Virus detection system and its use

Abstract

The systems, methods, devices, and kits disclosed herein are used to determine the presence and / or level of a target analyte in a biological sample, where the analyte is associated with an altered physiological state due to a pathogen (e.g., a viral antigen or whole virus) or otherwise (e.g., pregnancy). In certain embodiments, the systems, methods, devices, and kits offer one or more improved characteristics compared to art-known RT-PCT and lateral flow antibody assays for detecting SARS-CoV-2, including, but not limited to, assay time, ease of use, risk of infection, accuracy, specificity, selectivity, assay detection limit, quantitative detection, and the impact of common interferents on sensor output, cost, simplicity, or a combination thereof. TIFF2023534958000015.tif84158

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 053,048, filed on 21 July 2020, and U.S. Provisional Patent Application No. 63 / 084,814, filed on 29 September 2020, under Section 119(e) of the U.S. Patent Act, all of which are incorporated herein by reference.

[0002] Support from the U.S. government This invention was made with the support of the U.S. Government, authorized by the National Institutes of Health under grant number AI151559 and by the Department of Defense under contract number TTW-27-9661. The U.S. Government has certain rights to this invention.

[0003] Sequence List This application includes a sequence listing submitted in ASCII format via EFS-Web, the contents of which are incorporated herein by reference. A copy of the ASCII file, created on 15 July 2021, is named 701586-098060WOPT_SL.txt and has a size of 63,151 bytes.

[0004] Technical field This specification discloses systems, methods, devices, and kits for detecting and observing target analytes. In certain embodiments, the target analyte is associated with the outcome of a disease (e.g., a viral infection), but in other embodiments, the target analyte is a pathogen, (even Ba The virus itself ) In certain embodiments, a rapid quantitative point-of-care (POC) system, a single-user system, or a home-use system and a method for detecting SARS-CoV-2, influenza, or other viruses are disclosed, the system and method comprising a glucose oxidase-based amperometric sensor. [Background technology]

[0005] Background Infectious diseases, including pathogens that emerge and re-emerge over the long term, continue to pose significant public health and economic threats. One devastating example is the coronavirus pandemic (SARS-CoV-2), and interventions are being urgently explored. Despite the coronavirus pandemic, the rate of spread of infectious diseases was already on the rise, and demographic trends and climate change suggest that this problem may continue to worsen.

[0006] Comprehensive testing is an important approach for identifying and isolating infected individuals and / or providing early intervention. The current gold standard for SARS-CoV-2 testing is based on reverse transcription polymerase chain reaction (RT-PCR), which relies on obtaining viral nucleic acids from respiratory samples, experienced staff, large reagent stocks, expensive equipment, and has a very high rate of false negatives. In addition to the high false negative rate of RT-PCR, providing samples from the respiratory tract (e.g., nasal swabs) can be uncomfortable, so testing may be discouraged. Therefore, the number and usefulness of RT-PCR tests for SARS-CoV-2 are not optimal.

[0007] Alternatively, several lateral flow immunoassays for detecting SARS-CoV-2 antibodies have been developed. These tests look for the presence of three types of antibodies produced in the body as an immune response, namely IgG, IgM, and IgA. However, since the body typically takes 5 to 10 days after infection to produce these antibodies, these antibody-based assays are not as useful as RT-PCR for the early diagnosis of SARS-CoV-2 infection. Finally, antibody tests tend to have problems with sensitivity and specificity that result in a high false positive rate.

[0008] There remains a need to improve the number and usefulness of tests for SARS-CoV-2, as well as for other viral pathological altered physiological states. SUMMARY OF THE INVENTION

[0009] Summary The systems, methods, devices, and kits disclosed herein can be used to determine the presence and / or level of a target analyte in a biological sample, where the analyte is, for example, associated with a physiological state altered by a pathogen or other viral infection. In certain embodiments, the systems, methods, devices, and kits offer one or more improved characteristics relative to RT-PCR and lateral flow antibody assays known in the Art, including, but not limited to, assay time, ease of use, risk of infection, accuracy, specificity, selectivity, detection limit of the assay, quantitative detection, and the impact of common interferences on sensor output, cost, simplicity, or a combination thereof.

[0010] In one aspect, a system for detecting at least one target nucleic acid in a biological sample is disclosed, the system comprising (i) an assay of at least two binders, comprising first and second binders capable of forming a detectable complex with the at least one target analyte, and (ii) a detection device for detecting the detectable complex, the detection device being a glucometer or a glucose oxidase-based amperometric sensor, the biological sample being present in sweat, saliva, serum, mucus, or blood.

[0011] In one embodiment, the glucometer is a handheld portable glucose meter and includes a glucose sensor having a sensor output for glucose in a biological sample on a test strip. Specifically, the glucose sensor produces an output that correlates with the presence or concentration of a target analyte other than glucose in the biological sample.

[0012] In one embodiment, glucose oxidase catalyzes the oxidation of glucose to form hydrogen peroxide, which is then quantified by amperometry (e.g., change in current) using one or more electrodes. Since the amount of glucose in the biological sample is in excess and added for detection, amperometry is relevant to the quantification of the target analyte, such as SARS-CoV-2, H1N1, or others.

[0013] In one embodiment, the biological sample is saliva.

[0014] In other embodiments, biological samples include urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, sputum, pleural fluid, cerebral fluid, or nasopharyngeal specimens.

[0015] In one embodiment, the biological sample is mixed with glucose.

[0016] In other embodiments, the biological sample is mixed with glucose at a concentration of 0.01 mM to 1 M.

[0017] In other embodiments, the biological sample is mixed with sucrose, fructose, maltose, galactose, cellulose, or any combination thereof, at a concentration of 0.01 mM to 1 M (of the solubility limit), containing amylase or invertase.

[0018] In one embodiment of a conjugate capable of detecting a target analyte, the first and second binders are selected from aptamers, antibodies, or proteins.

[0019] In one embodiment of a complex capable of detecting the target analyte, 1 The first binder is selected from aptamers, antibodies, or proteins, and the second binder is selected from aptamers, antibodies, or proteins linked to an oxidase enzyme.

[0020] In certain embodiments of a complex capable of detecting the target analyte, 1The first binder is selected from aptamers, antibodies, or proteins, and the second binder is selected from aptamers, antibodies, or proteins that are linked to glucose oxidase and produced from a fermentation process.

[0021] In certain embodiments of a complex capable of detecting the target analyte, 1 The first binder is selected from aptamers, antibodies, or proteins, and the second binder is selected from aptamers, antibodies, or proteins linked to glucose oxidase, galactose oxidase, D-glucose:D-fructose oxidoreductase, and cellobiose oxidase.

[0022] In one embodiment of a complex capable of detecting the target analyte, 1 The first binder is selected from aptamers, antibodies, or proteins, and the second binder is selected from aptamers, antibodies, or proteins linked to a hydrogenase enzyme.

[0023] In certain embodiments of a complex capable of detecting the target analyte, 1 The first binder is selected from aptamers, antibodies, or proteins, and the second binder is selected from aptamers, antibodies, or proteins linked to glucose dehydrogenase, glucose-6-phosphate dehydrogenase, fructose dehydrogenase, sucrose dehydrogenase, glucoside dehydrogenase, alcohol dehydrogenase, sorbitol dehydrogenase, lactate dehydrogenase, and malate dehydrogenase.

[0024] In certain embodiments of a complex capable of detecting a target analyte, the first and second binders bind to a first site and a different second site on the target analyte, respectively.

[0025] In another embodiment of a complex capable of detecting the target analyte, the first and second binders each bind to the same site on the target analyte. Since the site on the target analyte exists as multiple copies (>100), there are enough sites for the first and second binders.

[0026] In certain embodiments, the binding affinity of the first binder to a first site on the target analyte is higher than the binding affinity of the second binder to a second site on the target analyte.

[0027] In certain embodiments, the binding affinity of the first binder to a first site on the target analyte is the same as the binding affinity of the second binder to a second site on the target analyte.

[0028] In certain embodiments, the binding affinity of the first binder to a first site on the target analyte is weaker than the binding affinity of the second binder to a second site on the target analyte.

[0029] In one embodiment, at least one target analyte is selected from a virus or an antibody produced from a viral infection.

[0030] In certain embodiments, at least one target analyte is a virus, more specifically a coronavirus such as a betacoronavirus, and even more specifically SARS-CoV-2.

[0031] In one embodiment, the first and second binders bind to different epitopes of the SARS-CoV-2 spike (S) protein. In a particular embodiment, at least one of the epitopes is located within the receptor-binding domain (RBD) of the S protein.

[0032] In one embodiment, one of the binders binds to the SARS-CoV-2 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.

[0033] In certain embodiments, at least one target analyte is a virus, more specifically a coronavirus such as a betacoronavirus, and even more specifically SARS-CoV.

[0034] In one embodiment, the first and second binders bind to different epitopes of the SARS-CoV spike (S) protein. In a particular embodiment, at least one of the epitopes is located within the receptor-binding domain (RBD) of the S1 protein.

[0035] In one embodiment, one of the binders binds to the 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.

[0036] In certain embodiments, at least one target analyte is a virus, and more specifically, a rhinovirus.

[0037] In one embodiment, the first and second binders bind to one of the four possible capsid proteins of the rhinovirus.

[0038] In certain embodiments, at least one target analyte is a virus, more specifically, a common human coronavirus including types 229E, NL63, OC43, and HKU1.

[0039] In one embodiment, the first and second binders bind to the spike protein, membrane protein, hemagglutinin protein, envelope, or envelope protein of a common human coronavirus.

[0040] In certain embodiments, at least one target analyte is a virus, more specifically, a polynuclear respiratory virus (RSV), parainfluenza (PIV), H1N1, or herpesvirus.

[0041] In one embodiment, the first and second binders bind to a fusion protein of respiratory multinuclear virus (RSV), parainfluenza (PIV), or H1N1, a membrane protein, a hemagglutinin protein, a neuraminidase protein, an envelope, or an envelope protein.

[0042] In certain embodiments, at least one target analyte is a virus, more specifically, a human metapneumovirus.

[0043] In one embodiment, the first and second binders bind to a human metapneumovirus fusion protein, SH protein, substrate protein, glycoprotein, envelope, or envelope protein.

[0044] In certain embodiments, at least one target analyte is a virus, more specifically, human immunodeficiency virus (HIV).

[0045] In one embodiment, the first and second binders bind to the MHC protein, p17 substrate protein, gp120 docking glycoprotein, gp41 transmembrane glycoprotein, envelope, or envelope protein of human immunodeficiency virus (HIV).

[0046] In certain embodiments, at least one target analyte is a virus, and more specifically, the Ebola virus.

[0047] In one embodiment, the first and second binders bind to the glycoprotein, substrate protein, nucleoprotein, envelope, or envelope protein of the Ebola virus.

[0048] In certain embodiments, at least one target analyte is a virus, more specifically, the Marburg virus.

[0049] In one embodiment, the first and second binders bind to the Marburg virus glycoprotein, VP40 substrate protein, nucleoprotein, envelope, or envelope protein.

[0050] In certain embodiments, at least one target analyte is a virus, and more specifically, a Lassa virus.

[0051] In one embodiment, the first and second binders bind to Lassa virus glycoprotein 1, glycoprotein 2, macroprotein, zinc protein, stable signaling peptide (SSP), nucleoprotein, envelope, or envelope protein.

[0052] In one embodiment, the binding assay allows for the detection of two or more target analytes, for example, two or more viruses.

[0053] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound to the test strip.

[0054] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound via a test strip and a polymer membrane placed on the strip.

[0055] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound to the test strip via a hydrophilic membrane, such as a nitrocellulose membrane, placed on the strip.

[0056] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound via a hydrophilic membrane, such as a nitrocellulose membrane, located directly above the test strip and the electrodes on the strip or between the two electrodes.

[0057] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound to the test strip and the strip via a hydrophilic membrane, such as a nitrocellulose membrane, which is fixed to the electrode on or between the two electrodes.

[0058] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound to a test strip via a hydrophilic membrane, the membrane also collects a biological sample and provides a sink area for the sample to flow from one point on the membrane to another.

[0059] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound to a test strip via a hydrophilic membrane, such as a nitrocellulose membrane, the membrane also collects a biological sample and provides a sink area for the sample to flow from one point on the membrane to another.

[0060] In another aspect, a system for detecting a single virus in a biological sample is disclosed, the system comprising (i) a two-conjugate assay comprising first and second conjugates capable of forming a detectable complex with the at least one virus, and (ii) a detection device for detecting the detectable complex, the detection device being a glucometer (also referred to herein as a glucose meter).

[0061] In one embodiment, the biological sample is saliva.

[0062] In other embodiments, the biological sample may be urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, nasal cavity samples, pleural fluid, cerebrospinal fluid, or nasopharyngeal specimens. In one embodiment, the biological sample is mixed with glucose.

[0063] In other embodiments, the biological sample is mixed with glucose at a concentration of 0.01 mM to 1 M.

[0064] In other embodiments, the biological sample is mixed with sucrose, fructose, maltose, galactose, cellulose, or any combination thereof, at concentrations of 0.01 mM to 1 M, containing amylase or invertase.

[0065] In one embodiment of a conjugate capable of detecting a target analyte, the first and second binders are selected from aptamers, antibodies, or proteins.

[0066] In one embodiment of a conjugate capable of detecting a target analyte, the first and second binders are selected from aptamers, antibodies, or proteins, and the second binder is selected from aptamers, antibodies, or proteins linked to an oxidase enzyme.

[0067] In certain embodiments of a complex capable of detecting a target analyte, the first and second binders are selected from aptamers, antibodies, or proteins, and the second binder is selected from aptamers, antibodies, or proteins that are linked to glucose oxidase and produced by a fermentation process.

[0068] In certain embodiments of a complex capable of detecting a target analyte, the first and second binders are selected from aptamers, antibodies, or proteins, and the second binder is selected from aptamers, antibodies, or proteins linked to glucose oxidase, galactose oxidase, D-glucose:D-fructose oxidoreductase, and cellobiose oxidase.

[0069] In one embodiment of a conjugate capable of detecting a target analyte, the first and second binders are selected from aptamers, antibodies, or proteins, and the second binder is selected from aptamers, antibodies, or proteins linked to a hydrogenase enzyme.

[0070] In certain embodiments of a complex capable of detecting a target analyte, the first and second binders are selected from aptamers, antibodies, or proteins, and the second binder is selected from aptamers, antibodies, or proteins linked to glucose dehydrogenase, glucose-6-phosphate dehydrogenase, fructose dehydrogenase, sucrose dehydrogenase, glucoside dehydrogenase, alcohol dehydrogenase, sorbitol dehydrogenase, lactate dehydrogenase, and malate dehydrogenase.

[0071] In certain embodiments of the complex capable of detecting the target analyte, the first and second binders bind to a first site and a different second site on the target analyte, respectively.

[0072] In another embodiment of a complex capable of detecting the target analyte, the first and second binders each bind to the same site on the target analyte. Since the site on the target analyte exists as multiple copies (>100), there are enough sites for the first and second binders.

[0073] In certain embodiments, the binding affinity of the first binder to a first site on the target analyte is higher than the binding affinity of the second binder to a second site on the target analyte.

[0074] In certain embodiments, the binding affinity of the first binder to a first site on the target analyte is the same as the binding affinity of the second binder to a second site on the target analyte.

[0075] In certain embodiments, the binding affinity of the first binder to a first site on the target analyte is weaker than the binding affinity of the second binder to a second site on the target analyte.

[0076] In one embodiment, the binding assay enables the detection of two or more viruses, specifically (i) coronaviruses such as beta-coronaviruses, more specifically SARS-CoV-2, and (ii) respiratory viruses, more specifically influenza.

[0077] In another aspect, a method for diagnostic evaluation is disclosed, which includes: (i) collecting a biological sample from a subject, wherein the biological sample is not blood; (ii) adding the biological sample to a test strip in the presence of glucose, wherein the test strip comprises first and second binders capable of forming a detectable complex with at least one target analyte if present in the biological sample; (iii) inserting the test strip into a detection device (such as a glucometer); (iv) incubating the biological sample with the test strip; (v) detecting the level of any detectable complex through a chemical reaction between glucose and glucose oxidase; and (vi) correlating the level of any detectable complex formed with the amount of any target analyte present in the at least one biological sample, thereby providing a diagnostic evaluation.

[0078] In another aspect, a method for diagnostic evaluation is disclosed, which includes: (i) collecting a biological sample from a subject, wherein the biological sample is not blood; (ii) adding the biological sample to a test strip in the presence of glucose, wherein the test strip comprises first and second binders capable of forming a detectable complex with at least one target analyte if present in the biological sample; (iii) incubating the biological sample with the test strip, or not incubating it; (iv) inserting the test strip into a detection device (such as a glucometer); (v) detecting the level of any detectable complex through a chemical reaction between glucose and glucose oxidase; and (vi) correlating the level of any detectable complex formed with the amount of any target analyte present in the at least one biological sample, thereby providing a diagnostic evaluation.

[0079] In another aspect, a method for diagnostic evaluation is disclosed, which includes: (i) collecting a biological sample from a subject, the biological sample being derived from urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, or a nasopharyngeal specimen; (ii) adding the biological sample to a test strip, the test strip comprising first and second binders capable of forming a detectable complex with at least one target analyte if present in the biological sample; (iv) inserting the test strip into a detection device such as a glucometer; (v) incubating the biological sample with the test strip; (vi) detecting the level of the detectable complex, if present, through a chemical reaction between glucose and glucose oxidase; and (vii) correlating the level of the detectable complex, if present, with the amount of the target analyte, if present in the at least one biological sample, thereby providing a diagnostic evaluation.

[0080] In another aspect, a method for diagnostic evaluation is disclosed, the method comprising: (i) collecting a biological sample from a subject, the biological sample being derived from urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, or nasopharyngeal specimens; (ii) diluting the collected sample 1 to 100 times in an aqueous solution / mixture in the presence of a second binder; (iii) adding the biological sample and the second binder to a test strip, the test strip being tested for at least one target analyte if present in the biological sample. The method includes the steps of: (iv) adding a first binder capable of forming a detectable complex; (v) incubating the biological sample with the test strip, or not incubating it; (v) inserting the test strip into a detection device; (vi) detecting the level of any detectable complex, if any, through a chemical reaction between glucose and glucose oxidase; and (vii) correlating the level of any detectable complex, if any, with the amount of any target analyte present in the at least one biological sample, thereby providing a diagnostic assessment.

[0081] In another aspect, this specification describes a method for diagnostic evaluation, the method comprising: (i) collecting a biological sample from a subject, the biological sample being derived from urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, or a nasopharyngeal specimen; (ii) adding the biological sample to a test strip, the test strip comprising first and second binders capable of forming a detectable complex with the biological sample if at least one target analyte is present therein; (iii) incubating the biological sample with the test strip, or not incubating it; (iv) inserting the test strip into a detection device such as a glucometer; (v) detecting the level of the detectable complex, if present, through a chemical reaction between glucose and glucose oxidase; and (vi) correlating the level of the detectable complex, if formed, with the amount of the target analyte in the at least one biological sample, thereby providing a diagnostic evaluation.

[0082] In another aspect, this specification describes a method for diagnostic evaluation, the method comprising: (i) collecting a biological sample from a subject, the biological sample being derived from urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, or nasopharyngeal specimens; (ii) diluting the collected sample 1 to 100 times in an aqueous solution / mixture in the presence of a second binder; (iii) adding the biological sample and the second binder to a test strip, the test strip being mixed with at least one target analyte if present in the biological sample. The method includes the steps of: (iv) incubating the biological sample with the test strip, or not incubating it; (v) inserting the test strip into a detection device such as a glucometer; (vi) detecting the level of any detectable complex through a chemical reaction between glucose and glucose oxidase; and (vii) correlating the level of any detectable complex formed with the amount of a target analyte in the at least one biological sample, thereby providing a diagnostic assessment.

[0083] In another aspect, this specification describes a method for diagnostic evaluation, the method comprising: (i) collecting a biological sample from a subject, the biological sample being derived from urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, or a nasopharyngeal specimen; (ii) adding the biological sample to a test strip, the test strip comprising first and second binders capable of forming a detectable complex with at least one target analyte if present in the biological sample; (iii) incubating the biological sample with the test strip; (iv) the test strip (v) inserting the sample into a detection device such as a glucometer; (v) detecting the level of any detectable complex through a chemical reaction between glucose and glucose oxidase; (vi) correlating the level of any detectable complex produced with the amount of any target analyte present in the at least one biological sample, thereby providing a diagnostic assessment; (vii) transmitting the diagnostic assessment or results to an electronic device, database, or cloud server for subsequent review by a clinician or trained healthcare provider; and (viii) transmitting the diagnostic assessment or outcome to the individual who performed the diagnostic assessment method.

[0084] In another aspect, this specification describes a method for diagnostic evaluation, the method comprising: (i) collecting a biological sample from a subject, the biological sample being derived from urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, or nasopharyngeal specimens; (ii) diluting the collected sample 1 to 100 times in an aqueous solution / mixture in the presence of a second binder; (iii) adding the biological sample to a test strip, the test strip comprising a first binder capable of forming a detectable complex with at least one target analyte if present in the biological sample; and (iv) adding the biological sample to the test strip (v) incubating the test strip in a glucose oxidase; (vi) inserting the test strip into a detection device such as a glucose meter; (vi) detecting the level of any detectable complexes present through a chemical reaction between glucose and glucose oxidase; (vii) correlating the level of any detectable complexes present, if any, with the amount of any target analytes present in the at least one biological sample, thereby providing a diagnostic assessment; (viii) transmitting the diagnostic assessment or results to an electronic device, database, or cloud server for subsequent review by a clinician or trained healthcare provider; and (ix) transmitting the diagnostic assessment or outcome to the individual who performed the diagnostic assessment method.

[0085] In another aspect, a test strip used for biological evaluation is disclosed, the test strip comprising: (i) one or more planar or coplanar electrodes made of carbon, iron, palladium, platinum, or gold; (ii) an electrode coated with an iron salt such as ferrous ferrocyanide as a mediator; (iii) an electrode coated with Prussian blue as a mediator; (iv) an electrode setup in which the electrode in contact with the electrolyte solution is a (semi)conducting solid; (v) the electrode setup comprising a working electrode, a reference electrode, and a pair or auxiliary electrode; (vi) current leads and sense leads connected, and acting Lead and sense of action LeadIt is connected to the working electrode, and is a reference Lead and against Lead (vii) A two-electrode setup in which the reference lead is connected to a second auxiliary, paired, or pseudo / pseudo-reference electrode; (vii) a paired reference lead Lead (viii) a three-electrode setup comprising: (viii) a four-electrode setup in which, in addition to the reference lead, the working sense lead is disconnected from the working electrode; and / or (ix) a no-resistance ammeter in which the working electrode lead and the counter electrode lead are short-circuited in a strip so that the voltage drop of the entire electrochemical cell is net zero.

[0086] Results obtained using the systems and methods described herein may be reported in a format selected from binary (e.g., yes / no), semi-quantitative (e.g., low, medium, high), or quantitative.

[0087] In another aspect, a test strip is disclosed comprising (i) a substrate, at least one of a first and a second binder, and two or more electrodes; (ii) both a substrate, both the first and a second binder, and two or more electrodes; (iii) at least one of the first and a second binder, and two or more electrodes; or (iv) both the first and a second binder, and two or more electrodes.

[0088] In one embodiment, the inspection strip further comprises an inspection area, wherein (i) the inspection area comprises a first binder; (ii) the inspection area comprises both the first and second binders; or (iii) the inspection area comprises a substrate and the first binder; or (iv) the inspection area comprises a substrate and both the first and second binders.

[0089] In another aspect, this specification describes a test strip used in a system described herein, the test strip comprising (i) a substrate, at least one of a first and a second binder, and two or more electrodes; (ii) a substrate, both of the first and a second binder, and two or more electrodes; (iii) at least one of the first and a second binder, and two or more electrodes; or (iv) both of the first and a second binder, and two or more electrodes.

[0090] In another aspect, a kit is disclosed, which includes test strips and optionally instructions for using the test strips. In certain embodiments, the kit further includes a glucometer.

[0091] In another context, localized or cloud-based software algorithms are disclosed, which trigger an electrochemical reaction in a detection system such that one or more detectable chemical species are the reaction product of a biological sample, a test strip, and a detection device.

[0092] In another aspect, this specification describes a method comprising: (i) preparing a biological sample derived from a subject; (ii) detecting the presence of a target analyte in the biological sample using a system described herein, wherein the target analyte is the SARS-CoV-2 virus or a component thereof; and (iii) optionally, treating the subject with a therapeutic agent (for example, one effective against the SARS-CoV-2 virus).

[0093] In another aspect, this specification describes a method comprising: (i) preparing a biological sample derived from a subject; (ii) detecting the presence of a target analyte in the biological sample using a system described herein, wherein the target analyte is the CoV virus or a component thereof; and (iii) optionally, treating the subject with a therapeutic agent.

[0094] In another aspect, a treatment is provided, which (for example, in a series of steps) includes the steps of (i) (a) preparing a biological sample derived from a subject, wherein the biological sample is saliva, and (b) detecting the presence of a target analyte in the biological sample using a system described herein, wherein the target analyte is the SARS-CoV-2 virus or a component thereof (for example, the S-1 protein); and (ii) treating the subject with a therapeutic agent selected from small molecule agents or biological agents (for example, one effective against the SARS-CoV-2 virus).

[0095] In another aspect, this specification describes a method comprising: (i) preparing a biological sample derived from a subject; (ii) detecting the presence of a target analyte in the biological sample using a system described herein, wherein the target analyte is an influenza virus or a component thereof; and (iii) optionally, treating the subject with a therapeutic agent (for example, one effective against the influenza virus).

[0096] In another aspect, this specification describes a method comprising: (i) preparing a biological sample derived from a subject; (ii) detecting the presence of a target analyte in the biological sample using a system described herein, wherein the target analyte is a hepatitis virus or a component thereof; and (iii) optionally, treating the subject with a therapeutic agent (for example, one effective against hepatitis viruses).

[0097] In another aspect, this specification describes a system for detecting at least one target nucleic acid in a biological sample, the system comprising (i) a sequence-specific endonuclease and guide nucleic acid that specifically bind to the target nucleic acid and cleave a collateral nucleic acid, (ii) a detection nucleic acid that can form a detectable complex with the cleaved collateral nucleic acid, and (iii) a detection device for detecting the detectable complex, the detection device being an oxidase-based amperometric sensor, the biological sample being present in sweat, saliva, serum, mucus, or blood.

[0098] In some aspects of any given situation, sequence-specific endonucleases are Cas enzymes.

[0099] In some aspects of any given situation, the sequence-specific endonuclease is Cas12a or Cas13.

[0100] In some aspects of any given situation, the guide nucleic acid is complementary or substantially complementary to at least a portion of the target nucleic acid.

[0101] In some aspects of any of these, the detected nucleic acid is complementary or substantially complementary to at least a portion of the cleaved collateral nucleic acid.

[0102] In some aspects of any given situation, the detected nucleic acid hybridizes with the cleaved collateral nucleic acid.

[0103] In some aspects of any given situation, the detected nucleic acid does not hybridize with the uncleaved collateral nucleic acid.

[0104] In some aspects of any given situation, the nucleic acid to be detected is linked to the test strip.

[0105] In some aspects of any given situation, collateral nucleic acids are linked to glucose oxidase.

[0106] In some aspects of any of these phases, the system further comprises an aptamer linked to glucose oxidase.

[0107] In some aspects of any of these, the aptamer specifically binds to at least a portion of the cleaved collateral nucleic acid.

[0108] In some aspects of any given scenario, the aptamer binds to the single-stranded portion of the cleaved collateral nucleic acid.

[0109] In some aspects of any of these phases, the aptamer binds to the double-stranded portion of the cleaved collateral nucleic acid that has hybridized with the detected nucleic acid.

[0110] In some aspects of any of these phases, the system further includes an antibody linked to glucose oxidase.

[0111] In some aspects of any of these, the antibody specifically binds to at least a portion of the cleaved collateral nucleic acid.

[0112] In some aspects of any given situation, collateral nucleic acids are linked to antibodies that specifically bind to glucose oxidase.

[0113] In some aspects of any of these situations, the collateral nucleic acid is linked to the first member of the affinity pair.

[0114] In some aspects of any of these phases, the system further comprises glucose oxidase linked to a second member of the affinity pair.

[0115] In some aspects of any aspect, the first and second members of an affinity pair are selected from the group consisting of: combinations of haptenic or antigenic compounds with corresponding antibodies or their binding portions or fragments; digoxigenin and anti-digoxigenin; mouse immunoglobulins and goat anti-mouse immunoglobulins; non-immunological binding pairs; biotin and avidin; biotin and streptavidin; hormones and hormone-binding proteins; thyroxine and cortisol hormone-binding proteins; receptors and receptor agonists; receptors and receptor antagonists; acetylcholine receptors and acetylcholine or their analogues; IgG and protein A; lectins and carbohydrates; enzymes and enzyme cofactors; enzymes and enzyme inhibitors; complementary oligonucleotide pairs capable of forming nucleic acid doubles; and a first molecule having a negative charge and a second molecule having a positive charge.

[0116] In some aspects of any given situation, the first and second members of the affinity pair are streptavidin and biotin.

[0117] In some aspects of either of these, streptavidin is linked to collateral nucleic acids, and biotin is linked to glucose oxidase.

[0118] In some aspects of either of these, biotin is linked to collateral nucleic acids, and streptavidin is linked to glucose oxidase.

[0119] In some aspects of any given situation, the target nucleic acid is viral nucleic acid.

[0120] In another aspect, this specification describes a method for detecting a target nucleic acid using a nucleic acid detection system described herein, the method comprising: (i) collecting a biological sample from a subject and optionally extracting nucleic acids from the biological sample; (ii) contacting the biological sample with a sequence-specific endonuclease, a guide nucleic acid, and a collateral nucleic acid, wherein, if the target nucleic acid is present, the collateral nucleic acid is cleaved as a result of such contact; (iii) adding the biological sample to a test strip in the presence of glucose, wherein the test strip is cleaved of the cleaved collateral nucleic acid. The method includes: (iv) incubating the biological sample with the test strip, or not incubating it; (v) inserting the test strip into a detection device; (vi) detecting the level of any detectable complex, if present, through a chemical reaction between glucose and glucose oxidase; and (vii) correlating the level of any detectable complex, if formed, with the amount of a target analyte in the at least one biological sample, thereby providing a diagnostic assessment.

[0121] In some aspects of any given situation, the biological sample is saliva.

[0122] In some aspects of any of these situations, the detection device is a glucose meter.

[0123] In some aspects of any of these aspects, the method further includes (viii) transmitting the diagnostic assessment or results to an electronic device, database, or cloud server for subsequent review by a clinician or trained healthcare provider; and (ix) providing the diagnostic assessment to the individual who performed the diagnostic assessment method.

[0124] In some aspects of any given situation, the individual is the subject.

[0125] In some aspects of any aspect, the method further includes (viii) recommending, instructing, and / or administering one or more treatment regimens to the subject in accordance with the diagnostic assessment.

[0126] In another context, this specification describes a test strip linked to a nucleic acid to be detected.

