, if any, in the form of hydrogen peroxide generated from the oxidation of glucose in the presence of excess glucose; and (vii) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method comprises (i) collecting a biological sample from a subject in a tube that dilutes the biological sample by 1X to 1,000,000,000X and contains a second binding reagent, wherein the biological sample is blood; (ii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent that competes with a target analyte conjugated to glucose oxidase, which is capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) introducing the test strip into a blood glucose meter or similar device; (iv) incubating the biological sample with the test strip; (v) Detecting the level of a detectable complex, if any, in the form of hydrogen peroxide generated from the oxidation of glucose in the presence of excess glucose; and (vi) correlating the level of detectable complex (if produced) with the amount of target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method comprises (i) collecting a biological sample from a subject in a tube diluted 1X to 1,000,000,000X and containing a second binding agent, wherein the biological sample is urine, sweat, ocular fluid including aqueous humor, blood, fecal matter, sebum, respiratory droplets, semen, vaginal mucus, cerumen, epidermal cells, or nasopharyngeal samples; (ii) wiping the biological sample; (iii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent that competes with a target analyte conjugated to glucose oxidase, which is capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iv) Introducing the test strip into a blood glucose meter or a chronoamperometric device; (v) incubating the biological sample with the test strip; (vi) Detecting the level of a detectable complex, if any, in the form of hydrogen peroxide generated from the oxidation of glucose in the presence of excess glucose; and (vii) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method includes obtaining multiple test results obtained at different times with respect to the same user and comparing the results to monitor or predict or track the likely progression of a disease or condition. In a particular embodiment, the method comprises obtaining at least two, at least three, at least four, or at least five test results.

In some embodiments, one or more results of the method may be transmitted to a remote entity continuously or periodically to determine whether the one or more results are above a threshold level or cut point.

In certain embodiments, the results may be compared to a predetermined reference level. The predetermined level may be obtained from a general population or a selected population of subjects. For example, the selected population may be comprised of apparently healthy patients, such as individuals who have not previously had any signs or symptoms indicative of the presence of a disease, such as an infection. The "predetermined reference level μ" may be determined, for example, by determining the expression level of the target analyte in a corresponding biological sample obtained from one or more control subjects (e.g., not suffering from an infection or known to be not susceptible to such a disease). When such predetermined reference levels are used, a higher or increased level determined in a biological sample (i.e., a test sample obtained from a subject) is indicative of, for example, the patient being at risk of developing a disease

Optionally, the method may further comprise a step of advice or guidance regarding the treatment and/or administration of the treatment. In one embodiment, the method comprises identifying a subject as having a target analyte level above a cutoff level threshold, and determining that the subject is thus a candidate for preventing and/or treating, for example, an infection or pathological condition. The "determining" step encompasses detection or quantification, wherein "detecting" means determining whether the target analyte is present in the biological sample, and "quantifying" means determining the amount of the target analyte present in the biological sample.

The method of the invention may have therapeutic use, for example it may be used to detect various pathological conditions or may be used to monitor the disease stage of a subject or its response to treatment.

In certain embodiments, the method may further comprise using statistical methods to predict the likelihood that detection of the target analyte results in a disease or disease progression and/or allows for disease prognosis (i.e., predicts the course of a disease).

In certain embodiments, the method may be performed across a group of patient populations, e.g., to allow stratification of their treatment methods or to meet public health or other monitoring objectives.

In one embodiment, a method for monitoring the efficiency of a therapeutic regimen in a subject having a pathological condition is disclosed, comprising using the methods and/or systems disclosed herein, wherein the target molecule is an antigen associated with the pathological condition, and wherein the amount of the detectable moiety is indicative of the level of the pathological condition, and thereby the efficiency of the therapeutic regimen in the subject.

In certain embodiments, the method includes monitoring the effectiveness of one or more therapeutic agents (e.g., antiviral agents, anticancer agents, etc.) over a period of time (e.g., days, weeks), and if the therapeutic agent is not sufficiently effective over a period of time, allowing the user to seek alternative treatment methods.

In one embodiment, if the treatment regimen does not produce a reduction in condition for a defined period of time (e.g., days), the user may interrupt the treatment regimen, switch to an alternative treatment regimen, or in some embodiments, supplement the treatment regimen with a second treatment regimen. In one embodiment, the system allows two or more results, three or more results, or five or more results to be obtained for the same user at different times with respect to the amount of target analyte to allow monitoring of trends in analyte levels over time.

In one embodiment, a method comprises (i) providing a biological sample from a subject, wherein the biological sample is blood; (ii) Adding a biological sample to a test strip, wherein the strip contains a first binding reagent and a second binding reagent capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) Incubating the detectable complex with horseradish peroxidase and a dye, such as 3,3', 5-tetramethylbenzidine, on the strip; (iv) Detecting the level of the detectable complex (if any) in the form of a color change, and (v) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, a method comprises (i) collecting a biological sample from a subject, wherein the biological sample is urine, saliva, sweat, ocular fluid including aqueous humor, blood, fecal matter, sebum, respiratory tract droplets, semen, vaginal mucus, cerumen, epidermal cells, nasal cavity sample, cerebrospinal fluid, pleural effusion, or nasopharyngeal sample; (ii) wiping the biological sample; (iii) Adding a biological sample to the strip, wherein the test strip contains a first binding reagent and a second binding reagent capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iv) Incubating the detectable complex with horseradish peroxidase and a dye, such as 3,3', 5-tetramethylbenzidine, on the strip; (v) Detecting the level of detectable complex (if any) in the form of a color change, and (vi) correlating the level of detectable complex (if produced) with the amount of target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method comprises (i) collecting a biological sample from a subject in a tube that dilutes the biological sample by 1X to 1,000,000,000X and contains a second binding reagent, wherein the biological sample is blood; (ii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) Incubating the detectable complex with horseradish peroxidase and a dye, such as 3,3', 5-tetramethylbenzidine, on the strip; (iv) Detecting the level of detectable complex (if any) in the form of a color change, and (v) correlating the level of detectable complex (if produced) with the amount of target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method comprises (i) collecting a biological sample from a subject in a tube diluted 1X to 1,000,000,000X and containing a second binding agent, wherein the biological sample is urine, sweat, ocular fluid including aqueous humor, blood, fecal matter, sebum, respiratory droplets, semen, vaginal mucus, cerumen, epidermal cells, or nasopharyngeal samples; (ii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) Incubating the detectable complex with horseradish peroxidase and a dye, such as 3,3', 5-tetramethylbenzidine, on the strip; (iv) Detecting the level of the detectable complex (if any) in the form of a color change, and (v) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, a method comprises (i) providing a biological sample from a subject, wherein the biological sample is blood; (ii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent that competes with a target analyte conjugated to glucose oxidase, which is capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) Incubating the detectable complex with horseradish peroxidase and a dye, such as 3,3', 5-tetramethylbenzidine, on the strip; (iv) Detecting the level of the detectable complex (if any) in the form of a color change, and (v) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, a method comprises (i) collecting a biological sample from a subject, wherein the biological sample is urine, saliva, sweat, ocular fluid including aqueous humor, blood, fecal matter, sebum, respiratory tract droplets, semen, vaginal mucus, cerumen, epidermal cells, nasal cavity sample, cerebrospinal fluid, pleural effusion, or nasopharyngeal sample; (ii) wiping the biological sample; (iii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent that competes with a target analyte conjugated to glucose oxidase, which is capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iv) Incubating the detectable complex with horseradish peroxidase and a dye, such as 3,3', 5-tetramethylbenzidine, on the strip; (v) Detecting the level of detectable complex (if any) in the form of a color change, and (vi) calibrating the level of detectable complex (if produced) with the amount of target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method comprises (i) collecting a biological sample from a subject in a tube that dilutes the biological sample by 1X to 1,000,000,000X and contains a second binding reagent, wherein the biological sample is blood; (ii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent that competes with a target analyte conjugated to glucose oxidase, which is capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) Incubating the detectable complex with horseradish peroxidase and a dye, such as 3,3', 5-tetramethylbenzidine, on the strip; (iv) Detecting the level of the detectable complex (if any) in the form of a color change, and (v) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method comprises (i) collecting a biological sample from a subject in a tube diluted 1X to 1,000,000,000X and containing a second binding agent, wherein the biological sample is urine, sweat, ocular fluid including aqueous humor, blood, fecal matter, sebum, respiratory droplets, semen, vaginal mucus, cerumen, epidermal cells, or nasopharyngeal samples; (ii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent that competes with a target analyte conjugated to glucose oxidase, which is capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) Incubating the detectable complex with horseradish peroxidase and a dye, such as 3,3', 5-tetramethylbenzidine, on the strip; (iv) Detecting the level of the detectable complex (if any) in the form of a color change, and (v) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method includes obtaining multiple test results obtained at different times with respect to the same user and comparing the results to monitor or predict or track the likely progression of a disease or condition. In a particular embodiment, the method comprises obtaining at least two, at least three, at least four, or at least five test results.

