Apparatus and method for rapid detection of target analytes in biological samples

Abstract

A rapid lateral flow device for detecting a target analyte in a liquid biological sample is provided.A method for rapidly detecting a target analyte in a liquid biological sample is also provided. [Solution] The device comprises a membrane strip, which includes a matrix, a conjugate pad having at least one reporter vitrified in the matrix, one or more test sites containing covalently or electrostatically bound capture agents vitrified in or on the matrix, and optionally a control line containing one or more capture agents vitrified in or on the matrix.

Description

Technical Field

[0001] <Cross - Reference to Related Applications> This application claims priority to U.S. Provisional Application No. 63 / 002,873, filed Mar. 31, 2020, and U.S. Provisional Application No. 63 / 051,668, filed Jul. 14, 2020, the entire contents of which are hereby incorporated by reference.

[0002] This disclosure relates to rapid diagnostic devices and methods for detecting the presence or absence of one or more target analytes in a biological sample.

Background Art

[0003] Standard serological diagnostic methods for detecting the presence of antibodies, enzymes, pathogens, etc. in biological samples often require cold - chain transportation and storage, special equipment, and skilled operators to perform complex experimental procedures. Methods such as enzyme - linked immunosorbent assay (ELISA), polymerase chain reaction, bead - based profiling kits (such as Luminex® assay, etc.) take at least three hours or more to obtain test results, and the accuracy may decrease due to sample degradation and contamination, leading to false positives or false negatives. During a pandemic or an epidemic, when the number of test samples overwhelms the staff and resources of the laboratory, patients may be waited for days to weeks to obtain test results. If it takes a long time to obtain accurate test results, it promotes the infection of pathogens, and as a result, the risk of further exhaustion of medical facilities and impairment of patient treatment is increased.

[0004] There is a need for an assay that can rapidly and reliably detect one or more analytes, such as antibodies and pathogens, in a biological sample.

Summary of the Invention

Means for Solving the Problems

[0005] The following summary is provided to facilitate understanding of some of the innovative features unique to this disclosure and is not intended to be a complete description. A complete understanding of the various aspects of this disclosure can be obtained by considering the specification, claims, drawings, and abstract as a whole.

[0006] Provided herein are apparatuses and methods for the rapid detection of one or more target analytes in a liquid biological sample, thereby eliminating the need for cold-chain storage of the assay material, ensuring robustness and maintaining activity even after long-term storage at temperatures above 55°C for several weeks or months, allowing for direct use at the point of care, and optionally eliminating the use of complex detection instruments other than the human eye. The apparatus of this disclosure comprises a membrane containing a vitrified matrix, the vitrified matrix having a capture agent and one or more desired reporter molecules, the capture agent optionally being covalently bound to the matrix or not, the capture agent itself optionally containing a detectable label (e.g., an enzyme), and the reporter molecule (e.g., an antibody, nucleic acid, etc.) optionally being vitrified to the matrix or other matrix layers of the apparatus, thereby allowing the vitrified matrix and / or other matrix layers to come into contact with and rehydrate the liquid biological sample, releasing the reporter, the target analytes in the sample binding to the capture agent and the reporter, and the presence or absence of the target analytes to be detected. In some embodiments, the apparatus and methods described herein provide qualitative and / or quantitative detection results. The apparatus of this disclosure is stable to time and temperature and does not require cold chain transport or storage. Furthermore, according to some embodiments, the disclosed apparatus and methods advantageously enable rapid point-of-care diagnostic results within about 20 minutes.

[0007] In some embodiments, a lateral flow immunoassay analyzer comprising a membrane strip is provided. The membrane strip comprises a matrix, a conjugate pad, one or more test regions, and optionally a control region. The conjugate pad comprises at least one reporter, which may comprise one or more detectable labels and an antigen or antibody against a target analyte, optionally comprising a detectable label or labeling molecule. The reporter is vitrified into the matrix. One or more test regions optionally comprise a covalently bound capture antibody or aptamer specific to a type of target analyte, which is vitrified into the matrix. The control region comprises one or more second capture agents, which are vitrified into the matrix, and the second capture agents in the control region are suitable for binding control reagents, which may optionally be further included in the conjugate pad.

[0008] These and other purposes, features, and aspects of this disclosure will be further understood by referring to the drawings and detailed description provided herein. [Brief explanation of the drawing]