[0127] In another context, this specification describes a kit comprising a test strip linked to a nucleic acid to be detected. [Brief explanation of the drawing]

[0128] [Figure 1] Figure 1 shows an exemplary repurposed glucometer-based SARS-CoV-2 sensor. [Figure 2] Figure 2 shows an exemplary test strip design and the signal output when the test strip is placed in a glucometer. [Figure 3] Figure 3 is a schematic diagram illustrating the sequence of events occurring in the glucose biosensor system. Glucose oxidation by GOx produces D-glucono-δ-lactone. The reduction of H2O2 in the Prussian blue (PB) film is measured by electrons transferred from the working electrode. [Figure 4] Figure 4 shows line graphs (left panel) illustrating the current over time for different virion (e.g., H1N1) concentrations. The bar graph (right panel) shows the area under the curve of the current vs. time plot (e.g., see left panel) for different virion concentrations in the buffer. [Figure 5]Figure 5 shows a schematic diagram of the CoV test strip described herein for use with a glucometer. Labels (1) to (16) correspond to different functional parts of the strip, and labels (A) to (H) correspond to layers of the strip. In one embodiment, the sensor strip includes (from bottom to top) (A) a base substrate; (B) a conductive layer containing three electrodes; (C) an insulating layer exposing only the portion of the electrode to which the sample to be tested is dropped; (D) a reagent layer containing a mediator to facilitate electron exchange; (E) an adhesive layer; (F) a hydrophilic nitrocellulose membrane, the proximal membrane containing an aptamer and lyophilized glucose for capturing the antigen, and the distal end being a paper sink (13); (G) lyophilized Ab-GOx; and (H) an upper layer. [Figure 6] Figure 6 shows line graphs illustrating the current over time for human saliva to which different H1N1 virion concentrations were added and which was diluted 10-fold. [Figure 7] Figure 7 shows a schematic diagram of a localized software algorithm used to trigger a detectable chemical change in a detection device. [Figure 8] Figures 8A-8B show schematic diagrams of cloud-based software algorithms on external servers used to process data and trigger chemical changes in detection devices. [Figure 9] Figure 9 shows the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein sequence aligned with other related coronaviruses. Specific sequence alignments of the interaction domains of SARS-CoV-2, SARS-CoV, and MERS-CoV are shown (see, for example, SEQ ID NO: 1-9). See, for example, W. Tai, et al., Cellular & Molecular Immunology, (2020) 17:613-620, the entire content of which is incorporated herein by reference. [Figure 10] Figure 10 shows a line graph illustrating the current over time in an assay pre-incubated with H1N1 virus and Ab-GOx. [Figure 11]Figure 11 shows a line graph illustrating the time-dependent current for the VSV-CoV-2 pseudotype virus. [Figure 12] Figure 12 shows a series of line graphs illustrating the time-dependent currents for the SARS-CoV-2 virus using rabbit polyclonal antibody-GOx conjugates (left panel) or membrane antibody-GOx conjugates (right panel). [Figure 13] Figure 13 shows line and bar graphs measuring the cross-reactivity of aptamers and antibodies targeting SARS-CoV-2 against the H1N1 virus. There was no significant difference between the signals detected from 104 H1N1 virions and those detected from non-virions. [Figure 14] Figure 14 is a schematic diagram illustrating the detection of one or two viruses. To detect one virus, the test strip has one aptamer (or antibody) against that virus on its surface. Urine, saliva, or other material is added to the strip, and the strip is inserted into the glucometer. For the detection of two viruses (for example, SarsCoV-2 and influenza A H1N1), one portion of the test strip has an aptamer (or antibody) against one virus, and the other portion has an aptamer (or antibody) against a second virus. Urine, saliva, or other material is added to the strip, the strip is divided into two strips by a dashed line, one strip is inserted into the glucometer and read, then the first strip is removed, and the second strip is inserted and read. [Figure 15A] Figures 15A–15F are a series of schematic diagrams illustrating alternative designs for the detection devices described herein (see, for example, Example 8). Figure 15A shows nucleic acid detection using collateral cleavage by an endonuclease, where the collateral nucleic acid is linked to glucose oxidase. [Figure 15B]Figures 15A–15F are a series of schematic diagrams illustrating alternative designs for the detection devices described herein (see, for example, Example 8). Figures 15B–15C illustrate collateral cleavage nucleic acid detection, where an aptamer that binds to a single-stranded (Figure 15B) or double-stranded (Figure 15C) region of the collateral nucleic acid is ligated to glucose oxidase. [Figure 15C] Figures 15A–15F are a series of schematic diagrams illustrating alternative designs for the detection devices described herein (see, for example, Example 8). Figures 15B–15C illustrate collateral cleavage nucleic acid detection, where an aptamer that binds to a single-stranded (Figure 15B) or double-stranded (Figure 15C) region of the collateral nucleic acid is ligated to glucose oxidase. [Figure 15D] Figures 15A–15F are a series of schematic diagrams illustrating alternative designs for the detection devices described herein (see, for example, Example 8). Figure 15D shows nucleic acid detection, where an antibody conjugating a collateral nucleic acid is linked to glucose oxidase. [Figure 15E] Figures 15A–15F are a series of schematic diagrams illustrating alternative designs for the detection devices described herein (see, for example, Example 8). Figure 15E shows nucleic acid detection, where collateral nucleic acids are linked to an antibody that specifically binds to glucose oxidase. [Figure 15F] Figures 15A–15F are a series of schematic diagrams illustrating alternative designs for the detection devices described herein (see, for example, Example 8). Figure 15F shows nucleic acid detection, where a collateral nucleic acid is linked to an affinity pair, one member of which is linked to glucose oxidase. [Figure 16]Figure 16 shows an example process and an example of the system overview of several embodiments of this disclosure. The upper half of Figure 16 is a flowchart of an example process for detecting a target analyte in a sample using the test strip and detection device described herein. In some embodiments of any aspect, a test sample 110 is received (e.g., from a subject 100). Further samples may include a negative control 111 and a positive control 112. In some embodiments of any aspect, the sample is optionally processed 120 (e.g., protein or nucleic acid extraction; e.g., dilution). The biological sample is then added to the test strip 135 in the presence of glucose 130. The test may include at least one of the detection reagents described herein (e.g., an antibody, an aptamer, a detection nucleic acid). The test strip is then inserted into the detection device 140 (e.g., a glucometer). The lower half of Figure 16 shows an example of the system overview of several embodiments of this disclosure. The test strip is input to a detection device 150, which is part of a system that includes a network 160, a computer device 170, a display 175, a server 180, and / or a database 185. [Modes for carrying out the invention]

[0129] Detailed explanation The above summary of the present invention does not describe all or any of the embodiments disclosed. The following description provides more specific examples of exemplary embodiments. In several places in this specification, we provide guidance through lists of examples, which can be used in various combinations. In any example, the listed examples serve only as a representative group and should not be interpreted as an exclusive list.

[0130] For reference, the meanings of some terms and phrases used in this specification, the examples, and the appended claims are given below. Unless otherwise specified or implied by the context, the following terms and phrases have the meanings set forth below. These definitions are intended to help describe specific embodiments and do not limit the claimed invention, for the scope of the invention is limited solely by the claims. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art in which the invention pertains. If there is any clear discrepancy between the usage of a term in the art and the definitions provided herein, the definitions provided herein shall prevail.

[0131] Where used herein, the singular forms “a,” “an,” and “the” also include the plural forms unless the context explicitly negates them. Similarly, the word “or” also includes “and” unless the context explicitly negates it. In the implementation or testing of this disclosure, methods and materials similar or equivalent to those described herein may be used, but preferred methods and materials are described below. “eg” is an abbreviation derived from the Latin “exempli gratia” and is used herein to indicate an unrestricted example. Thus, the abbreviation “eg” is synonymous with the term “for example.”

[0132] When the term "approximately" is used herein in relation to any value (including the upper and lower limits of a numerical range), it means ±0.5% to ±20% (and values ​​in between, for example, ±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5%, ±5%, ±5.5%, ±6%, ±6.5%, ±7%, ±7.5%, ±8%, ±8.5%, ± This refers to any value having an acceptable deviation range of 9%, ±9.5%, ±10%, ±10.5%, ±11%, ±11.5%, ±12%, ±12.5%, ±13%, ±13.5%, ±14%, ±14.5%, ±15%, ±15.5%, ±16%, ±16.5%, ±17%, ±17.5%, ±18%, ±18.5%, ±19%, ±19.5%, and ±20%. Unless otherwise indicated in the examples of work, all numbers representing the amounts of ingredients or reaction conditions used herein are understood to be "approximately". When the term "approximately" is used in relation to a percentage, it can mean ±1%, or ±0.5% to 20% as described above.

[0133] As used herein, the term "affinity" refers to a measure of the strength of the binding between a target molecule and a binder. Affinity is typically expressed by the dissociation constant (Kd). Generally, it is approximately 10 -6 Kd molecules larger than M are thought to exhibit nonspecific binding.

[0134] As used herein, the term "amperometry" refers to a chemical titration in which the current flowing between two electrodes in a solution, under an applied potential difference, is measured and used to detect an endpoint.

[0135] The term "analyte," as used herein, is a broad term used to refer to a substance or chemical component in a liquid, such as a biological fluid. Analytes may include natural substances, artificial substances, metabolites, and / or reaction products. Analytes may be naturally occurring, i.e., endogenous, in biological fluids, such as metabolites, hormones, antigens, antibodies, etc. Alternatively, analytes may be introduced into the body, i.e., exogenous.

[0136] The terms “antibody” or “immunoglobulin,” when used interchangeably herein, include the whole antibody and its antigen-binding fragment (antigen-binding portion) or single-chain homologue. An “antibody” comprises at least one heavy (H) chain and one light (L) chain. For example, in natural IgG, these heavy and light chains are linked to each other by disulfide bonds, resulting in two pairs of heavy-light chains, also linked to each other 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 consists of one domain, CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), with more conserved regions interspersed between them, called framework regions (FRs) or linking (J) regions (JH for the heavy chain and JL for the light chain). Each VH and VL consists of three CDRs, three FRs, and one J domain, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, J. The variable regions of the heavy and light chains bind to antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (e.g., effector cells) or humoral factors such as the first component of the classical complement system (Clq). The term "antibody" is used in its broadest sense herein and includes a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0137] As used herein, the term “antigen” refers to a substance to which an antibody binds (e.g., a protein or peptide). In certain embodiments, the antigen is a coronavirus protein (e.g., the spike protein), or a derivative, fragment, analog, homolog, or ortholog thereof, which acts as an antigen in the systems and methods disclosed herein.

[0138] The term "antigen-binding region" refers to a portion of a binder (e.g., an antibody, an aptamer) that interacts with a target molecule (e.g., an antigen) and imparts specificity and affinity to the binder for the target molecule.

[0139] As used herein, the term “antiviral drug” broadly refers to any anti-infective drug or treatment used to treat or induce remission of the viral infection in question.

[0140] As used herein, the term "aptamer" refers to an oligonucleotide (DNA or RNA) that is three-dimensionally adapted to bind with another molecule with high affinity in nanomolar or sub-nanomolar ranges. Examples of nucleic acid molecules or polynucleotides constituting such aptamers include, but are not limited to, D- or L-nucleic acids, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA) (including LNA having a beta-D-ribo configuration, alpha-LNA having an alpha-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having 2'-amino functionalization, and 2'-amino-alpha-LNA having 2'-amino functionalization), or hybrids thereof. Aptamers may also bind to other molecules, including small molecules, proteins, nucleic acids, as well as cells, tissues, and organisms (e.g., all viruses), and may be monovalent or polyvalent. The aptamers used in the disclosed portion may be obtained by selecting them from a large random sequence library using methods such as the Synthetic Evolution of Ligands by Exponential Enrichment (SELEX), which are well known in the art.

[0141] As used herein, the term “binding agent” refers to a molecule that binds to a congener ligand with high affinity and specificity. Binding agents are typically used to identify the presence of their congener ligand and may be detectably labeled to enable identification. “X binding agent” means a molecule that binds to “X” with high affinity and specificity. Examples of “X” binding agents include, for example, aptamers, antibodies, receptor ligands, or molecular imprinting polymers.

[0142] As used herein, the term “binding pair” refers to a pair of molecules that bind to each other with high affinity and specificity. A “binding pair member” refers to one molecule of a binding pair. For example, streptavidin and biotin are binding pair members that are non-covalently bound to each other. Further non-limiting examples of the first and second members of a binding pair (also called an affinity pair) include: combinations of haptenic or antigenic compounds with corresponding antibodies or their binding portion or fragments; digoxigenin and anti-digoxigenin; mouse immunoglobulins and goat anti-mouse immunoglobulins; non-immunological binding pairs; biotin and avidin; biotin and streptavidin; hormones and hormone-binding proteins; thyroxine and cortisol hormone-binding proteins; receptors and receptor agonists; receptors and receptor antagonists; acetylcholine receptors and acetylcholine or their analogues; IgG and protein A; lectins and carbohydrates; enzymes and enzyme cofactors; enzymes and enzyme inhibitors; complementary oligonucleotide pairs capable of forming nucleic acid doubles; and a negatively charged first molecule and a positively charged second molecule.

[0143] The terms “sample” or “test sample,” as used herein, refer to a sample taken from or isolated from a living organism, such as a blood or plasma sample derived from the subject. In one aspect, the term “biological sample,” as used herein, refers to saliva. In other aspects, a biological sample is urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, sputum, pleural fluid, cerebral fluid, or nasopharyngeal specimens. Further exemplary biological samples include, but are not limited to, biopsies; tumor samples; biological fluid samples; blood; serum; plasma; urine; semen; mucus; tissue biopsies; organ biopsies; synovial fluid; bile; cerebrospinal fluid; mucous secretions; exudates; sweat; saliva; and / or tissue samples, etc. The term also includes mixtures of the above samples. The term “test sample” also includes untreated or pretreated (pre-processed) biological samples. In some aspects of either aspect, a test sample may include cells derived from the subject. Test samples can be obtained by removing the sample from the subject, but they can also be obtained using samples that have been isolated in the past (for example, isolated from the person or another person at some point in the past).

[0144] In some aspects of any aspect, the test sample may be an untreated test sample. As used herein, “untreated test sample” means a test sample that has not undergone any prior sample pretreatment, except for dilution and / or suspension in solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing, and thawing, as well as combinations thereof. In some aspects of any aspect, the test sample may be a frozen test sample, for example, a frozen tissue. A frozen sample may be thawed before use in the methods, assays, and systems described herein. After thawing, a frozen sample may be centrifuged before being subjected to the methods, assays, and systems described herein. In some aspects of any aspect, the test sample is a clarified test sample, for example, by centrifugation and collection of the supernatant containing the clarified test sample. In some aspects of any aspect, the test sample may be a pre-treated test sample, which may be a supernatant or filtrate obtained from a treatment selected from the group consisting of centrifugation, homogenization, sonication, filtration, melting, purification, and any combination thereof. In some aspects of any aspect, the test sample may be treated with chemical and / or biological reagents. Chemical and / or biological reagents may be used, for example, to protect and / or maintain the stability of the sample (including the biomolecules contained therein, e.g., nucleic acids and proteins) during treatment. One exemplary reagent is a protease inhibitor, which is commonly used to protect or maintain the stability of proteins during treatment. Those skilled in the art will be familiar with suitable methods and treatments for the pretreatment of biological samples required for determining the levels of expression products as described herein.

[0145] The term "biological sample" encompasses a variety of sample types obtained from an individual that may be used in diagnostic or observational assays. This definition includes blood and other biological fluid samples, solid tissue samples such as biopsy specimens or tissue cultures, or cells or their progeny derived therefrom. This definition also includes samples that have undergone some manipulation after procurement, such as treatment with reagents, solubilization, or concentration of specific components such as polynucleotides. The term "biological sample" includes, but is not limited to, clinical samples, and also includes cells in culture, cell supernatants, cell lysates, tissues, peripheral blood, serum, plasma, urine, cerebrospinal fluid, biological fluids, and tissue samples. Samples may be pre-treated by dilution with appropriate buffer solutions as needed, or by concentration as desired. One of several standard buffer solutions, preferably at physiological pH, may be used, using one of various buffers such as phosphate or Tris. Biological samples can be obtained from patients using well-known techniques such as venipuncture, lumbar puncture, fluid collection such as saliva or urine, or tissue biopsy. In certain embodiments, the sample is a body sample from any animal; in one embodiment, it is from a mammal; in another embodiment, it is from a human subject; and in yet another embodiment, it is from a non-human animal (e.g., an insect or a bat).

[0146] As used herein, the term "binding affinity" refers to the tendency of a binder to bind to or not bind to a target, and describes a measure of the strength or affinity of a binder to bind to a target molecule.

[0147] As used herein, the term "capture reagent" refers to a reagent capable of binding to and capturing a target molecule in a sample. Typically, capture reagents are immobilized or can be immobilized. In sandwich immunoassays, the capture reagent may be, for example, an aptamer or an antibody.

[0148] As used herein, the term "chronoamperometry" refers to an electrochemical measurement method for electrochemical analysis or for determining the kinetics and mechanisms of an electrode reaction. A rapidly rising potential pulse is applied to the working (or reference) electrode of an electrochemical cell, and the current flowing through this electrode is measured as a function of time.

[0149] As used herein, the term “complex” refers to a substance in which two or more molecules are bound or associated with at least one other molecule, for example, by chemical association. Thus, the term “matrix-aptamer-target molecule complex” refers to the association of the matrix, aptamer, and target molecule. The term “biotinylated second binder streptavidin (or β-binder-SA) complex” refers to the association of biotin, a second binder, and streptavidin.

[0150] The terms "correlated with" or "associated with" refer to levels of an analyte or fragment in a biological sample of interest that are statistically significant in correlation with a physiological state, such as the severity of a disease or illness, response to treatment, and survival. The strength of the correlation between the level of the analyte or fragment and the presence or absence of a particular physiological state may be determined by a statistical significance test.

[0151] The terms "statistically significant" or "significantly significant" refer to statistical significance, generally meaning a difference of two standard deviations (2SD) or more.

[0152] As used herein, the term "cross-reactivity" refers to the ability of a binder (e.g., an aptamer, antibody) directed towards one target molecule to successfully bind to another different molecule, i.e., a non-target molecule. The degree of cross-reactivity can vary. In certain embodiments, the target molecule and the non-target molecule share a common epitope, i.e., a highly conserved feature across species.

[0153] As used herein, the term "cut point" refers to the threshold used to distinguish between negative and positive responses in an assay. It is a constant value statistically determined by analyzing the assay responses of a set of samples from affected individuals who have never received the drug.

[0154] The term "detectable label" refers to a portion, molecule, or compound, or group of molecules or compounds, bound to a binder, and is used to identify the binder. The signal from a detectable label can be detected by various means and will depend on the properties of the detectable label. Detectable labels may be isotopes, fluorescent moieties, colorants, enzymes, enzyme substrates, etc. Examples of means for detecting a detectable label include, but are not limited to, spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifusion, chemiluminescence, or any other suitable means.

[0155] The term "drop casting" refers to a method of forming a thin solid film by dropping a solution onto a flat surface and then evaporating the solution.

[0156] The term "electrochemical system," as used herein, refers to a system for determining the presence and / or amount of a redox analyte by measuring an electrical signal between a working electrode and a counter electrode in a solution, such as one induced by a redox reaction or potential, resulting from the release or absorption of ions. A redox reaction refers to the loss of electrons (oxidation) or gain of electrons (reduction) that a substance undergoes in response to an electrical stimulus, such as the application of an electrical potential. The redox reaction occurs at the working electrode, which is typically made of an inert material such as platinum or carbon for chemical detection. The potential of the working electrode is measured relative to a reference electrode, which is typically a stable, settled electrochemical half-cell, such as silver / silver chloride. Electrochemical systems can be used to support many different techniques for determining the presence and / or concentration of target biomolecules, including, but not limited to, various types of voltammetry, amperometry, potentiometry, coulometry, conductivity measurements, and electrical conductivity measurements, such as AC voltammetry, differential pulse voltammetry, square wave voltammetry, electrochemical impedance spectroscopy, anode stripping voltammetry, cyclic voltammetry, and fast scan cyclic voltammetry. An electrochemical system may further include one or more negative and positive reference electrodes. In the context of this invention, a single electrochemical system can be used to quantify two or more types of analytes.

[0157] The terms “epitope” or “antigenic determinant” are used interchangeably herein to refer to a portion of a molecule, such as an antigen, that can be recognized and specifically bound by a particular binder (e.g., an antibody or aptamer). When the antigen is a polypeptide, the epitope can be formed by tertiary folding of the protein from both juxtaposed continuous and discontinuous amino acids. Epitopes formed from continuous amino acids are typically retained after protein denaturation, while epitopes formed by tertiary folding are typically lost after protein denaturation. Epitopes typically contain at least three, more commonly five to eight-ten, amino acids in a unique spatial arrangement. Antigenic determinants can compete for binding to antibodies with intact antigens (i.e., “immunogens” used to induce an immune response).

[0158] As used herein, the term "false negative" refers to a sample that is misidentified as not containing one or more analytes, such as a virus.

[0159] As used herein, the term "false positive" refers to a sample that has been misidentified as containing one or more analytes, such as a virus.

[0160] As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 (n minus 1) relative to a full-length polypeptide or polynucleotide (length = n). The length of the fragment can be appropriately varied depending on its purpose. Examples of lower limits for the length of a fragment include, in the case of polypeptides, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more amino acids, and lengths represented by integers not specifically listed here (e.g., 11) may also be appropriate as lower limits. Furthermore, in the case of polynucleotides, examples of lower limits for the length include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides, and lengths represented by integers not specifically listed here (e.g., 11) may also be appropriate as lower limits.

[0161] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) may be functional fragments of one of the amino acid sequences described herein. As used herein, “functional fragment” is a fragment or segment of a polypeptide that retains at least 50% of the activity of a wild-type reference polypeptide according to the assays described herein below. Functional fragments may include conservative substitutions of the sequences disclosed herein.

[0162] As used herein, the term "glucometer" refers to a medical device widely used for self-monitoring of blood glucose in diabetic patients. Many glucometers employ an electrochemical method based on a test medium, such as a test strip. In the context of diabetes observation, the test strip is a consumable element containing a chemical that reacts with glucose in a drop of blood used for each measurement. Specifically, a chemical reaction occurs, and the meter indicates a glucose level expressed as mg / dl or mmol / l. Glucometers are usually portable and used at home, although professional-grade glucometers are also known.

[0163] As used herein, the term "glucose" refers to a monosaccharide, a common hexose monosaccharide.

[0164] As used herein, the term "high affinity" means at least 10 -8 M, about 10 -8 ~ about 10 -12 、or more specifically, about 10 -8 M, about 10 -9 M; about 10 -10 M, about 10 -11 M, or about 10 -12 M of binding affinity.

[0165] As used herein, the terms "isolated", "purified", or "biologically pure" refer to a substance that is substantially or essentially free of components that are normally associated with it in its natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0166] The term "K D " as used herein refers to the equilibrium dissociation constant of the interaction between a specific binder and a target molecule.

[0167] The terms "measuring" and "determining" are used interchangeably throughout this specification and refer to methods that include obtaining a patient sample and / or detecting the level of a biomarker in a biological sample. In one aspect, these terms refer to obtaining a patient sample and detecting the level of one or more biomarkers in the sample. In another aspect, the terms "measuring" and "determining" mean detecting the level of one or more biomarkers in a biological sample. The term "measuring" is also used interchangeably throughout this specification with the term "detecting".

[0168] As used herein, the term "molecule" is used broadly to refer to natural, synthetic, or semi-synthetic molecules or compounds.

[0169] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies constituting the population are identical except for any naturally occurring variants that may be present in very small quantities.

[0170] The term "mutation" refers to a change in the amino acid sequence of a native protein. Mutations can be described by using the native sequence and identifying the specific acid that has been altered. A "mutant" or "variant" refers to a protein that contains a mutation. A full-length mutant sequence refers to the entire amino acid sequence of a mutant protein, instead of describing the mutant as having different amino acids from the native protein.

[0171] The term "native protein" refers to a protein that is native or in its natural state and has not been modified by denaturing agents such as heat, chemical mutation, or enzymatic reactions.

[0172] As used herein, the term "non-target molecule" refers to a molecule that is not a biomarker of interest. Specifically, a non-target molecule may be a molecule that is structurally similar to a biomarker of interest.

[0173] The term "pathogen" is not limited to any factor that causes disease, including viruses, bacteria, or other microorganisms. Self-replicating pathogens (e.g., viruses and bacteria) are organisms that cause disease by replicating using the body's resources while largely evading the body's immune response.

[0174] The term “point-of-care testing” or “POCT” is used herein to refer to the assay of a biological specimen in or near the patient, with the understanding that results will be obtained immediately or very quickly, in order to support caregivers with rapid diagnosis and / or clinical intervention. See, for example, Ehrmeyer SS et al. (2007) Clin Chem Lab Med 45:766-773. This term is not intended to be limited to patient and home use, but also includes a variety of settings (e.g., community, clinic, surrounding laboratories and hospitals) and users (e.g., dental technicians and caregivers). Depending on the setting and user, the purpose of POC testing can also vary, from triage and referral to diagnosis, treatment, and observation.

[0175] The term "potentiostat," as used herein, is a broad and non-limiting term, but is used in its original sense to include electrical devices that control the potential between the working electrode and the reference electrode of a three-electrode cell to a predetermined value. A potentiostat maintains a desirable potential for the current that needs to flow between the working electrode and the counter electrode, provided that the required cell voltage and current do not exceed its compliance limits.

[0176] The term “predetermined threshold” refers to a numerical threshold value at which a classifier gives a desirable balance between false negatives and false positives (and their costs). In some aspects, the “predetermined threshold” is statistically (and clinically) determined, scrutinized, adjusted, and / or confirmed through clinical trials and their outcome analyses (collectively, “clinical data”) and / or preclinical or nonclinical trials (collectively, “nonclinical data”), in order to minimize the undesirable effects of false positives and false negatives.

[0177] The terms “prevent,” “prevention,” or “prevention” refer to the inhibition of the manifestation of symptoms or signs of a disease, such as symptoms or signs of a viral infection.

[0178] As used herein, the term “processor” is used broadly to refer to programmable or non-programmable processing devices, such as microprocessors, microcontrollers, application-specific integrated circuits (ASICS), programmable logic devices (PLDs), field-programmable gate arrays (FPGAs), and others. The term “processor” may also include multi-processing devices that work together.

[0179] As used herein, the term "point mutation" refers to a manipulation of a polynucleotide resulting from the substitution or exchange, deletion or insertion of one or more single (discontinuous) or two different amino acids, resulting in the expression of an amino acid sequence different from the unmanipulated amino acid sequence.

[0180] The terms “protein,” “peptide,” and “polypeptide” are interchangeable to refer to amino acid polymers, or a set of two or more interacting or bonded amino acid polymers in which, for example, the alpha-amino and carboxyl groups of adjacent residues are linked to each other by peptide bonds. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding natural amino acids, as well as natural amino acid polymers and non-natural amino acid polymers that include modified residues. The terms “protein” and “polypeptide” refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. While “protein” and “polypeptide” are often used in reference to relatively large polypeptides, the term “peptide” is often used in reference to smaller polypeptides, but the usage of these terms overlaps in the art. The terms “protein” and “polypeptide” are interchangeable herein when referring to gene products and their fragments. Thus, exemplary polypeptides or proteins include gene products, natural proteins, their homologs, orthologues, paralogs, fragments and other equivalents, variants, fragments, and analogs.

[0181] The term “reference value” may be “threshold” or “cutoff value” as used herein. Typically, “threshold” or “cutoff value” may be determined experimentally, empirically, or theoretically.

[0182] The term “risk,” as used herein, refers to the probability that an event will occur after a certain period of time, such as a positive conversion of a Covid-19 test result, and may mean either the “absolute” or “relative” risk of the subject. Absolute risk may be measured by referring to actual post-measurement observations of an appropriate time cohort, or by referring to an index value constructed from a statistically valid historical cohort followed for an appropriate period. Relative risk refers to the ratio of the absolute risk of the subject to the absolute risk of a low-risk cohort or the mean population risk, and may vary depending on how the clinical risk factors are assessed. The odds ratio is the ratio of positive events to negative events for a given test result and is also widely used (odds are given by the formula p / (1-p) [where p is the probability of the event and (1-p) is the probability of no event to no conversion]). An alternative continuous measure that may be assessed in the context of the present invention is the ratio of time versus the reduction in conversion risk.

[0183] As used herein, the term "selectivity" refers to the ability of a system or method to distinguish a particular analyte in a complex mixture without interference from other components.

[0184] As used herein, the term “sensor” refers to a means used to detect an analyte. A “sensor system” includes, for example, elements, structures, and architectures intended to facilitate the use and function of a sensor. A sensor system may include, for example, compositions having selected material properties, as well as electronic components such as elements and devices used for signal detection and analysis (e.g., current detectors, monitors, processors, etc.).

[0185] The terms "specific binding," "specific binding," "selective binding," and "selective binding" mean that the binder (e.g., antibody, aptamer) exhibits sufficient affinity for the target molecule but generally does not show significant cross-reactivity with non-target molecules, which in certain embodiments means at least about 1 x 10⁻¹⁶. -8It has an equilibrium dissociation constant less than or equal to M (for example, a smaller K). D This means that they exhibit a stronger 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.

[0186] Specifically, as used herein, the term “specific binding” refers to a chemical or physical interaction between two molecules, compounds, cells, and / or particles, such that a first substance binds to a target second substance with higher specificity and affinity than it binds to a third, non-target substance. In some embodiments, specific binding may refer to an affinity of the first substance for the target second substance that is at least 10, at least 50, at least 100, at least 500, at least 1000, or more than its affinity for the third, non-target substance. A reagent specific to a given target is a reagent that exhibits specific binding to the target under the conditions of the assay being used.

[0187] As used herein, the term "sensitivity" refers to the proportion of positive individuals correctly identified (e.g., the percentage of SARS-CoV-2 positive individuals identified by a system or method). Highly sensitive systems or methods have a low rate of false negatives.

[0188] As used herein, the term "specificity" refers to the proportion of people who are correctly identified as negative (for example, the percentage of people who are correctly identified as not being infected with SARS-CoV-2 by a system or method). Highly specific systems or methods have a low rate of false positives.

[0189] As used herein, the term "screen printing" refers to a technique for manufacturing electrochemical measuring devices by printing different types of inks onto plastic or ceramic substrates, enabling rapid in-situ analysis with high reproducibility, sensitivity, and accuracy. The selectivity and sensitivity can be determined by the composition of the different inks used in the manufacture of the electrodes (e.g., carbon, silver, gold, platinum). Screen printing makes it possible to manufacture high-quality disposable electrodes at low cost and in a reproducible manner. Other printing methods or other electrode manufacturing methods are known in the art.

[0190] The term “subject” refers to mammals such as humans. Typically, animals are vertebrates such as apes, rodents, domesticated animals, or game animals. Examples of apes include chimpanzees, crab-eating macaques, spider monkeys, and macaques, such as rhesus macaques. Examples of rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Examples of domesticated animals and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, foxes, wolves, birds such as chickens, emus, ostriches, and fish such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as an ape, such as a human. The terms “individual,” “patient,” and “subject” are used interchangeably herein.

[0191] Preferably, the subject is a mammal. Mammals may be, but are not limited to, humans, non-human apes, mice, rats, dogs, cats, horses, or cattle. Non-human mammals may be advantageously used, for example, as representative subjects for animal models of infectious diseases. The subject may be male or female.