In some embodiments, one or more results of the method may be transmitted to a remote entity continuously or periodically to determine whether the one or more results are above a threshold level or cut point.

In certain embodiments, the results may be compared to a predetermined reference level. The predetermined level may be obtained from a general population or a selected population of subjects. For example, the selected population may be comprised of apparently healthy patients, such as individuals who have not previously had any signs or symptoms indicative of the presence of a disease, such as an infection. The "predetermined reference level" may be determined, for example, by determining the expression level of the target analyte in a corresponding biological sample obtained from one or more control subjects (e.g., not suffering from an infection or known to be not susceptible to such a disease). When such predetermined reference levels are used, a higher or increased level determined in a biological sample (i.e., a test sample obtained from a subject) is indicative of, for example, the patient being at risk of developing a disease

Optionally, the method may further comprise a step of advice or guidance regarding the treatment and/or administration of the treatment. In one embodiment, the method comprises identifying a subject as having a target analyte level above a cutoff level threshold, and determining that the subject is thus a candidate for preventing and/or treating, for example, an infection or pathological condition. The "determining" step encompasses detection or quantification, wherein "detecting" means determining whether the target analyte is present in the biological sample, and "quantifying" means determining the amount of the target analyte present in the biological sample.

The method of the invention may have therapeutic use, for example it may be used to detect various pathological conditions or may be used to monitor the disease stage of a subject or its response to treatment.

In certain embodiments, the method may further comprise using statistical methods to predict the likelihood that detection of the target analyte results in a disease or disease progression and/or allows for disease prognosis (i.e., predicts the course of a disease).

In certain embodiments, the method may be performed across a group of patient populations, e.g., to allow stratification of their treatment methods or to meet public health or other monitoring objectives.

In one embodiment, a method for monitoring the efficiency of a therapeutic regimen in a subject having a pathological condition is disclosed, comprising using the methods and/or systems disclosed herein, wherein the target molecule is an antigen associated with the pathological condition, and wherein the amount of the detectable moiety is indicative of the level of the pathological condition, and thereby the efficiency of the therapeutic regimen in the subject.

In certain embodiments, the method includes monitoring the effectiveness of one or more therapeutic agents (e.g., antiviral agents, anticancer agents, etc.) over a period of time (e.g., days, weeks), and if the therapeutic agent is not sufficiently effective over a period of time, allowing the user to seek alternative treatment methods.

In one embodiment, if the treatment regimen does not produce a reduction in condition for a defined period of time (e.g., days), the user may interrupt the treatment regimen, switch to an alternative treatment regimen, or in some embodiments, supplement the treatment regimen with a second treatment regimen. In one embodiment, the system allows two or more results, three or more results, or five or more results to be obtained for the same user at different times with respect to the amount of target analyte to allow monitoring of trends in analyte levels over time.

In one embodiment, a method comprises (i) providing a biological sample from a subject, wherein the biological sample is blood; (ii) Adding a biological sample to a test strip, wherein the strip contains a first binding reagent, a second binding reagent, and a third binding reagent capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) Incubating the detectable complex with a dye on the strip, such as 3,3', 5-tetramethylbenzidine; (iv) Detecting the level of the detectable complex (if any) in the form of a color change, and (v) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, a method comprises (i) collecting a biological sample from a subject, wherein the biological sample is urine, saliva, sweat, ocular fluid including aqueous humor, blood, fecal matter, sebum, respiratory tract droplets, semen, vaginal mucus, cerumen, epidermal cells, nasal cavity sample, cerebrospinal fluid, pleural effusion, or nasopharyngeal sample; (ii) wiping the biological sample; (iii) Adding a biological sample to a test strip, wherein the strip contains a first binding reagent, a second binding reagent, and a third binding reagent capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iv) Incubating the detectable complex with a dye on the strip, such as 3,3', 5-tetramethylbenzidine; (v) Detecting the level of detectable complex (if any) in the form of a color change, and (vi) calibrating the level of detectable complex (if produced) with the amount of target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method comprises (i) collecting a biological sample from a subject in a tube that dilutes the biological sample by 1X to 1,000,000,000X and contains a second binding reagent, wherein the biological sample is blood; (ii) Adding a biological sample to a test strip, wherein the strip contains a first binding reagent, a second binding reagent, and a third binding reagent capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) Incubating the detectable complex with a dye on the strip, such as 3,3', 5-tetramethylbenzidine; (iv) Detecting the level of the detectable complex (if any) in the form of a color change, and (v) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method comprises (i) collecting a biological sample from a subject in a tube diluted 1X to 1,000,000,000X and containing a second binding agent, wherein the biological sample is urine, sweat, ocular fluid including aqueous humor, blood, fecal matter, sebum, respiratory droplets, semen, vaginal mucus, cerumen, epidermal cells, or nasopharyngeal samples; (ii) Adding a biological sample to a test strip, wherein the strip contains a first binding reagent, a second binding reagent, and a third binding reagent capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) Incubating the detectable complex with a dye on the strip, such as 3,3', 5-tetramethylbenzidine; (iv) Detecting the level of the detectable complex (if any) in the form of a color change, and (v) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, a method comprises (i) providing a biological sample from a subject, wherein the biological sample is blood; (ii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent and a third binding reagent that compete with a target analyte conjugated to glucose oxidase, which are capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) Incubating the detectable complex with a dye on the strip, such as 3,3', 5-tetramethylbenzidine; (iv) Detecting the level of the detectable complex (if any) in the form of a color change, and (v) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, a method comprises (i) collecting a biological sample from a subject, wherein the biological sample is urine, saliva, sweat, ocular fluid including aqueous humor, blood, fecal matter, sebum, respiratory tract droplets, semen, vaginal mucus, cerumen, epidermal cells, nasal cavity sample, cerebrospinal fluid, pleural effusion, or nasopharyngeal sample; (ii) wiping the biological sample; (iii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent and a third binding reagent that compete with a target analyte conjugated to glucose oxidase, which are capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iv) Incubating the detectable complex with a dye on the strip, such as 3,3', 5-tetramethylbenzidine; (v) Detecting the level of detectable complex (if any) in the form of a color change, and (vi) calibrating the level of detectable complex (if produced) with the amount of target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method comprises (i) collecting a biological sample from a subject in a tube that dilutes the biological sample by 1X to 1,000,000,000X and contains a second binding reagent, wherein the biological sample is blood; (ii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent and a third binding reagent that compete with a target analyte conjugated to glucose oxidase, which are capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) Incubating the detectable complex with horseradish peroxidase and a dye, such as 3,3', 5-tetramethylbenzidine, on the strip; (iv) Detecting the level of the detectable complex (if any) in the form of a color change, and (v) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

In one embodiment, the method comprises (i) collecting a biological sample from a subject in a tube diluted 1X to 1,000,000,000X and containing a second binding agent, wherein the biological sample is urine, sweat, ocular fluid including aqueous humor, blood, fecal matter, sebum, respiratory droplets, semen, vaginal mucus, cerumen, epidermal cells, or nasopharyngeal samples; (ii) Adding a biological sample to a test strip, wherein the test strip contains a first binding reagent and a third binding reagent that compete with a target analyte conjugated to glucose oxidase, which are capable of producing a detectable complex with at least one target analyte (if present) in the biological sample; (iii) Incubating the detectable complex with horseradish peroxidase and a dye, such as 3,3', 5-tetramethylbenzidine, on the strip; (iv) Detecting the level of the detectable complex (if any) in the form of a color change, and (v) calibrating the level of the detectable complex (if produced) with the amount of the target analyte (if any) in the at least one biological sample, thereby providing a diagnostic assessment.