[0009] The embodiments described in the drawings are essentially illustrative and exemplary, and are not intended to limit the subject matter defined by the claims. The following detailed description of the exemplary embodiments can be understood by reading in conjunction with the following drawings, where similar structures are indicated by similar reference figures. [Figure 1] Figure 1 shows an exemplary embodiment of the apparatus according to the present disclosure, in which the capture antigen and reporter antibody are vitrified in a membrane matrix. [Figure 2A] Figure 2A illustrates the method steps according to this disclosure, in which the apparatus shown in Figure 1 is brought into contact with a liquid biological sample containing the analyte. [Figure 2B] Figure 2B illustrates the method steps according to this disclosure, in which the target analyte is conjugated with a capture antigen and a reporter antibody in a membrane layer. [Figure 2C] Figure 2C illustrates the method steps according to this disclosure, in which the substrate is added to the apparatus and a signal is detected. [Figure 3] Figure 3 shows an exemplary embodiment of the apparatus according to this disclosure, in which the capture antigen is vitrified in a membrane layer and the reporter antibody is incorporated into a contaminant screen layer. [Figure 4] Figure 4 shows the method steps according to this disclosure, and the apparatus shown in Figure 3 is brought into contact with the liquid biological sample. [Figure 5] Figure 5 shows an exemplary embodiment of the apparatus according to the present disclosure, in which a removable separator layer is positioned between two channels. [Figure 6] Figure 6 illustrates the exemplary comparative advantages of the apparatus and methods disclosed herein compared to standard available methods for ELISA and Luminex assays. [Figure 7A] Figure 7A shows an exemplary embodiment of a lateral flow membrane strip according to the present disclosure. [Figure 7B] Figure 7B shows an exemplary embodiment of a lateral flow membrane strip according to the present disclosure. [Figure 7C] Figure 7C shows an exemplary embodiment of a lateral flow membrane strip according to the present disclosure. [Figure 8] Figure 8 shows an exemplary embodiment of a lateral flow cassette according to the present disclosure. [Figure 9A] Figure 9A is an exploded perspective view of the lateral flow cassette according to this disclosure. [Figure 9B] Figure 9B is an exploded perspective view of the lateral flow cassette according to this disclosure. [Figure 10] Figure 10 shows an exemplary embodiment of a lateral flow membrane strip according to the present disclosure. [Figure 11A] Figure 11A shows the method steps according to this disclosure, in which the lateral flow membrane strip according to Figure 10 is brought into contact with the liquid biological sample. [Figure 11B]FIG. 11B shows method steps according to the present disclosure, where an analyte of interest and unbound reporter antibody are captured and detected on a lateral flow test strip. [Figure 12] FIG. 12 is a diagram showing an exemplary embodiment of a cassette according to the present disclosure that includes a plurality of lateral flow membrane strips. [Figure 13A] FIG. 13A is an exploded perspective view of an exemplary embodiment of a cassette according to the present disclosure that includes a plurality of lateral flow membrane strips. [Figure 13B] FIG. 13B is an exploded perspective view of an exemplary embodiment of a cassette according to the present disclosure that includes a plurality of lateral flow membrane strips. [Figure 14] FIG. 14 is a diagram showing an exemplary multi-strip lateral flow assay according to the present disclosure. [Figure 15A] FIG. 15A shows a lateral flow cassette having a plurality of membrane strips according to the present disclosure, where each strip is provided for serial dilution of the captured target analyte to determine the concentration of the target analyte. [Figure 15B] FIG. 15B is a diagram showing the plurality of membrane strips of the cassette according to FIG. 15A. [Figure 16] FIG. 16 shows an exemplary membrane strip according to some embodiments of the present disclosure that has a plurality of test regions on a single strip. [Figure 17] FIG. 17 is a diagram showing an exemplary test strip according to some embodiments as provided, where A shows various regions of an exemplary membrane strip, B shows an exemplary fluid flow rate and total flow rate through the membrane strip, and C shows a desirable linear front line after application of a substrate to the device. [Figure 18A] FIG. 18A is a diagram showing a device according to some embodiments provided herein that includes a substrate membrane on a lever for application of a substrate to a membrane layer. [Figure 18B] FIG. 18B is a diagram showing the device of FIG. 17A showing the position of the substrate membrane and lever with respect to a membrane strip according to some embodiments. [Figure 18C]FIG. 18C is a diagram showing the device of FIG. 17A, which shows the positions of the substrate membrane and the lever with respect to the membrane strip in several embodiments when the lever is actuated to facilitate contact between the membrane strip and the substrate pad. [Figure 18D] FIG. 18D shows an exemplary fully assembled device in several embodiments. [Figure 18E] FIG. 18E is an extended view showing another example of an exemplary fully assembled device provided herein, where the lever may be an enlarged area or may include an enlarged area. [Figure 19] FIG. 19 is a diagram showing a device provided according to several embodiments provided herein, which is treated with recombinant SARS-CoV-2 S protein as a sample and detected to be less than 10 pg / ml. [Figure 20] FIG. 20 is a diagram showing a device provided according to several embodiments provided herein, which is treated with recombinant SARS-CoV-2 S protein as a sample and detected to be less than 1 pg / ml when tested with substrate TMBMX. [Figure 21] FIG. 21 is a diagram showing a device provided according to several embodiments provided herein, where when treated with heat-inactivated SARS-CoV-2 as a sample and tested with substrate TMBMX, the S protein was detected at a load more than 100 times lower than expected in a biological sample. [Figure 22] FIG. 22 is a diagram showing a device provided according to several embodiments provided herein, which compares directly binding a capture agent to the test site (A) and pre-coating the test site with a molecule to which the capture agent binds (B).