[0192] Subjects may be those who have been diagnosed with or identified as having a condition requiring treatment for one or more complications related to one or more of the diseases or disorders described herein (e.g., infectious diseases), and who have optionally already received treatment for one or more of the diseases or disorders described herein (e.g., infectious diseases) or one or more complications related to one or more of the diseases or disorders described herein (e.g., infectious diseases). Alternatively, subjects may never have been diagnosed with one or more complications related to one or more of the diseases or disorders described herein (e.g., infectious diseases). For example, subjects may exhibit one or more risk factors for one or more of the diseases or disorders described herein (e.g., infectious diseases) or one or more complications related to one or more of the diseases or disorders described herein (e.g., infectious diseases), or subjects may not exhibit any risk factors.

[0193] A person who "needs treatment" for a particular condition may be a person who has that condition, has been diagnosed with that condition, or is at risk of developing that condition.

[0194] As used herein, the term "system noise" refers to unwanted electronic or diffusion-related noise, but is not limited to, such noises including Gaussian noise, motion-related noise, flicker, dynamic noise, or other white noise.

[0195] As used herein, the term "target molecule" refers to a molecule that may be present in a test sample and that can bind to a binder.

[0196] As used herein, the terms “to treat,” “treatment,” or “the act of treating” refer to various types of treatments aimed at preventing, inhibiting, alleviating, reversing, relieving, delaying, or stopping the progression or severity of a disorder or disease, such as a condition and symptoms associated with an infectious disease, such as COVID-19. The term “to treat” includes reducing or alleviating at least one adverse event or symptom of a condition, disease, or disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Or, treatment is “effective” if the progression of the disease is reduced or stopped. In other words, “treatment” includes not only improvement of symptoms or markers, but also a cessation or at least a slowing of the progression or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desirable clinical outcomes include, but are not limited to, relief of one or more symptoms, whether detectable or undetectable; reduction of disease extent; stabilization of the disease (i.e., no worsening); delay or slowing of disease progression; remission or relief of the disease; improvement (in part or in whole); and / or reduction of mortality. The term “treatment” of a disease also includes relief from the symptoms or side effects of the disease (including palliative care).

[0197] The term "therapeutic dose," as used herein, refers to the amount of an active compound or agent (e.g., an antiviral agent) that elicits a biological or medical response to a target sought by a researcher, veterinarian, physician, or other clinician, such response may include prevention, remission, or mitigation of symptoms of the disease or disorder being treated. Methods for determining the therapeutic dose of the pharmaceutical composition are known in the art.

[0198] The term "dual-binding assay" refers to an assay in which a target molecule attached to a first binding agent bound to a matrix is ​​further incubated in the presence of a second binding agent associated with a chemically reactive group.

[0199] The term “variant,” as used herein, is a relative term describing the relationship between a particular polypeptide of interest and a “parent” or “reference” polypeptide whose sequence is being compared. A polypeptide of interest is considered a “variant” of a parent or reference polypeptide if it has an amino acid sequence identical to that of the parent, except for a few sequence changes at specific positions. Examples of variants include substitution, insertion, or deletion variants. Typically, a variant has less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of residues substituted compared to the parent. In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue compared to the parent. Often, a variant has a very small number (e.g., fewer than 5, 4, 3, 2, or 1) substituted functional residues (i.e., specific residues that contribute to biological activity). Furthermore, variants typically have only 5, 4, 3, 2, or 1 addition or deletion compared to the parent, and often have no additions or deletions at all. Moreover, any addition or deletion is typically less than approximately 25, 20, 19, 18, 17, 16, 15, 14, 13, 10, 9, 8, 7, or 6 residues, and generally less than approximately 5, 4, 3, or 2 residues. In some embodiments, the parent or reference polypeptide is a naturally occurring polypeptide. As those skilled in the art will understand, multiple variants of a particular polypeptide of interest are generally found in nature, especially if the polypeptide of interest is an infectious polypeptide. In certain embodiments, the variant is a viral protein (e.g., a spike protein) that is particularly similar in function to the reference viral protein but differs from the wild-type viral protein in one or more positions due to a mutation in its amino acid sequence.

[0200] In the various embodiments described herein, it is also assumed that variants (natural or otherwise), alleles, homologs, conserved modified variants, and / or conserved substitution variants of any particular polypeptide described herein may be included. With respect to amino acid sequences, as those skilled in the art will recognize, individual substitutions, deletions, or additions to the sequence of a nucleic acid, peptide, polypeptide, or protein that alter a single amino acid or a very small percentage of amino acids in the encoded sequence are “conserved modified variants,” where the alteration involves substituting an amino acid with a chemically similar amino acid, but preserving the desired activity of the polypeptide. Such conserved modified variants are added to, and not excluded from, the polymorphic variants, interspecific homologs, and alleles in accordance with this disclosure.

[0201] A given amino acid can be replaced with a residue having similar physiological and chemical characteristics, for example, by substituting one aliphatic residue with another aliphatic residue (e.g., Ile, Val, Leu, or Ala mutually), or by substituting one polar residue with another polar residue (e.g., Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, such as substitutions of entire regions having similar hydrophobic characteristics, are well known. Polypeptides containing conservative amino acid substitutions can be tested with any of the assays described herein to confirm that the desired activity, such as binding activity and specificity to the native or reference polypeptide target, is preserved.

[0202] Amino acids can be grouped according to the similarity of their side chain attributes (from AL Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) Nonpolar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M); (2) Uncharged polar: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q); (3) Acidic: Asp(D), Glu(E); (4) Basic: Lys(K), Arg(R), His(H). Alternatively, natural residues can be grouped based on common side-chain attributes: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging members between one of these classes and another. Specific examples of conservative substitutions include, for instance, Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0203] In some embodiments, the polypeptides described herein may be variants of the sequences described herein. In some embodiments, the variants are conserved modified variants. Conservative substitution variants may be obtained, for example, by mutations in the native nucleotide sequence. As used herein, “variant” is a polypeptide that is substantially homologous to the native or reference polypeptide but has a different amino acid sequence from the native or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequence encoding the variant polypeptide includes a sequence encoding a variant protein or a fragment thereof that contains one or more nucleotide additions, deletions, or substitutions compared to the native or reference DNA sequence, but retains activity. Various PCR-based site-directed mutagenesis approaches are known in the art and may be used by those skilled in the art to prepare and test artificial variants.

[0204] A variant amino acid or DNA sequence may be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to the native or reference sequence. The degree of homology (percentage of identity) between a native sequence and a variant sequence can be determined, for example, by comparing the two sequences using a free computer program widely used on the World Wide Web for sequence comparison purposes (e.g., BLASTp or BLASTn, with default settings).

[0205] A variant amino acid sequence may be similar to a native or reference sequence by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more. As used herein, “similarity” refers to identical or conservedly substituted amino acids as described herein. Thus, the percentage of “sequence similarity” is the percentage of amino acids that are identical or conservatively modified, for example, “sequence similarity” = (sequence identity %) + (conservative modification %). It should be understood that a sequence having a particular percentage of similarity to a reference sequence necessarily contains sequences having the same particular percentage of identity to that reference sequence. Those skilled in the art will know of several computer programs using various mathematical algorithms that are available for determining identity or similarity between two sequences. For example, a computer program using the Needleman-Wunsch algorithm (Needleman et al. (1970)); the GAP program in the Accelrys GCG software package (Accelerys Inc., San Diego, USA); the E. Meyers and W. Miller algorithm (Meyers et al. (1989)) incorporated into the ALIGN program (version 2.0); or more preferably BLAST (an acronym for Basic Local AlignmentTool, using default parameters) can be used; see, for example, U.S. Patent No. 10,023,890, the entire contents of which are incorporated herein by reference.

[0206] Modification of the native amino acid sequence can be achieved by any of the numerous techniques known to those skilled in the art. Mutations can be introduced, for example, at specific loci by synthesizing an oligonucleotide containing a mutant sequence adjacent to a restriction site that allows ligation to a fragment of the native sequence. The reconstructed sequence obtained after ligation encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, an oligonucleotide-dependent site-directed mutagenesis procedure can be used to obtain a modified nucleotide sequence having specific codons altered according to the required substitution, deletion, or insertion. Techniques for making such modifications are well-established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patents 4,518,584 and 4,737,462, the entire contents of which are incorporated herein by reference. All cysteine ​​residues that do not participate in maintaining the proper three-dimensional structure of the polypeptide can also be substituted with serine, thereby improving the oxidative stability of the molecule and preventing abnormal crosslinking. Conversely, cysteine ​​bonds may be added to polypeptides to improve their stability or promote oligomerization.

[0207] The term "wild-type," as used herein, refers to a protein or nucleic acid in its native, full-length form as found in nature. The term "full-length native protein sequence," as used herein, refers to the amino acid sequence found in a full-length native protein. Wild-type proteins can be obtained, for example, from biological samples.

[0208] In any method disclosed herein, including separate steps, the steps may be carried out in any feasible order. Furthermore, two or more steps, optionally combined, may be carried out simultaneously.

[0209] The terms “decrease,” “reduced,” “decrease,” or “inhibition” are all used herein to mean a statistically significant reduction. In some embodiments, “decrease,” “reduced,” or “decrease,” or “inhibition” typically mean a reduction of at least 10% compared to a baseline level (e.g., the absence of a given treatment or agent), and may include reductions of, for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or greater. As used herein, “decrease” or “inhibition” does not include complete inhibition or reduction compared to a baseline level. “Complete inhibition” is 100% inhibition compared to a baseline level. The reduction may preferably be to a level that is acceptable to a person without a given disability as being within the normal range.

[0210] The terms “increased,” “enhance,” “strengthen,” or “activate” are all used herein to mean an increase of a statistically significant amount. In some aspects, the terms “increased,” “enhance,” “strengthen,” or “activate” may mean an increase of at least 10% compared to a baseline level, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100% compared to a baseline level, or an increase of 10 to 100%, or an increase of at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, or at least about 10 times compared to a baseline level, or an increase of 2 to 10 times or more. In the context of markers or symptoms, “increase” is a statistically significant increase of such a level.

[0211] As used herein, the terms “nucleic acid” or “nucleic acid sequence” refer to any molecule, preferably a polymer containing multiple units of ribonucleic acid, deoxyribonucleic acid, or analogues thereof. Nucleic acids can be single-stranded or double-stranded. A single-stranded nucleic acid may be a single nucleic acid strand of denatured double-stranded DNA. Alternatively, it may be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, a nucleic acid may be DNA. In another aspect, a nucleic acid may be RNA. Suitable nucleic acids include guide nucleic acids, collateral nucleic acids, and / or detection nucleic acids. Suitable DNA may include, for example, viral DNA, genomic DNA, or cDNA. Suitable RNA may include, for example, mRNA or viral RNA.

[0212] The term “expression” refers to cellular processes involved in the production of RNA and proteins, and, where appropriate, the secretion of proteins, and, where appropriate, includes, but is not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing. Expression may refer to the transcription and stable accumulation of sense RNA (e.g., mRNA) or antisense RNA derived from one or more nucleic acid fragments, and / or translation from mRNA to polypeptides.

[0213] In some embodiments, the expression of biomarkers, targets, or genes / polypeptides described herein is tissue-specific. In some embodiments, the expression of biomarkers, targets, or genes / polypeptides described herein is general. In some embodiments, the expression of biomarkers, targets, or genes / polypeptides described herein is systemic.

[0214] Expression products include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence (DNA) that, when functionally linked with appropriate regulatory sequences, is transcribed into RNA in vitro or in vivo. A gene may or may not include the regions before and after the coding region, such as the 5' untranslated (5'UTR) or "leader" sequence and the 3'UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0215] In the context of this invention, “marker” refers to an expression product, such as a nucleic acid or polypeptide, that is differentially present in a sample taken from a subject with an infectious disease (e.g., COVID-19) compared to a comparative sample taken from a control subject (e.g., a healthy subject). The term “biomarker” is interchangeable with the term “marker.”

[0216] In some aspects, the methods described herein relate to measuring, detecting, or determining the level of at least one marker. As used herein, the terms “detecting” or “measuring” refer to observing a signal from, for example, a probe, label, or target molecule that indicates the presence of an analyte in a sample. Any method known in the art for detecting a particular label portion may be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical, or chemical methods. In some aspects of any of these, measurement may be quantitative observation.

[0217] In some aspects of any aspect, the polypeptides, nucleic acids, or cells described herein may be engineered. As used herein, “engineered” refers to an aspect that has been manipulated by human hands. For example, a polypeptide is considered “engineered” if at least one aspect of it, such as its sequence, has been manipulated by human hands, thereby differing from that aspect as it would be in its natural state. By convention, and as those skilled in the art will understand, the offspring of an engineered cell are also typically said to be “engineered,” although the actual manipulation has been carried out relative to the previous generation.

[0218] As used herein, the term “pharmaceutical composition” refers to an active agent in combination with a pharmaceutically acceptable carrier, such as a carrier commonly used in the pharmaceutical industry. The term “pharmaceutically acceptable” is used herein to mean a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, given a reasonable merit / risk ratio. In some aspects of any aspect, a pharmaceutically acceptable carrier may be a carrier other than water. In some aspects of any aspect, a pharmaceutically acceptable carrier may be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some aspects of any aspect, a pharmaceutically acceptable carrier may be an artificial or manipulated carrier, such as one in which the active ingredient is not found or occurs naturally.

[0219] As used herein, the term “administer” means placing a compound disclosed herein into a subject body by a method or route that results in the delivery of the agent at least partially to a desired site. A pharmaceutical composition comprising a compound disclosed herein may be administered by any suitable route that results in an effective treatment in a subject. In some embodiments, administration involves human physical activity, including, for example, injection, ingestion, topical application, and / or operation of a delivery device or machine. Such activity may be performed, for example, by a medical professional and / or the subject under treatment.

[0220] As used herein, “contact” means any preferred means for delivering or exposing an agent to at least one cell. Illustrative delivery methods include, but are not limited to, direct delivery to cell culture media, transfection, transduction, perfusion, injection, or other delivery methods known to those skilled in the art. In some embodiments, contact includes, for example, injection; acts of dispensing, mixing, and / or decanting; and / or operation of a delivery device or machine, including human physical activity.

[0221] Levels below the baseline may be at least approximately 10%, at least approximately 20%, at least approximately 50%, at least approximately 60%, at least approximately 80%, at least approximately 90%, or lower relative to the baseline. In some aspects of any of these, levels below the baseline may be statistically significant.

[0222] Levels above the baseline level may be at least approximately 10%, at least approximately 20%, at least approximately 50%, at least approximately 60%, at least approximately 80%, at least approximately 90%, at least approximately 100%, at least approximately 200%, at least approximately 300%, at least approximately 500%, or higher than the baseline level. In some aspects of any of these, levels above the baseline level may be statistically significant.

[0223] In some aspects of any aspect, the criterion may be the level of the target molecule in a population of subjects who have not been diagnosed with, or are not diagnosed with, and / or exhibit signs or symptoms of a disease or disorder described herein (e.g., an infectious disease such as COVID-19). In some aspects of any aspect, the criterion may also be the expression level of the target molecule in a control sample or a pool of control samples, or a numerical value or numerical range based thereon. In some aspects of any aspect, the criterion may be the level of the target molecule in a sample taken from the same subject at an earlier point in time, and for example, using the method described herein, it may be possible to determine whether the subject's sensitivity or response to a given treatment has changed over time.

[0224] In some embodiments, the reference level may be the level in a sample obtained from a subject of similar cell type, sample type, sample processing, and / or similar age, sex, and other demographic parameters as the sample / subject whose level of the target analyte is to be determined. In some embodiments, the test sample and the control reference sample are of the same type, i.e., obtained from the same biological source and of the same composition, for example, consisting of the same number and type of cells.

[0225] As used herein, the term “including” means that other elements may be present in addition to the given elements described. The use of “including” indicates inclusion rather than limitation.

[0226] The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding elements not specified in the description of their embodiments.

[0227] As used herein, the term “essentially derived from” refers to elements necessary for a given embodiment. This term allows for the presence of additional elements that do not significantly affect the basic, novel, or functional features of that embodiment of the invention.

[0228] As used herein, the term "corresponding to" refers to an amino acid or nucleotide at a counted position in a first polypeptide or nucleic acid, or an amino acid or nucleotide in a second polypeptide or nucleic acid that corresponds to the counted amino acid or nucleotide. The corresponding counted amino acid or nucleotide can be determined by alignment of candidate sequences using various homology programs known in the art, such as BLAST.

[0229] The group of alternative elements or embodiments of the Invention disclosed herein should not be construed as limiting. Each member of the group may be referred to individually or in any combination with other members of the group or other elements described herein, and may be claimed. One or more members of the group may be included in or excluded from the group for convenience and / or patentability reasons. Where such inclusion or exclusion occurs, the description herein shall be deemed to include the group as amended and shall thus satisfy all descriptions of the Markush group used in the appended claims.

[0230] Unless otherwise defined herein, scientific and technical terms used in connection with this application have the meanings generally understood by those skilled in the art to which this disclosure pertains. It should be understood that the present invention is not limited to, and therefore may differ from, the specific methodologies, protocols, reagents, and other items described herein. The terminology used herein is for the sole purpose of describing specific embodiments and does not limit the scope of the present invention as defined solely by the claims. Definitions of general terms in cell biology, immunology, and molecular biology are found in: The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier. 2006;Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), WWNorton & Company, 2016 (ISBN 0815345054, 978-0815345053);Lewin's Genes XI, PublisherJones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning:A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414);Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X);Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN This information is found in Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), and the entire contents of all of these are incorporated herein by reference.

[0231] Other terms are defined herein in the descriptions of various aspects of the invention.

[0232] In some embodiments, a system for detecting one or more target analytes in a biological sample is disclosed. The system comprises a binding assay for analyzing a biological sample other than blood, and a detection means, the detection means being a glucometer or similar device for measuring or detecting glucose. In certain embodiments, the system is an electrochemical system.

[0233] In one aspect, a system for detecting at least one target analyte in a biological sample is disclosed, the system comprising a two-binding assay including (a) first and second binding agents capable of forming a detectable complex with the at least one target analyte, ( b ) comprising a detection device for detecting the detectable complex, wherein the detection device is a glucometer, and the biological sample is not blood.

[0234] In another aspect, a system for detecting at least one pathogen (e.g., a virus) in a biological sample is disclosed, the system comprising a two-conjugate assay, (a) a first and second conjugate capable of forming a detectable complex with the at least one pathogen, and ( b ) comprises a detection device for detecting the detectable complex, the detection device being a glucometer. In certain embodiments, the biological sample is saliva.

[0235] In one embodiment, the system provides a yes / no result. In other embodiments, the system provides a semi-quantitative or quantitative result (e.g., analyte level / volume, such as virus copy number / volume).

[0236] In certain embodiments, the first binder is a capture reagent immobilized on a test strip, and the second binder is a detectable binder, and when the first and second binders bind to a target analyte (e.g., a viral antigen or virus), a detectable complex is formed.

[0237] In certain embodiments, the second binder is a binder-glucose oxidase (Ab-GOx) conjugate, where the glucometer provides an electrochemical signal indicating the presence of the target analyte and / or correlates with the amount of the target analyte present in the biological sample, and the biological sample is not blood.

[0238] In certain embodiments, the second binder is a binder-glucose oxidase (Ab-GOx) conjugate, where the glucometer provides an electrochemical signal indicating the presence of the target analyte and / or correlates with the amount of the target analyte present in the biological sample, and the biological sample is urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, nasal cavity samples, pleural fluid, cerebrospinal fluid, or nasopharyngeal specimens.

[0239] In certain embodiments, the second binder is a binder-glucose oxidase (Ab-GOx) conjugate, where the glucometer provides an electrochemical signal indicating the presence of the target analyte and / or correlates with the amount of the target virus present in the biological sample, and the biological sample is not blood.

[0240] In certain embodiments, the second binder is a binder-glucose oxidase (Ab-GOx) conjugate, where the glucometer provides an electrochemical signal indicating the presence of the target analyte and / or correlates with the amount of the target virus present in the biological sample, and the biological sample is urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, or nasopharyngeal specimens.

[0241] In one embodiment, the biological sample is mixed with glucose.

[0242] In other embodiments, the biological sample is mixed with glucose at a concentration of 0.01 mM to 1 M.

[0243] In other embodiments, the biological sample is mixed with sucrose, fructose, maltose, galactose, cellulose, or any combination thereof, at concentrations of 0.01 mM to 1 M, containing amylase or invertase. In some embodiments of any aspect, the concentration of glucose, sucrose, fructose, maltose, galactose, or cellulose is at least 0.01 mM, at least 0.02 mM, at least 0.03 mM, at least 0.04 mM, at least 0.05 mM, at least 0.06 mM, at least 0.07 mM, at least 0.08 mM, at least 0.09 mM, at least 0.1 mM, at least 0.2 mM, at least 0.3 mM, at least 0.4 mM, at least 0.5 mM, at least 0.6 mM, at least 0.7 mM, at least 0.8 mM, at least 0.9 mM, or at least 1.0 mM or higher.

[0244] In one embodiment of a conjugate capable of detecting a target analyte, the first and second binders are selected from aptamers, antibodies, proteins, or combinations thereof.

[0245] In one embodiment of a conjugate capable of detecting a target analyte, the first and second binders are selected from aptamers, antibodies, or proteins, and the second binder is selected each time from aptamers, antibodies, or proteins linked to an oxidase enzyme.

[0246] In certain embodiments of a complex capable of detecting a target analyte, the first and second binders are selected from aptamers, antibodies, or proteins, and the second binder is selected each time from aptamers, antibodies, or proteins linked to glucose oxidase.

[0247] In certain embodiments of a complex capable of detecting a target analyte, the first and second binders are selected from aptamers, antibodies, or proteins, and the second binder is selected each time from aptamers, antibodies, or proteins linked to glucose oxidase, galactose oxidase, D-glucose:D-fructose oxidoreductase, or cellobiose oxidase.

[0248] In one embodiment of a conjugate capable of detecting a target analyte, the first and second binders are selected from aptamers, antibodies, or proteins, and the second binder is selected each time from aptamers, antibodies, or proteins linked to a hydrogenase enzyme.

[0249] In certain embodiments of a complex capable of detecting a target analyte, the first and second binders are selected from aptamers, antibodies, or proteins, and the second binder is selected each time from aptamers, antibodies, or proteins linked to glucose dehydrogenase, glucose-6-phosphate dehydrogenase, fructose dehydrogenase, sucrose dehydrogenase, glucoside dehydrogenase, alcohol dehydrogenase, sorbitol dehydrogenase, lactate dehydrogenase, or malate dehydrogenase.

[0250] In other embodiments, the system comprises a single binder.

[0251] In a particular embodiment, the single binder is an aptamer, which is bound to cellobiose oxidase. The target analyte binds to the aptamer, and the aptamer releases cellobiose, thus freeing the oxidase into the solution.

[0252] In a particular embodiment, a single binder is an aptamer, which binds to the target analyte, and the target analyte binds to the antibody-GOX complex. A signaling molecule then binds and replaces the target analyte, resulting in a decrease in signal.

[0253] Currently, there is no FDA-approved POC device that has the low detection limits and high accuracy necessary to be truly useful for the detection of SARS-CoV-2 or other viruses, and that allows a subject to rapidly self-test at home, or equally in the workplace, or in the field, or in resource-limited environments, with test results provided within 15 minutes, 10 minutes, 5 minutes, 1 minute, 0.5 minutes, or 0.1 minutes. POC testing can be important for promoting better disease diagnosis, surveillance, and management by enabling rapid detection of infection at an early stage.

[0254] In certain aspects, the systems disclosed herein are portable and suitable for use in a number of environments, including at home, in the workplace, in hospitals, in emergency rooms, or on the battlefield, and have one or more properties equivalent to, or preferably relatively improved over, other systems or devices for detecting a target analyte, including systems or devices for detecting pathogens such as respiratory viruses and coronaviruses, and more specifically beta-coronaviruses such as SARS-CoV-2. These properties can include, but are not limited to, speed and duration of sensing (<1 minute), specificity (>90%), selectivity (>90%), assay detection limit (1 target analyte / milliliter, or >100,000), quantitative detection (accuracy >90%, and precision >90%), effect of common interferents on sensor output, cross-reactivity (selectivity for target analyte >90%) with related viruses such as SARS-CoV-1 and SARS-CoV-2, dynamic range, coefficient of variation of repeated measurements (variance <10%), operational stability, or combinations thereof. In one aspect, in a five-variable analysis of variance, convergence greater than 0.95 can be obtained with five measurements depending on the statistical method used. Standardizing manufacturing and reducing the number of variables to one or two can achieve this confidence level with two measurements.

[0255] In one aspect, the system enables a sensitivity of about 90% or more, 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, or about 99% or more.

[0256] In certain aspects, the system enables a result of 9 true positives and 1 false negative out of 10 tests, where the true positives are subjects infected with or having been previously infected with a pathogen (e.g., a virus).

[0257] In one aspect, the system enables a sensitivity of about 90% or more, 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, or about 99% or more.

[0258] In certain aspects, the system enables a result of 9 true negatives and 1 false negative out of 10 tests.

[0259] Other such characteristics can include the scale of testing, assay time, ease of use, and reduction of secondary (e.g., healthcare worker) infections. In particular, accuracy is of paramount importance because a false negative result could cause an infectious person to believe they are not infected with a disease (e.g., SARS-CoV-2) and thus potentially infect others without knowing it.

[0260] In certain aspects, the systems disclosed herein have one or more characteristics that are improved relative to RT-PCR assays performed on purchased human saliva and nasal samples or samples taken directly from an individual. In one aspect, The systems disclosed herein areLower false positive rates than those performed on commercially available nasal samples, specifically around 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, or 2%, or lower. It holds.

[0261] In one embodiment, the system disclosed herein provides the user with results within approximately 10 minutes or less, more specifically within approximately 5 minutes or less, within approximately 2 minutes or less, or within approximately 1 minute or less, after the addition of a biological (e.g., saliva) sample. In a particular embodiment, the system enables the user to receive results within approximately 1 to 2 minutes.

[0262] In one embodiment, the systems disclosed herein have a false positive rate of less than about 33%. In certain embodiments, the false positive rates are 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.

[0263] In another embodiment, the systems disclosed herein have false negative rates of about 20%, about 18%, about 16%, about 14%, about 12%, about 10%, about 8%, about 6%, about 4%, or about 2%, or less.

[0264] In one embodiment, the system disclosed herein is approximately 10 1 ~about 10 11 This enables the detection of a range of virus copies / mL. In one embodiment, the system disclosed herein has at least 10 per mL. 1 , at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , or at least 1011 This enables the detection of, or even more than, copies of the virus.

[0265] In one embodiment, the system disclosed herein enables the detection of about 10 to 1000 viruses in a solution, far below the clinical range of current interest. In another embodiment, the system enables the detection of about 10 to 100 viruses in a solution, or about 10 to 50, more specifically about 10 to 20 viruses in a solution.

[0266] In one embodiment, the system disclosed herein has a detection limit of approximately 10 virus copies / mL, or 10 analytes / mL, or a similar concentration.

[0267] In one embodiment, the system disclosed herein enables 95% sensitivity and 95% specificity (for example, within a 95% confidence interval).

[0268] In another embodiment, the system disclosed herein enables a minimum target clinical sensitivity of approximately 90% and an optimal target sensitivity of approximately 98%. In yet another embodiment, the system disclosed herein enables a minimum target specificity of approximately 90% and an optimal target specificity of >98%.

[0269] In one embodiment, the systems disclosed herein enable improved disease diagnosis, observation, management, or a combination thereof.

[0270] In certain embodiments, the system stores multiple test results from the same user taken at different times and compares them to observe or predict the possible progression of a disease or condition (e.g., COVID-19). In one embodiment, the system allows obtaining two or more, three or more, or five or more results for the amount of target analytes from the same user at different times, enabling observation of trends in analyte levels over time.

[0271] In certain embodiments, the systems disclosed herein enable the selection of a mode of treatment for the prevention or treatment of a disease (e.g., COVID-19). Modes of treatment can vary and may include, for example, small molecule therapeutics and biological agents (e.g., proteins, antibodies, therapeutic vaccines).

[0272] In certain embodiments, the systems disclosed herein enable the observation of the effects of one or more therapeutic agents (e.g., antiviral agents) and, if the therapeutic agent is not sufficiently effective over a period of time, enable the user to explore alternative therapeutic approaches. In certain embodiments, the system enables the storage of multiple test results from the same user taken at different times and the comparison of these test results to observe therapeutic regimens associated with fluctuations in the levels of a given analyte. In one embodiment, if a therapeutic regimen does not result in a decrease in the level of an analyte (e.g., viral count) within a predetermined period (e.g., several days), the user may discontinue the therapeutic regimen and choose an alternative therapeutic regimen, or, in certain embodiments, supplement the therapeutic regimen with a second therapeutic regimen. In one embodiment, the system enables the observation of trends in analyte levels over time by enabling the acquisition of two or more, three or more, or five or more results for the amount of a target analyte from the same user at different times.

[0273] In certain embodiments, the systems disclosed herein advantageously enable one or more of the following: (i) detection of viral antigens in saliva (i.e., avoiding the use of unpleasant sample collection methods); (ii) straightforward saliva sample collection; (iii) use of existing glucomometer technologies that are widely available and relatively inexpensive; or (iv) test strips that can also be applied to the detection of other pathogens (e.g., viruses).

[0274] In another aspect, the present specification describes a system for detecting at least one target nucleic acid in a biological sample (referred to herein as a nucleic acid detection system; see, for example, FIGS. 15A - 15F), which system comprises: (i) a sequence - specific endonuclease and a guide nucleic acid that cleave collateral nucleic acids when specifically bound to the target nucleic acid; (ii) a detection nucleic acid capable of forming a detectable complex with the cleaved collateral nucleic acid; and (iii) a detection device for detecting the detectable complex, the detection device being an oxidase - based amperometric sensor, and the biological sample being present in sweat, saliva, serum, mucus, or blood.

[0275] One or more targets detected or observed using the systems and methods herein may be present in a biological sample (e.g., a liquid biological sample) collected from a subject, such as a human subject.

[0276] Biological samples can be diverse and can include, for example, blood, serum, milk, sweat, semen, ejaculate, mucus, tears, saliva, plasma, urogenital secretions, lymph fluid, urine, white blood cells, pleural fluid, ascites, sputum, peritoneal fluid, cerebrospinal fluid, pleural effusion, pericardial fluid, amniotic fluid, synovial fluid, interstitial fluid, feces, sebum, respiratory - derived droplets, semen, vaginal mucus, earwax, epidermal cells, and samples of any combination or mixture thereof.

[0277] In one aspect, the biological sample is not blood. In certain aspects, the biological sample is saliva. Saliva is a viscous, thick, stringy liquid that essentially contains microorganisms such as bacteria and fungi, intact human cells, cell debris, as well as many soluble substances 、 enzymes, hormones, antibodies, and other molecules.

[0278] Saliva specimens can be easily collected from a subject in any suitable manner and, in certain aspects, without using a dedicated tool, for example, by the subject spitting into a container handThe saliva can be collected by having someone spit it out, then the contents of the container are diluted and added to the test strip, or the saliva is spit directly onto the test strip. See, for example, Navazesn M (1993). Methods for collecting saliva. Ann NY Acad Sci 694:72-77.