Therapeutic agents may vary. In one embodiment, the therapeutic agent is a pharmaceutical agent such as a small molecule, protein, virus, bacterial nucleic acid, or biological agent.

In one embodiment of the methods disclosed herein, the result has a specificity of about 90% or more, or more specifically, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more.

In one embodiment of the methods disclosed herein, the results have a selectivity of about 90% or greater, or more specifically, about 91% or greater, about 92% or greater, about 93% or greater, about 94% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, about 99% or greater.

In one embodiment of the methods disclosed herein, the results have an accuracy of about 90% or greater, or more specifically, about 91% or greater, about 92% or greater, about 93% or greater, about 94% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, about 99% or greater.

The methods disclosed herein are not limited to the steps described above, and may include additional steps performed before, after, or between the steps.

III preparation method

Also disclosed are methods of preparing universal test strips or substrates for use in the systems and assays disclosed herein. The test strip may be manufactured using any suitable method. In one embodiment, the test cartridge or strip (trip) is manufactured using an injection molding or roll-to-roll process, a screen printing process, a drop casting process, or a combination thereof.

In one embodiment, a method for producing a test strip for use in a diagnostic or detection system or assay such as those disclosed herein is provided that includes (i) providing a membrane; (ii) Binding the first binding reagent to a membrane (e.g., streptavidin), and (iii) optionally, crosslinking the first binding reagent with one or more additional first binding reagents.

In a specific embodiment, the first binding agent is crosslinked with one or more additional binding agents by means of a polymer (e.g., PEG).

IV. kit

Kits for practicing the methods disclosed herein are also disclosed.

In one embodiment, the kit comprises the assays disclosed herein, either as a stand-alone assay or as part of the disclosed system. In certain embodiments, the kit comprises a multiplex assay. In certain embodiments, the kit includes a detection device (e.g., a blood glucose meter).

The test kit or strip compositions as disclosed herein may be combined with other ingredients or reagents, or prepared as a kit or component of other retail products for commercial sale or distribution.

Kits of the invention may comprise a test strip or other solid support. In certain embodiments, the kit comprises a lateral flow test strip or a vertical flow test strip, which may be provided as a separate element or on one or more binding reagents that have been discovered. In one embodiment, the lateral flow strip or vertical flow strip may comprise a first binding reagent (e.g., streptavidin or avidin) bound thereto. Optionally, the first binding reagent may be present in cross-linked form on a lateral or vertical flow strip provided in the kit.

Typically, the above-described kits will also include one or more additional containers containing, for example, wash reagents and/or other reagents capable of quantitatively detecting the presence of the bound target analyte.

The detection reagent and optional reporter reagent may be unlabeled or labeled.

When the label is an enzyme, the kit may include substrates and cofactors required by the enzyme. In certain embodiments, the kit may contain an amount of sugar (e.g., glucose) to be added to the assay by the user. If the label is a fluorophore, the kit may include a dye precursor that provides a detectable chromophore.

In one particular embodiment, a sealed disposable cup is included with a dry identification element (e.g., capture reagent, detection reagent) therein. Compartmentalized kit includes any kit in which reagents are contained in separate containers, e.g., plastic containers. Such containers may allow for efficient transfer of reagents from one chamber to another while avoiding cross-contamination of the sample and reagents, and the reagents or solutions of each container are added from one chamber to the other in a quantitative manner. Such kits may also include containers for receiving test samples, containers containing antibodies for use in the assay, containers containing wash reagents (e.g., phosphate buffered saline, tris buffer, etc.), and containers containing detection reagents.

In certain embodiments, the kit may include disposable pipettes, dropper bottles for buffers, reagents, blotting/wicking materials, and disposable tests.

In certain embodiments, the kit components may be foil or paper packaged and may include a desiccant.

The kit may also contain instructions and/or reference standards for performing the assay (e.g., purified collagen VII, e.g., recombinantly produced collagen VII), as well as other additives such as stabilizers, wash and incubation buffers, and the like. The kit will also contain instructions for administration and/or use of the kit.

The kit may also contain a reader.

In certain embodiments, the kit allows (i) detection of at least one target analyte at a detection limit of about 1 target analyte per milliliter to > 100,000 target analytes per milliliter; (i) Detecting at least one target analyte with an accuracy of about 90%, and/or (iii) providing a result in about 10 minutes or less, about 5 minutes or less, about 2 minutes or less, or about 1 minute or less.

Examples

Example 1: electrochemical detection of H1N1

As shown in fig. 14, a scheme is described that enables electrochemical detection of H1N1 via a functionalized nitrocellulose strip over a working electrode in a 3-electrode detection system. The physical settings are shown in the lower left hand corner and the chronoamperometric results are shown in the lower right hand corner. Operation with virus indicated a higher current than the control curve in which no virus was present.

Example 2: electrochemical detection of SARS-CoV-2

As shown in fig. 15, electrochemical detection of SARS-CoV-2 via functionalized nitrocellulose strips over the working electrode in a 3-electrode detection system revealed a higher accumulated charge, as calculated by area under the chronoamperometric curve with higher viral titer.

Example 3: electrochemical detection of SARS-CoV-2

As shown in fig. 16, the specificity of electrochemical detection of SARS-CoV-2 via functionalized nitrocellulose strips over the working electrode in a 3-electrode detection system was compared by using the same assay with respect to cross-reactivity against RSV, OC43 and H1N1.

Example 4: colorimetric detection of H1N1

As shown in fig. 17, H1N1 was detected colorimetrically using the same assay as described in example 1 by adding redox mediator and dye to the glucose solution described herein. The glucose solution results in the formation of hydrogen peroxide when in the presence of target-bound oxidase. In contrast to charge detection via electrochemical means, for example, the addition of HRP and duplex Red results in a pink color.

Example 5: colorimetric detection of H1N1

As shown in fig. 18, specific conditions for colorimetric detection of H1N1 by adding a redox mediator and dye to the glucose solutions described herein are described. The glucose solution results in the formation of hydrogen peroxide when in the presence of target-bound oxidase. In contrast to charge detection via electrochemical means, for example, the addition of HRP and TMB or ampliex Red results in blue or pink, respectively.

Example 6: colorimetric detection of OPN

As shown in fig. 19, specific conditions for colorimetric detection of osteopontin by adding TMB and HRP to a glucose solution over a sandwich-bound target complex are described.

Example 7: colorimetric detection of OPN

As shown in fig. 20, more specific conditions for colorimetric detection of osteopontin by adding TMB and HRP to a glucose solution over a sandwich-bound target complex are described.

Example 8: crosslinked streptavidin

For electrochemical detection of analytes using sandwich sensing assays, streptavidin-coated beads were loaded into nitrocellulose membranes (e.g., membranes purchased from Thermoscientific with 0.45 μm pores) by drop casting 10 μl of PBS solution. Next, PEG chemically modified to 3400Mw containing two terminal biotin was added to the nitrocellulose membrane. The ratio of streptavidin to biotin was 1:0.25. Next, the membrane will be washed with 200 μl PBST and transferred to DropSens 710 electrode. Antibodies to the analyte will be functionalized with biotin, while a second, different antibody to the analyte will be conjugated to GOx (Ab-GOx) synthesized using Abcam's Lightning-Link (Gox conjugation kit, # Ab 102887). The analyte and the two antibodies were mixed together, then 5 μl of the solution was drop cast onto the membrane surface and spread thereon, and incubated for 5 minutes. The membranes will be washed using 200 μl PBST and chronoamperometric measurements performed after the addition of 50 μl 500mM glucose solution.