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] This application provides an apparatus and method for the rapid and diagnostic detection of a target analyte in a liquid biological sample. As detailed herein, vitrification of a bound capture agent and optionally a reporter into a matrix allows for rapid reconstitution of the matrix upon contact with the liquid biological sample, thereby releasing the reporter and capture agent, enabling rapid point-of-care immunoassay detection of the target analyte without the need for cold chain transport or storage. It has been found that by embedding specific molecules in a membrane, covalently or electrostatically bonding them, or otherwise associating them with the membrane, and then performing a vitrification process, the molecules can be effectively stabilized, thereby maintaining their activity even during subsequent storage under harsh conditions, and eliminating the need for conventional cold chain storage processes. All analytical materials may optionally be vitrified in or using an apparatus such as those provided herein, in or with the apparatus provided herein, by reconstituting them with a liquid biological sample (e.g., blood, plasma, serum, saliva, bronchoalveolar lavage fluid, sputum, nasal fluid, skin secretions, buffer, water, or other liquids containing or being part of a biological sample) or other liquid, with little or no loss of activity, thereby providing a robust measurement system for detecting one or more analytes, whether or not they are present in the biological sample.

[0011] Detecting the presence of infectious agents (as an example of a target analyte) in subjects, particularly human subjects, presents many challenges for diagnosticians. For example, detecting viral factors themselves can be difficult, especially when the infection is recent and the viral load is low, or when an active infection has subsided but it is desired to know whether a previous infection occurred or whether the subject has persistent immunity. Viral infection can lead to the production of antibodies against the virus in the subject's body. Detecting these antibodies can provide information related to past or current exposure to the virus. As a non-limiting example, infections with betacoronaviruses, such as the human coronavirus SARS-CoV (causing severe acute respiratory syndrome; SARS) and MERS-CoV (causing Middle East respiratory syndrome; MERS), present several challenges due to the increase in new strains. Detecting these new strains of the highly infectious novel coronavirus SARS-CoV-2 (causing COVID-19; COVID-19) required the development of diagnostic methods specific to this new virus.

[0012] The apparatus and methods provided herein provide for the rapid, reproducible, and selectively quantitative detection of one or more target analytes in a biological sample. As provided herein, the apparatus comprises a housing and a substantially dry membrane layer within the housing, the membrane layer comprising a first matrix and a vitrified capture agent in or on the first matrix at a first test site. The apparatus may be in the form of a lateral flow assay in which a liquid biological sample (or a target analyte in liquid) is applied at a sample application site, and the liquid wets the membrane and moves toward one or more test sites at locations other than the sample application site. The liquid between the sample application site and the test sites may wet and rehydrate a reporter and / or capture agent (or other desired molecule) that can effectively localize the target analyte at the test sites and detect the presence or absence of the target analyte in the biological sample.

[0013] In some embodiments, the apparatus is a lateral flow apparatus comprising a substantially dry membrane, the membrane comprising a matrix, a conjugate pad comprising one or more test areas comprising a covalent or electrostatic capture agent (e.g., antigen, aptamer, nucleotide, or antibody) vitrified to the matrix, and optionally a conjugate pad comprising a control area comprising at least one reporter antibody vitrified to the matrix, and optionally one or more second capture agents vitrified to the matrix.

[0014] In some embodiments, a rapid diagnostic device is provided for detecting an analyte of interest in a liquid biological sample, comprising a membrane layer, the membrane layer comprising a first matrix, a capture agent vitrified to the first matrix, and at least one reporter vitrified to the first matrix. Optionally, the device further comprises a screen layer positioned on or otherwise in contact with the membrane layer. The screen layer, if present, may include a second matrix and is configured to function as a filter for substantially capturing and isolating contaminants (e.g., molecules other than the analyte of interest) in the liquid biological sample.

[0015] In some embodiments, a rapid diagnostic device is provided for detecting an analyte of interest in a liquid biological sample, the rapid diagnostic device comprising a membrane layer and a screen layer, the membrane layer comprising a first matrix and a capture agent vitrified to the first matrix, the screen layer comprising a second matrix positioned on or otherwise in contact with the membrane layer, the screen layer comprising a second matrix configured to function as a filter for substantially capturing and isolating contaminants in a liquid biological sample, and optionally, at least one reporter vitrified to the first matrix, the second matrix, or both.

[0016] In some embodiments, a rapid diagnostic device is provided for detecting an analyte of interest in a liquid biological sample, comprising a membrane layer and a substantially dry substrate membrane containing a vitrified substrate in or on the substrate membrane. The membrane layer comprises a first matrix and a vitrified capture agent in the first matrix. The substrate membrane is associated with a housing and configured to contact the membrane layer when desired, and optionally, after the membrane layer has been wetted with a liquid, optionally, a liquid biological sample alone or together with a further wetting agent.

[0017] The film layers or entire apparatus provided herein are optionally substantially dry. "Substantially dry" is defined as having a moisture content ratio (grams H2O / gram dry weight) of 0.1 or less, optionally 0.05 or less, optionally 0.04 or less, optionally 0.03 or less, optionally 0.02 or less, or optionally 0.01 or less.

[0018] "Amorphous" or "glass" refers to an crystalline material that lacks long-range order in the positions of atoms with an order parameter of 0.3 or less. The conversion from liquid to glassy solid occurs at the glass transition temperature Tg. In some embodiments, the glass-solidifying medium may be an amorphous (non-crystalline) material or may form an amorphous material. In other embodiments, the biomaterial may be an amorphous material.