[0279] The amount of biological sample can vary. In one embodiment, the amount of biological sample is approximately 1 μL, 10 μL, 20 μL, 50 μL, or 100 μL to approximately 2000 μL, more specifically, approximately 100 μL, approximately 150 μL, approximately 200 μL, approximately 250 μL, approximately 300 μL, approximately 350 μL, approximately 400 μL, approximately 450 μL, approximately 500 μL, approximately 550 μL, approximately 600 μL, approximately 650 μL, approximately 700 μL, approximately 750 μL, approximately 800 μL, approximately 850 μL, approximately 900 μL, approximately 950 μL, approximately 1000 μL, approximately 1250 μL, approximately 1500 μL, approximately 1750 μL, or approximately 2000 μL.

[0280] In certain embodiments, biological samples are pre-treated before use in the systems and methods disclosed herein. For example, saliva may be treated (e.g., by centrifugation) to obtain a cell-free liquid phase.

[0281] The systems and methods disclosed herein are designed to detect one or more targets. Typical non-limiting targets include viral pathogens.

[0282] In some embodiments, one or more target molecules are viral antigens. In the context of the present invention, the term “viral antigen” should be understood as a protein encoded by a viral genome, its subunits or fragments thereof, or a virus-related nucleic acid (e.g., a viral genome or viral transcript).

[0283] Viruses can be diverse, but include respiratory viruses and coronaviruses.

[0284] In one embodiment, the target molecule is a coronavirus-related viral antigen. Coronaviruses comprise a large and diverse family of enveloped, positive-sense single-stranded RNA viruses. Each coronavirus contains four structural proteins, such as the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. Of these, the S protein plays the most important role in viral attachment, fusion, and entry.

[0285] The S protein is a trimer type I transmembrane glycoprotein that forms a characteristic large, spike-like crown (corona) on the virion surface, mediating binding to host cell receptors and fusion with the host cell membrane. In many coronaviruses, S is posttranslationally cleaved into two subunits called S1 and S2, which trimerize and fold to form a metastable pre-fusion structure. The S1 subunit forms the "head" of the spike and contains two domains: an amino (N)-terminal domain (NTD) and a carboxy (C)-terminal domain (CTD), the latter usually containing a receptor-binding domain (RBD). The S2 subunit contains two 7-amino acid repeat (HR) regions. When S1 recognizes and binds to the corresponding host receptor, the conformation of S2 changes, extending from a compressed form to a nail-like shape called the fused state. This allows the viral envelope to fuse with the outer membrane, enabling the accumulation of viral genetic material within the cell. Subsequently, the viral life cycle proceeds to biosynthesis, viral construction, and viral release.

[0286] In one embodiment, the target molecule is S1 or S2, more specifically, NTD, RBD, CTD1, CTD2, S1 / S2, S1 / S2 cleavage site, S2', S2' cleavage site, fusion peptide, fusion peptide proximal region (FPPR), 7-amino acid repeat 1 (HR1), 7-amino acid repeat 1, central helix region (CHD), connector domain (CD), 7-amino acid repeat 2 (7-amino acid repeat 2), transmembrane anchor (TM), or cytoplasmic end (CT), or a combination thereof.

[0287] The diversity of coronaviruses is reflected in their variable S proteins, which have evolved into various forms with different receptor interactions. These responses to diverse environments trigger fusion between the virus and the cell membrane. In particular, the RBD of the S protein is the most variable genomic region of the beta-coronavirus group.

[0288] Four serologically distinct groups of coronaviruses have been described: alpha, beta (formerly called the second group), delta, and gamma. Within each group, viruses are characterized by their host range and genome sequence. Alpha and beta coronaviruses infect only mammals, while gamma and delta coronaviruses primarily infect birds, although some can also infect mammals. Mammalian novel coronaviruses have now been frequently identified (see, for example, Su et al., Trends Microbiol. 2016;24:490-502). Beta coronaviruses (beta-CoVs) known to be clinically important to humans include viruses of lineages A, B, and C, more specifically lineage A: OC43 (which can cause the common cold) and HKU1; lineage B: SARS-CoV and SARS-CoV-2 (which cause COVID-19); and lineage C: MERS-CoV.

[0289] In one embodiment, the system disclosed herein is directed toward the detection of beta-coronavirus infections, more specifically, A, B, or C lineage coronavirus infections. These are viruses of approximately 32 kb with positive-sense single-stranded RNA, encoding several structural and non-structural proteins. The viral particle contains four major structural proteins: spike, membrane, envelope proteins, and nucleocapsid. The spike protein protrudes from the virion's envelope and plays a major role in receptor host selectivity and cell adhesion. While beta-coronaviruses share many similarities in the ORF1ab polyprotein and most structural proteins, the spike and accessory proteins are quite diverse. Mutations in the spike protein can alter viroptosis, including novel host or increased pathogenicity.

[0290] In other specific embodiments, the systems and methods disclosed herein are directed toward the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. SARS-CoV-2 (also known as 2019-nCoV), also known as Covid-19, was identified in January 2020 as the causative agent of severe acute respiratory syndrome 2. The infection caused by the novel coronavirus quickly became a pandemic, and the World Health Organization (WHO) declared Covid-19 a pandemic in March 2020. As of June 2021, the virus had infected more than 178 million people and killed more than 3.85 million. People who live or work in close proximity to others (e.g., military personnel) are at particular risk.

[0291] Clinical signs associated with SARS-CoV-2 include pneumonia, fever, dry cough, headache, and dyspnea, which can eventually lead to respiratory failure and death. The incubation period for SARS-CoV-2 is 2 to 14 days, which can be longer than that of SARS-CoV and MERS-CoV, which have an average incubation period of 5 to 7 days.

[0292] SARS-CoV-2 was sequenced and isolated in January 2020 (see, e.g., Zhou N. N Engl J Med., 382 (2020), pp. 727-733). Since then, several sequences of SARS-CoV-2 have been published. In some aspects of any of these, the target analyte (e.g., protein, glycoprotein, or nucleic acid) contains at least a portion of the severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2 (see, e.g., the complete genome of SARS-CoV-2 Jan. 2020 / NC_045512.2 Assembly (wuhCor1)).

[0293] In some aspects of any aspect, the target analyte includes SEQ ID NO:1 or SEQ ID NO:2 (Severe Acute Respiratory Syndrome Coronavirus 2 isolate SARS-CoV-2, S gene). In some aspects of any aspect, the target nucleic acid includes SEQ ID NO:1, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1 and maintains the same function, or a codon-optimized version of SEQ ID NO:1. In some aspects of any aspect, the target nucleic acid includes SEQ ID NO:1, or a nucleic acid sequence that is at least 95% identical to SEQ ID NO:1 and maintains the same function, or a codon-optimized version of SEQ ID NO:1.

[0294] In some aspects of any aspect, the target polypeptide contains an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2 or SEQ ID NO:3, or one of SEQ ID NO:2-3, and maintains the same function. In some aspects of any aspect, the target polypeptide contains an amino acid sequence that is at least 95% identical to one of SEQ ID NO:2-3, or SEQ ID NO:2-3, and maintains the same function.

[0295] SEQ ID NO:1, Severe Acute Respiratory Syndrome Coronavirus 2 isolate Wuhan-Hu-1, S surface glycoprotein, Gene ID:43740568, 3822 bp ss-RNA, NC_045512 region:21563-25384 TIFF0007842729000001.tif238166

[0296] SEQ ID NO:2, Severe Acute Respiratory Syndrome Coronavirus 2 isolate Wuhan-Hu-1, S surface glycoprotein, Gene ID:43740568, 1273 aa TIFF0007842729000002.tif130166

[0297] SEQ ID NO:3, SARS-CoV-2, receptor-binding domain (RBD) of the spike protein, 194 aa (see, for example, Figure 9); corresponds to amino acids (aa) 331-524 of SEQ ID NO:2. TIFF0007842729000003.tif18165

[0298] Like other coronaviruses, the spike (S) protein is the major glycoprotein on the surface of the SARS-CoV-2 virus. SARS-CoV-2 appears to possess a receptor-binding domain (RBD) that binds with high affinity to ACE2 derived from humans, ferrets, cats, and other species due to high receptor homology (Wan et al., (2020) J. Virol. doi.org / 10.1128 / JVI.00127-20).

[0299] In some embodiments, the SARS-CoV-2 S1 RBD is 194 amino acids long (e.g., N331-V524 in SEQ ID NO:2; see e.g., SEQ ID NO:3). In some embodiments, the SARS-CoV-2 S1 RBD is the region corresponding to the SARS-CoV S RBD and is 193 amino acids long (e.g., N318-V510 in SEQ ID NO:5; see e.g., SEQ ID NO:6).

[0300] According to reports, the SARS-CoV-2 S protein has 76% amino acid sequence identity with SARS-CoV S Urbani and 80% amino acid sequence identity with bat SARSr-CoV ZXC21 S and ZC45 S glycoproteins. Figure 9 shows the sequence alignment of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 and other related coronaviruses. Sequence alignments for the interaction domains of SARS-CoV-2 (see, e.g., NCBI accession number MN938384), Bat-CoV (see, e.g., NCBI accession numbers MN996532 or MG772933), and SARS-CoV (see, e.g., NCBI accession number NC004718) are available. The RBD of SARS-CoV-2 differs from that of SARS-CoV mainly in its C-terminal residues.

[0301] The S1 subunit of SARS-CoV-2 contains a receptor-binding domain (RBD), while the S2 subunit contains a hydrophobic fusion peptide and two 7-amino acid repeat regions. S1 contains two structurally independent domains: an N-terminal domain (NTD) and a C-terminal domain (C-domain). In some viruses, either the NTD domain or the C-domain may function as the receptor-binding domain (RBD).

[0302] In one embodiment, the systems and methods disclosed herein enable the detection of one or more epitopes of the SARS-CoV-2 S protein, or its subunits or fragments, more specifically, but not limited to, RBD, S1 amino-terminal domain (S1-NTD), ORF3 (3a and 3b), and accessory gene ORF8.

[0303] In one embodiment, the systems and methods described herein enable the detection of the entire virus, i.e., SARS-CoV-2 particles.

[0304] In one embodiment, the systems and methods described herein enable the detection of one or more epitopes in the N-terminal domain (NTD) and C-terminal domain (C-domain) of SARS-CoV-2.

[0305] In one embodiment, the systems and methods disclosed herein enable the detection of one or more epitopes of the SARS-CoV-2 S protein, or its subunits or fragments, more specifically, including, but not limited to, RBD.

[0306] In one embodiment, the systems and methods described herein enable the detection of one or more epitopes in the SARS-CoV-2 RBD, more specifically, one or more epitope residues between residues 331 to 524 of the RBD (see, for example, SEQ ID NO:3).

[0307] In one embodiment, the systems and methods described herein enable the detection of one or more epitopes in the RBD of SARS-CoV-2, more specifically, one or more epitope residues between residues 318 and 510 of the RBD.

[0308] In one embodiment, the systems and methods described herein enable the detection of one or more epitopes in the RBD of SARS-CoV-2, more specifically, one or more epitope residues between residues 319 and 510 of the RBD.

[0309] In certain embodiments, the systems and methods disclosed herein are directed toward the detection of SARS-CoV infection. SARS-CoV was identified in April 2003 as the causative agent of severe acute respiratory syndrome (SARS) (see, e.g., Drosten et al., New Engl. J. Med. 2003;348:1967-1976). Clinically, SARS-CoV presents a biphasic course, namely, an initial high fever and parainfluenza syndrome followed by increased respiratory tachycardia. Droplets play a major role in transmission. Diagnosis is supported by positive hematological tests, PCR, or the presence of the virus in cell cultures, based on clinical imaging and epidemiological data. Shortly thereafter, the consensus genome sequence of SARS-CoV, which most closely resembles the beta-coronavirus of group B, was published (see, for example, Marra et al., Science. 2003;300:1399-14040; Ruan et al., Lancet. 2003;361:1779-1785).

[0310] The SARS-CoV spike protein is known to consist of two functional domains: S1 (amino acids 12-680) and S2 (amino acids 681-1255) (see, for example, Li et al., Science. 2005;309:1864-1868). The RBD is located within the S1 subunit and maps to a fragment of the S1 domain consisting of amino acids (aa) 318-510 (see, for example, Wong et al., J Biol Chem. 2004;279:3197-3201).

[0311] In one embodiment, the systems and methods disclosed herein enable the detection of one or more epitopes of the SARS-CoV S protein, including, but not limited to, RBD, the SARS-CoV S protein, or its subunits or fragments.

[0312] In one embodiment, the systems and methods described herein enable the detection of one or more epitopes in the SARS-CoV RBD, more specifically, one or more epitope residues between residues 318 and 510 of the RBD.

[0313] In some aspects of any of these, the target analyte (e.g., a protein, glycoprotein, or nucleic acid) contains at least a portion of SARS-CoV (e.g., see the complete genome of NCBI reference sequence: NC_004718).

[0314] In some aspects of any aspect, the target analyte includes one of SEQ ID NO:4-6 (SARS-CoV S gene). In some aspects of any aspect, the target nucleic acid includes SEQ ID NO:4, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:4 and maintains the same function, or a codon-optimized version of SEQ ID NO:4. In some aspects of any aspect, the target nucleic acid includes SEQ ID NO:4, or a nucleic acid sequence that is at least 95% identical to SEQ ID NO:4 and maintains the same function, or a codon-optimized version of SEQ ID NO:4.

[0315] In some aspects of any aspect, the target polypeptide contains an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:5 or SEQ ID NO:6, or one of SEQ ID NO:5-6, and maintains the same function. In some aspects of any aspect, the target polypeptide contains an amino acid sequence that is at least 95% identical to one of SEQ ID NO:5-6, or one of SEQ ID NO:5-6, and maintains the same function.

[0316] SEQ ID NO: 4, spike glycoprotein of SARS coronavirus Tor2, NCBI reference sequence: NC_004718.3 region: 21492-25259, 3768 bp TIFF0007842729000004.tif238166

[0317] SEQ ID NO: 5, spike glycoprotein SARS coronavirus Tor2, NCBI reference sequence: YP_009825051.1, 1255 aa TIFF0007842729000005.tif131166

[0318] SEQ ID NO: 6, SARS-CoV, receptor-binding domain (RBD) of the spike protein, 193 aa (see, for example, Figure 9); corresponds to amino acids (aa) 318-510 of SEQ ID NO: 5. TIFF0007842729000006.tif23164

[0319] In certain embodiments, the systems and methods disclosed herein are directed toward the detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection. MERS-CoV is a newly emerged beta-coronavirus that causes severe acute respiratory illness. It was first isolated in Saudi Arabia in 2012 (see, e.g., Zaki et al 2012, NEJM 367:1814-1820) and has since spread to approximately 18 countries, although most cases are within Saudi Arabia and the United Arab Emirates. The clinical features of human MERS-CoV infection range from asymptomatic infection to very severe pneumonia, and can lead to acute respiratory distress syndrome, septic shock, and multiple organ failure, potentially resulting in death. The virus uses its spike protein to interact with cell receptors and enter target cells. The virus is known to bind to dipeptidyl peptidase 4 (DPP4) on human epithelial and endothelial cells via the receptor-binding domain of its spike protein (see, for example, Raj et al 2013, Nature 495:251-256). The MERS-CoV receptor-binding domain consists of a core and a receptor-binding subdomain that interacts with DPP4 (see, for example, Lu et al 2013, Nature 500:227-231).

[0320] The MERS-CoV spike protein is a 1353-amino acid type I membrane glycoprotein that associates to form trimers, creating a spike or peplomer on the surface of enveloped MERS coronavirus particles. The protein has two essential functions: host receptor binding and membrane fusion, which are located at the N-terminus (S1, amino acid residues 1-751) and C-terminus (S2, amino acid residues 752-1353), which are halves of the S protein. MERS-CoV-S binds to its homologous receptor, dipeptidyl peptidase 4 (DPP4), via a receptor-binding domain (RBD) of approximately 230 amino acids located in the S1 subunit. The MERS-CoV RBD is located within residues 358-588 of the spike protein (see, for example, Mou et al (2013) J. Virology vol 87, pp. 9379-9383). The amino acid sequence of the full-length MERS-CoV spike protein is exemplified by the amino acid sequence of the spike protein from the MERS-CoV isolate EMC / 2012, which is provided in GenBank under accession number AFS88936.1 (SEQ ID NO: 8).

[0321] In some aspects of any of these, the target analyte (e.g., protein, glycoprotein, or nucleic acid) contains at least a portion of MERS-CoV (e.g., NCBI reference sequence: NC_019843.3, see complete sequence of isolate HCoV-EMC / 2012).

[0322] In some aspects of any aspect, the target analyte includes one of SEQ ID NO:7-9 (MERS-CoV S gene). In some aspects of any aspect, the target nucleic acid includes SEQ ID NO:7, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7 and maintains the same function, or a codon-optimized version of SEQ ID NO:7. In some aspects of any aspect, the target nucleic acid includes SEQ ID NO:7, or a nucleic acid sequence that is at least 95% identical to SEQ ID NO:7 and maintains the same function, or a codon-optimized version of SEQ ID NO:7.

[0323] In some aspects of any aspect, the target polypeptide contains an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8 or SEQ ID NO:9, or one of SEQ ID NO:8-9, and maintains the same function. In some aspects of any aspect, the target polypeptide contains an amino acid sequence that is at least 95% identical to one of SEQ ID NO:8-9, or one of SEQ ID NO:8-9, and maintains the same function.

[0324] SEQ ID NO: 7, S protein [MERS, human beta coronavirus 2c EMC / 2012], NCBI reference sequence: NC_019843.3, region: 21456-25514, 4059 bp TIFF0007842729000007.tif255164

[0325] SEQ ID NO:8, S protein [MERS, human beta coronavirus 2c EMC / 2012], GenBank:AFS88936.1, 1353 aa TIFF0007842729000008.tif137166

[0326] SEQ ID NO:9, MERS-CoV, receptor-binding domain (RBD) of the spike protein, 212 aa (see, for example, Figure 9); corresponds to amino acids (aa) 377-588 of SEQ ID NO:8. TIFF0007842729000009.tif24164

[0327] The term "MERS-CoV-S" refers to various MERS-CoV isolates, such as Jordan-N3 / 2012, England-Qatar / 2012, Al-Hasa_1_2013, Al-Hasa_2_2013, Al-Hasa_3_2013, Al-Has a_4_2013, Al-Hasa_12, Al-Hasa_15, Al-Hasa_16, Al-Hasa_17, Al-Hasa_18, Al-Hasa_19, Al-Hasa_21, Al-Hasa_25, Bisha_1, Buraidah_1, England 1, Hafr-Al-Batin_1, Hafr-Al-Batin_2, Hafr-Al-Batin_6, Jeddah_1, KFU-HKU 1, KFU-HKU This also includes protein variants of the MERS-CoV spike protein isolated from 13, Munich, Qatar3, Qatar4, Riyadh_1, Riyadh_2, Riyadh_3, Riyadh_3, Riyadh_4, Riyadh_5, Riyadh_9, Riyadh_14, Taif_1, UAE, and Wadi-Ad-Dawasir. The term "MERS-CoV-S" includes recombinant MERS-CoV spike protein or fragments thereof.

[0328] The systems and methods described herein also include virus-related target molecules other than coronaviruses. Non-limiting examples of target molecules include antigens associated with seasonal influenza, highly pathogenic influenza, HIV, Ebola virus, herpes simplex virus 1, herpes simplex virus 2, human papillomavirus, Marburg virus, Lassa virus, and polynuclear respiratory virus (RSV).

[0329] In one embodiment, one of the binders binds to the SARS-CoV-2 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.

[0330] In certain embodiments, at least one target analyte is a virus, more specifically a coronavirus such as a betacoronavirus, and even more specifically SARS-CoV-1.

[0331] In one embodiment, the first and second binders bind to different epitopes of the SARS-CoV-1 spike (S) protein. In a particular embodiment, at least one of the epitopes is located within the receptor-binding domain (RBD) of the S1 protein.

[0332] In one embodiment, one of the binders binds to the 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.

[0333] In a particular embodiment, at least one target analyte is a virus, more specifically, a rhinovirus. In one embodiment, the first and second binders bind to one of the four possible capsid proteins of the rhinovirus.

[0334] In certain embodiments, at least one target analyte is a virus, more specifically a common human coronavirus including types 229E, NL63, OC43, and HKU1. In one embodiment, the first and second binders bind to the spike protein, membrane protein, hemagglutinin protein, envelope, or envelope protein of a common human coronavirus (e.g., types 229E, NL63, OC43, and HKU1).

[0335] In certain embodiments, at least one target analyte is a virus, more specifically, a polynuclear respiratory virus (RSV), parainfluenza virus (PIV), or H1N1. In one embodiment, the first and second binders bind to a fusion protein, membrane protein, hemagglutinin protein, neuraminidase protein, envelope, or envelope protein of polynuclear respiratory virus (RSV), parainfluenza virus (PIV), or H1N1.

[0336] In certain embodiments, at least one target analyte is a virus, more specifically, a human metapneumovirus. In one embodiment, the first and second binders bind to a fusion protein, SH protein, substrate protein, glycoprotein, envelope, or envelope protein of the human metapneumovirus.

[0337] In certain embodiments, at least one target analyte is a virus, more specifically, human immunodeficiency virus (HIV). In one embodiment, the first and second binders bind to the MHC protein, p17 substrate protein, gp120 docking glycoprotein, gp41 transmembrane glycoprotein, envelope, or envelope protein of human immunodeficiency virus (HIV).

[0338] In a particular embodiment, at least one target analyte is a virus, more specifically, the Ebola virus. In one embodiment, the first and second binders bind to the glycoprotein, substrate protein, nucleoprotein, envelope, or envelope protein of the Ebola virus.

[0339] In certain embodiments, at least one target analyte is a virus, more specifically, a Marburg virus. In one embodiment, the first and second binders bind to the glycoprotein, VP40 substrate protein, nucleoprotein, envelope, or envelope protein of the Marburg virus.

[0340] In a particular embodiment, at least one target analyte is a virus, more specifically, a Lassa virus. In one embodiment, the first and second binders bind to Lassa virus glycoprotein 1, glycoprotein 2, macroprotein, zinc protein, stable signaling peptide (SSP), nucleoprotein, envelope, or envelope protein.

[0341] In certain embodiments, at least one target analyte is a parasite, more specifically, a species of malaria Plasmodium (e.g., Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, or Plasmodium knowlesi). In one embodiment, the first and second binders bind to the TRAP protein, SPECT protein, MAEBL protein, PPLP protein, LSA protein, STARP protein, CS protein, SALSA protein, SPATR protein, PxSR protein, or PfEMP3 protein of the Plasmodium malaria species.

[0342] In certain embodiments, one or more target analytes or pathogens are found in biological samples of non-human animal origin, such as West Nile virus and zoonotic pathogens found in bats.

[0343] In some aspects of any aspect, the target analyte is an analyte derived from an RNA virus or a DNA virus (e.g., a protein, glycoprotein, nucleic acid). As used herein, “RNA virus” refers to a virus containing an RNA genome. In some aspects of any aspect, an RNA virus is a double-stranded RNA virus, a positive-sense RNA virus, a negative-sense RNA virus, or a reverse-transcribed virus (e.g., a retrovirus). As used herein, the term “DNA virus” refers to a virus containing a DNA genome. In some aspects of any aspect, a DNA virus is a group I (dsDNA) virus, a group II (ssDNA) virus, or a group VII (dsDNA-RT) virus.

[0344] In some aspects of any given situation, RNA viruses are Group III (i.e., double-stranded RNA (dsRNA)) viruses. In some aspects of any given situation, Group III RNA viruses belong to a viridae selected from the group consisting of amalgaviridae, birnaviruses, chrysoviruses, cystviruses, endornaviridae, hypoviruses, megabirnaviridae, partitiviruses, picobirnaviridae, reoviruses (e.g., rotavirus), totiviruses, and quadriviruses. In some aspects of any given situation, Group III RNA viruses belong to the genus Botybirnavirus. In some aspects of any given situation, Group III RNA viruses are unclassified species selected from a group consisting of Botrytis porri RNA virus 1, Circulifer tenellus virus 1, Colletotrichum camelliae filamentous virus 1, Cucurbit yellows-associated virus, Sclerotinia sclerotiorum debilitation-associated virus, and Spissistilus festinus virus 1.

[0345] In some aspects of any given situation, RNA viruses are group IV (i.e., positive sense single-stranded (ssRNA)) viruses. In some aspects of any given situation, group IV RNA viruses belong to a viral order selected from the group consisting of nidoviruses, picornaviruses, and tymovirales. In some aspects of any given situation, group IV RNA viruses include Arteriviruses, Coronaviruses (e.g., coronavirus, SARS-CoV), Mesoniviruses, Roniviruses, Dicistroviridae, Iflaviruses, Marnaviruses, Picornaviridae (e.g., poliovirus, rhinovirus (common cold virus), hepatitis A virus), Secoviruses (e.g., subcomoviruses), and Alphaflexiviruses (Alp Haflexiviridae, Betaflexivirus, Gammaflexivirus, Tymoviridae, Alphatetravirus, Alvernavirus, Astrovirus, Barnavirus, Benyvirus, Bromovirus, Calicivirus (e.g., Norwalk virus), Carmotetravirus, Closterovirus, Flavivirus (e.g., Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus)Fever virus, Zika virus, Fusarivirus (Fusariviridae), Hepevirus (Hepeviridae), Hypovirus, Levivirus (Leviviridae), Luteovirus (Luteoviridae) (e.g., Barley yellow dwarf virus), Polycipivirus (Polycipiviridae), Narnavirus (Narnaviridae), Nodavirus (Nodaviridae), Permutotetravirus (Permutotetraviridae), Potyvirus (Potyviridae), Sarsurovirus (Sarthroviridae), Statovirus (Statovirus), Togavirus (Togaviridae) (e.g., Rubella virus, Ross River virus) Group IV RNA viruses belong to a family of viruses selected from the group consisting of River virus, Sindbis virus, Chikungunya virus, Tombusvirus (Tombusviridae), and Virgavirus (Virgaviridae). In some aspects of any part of the spectrum, group IV RNA viruses belong to a genera of viruses selected from the group consisting of Bacillariornavirus, Dicipivirus, Labyrnavirus, Sequiviridae, Blunervirus, Cilevirus, Higrevirus, Idaeovirus, Negevirus, Ourmiavirus, Polemovirus, Sinaivirus, and Sobemovirus. In some aspects of any of these, group IV RNA viruses include Acyrthosiphon pisum virus, Bastrovirus, Blackford virus, and Blueberry necrotic ring blotch virus.blotch virus, Cadicistrovirus, Chara australis virus, Extra small virus, Goji berry chlorosis virus, Hepelivirus, Jingmen tick virus, Le Blanc virus, Nedicistrovirus, Nesidiocoris tenuis virus 1, Niflavirus, Nylanderia fulva virus 1, Orsay virus, Osedax japonicus RNA virus 1, Picalivirus, Plasmopara halstedii virus, Rosellinia necatrix It is an unclassified species selected from the group consisting of fusarivirus1, Santeuil virus, Secalivirus, Solenopsis invicta virus3, and Wuhan large pig roundworm virus. In some aspects of any of these, group IV RNA viruses are satellite viruses selected from the group consisting of Sarsuroviridae, Albetovirus, Aumaivirus, Papanivirus, Virtovirus, and Chronic bee paralysis virus.

[0346] In some aspects of any given context, RNA viruses are group V (i.e., negative sense ssRNA) viruses. In some aspects of any given context, group V RNA viruses belong to a phylum or subphylum of Virovirus selected from the group consisting of Negarnaviricota, Haploviricotina, and Polyploviricotina. In some aspects of any given context, group V RNA viruses belong to a class of Virovirus selected from the group consisting of Chunqiuviricetes, Ellioviricetes, Insthoviricetes, Milneviricetes, Monjiviricetes, and Yunchangviricetes. In some aspects of any given situation, group V RNA viruses belong to a viridacea selected from the group consisting of articulaviruses, bunyaviruses, goujianviruses, jingchuviruses, mononegaviruses, muvirals, and serpentovirales.In some aspects of any of these, group V RNA viruses include amnoonviruses (e.g., Taastrup virus), arenaviruses (e.g., Lassa virus), aspiviruses (e.g., Aspiviridae), and bornaviruses (e.g., Borna disease). Disease viruses include: Chuviridae, Cruliviridae, Feraviridae, Filoviridae (e.g., Ebola virus, Marburg virus), Fimovirus, Hantavirus, Jonviridae, Mymonaviridae, Nairoviridae, Nyamiviridae, Orthomyxovirus (e.g., influenza virus), Paramyxovirus (e.g., measles virus). It belongs to a viridae family selected from the group consisting of mumps virus, nipah virus, hendra virus, and NDV, peribunyaviridae, phasmavirus, fenuivirus, pneumovirus (e.g., RSV and metapneumovirus), qinvirus, rhabdovirus (e.g., rabies virus), sunvirus, tospovirus, and yueviridae.In some aspects of any given situation, group V RNA viruses belong to a virus genera selected from the group consisting of anphevirus, arlivirus, chentivirus, crustavirus, tilapinevirus, wastrivirus, and deltavirus (e.g., hepatitis D virus).

[0347] In some aspects of any given context, RNA viruses are group VI RNA viruses and contain a viral encoding reverse transcriptase. In some aspects of any given context, group VI RNA viruses belong to the order Ortervirales. In some aspects of any given context, group VI RNA viruses belong to a family or subfamily selected from the group consisting of Belpaoviridae, Caulimoviridae, Metaviridae, Pseudoviridae, Retroviruses (e.g., retroviruses, e.g., HIV), Orthoretrovirinae, and Spumaretrovirinae.In some aspects of any of these, group VI RNA viruses include alpha-retroviruses (e.g., avian leukosis virus; Rous sarcoma virus), beta-retroviruses (e.g., mouse mammary tumor virus), bovispumaviruses (e.g., bovine foamy virus), delta-retroviruses (e.g., bovine leukemia virus; human T-lymphotropic virus), epsilon-retroviruses (e.g., walleye dermal sarcoma virus), and equispumaviruses (e.g., equine foamy virus). They belong to a genus of viruses selected from the group consisting of foamy viruses, Felispumavirus (e.g., Feline foamy virus), Gammaretrovirus (e.g., Murine leukemia virus; Feline leukemia virus), Lentivirus (e.g., Human immunodeficiency virus 1; Monkey immunodeficiency virus; Feline immunodeficiency virus), Prosimiispumavirus (e.g., Brown greater galago prosimian foamy virus), and Simiispumavirus (e.g., Eastern chimpanzee simian foamy virus).In some aspects of any given situation, the virus is an endogenous retrovirus (ERV; for example, endogenous retrovirus group W envelope member 1 (ERVWE1); HCP5 (HLA complex P5); human teratoma-derived virus), which is a genomic endogenous viral element that closely resembles and may be derived from retroviruses.