PEG molecular weights of 1000 to 50,000 can be used, but a preferred Mw is 3000-10,000. Similarly, linear PEG with 2 biotins on the ends or star PEG with 4 biotins on the ends may be used.

For electrochemical detection of analytes using sandwich sensing assays, nitrocellulose membranes both surface and interior (4 mm diameter discs) will be functionalized with streptavidin (e.g., membranes purchased from Thermoscientific with 0.45 μm pores). Next, PEG chemically modified to 3400Mw containing two terminal biotin was added to the nitrocellulose membrane. The ratio of streptavidin to biotin was 1:0.25. Next, the membrane will be washed with 200 μl PBST and transferred to DropSens 710 electrode. Antibodies to the analyte will be functionalized with biotin, while a second, different antibody to the analyte will be conjugated to GOx (Ab-GOx) synthesized using Abcam's Lightning-Link (Gox conjugation kit, # Ab 102887). The analyte and the two antibodies were mixed together, then 5 μl of the solution was drop cast onto the membrane surface and spread thereon, and incubated for 5 minutes. The membranes will be washed using 200 μl PBST and chronoamperometric measurements performed after the addition of 50 μl 500mM glucose solution. PEG molecular weights of 1000 to 50,000 can be used, but a preferred Mw is 3000-10,000. Similarly, linear PEG with 2 biotins on the ends or star PEG with 4 biotins on the ends may be used.

For optical detection of analytes using sandwich sensing assays, nitrocellulose membranes both surface and interior (4 mm diameter discs) will be functionalized with streptavidin (e.g., membranes purchased from Thermoscientific with 0.45 μm pores). Next, PEG chemically modified to 3400Mw containing two terminal biotin was added to the nitrocellulose membrane. The ratio of streptavidin to biotin was 1:0.25. Next, the membranes will be washed using 200 μl PBST. Antibodies to the analyte will be functionalized with biotin, while a second, different antibody to the analyte will be conjugated to GOx (Ab-GOx) synthesized using Abcam's Lightning-Link (Gox conjugation kit, # Ab 102887). The analyte and the two antibodies were mixed together, then 5 μl of the solution was drop cast onto the membrane surface and spread thereon, and incubated for 5 minutes. The membranes will be washed using 200 μl PBST and optical measurements performed after the addition of 50 μl 500mM glucose solution and HRP and duplex Red. A pink/violet color is formed that indicates the presence of the analyte. PEG molecular weights of 1000 to 50,000 can be used, but a preferred Mw is 3000-10,000. Similarly, linear PEG with 2 biotins on the ends or star PEG with 4 biotins on the ends may be used.

For optical detection of analytes using sandwich sensing assays, streptavidin-coated beads were loaded into nitrocellulose membranes (e.g., membranes purchased from Thermoscientific with 0.45 μm wells) by drop casting 10 μl of PBS solution. Next, PEG chemically modified to 3400Mw containing two terminal biotin was added to the nitrocellulose membrane. The ratio of streptavidin to biotin was 1:0.25. Next, the membranes will be washed using 200 μl PBST. Antibodies to the analyte will be functionalized with biotin, while a second, different antibody to the analyte will be conjugated to GOx (Ab-GOx) synthesized using Abcam's Lightning-Link (Gox conjugation kit, # Ab 102887). The analyte and the two antibodies were mixed together, then 5 μl of the solution was drop cast onto the membrane surface and spread thereon, and incubated for 5 minutes. The membranes will be washed using 200 μl PBST and optical measurements performed after the addition of 50 μl 500mM glucose solution and HRP and duplex Red. A pink/violet color is formed that indicates the presence of the analyte. PEG molecular weights of 1000 to 50,000 can be used, but a preferred Mw is 3000-10,000. Similarly, linear PEG with 2 biotins on the ends or star PEG with 4 biotins on the ends may be used.

Example 9: crosslinked capture reagent

For electrochemical detection of analytes using sandwich sensing assays, streptavidin-coated beads were loaded into nitrocellulose membranes (e.g., membranes purchased from Thermoscientific with 0.45 μm pores) by drop casting 10 μl of PBS solution. Next, the membrane will be washed with 200 μl PBST and transferred to DropSens 710 electrode. Antibodies to the analyte will be functionalized with at least two biotins, while a second, different antibody to the analyte will be conjugated to GOx (Ab-GOx) synthesized using Abcam's lighting-Link (Gox conjugation kit, # Ab 102887). The analyte and the two antibodies were mixed together, then 5 μl of the solution was drop cast onto the membrane surface and spread thereon, and incubated for 5 minutes. The membranes will be washed using 200 μl PBST and chronoamperometric measurements performed after the addition of 50 μl 500mM glucose solution.

For electrochemical detection of analytes using sandwich sensing assays, nitrocellulose membranes both surface and interior (4 mm diameter discs) will be functionalized with streptavidin (e.g., membranes purchased from Thermoscientific with 0.45 μm pores). Next, the membrane will be washed with 200 μl PBST and transferred to DropSens 710 electrode. Antibodies to the analyte will be functionalized with at least two biotins, while a second, different antibody to the analyte will be conjugated to GOx (Ab-GOx) synthesized using Abcam's lighting-Link (Gox conjugation kit, # Ab 102887). The analyte and the two antibodies were mixed together, then 5 μl of the solution was drop cast onto the membrane surface and spread thereon, and incubated for 5 minutes. The membranes will be washed using 200 μl PBST and chronoamperometric measurements performed after the addition of 50 μl 500mM glucose solution.

For optical detection of analytes using sandwich sensing assays, nitrocellulose membranes both surface and interior (4 mm diameter discs) will be functionalized with streptavidin (e.g., membranes purchased from Thermoscientific with 0.45 μm pores). Next, the membranes will be washed using 200 μl PBST. Antibodies to the analyte will be functionalized with at least two biotins, while a second, different antibody to the analyte will be conjugated to GOx (Ab-GOx) synthesized using Abcam's lighting-Link (Gox conjugation kit, # Ab 102887). The analyte and the two antibodies were mixed together, then 5 μl of the solution was drop cast onto the membrane surface and spread thereon, and incubated for 5 minutes. The membranes will be washed using 200 μl PBST and optical measurements performed after the addition of 50 μl 500mM glucose solution and HRP and duplex Red. A pink/violet color is formed that indicates the presence of the analyte.

For optical detection of analytes using sandwich sensing assays, streptavidin-coated beads were loaded into nitrocellulose membranes (e.g., membranes purchased from Thermoscientific with 0.45 μm wells) by drop casting 10 μl of PBS solution. Next, the membranes will be washed using 200 μl PBST. Antibodies to the analyte will be functionalized with at least two biotins, while a second, different antibody to the analyte will be conjugated to GOx (Ab-GOx) synthesized using Abcam's lighting-Link (Gox conjugation kit, # Ab 102887). The analyte and the two antibodies were mixed together, then 5 μl of the solution was drop cast onto the membrane surface and spread thereon, and incubated for 5 minutes. The membranes will be washed using 200 μl PBST and optical measurements performed after the addition of 50 μl 500mM glucose solution and HRP and duplex Red. A pink/violet color is formed that indicates the presence of the analyte.

Example 10 electrochemical prototype

As shown in fig. 21, a schematic diagram of a top view of an electrochemical disposable test prototype. The schematic includes a waterproof barrier surrounding a 3-electrode system on a waterproof base and a fixed area on top of the working electrode.

EXAMPLE 11 optical prototype

As shown in fig. 22, a schematic diagram of a top view of an optical disposable test prototype. The schematic includes a disposable optical/colorimetric test prototype that includes a waterproof barrier surrounding a fixed area on top of a waterproof base material. The waterproof barrier creates a chamber within the waterproof barrier.

Example 12 optical prototype

As shown in fig. 23, a schematic diagram of a top view of another optical disposable test prototype. The schematic includes a disposable optical/colorimetric test prototype that includes a waterproof barrier surrounding a fixed area placed on a waterproof base. The waterproof barrier creates a chamber within the waterproof barrier.