[0019] The "glass transition temperature" refers to the temperature at which a material behaves like a liquid but below the temperature at which it behaves like a solid and enters an amorphous / glassy state. This is not a fixed temperature point, but rather varies depending on the timescale of the measurement used. In some embodiments, the glassy state may refer to the state entered when the biological composition decreases below its glass transition temperature. In other embodiments, the glassy state may refer to the state entered when the vitrification mixture and / or vitrifying agent decreases below its glass transition temperature. In yet another embodiment, the glassy state may have the mechanical rigidity of a crystal or gel, but with the random and disordered arrangement of molecules characteristic of a liquid.

[0020] A "crystal" is a three-dimensional structure of atoms, ions, or molecules consisting of a specific geometric arrangement that repeats periodically, and is called a lattice or unit cell.

[0021] "Crystalline" refers to a form of material that, unlike glassy or amorphous materials, contains constituent elements arranged regularly at the atomic level. Crystalline solids solidify at the crystallization temperature Tc.

[0022] As used herein, "vitrification" is a process that converts a material into an amorphous material. The amorphous solid does not have to have any crystalline structure.

[0023] As used herein, “vitrification mixture” means a heterogeneous mixture of biological material and vitrification medium, comprising one or more vitrifying agents, optionally a solvent, and optionally other materials.

[0024] As used herein, “biomaterial” or “biological sample” refers to material that may be isolated from or derived from a living organism. Examples of biomaterials include, but are not limited to, proteins (e.g., enzymes, or non-enzymatically functioning proteins), cells, tissues, organs, cell-based constructs, saliva or parts thereof, blood or parts thereof, nucleic acids, or combinations thereof. In some embodiments, biomaterial may refer to mammalian cells. In other embodiments, biomaterial may refer to human mesenchymal stem cells, mouse fibroblasts, leukocytes, erythrocytes, platelets, bacteria, viruses, mammalian cells, liposomes, enzymes, tissues (e.g., intestines, liver, neurons, or others), or combinations thereof. In other embodiments, biomaterial may refer to germ cells, including spermatocytes, spermatocytes, oocytes, embryos, blastocysts, or combinations thereof. In other embodiments, biomaterial may refer to whole blood, erythrocytes, leukocytes, platelets, plasma, blood serum, saliva, gastric aspirate, nasal fluid, algae, fungi, or combinations thereof.

[0025] As used herein, “vitrifying agent” refers to a material that forms an amorphous structure when a mixture of the vitrifying agent and other materials is cooled or dried, or a material that inhibits crystal formation in the other materials. The vitrifying agent may also provide osmotic protection or allow the survival of cells otherwise dehydrated. In some embodiments, the vitrifying agent may be any water-soluble solution that results in an amorphous structure suitable for the storage of biomaterials. In other embodiments, the vitrifying agent may be absorbed into cells, tissues, or organs.

[0026] As used herein, “storable,” “storage,” or “storage stable” refers to the ability of a biomaterial to be preserved and remain viable for use at a later time.

[0027] As used herein, "above extremely low temperatures" refers to temperatures above -80°C. As used herein, "room temperature" refers to a temperature range between 18°C ​​and 37°C.

[0028] "Hydrophilicity" means attracting water molecules or preferentially associating with water molecules. Hydrophilic materials, which have a special affinity for water, maximize contact with water and have a smaller contact angle with water.

[0029] "Hydrophobic" means having a low affinity for water. Hydrophobic materials inherently repel water, making it easy for water droplets to form, and they have a small contact angle with water.

[0030] As used herein, "ambient temperature" ("environmental temperature") refers to a temperature between approximately 16°C and approximately 30°C.

[0031] As used herein, “liquid biological sample” refers to a liquid sample that may contain the target analyte of interest. In some embodiments, the liquid biological sample is obtained from a subject, exemplary, a human patient. In some embodiments, the subject is suspected to have a disease or condition such as SARS-CoV-2 infection (i.e., COVID-19 disease), or any other infectious disease such as influenza or other viral, bacterial, or fungal infections. The liquid biological sample may be any sample that can flow through the matrix described herein and potentially contain the analyte of interest. In some embodiments, the liquid biological sample includes saliva, blood, serum, plasma, bronchoalveolar lavage fluid, sputum, nasal fluid, skin secretions, or a combination thereof.

[0032] As used herein, “subject” is an animal, optionally human, non-human primate, horse, cattle, mouse, sheep, pig, rabbit, or other mammal.

[0033] Apparatus and methods provided herein may be used to detect the presence or absence of a target analyte in a biological sample. The target analyte may be any biological material. Optionally, the target analyte may be a virus or a part thereof. In other embodiments, the target analyte may be a bacterium or a part thereof. Optionally, the target analyte may be a protein, optionally an enzymatically active protein. The exemplary virus used and illustrated throughout this disclosure for illustrative purposes only is human coronavirus. Apparatus and methods provided herein may be similarly described with respect to any other virus, bacterium, fungus, or a part thereof. Human coronaviruses (HCoVs) are positive-sense, long (30,000 bp) single-stranded RNA viruses. HCoVs are characterized by structural proteins such as the spike (S), nucleocapsid (N), membrane glycoprotein (M), and envelope (E), as well as non-structural proteins such as proteases (nsp3, nsp5), helicases (nsp13), and RNA-dependent RNA polymerases (RdRp, nsp12). The spike protein is known to be an important recognition factor for viral attachment and entry into host cells. This disclosure provides an apparatus and method that can specifically detect the presence or absence of any infectious substance or other target analyte in a specific and robust system. In some embodiments, this system is applied to the detection of one or more HCoVs, and optionally SARS-CoV-2.