[0348] In some aspects of any given context, DNA viruses are Group I (i.e., dsDNA) viruses. In some aspects of any given context, Group I dsDNA viruses belong to a viral order selected from the group consisting of caudoviruses; herpesviruses; and ligamenuviruses. In some aspects of any of these, Group I dsDNA viruses include adenoviruses (e.g., adenovirus), alloherpesviridae, ampullaviridae, ascovirus, asfarviridae (e.g., African swine fever virus), baculovirus, bicaudaviridae, clavavirus, corticovirus, fuselloviridae, globulovirus, guttavirus, herpesvirus (e.g., human herpesvirus, varicella).Zoster virus, histrosavirus (Hytrosaviridae), iridovirus (Iridoviridae), lavidaviridae, liposlipsvirus (Lipothrixviridae), malacoherpesvirus (Malacoherpesviridae), Marseillevirus (Marseilleviridae), mimivirus (Mimiviridae), myovirus (Myoviridae) (e.g., enterobacteria phage T4), nimavirus (Nimaviridae), nudivirus (Nudiviridae), pandoravirus (Pandoraviridae), papillomavirus (Papillomaviridae), phycodnavirus (Phycodnaviridae), plasmavirus (Plasmaviridae) It belongs to a viridae family selected from the group consisting of ae), podoviruses (e.g., enterobacteriaceae T7), polidnaviruses, polyomaviruses (e.g., Simian virus 40, JC virus, BK virus), poxviruses (e.g., cowpox virus, smallpox), rudiviruses (rudiviridae), siphoviruses (e.g., enterobacteriaceae λ), spherolipoviruses (sphaerolipoviridae), tectiviruses (tectiviridae), tristromaviruses (tristromaviridae), and turriviruses (turriviridae). In some aspects of any given situation, group I dsDNA viruses belong to a virus genera selected from the group consisting of Dinodnavirus, Rhizidiovirus, and Salterprovirus. In some aspects of any given situation, group I dsDNA viruses include Abalone shriveling syndrome-related viruses and Apis mellifera filamentus.It belongs to an unclassified virus species selected from the group consisting of filamentous viruses, Bandicoot papillomatosis carcinomatosis virus, Cedratvirus, Kaumoebavirus, KIs-V, Lentille virus, Leptopilina boulardi filamentous virus, Megavirus, Metallosphaera turreted icosahedral virus, Methanosarcina spherical virus, Mollivirus sibericum virus, Orpheovirus IHUMI-LCC2, Phaeocystis globosa virus, and Pithovirus. In some aspects of any given situation, the group I dsDNA viruses are virophages selected from the group consisting of Organic Lake virophage, Ace Lake Mavirus virophage, Dishui Lake virophage 1, Guarani virophage, Phaeocystis globosavirus virophage, Rio Negro virophage, Sputnik virophage 2, Yellowstone Lake virophage 1, Yellowstone Lake virophage 2, Yellowstone Lake virophage 3, Yellowstone Lake virophage 4, Yellowstone Lake virophage 5, Yellowstone Lake virophage 6, Yellowstone Lake virophage 7, and Zamilon virophage 2.

[0349] In some aspects of any given situation, DNA viruses are Group II (i.e., ssDNA) viruses. In some aspects of any given situation, Group II ssDNA viruses belong to a viridae family selected from the group consisting of anelloviruses (Anelloviridae), bacilladnaviridae, bidnaviridae, circoviruses (Circoviridae), geminiviruses (Geminiviridae), genomoviruses (Genomoviridae), inoInoviridae, microviruses (Microviridae), nanoviruses (Nanoviridae), parvoviruses (Parvoviridae), smacoviruses (Smacoviridae), and spiraviruses (Spiraviridae).

[0350] In some aspects of any given situation, the DNA virus is a group VII (i.e., dsDNA-RT) virus. In some aspects of any given situation, the group VII dsDNA-RT virus belongs to the order Ortervirales. In some aspects of any given situation, the group VII dsDNA-RT virus belongs to the family Kalimoviridae or Hepadnaviridae (e.g., Hepatitis B virus). In some aspects of any given situation, group VII dsDNA-RT viruses belong to a virus genus selected from the group consisting of Badnavirus, Caulimovirus, Cavemovirus, Petuvirus, Rosadnavirus, Solendovirus, Soymovirus, Tungrovirus, Avihepadnavirus, and Orthohepadnavirus.

[0351] In some aspects of any given situation, the target analytes are derived from coronaviruses. The scientific name for coronaviruses is Orthocoronavirinae or Coronavirus. Coronaviruses belong to the family Coronaviridae, order Nidovirales, kingdom Riboviria. Coronaviruses are divided into alpha-coronaviruses and beta-coronaviruses, which infect mammals, and gamma-coronaviruses and delta-coronaviruses, which primarily infect birds. Non-specific examples of alpha-coronaviruses include human coronavirus 229E, human coronavirus NL63, Miniopterus bat coronavirus 1, Miniopterus bat coronavirus HKU8, porcine epidemic diarrhea virus, Rhinolophus bat coronavirus HKU2, Scotophilus bat coronavirus 512, and feline infectious peritonitis virus (FIPV, also known as feline infectious hepatitis virus). Non-limiting examples of beta-coronaviruses include beta-coronavirus 1 (e.g., bovine coronavirus, human coronavirus OC43), human coronavirus HKU1, mouse coronavirus (also known as mouse hepatitis virus (MHV)), pipistrellus bat coronavirus HKU5, rosettus bat coronavirus HKU9, severe acute respiratory syndrome-associated coronaviruses (e.g., SARS-CoV, SARS-CoV-2), bamboo bat coronavirus HKU4, Middle East respiratory syndrome (MERS)-associated coronaviruses, and hedgehog coronavirus 1 (EriCoV). Non-limiting examples of gamma-coronaviruses include beluga whale coronavirus SW1 and infectious bronchitis virus. Non-limiting examples of delta-coronaviruses include bulrulose coronavirus HKU11 and swine coronavirus HKU15.

[0352] In some aspects of any given context, the target nucleic acid is a nucleic acid (see, for example, Figures 15A–15F). In some aspects of any given context, the target nucleic acid is a viral nucleic acid, such as a viral DNA or RNA genome, or a viral RNA transcript.

[0353] This specification discloses binding assays that constitute components of the systems and methods disclosed herein. The objective of a binding assay is to bind to at least one target analyte or target molecule and to generate a detectable signal.

[0354] In one embodiment, the binding assay utilizes standard electrophoresis and immunodiagnostic methods, including immunoassays such as competitive, direct reaction, or sandwich-type assays. Such assays include, but are not limited to, Western blotting, agglutination tests, enzyme-labeled and mediated immunoassays (e.g., ELISA), biotin / avidin-type assays, and radioimmunosytases. In one embodiment, the binding assay utilizes sequence-specific endonucleases and guide nucleic acids, collateral nucleic acids, and / or detection nucleic acids.

[0355] Specific recognition of the target molecule is achieved using at least one binder. A binder suitable for use with one or more target analytes or any one or more molecules capable of associating with or binding to one or more target molecules. The binder has at least one binding site specific to the target analyte.

[0356] In one embodiment, at least one binder is selected from aptamers, antibodies, receptor ligands, proteins, or molecular imprinting polymers.

[0357] In one embodiment, the specific binder is an aptamer with a size of approximately 10-15 kDa (20-45 nucleotides), which binds to the target molecule with at least micromolar affinity and distinguishes it from closely related target molecules.

[0358] In one embodiment, the specific binder is an aptamer of approximately 10-15 kDa size (20-45 nucleotides) that binds to the target molecule with at least nanomolar affinity and / or distinguishes it from closely related target molecules.

[0359] In certain embodiments, the binder is an aptamer, and the Kd of the aptamer relative to the target molecule is 10 nM or less, or 5 nM or less, and may be as low as 100 pM.

[0360] In certain embodiments, the system comprises a two-conjugate assay, where the first and second conjugates are selected from antibodies (e.g., monoclonal antibodies) and aptamers, or combinations thereof. In one embodiment, the first conjugate is an aptamer and the second conjugate is an antibody (e.g., a monoclonal antibody). In another embodiment, the first conjugate is an antibody (e.g., a monoclonal antibody) and the second conjugate is an aptamer.

[0361] In a particular embodiment, the system comprises a two-conjugate assay, where the first and second conjugates are selected from various antibodies (e.g., monoclonal antibodies). In one embodiment, the first conjugate is an antibody, and the second conjugate is an antibody (e.g., a monoclonal antibody), and these antibodies may be the same or different, or the targets of these antibodies may be the same or different.

[0362] In certain embodiments, the binding assay comprises a first and a second binder, the first binder binding to a first site of the target analyte, and the second binder binding to a second (different) site of the target analyte or molecule.

[0363] In certain embodiments, the binding assay comprises a first and a second binder, the first binder binding to a first site on the target analyte, and the second binder binding to the same site on the target analyte. Since there is an excess of copies of the site on the target, both the first and second binders can bind to the target.

[0364] In certain embodiments, the affinity of the first binder to the first epitope is higher than the affinity of the second binder to the second epitope. The ratio of the Kd of the first epitope to the Kd of the second epitope may be 1:10,000 to 10,000:1.

[0365] In a particular embodiment, the first conjugate is an aptamer, and the second conjugate is an antibody, more specifically, a detectably labeled antibody.

[0366] In one embodiment, an antibody is combined with or linked to an enzyme (e.g., glucose oxidase) in a fixed integer ratio (for example, 1 antibody to 1, 2, 3, 4, 5, or more enzymes).

[0367] In one particular embodiment, an antibody is combined with glucose oxidase to obtain an antibody-GOx conjugate. In another embodiment, an alternative conjugate strategy is utilized that uses a chemical linker for site-specific conjugation with GOx, such as the linkage of an uncleavable thioether to a peptide. As shown in Figure 2, the aptamer captures the target analyte (e.g., a viral antigen), and Ab-GOx binds to the viral antigen if present. When a constant potential is applied, GOx oxidizes glucose, transferring electrons to oxygen, producing hydrogen peroxide, which then reacts with the electrode to generate a current output.

[0368] Glucose oxidase (Enzyme Commission number (EC) 1.1.3.4) catalyzes the oxidation of beta-D-glucose to D-glucono-delta-lactone using an oxygen molecule as an electron acceptor, simultaneously producing hydrogen peroxide (see, for example, Figure 3). Glucose oxidase functions as a homodimer. Glucose oxidase may also be called beta-D-glucose:oxygen 1-oxidoreductase; notatin; glucose oxyhydrase; corirophyllin; penatin; glucose aerodehydrogenase; microcid; beta-D-glucose oxidase; D-glucose oxidase; D-glucose-1-oxidase; beta-D-glucose:quinone oxidoreductase; glucose oxyhydrase; deoxin-1; GOD; or GOx. In some aspects of any of these, glucose oxidase is microbial (e.g., fungal or bacterial) glucose oxidase. In some aspects of any of these, glucose oxidase is derived from Aspergillus niger (e.g., any sequence available from UniProtKB - P13006 (GOX_ASPNG), or NCBI gene IDs: 37106576, 4977376, 4985693, 4984787 or 4981316, or any related orthologue or homolog; see, for example, SEQ ID NO: 10), Penicillium chrysogenum (also known as Penicillium notatum; see, for example, any sequence available from UniProtKB - K9L4P7 (K9L4P7_PENCH), or any related orthologue or homolog), or Penicillium amagasakiense (e.g., UniProtKB - It originates from any sequence available from P81156 (GOX_PENAG), or any related orthologue or homolog reference.For example, see Raba et al., “Glucose Oxydase as an Analytical Reagent,” Critical Reviews in Analytical Chemistry, 25(1):1-42 (1995), the entire contents of which are incorporated herein by reference.

[0369] In some aspects of any aspect, glucose oxidase includes an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:10 and maintains the same function (e.g., glucose oxidation and / or hydrogen peroxide production). In some aspects of any aspect, glucose oxidase includes an amino acid sequence that is at least 95% identical to SEQ ID NO:10 and maintains the same function (e.g., glucose oxidation and / or hydrogen peroxide production).

[0370] SEQ ID NO:10, glucose oxidase precursor, Aspergillus oryzae, UniProtKB-P13006, 605 amino acids (aa) TIFF0007842729000010.tif64166

[0371] In certain embodiments, antibodies are combined with oxidases to obtain antibody-Ox conjugates made of galactose oxidase (see, e.g., EC 1.1.3.9); D-glucose:D-fructose oxidoreductase (see, e.g., EC 1.1.99.28); or cellobiose oxidase (see, e.g., EC 1.1.3.25).

[0372] In certain embodiments, antibodies are combined with dehydrogenases to obtain antibody-DH conjugates made of glucose dehydrogenase (e.g., see EC 1.1.1.47); glucose 6-phosphate dehydrogenase (e.g., see EC 1.1.1.49); fructose dehydrogenase (e.g., see EC 1.1.99.11); sucrose dehydrogenase (also known as glucoside 3-dehydrogenase; e.g., see EC 1.1.99.13); glucoside dehydrogenase (e.g., see EC 1.1.99.13); alcohol dehydrogenase (e.g., see EC 1.1.1.1); sorbitol dehydrogenase (e.g., see EC 1.1.99.21); lactate dehydrogenase (e.g., see EC 1.1.1.27); or malate dehydrogenase (e.g., see 1.1.1.37).

[0373] In embodiments of this two-conjugate assay, the first and second conjugates may be specific to at least one viral antigen associated with a coronavirus or another virus of interest, for example, the S protein of a coronavirus, or its subunit, fragment, or epitope. The at least one viral antigen may be the S-1 subunit of a beta-coronavirus, more specifically a type C beta-coronavirus such as SARS-CoV-2 or SARS-CoV, or one or more epitopes thereof.

[0374] In certain embodiments, the aptamers are those disclosed in Song, Y. et al. Discovery of Aptamers Targeting Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein. (2020). doi:10.26434 / chemrxiv.12053535.v2, or Song et al., Analytical Chemistry, 02 Jul 2020, 92(14):9895-9900, the contents of which are incorporated herein by reference.

[0375] In another specific embodiment, the antibody is the antibody disclosed in Yuan, M. et al. A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV. Science 633, eabb7269 (2020), the entire contents of which are incorporated herein by reference.

[0376] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound to the test strip.

[0377] In another specific embodiment, the first binder is an aptamer bound to a test strip, more specifically, to a hydrophilic membrane such as a nitrocellulose membrane.

[0378] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound via a test strip and a polymer membrane placed on the strip.

[0379] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound to the test strip via a hydrophilic membrane, such as a nitrocellulose membrane, placed on the strip.

[0380] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound via a hydrophilic membrane, such as a nitrocellulose membrane, located directly above the test strip and the electrodes on the strip or between the two electrodes.

[0381] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound to the test strip and the strip via a hydrophilic membrane, such as a nitrocellulose membrane, which is fixed to the electrode on or between the two electrodes.

[0382] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound to a test strip via a hydrophilic membrane, the membrane also collects a biological sample and provides a sink area for the sample to flow from one point on the membrane to another.

[0383] In another specific embodiment, the first binder is an aptamer, antibody, or protein bound to a test strip via a hydrophilic membrane, such as a nitrocellulose membrane, the membrane also collects a biological sample and provides a sink area for the sample to flow from one point on the membrane to another.

[0384] In some aspects of any aspect, the target analyte is an antibody (e.g., IgG, IgM, and IgA). In some aspects of any aspect, the first conjugate is a protein bound to the test strip, to which the target analyte (e.g., antibody) specifically binds. In some aspects of any aspect, the first conjugate is a viral protein (e.g., Flu H1N1 HA or NA; SARS-CoV-2 S spike protein). In some aspects of any aspect, the second conjugate is an aptamer or antibody that specifically binds to the target analyte (e.g., antibody). In some aspects of any aspect, the second conjugate is an anti-antibody antibody. In some aspects of any aspect, the second conjugate is an anti-antibody antibody linked to glucose oxidase. In some aspects of any aspect, the second conjugate is an anti-IgG, anti-IgM, or anti-IgA antibody. In some aspects of any of these aspects, the second binder is an anti-human IgG, anti-human IgM, or anti-human IgA antibody.

[0385] Antibody reagents specific to the targets described herein, such as influenza neuraminidase protein, influenza hemagglutinin protein, SARS-CoV-2 spike or membrane protein, and glucose oxidase, are known in the art. For example, such reagents are readily available. In some aspects of any aspect, an antibody reagent specific to the targets described herein (e.g., a viral antigen such as Flu HA or NA, or SARS-CoV-2 spike protein or membrane protein, or glucose oxidase) may be an antibody reagent containing one or more (e.g., one, two, three, four, five, or six) CDRs of any one of the antibodies listed in Table 2. In some aspects of any aspect, an antibody reagent that is specific to the target described herein (for example, a viral antigen such as Flu HA or NA, or SARS-CoV-2 spike protein or membrane protein, or glucose oxidase) may be an antibody reagent comprising three heavy chain CDRs of any one antibody listed in Table 2.In some aspects of any aspect, an antibody reagent that is specific to the target described herein (for example, a viral antigen such as Flu HA or NA, or SARS-CoV-2 spike protein or membrane protein, or glucose oxidase) may be an antibody reagent comprising the VH and / or VL domains of any one of the antibodies listed in Table 2. Such an antibody reagent is specifically intended for use in the methods, systems, and / or kits described herein.

[0386] (Table 2) Exemplary antibody reagents TIFF0007842729000011.tif70169

[0387] Aptamer reagents specific to the targets described herein, such as influenza neuraminidase protein, influenza hemagglutinin protein, SARS-CoV-2 spike or membrane protein, and glucose oxidase, are known in the art. For example, such reagents are readily available for purchase. In some embodiments, aptamers are described as follows: Song, Y. et al. Discovery of Aptamers Targeting Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein. (2020). doi:10.26434 / chemrxiv.12053535.v2, or Song et al., Analytical Chemistry, 02 Jul 2020, 92(14):9895-9900; International Patent Application WO2013183383A1; U.S. Patent Publication US20150167106A1; Gopinath et al., Aptamers that bind to the hemagglutinin of the recent pandemic influenza virus H1N1 and efficiently inhibit agglutination, Acta Biomater. 2013. Aptamers may be selected from those described in Nov;9(11):8932-41, or designed using the methods described therein, the contents of which are all incorporated herein by reference. In some aspects of any aspect, the aptamer contains biotin ligated to the 5' end (5Biosg) or 3' end (3Biosg).

[0388] In some aspects of any aspect, the aptamer is selected from Table 3. In some aspects of any aspect, the aptamer contains a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NO: 11–19 and maintains the same function (e.g., binding to the target analyte). In some aspects of any aspect, the aptamer contains a nucleic acid sequence that is at least 95% identical to one of SEQ ID NO: 11–19 and maintains the same function (e.g., binding to the target analyte).

[0389] In some aspects of any aspect, the aptamer includes a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NO: 11-12 and maintains the same function (e.g., binding to the target analyte). In some aspects of any aspect, the aptamer includes a nucleic acid sequence that is at least 95% identical to one of SEQ ID NO: 11-12 or at least 95% identical to one of SEQ ID NO: 11-12 and maintains the same function (e.g., binding to the target analyte).

[0390] (Table 3) Exemplary aptamer reagents TIFF0007842729000012.tif81170

[0391] In another context, this specification describes a system for detecting at least one target nucleic acid in a biological sample (hereinafter referred to as a nucleic acid detection system; see, for example, Figures 15A–15F), the system comprising (i) a sequence-specific endonuclease and guide nucleic acid that specifically bind to the target nucleic acid and cleave a collateral nucleic acid; (ii) a detection nucleic acid that can form a detectable complex with the cleaved collateral nucleic acid; and (iii) a detection device for detecting the detectable complex, the detection device being an oxidase-based amperometric sensor, the biological sample being present in sweat, saliva, serum, mucus, or blood.

[0392] In some aspects of any aspect, the sequence-specific endonuclease is a Cas enzyme. In some aspects of any aspect, the sequence-specific endonuclease has the ability to cleave collateral nucleic acids when the endonuclease and guide nucleic acid bind to the target nucleic acid. In some aspects of any aspect, the sequence-specific endonuclease is Cas13a (formerly known as C2c2), Cas13b, Cas13c, Cas12a, and / or Csm6. In some aspects of any aspect, the sequence-specific endonuclease is Cas12a or Cas13. For example, see U.S. Patent Application US20190241954; PCT Patent Application WO2020028729; Chen et al., CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity, Science. 2018 Apr 27, 360(6387):436-439, the contents of which are incorporated herein by reference.

[0393] In some aspects of the various aspects described herein, a sequence-specific endonuclease binds to a guide nucleic acid (gNA), for example, in the presence of a target nucleic acid. As used herein, the terms “guide nucleic acid,” “guide sequence,” “crRNA,” “guide RNA,” or “single guide RNA,” or “gRNA” refer to a polynucleotide containing any polynucleotide sequence. Generally, the guide nucleic acid sequence is selected so as to have sufficient complementarity with the target nucleic acid sequence to hybridize with it, and so as to guide sequence-specific binding of the fusion protein, i.e., the sequence-specific endonuclease, to the target nucleic acid sequence.

[0394] The full-length guide nucleic acid, collateral nucleic acid, and / or detection nucleic acid strands may be of any length. For example, the guide nucleic acid, collateral nucleic acid, and / or detection nucleic acid strands may have nucleotide lengths of about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 35, about 40, about 45, about 50, about 75, or more, or longer. In some aspects of the various aspects described herein, the guide nucleic acid, collateral nucleic acid, and / or detection nucleic acid strands may have nucleotide lengths of less than about 75, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 12, or shorter. For example, the strands of the guide nucleic acid, collateral nucleic acid, and / or detection nucleic acid are 10 to 30 nucleotides long. In some aspects of the various aspects described herein, the guide nucleic acid is designed using guide design tools (e.g., Benchling™; Broad Institute GPP™; CasOFFinder™; CHOPCHOP™; CRISPOR™; Deskgen™; E-CRISP™; Geneious™; GenHub™; GUIDES™ (e.g., for library design); Horizon Discovery™; IDT™; Off-Spotter™; and Synthego™, available on the World Wide Web).

[0395] In some aspects of any aspect, the guide nucleic acid is complementary or substantially complementary to at least a portion of the target nucleic acid. In some aspects of any aspect, the detection nucleic acid is complementary or substantially complementary to at least a portion of the cleaved collateral nucleic acid. The terms “substantially complementary” or “substantially complementary,” as used herein, refer to both complete complementarity of the binding nucleic acids, which in some cases may be called identical sequences, and sufficient complementarity to enable the binding of the desired nucleic acid. Similarly, the term “complementary hybrid” includes substantially complementary hybrids.

[0396] In some aspects of any aspect, the detected nucleic acid hybridizes with cleaved collateral nucleic acid. In some aspects of any aspect, the detected nucleic acid does not hybridize with uncleaved collateral nucleic acid. In some aspects of any aspect, the detected nucleic acid is ligated to a test strip, for example, at the 5' or 3' end of the detected nucleic acid.

[0397] In some aspects of any of these, the collateral nucleic acid is ligated with glucose oxidase, for example, at the 5' or 3' end of the collateral nucleic acid (see, for example, Figure 15A).

[0398] In some aspects of any aspect, the system further comprises an aptamer linked to glucose oxidase (see, for example, Figures 15B–15C). The aptamer may be linked to glucose oxidase using any linkage known in the art, as further described herein. The 3' end, 5' end, or internal region of the aptamer may be linked to glucose oxidase.

[0399] In some aspects of any given aspect, the aptamer specifically binds to at least a portion of the cleaved collateral nucleic acid. In some aspects of any given aspect, the aptamer binds to the single-stranded portion of the cleaved collateral nucleic acid (see, for example, Figure 15B). In some aspects of any given aspect, the aptamer binds to the double-stranded portion of the cleaved collateral nucleic acid hybridized with the detected nucleic acid (see, for example, Figure 15C).

[0400] In some aspects of any aspect, the system further comprises an antibody conjugated to glucose oxidase (see, for example, Figure 15D). The antibody may be conjugated to glucose oxidase using any conjugation known in the art, as further described herein. The N-terminus, C-terminus, or internal region of the light or heavy chain of the antibody may be conjugated to glucose oxidase. In some aspects of any aspect, the antibody specifically binds to at least a portion of the cleaved collateral nucleic acid.

[0401] In some aspects of any given scenario, the collateral nucleic acid is linked to an antibody that specifically binds to glucose oxidase (see, for example, Figure 15E). Antibody reagents specific to glucose oxidase are known in the art. For example, such reagents are readily available (see, for example, Table 2).

[0402] In some aspects of any aspect, a collateral nucleic acid is linked to a first member of an affinity pair (see, for example, Figure 15F). In some aspects of any aspect, the system further comprises glucose oxidase linked to a second member of an affinity pair. In some aspects of any aspect, the first and second members of the affinity pair are selected from the group consisting of haptenic or antigenic compounds and corresponding antibodies or their binding portions or fragments; digoxigenin and anti-digoxigenin; mouse immunoglobulins and goat anti-mouse immunoglobulins; non-immunological binding pairs; biotin and avidin; biotin and streptavidin; hormones and hormone-binding proteins; thyroxine and cortisol hormone-binding proteins; receptors and receptor agonists; receptors and receptor antagonists; acetylcholine receptors and acetylcholine or their analogues; IgG and protein A; lectins and carbohydrates; enzymes and enzyme cofactors; enzymes and enzyme inhibitors; complementary oligonucleotide pairs capable of forming nucleic acid doubles; and a first molecule having a negative charge and a second molecule having a positive charge.

[0403] In some aspects of any aspect, the first and second members of the affinity pair are streptavidin and biotin. In some aspects of any aspect, streptavidin is linked to a collateral nucleic acid and biotin is linked to glucose oxidase. In some aspects of any aspect, biotin is linked to a collateral nucleic acid and streptavidin is linked to glucose oxidase.

[0404] This specification also discloses detection devices that are components of the systems and methods disclosed herein, which detect signals generated by a binding assay.

[0405] In certain embodiments, the detection device is a portable (e.g., handheld) battery-powered device.

[0406] In one embodiment, the detection device used in the systems and methods described herein is a glucometer, such as a personal glucose meter (PGM). Conventionally, a PGM is a portable, handheld device used by users with type 1 or type 2 diabetes to measure blood glucose levels. Typically, the user purchases a small strip (e.g., about 20-30 mm x about 5-9 mm) to contact with the PGM. The user uses a lancet to draw a small amount of blood (e.g., a few microliters) from a finger or other site, applies the blood droplet sample to the exposed end of the strip, and then inserts the connector end of the strip into the PGM connector port. A chemical reaction occurs between the blood sample and the chemicals on the strip, which the PGM measures to determine the blood glucose level in units of mg / dL, mmol / L, or kg / L. After repeatedly measuring blood glucose levels, the used test strip is removed from the PGM and a new test strip is loaded into the connector port.

[0407] In one embodiment, the glucometer in the systems and methods described herein is a standard commercially available handheld glucometer. Non-limiting examples of commercially available glucometers include Accu Chek® (ROCHE DIABETES CARE, INC., Indianapolis, Indiana), Van Touch®, Bionime® Presto® (AGAMATRIX, Salem, New Hampshire), Wavesense Presto® (AGAMATRIX, Salem, New Hampshire), Counter® (ASCENSIA, Basel, Switzerland), CounterPlus® (ASCENSIA, Basel, Switzerland), FreeStyle® (ABBOTT DIABETES CARE INC., Abbott Park, Illinois), and True® (TRIVIDIA HEALTH, Fort Lauderdale, Florida).

[0408] In certain embodiments, a glucometer is a glucometer with usage restrictions or is disposable.

[0409] A glucometer typically includes a base unit that houses the control and testing electronics necessary for testing blood glucose levels in a blood sample. In other embodiments, the glucometer is modified with one or more modifications to enhance its ability to detect general or saliva-derived analytes.

[0410] In a particular embodiment, the detection device is a glucometer having a base unit having a test strip slot and a reader configured to analyze a biological sample (e.g., a saliva sample). In one embodiment, the glucometer measures a glucose signal (e.g., quantitatively). The base unit may vary in shape and size. The test strip slot is configured to accept a glucose test strip, such as those described herein, which can be removably inserted into the test strip slot. The glucometer may also have means for storing and transmitting data.

[0411] Glucose measurement can be performed by glucose detection using glucose oxidase by standard amperometry. In this embodiment, a sensor is used to convert the glucose concentration in a biological fluid into a voltage or current signal. The sensor uses platinum and silver electrodes that form part of an electrical circuit, where hydrogen peroxide is electrolyzed. Hydrogen peroxide is produced as a result of glucose oxidation on a glucose oxide film. The current flowing through the circuit provides a measurement of the hydrogen peroxide concentration, and the glucose concentration is obtained.

[0412] Glucose measurement can be performed by detecting glucose using glucose oxidase by standard amperometry. In this embodiment, a sensor is used to convert the glucose concentration in a biological fluid into a voltage or current signal. The sensor uses carbon electrodes that form part of an electrical circuit, where hydrogen peroxide is electrolyzed. Hydrogen peroxide is produced as a result of glucose oxidation on a glucose oxide film. The current flowing through the circuit provides a measurement of the hydrogen peroxide concentration, and the glucose concentration is obtained.

[0413] In certain embodiments, the system includes one or more signal processing applications or electronic amplifiers within the circuit for amplifying signals.

[0414] In one embodiment, H2O2 can be obtained at these electrodes at a low applied potential (for example, about -0.2V relative to Ag / AgCl; for example, Ag / AgCl may be the reference electrode).

[0415] In one embodiment, the biological sample is mixed with glucose at a concentration of approximately 0.01 mM to approximately 1 M.

[0416] In other embodiments, the biological sample is mixed with sucrose, fructose, maltose, galactose, cellulose, or any combination thereof, at concentrations of 0.01 mM to 1 M, containing amylase or invertase. In some embodiments of any aspect, the concentration of glucose, sucrose, fructose, maltose, galactose, or cellulose is at least 0.01 mM, at least 0.02 mM, at least 0.03 mM, at least 0.04 mM, at least 0.05 mM, at least 0.06 mM, at least 0.07 mM, at least 0.08 mM, at least 0.09 mM, at least 0.1 mM, at least 0.2 mM, at least 0.3 mM, at least 0.4 mM, at least 0.5 mM, at least 0.6 mM, at least 0.7 mM, at least 0.8 mM, at least 0.9 mM, or at least 1.0 mM or higher.

[0417] In one embodiment, the device includes a display unit for displaying results. The display can show the most recent test and optionally, past tests. In certain embodiments, the glucometer includes a voice control function for ease of use by visually impaired individuals. The glucometer may include the measurement of other features unrelated to glucose measurement, such as other physiological functions. The glucose reading displayed on the glucometer may be positively correlated with the enzyme concentration on the sensor surface, and the enzyme concentration may correlate with the number of analytes (e.g., viral particles) present in the biological sample.

[0418] Glucometers may have software components. Various software algorithms for glucometers are known.

[0419] In one embodiment, the glucometer has a wireless transmitter configured to communicate a message to a second device, such as a mobile device like a cell phone or tablet computer. In one embodiment, the message is sent to the second device using a short-range communication protocol, such as the Bluetooth protocol. The message may also be, for example, a text message or an email.

[0420] In one embodiment, the glucometer presents the results immediately after the test begins, for example, in less than approximately 5 minutes, less than approximately 1 minute 30 seconds, less than approximately 15 seconds, or less than approximately 5 seconds.

[0421] The accuracy of a glucometer can vary, but generally does not exceed a 20% error, more specifically, not exceeding an error of about 15%, 10%, 5%, or less than 5%, for example, not exceeding an error of about 4%, 3%, 2%, or 1%, or less. In certain embodiments, the cross-sensitivity of the glucometer is reduced or limited based on the determination and validation of new correction factors by experiment. In one embodiment, the accuracy of the glucometer is in the range of about 85% to about 95%.

[0422] In one embodiment, the glucometer allows the user to save the most recent test value and calculate the average glucose value over a certain period (e.g., at least two weeks), thereby enabling observation over time.

[0423] In certain embodiments, the glucose meter is "display-less" (i.e., does not include a display) to minimize the complexity and cost of the meter unit. In this embodiment, the glucose meter turns on wirelessly and transmits results or readings to a second device, such as a mobile phone or personal computer.