EXAMPLE 13 optical prototype

As shown in fig. 24, a schematic diagram of a top view of the fixation area is shown. The immobilization region may have, for example, streptavidin as a universal binding site, which may be in the form of lines, whole regions, gradients, patterns, topologies, etc., which may aid in quantification of the target.

EXAMPLE 14 optical prototype

As shown in fig. 25, a schematic diagram of a top view of another fixation area. The immobilization region may include a universal binding site, such as a polystyrene bead coated via direct immobilization of streptavidin or via streptavidin. The universal binding sites on the immobilized region may be exposed to a solution of antibodies specific for the analyte of interest, such as biotinylated antibodies and enzyme-labeled antibodies. If the analyte of interest is included in the solution/sample solution, the antibodies can both bind the target analyte and generate a detectable complex on/over the immobilized area. If the analyte of interest is not included, the detectable complex is not immobilized to the immobilization region.

Example 15.

A solution containing 1. Mu.g/mL of the IL-6 antibody bound to alkaline phosphatase, 1. Mu.g/mL of the biotinylated IL-6 antibody, and IL-6 protein at 1ng/mL or 0ng/mL was prepared in the assay buffer. The area of streptavidin coated beads embedded in the lateral flow membrane is located on top of the electrode. 15ul of test or control solution was added to the lateral flow membrane (FIG. 36). The solution flows through the membrane. After 2 minutes, the membrane was washed with 200uL of buffer, which was collected via a wicking pad. 100uL of 20mM 4-aminophenylphosphate/diethanolamine substrate (pH 9.6) was then deposited onto the membrane area on top of the electrode and the chronoamperometry was run. The first graph indicates the chronoamperometric results collected over the course of 10 minutes (2 test runs and 2 control runs were performed; fig. 37). The second plot indicates the chronoamperometric results collected on the same electrode after 20 minutes (fig. 38).

EXAMPLE 16 prototype cartridge

A prototype cartridge is shown in fig. 39, which provides sample collection, flow toward the target binding site in contact with the electrode, waste and substrate solution reservoirs. The cartridge may be inserted into a virometer for electrochemical detection.

Example 17.

Alkaline phosphatase (ALP) was dissolved in Diethanolamine (DEA) buffer to ten times the targeted measurement concentration. 5uL of the solution was applied to the electrode followed by 45uL of the substrate solution (10 mM 4-Aminophenylphosphate (APP)/DEA (1M) +5mM MgCl) ₂ . The enzyme and substrate (ALP and APP) were incubated for various detection intervals ranging from 30 seconds to 5 minutes followed by a 10 second chronoamperometric detection. For 30 second intervals, a potential of 0.2V (relative to Ag) was applied for 10 seconds, and the current was measured after the initial 30 second incubation. After the measurement, the battery remains open for 20 seconds or more before repeating the 10 second measurement. This detection pattern was repeated for a period of up to 240 seconds (eight intervals). For a 2 minute detection interval incubation, a 10 second chronoamperometric measurement is performed every 2 minutes for a period of up to 240 seconds, or two intervals with the cell in between for 110 seconds open. For a 5 minute test interval, only one round of 10 second chronoamperometric test was performed. The final (termination) current at 10 seconds was plotted against the total incubation time of 0 or 0.1ng/ml ALP activity in the APP/DEA buffer system (FIG. 26). Note that the background (0 ng/ml) termination current decreases over time, whereas the enzyme activity may initially decrease but then increase over time. Repeated timing of the current detection shortens the total measurement time required to reach the required current discrimination, as shown in fig. 27, comparing the accumulated current in nA according to the total measurement time and the measurement time interval.

Example 18.

A solution containing 1. Mu.g/mL of the anti-IL-6 antibody bound to alkaline phosphatase, 1. Mu.g/mL of the biotinylated anti-IL-6 antibody, and 2ng/mL, 0.1ng/mL, or 0ng/mL of IL-6 protein was prepared in the assay buffer. The area of streptavidin coated beads embedded in the lateral flow membrane is located on top of the electrode. 100ul of test or control solution was added to the crossflow membrane. The solution flows through the membrane. After 2 minutes, the membrane was washed with 200uL of buffer, which was collected via a wicking pad. 60uL of 10mM 4-aminophenylphosphate/diethanolamine substrate (pH 9.8) was then deposited onto the membrane area on top of the electrode to measure the concentration of IL-6 captured in the sandwich configuration (FIG. 28). In one case, the chronoamperometry is run for 10 seconds after 5 minutes of substrate incubation. In another case, after introduction of the substrate solution, the modified chronoamperometry (measuring the current in these 10 seconds) was run by repeatedly applying a potential of 0.2V (vs Ag) every 30 seconds for a total duration of 310 seconds (fig. 29). The results show a cumulative current resolution of 2.5 times at 0.1ng/ml for the modified chronoamperometric detection and a cumulative current resolution of 3.8 times for 2ng/ml compared to a single 10 second detection after 5 minutes incubation (fig. 30).

Example 19.

Bovine-derived alkaline phosphatase (ALP) was dissolved in Diethanolamine (DEA) buffer to ten times the targeted measurement concentration. Applying 5uL solution to the electrode followed by 45uL substrate solution (10 mM 4-Aminophenylphosphate (APP)/DEA or APP/2- (ethylamino) ethanol (EAE) with varying amounts of MgCl2. Incubating the enzyme and substrate (ALP and APP) for 5 min; followed by 5 min chronoamperometric collection. A comparison of ALP activity (in. Mu.C charge) in the APP/DEA and APP/EAE substrate/buffer systems based on MgCl2 concentration indicates MgCl ₂ The concentration significantly affected the charge change for each given enzyme concentration as shown in figure 31.

Example 20.

Calf intestine-derived alkaline phosphatase is used in a sandwich assay for measuring IL-6 concentration. A solution containing 1. Mu.g/mL of the anti-IL-6 antibody bound to alkaline phosphatase, 1. Mu.g/mL of the biotinylated anti-IL-6 antibody, and 2ng/mL, 0.1ng/mL, or 0ng/mL of IL-6 protein was prepared in the assay buffer. The area of streptavidin coated beads embedded in the lateral flow membrane is located on top of the electrode. 100ul of test or control solution was added to the crossflow membrane. The solution flows through the membrane. After 2 minutes, the membrane was washed with 200uL of buffer, which was collected via a wicking pad. 60uL 10mM APP- DEA substrate (pH 9.8) (with or without 5mM MgCl) ₂ ) Or 10mM APP/EAE substrate (pH 9.8) (containing 5mM MgCl) ₂ ) Deposited onto the membrane area on top of the electrode to measure the concentration of the captured IL-6 in the sandwich configuration. Modification of substrate solution/addition of MgCl ₂ The results of the IL-6 assay were not significantly affected (FIG. 32).

The results indicate that a) enzymes from different sources and/or b) modified for conjugation in commercially available kits may behave differently and their optimal concentration or optimal solution for running a chronoamperometric measurement may promote more sensitive detection of the target analyte.

Example 21.

A solution containing 1. Mu.g/mL of the anti-IL-6 antibody bound to alkaline phosphatase, 1. Mu.g/mL of the biotinylated anti-IL-6 antibody, and 2ng/mL, 0.1ng/mL, or 0ng/mL of IL-6 protein was prepared in the assay buffer. The area of streptavidin coated beads embedded in the lateral flow membrane is located on top of the electrode. 100ul of test or control solution was added to the crossflow membrane. The solution flows through the membrane. After 2 minutes, the membrane was washed with 200uL of buffer, which was collected via a wicking pad. 60uL of 10mM 4-aminophenylphosphate/diethanolamine substrate (pH 9.8) was then deposited onto the membrane area on top of the electrode to measure the concentration of IL-6 captured in the sandwich configuration (FIG. 28). In one case, the chronoamperometry is run for 10 seconds after 5 minutes of substrate incubation. In another case, after introduction of the substrate solution, the modified chronoamperometry (measuring the current in these 10 seconds) was run by repeatedly applying a potential of 0.2V (relative to Ag) every 30 seconds for a total duration of 310 seconds (fig. 27). After current versus time data for the modified recurring timing current sense is obtained, the results are analyzed by three methods. First, the sum of the termination currents after every 10 seconds of detection period in the entire 31-0 second detection is passed (fig. 33). Next, the charge after each 10 second detection period is obtained by integrating the timed current curve and summing each charge measurement over the entire 310 second detection (fig. 34). And third, by comparing the slope of the termination current at 10 seconds with respect to the total time since the addition of substrate (310 seconds). These three data analysis methods are depicted in fig. 33 (fig. 35).