[0034] The capture agents used herein are optionally antigens, enzymes, antibodies, or aptamers that bind (optionally, selectively) to a target analyte. In some embodiments, the capture agent is an antibody. Optionally, the capture agent is an aptamer. Optionally, the capture agent is an antigen, which optionally takes the form of a protein, nucleic acid, or fatty substance. Optionally, the capture agent is an enzyme. Optionally, the capture agent is an antibody, such as an antibody specific to the target analyte, which optionally recognizes a binding site on the target analyte different from the binding site of the reporter. Optionally, the capture agent is an antigen, such as an antigen, which can be specifically recognized by an antibody as the target analyte, and the antibody may be specific to infectious agents in a biological sample indicating the presence or past presence of infectious agents in the subject. In other embodiments, the capture agent is an aptamer induced or selected for one or more target analytes.

[0035] Recognition of the target analyte may also be achieved by forming a sandwich between the capture agent, the target analyte, and the reporter, so that both the capture agent and the reporter bind to the target analyte so that the target analyte is isolated at the location of the capture agent, and the reporter can emit or produce an observable signal to identify the presence of the target analyte bound to the capture agent. It is understood that the apparatus and methods provided herein may also be used in competitive assays in which the presence of the target analyte prevents association between the reporter and the capture agent (e.g., competition), thereby detecting the presence of the target analyte in a biological sample by the absence of a signal at the test site.

[0036] The target analyte is optionally a virus or a part thereof, an antibody directed at one or more protein molecules present on the surface of a virus, or a viral protein or viral antigen. Optionally, it may be an enveloped virus, or optionally, a virus from the families Arenaviridae, Herpesviridae, Retroviridae, Coronaviridae, Poxviridae, Togaviridae, Flaviviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, or Rhabdoviridae. The Coronaviridae family may be selected from Kolacovirus, Decacovirus, Dubinacovirus, Luchacovirus, Minacovirus, Minunacovirus, Myotacovirus, Niktacovirus, Pedacovirus, Linacovirus, Cetracovirus, Soracovirus, Sunacovirus, Tegacovirus, Envecovirus, Hibecovirus, Mabecovirus, Novecovirus, Surveycovirus, Brangacovirus, Brangacovirus, Igacovirus, Andecovirus, Brudeccovirus, or Herdeccovirus. Optionally, the Coronaviridae family may be betacoronaviruses, optionally severe acute respiratory syndrome-associated coronaviruses, optionally severe acute respiratory syndrome coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or other related elements. Optionally, the Arenaviridae family may be Lassavirus, Juninvirus, or lymphocytic choriomeningitis virus (LCMV). Optionally, the Herpesviridae family may be herpes simplex virus, hepatitis B virus, varicella-zoster virus, cytomegalovirus, or Epstein-Barr virus. Optionally, the Retroviridae family may be human immunodeficiency virus or human T-lymphophilic virus.Other exemplary virus-targeting agents or agents that induce antibody production in subjects include, but are not limited to, smallpox virus, rubella virus, hepatitis C virus, Zika virus, West Nile virus, dengue virus, yellow fever virus, TBE virus, Semryki Forest virus, Rift Valley fever virus, sandfly fever virus, Hantan virus, Marburg virus, Ebola virus, influenza virus A, influenza virus B, influenza virus C, Nipah virus, human parainfluenza virus, measles virus, mumps virus, varicella stomatitis virus, or rabies virus.

[0037] In some embodiments, the virus is a non-enveloped virus, optionally belonging to the adenoviridae, polyomaviridae, papillomaviridae, hepaviridae, or rhinovirus family.

[0038] The target viral analytes are optionally HCoV and optionally SARS-CoV-2.

[0039] In some embodiments provided herein, antibody-targeted analytes in biological samples from subjects may be detected. In some embodiments, such as the detection of a subject's viral infection, detection of antibodies produced by the subject against a virus may be included. Antibody-specific capture agents that selectively bind to viruses, bacteria, fungi, or other infectious agents can be covalently or electrostatically attached to the matrix of a membrane at a test site or region, such as a test line or well. Anti-IgG, anti-IgM, anti-IgA, or other reporters, conjugated to or containing a reporter molecule (e.g., a fluorescent, chromogenic agent, nanoparticle, enzyme, or other detection agent), may also be included in the matrix in or near the sample deposition region.

[0040] The capture agent and an optional reporter molecule are vitrified in the matrix. When a liquid biological sample is brought into contact with the matrix, the vitrified molecules are reconstituted. The target analyte in the biological sample binds to the reporter and is transported along the membrane to the test site by optional capillary action or other mechanisms. An optional capture agent specific to the target analyte localizes the analyte, thereby allowing the reporter to detect its presence and localize it at the test site. As a control, a control capture agent covalently or electrostatically coupled within a control strip may be located downstream of the test area, and a positive control is possible by detecting the movement of the capture agent past the test area. A positive result is achieved by detecting a signal emitted from the reporter localized in the test area.