[0424] Optionally, the glucometer also includes a transmitter configured to wirelessly transmit data relating to the analysis results encoded in an audio signal, and a controller configured to facilitate such encoding.

[0425] This specification also discloses a glucometer and a remote computing device that may be used in the system and method herein, which is a remote computing device. In one embodiment, the remote computing device may be any other suitable device, such as a smartphone or a communication device, and may constitute an output device.

[0426] The glucometer transmits the measurement values ​​to a remote computing device, for example, via a wireless voice-based channel, through a transmitting unit.

[0427] The remote computing device may also communicate information to other remote devices, such as a central repository device, and to recipient lists via a network, such as the internet or mobile-based devices. For example, a detection device may transmit medical data through the remote computing device. This data can then be communicated to a remote caregiver, for example, via a computer or a handheld device such as a smartphone.

[0428] In this embodiment, a software algorithm is disclosed which triggers an electrochemical reaction in a detection system such that one or more detectable chemical species are the reaction product of a biological sample, a test strip, and a detection device.

[0429] In one embodiment, mathematical calculations are performed on the detection device using a localized computing algorithm to cause a chemical reaction to take place between the biological sample, the test strip, and the detection device, yielding a detectable reaction product.

[0430] In one embodiment, mathematical calculations are performed using cloud computing on a server located physically outside the detection device's location, and a chemical reaction proceeds between the biological sample, the test strip, and the detection device, yielding a detectable reaction product.

[0431] In one embodiment, a data card containing additional algorithms that are not initially programmed into the detection device is inserted into the data card slot of the detection device to generate a chemical reaction that gives a detectable reaction product that proceeds between the biological sample, the test strip, and the detection device.

[0432] In one embodiment, a non-temporary computer-readable storage medium is disclosed on which executable instructions to be executed by a processor to detect a target analyte are encoded.

[0433] Figure 16 shows an example of a system for implementing the technology described herein. The system includes a detection device 150 (e.g., a glucometer) into which a test strip 135 is inserted (130). Data output from the detection device 150 may be input into a program, which may be stored in a database 185.

[0434] The computer device 170 and the server 180 may be connected by a network 160, which may be connected to various other devices, servers, or network equipment for implementing the disclosure. The computer device 170 may be connected to a display 175. The computer device 170 may be any suitable computer device, including a desktop computer, a server (including a remote server), a mobile device, or other suitable computer device. In some examples, the algorithms and other software described herein may be stored in a database 185 and executed on the server 180. In addition, mass spectrometer data (e.g., mass spectra) and data processed or generated by the algorithm or program (e.g., processed profiles, scores, output tables, etc.) may be stored in the database 185.

[0435] First, it should be understood that the technologies described herein may be implemented using any type of hardware and / or software, and may also be pre-programmed general-purpose computing devices. For example, a system may be implemented using a server, a personal computer, a portable computer, a thin client, or any suitable one or more devices. The technologies and / or components described herein may be a single device in one location, or multiple devices in multiple locations, connected to each other by any communication medium such as electrical cables, fiber optic cables, or wirelessly using any suitable communication protocol.

[0436] It should also be understood that the technologies described herein are shown and discussed as having multiple modules that perform a particular function. It should be understood that these modules are shown schematically based on their function for clarity only and do not necessarily represent specific hardware or software. In this regard, these modules may be hardware and / or software implemented to substantially perform the particular function discussed. Furthermore, within the scope of the technologies described herein, modules may be combined with each other or divided into further modules based on a particular desired function. Therefore, the technologies described herein should not be interpreted as limiting the technologies disclosed herein, but rather as merely illustrating one example of such implementation.

[0437] A computer system may include clients and servers. Clients and servers are generally geographically separated from each other and typically communicate through a communication network. The relationship between a client and a server arises from computer programs running on each computer that have a client-server relationship with each other. In some implementations, the server sends data (e.g., an HTML page) to a client device (for example, for the purpose of displaying data to a user interacting with the client device and receiving user input from that user). Data generated by the client device and received from that client device (e.g., the results of user interactions) may be received by the server.

[0438] Implementations of the subject matter described herein may be implemented in a computer system that includes, for example, a backend component as a data server, or a middleware component, for example, an application server, or a frontend component, for example, a client computer having a graphical user interface or a web browser through which a user can interact with implementations of the subject matter described herein, or any combination of one or more such backend, middleware, or frontend components. The components of the system may be connected to one another by any form or medium of digital data communication, for example, a communication network. Examples of communication networks include local area networks ("LANs") and wide area networks ("WANs"), internetworks (for example, the Internet), and peer-to-peer networks (for example, ad-hoc peer-to-peer networks).

[0439] The subjects and operations described herein may be implemented in digital electrical circuits or in computer software, firmware, or hardware, including the structures disclosed herein and their structural equivalents, or one or more combinations thereof. The subjects described herein may be implemented as one or more computer programs, i.e., as computer program instructions for one or more modules to be executed by or control the operation of a data processing device, encoded on a computer storage medium. Alternatively, or in addition, program instructions may be encoded on a propagating signal, such as a machine-generated electrical, optical, or electromagnetic signal, which is artificially generated to encode information and transmit it to a suitable receiving device for execution by a data processing device. The computer storage medium may be, or may be, a computer-readable storage device, a computer-readable storage board, a random or serial access memory array or device, or one or more combinations thereof. Furthermore, although the computer storage medium is not a propagating signal, it may be a source or destination for computer program instructions encoded within an artificially generated propagating signal. Computer storage media may also be, or be contained within, one or more distinct physical components or media (for example, CDs, disks, or other storage devices).

[0440] The operations described herein may be performed as operations carried out by a “data processing device” with respect to data stored in one or more computer-readable storage devices or received from other sources.

[0441] The term "data processing device" includes, for example, any device, machine, or apparatus for processing data, including programmable processors, computers, systems on a chip, or a combination of these. A device may include special-purpose logic circuits, such as FPGAs (Field-Programmable Gate Arrays) or ASICs (Application-Specific Integrated Circuits). In addition to hardware, a device may also include code that constitutes the execution environment for the computer program, such as processor firmware, protocol stacks, database management systems, operating systems, cross-platform runtime environments, virtual machines, or a combination of these. Devices and execution environments can provide infrastructure for a wide variety of computing models, including various web services, distributed computing, and grid computing infrastructure.

[0442] A computer program (also known as a program, software, software application, script, or code) can be written in any form of programming language, including compiled or interpreted languages, declarative or procedural languages, and can be deployed in any form, for example, as a standalone program, or as a module, component, subroutine, object, or other unit suitable for use in a computing environment. A computer program may, though not required, correspond to a file in a file system. A program may be stored as part of a file containing other programs or data (for example, one or more scripts stored in a markup language document), in a single file dedicated to that program, or in multiple coordinate files (for example, a file storing one or more modules, subprograms, or code sections). A computer program can be deployed and executed on one computer, or on multiple computers located at one site, or on multiple sites connected to each other by a communication network.

[0443] The processing and logic flows described herein may be implemented by one or more programmable processors that execute one or more computer programs that perform operations by arithmetic operations on input data and generating outputs. The processing and logic flows may also be implemented by special-purpose logic circuits, such as the FPGAs or ASICs described above, and the device may be implemented as such circuits.

[0444] Processors suitable for executing computer programs include, for example, both general-purpose and specialized microprocessors, as well as any one or more processors in any type of digital computer. Generally, a processor receives instructions and data from read-only memory, random-access memory, or both. Essential elements of a computer are a processor that performs actions according to instructions, and one or more memory devices that store instructions and data. Generally, a computer has one or more mass storage devices for data storage, including, or functionally coupled with, magnetic disks, magneto-optical disks, or optical disks to receive, transmit, or receive data. However, a computer does not necessarily have such devices. Furthermore, a computer may be built into another device, for example, a mobile phone, a personal digital assistant (PDA), a mobile voice or video player, a game console, a Global Positioning System (GPS) receiver, or a portable storage device (for example, a Universal Serial Bus (USB) flash drive). Suitable devices for storing computer program instructions and data include all forms of non-volatile or non-temporary memory, media, and memory devices, examples of which include semiconductor memory devices such as EPROM, EEPROM, and flash memory devices; magnetic disks such as internal hard disks or removable disks; magneto-optical disks; and CD-ROM and DVD-ROM disks. Processors and memory may be supplemented with or incorporated into special-purpose logic circuits.

[0445] Inspection strips used in the systems and methods disclosed herein are also disclosed.

[0446] In one embodiment, the test strip is a disposable electrochemical test strip that is brought into contact with a small amount of biological sample. The test strip, together with a test meter, generates an electric current proportional to the glucose concentration in the biological sample, and this concentration correlates with the concentration of the analyte or virus.

[0447] The test strip includes an insertion portion and an exposed portion. The exposed portion of the test strip is designed to receive a biological sample from the subject (e.g., saliva).

[0448] In one embodiment, the inspection strip includes a substrate comprising at least one inspection area, two or more electrodes (e.g., an working electrode and a reference electrode), and means for bringing these electrodes into contact with a meter.

[0449] The substrate can be any suitable substrate, such as plastic, ceramic, metal, or polymer material (e.g., hydrogel).

[0450] 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., beads or microtiter plate), polyvinylidene fluoride, diazotized paper, nylon membrane, activated beads, or magnetically responsive beads.

[0451] In one embodiment, the substrate is an anionic polymer such as a nitrocellulose membrane. In another embodiment, the substrate is sulfonated tetrafluoroethylene, poly(acrylic acid), or poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (polyAMPS).

[0452] The first binder may be immobilized on a test strip to provide a test site. The binder may be an aptamer, antibody, receptor ligand, or molecular imprinting polymer as discussed herein. In certain embodiments, the first binder (e.g., an aptamer) is immobilized directly on an electrode (e.g., a screen-printed electrode).

[0453] In another embodiment, the first binder may, but not limited to, be immobilized on a membrane such as nitrocellulose in a test strip and inserted on an electrode to provide a test site. The binder may be an aptamer, antibody, receptor ligand, protein, or molecular imprinting polymer as used herein.

[0454] In another embodiment, the first binder may, but is not limited to, be immobilized on a membrane such as nitrocellulose in a test strip and inserted between two electrodes to provide a test site. The binder may be an aptamer, antibody, receptor ligand, protein, or molecular imprinting polymer as used herein.

[0455] Adding a second binder linked to an oxidase (such as glucose oxidase, though not limited to this) allows it to bind to the virus or target analyte captured by the first binder.

[0456] A current is generated by the selective oxidation of glucose, which is catalyzed by two pre-coated reagents within the test strip: (1) an enzyme and (2) a mediator molecule. The enzyme reacts directly with glucose molecules to produce hydrogen peroxide, and the mediator molecule reacts with electrons to transfer them to the working electrode.

[0457] The enzyme may be, for example, glucose oxidase, PQQ-glucose dehydrogenase, NAD-glucose dehydrogenase, or FAD-glucose dehydrogenase.

[0458] The mediator molecule may be, for example, ferricyanide; hexacyanoferrate III / hexacyanoferrate II; 1,10-phenanthroline quinone; quinone imine / phenylenediamine; or an osmium-based mediator.

[0459] When a voltage is applied between two electrodes, free electrons can move within the circuit. Each enzyme and mediator molecule can repeat this movement multiple times as needed. The amount of charge moving within the circuit represents the glucose level in the system, which reflects the concentration of the analyte in the sample.

[0460] In a particular embodiment, glucose oxidase is used as the enzyme, and the resulting electrochemical reaction is given by the following equation I: Formula I: Glucose + O2D-glucono-1,5-lactone + H2O2 This is shown.

[0461] This oxidation reaction generates an electric current. The amplitude of this current is directly related to the blood glucose concentration. Figure 3 is a schematic diagram of the sequence of events occurring in the glucose biosensor system. Glucose oxidation by GOx produces D-glucono-δ-lactone. The reduction of H2O2 in the Prussian blue (PB) film is measured by electrons transferred from the working electrode.

[0462] In one embodiment, the reagents used in the test strip are storage-stable. In a particular embodiment, the reagents used with the test strip are freeze-dried to extend the shelf life of the test strip.

[0463] The test strip typically contains layers of conductive and non-conductive components that overlap each other to form a sensor structure.

[0464] In one embodiment, the test strip comprises 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 binder (e.g., an aptamer) for capturing a target analyte (e.g., an antigen) is attached; a lyophilized, detectably labeled second binder (e.g., Ab-GOx) and glucose; and a top layer.

[0465] In another embodiment, the test strip comprises a base substrate; a conductive layer; an insulating layer; a reagent layer; an adhesive layer; and a hydrophilic (e.g., nitrocellulose) membrane to which a first binder (e.g., an aptamer) for capturing a target analyte (e.g., an antigen) is attached, and lyophilized glucose and / or labeled second binder (e.g., Ab-GOx) is added to a biological sample containing the target analyte.

[0466] The base substrate forms a matrix of multiple constituent elements that stack on top of each other to constitute a functional sensor. These base constituent elements can be made from a variety of materials having desirable properties such as dielectric properties, water impermeability, air impermeability, and sealing properties. Some materials include metallic and / or ceramic and / or polymer substrates.

[0467] The conductive layer is mounted on a base substrate and includes at least one electrode (e.g., one, two, or three electrodes) containing a conductive material that comes into contact with the analyte being assayed or its by-products (e.g., oxygen and / or hydrogen peroxide). The one or more electrodes may include one or more working electrodes, and one or more counter electrodes, reference electrodes, and / or a pair / reference electrode.

[0468] The electrodes may be screen-printed electrodes, for example, screen-printed using conductive carbon ink. A variety of materials may be used. Convenient conductive ink compositions for the glucose sensor systems described herein include, but are not limited to, silver, carbon, or blended conductive inks. Convenient inks for printing the working electrodes include, but are not limited to, carbon, platinum, carbon / platinum, carbon nanotubes, or other conductive materials suitable for detecting peroxides in the sample.

[0469] The electrodes used and the required sensitivity generally determine the enzyme chemistry that can be employed. For example, a second binder linked to glucose oxidase requires excess glucose to detect the analyte in the sample.

[0470] The "working electrode" is the electrode on which the analyte is electrooxidized or electroreduced, with or without the mediation of a redox mediator. The working electrode can measure an increase or decrease in current in response to exposure to stimuli, such as changes in the concentration of the target analyte or molecule or its byproducts. The electrode provides a detectable signal in the presence of variable concentrations of molecules such as hydrogen peroxide or oxygen.

[0471] In addition to the working electrode, the conductive layer may also include a reference electrode (RE) or a combined reference / counter electrode (also called a pseudo-reference electrode or pair / reference electrode).

[0472] In one embodiment, the electrode has a minimum sensitivity of at least about 50 micromoles of glucose concentration and about 10 nA / mm². 2 It provides a noise level below a certain level.

[0473] In one embodiment, the insulating layer may be a thin film of an insulating (e.g., electrically insulating or water-impermeable) material such as poly(vinyl chloride), polyethylene, polypropylene, aromatic and aliphatic polyurethenes, aromatic and aliphatic polyurethanes, poly(butylene terephthalate), polybutadiene, silicone rubber, thiol-en copolymer, or poly(ethylene-co-vinyl acetate).

[0474] In one embodiment, the reagent layer includes a mediator to facilitate electron exchange. In one embodiment, the reagent layer includes a binder; silica; ferricyanide; ferricyanide; 1,10-phenanthroline quinone; or an osmium-based mediator.

[0475] In one embodiment, the adhesive layer may be an acrylic copolymer such as poly(ethyl acrylate), poly(cyanoacrylate), poly(butyl acrylate), poly(2-ethylhexyl acrylate), or urethane acrylate copolymer.

[0476] In one embodiment, the hydrophilic membrane may be composed of an anionic hydrophilic copolymer such as nitrocellulose, sulfonated tetrafluoroethylene, poly(acrylic acid), or poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (polyAMPS). The membrane may be coated with streptavidin-NC and a first binder (e.g., a biotinylated aptamer), the latter adhering to the membrane and acting as a capture agent for the target analyte or molecule. Streptavidin-NC is streptavidin modified to bind to nitrocellulose.

[0477] In a particular embodiment, the test strip comprises (A) a base substrate; (B) a conductive layer containing three electrodes; (C) an insulating layer exposing only the portion of the electrode onto which the sample to be tested is dropped; (D) a reagent layer containing a mediator to facilitate electron exchange; (E) an adhesive layer; (F) a hydrophilic nitrocellulose membrane, the proximal membrane containing a first binder (e.g., an aptamer) and lyophilized glucose for capturing the target analyte (e.g., an antigen), and the distal end being a paper sink (13); (G) lyophilized Ab-GOx; and (H) a top layer. See, for example, Figure 5, and the labels (A)-(H) and (1)-(16) in the same figure.

[0478] In a particular embodiment, the test strip comprises (A) a base substrate; (B) a conductive layer containing two electrodes; (C) an insulating layer exposing only the portion of the electrode onto which the sample to be tested is dropped; (D) a reagent layer containing a mediator to facilitate electron exchange; (E) an adhesive layer; (F) a hydrophilic nitrocellulose membrane, the proximal membrane containing a first binder (e.g., an aptamer) and lyophilized glucose for capturing a target analyte (e.g., an antigen), and the distal end being a paper sink (13); (G) lyophilized Ab-GOx; and (H) an upper layer.

[0479] In this embodiment, the base substrate is polyester and is coated with an acrylic coating to improve ink adhesion. The electrode mask is laser-processed onto the base substrate using a CAD (computer-aided design) model of the electrode mask. The electrodes are then screen-printed using conductive carbon ink (ERCON INC), followed by an insulating layer (ERCON INC, INSULAYER INK). Each of the two working electrodes is 0.6 mm thick. 2 It may have a surface area of ​​1.2 mm, and the reference electrode is 1.2 mm 2It may have a surface area of ​​the above. In one embodiment, the reagent layer is a mediator layer consisting of a binder, silica, and ferricyanide. This reagent layer is screen-printed onto the working electrode in two cycles. In one embodiment, the adhesive layer above it is an acrylic copolymer, and the hydrophilic membrane is a nitrocellulose membrane having streptavidin-NC, with a biotinylated aptamer (e.g., to streptavidin) bound to capture the antigen (e.g., viral antigen). In one embodiment, the top layer is PET (polyethylene terephthalate) with a small transparent section for observing the movement of the sample on the strip. The overall dimensions are similar to those described for other test strips to ensure compatibility with glucometers such as LIFESCAN readers, and the test strip can also be modified to be compatible with other commercial glucometers.

[0480] In one embodiment, dropcast GOx is directly dropcast to the working electrode before the addition of freeze-dried biological reagents and aptamer immobilization.

[0481] In certain embodiments, the test strip is pre-blocked with any suitable blocking agent to reduce or eliminate nonspecific binding. Non-limiting examples of coating or blocking materials include proteins, acrylamides, synthetic polymers, or polysaccharides. In one embodiment, BSA is used as the blocking agent. In another embodiment, milk protein, TWEEN, or other surfactants are used as the blocking agent.

[0482] In certain embodiments, the system limits transient non-glucose-related signal noise, for example, enabling a low signal-to-noise ratio. The composition of the base layer, the method used for electrode deposition, the electrode configuration, the electrode material, the enzyme chemistry used, and other design factors all contribute to the system's noise.

[0483] In one embodiment, the strip has a shelf life of more than one year, or more than two years, or more than three years.

[0484] In some embodiments, analyte detection is performed at temperatures between 5°C and 30°C. In one embodiment, analyte detection is performed at temperatures between 17°C and 25°C. In one embodiment, analyte detection is performed at temperatures of at least 5°C, at least 6°C, at least 7°C, at least 8°C, at least 9°C, at least 10°C, at least 11°C, at least 12°C, at least 13°C, at least 14°C, at least 15°C, at least 16°C, at least 17°C, at least 18°C, at least 19°C, at least 20°C, at least 21°C, at least 22°C, at least 23°C, at least 24°C, at least 25°C, at least 26°C, at least 27°C, at least 28°C, at least 29°C, or at least 30°C or higher.

[0485] In another context, this specification describes a test strip linked to a detection nucleic acid using any linking method known in the art (e.g., UV crosslinking, or using an affinity pair in which one member is linked to the detection nucleic acid and the second member is linked to the test strip (e.g., streptavidin and biotin)). acid The nucleic acid may be ligated to the test strip at its 3' or 5' end. In some aspects of any aspect, the detection nucleic acid is ligated to a hydrophilic nitrocellulose membrane (e.g., layer F in Figure 5). In some aspects of any aspect, the detection nucleic acid is ligated to the proximal membrane of the test strip (e.g., layer F in Figure 5). In some aspects of any aspect, the sequence-specific endonuclease, guide nucleic acid, and / or collateral nucleic acid are present in the test strip but are not necessarily ligated to the test strip (e.g., layer G in Figure 5; e.g., as lyophilized reagents).

[0486] This specification also discloses a method for detecting, optionally over time, the presence of at least one target analyte, more specifically, at least one target analyte, such as a pathogen or its components (e.g., a viral antigen), using the system described herein.

[0487] In one embodiment, the method includes the steps of: (i) preparing a biological sample derived from a subject, wherein the biological sample is not blood; (ii) adding the biological sample to a test strip, wherein the test strip comprises first and second binders capable of forming a detectable complex with at least one target analyte if present in the biological sample; (iii) inserting the test strip into a glucometer; (iv) incubating the biological sample with the test strip; (v) detecting the level of any detectable complex in the form of hydrogen peroxide produced from the glucose oxidation of any excess glucose present; and (vi) correlating the level of any detectable complex formed with the amount of any target analyte present in the at least one biological sample, thereby providing a diagnostic assessment.

[0488] In one embodiment, the method includes the steps of: (i) collecting a biological sample from a subject, the biological sample being urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, nasal cavity sample, cerebrospinal fluid, pleural fluid, or nasopharyngeal specimen; (ii) adding the biological sample to a test strip, the test strip comprising first and second binders capable of forming a detectable complex with at least one target analyte if present in the biological sample; (iii) inserting the test strip into a glucometer; (iv) incubating the biological sample with the test strip; (v) detecting the level of the detectable complex, if present, in the form of hydrogen peroxide produced from the glucose oxidation of any excess glucose present; and (vi) correlating the level of the detectable complex, if formed, with the amount of the target analyte, if present in the at least one biological sample, thereby providing a diagnostic assessment.

[0489] In one embodiment, the method includes the steps of: (i) collecting a biological sample from a subject into a tube containing a second binder and a 1- to 1,000,000,000-fold dilution of the biological sample, wherein the biological sample is not blood; (ii) adding the biological sample to a test strip, wherein the test strip contains a first binder capable of forming a detectable complex with at least one target analyte if present in the biological sample; (iii) inserting the test strip into a glucometer; (iv) incubating the biological sample with the test strip; (v) detecting the level of any detectable complex in the form of hydrogen peroxide produced from the glucose oxidation of any excess glucose present; and (vi) correlating the level of any detectable complex formed with the amount of any target analyte present in the at least one biological sample, thereby providing a diagnostic assessment.

[0490] In one embodiment, the method comprises: (i) collecting a biological sample from a subject into a tube containing a second binder and diluted 1 to 1,000,000,000 times, wherein the biological sample is urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, or a nasopharyngeal specimen; and (ii) adding the biological sample to a test strip, wherein the test strip forms a detectable complex with at least one target analyte present in the biological sample. The present invention includes the steps of: (iii) inserting the test strip into a glucometer; (iv) incubating the biological sample with the test strip; (v) detecting the level of any detectable complex in the form of hydrogen peroxide produced from the glucose oxidation of any excess glucose present; and (vi) correlating the level of any detectable complex produced with the amount of any target analyte present in the at least one biological sample, thereby providing a diagnostic assessment.

[0491] In one embodiment, the biological sample is diluted, for example, by a diluent in a sample collection tube to at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, and at least 10-fold. 2 double, at least 10 3 double, at least 10 4 double, at least 10 5 double, at least 10 6 double, at least 10 7 double, at least 10 8 double, or at least 10 8 The sample is diluted by a factor of 10 or more. In one embodiment, a biological sample (e.g., saliva) is diluted at least 10 times by a diluent in a sample collection tube, for example. In one embodiment, the diluent is DMEM, PBS, PBST, or another buffer, or a cell medium. Dilution may be performed to reduce nonspecific interactions from various proteins present in the biological sample (e.g., saliva).

[0492] In some embodiments, the method further includes a washing step to remove any excess reagent, such as excess binders (e.g., first and second binders; e.g., glucose oxidase linked to an aptamer, or glucose oxidase linked to an antibody). In some embodiments, the washing step is performed before inserting the test strip into the glucometer. In some embodiments, the washing step may be performed using a diluent described herein (e.g., DMEM, PBS, PBST, or another buffer, or cell medium) or a blocking agent (e.g., BSA, milk protein, TWEEN, or other surfactant). In some embodiments, the method does not include a washing step.

[0493] In one embodiment, the method includes a step of obtaining multiple test results from the same user taken at different times and comparing them to observe or predict the possible progression of a disease or condition (e.g., COVID-19). In a particular embodiment, the method includes a step of obtaining at least two, at least three, at least four, or at least five or more test results.

[0494] In certain embodiments, one or more results of the method may be communicated to a remote entity continuously or periodically to determine whether the one or more results exceed a threshold level or cut point.

[0495] In certain embodiments, results may be compared to a predetermined reference level. The predetermined level may be obtained from the general population or from a selection population. For example, the selection population may consist of patients who appear healthy, such as those who have never had any signs or symptoms indicating the presence of a disease, such as an infectious disease. The “predetermined reference level” may be determined, for example, by determining the expression level of the target analyte in corresponding biological samples obtained from one or more control subjects (e.g., those who have not had an infectious disease or are not known to be susceptible to such a disease). When such a predetermined reference level is used, a higher or increased level determined in the biological sample (i.e., the test sample obtained from the subject) compared to the predetermined reference level indicates, for example, that the patient is at risk of developing a disease (e.g., COVID-19 infection) or has such a disease.

[0496] In some embodiments, the method may further include the step of recommending a treatment instruction and / or administering a treatment. In one embodiment, the method includes the step of identifying that a subject has a level of target analyte above a threshold or cutoff level, and thus determining that the subject is a candidate for the prevention and / or treatment of, for example, an infection or disease. The “determining” step includes detecting and quantifying, where “detecting” means determining whether or not the target analyte is present in the biological sample, and “quantifying” means determining the amount of the target analyte present in the biological sample.

[0497] The method of the present invention may have therapeutic applications, for example, it may be used to detect various pathological conditions or to observe the disease stage or treatment response of a target.

[0498] In certain embodiments, the method may further include a step that uses statistical methods to predict the likelihood of detecting a target analyte that causes a disease or disease progression, and / or to predict the prognosis of the disease (i.e., the course of the disease).

[0499] In certain embodiments, the method may be performed on a single patient population, for example, to stratify the treatment approach for that population or to satisfy public health or other observational purposes.

[0500] In one embodiment, a method is disclosed for observing the effectiveness of a treatment regimen in a subject suffering from a disease, comprising using the methods and / or systems disclosed herein, wherein the target molecule is an antigen associated with the disease, and the amount of the detectable portion indicates the level of the disease and therefore indicates the effectiveness of the treatment regimen in the subject.

[0501] In certain embodiments, the method involves observing the effects of one or more therapeutic agents (e.g., antiviral agents) over a period of time (e.g., several days, several weeks), and if the therapeutic agent is not sufficiently effective over that period, allowing the user to explore alternative therapeutic approaches.

[0502] In one embodiment, if a treatment regimen does not result in a decrease in viral count within a predetermined period (e.g., several days), the user may discontinue the treatment regimen and choose an alternative treatment regimen, or, in certain embodiments, supplement the treatment regimen with a second treatment regimen. In one embodiment, the system enables the observation of trends in analyte levels over time by enabling the acquisition of two or more results, three or more results, or five or more results for the amount of target analyte of the same user at different times.

[0503] The therapeutic agent can vary. In one embodiment, the therapeutic agent is an antiviral agent, such as a small molecule or biological antiviral agent. In a specific embodiment, the therapeutic agent is an anti-SARS-CoV-2 agent or an anti-influenza agent.

[0504] In another aspect, this specification describes a method for detecting a target nucleic acid using a nucleic acid detection system described herein, the method comprising: (i) collecting a biological sample from a subject and optionally extracting nucleic acids from the biological sample; (ii) contacting the biological sample with a sequence-specific endonuclease, a guide nucleic acid, and a collateral nucleic acid, wherein, if the target nucleic acid is present, the collateral nucleic acid is cleaved as a result of such contact; (iii) adding the biological sample to a test strip in the presence of glucose, wherein the test strip reacts with the cleaved collateral nucleic acid The method includes the steps of: (iv) incubating the biological sample with the test strip, or not incubating it; (v) inserting the test strip into a detection device; (vi) detecting the level of any detectable complex through a chemical reaction between glucose and glucose oxidase; and (vii) correlating the level of any detectable complex formed with the amount of a target analyte in the at least one biological sample, thereby providing a diagnostic assessment.

[0505] In some aspects of any given situation, the biological sample is saliva. In some aspects of any given situation, the biological sample is not blood. In some aspects of any given situation, the detection device is a glucose meter.

[0506] In some aspects of any aspect, the method further includes (viii) transmitting the diagnostic assessment or results to an electronic device, database, or cloud server for subsequent review by a clinician or trained healthcare provider; and (ix) providing the diagnostic assessment to the individual who performed the diagnostic assessment method. In some aspects of any aspect, the individual is the subject. In some aspects of any aspect, the method further includes (viii) recommending, directing, and / or administering one or more treatment regimens to the subject in accordance with the diagnostic assessment.

[0507] Methods for manufacturing the systems and test strips disclosed herein are also disclosed.

[0508] The inspection strips may be manufactured using any preferred method. In one embodiment, the inspection strips are manufactured using a roll-to-roll method, a screen printing method, a drop casting method, or a combination thereof. Exemplary manufacturing methods are provided in the Examples section.

[0509] The test strip compositions disclosed herein may be combined with other components or reagents, or may be provided as components of a kit or other commercially available or retail product. The kit may also include instructions or informational materials relating to the administration and / or use of the kit. The kit may also include a reader or detection device described herein.

[0510] In one embodiment, the kit comprises a glucose meter, at least one test strip as described herein, and a container or pouch for storing the at least one test strip during transport. In some embodiments, the kit comprises a test strip comprising first and second binders (e.g., one aptamer and one antibody, or two antibodies, or two aptamers) capable of forming a detectable complex with at least one target analyte. In some embodiments, the kit comprises a test strip comprising a detection nucleic acid (e.g., linked to the test strip), the test strip of the kit may further comprise a sequence-specific endonuclease, a guide nucleic acid, and / or a collateral nucleic acid, or the sequence-specific endonuclease, guide nucleic acid, and / or collateral nucleic acid may be provided separately in the kit from the test strip.

[0511] In some embodiments, the kit contains an effective amount of glucose. In some embodiments, the kit contains an effective amount of sequence-specific endonuclease, guide nucleic acid, and collateral nucleic acid, and the test strip contains the detection nucleic acid.

[0512] In some embodiments, the components described herein may be provided individually or in any combination as a kit. Such a kit may optionally include one or more agents that enable the detection of the detectable complex described herein.

[0513] In some embodiments, the components within the kit may be supplied in watertight or airtight containers that, in some embodiments, substantially contain less of the other components of the kit. For example, the test strips may be supplied in two or more containers, for example, in containers containing a predetermined number of reagents sufficient for one, two, three, or more detection reactions. One or more components described herein may be supplied in any form, for example, in liquid, dry, or lyophilized form. The components described herein are preferably substantially pure and / or sterile. If the components described herein are supplied in a solution, the solution is preferably an aqueous solution, and preferably a sterile aqueous solution.