Example 22: methods for rapid immunoassays by membrane running followed by subsequent electrochemical detection of the immunoassay target.

Solutions containing 1 μg/mL of anti-nucleocapsid antibody 1 conjugated to alkaline phosphatase, 1 μg/mL of biotinylated anti-nucleocapsid antibody 2 (matching a different target epitope than antibody 1), and nucleocapsid protein at 5ng/mL or 0ng/mL were prepared in assay buffer in tubes. As shown in fig. 40, this is formulated to simulate the addition of biological samples, which may be diluted in a buffer, to a vessel containing reagents and capture materials and/or anchoring materials. The mixed solution containing 500 or 0pg of nucleocapsid protein was transferred from above onto a blank test membrane (blocked or unblocked, but not functionalized with any capture reagent) and then washed with buffer collected via a wicking material. In this embodiment the membrane is located on top of the plastic ring to raise it over the wicking pad, but the membrane may be located directly on the wicking pad, the membrane may be located on top of another membrane material, or the membrane may be suspended over a collection device, etc. 60uL of 10mM 4-aminophenylphosphate/diethanolamine substrate (pH 9.8) was then deposited onto the membrane area on top of the electrode to measure the concentration of target present and captured. After substrate addition, the chronoamperometry was run for 2 seconds, 30 seconds, and then again at 90 seconds, 180 seconds, and 300 seconds by repeatedly applying a potential of 0.2V (relative to Ag). The results show nucleocapsid protein detection via this method (fig. 41).

Example 23: methods for running an immunoassay along a membrane with subsequent electrochemical detection of the immunoassay target.

A solution containing 1. Mu.g/mL of anti-IL-6 antibody 1 bound to alkaline phosphatase, 1. Mu.g/mL of biotinylated anti-IL-6 antibody 2 (matching a different target epitope than antibody 1), and IL-6 protein at 0.1ng/mL or 0ng/mL was prepared in assay buffer. 100uL of test or control solution was added to a lateral flow membrane containing embedded streptavidin coated beads. The solution (containing 10 or 0pg of IL-6 protein) was passed through the membrane and the membrane was then washed with buffer. Excess solution was collected via a wicking pad. Then 60uL 10mM 4-AP/DEA substrate was deposited on the membrane area on top of the electrode to measure the concentration of IL-6 captured in the sandwich configuration (FIG. 42). The test protocol included repeated chronoamperometric runs every 30 seconds (each data point shows 2 seconds of current) for a total of 300 seconds (fig. 43). The rapid detection was clearly completed in 2-3 minutes.

Example 24: modified chronoamperometry was used in conjunction with immunoassays to detect IL-6 proteins as low as 20 pg/ml.

Solutions containing 1. Mu.g/mL of anti-IL-6 antibody 1 conjugated to alkaline phosphatase, 1. Mu.g/mL of biotinylated anti-IL-6 antibody 2 (matching a different target epitope than antibody 1), and IL-6 protein at 500, 100, 20 or 0pg/mL were prepared in assay buffer. 100uL of test or control solution was added to a lateral flow membrane containing embedded streptavidin coated beads. The solution (containing 50, 10, 2 or 0pg of IL-6 protein) was passed through the membrane and the membrane was then washed with buffer. Excess solution was collected via a wicking pad. The membrane was transferred to the surface of the electrode, and then 60ul of 10mm 4-AP/DEA substrate was deposited onto the membrane to measure the concentration of IL-6 captured in the sandwich configuration (fig. 28). Electrochemical detection included 2 seconds chronoamperometric operation at 30 seconds, 90 seconds, 180 seconds, 300 seconds, then every 300 seconds up to 1500 seconds. Each data point in fig. 44 shows a 2 second current average and standard deviation. The slope of the termination current according to concentration is shown in fig. 45.

Example 25: modified chronoamperometry was used in conjunction with immunoassays to detect prolactin proteins as low as 200 pg/ml.

Solutions containing 1 μg/mL of anti-prolactin antibody 1 conjugated to alkaline phosphatase, 1 μg/mL of biotinylated anti-prolactin antibody 2 (matching a different target epitope than antibody 1), and 20, 2, 0.2, 0.1 or 0ng/mL of prolactin protein were prepared in assay buffer. 100uL of test or control solution was added to a lateral flow membrane containing embedded streptavidin coated beads. The solution (containing 2 or 0ng prolactin protein) was passed through the membrane and the membrane was then washed with buffer. Excess solution was collected via a wicking pad. The lateral flow membrane was transferred to the surface of the electrode, and then 60ul of 10mm 4-AP/DEA substrate was deposited onto the membrane to measure the concentration of IL-6 captured in the sandwich configuration (fig. 28). The detection scheme includes 2 second chronoamperometric operation at 30 seconds, 90 seconds, 180 seconds, 300 seconds, 600 seconds. Each data point below shows the 2 second current mean and standard deviation; the slope of the termination current as a function of concentration is also shown (fig. 46).

Example 26:

modified chronoamperometry was used in conjunction with competitive immunoassays to detect biotin as low as 2.4ng/m and confirm the competitive assay. Solutions containing 1nM biotin-conjugated alkaline phosphatase in assay buffer (control) and 1nM biotin-conjugated alkaline phosphatase plus 10nM or 100nM biotin in assay buffer (test) were prepared. 100ul of test or control solution was added to a lateral flow membrane containing embedded streptavidin coated beads. The solution (containing 2.4ng or 240pg biotin) was passed through the membrane and the membrane was then washed with buffer. Excess solution was collected via a wicking pad. The lateral flow membrane was transferred to the surface of the electrode, and then 60ul of 10mm 4-AP/DEA substrate was deposited on the membrane to measure the concentration of protein based on the current decrease (competitive detection scheme depicted in fig. 43). Electrochemical detection included 2 second chronoamperometric operation at 30 seconds, 90 seconds, 180 seconds, 300 seconds, and 600 seconds. Each data point in fig. 48 shows a 2 second current average and standard deviation.

Example 27: modified chronoamperometry was used in conjunction with immunoassays to detect protein conjugates as low as 80 pg/ml.

Solutions containing biotin-conjugated alkaline phosphatase in assay buffer were prepared at 200pg/mL, 80pg/mL, or 0 ng/mL. 100uL of test or control solution was added to a lateral flow membrane containing embedded streptavidin coated beads. The solution (containing 20, 8 or 0pg protein conjugate) was passed through the membrane and the membrane was then washed with buffer. Excess solution was collected via a wicking pad. Transfer lateral flow membrane to surface of electrode, then 60uL including 5mM MgCl ₂ To measure the concentration of conjugated protein. Detection scheme packageIncluding 2 seconds chronoamperometric operation at 30 seconds, 90 seconds, 180 seconds, 300 seconds, 600 seconds. Each data point in fig. 50 shows a 2 second current average and standard deviation.

Example 28: modified chronoamperometry was used in conjunction with immunoassays to detect osteopontin as low as 100 pg/ml.

Solutions containing 1 μg/mL of anti-osteopontin antibody 1 conjugated to alkaline phosphatase, 1 μg/mL of biotinylated anti-osteopontin antibody 2 (matching a different target epitope than antibody 1), and 100, 10, 0.5, 0.2, 0.1, or 0ng/mL of osteopontin were prepared in assay buffer. 100uL of test or control solution was added to a lateral flow membrane containing embedded streptavidin coated beads. The solution (containing 100, 20 or 0pg osteopontin) was passed through the membrane and the membrane was then washed with buffer. Excess solution was collected via a wicking pad. The lateral flow membrane was transferred to the surface of the electrode, and then 50ul of 10mm 4-AP/DEA substrate was deposited onto the membrane to measure the concentration of osteopontin captured in the sandwich configuration. The detection protocol included 2 second chronoamperometric operation at 30 seconds, 90 seconds, 120 seconds, 180 seconds, and 300 seconds. Each data point below shows the 2 second current mean and standard deviation; the slope of the termination current as a function of concentration is also shown (figures 50-53).