[0041] In some embodiments, the apparatus and method enable the detection of the presence of the infectious agent itself. In non-limiting examples, a viral coat protein, exemplified by a spike protein or nucleocapsid protein, or a capture agent specifically binding to a portion thereof, is covalently or electrostatically attached to the matrix of a membrane in a test area such as a test line or well. A reporter antibody is provided that also binds to the infectious agent in a different region than the capture antibody. Thereafter, the reporter antibody, bound to a reporter molecule (e.g., a fluorescent, chromogenic agent, enzyme, or other detection), is also included in the matrix in or near the sample deposition area, or is separated from the system and retained before sample application. When a liquid biological sample is brought into contact with the matrix, the vitrified molecules are reconstituted. Any target analyte in the biological sample binds to the reporter antibody and travels along the membrane to the test site, optionally by capillary action or other means, where any capture antibody specific to the target analyte of interest localizes the analyte to the test site. As an optional control, a covalently or electrostatically bound antibody capable of binding to the reporter antibody in a control strip is located downstream of the test area, allowing for a positive control. A positive result is achieved by detecting a signal emitted from or generated by a reporter located at the test site.

[0042] The apparatus provided herein includes a matrix, in which one or more scavengers may be associated at one or more positions within or on the matrix. Exemplary examples of matrix materials include polymers, optionally collagen, elastin, hyaluronic acid and derivatives, sodium alginate and derivatives, chitosan and derivatives, gelatin, starch, cellulose polymers (e.g., nitrocellulose (e.g., Sartorius) (CN95, etc.), methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropylmethylcellulose phthalate), poly(diol citrate) (e.g., poly(octanediol citrate), etc.), casein, dextran and its derivatives, poly(caprolactone), poly(hydroxyl acid), poly(L-lactide), poly(D,L-lactide), poly(D,L-lactide-co-glycolide), poly(L-lactide-co-glycolide), copolymers of lactic acid and glycolic acid, copolymers of ε-caprolactone and lactide, copolymers of glycolide and ε-caprolactone, lactide and 1,4-dioxy Examples include copolymers of sun-2-one, polymers and copolymers comprising one or more residual units from the monomers D-lactide, L-lactide, D,L-lactide, glycolide, ε-caprolactone, trimethylene carbonate, 1,4-dioxan-2-one or 1,5-dioxepant-2-one, poly(glycolide), poly(hydroxybutyrate), poly(alkyl carbonate and poly(orthoester), polyester, poly(hydroxyvaleric acid), polydioxanone, poly(ethylene terephthalate), poly(malic acid), poly(tartonic acid), polyanhydride, polyphosphazene, and poly(amino acid), as well as copolymers of the above polymers, and mixtures and combinations of the above polymers.Exemplary examples of such polymers are described in U.S. Patent Application Publication No. 2018 / 0125990 and the literature cited herein, such as Polymers in Controlled Drug Delivery (Illum, L. & Davids, SS, eds., Wright, Bristol 1987); Arshady, J. Controlled Release 17:1-22 (1991); Pitt, Int. J. Phar. 59:173-96 (1990); and Holland et al., J. Controlled Release 4:155-80 (1986). In certain embodiments, the first and second matrices are independently selected from the group consisting of collagen, polycaprolactone, polylactic acid, lactic acid-glycolic acid copolymers, or combinations thereof.

[0043] Optionally, the matrix exists in the form of a fibrous, random or ordered mesh defining channels or other continuous or discontinuous access paths for the fluid or target analyte. Optionally, the matrix exists in the form of a nonwoven fibrous mesh with high porosity (e.g., greater than 50% by volume).

[0044] The polymers that may be used in the matrix herein may optionally be formed into porous meshes, such as filters, symmetrical meshes, or other such porous sheet materials. Exemplarily, polymers may be formed into fibrous networks by methods including electrospinning. In electrospinning, the desired polymer is placed in a desired solvent (e.g., 2,2,2-trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP)) and subjected to an electrospinning process to form fibers of desired cross-sectional dimensions and length, which are arranged in a desired direction (optionally, randomly) so that a pore size (average distance between strands) is achieved, allowing materials, analytes, activators, etc., to pass through or be retained within the polymer network.

[0045] Optionally, the matrix may contain two or more polymer layers, and optionally, three, four, five, six or more polymer layers.

[0046] Optionally, the matrix is ​​made of a hydrophilic material. Optionally, the matrix is ​​made of a hydrophobic material. The relative hydrophobicity of the matrix can be adjusted to a desired level by mixing two or more matrix materials and / or by post-formation treatment of one or more matrix materials.

[0047] In some embodiments, the matrix includes pore size, which is optionally suitable for controlling sample flow or regulating the size of molecules that can pass through the system. In some embodiments, the pore size in the matrix is ​​approximately 5 microns to approximately 500 micrometers (μm), or any value or range in between. The resulting matrix pore diameter is optionally in the range of 10 μm to 200 μm, optionally 10 μm to 40 μm, optionally 10 μm to 35 μm, optionally 10 μm to 30 μm, optionally 10 μm to 30 μm, and optionally 10 μm to 25 μm. In some embodiments, the matrix pore diameter is selected to allow liquid biological samples and reporters or target analytes present in the biological samples to flow. In multilayer embodiments, different layers of the matrix component may consist of different pore diameters. For example, the screen layer may include a matrix having pore sizes selected to capture and isolate cells (if desired) or other contaminants, while allowing liquids, antibodies, or other target analytes to pass from the screen layer to the membrane layer. Exemplary matrices can be found in International Patent Application Publication No. WO2020 / 086812, and methods for vitrifying or incorporating molecules into the matrix are also taught. Optionally, the matrix includes a layer of fibrous mesh, which is optionally a nonwoven fabric. The creation of the fibrous mesh may be achieved by creating fibers having fiber diameters of 0.1 μm to 200 μm. Optionally, the polymer is present in fibers with fiber diameters greater than 0.5 μm, optionally 0.5 μm to 4 μm, optionally 0.5 μm to 3 μm, and optionally 0.5 μm to 2 μm.