[0514] Informational materials may be explanatory, instructional, marketing, or other materials relating to the methods described herein. The form of the kit's informational materials is not limited. In one embodiment, the informational materials may include information about the manufacture, concentration, expiration date, batch, or place of manufacture of the test strips. In one embodiment, the informational materials relate to methods of using or administering the components of the kit.

[0515] The kit may include components for detecting the target analyte. In addition, the kit may include the target analyte Combine It may contain one or more aptamers or antibodies. The aptamers or antibodies may be supplied, for example, lyophilized and in a dry preparation, or in a solution. The antibody or other detection reagent may be coupled with a label used for detection, such as a radioactive, fluorescent (e.g., GFP), or chromogenic label.

[0516] The kit is typically supplied with its various components contained in a single package, for example, a fiber-based box, such as a corrugated cardboard box, or a polymer box, such as a styrofoam box. This enclosure may be configured to maintain a temperature difference between the inside and outside, for example, by providing insulating properties to keep reagents at a predetermined temperature for a predetermined period of time.

[0517] All patents and other publications cited herein, including literature, granted patents, patent application gazettes, and concurrently pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, various methods that may be used in connection with the technology described herein, as described in such publications. These publications are provided simply because their disclosures predate the filing date of this application. This should not be interpreted in any way as the inventors acknowledging that such disclosures do not qualify as prior art or for any other reason. All statements regarding dates or expressions concerning the contents of these documents are based on information available to the applicants and do not constitute any endorsement of the accuracy of the dates or contents of these documents.

[0518] The descriptions of the embodiments of this disclosure are not intended to be exclusive, nor to limit the disclosure to the embodiments themselves. Specific embodiments and examples of the disclosure are described herein for illustrative purposes; however, as will be apparent to those skilled in the art, various equivalent modifications are possible within the scope of this disclosure. For example, while the steps or functions of a method are presented in a given order, alternative embodiments may perform the functions in a different order, or even substantially simultaneously. The teachings of the disclosure provided herein may be applied to other procedures or methods as appropriate. Further embodiments may be provided by combining the various embodiments described herein. Where necessary, aspects of this disclosure may be modified to use the compositions, functions, and concepts of the above-mentioned references or applications to provide further embodiments of this disclosure. Furthermore, due to biological functional equivalence, several modifications may be made to the type or amount of protein structure without affecting biological or chemical activity. These and other modifications may be made in view of the detailed description. All such modifications shall be included within the scope of the appended claims.

[0519] Certain elements of any of the above embodiments may be combined with or replaced with elements of other embodiments. Furthermore, while the advantages of certain embodiments of this disclosure have been described in the context of those embodiments, and other embodiments may also exhibit such advantages, not all embodiments are necessarily required to exhibit such advantages in order to be included in the scope of this disclosure.

[0520] The technologies described herein are further illustrated by the following examples, but these should not be construed as further limitations.

[0521] Some aspects of the technology described herein may be defined according to any of the following numbered paragraphs. 1. A system for detecting at least one target analyte in a biological sample, (i) A two-binding assay comprising first and second binding agents capable of forming a detectable complex with at least one target analyte; and (ii) A detection device for detecting the detectable complex Includes, The detection device is an oxidase-based amperometric sensor, and the biological sample is present in sweat, saliva, serum, mucus, or blood. system. 2. The amperometric sensor is an oxidase-based amperometric sensor, as in the system of paragraph 1. 3. The amperometric sensor is a hydrogenase or dehydrogenase-based amperometric system as described in paragraph 2. 4. The biological sample is any of the systems described in the preceding paragraph, such as sweat, saliva, serum, mucus, or blood. 5. The biological sample is saliva, one of the systems described in the preceding paragraph. 6. The detection device is a glucose meter, as per any of the systems in the preceding paragraphs. 7. The glucose meter system of paragraph 6 includes a glucose sensor having a sensor output relating to glucose in a biological sample on a test strip. 8. The biological sample is mixed with a sugar such as glucose, or any of the systems described in the preceding paragraph, but is not limited to those described above. 9. The biological sample is mixed with a sugar, such as glucose, at a concentration of approximately 0.01 mM to approximately 1 M, in the system of paragraph 8. 10. The first and second binders are selected from the group consisting of aptamers, antibodies, proteins, or combinations thereof, according to any of the systems in the preceding paragraph. 11. Any system of the preceding paragraph, wherein the first binder is an aptamer and the second binder is an antibody, the antibody being linked to glucose oxidase. 12. The first and second binders are any of the systems described in the preceding paragraph, which bind to different sites on the target analyte. 13. The system of paragraph 12, wherein the first and second binders have a Kd ratio of about 1:1000 to about 1000:1 with respect to the target analyte. 14. The system of paragraph 12 or 13, wherein the binding affinity of the first binder is weaker than the binding affinity of the second binder. 15. The target analyte is any system of the preceding paragraph, which is the whole virus or a component thereof. 16. The virus is a beta-coronavirus, influenza virus, HIV virus, or hepatitis virus, according to the system of paragraph 15. 17. The target coronavirus analyte is SARS-CoV-2 or a system of its components as described in paragraph 16. 18. The system of paragraph 17, wherein the target analyte is a component of SARS-CoV-2 selected from the group consisting of spike proteins, membrane proteins, hemagglutinin proteins, or envelope proteins. 19. The target analyte is selected from the group consisting of IgG, IgM, and IgA, according to any system from paragraphs 1 to 14. 20. A system for detecting at least one virus in a biological sample, (i) A two-conjugate assay comprising first and second conjugates capable of forming a detectable complex with at least one virus; and (ii) A detection device for detecting the detectable complex Includes, The detection device is a glucose meter, in this system. 21. The glucose meter system of paragraph 20 includes a glucose sensor having a sensor output relating to glucose in a biological sample on a test strip. 22. The biological sample is mixed with a sugar such as glucose, but is not limited to, the system of paragraph 20. 23. The biological sample is mixed with a sugar, such as glucose, at a concentration of approximately 0.01 mM to approximately 1 M, according to the system of paragraph 22. 24. The first and second binders are selected from the group consisting of aptamers, antibodies, proteins, or combinations thereof, according to any system of paragraphs 20-23. 25. Any system of paragraphs 20-24, wherein the first binder is an aptamer and the second binder is an antibody, the antibody being linked to glucose oxidase. 26. The first and second binders are systems of any of paragraphs 20 to 25, wherein they bind to different sites on the target analyte. 27. The system of paragraph 26, wherein the first and second binders have a Kd ratio of about 1:1000 to about 1000:1 with respect to the target analyte. 28. The system of paragraph 26 or 27, wherein the binding affinity of the first binder is weaker than the binding affinity of the second binder. 29. The target analyte is any system of paragraphs 20-28, which is the whole virus or a component thereof. 30. The aforementioned virus is the betacoronavirus system, as described in paragraph 29. 31. The target analyte is SARS-CoV-2 or a component thereof, as described in paragraph 30. 32. The system of paragraph 31, wherein the target analyte is a component of SARS-CoV-2 selected from the group consisting of spike proteins, membrane proteins, hemagglutinin proteins, or envelope proteins. 33. The target analyte is selected from the group consisting of IgG, IgM, and IgA, according to any system described in paragraphs 20 to 28. 34. Methods for diagnostic evaluation, (i) A step of collecting a biological sample from a subject, wherein the biological sample is not blood; (ii) A step of adding the biological sample to a test strip in the presence of glucose, wherein the test strip comprises first and second binders capable of forming a detectable complex with at least one target analyte if present in the biological sample; (iii) The step of incubating the biological sample with the test strip, or not incubating it; (iv) Inserting the inspection strip into the detection device; (v) A step in which the level of any detectable complex is detected through a chemical reaction between glucose and glucose oxidase; and (vi) A step of correlating the level of any detectable complex formed with the amount of the target analyte present in the at least one biological sample, thereby providing a diagnostic assessment. Methods that include... 35. Methods for diagnostic evaluation, (i) A step of collecting a biological sample from a subject, wherein the biological sample is derived from urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, or nasopharyngeal specimens; (ii) Diluting the collected sample 1 to 100 times in an aqueous solution / mixture in the presence of a second binder; (iii) A step of adding the biological sample and a second binder to a test strip, wherein the test strip contains a first binder capable of forming a detectable complex with at least one target analyte if present in the biological sample; (iv) The step of incubating the biological sample with the test strip, or not incubating it; (v) Inserting the inspection strip into the detection device; (vi) A step in which the level of any detectable complex is detected through a chemical reaction between glucose and glucose oxidase; and (vii) A step of correlating the level of any detectable complex formed with the amount of the target analyte present in the at least one biological sample, thereby providing a diagnostic assessment. Methods that include... 36. The biological sample is saliva, according to the methods of paragraphs 34 and 35. 37. The method of paragraphs 34 and 35, wherein the detection device is a glucose meter. 38. (viii) The step of transmitting the diagnostic assessment or results to an electronic device, database, or cloud server for subsequent review by a clinician or trained healthcare provider; and (ix) The step of providing the diagnostic assessment to an individual who has undergone the diagnostic assessment method described above. The methods described in paragraphs 34 and 35, further include the methods described therein. 39. The aforementioned individual is the subject, in the manner of paragraph 38. 40. (vi, viii) The stage of recommending, instructing, and / or administering one or more treatment regimens to the subject in accordance with the diagnostic evaluation. Any of the methods described in paragraphs 34-37, including the above. 41. Methods for diagnostic evaluation, (i) A step of collecting a biological sample from a subject, wherein the biological sample is derived from urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, or nasopharyngeal specimens; (ii) A step of adding the biological sample to a test strip, wherein the test strip comprises first and second binders capable of forming a detectable complex with at least one target analyte if present in the biological sample; (iii) The step of incubating the biological sample with the test strip, or not incubating it; (iv) Inserting the test strip into a detection device such as a glucometer; (v) A step in which the level of any detectable complex is detected through a chemical reaction between glucose and glucose oxidase; and (vi) A step of correlating the level of any detectable complex formed thereon with the amount of the target analyte in the at least one biological sample, thereby providing a diagnostic assessment. Methods that include... 42. Methods for diagnostic evaluation, (i) A step of collecting a biological sample from a subject, wherein the biological sample is derived from urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, or nasopharyngeal specimens; (ii) Diluting the collected sample 1 to 100 times in an aqueous solution / mixture in the presence of a second binder; (iii) A step of adding the biological sample and a second binder to a test strip, wherein the test strip contains a first binder capable of forming a detectable complex with at least one target analyte if present in the biological sample; (iv) The step of incubating the biological sample with the test strip, or not incubating it; (v) Inserting the test strip into a detection device such as a glucometer; (vi) A step in which the level of any detectable complex is detected through a chemical reaction between glucose and glucose oxidase; and (vii) A step of correlating the level of any detectable complex formed with the amount of the target analyte in the at least one biological sample, thereby providing a diagnostic assessment. Methods that include... 43. (viii) The stage of recommending, instructing, and / or administering one or more treatment regimens in accordance with the diagnostic evaluation. The methods described in paragraphs 41 and 42, further including the methods described therein. 44. Methods for diagnostic evaluation, (i) A step of collecting a biological sample from a subject, wherein the biological sample is derived from urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, or nasopharyngeal specimens; (ii) A step of adding the biological sample to a test strip, wherein the test strip comprises first and second binders capable of forming a detectable complex with at least one target analyte if present in the biological sample; (iii) The step of incubating the biological sample with the test strip; (iv) Inserting the test strip into a detection device such as a glucometer; (v) A step in which the level of any detectable complex is detected through a chemical reaction between glucose and glucose oxidase; (vi) A step of correlating the level of any detectable complex formed with the amount of the target analyte present in the at least one biological sample, thereby providing a diagnostic assessment; (vii) the step of transmitting the diagnostic assessment or results to an electronic device, database, or cloud server for subsequent review by a clinician or trained healthcare provider; and (viii) Sending the diagnostic assessment or outcome to the individual who has undergone the diagnostic assessment method. Methods that include... 45. Methods for diagnostic evaluation, (i) A step of collecting a biological sample from a subject, wherein the biological sample is derived from urine, sweat, ocular fluid including aqueous humor, blood, feces, sebum, respiratory droplets, semen, vaginal mucus, earwax, epidermal cells, or nasopharyngeal specimens; (ii) Diluting the collected sample 1 to 100 times in an aqueous solution / mixture in the presence of a second binder; (iii) A step of adding the biological sample to a test strip, wherein the test strip comprises a first binder capable of forming a detectable complex with at least one target analyte if present in the biological sample; (iv) Incubating the biological sample with the test strip; (v) Inserting the test strip into a detection device such as a glucometer; (vi) A step in which, if any detectable complexes exist, their levels are detected through a chemical reaction between glucose and glucose oxidase; (vii) A step of correlating the level of any detectable complex formed with the amount of the target analyte present in the at least one biological sample, thereby providing a diagnostic assessment; (viii) the step of transmitting the diagnostic assessment or results to an electronic device, database or cloud server for subsequent review by a clinician or trained healthcare provider; and (ix) Sending the diagnostic assessment or outcome to the individual who underwent the diagnostic assessment method described above. Methods that include... 46. ​​The person is subject to the methods of paragraphs 44 and 45. 47. (ix, x) The stage of recommending, instructing, and / or administering one or more treatment regimens to the subject in accordance with the diagnostic evaluation. The methods described in paragraphs 44 and 45, further include the methods described therein. 48. A system of any of paragraphs 1-33 or 76-98, or a method of any of paragraphs 34-47, 56-75 or 99-104, in which one or more different target analytes are evaluated simultaneously or sequentially. 49. An inspection strip used in any of the systems described in paragraphs 1 to 33 or in any of the methods described in paragraphs 34 to 47 or 56 to 75, (i) a substrate, at least one of the first and second binders, and two or more electrodes; (ii) the substrate, both the first and second binders, and two or more electrodes; (iii) at least one of the first and second binders, and two or more electrodes; or (iv) Both the first and second binders, and two or more electrodes A test strip containing at least one of the following. 50. (i) One or more planar or coplanar electrodes made of carbon, iron, palladium, platinum, or gold; (ii) Electrodes coated with iron salts such as ferrous ferrocyanide as mediators; (iii) Electrodes coated with Prussian blue as a mediator; (iv) An n-electrode setup in which the electrode in contact with the electrolyte solution is a (semi)conducting solid; (v) The electrode setup includes a working electrode, a reference electrode, and a pair or auxiliary electrode; (vi) The current lead and the sense lead are connected and the action Lead and sense of action Lead It is connected to the working electrode, and is a reference Lead and against Lead A two-electrode setup in which one is connected to a second auxiliary, paired, or pseudo / pseudo-reference electrode; (vii) The reference lead is LeadA three-electrode setup in which a third electrode is connected to the working electrode, which is separated from the working electrode and is typically positioned to measure the nearest point to the working electrode, to which both the working and working sense leads are attached; (viii) A four-electrode setup in which, in addition to the reference lead, the working sense lead is disconnected from the working electrode; and / or (ix) A non-resistivity ammeter in which the working electrode lead and the counter electrode lead are both short-circuited within the strip so that the overall voltage drop of the electrochemical cell is zero. A test strip containing at least one of the following. 51. The inspection strip of paragraph 49 or 50, wherein the first binder is immobilized on a porous polymer, metal, or ceramic film, and the film is placed on or between electrodes. 52. The inspection strip of paragraph 51, wherein the membrane is nitrocellulose. 53. A kit containing one of the test strips from paragraphs 49, 50, 51, or 52. 54. The kit of paragraph 53, including instructions for using the aforementioned test strips. 55. The kit of paragraph 53, further comprising a glucometer or other electrochemical detection device. 56. (i) The step of preparing a biological sample derived from the subject, (ii) A step of detecting the presence of a target analyte in the biological sample using the system described in the paragraph above, wherein the target analyte is the SARS-CoV-2 virus or a component thereof; and (iii) Optionally, the step of treating the subject with a therapeutic agent. Methods that include... 57. The biological sample is saliva, according to the method of paragraph 56. 58. The target analyte is the SARS-CoV-2 virus, by any of the methods in paragraphs 56-57. 59. The target analyte is a component of the SARS-CoV-2 virus, as described in any of paragraphs 56-57. 60. The component is an S protein or a fragment thereof, according to any of the methods in paragraphs 56-59. 61. (i) The step of preparing a biological sample derived from the subject, (ii) A step of detecting the presence of a target analyte in the biological sample using the system described in the paragraph above, wherein the target analyte is the CoV virus or a component thereof, and (iii) Optionally, the step of treating the subject with a therapeutic agent. Methods that include... 62. The biological sample is saliva, according to the method of paragraph 61. 63. The method of paragraph 61 or 62, wherein the target analyte is the CoV virus. 64. (i) The step of preparing a biological sample derived from the subject, (ii) A step of detecting the presence of a target analyte in the biological sample using the system described in the paragraph above, wherein the target analyte is an influenza virus or a component thereof, and (iii) Optionally, the step of treating the subject with a therapeutic agent. Methods that include... 65. The biological sample is saliva, according to the method of paragraph 64. 66. The method of paragraph 64 or 65, wherein the target analyte is influenza virus. 67. The method of paragraph 64 or 65, wherein the target analyte is a component of the influenza virus. 68. (i) The step of preparing a biological sample derived from the subject, (ii) A step of detecting the presence of a target analyte in the biological sample using the system described in the paragraph above, wherein the target analyte is the HIV virus or a component thereof, and (iii) Optionally, the step of treating the subject with a therapeutic agent. Methods that include... 69. The biological sample is saliva, according to the method of paragraph 68. 70. The method of paragraph 68 or 69, wherein the target analyte is the HIV virus. 71. The method of paragraph 68 or 69, wherein the target analyte is a component of the HIV virus. 72. (i) The step of preparing a biological sample derived from the subject, (ii) A step of detecting the presence of a target analyte in the biological sample using the system described in the paragraph above, wherein the target analyte is a hepatitis virus or a component thereof, and (iii) Optionally, the step of treating the subject with a therapeutic agent. Methods that include... 73. The biological sample is saliva, according to the method of paragraph 72. 74. The method of paragraph 72 or 73, wherein the target analyte is a hepatitis virus. 75. The method of paragraph 72 or 73, wherein the target analyte is a component of a hepatitis virus. 76. A system for detecting at least one target nucleic acid in a biological sample, (i) A sequence-specific endonuclease and a guide nucleic acid that, when the target nucleic acid specifically binds to the sequence-specific endonuclease and guide nucleic acid, cleaves the collateral nucleic acid; (ii) A detection nucleic acid that can form a detectable complex with the cleaved collateral nucleic acid; and (iii) A detection device for detecting the detectable complex Includes, The detection device is an oxidase-based amperometric sensor, and the biological sample is present in sweat, saliva, serum, mucus, or blood. system. 77. The sequence-specific endonuclease is a Cas enzyme, as described in paragraph 76. 78. The sequence-specific endonuclease is Cas12a or Cas13, according to the system of paragraph 76 or 77. 79. The guide nucleic acid is a system of any of paragraphs 76-78, wherein the guide nucleic acid is complementary or substantially complementary to at least a portion of the target nucleic acid. 80. The detection nucleic acid is complementary or substantially complementary to at least a portion of the cleaved collateral nucleic acid, according to any system of paragraphs 76-79. 81. The detected nucleic acid hybridizes with the cleaved collateral nucleic acid, according to any system of paragraphs 76-80. 82. The detected nucleic acid does not hybridize with the uncleaved collateral nucleic acid, according to any of the systems in paragraphs 76-81. 83. The detection nucleic acid is linked to a test strip in any of the systems described in paragraphs 76-82. 84. The collateral nucleic acid is linked to glucose oxidase in any of the systems described in paragraphs 76-83. 85. Any system from paragraphs 76–84, further comprising an aptamer linked to glucose oxidase. 86. The system of paragraph 85 wherein the aptamer specifically binds to at least a portion of the cleaved collateral nucleic acid. 87. The system of paragraph 86, wherein the aptamer binds to the single-stranded portion of the cleaved collateral nucleic acid. 88. The system of paragraph 86, wherein the aptamer binds to the double-stranded portion of the cleaved collateral nucleic acid hybridized with the detected nucleic acid. 89. Any system from paragraphs 76-88, further comprising an antibody linked to glucose oxidase. 90. The antibody specifically binds to at least a portion of the cleaved collateral nucleic acid in the system of paragraph 89. 91. The collateral nucleic acid is linked to an antibody that specifically binds to glucose oxidase, in any of the systems described in paragraphs 76-90. 92. The collateral nucleic acid is linked to the first member of the affinity pair in any of the systems of paragraphs 76-91. 93. The system of paragraph 92, further comprising glucose oxidase linked to the second member of the affinity pair. 94. The first and second members of the affinity pair, Combinations of haptenic or antigenic compounds with corresponding antibodies or their binding sites or fragments; digoxigenin and anti-digoxigenin; mouse immunoglobulins and goat anti-mouse immunoglobulins; non-immunological binding pairs; biotin and avidin; biotin and streptavidin; hormones and hormone-binding proteins; thyroxine and cortisol hormone-binding proteins; receptors and receptor agonists; receptors and receptor antagonists; acetylcholine receptors and acetylcholine or their analogues; IgG and protein A; lectins and carbohydrates; enzymes and enzyme cofactors; enzymes and enzyme inhibitors; complementary oligonucleotide pairs capable of forming nucleic acid doubles; as well as a negatively charged first molecule and a positively charged second molecule. A system of paragraphs 92 or 93, selected from the group consisting of the following. 95. The system of paragraph 92 or 93, in which the first and second members of the affinity pair are streptavidin and biotin. 96. Any system from paragraphs 92-95, wherein streptavidin is linked to the collateral nucleic acid and biotin is linked to the glucose oxidase. 97. Any system from paragraphs 92-95, wherein biotin is linked to the collateral nucleic acid and streptavidin is linked to the glucose oxidase. 98. The target nucleic acid is a viral nucleic acid, one of the systems described in paragraphs 76-97. 99. A method for detecting a target nucleic acid using any of the systems described in paragraphs 76-98, (i) The step of collecting a biological sample from the subject and optionally extracting nucleic acids from the biological sample; (ii) A step of contacting the biological sample with a sequence-specific endonuclease, a guide nucleic acid, and a collateral nucleic acid, wherein, if the target nucleic acid is present, such contact results in the cleavage of the collateral nucleic acid; (iii) A step of adding the biological sample to a test strip in the presence of glucose, wherein the test strip contains a detection nucleic acid that can form a detectable complex with the cleaved collateral nucleic acid, if present; (iv) The step of incubating the biological sample with the test strip, or not incubating it; (v) Inserting the inspection strip into the detection device; (vi) A step in which the level of any detectable complex is detected through a chemical reaction between glucose and glucose oxidase; and (vii) A step of correlating the level of any detectable complex formed with the amount of the target analyte in the at least one biological sample, thereby providing a diagnostic assessment. Methods that include... 100. The biological sample is saliva, according to the method of paragraph 99. 101. The detection device is a glucose meter, according to the method of paragraph 99 or 100. 102. (viii) The step of transmitting the diagnostic assessment or results to an electronic device, database, or cloud server for subsequent review by a clinician or trained healthcare provider; and (ix) The step of providing the diagnostic assessment to an individual who has undergone the diagnostic assessment method described above. Any of the methods described in paragraphs 99–101, including further, 103. The said individual is the subject in any of the manner described in paragraphs 99-102. 104. (viii) The stage of recommending, instructing, and / or administering one or more treatment regimens to the subject in accordance with the diagnostic evaluation. Any of the methods described in paragraphs 99–103, including further details. 105. A test strip from any of paragraphs 49-52, linked to the nucleic acid to be detected. 106. A kit containing the test strips from paragraph 105. 107. A sensor for detecting at least one target analyte in a biological sample, (i) A two-binding assay comprising first and second binding agents capable of forming a detectable complex with at least one target analyte; and (ii) A detection device for detecting the detectable complex Includes, The detection device is an oxidase-based amperometric sensor, and the biological sample is present in sweat, saliva, serum, mucus, or blood. sensor. 108. A sensor for detecting at least one virus in a biological sample, (i) A two-conjugate assay comprising first and second conjugates capable of forming a detectable complex with at least one virus; and (ii) A detection device for detecting the detectable complex Includes, The detection device is a glucose meter, which is a sensor. 109. A sensor for detecting at least one target nucleic acid in a biological sample, (i) A sequence-specific endonuclease and a guide nucleic acid that, when the target nucleic acid specifically binds to the sequence-specific endonuclease and guide nucleic acid, cleaves the collateral nucleic acid; (ii) A detection nucleic acid that can form a detectable complex with the cleaved collateral nucleic acid; and (iii) A detection device for detecting the detectable complex Includes, The detection device is an oxidase-based amperometric sensor, and the biological sample is present in sweat, saliva, serum, mucus, or blood. sensor. [Examples]

[0522] Example 1: Detection of influenza Influenza virus culture. Cell culture and subculturing were performed according to the WHO MDCK (Madin-Darby Canine Kidney) cell culture protocol. When the cells reached 70%-80% confluence, DMEM medium (GIBCO Dulbecco's modified Eagle medium) was aspirated, and the cells were washed three times with 5 mL of lx PBS (phosphate-buffered saline). The virion stock of influenza H1N1 A / Puerto Rico / 8 / 34 (A / PR / 8 / 34) was thawed in a 37°C bath, and 500 μL-1000 μL of virion sample was inoculated into a T-75 flask. The flask was slowly tilted and rotated to spread the virus inoculum. The inoculum was adsorbed in a 37°C incubator for 30 minutes. 12 mL of viral proliferation DMEM (containing TPCK-trypsin) was added to this T75 flask (tosylphenylalanyl chloromethyl ketone (TPCK) irreversibly inhibits the serine protease α-chymotrypsin). This was incubated at 37°C, and the cytotoxic effect (e.g., blackening of the flask due to lysis) was checked daily. When the cytotoxic effect reached approximately 75% to 100%, 12 mL of the supernatant was collected using a serum pipette. 15% glycerol was added to the final solution, and aliquots were frozen and stored at -80°C. The stock yielded 5 x 10⁶ saturates when measured by plaque assay. 6 It has a concentration of PFU / mL (plaque-forming units / milliliter) and when measured by RT-PCR (reverse transcription polymerase chain reaction) assay, it yields 1.64 x 10⁻⁴. 8 It had a concentration of RNA copies / mL.

[0523] Sensor Design. A sandwich-type electrochemical detection mechanism consisting of an aptamer and a glucose oxidase (GOx)-labeled antibody (both binding to the virus of interest) was designed and constructed. As shown in Figure 2, the aptamer (bound to a nitrocellulose membrane) captures the viral antigen, and Ab-GOx binds to the viral antigen if present. When a constant potential is applied, GOx oxidizes glucose (e.g., 500 mM), transferring electrons to oxygen, producing hydrogen peroxide, which then generates a current output via an electrode reacting with the hydrogen peroxide. The sensor electrode contains a mediator layer (e.g., Prussian blue) which reduces the overpotential of hydrogen peroxide production, generating an output current at lower potentials. Detection at higher potentials can be achieved without the mediator layer.

[0524] Detection of H1N1 influenza. In the sandwich detection assay, anti-influenza A H1N1 neuraminidase antibody (ABCAM) was used. This antibody has high affinity for the neuraminidase protein on the viral membrane. Similarly, an aptamer with affinity for hemagglutinin protein was used (IDT TECHNOLOGIES). A nitrocellulose membrane with 0.45 μm pores was used (THERMOSCIENTIFIC). 4 mm diameter holes were made in the membrane. The membrane was functionalized with streptavidin-NC by dropcasting streptavidin (0.5 mg / mL in PBS) onto the membrane. The membrane was dried at 37°C and stored overnight under dry conditions before use. Next, 10 μL of 20 μM biotinylated aptamer was dropcast over 30 minutes and then washed with PBS buffer. After aptamer immobilization, 1% BSA (bovine serum albumin) was added to the surface to block the surface, and then washed. The virion stock was thawed at -80°C and centrifuged at 3000 rpm to remove cell debris. The supernatant virion stock was diluted 10-fold twice, and 10 5 PFU / mL and 10 4A virion concentration of PFU / mL was obtained. 10 μL of these solutions were added as droplets to two different membranes containing aptamers and incubated for 15 minutes. GOx conjugate antibody synthesized using ABCAM's LIGHTNING-LINK (GOx conjugate kit, #ab102887) was diluted in PBST (e.g., 1 mg / mL of GOx conjugate antibody in PBS containing 0.05% detergent TWEEN 20), drop-cast onto the membrane surface, and incubated for 15 minutes. The membrane was then washed with 200 μL of PBST and transferred to a DROPSENS 710 electrode, which was then placed on the electrode. After adding 50 μL of 500 mM glucose solution, chronoamperometry measurements were performed. For the negative control sample, instead of adding virion stock, a virus-free DMEM solution (0.2% BSA, 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), 2 μg / mL TPCK-trypsin) was used. Using a self-made potentiostat, chronoamperometry measurements were performed at a potential of -0.2 V relative to Ag / AgCl (e.g., reference electrode), and the current over time was observed. As shown in Figure 4, when virions were present in the sample, bound glucose oxidase oxidized glucose, generating a current signal, whereas in the control sample (virions were absent), a negligible or background current was measured. The area under the curve of the current vs. time plot indicates total charge transfer, and as shown in Figure 4, when virions were present, the charge transfer was 10 times higher than in the control sample. Thus, a physiologically reasonable virus concentration (10 4 A significant current signal was observed in the presence of pfu / mL, and this can be reproduced for other viruses using the corresponding antibody-aptamer combination.

[0525] Calibration plot for H1N1 detection. The sensor was prepared by adding 10 μL of 1 mg / mL streptavidin-NC followed by 10 μL of a 40 μM aptamer targeting the H1N1 hemagglutinin protein. The blocking agent was 5 μL of 3% BSA. The antibody-GOx concentration used in this experiment was 0.1 mg / mL in PBST. 10 5 Different virus concentrations ranging from pfu / mL to 10 pfu / mL were prepared by diluting with DMEM buffer. A control sample of virus-free DMEM buffer was also assessed. The virions were incubated for 15 minutes, then Ab-GOx was added, followed by PBST (0.05% TWEEN-20). ) The samples were cleaned using [method / method]. Chronoamperometry measurements were performed at -0.2 V for 1500 seconds. The area under the curve of the current vs. time graph was plotted for different virion concentrations. 10 5 ~10 2 There was a statistically significant difference between the pfu / mL virus concentration and the control sample (see, for example, Figure 4).

[0526] Detection of H1N1 in human saliva. Human saliva that had tested negative for infectious diseases was used (LEE BIOSOLUTIONS, catalog # 991-05-S). The saliva samples contained very high protein concentrations. Therefore, specific antibody-virus binding was inhibited. The saliva samples were then diluted 10-fold and 100-fold in DMEM buffer before being added to the membrane surface. An antibody concentration of 1 mg / mL was used. When the saliva sample was diluted 10-fold (i.e., saliva diluted 10-fold in buffer), a significant difference in current was observed compared to the control sample. From this point onward, 10-fold saliva dilutions were used in further experiments. Dilution was performed to reduce nonspecific interactions from different proteins present in the saliva. The same procedure as above was followed to obtain the current output. Different virus concentrations in human saliva were used. 4 ~10 2PFU / mL was added, the sample was diluted 10-fold, and current vs. time was plotted. Significantly higher currents were observed in the viral saliva sample compared to the control sample (i.e., 10-fold diluted saliva without the virus).