Example 29: full virus detection of inactivated coronavirus is detected electrochemically via a lateral flow based sandwich assay targeting both spike virus proteins and membrane virus proteins.

A solution containing 1mg/mL of anti-SARS-CoV-2 membrane (matrix) protein antibody conjugated to glucose oxidase, 1mg/mL of biotinylated anti-SARS-CoV-2S 1 spike protein antibody, and SARS-CoV-2 virus at 12900TCID50/mL, 1290TCID50/mL, 129TCID50/mL or 0TCID50/mL was prepared in assay buffer in a tube. The final virus concentration was formulated to simulate the addition of biological samples (1290 TCID50/mL, 129TCID50/mL, 12.9TCID50/mL or 0TCID 50/mL) that could be diluted in buffer to a vessel containing reagents and capture material and/or anchor material. The mixed solution containing 1290TCID50/mL, 129TCID50/mL, 12.9TCID50/mL or 0TCID50/mL SARS-CoV-2 virus was transferred from above onto a blank test membrane (blocked or unblocked, but not functionalized with any capture reagent) and then washed with buffer collected via a wicking material. In this embodiment, the membrane is positioned on top of the electrode with the wicking pad at one end of the test membrane and the solution flows laterally. Then 50 μl of 500mM glucose (pH 7.4) was deposited onto the membrane area on top of the electrode to measure the concentration of target present and captured. The chronoamperometry was run for 300 seconds 1 second after substrate addition by applying a potential of-0.2V (vs Ag/AgCl). The results show SARS-CoV-2 virus detection via this method (FIGS. 54-61).

Example 30: detection of IgG.

Solutions containing 1. Mu.g/mL of anti-IgG antibody conjugated to alkaline phosphatase, 1. Mu.g/mL of biotinylated anti-IgG antibody, and 100ng/mL, 50ng/mL, or 10ng/mL of IgG protein were prepared in assay buffer. IgG is produced in response to a covd infection. The area of streptavidin coated beads embedded in the lateral flow membrane is located on top of the electrode. 100ul of test or control solution was added to the crossflow membrane. The solution flows through the membrane. After 2 minutes, the membrane was washed with 200uL of buffer, which was collected via a wicking pad. 60uL of 10mM 4-aminophenylphosphate/diethanolamine substrate (pH 9.8) was then deposited onto the membrane area on top of the electrode to measure the concentration of captured IgG in the sandwich configuration (FIG. 28). In one case, the chronoamperometry is run for 10 seconds after 5 minutes of substrate incubation. In another case, the modified chronoamperometry (measuring current in these 10 seconds) is run by repeatedly applying a potential of 0.2V (relative to Ag) every 30 seconds after the introduction of the substrate solution, for a total duration of 310 seconds. Both chronoamperometric runs showed detection of IgG compared to control solutions without IgG.

Example 31: and (5) a detection limit.

In the above examples of detection of proteins, small molecules or viruses, the limit of detection (LoD) is defined as the value of three standard deviations above the 0ng/mL baseline (control assay without analyte added). LoD may also be defined as a value of two standard deviations above the 0ng/mL baseline. LoD is also calculated based on the standard deviation of the curve response (Sy) and the slope of the calibration curve (S) (e.g., lod=3.3 (Sy/S)).

Claims (108)

1. A system for detecting at least one target analyte in a biological sample added to the system, comprising: (i) An assay comprising at least one capture reagent and at least one detection reagent, said reagents being capable of producing a detectable complex with at least one target analyte in the presence of added substrate, if present; and (ii) a detection device for detecting the detectable complex, wherein the detection device comprises an enzyme-based amperometric sensor comprising at least one electrode, wherein the detectable complex forms over at least one electrode or migrates within the system to become positioned over at least one electrode.

2. The system of claim 1, wherein the detectable complex is detected in about 30 minutes or less.

3. The system of claim 1, wherein the detectable complex is detected within a time frame selected from about 10 minutes or less, about 5 minutes or less, about 2 minutes or less, or about 1 minute or less.

4. The system of claims 1-3, wherein the target analyte is a protein or peptide.

5. The system of claims 1-3, wherein the target analyte is a viral protein or peptide.

6. The system of claims 1-3, wherein the target analyte is a nucleocapsid (N) protein of a coronavirus.

7. The system of claims 1-3, wherein the target analyte is an epitope of the N protein of a coronavirus.

8. The system of claims 1-3, wherein the target analyte is the N protein of SARS-CoV-2 or a variant thereof.

9. The system of claims 1-3, wherein the target analyte is an epitope of the N protein of SARS-CoV-2 or a variant thereof.

10. The system of claims 1-3, wherein the system detects two or more target analytes in the biological sample.

11. The system of claims 1-3, wherein the system detects two or more viral species in the biological sample.

12. The system of claims 1-3, wherein the target analyte is a cytokine.

13. The system of claims 1-3, wherein the target analyte is an interleukin or an interferon.

14. The system of claims 1-3, wherein the target analyte is a hormone.

15. The system of claims 1-3, wherein the target analyte is a small molecule.

16. The system of claims 1-11, wherein the system has a limit of detection (LOD) of about 1ng/ML or less.

17. The system of claims 1-15, wherein the system has a LOD of about 500pg/mL or less.

18. The system of claims 1-15, wherein the system has a LOD of about 100pg/mL or less.

19. The system of claims 1-15, wherein the system has a LOD of about 100 target analytes/mL or less.

20. The system of claims 1-14, wherein the system has an LOD selected from about 20, about 10, or about 5 target analytes/mL or less.

21. The system of claims 1-3, wherein the target analyte is a whole virus.

22. The system of claim 21, wherein the virus is SARS-CoV-2 or a variant thereof.

23. The system of claims 1-3, wherein the system has an LOD of about 10000TCID50/mL or less.

24. The system of claims 1-3, wherein the system has an LOD of less than about 100 or less, about 50 or less, about 10 or less, or about 5 or less TCID 50/mL.

25. The system of claims 1-24, wherein the capture reagent and detection reagent are selected from the group consisting of an aptamer, an antibody, a protein, or a combination thereof.

26. The system of claims 1-25, wherein the detection reagent is labeled with an enzyme.

27. The system of claim 25, wherein the enzyme is an oxidoreductase.

28. The system of claim 26, wherein the enzyme is selected from the group consisting of oxidase, peroxidase, hydrogenase, catalase, dehydrogenase, or phosphatase.

29. The system of claim 25, wherein the enzyme is alkaline phosphatase and the added substrate is selected from pyridoxal 5' -phosphate (PLP), 5-bromo-4-chloro-3-indolyl-phosphate, L-ascorbic acid-2-phosphate, acetaminophen phosphate, 4-acetamidophenyl phosphate, 4-aminophenyl phosphate in Diethanolamine (DEA), 1-amino-2-propanol, N-methyl-D-glucosamine, or tris buffer.

30. The system of claim 25, wherein the enzyme is glucose oxidase and the added substrate is glucose.

31. The system of claims 1-30, wherein the capture reagent is immobilized to a solid or porous support, thereby providing a test site.

32. The system of claim 31, wherein the capture reagent is immobilized to the solid support by means of a first binding reagent.

33. The system of claims 31-32, wherein the capture reagent is conjugated to a second binding reagent, wherein the second binding reagent binds to the first binding reagent.

34. The system of claims 31-34, wherein the first binding reagent is crosslinked with one or more additional first binding reagents.

35. The system of claims 31-34, wherein the capture reagent is crosslinked with one or more additional capture reagents.

36. The system of claims 32-35, wherein the first binding reagent is selected from the group consisting of streptavidin, gold, silver, maleimide, acrylate, amine, carboxylic acid, vinyl sulfone, thiol, silane, and epoxide.

37. The system of claims 31-36, wherein the solid support further comprises a control site.

38. The system of claims 1-30, wherein at least one of the at least one capture reagent and the at least one detection reagent is added to the system by a user.