[0048] The matrix is ​​optionally 0.1 μm or thicker, optionally 0.5 μm, optionally 1 μm, optionally 2 μm, optionally 5 μm, optionally 10 μm, optionally 20 μm, optionally 50 μm, optionally 100 μm, optionally 200 μm, optionally 500 μm, or thicker.

[0049] In some embodiments, the matrix comprises polycaprolactone (PCL), collagen, or a combination thereof. A key feature of such water-stable polymers is their ability to form networks or fibers on which one or more scavengers can be vitrified or contained. Therefore, the matrix needs to be sufficiently stable in an aqueous environment to function as a suitable surface for vitrification of an aqueous vitrification medium containing one or more desired molecules, such as a reporter and / or scavenger. Exemplarily, the matrix comprises PCL with a Mn of approximately 80,000 Da.

[0050] In some embodiments, the capture agent (e.g., antigen, aptamer, or antibody) is optionally covalently bound to the matrix at the test site or control site. Alternatively, the capture agent is electrostatically bound to the matrix. Covalent binding to the PCL water-stable polymer can be, for example, a capture agent (e.g., SARS-CoV-2 spike (S) protein or nucleocapsid (N) protein), an aptamer (e.g., ACE-2 peptide or protein), or an antibody (e.g., anti-human SARS coronavirus nucleoprotein mouse MAb (40143-MM05, Sino Biologicals) or anti-human SARS coronavirus nucleoprotein rabbit MAb (40143-R001, Sino Biologicals). This can be achieved by acrylicating the capture agent by reacting a product (manufactured by Biologicals) with polyethylene glycol diacrylate in phosphate-buffered saline containing ACRL-PEG-SVA5000 and the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone. This can be coated on one or both sides and reacted with a PCL layer, and the coated layer can be irradiated with UV light to covalently bind the capture agent to the matrix. This covalently binds the antigen, aptamer, or antibody to the matrix. The resulting material can then be used to vitrify one or more additional activators into the matrix, or can be used in a multilayer system having another layer containing one or more vitrified activators into the matrix as described herein. Optionally, the capture agent does not need to be covalently bound to the matrix or any part thereof.

[0051] Optionally, when the capture agent is an antibody, it has been found that pre-coating the test site (or other desired site) with an anti-Fc antibody and then localizing the capture antibody to that site improves the ability of the capture antibody to react with the analyte in the sample. Therefore, the test site, control site, or other site can be optionally formed by first contacting the membrane with an anti-Fc antibody, drying it, and then contacting the same site with the desired capture antibody. As an example, 2 mg / mL of goat-produced anti-mouse IgG (Fc-specific) antibody (Sigma Aldrich M4280-1ML) is striped onto a CN95 membrane at a distribution / conversion rate of 0.291 μL / mm using an automated lateral flow reagent dispenser (ALFRD) from Claremont Bio (Upland, California). After the first layer has dried, the desired monoclonal antibody (e.g., 1 mg / mL in 5% disaccharide) is coated onto the first layer and vitrified according to the instructions of U.S. Patent No. 10,433,540.

[0052] It has been found that the scavenger can be directly vitrified during the electrospinning process, forming a vitrified material layer that may be associated with or present within the matrix or a portion thereof. By mixing pullulan, trehalose (as an exemplary vitrifying agent), and a desired reporter, substrate, scavenger, or combination thereof within the electrospinning apparatus, pullulan serves as the foundation for fiber formation. In this way, the scavenger, reporter, substrate, or combination thereof does not crystallize during the electrospinning process, but instead transitions to a vitrified glassy state so that the activator can be incorporated into the matrix or a portion thereof so that it is stable with heat and storage time.

[0053] The capture agent or reporter is optionally an antibody. As used herein, the term “antibody” (and “antibodies”) includes monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single-chain antibodies, bispecific antibodies, simianized antibodies, and humanized antibodies, as well as Fab fragments, including products of Fab immunoglobulin expression libraries. Intact antibodies, fragments thereof (e.g., Fab or F(ab')2), or their engineered variants (e.g., sFv) may also be used. Optionally, the antibody is humanized as recognized in the art. Optionally, the antibody is not humanized. The antibody may be any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any of their subclasses.

[0054] Optionally, the capture agent is an aptamer. In some embodiments, the capture aptamer is the human angiotensin-converting enzyme 2 (ACE2) protein, or a peptide or fragment thereof, or (in the case of a nucleotide aptamer, for example) a mimic thereof. The human ACE2 protein is available from various commercial vendors, including, for example, RayBiotech (Peachtree Corners, Virginia) and Novus Biologicals (Centennial, Colorado). The full-length human ACE2 sequence is available under UniProt Accession No. Q9BYF1.