[0527] Comparison of different antibody targets for H1N1 detection. Antibodies targeting the hemagglutinin (HA) protein on the H1N1 virus membrane surface (e.g., anti-HA mouse monoclonal antibody) and antibodies targeting the neuraminidase (NA) protein (anti-NA rabbit polyclonal antibody) were also tested. The antibodies used were ab 128412 (anti-influenza A H1N1 hemagglutinin antibody [C102(IV.C102)], ABCAM) and ab 91646 (anti-influenza A H1N1 neuraminidase antibody, ABCAM). Higher current signals were observed for antibodies as aptamers targeting NA, and for antibodies targeting different proteins on the virus membrane surface (e.g., HA), but the aptamer target may also be the HA protein, and the antibody target may be the NA protein (see, for example, Figure 6). When both the aptamer and antibody target the same protein (e.g., HA), the binding affinity of the aptamer and antibody plays a crucial role. Antibodies with higher affinity can strip the virus from the sensor surface; therefore, to acquire a signal and detect the virus, a lower concentration of the antibody must be used (see, for example, Figure 10).

[0528] The washing step was eliminated. The virus and antibody-GOx were pre-mixed to a total volume of 200 μL (0.05 mg / mL Ab-GOx + virion in PBST). 200 μL of this solution was dropped onto a membrane disc and incubated for 5 minutes. The control sample did not contain the virus. The membrane was moved to an electrode, 50 μL of glucose solution (e.g., 500 mM) was added, and chronoamperometry was performed. In the case of the virus sample, as in the control sample, bound Ab-GOx in solution was closer to the electrode surface than suspended Ab-GOx. A higher current signal was observed in the case of the virus sample, indicating that the proximity of glucose oxidase to the electrode surface generated a higher current signal.

[0529] Therefore, in some embodiments, the washing step (e.g., removal of suspended Ab-GOx in the solution) can be omitted. In some embodiments, in a lateral flow system, without a separate washing step, the analyte would flow from the detection area to the suction pad.

[0530] Example 2: Detection of rVSV-CoV-2 and SARS-CoV-2 rVSV-CoV-2 virus culture: ATCC SARS-CoV-2 S protein was transfected into HEK293T cells using PEI (polyethyleneimine) over approximately 2 hours. Cells were washed twice with culture medium to remove VSV (vesicular stomatitis virus). Once the cells showed adequate cytotoxicity (approximately 48 hours after infection), the culture medium was collected and spun down to remove cell debris. The supernatant containing the virus was then aliquoted and stored at -80°C.

[0531] SARS-CoV-2 Viral Culture: The first-choice cell type for viral culture was Vero cells due to their susceptibility to SARS-CoV-2 infection. Other cells, such as HuH7, or other human cell types, which are also susceptible to infection, can be used. Vero E6 cells were infected in phosphate-buffered saline (PBS) containing 50 μg / mL DEAE-dextran (diethylaminoethyl-dextran) and 2% fetal bovine serum (FCS; BODINCO). After adding the inoculum to the cells at 37°C for 1 hour, the cells were washed twice with PBS and maintained in Eagle's Minimal Essential Medium (EMEM; LONZA) containing 2% FCS, 2 mM L-glutamine (PAA LABORATORIES), and antibiotic (SIGMA). Viral titration was determined by plaque assay of Vero E6 cells.

[0532] Detection: In the sandwich sensing assay, a rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (ABCAM ab 272504) was used. This antibody targets the SARS-CoV-2 spike (S) protein. Similarly, aptamers targeting the S protein (e.g., as described by Song et al.) were used (IDT technologies). A nitrocellulose membrane with 0.45 μm pores was used (THERMOSCIENTIFIC). The membrane surface (e.g., a 4 mm diameter disc) was functionalized with streptavidin-NC (ENQUIRE BIOREAGENTS) by drop-casting 10 μL of solution (1 mg / mL streptavidin-NC in PBS) onto the membrane. The membrane was dried at 37°C for 1 hour. The aptamer (100 μM) was folded in 1 mM MgCl2 in PBS at 95°C for 5 minutes and allowed to cool to room temperature for 15 minutes. Next, the aptamer was diluted to working concentration in PBS, and 10 μL of 20 μM biotinylated aptamer was dropcast onto the membrane over 1 hour. After aptamer immobilization, the surface was blocked with 5 μL of 3% BSA to prevent nonspecific binding, and then washed with 200 μL of PBST (PBS + 0.05% TWEEN 20). The virion stock was diluted to the desired concentration in DMEM (THERMOFISHER, #21063029; e.g., 1000, 100, or 10 virus particles / mL). 10 μL of virus sample was added as a droplet to the membrane containing the aptamer and incubated for 15 minutes. The GOx conjugate antibody (Ab-GOx), synthesized using ABCAM's LIGHTNING-LINK (GOx conjugate kit, #ab102887), was diluted in PBST, and 5 μL of the solution was dropcast onto the membrane surface and incubated for 15 minutes. Next, the membrane was washed with 200 μL of PBST and transferred to a DROPSENS 710 electrode. After adding 50 μL of 500 mM glucose solution, chronoamperometry was performed. DMEM buffer (i.e., virion-free) was used as the negative control sample.Chronoamperometry measurements were performed at a potential of -0.2 V using a portable potentiostat, and the current over time was observed. A significantly larger current was observed compared to the background current.

[0533] Detection of VSV-CoV-2 pseudotypes in buffer: For the detection of rVSV viruses, which have the same spike protein on their surface as SARS-CoV-2, the same procedure as above was followed. In the sandwich sensing assay, rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (ABCAM ab 272504) was used. This antibody targets the SARS-CoV-2 spike (S) protein. Similarly, aptamers targeting the S protein (e.g., as described in Song et al.) were used (IDT TECHNOLOGIES). A nitrocellulose membrane with 0.45 μm pores was used (THERMOSCIENTIFIC). The membrane surface (e.g., a 4 mm diameter disc) was functionalized with streptavidin-NC (ENQUIRE BIOREAGENTS) by drop-casting 10 μL of solution (1 mg / mL streptavidin-NC in PBS) onto the membrane. The membrane was dried at 37°C for 1 hour. The aptamer (100 μM) was folded in 1 mM MgCl2 in PBS at 95°C for 5 minutes and allowed to cool to room temperature for 15 minutes. Next, the aptamer was diluted to working concentration in PBS, and 10 μL of 20 μM biotinylated aptamer was dropcast onto the membrane over 1 hour. After aptamer immobilization, the surface was blocked with 5 μL of 3% BSA to prevent nonspecific binding, and then washed with 200 μL of PBST (PBS + 0.05% TWEEN 20). The virion stock was diluted to the desired concentration in DMEM (THERMOFISHER, #21063029; e.g., 1000, 100, or 10 virus particles / mL). 10 μL of virus sample was added as a droplet to the membrane containing the aptamer and incubated for 15 minutes. GOx conjugate antibody (Ab-GOx) synthesized using ABCAM's LIGHTNING-LINK (GOx conjugate kit, #ab102887) was diluted with PBST, and 5 μL of the solution was drop-cast onto the membrane surface and incubated for 15 minutes. Next, the membrane was washed with 200 μL of PBST and transferred to a DROPSENS 710 electrode. After adding 50 μL of 500 mM glucose solution, chronoamperometry was performed.Without a washing step, no significant difference was observed between the control sample and the virion sample. Even with a washing step using a weak detergent (NP-40), no difference was observed between the control sample and the virion sample. When washing with PBST (0.5% tween) was used, a significant difference in current signals was observed between the virus-containing sample and the control sample. A significantly larger current was observed compared to the background current.

[0534] Antibody concentration: When aptamers and antibodies target the same protein, antibody concentration is important. The binding affinity of aptamers to spike proteins is in the nM range, while the binding affinity of antibodies to spike proteins is in the pM range. At a certain concentration of Ab-GOx (0.5 mg / mL), no significant difference was observed between the control sample and the virus sample. Further addition of Ab-GOx (1 mg / mL) to detect the virus did not result in any difference in signal. Addition of a lower concentration of Ab-GOx reagent (0.1 mg / mL) resulted in a signal and detection of the virus. These results demonstrate the importance of the relative binding affinity of aptamers and antibodies to the virus. If the antibody has a higher binding affinity to the target protein than the aptamer, the virus particles will be stripped from the aptamer along with the Ab-GOx during the washing process. Similarly, although the binding affinity is similar, if Ab-GOx is used in excess, the virus particles will be detached from the aptamer along with the Ab-GOx during the washing process. Therefore, the concentrations of the capture aptamer and Ab-GOx used must be determined through extensive experimentation and optimization to find the optimal performance concentration. In the detection of SARS-CoV-2 spike protein, optimal performance is achieved when Ab-GOx has a lower affinity for the target antigen than the aptamer or antibody that captures the virus particles bound to the test strip.

[0535] Comparison of Different Antibodies: Different antibodies and proteins targeting spike proteins and membrane proteins were used. First, rabbit polyclonal antibodies (ABCAM ab272504) and monoclonal antibodies (ABCAM ab273433, 1A9) targeting the SARS-CoV-2 spike protein were used. Second, since the SARS-CoV-2 spike protein binds to ACE2, which mediates entry into host cells, an ACE2 (angiotensin-converting enzyme-2)-Fc chimera (ABCAM #273687) was used and tested against cultured SARS-CoV-2. Third, an antibody targeting the SARS-CoV-2 membrane protein (NOVUS BIOLOGICALS, catalog # NB100-56569) was used. All of these antibodies and ACE2 were tested against SARS-CoV-2. In all combinations, a signal to be detected was obtained compared to a virion-free control (see, for example, Figures 11-13). Rabbit polyclonal antibodies induced the maximum signal change from the control baseline (see, for example, Figures 11–13). The rabbit polyclonal antibodies bind to the C-terminus of the SARS-CoV-2 spike protein, and the aptamer targets the N-terminal reception-binding domain of the spike protein. A stronger signal is generated when the aptamer epitope differs from the antibody epitope.

[0536] Aptamer Immobilization Strategies: Two aptamer immobilization methods were investigated. One was immobilization onto a nitrocellulose membrane, and the other was direct immobilization onto a screen-printed electrode. The performance of each system can be assessed by measuring the current generated by glucose oxidation using glucose oxidase under these conditions.

[0537] Immobilization Method 1: When immobilizing directly onto the electrode, a thiolated aptamer may be used, and the aptamer can be immobilized onto the gold electrode by utilizing the thiol-gold interaction. For aptamer immobilization, the electrode has both a mediator layer and gold, so the mediator layer may be electrodeposited first, and then gold nanoparticles may be electrodeposited. A DROPSENS (DRP 510) electrode (working electrode (WE): carbon, counter electrode (CE): Pt, reference electrode (Ref): Ag / AgCl) may be used, and a Prussian blue layer can be fixed by immersing the electrode in 2.5 mM FeCl3 and 2.5 mM potassium ferricyanide in 0.1 M HCl and applying a potential of 0.4 V for 40 seconds. The electrode can then be adjusted by applying a cyclic potential of -0.5 V to 0.35 V in a 0.1 M HCl + 0.1 M KCl solution at 50 mV / s for 25 cycles. Gold nanoparticles can be electrodeposited by immersing them in a 100 mg / mL HAuCl4 solution and applying a potential of -0.2 V for 30 seconds. After electrode preparation, the desired concentration of aptamer can be drop-cast and incubated in a humidity chamber for 16 hours.

[0538] Immobilization Method 2: Immobilization to NC uses streptavidin-NC (streptavidin engineered to bind to nitrocellulose) and follows the procedure described in the first section. Immobilization to nitrocellulose (NC) can provide a larger surface area and more specific and strong aptamer binding to the surface, but may consequently add electrical resistance to the system.

[0539] Immobilization Method 3: A cellulose nitrate solution containing the mediator (SIGMA catalog # 09986-500ML) can be dropped onto the working electrode and dried as is. Streptabidin-NC can be dropped onto the electrode, and other reagents can be drop-cast as described in Immobilization Method 2. The liquid cellulose solution can assist electron transfer between the enzyme and mediator complex, and the overall system resistance may be lower compared to a solid membrane. The optimal immobilization strategy can be determined by comparing the magnitude of the current output under the same concentration of aptamer-virion-Ab-GOx.

[0540] Comparison of Different Mediators: Various mediators are used for glucose detection from human samples. Some examples of mediator complexes are those primarily composed of iron, osmium, and ruthenium. A thorough comparison of different mediators can be performed by observing the generated current. Mediators that provide a rapid response and high current, while requiring low power, can be used in the fabrication of sensor strips.

[0541] Evaluation and validation of biosensor performance: This specification assumes that the expected performance of the sensor can be validated in terms of current flux magnitude and velocity, appropriate dependence on redox enzymes, specificity, and dynamic range. The sensor can be optimized for key performance indicators, including analytical sensitivity and specificity, cross-reactivity, dynamic range, detection limit, sensing speed and duration, coefficient of variation for repeated measures, and operational stability. Interference from various compounds present in saliva can be tested for the sensor using artificial saliva samples. The performance of the device can be tested with purchased human saliva samples, followed by the addition of viral antigens at known concentrations, and then a calibration curve can be created. The detection limit (LOD) and sensitivity of the sensor using saliva samples can be determined using the samples. Statistical methods can be used to design a sample size with sufficient statistical power, determine confidence intervals, and assess significance. Finally, tests can be performed to evaluate and calibrate the sensor's performance within a range of humidity levels, pH, and temperature.

[0542] Example 3: A commercial glucometer was repurposed to detect SARS-CoV-2 using a test strip. For example, the optimization results of Examples 1-2 can be used to fabricate SARS-CoV-2 test strips for use in commercial glucometers to detect SARS-CoV-2. Laser cutting and screen printing methods can be used to fabricate the test strips. Before testing with human samples, the electrode strips may undergo several rounds of optimization using known virus concentrations in artificial saliva (PICKERING SOLUTIONS, #1700-0313) to meet design requirements. The test strips may first be tested using a laboratory potentiostat, then compared to commercially available electrodes, and then incorporated into a glucometer. The glucometer can read glucose values ​​that reflect the concentration of glucose oxidase enzyme present on the sensor surface. Different GOx concentrations relative to a fixed glucose concentration may produce different glucose outputs on the glucometer. The glucose value displayed on the glucometer (proportional to the current output) can be correlated with the concentration of present virus particles.

[0543] Point-of-care detection using commercial glucose meters can be used to detect viral infections. Since the sensor output depends on the concentration of the bound antibody-GOx conjugate, it will depend on the viral...

Claims

1. A system for detecting at least one target analyte in a biological sample, (i) a two-binding assay comprising first and second binding agents capable of forming a detectable complex with at least one target analyte; and (ii) A detection device for detecting the detectable complex Includes, The detection device is a glucose oxidase-based amperometric sensor, and the biological sample is present in saliva. The first binder is an aptamer, and the second binder is an antibody, wherein the antibody is linked to glucose oxidase. system.

2. The system according to claim 1, wherein the detection device is a glucose meter.

3. The system according to claim 2, wherein the glucose meter includes a glucose sensor having a sensor output relating to glucose in a biological sample on a test strip.

4. The system according to any one of claims 1 to 3, wherein the biological sample is mixed with sugar.

5. The system according to claim 4, wherein the sugar is glucose.

6. The system according to claim 5, wherein the glucose is at a concentration of approximately 0.01 mM to approximately 1 M.

7. The system according to any one of claims 1 to 6, wherein the first and second binders bind to different sites on the target analyte.

8. The system according to claim 7, wherein the first and second binders have a Kd ratio of about 1:1000 to about 1000:1 with respect to the target analyte.

9. The binding affinity of the first binder to the first site on the target analyte is higher than the binding affinity of the second binder to the second site on the target analyte. The first binder is a capture reagent immobilized on a test strip, and the second binder is a detectable binder, and when the first and second binders bind to the target analyte, a detectable complex is formed. The system according to claim 7 or 8.

10. The system according to any one of claims 1 to 9, wherein the target analyte is the whole virus or its components.

11. The system according to claim 10, wherein the virus is a beta coronavirus, an influenza virus, a human immunodeficiency virus (HIV), or a hepatitis virus.

12. The system according to claim 10, wherein the target analyte is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a component thereof.

13. The system according to claim 12, wherein the target analyte is a component of SARS-CoV-2 selected from the group consisting of spike proteins, membrane proteins, hemagglutinin proteins, or envelope proteins.

14. The system according to any one of claims 1 to 9, wherein the target analyte is selected from the group consisting of IgG, IgM, and IgA.

15. A system for detecting target analytes of at least one virus in a biological sample, (i) a two-conjugate assay comprising first and second conjugates capable of forming a detectable complex with at least one virus; and (ii) A detection device for detecting the detectable complex Includes, The detection device is a glucose meter. The aforementioned biological sample is present in saliva. The first binder is an aptamer, and the second binder is an antibody, wherein the antibody is linked to glucose oxidase. system.

16. The system according to claim 15, wherein the glucose meter includes a glucose sensor having a sensor output relating to glucose in a biological sample on a test strip.

17. The system according to claim 15, wherein the biological sample is mixed with sugar.

18. The system according to claim 17, wherein the sugar is glucose.

19. The system according to claim 18, wherein the glucose is present at a concentration of approximately 0.01 mM to approximately 1 M.

20. The system according to any one of claims 15 to 19, wherein the first and second binders bind to different sites on the target analyte.

21. The system according to claim 20, wherein the first and second binders have a Kd ratio of about 1:1000 to about 1000:1 with respect to the target analyte.

22. The binding affinity of the first binder to the first site on the target analyte is higher than the binding affinity of the second binder to the second site on the target analyte. The first binder is a capture reagent immobilized on a test strip, and the second binder is a detectable binder, and when the first and second binders bind to the target analyte, a detectable complex is formed. The system according to claim 20 or 21.

23. The system according to any one of claims 15 to 22, wherein the target analyte is the whole virus or its components.

24. The system according to claim 23, wherein the virus is a betacoronavirus.

25. The system according to claim 23, wherein the target analyte is SARS-CoV-2 or a component thereof.

26. The system according to claim 25, wherein the target analyte is a component of SARS-CoV-2 selected from the group consisting of spike proteins, membrane proteins, hemagglutinin proteins, or envelope proteins.

27. (i) A step of preparing a biological sample collected from a subject, wherein the biological sample is saliva; (ii) A step of adding the biological sample to a test strip in the presence of glucose, wherein the test strip comprises first and second binders capable of forming a detectable complex with at least one target analyte if present in the biological sample, the first binder being an aptamer and the second binder being an antibody, the antibody being linked to glucose oxidase; (iii) The step of incubating the biological sample with the test strip, or not incubating it; (iv) Inserting the inspection strip into the detection device. A method including, If a detectable complex exists, its level is detected through a chemical reaction between glucose and glucose oxidase, and If a detectable complex is generated, its level is correlated with the amount of the target analyte present in at least one biological sample, thereby providing a diagnostic assessment. method.

28. (i) A step of preparing a biological sample collected from a subject, wherein the biological sample is derived from saliva; (ii) Diluting the collected sample 1 to 100 times in an aqueous solution / mixture in the presence of a second binder; (iii) A step of adding the biological sample and a second binder to a test strip, wherein the test strip comprises a first binder capable of forming a detectable complex with at least one target analyte if present in the biological sample, the first binder being an aptamer and the second binder being an antibody, the antibody being linked to glucose oxidase; (iv) The step of incubating the biological sample with the test strip, or not incubating it; (v) Inserting the inspection strip into the detection device A method including, If a detectable complex exists, its level is detected through a chemical reaction between glucose and glucose oxidase, and If a detectable complex is generated, its level is correlated with the amount of the target analyte present in at least one biological sample, thereby providing a diagnostic assessment. method.

29. The method according to claim 27 or 28, wherein the detection device is a glucose meter.

30. For subsequent review by a clinician or trained healthcare provider, the diagnostic assessment or results are transmitted to an electronic device, database, or cloud server, and The method according to claim 27 or 28, wherein the diagnostic evaluation is provided to an individual who has undergone the method.

31. The aforementioned individual is the subject of the method according to claim 30.

32. The method according to any one of claims 27 to 29, wherein one or more treatment regimens are recommended, instructed, and / or implemented for the subject in accordance with the diagnostic evaluation.

33. (i) A step of preparing a biological sample collected from a subject, wherein the biological sample is derived from saliva; (ii) A step of adding the biological sample to a test strip, wherein the test strip comprises first and second binders capable of forming a detectable complex with at least one target analyte if present in the biological sample, the first binder being an aptamer and the second binder being an antibody, the antibody being linked to glucose oxidase; (iii) The step of incubating the biological sample with the test strip, or not incubating it; (iv) Inserting the test strip into the glucomometer detection device. A method including, If a detectable complex exists, its level is detected through a chemical reaction between glucose and glucose oxidase, and A method wherein, if a detectable complex is formed, its level is correlated with the amount of the target analyte in the at least one biological sample, thereby providing a diagnostic assessment.

34. (i) A step of preparing a biological sample collected from a subject, wherein the biological sample is derived from saliva; (ii) Diluting the collected sample 1 to 100 times in an aqueous solution / mixture in the presence of a second binder; (iii) A step of adding the biological sample and a second binder to a test strip, wherein the test strip comprises a first binder capable of forming a detectable complex with at least one target analyte if present in the biological sample, the first binder being an aptamer and the second binder being an antibody, the antibody being linked to glucose oxidase; (iv) The step of incubating the biological sample with the test strip, or not incubating it; (v) Inserting the test strip into the glucomometer detection device. A method including, If a detectable complex exists, its level is detected through a chemical reaction between glucose and glucose oxidase, and A method wherein, if a detectable complex is formed, its level is correlated with the amount of the target analyte in the at least one biological sample, thereby providing a diagnostic assessment.

35. The method according to claim 33 or 34, wherein one or more treatment regimens are recommended, instructed, and / or implemented in accordance with the diagnostic evaluation.

36. (i) A step of preparing a biological sample collected from a subject, wherein the biological sample is derived from saliva; (ii) A step of adding the biological sample to a test strip, wherein the test strip comprises first and second binders capable of forming a detectable complex with at least one target analyte if present in the biological sample, the first binder being an aptamer and the second binder being an antibody, the antibody being linked to glucose oxidase; (iii) The step of incubating the biological sample with the test strip; (iv) Inserting the test strip into the glucomometer detection device. A method including, If a detectable complex exists, its level is detected through a chemical reaction between glucose and glucose oxidase. If a detectable complex is generated, its level is correlated with the amount of the target analyte present in at least one biological sample, thereby providing a diagnostic assessment. For subsequent review by a clinician or trained healthcare provider, the diagnostic assessment or results are transmitted to an electronic device, database, or cloud server, and The diagnostic assessment or outcome is transmitted to the individual who has completed steps (i) through (iv) above. method.

37. (i) A step of preparing a biological sample collected from a subject, wherein the biological sample is derived from saliva; (ii) Diluting the collected sample 1 to 100 times in an aqueous solution / mixture in the presence of a second binder; (iii) A step of adding the biological sample to a test strip, wherein the test strip comprises a first binder capable of forming a detectable complex with at least one target analyte if present in the biological sample, the first binder being an aptamer and the second binder being an antibody, the antibody being linked to glucose oxidase; (iv) The step of incubating the biological sample with the test strip; (v) Inserting the test strip into the glucomometer detection device. A method including, If a detectable complex exists, its level is detected through a chemical reaction between glucose and glucose oxidase. If a detectable complex is generated, its level is correlated with the amount of the target analyte present in at least one biological sample, thereby providing a diagnostic assessment. For subsequent review by a clinician or trained healthcare provider, the diagnostic assessment or results are transmitted to an electronic device, database, or cloud server, and The diagnostic assessment or outcome is transmitted to the individual who has completed steps (i) through (v) above. method.

38. The aforementioned individual is subject to the method according to claim 36 or 37.

39. The method according to claim 36 or 37, wherein one or more treatment regimens are recommended, instructed, and / or implemented for the subject in accordance with the diagnostic evaluation.

40. The system according to any one of claims 1 to 26, wherein one or more different target analytes are evaluated simultaneously or sequentially.

41. An inspection strip used in a system according to any one of claims 1 to 26, (i) a substrate, at least one of the first and second binders, and two or more electrodes; (ii) the substrate, both the first and second binders, and two or more electrodes; (iii) at least one of the first and second binders, and two or more electrodes; or (iv) Both the first and second binders, and two or more electrodes comprising at least one of the following, wherein the first binder is an aptamer and the second binder is an antibody, the antibody is linked to glucose oxidase, Test strip.

42. (i) One or more planar or coplanar electrodes made of carbon, iron, palladium, platinum, or gold; (ii) Electrodes coated with iron salt as a mediator; (iii) Electrodes coated with Prussian blue as a mediator; (iv) Electrode setups including electrodes that are conductive solids or semiconductor solids in contact with the electrolyte solution; (v) an electrode setup including a working electrode, a reference electrode, and a pair or auxiliary electrode (iv); (vi) A two-electrode setup in which the current lead and sense lead are connected, the working lead and working sense lead are connected to the working electrode, and the reference lead and pair lead are connected to a second auxiliary, pair, or pseudo / pseudo-reference electrode; (vii) A three-electrode setup in which the reference lead is separated from the paired lead and connected to a third electrode; (viii) A four-electrode setup in which, in addition to the reference lead, the working sense lead is disconnected from the working electrode; and / or (ix) A non-resistive ammeter in which the working electrode lead and the counter electrode lead are both short-circuited within the strip so that the overall voltage drop of the electrochemical cell is zero. The inspection strip according to claim 41, comprising at least one of the following.

43. The test strip according to claim 42, wherein in the electrode setup of (vii), the third electrode is positioned to measure the nearest point of the working electrode to which both the working and working sense leads are attached.

44. The inspection strip according to claim 42 or 43, wherein the iron salt is a ferrous ferrocyanide.

45. The inspection strip according to any one of claims 41 to 44, wherein the first binder is immobilized on a porous polymer, a metal, or a ceramic film, and the film is positioned on or between electrodes.

46. The inspection strip according to claim 45, wherein the membrane is nitrocellulose.

47. A kit comprising the test strip described in any one of claims 41 to 46.

48. The kit according to claim 47, including instructions for using the aforementioned test strip.

49. The kit according to claim 47, further comprising a glucometer or other electrochemical detection device.

50. (i) the step of preparing a biological sample derived from the subject, and (ii) A step of detecting the presence of a target analyte in the biological sample using the system according to any one of claims 1 to 26, wherein the target analyte is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a component thereof. A method comprising the biological sample being saliva.

51. The method according to claim 50, wherein the target analyte is SARS-CoV-2.

52. The method according to claim 50, wherein the target analyte is a component of SARS-CoV-2.

53. The method according to any one of claims 50 to 52, wherein the component is an S protein or a fragment thereof.

54. (i) the step of preparing a biological sample derived from the subject, and (ii) A step of detecting the presence of a target analyte in the biological sample using the system according to any one of claims 1 to 26, wherein the target analyte is coronavirus (CoV) or a component thereof. A method comprising the biological sample being saliva.

55. The method according to claim 54, wherein the target analyte is CoV.

56. The method according to claim 54, wherein the target analyte is a component of CoV.

57. (i) the step of preparing a biological sample derived from the subject, and (ii) A step of detecting the presence of a target analyte in the biological sample using the system according to any one of claims 1 to 26, wherein the target analyte is an influenza virus or a component thereof. A method comprising the biological sample being saliva.

58. The method according to claim 57, wherein the target analyte is influenza virus.

59. The method according to claim 57, wherein the target analyte is a component of the influenza virus.

60. (i) the step of preparing a biological sample derived from the subject, and (ii) A step of detecting the presence of a target analyte in the biological sample using the system according to any one of claims 1 to 26, wherein the target analyte is human immunodeficiency virus (HIV) or a component thereof. A method comprising the biological sample being saliva.

61. The method according to claim 60, wherein the target analyte is HIV.

62. The method according to claim 60, wherein the target analyte is a component of HIV.

63. (i) the step of preparing a biological sample derived from the subject, and (ii) A step of detecting the presence of a target analyte in the biological sample using the system according to any one of claims 1 to 26, wherein the target analyte is a hepatitis virus or a component thereof. A method comprising the biological sample being saliva.

64. The method according to claim 63, wherein the target analyte is a hepatitis virus.

65. The method according to claim 63, wherein the target analyte is a component of a hepatitis virus.

66. A sensor for detecting at least one target analyte in a biological sample, (i) a two-binding assay comprising first and second binding agents capable of forming a detectable complex with at least one target analyte; and (ii) A detection device for detecting the detectable complex Includes, The detection device is a glucose oxidase-based amperometric sensor, and the biological sample is present in saliva. The first binder is an aptamer, and the second binder is an antibody, wherein the antibody is linked to glucose oxidase. sensor.

67. A sensor for detecting at least one virus in a biological sample, (i) a two-conjugate assay comprising first and second conjugates capable of forming a detectable complex with at least one virus; and (ii) A detection device for detecting the detectable complex Includes, The detection device is a glucose meter. The first binder is an aptamer, and the second binder is an antibody, wherein the antibody is linked to glucose oxidase. The aforementioned biological sample is present in saliva. sensor.

68. The method according to any one of claims 27 to 39 or 50 to 65, wherein one or more different target analytes are evaluated simultaneously or sequentially.

69. An inspection strip used in the method according to any one of claims 27 to 39 or 50 to 65, (i) a substrate, at least one of the first and second binders, and two or more electrodes; (ii) the substrate, both the first and second binders, and two or more electrodes; (iii) at least one of the first and second binders, and two or more electrodes; or (iv) Both the first and second binders, and two or more electrodes comprising at least one of the following, wherein the first binder is an aptamer and the second binder is an antibody, the antibody is linked to glucose oxidase, Test strip.

70. (i) One or more planar or coplanar electrodes made of carbon, iron, palladium, platinum, or gold; (ii) Electrodes coated with iron salt as a mediator; (iii) Electrodes coated with Prussian blue as a mediator; (iv) Electrode setups including electrodes that are conductive solids or semiconductor solids in contact with the electrolyte solution; (v) an electrode setup including a working electrode, a reference electrode, and a pair or auxiliary electrode (iv); (vi) A two-electrode setup in which the current lead and sense lead are connected, the working lead and working sense lead are connected to the working electrode, and the reference lead and pair lead are connected to a second auxiliary, pair, or pseudo / pseudo-reference electrode; (vii) A three-electrode setup in which the reference lead is separated from the paired lead and connected to a third electrode; (viii) A four-electrode setup in which, in addition to the reference lead, the working sense lead is disconnected from the working electrode; and / or (ix) A non-resistive ammeter in which the working electrode lead and the counter electrode lead are both short-circuited within the strip so that the overall voltage drop of the electrochemical cell is zero. The inspection strip according to claim 69, comprising at least one of the following.

71. The test strip according to claim 70, wherein in the electrode setup of (vii), the third electrode is positioned to measure the nearest point of the working electrode to which both the working and working sense leads are attached.

72. The inspection strip according to claim 70 or 71, wherein the iron salt is a ferrous ferrocyanide.

73. The inspection strip according to any one of claims 69 to 72, wherein the first binder is immobilized on a porous polymer, metal, or ceramic film, and the film is positioned on or between electrodes.

74. The inspection strip according to claim 73, wherein the membrane is nitrocellulose.

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