39. The system of claims 1-38, wherein the assay is contained within a cartridge.

40. The system of claims 1-4, wherein the capture reagent and detection reagent have about 10 for the protein ^-10 Kd or less.

41. The system of claims 1-4, wherein the capture reagent and the detection reagent for the protein have about 10 for the protein ^-8 Kd or less.

42. The system of claims 1-4, wherein the capture reagent and detection reagent have about 10 for the protein ^-6 Or a smaller Kd.

43. The system of claims 1-42, wherein the system allows an accuracy of at least about 90% or greater.

44. The system of claims 1-42, wherein the system allows an accuracy of at least about 98% or greater.

45. The system of claims 1-43, wherein the system continuously generates an electrochemical signal.

46. The system of claims 1-45, wherein the electrochemical signal is discontinuously collected.

47. The system of claim 46, wherein the electrochemical signals are collected at intervals separated by waiting periods.

48. The system of claims 1-47, wherein the assay is a lateral flow assay.

49. The system of claims 1-46, wherein the assay is a vertical flow assay.

50. The system of claims 1-49, wherein the self-monitoring system.

51. The system of claims 1-50, wherein said detection is quantitative.

52. An assay system for detecting at least one target analyte in a sample comprising at least one capture reagent and at least one detection reagent, wherein the detection reagent is labeled with an enzyme label and the capture reagent and detection reagent form a detectable complex in the presence of the at least one target analyte and added substrate.

53. The assay system of claim 52, wherein said detectable complex is detected in about 30 minutes or less.

54. The assay system of claims 52-53, wherein said detectable complex is detected within a time frame selected from the group consisting of about 10 minutes or less, about 5 minutes or less, about 2 minutes or less, or about 1 minute or less.

55. The assay system of claims 52-54, wherein said target analyte is a protein or peptide.

56. The assay system of claims 52-54, wherein said target analyte is a viral protein or peptide.

57. The assay system of claims 52-54, wherein said target analyte is a nucleocapsid (N) protein of a coronavirus.

58. The assay system of claims 52-54, wherein said target analyte is an epitope of the N protein of coronavirus.

59. The assay system of claims 52-54, wherein said target analyte is the N protein of SARS-CoV-2 or a variant thereof.

60. The assay system of claims 52-54, wherein said target analyte is an epitope of the N protein of SARS-CoV-2 or a variant thereof.

61. The assay system of claims 52-54, wherein said system detects two or more viral species in said biological sample.

62. The assay system of claims 52-54, wherein the target analyte is a cytokine.

63. The assay system of claims 52-54, wherein said target analyte is an interleukin or an interferon.

64. The assay system of claims 52-54, wherein said target analyte is a hormone.

65. The assay system of claims 52-54, wherein said target analyte is a small molecule.

66. The assay system of claims 52-65, wherein said assay has a limit of detection (LOD) of about 1ng/ML or less.

67. The assay system of claims 52-65, wherein said assay has an LOD of about 500pg/mL or less.

68. The assay system of claims 52-65, wherein said assay has an LOD of about 100pg/mL or less.

69. The assay of claims 52-65, wherein the assay has a LOD of about 100 target analytes/mL or less.

70. The system of claims 52-65, wherein the assay has an LOD selected from 20, 10, or 5 target analytes/mL or less.

71. The assay system of claims 52-54, wherein said target analyte is a whole virus.

72. The assay system of claims 52-54, wherein said virus is SARS-CoV-2 or a variant thereof.

73. The assay system of claims 52-54, wherein said assay has an LOD of about 10000TCID50/mL or less.

74. The assay system of claims 52-54, wherein said assay has an LOD of about 100TCID50/mL or less, about 75TCID50/mL, about 50TCID50/mL or less, about 25TCID50/mL or less, about 10TCID50/mL or less, or about 5TCID50/mL or less.

75. The assay system of claims 52-74, wherein said capture and detection reagents are selected from the group consisting of aptamers, antibodies, proteins, or combinations thereof.

76. The assay system of claims 52-75, wherein said detection reagent is labeled with an enzyme.

77. The assay system of claim 76 wherein said enzyme is an oxidoreductase.

78. The assay system of claim 77, wherein said enzyme is selected from the group consisting of oxidase, peroxidase, hydrogenase, catalase, dehydrogenase, or phosphatase.

79. The assay system according to claim 76, wherein said enzyme is alkaline phosphatase and said added substrate is pyridoxal 5' -phosphate (PLP), 5-bromo-4-chloro-3-indolyl-phosphate, L-ascorbic acid-2-phosphate, acetaminophen phosphate, 4-acetamidophenyl phosphate, 4-aminophenyl phosphate in Diethanolamine (DEA), 1-amino-2-propanol, N-methyl-D-glucosamine or tris buffer.

80. The assay system of claim 76 wherein said enzyme is glucose oxidase and said added substrate is glucose.

81. The system of claims 52-80, wherein the capture reagent is immobilized to a solid or porous support, thereby providing a test site.

82. The assay system of claim 81, wherein the capture reagent is immobilized to a solid or porous support by means of a first binding reagent.

83. The assay system of claim 82, wherein the capture reagent is conjugated to a second binding reagent, wherein the second binding reagent binds to the first binding reagent.

84. The assay system of claims 82-83, wherein the first binding reagent is crosslinked with one or more additional first binding reagents.

85. The assay system of claims 81-84 wherein the capture reagent is crosslinked with one or more additional capture reagents.

86. The assay system of claim 83, wherein said first binding reagent is selected from the group consisting of streptavidin, gold, silver, maleimide, acrylate, amine, carboxylic acid, vinyl sulfone, thiol, silane, and epoxide.

87. The assay system of claims 82-86, wherein the solid support further comprises a control site.

88. The assay system of claims 52-80, wherein said at least one capture reagent and at least one detection reagent are added to said system by a user.

89. The assay system of claims 52-88, wherein the assay is contained within a cartridge.

90. The assay system of claims 52-89, wherein said capture reagent and assayThe test agent has about 10 for the protein ^-10 Or a smaller Kd.

91. The assay system of claims 52-89, wherein said capture and detection reagents have about 10 for said protein ^-8 Kd or less.

92. The assay system of claims 52-89, wherein said capture and detection reagents have about 10 for said protein ^-6 Kd or less.

93. The assay system of claims 52-92, wherein the assay allows an accuracy of at least about 90% or greater.

94. The assay system of claims 52-92, wherein the assay allows an accuracy of at least about 98% or greater.

95. The assay system of claims 52-92, wherein said assay is a lateral flow assay.

96. The assay system of claims 52-92, wherein said assay is a vertical flow assay.

97. The assay system of claims 52-92, wherein detection does not require a detection device.

98. A method for detecting at least one target analyte in a sample, comprising (i) providing a sample, (ii) optionally, treating the sample; (iii) Adding the sample to the system of claims 1-51 or the assay system of claims 52-97; and (iv) if at least one target analyte is present, detecting the target analyte.

99. The method of claim 98, further comprising transmitting the results to a third party for review and optional further action.

100. The method of claim 98, wherein the further action comprises diagnosing a disease or health state.

101. The method of claim 100, wherein the further action comprises diagnosing a diseased state, and optionally administering at least one therapeutic agent.

102. The method of claim 101, wherein the further action comprises diagnosing a disease state, and optionally interrupting administration of or adjusting the dosage of at least one therapeutic agent.

103. The method of claims 98-102, wherein the treating in (ii) comprises diluting the sample.

104. The method of claims 93-102, wherein the treating in (ii) comprises adding one or more capture reagents, detection reagents, or second binding reagents or substrates to the sample prior to adding the sample to the system or assay in (iii).

105. A kit comprising one or more components of the system of claims 1-51 and optionally instructions for use.

106. A kit comprising one or more components of the assay system of claims 52-97 and optionally instructions for use.

107. A blood glucose meter or chronoamperometer configured to read the results of an immunoassay.

108. The chronoamperometric detection system of claim 107 wherein the chronoamperometric detection system utilizes a modified chronoamperometric method.