[0055] The apparatus and methods of this disclosure are optionally suitable for the detection of a wide variety of target analytes, including sequences of diverse pathogens and toxins. In some embodiments, the capture agent includes nucleotides, antibodies, proteins, peptides, aptamers, or fragments thereof. In some embodiments, the capture agent is a viral or bacterial nucleotide, protein, peptide, or fragment thereof. In some embodiments, the capture agent is exemplary influenza virus antigen (e.g., hemagglutinin, neuraminidase, etc.), coronavirus antigen (e.g., SARS-CoV protein, SARS-CoV-2 protein, MERS-CoV protein, etc.), Ebola virus antigen, HIV antigen, poxvirus antigen, etc.; bacterial antigen; fungal antigen; toxin antigen such as bacterial toxins or their subunits (e.g., Bacillus anthrax); etc.

[0056] In some exemplary embodiments, the target analyte is a viral antigen (optionally, the SARS-CoV-2 viral antigen) or a host antibody against it (exemplarily, a host anti-SARS-CoV-2 IgG or IgM molecule present in the biological sample). Suitable capture agents for host antibodies against viral antigens include, for example, the SARS-CoV-2 spike (S) protein and the SARS-CoV-2 nucleocapsid (N) protein. SARS-CoV-2 and other viral N and S proteins are commercially available through various vendors, including, for example, RayBiotech (Peachtree Corners, Virginia). Such proteins may be full-length or comprise peptides or subunits of the full-length protein. For example, in some embodiments, the capture antigen may comprise the S1 or S2 subunit of the SARS-CoV-2 spike protein S. However, those skilled in the art will understand that other peptides or fragments thereof may be useful for capturing the target analyte. The SARS-CoV-2 spike protein is characterized by Ou, et al., Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV, Nature Communications 11, article 1620 (2020); and Ibrahim, et al, COVID-19 spike-host cell receptor GFP78 binding site prediction, J. Infect. S0163-4453 (20) (March 10, 2020), each of which is incorporated herein by reference in whole.

[0057] Optionally, the capture agent is an antibody specific to a virus, optionally to the SARS-CoV-2 virus, or optionally to the spike protein of the SARS-CoV-2 virus. Exemplary examples of such antibodies include, but are not limited to, anti-human SARS coronavirus nucleoprotein mouse MAb (40143-MM05, Sino Biological) and anti-human SARS coronavirus nucleoprotein rabbit MAb (40143-R001, Sino Biologicals). The capture antibody may be covalently or electrostatically coupled to one or more regions of a test strip, such as the test region used herein.

[0058] The reporter is optionally bound to a detectable label. Examples of detectable labels include, but are not limited to, enzymes, fluorescent labels, phosphorescent labels, chemiluminescent labels, colorimetric labels, detectable particles or ligands, biotin, etc. Examples of detectable labels for suitable reporter enzymes include, but are not limited to, urease, alkaline phosphatase, horseradish peroxidase (HRP), glucose oxidase, etc.

[0059] The reporter is optionally an antibody or contains an antibody. Reporter antibodies include, but are not limited to, anti-human antibody conjugates containing a detectable label, as used herein. In some embodiments, the reporter antibody contains an IgG or IgM antibody conjugated to a detectable label. Labeled reporter antibodies are available from various suppliers, such as Rockland Antibodies and Assays (Limerick, Pennsylvania) and Sigma-Aldrich (St. Louis, Missouri). Exemplarily, the labeled reporter antibody is optionally conjugated to an enzyme, horseradish peroxidase. Methods for conjugating antibodies to horseradish peroxidase or other enzymes are known in the art, and the conjugation of HRP to an antibody can be performed using the LYNX Rapid HRP Antibody Conjugation Kit, available from Bio-rad Antibodies (Hercules, California).

[0060] A capture agent, reporter, substrate, or any other desired molecule may be vitrified to or on any part of the apparatus in accordance with the teachings of U.S. Patent No. 10,433,540 and Patent Application No. PCT / US2021 / 019887. Briefly, a vitrification medium is combined containing one or more activators to be vitrified into a matrix and placed in or on the matrix. The vitrification medium may contain at least one vitrifying agent. Exemplary examples of vitrifying agents include, but are not limited to, dimethyl sulfoxide, glycerol, sugars (e.g., trehalose), polyhydric alcohols, methylamines, betin, antifreeze proteins, synthetic antinuclear agents, polyvinyl alcohol, cyclohexanetriol, cyclohexanediol, inorganic salts, organic salts, ionic liquids, or combinations thereof. In embodiments, one, two, three, four, or more vitrifying agents are included in the vitrification medium.

[0061] The vitrifying agent is included in the vitrification medium at a concentration dependent on the identity of the vitrifying agent. In some embodiments, the concentration of the vitrifying agent is below a concentration that is toxic to the biological sample being vitrified. As used herein, “toxicity” means that functional or biological viability is not achieved when the sample is subsequently used, or that the biological sample is unsuitable for subsequent analysis. In various embodiments, the concentration of the vitrifying agent is 500 micromoles (μM) or more, 6 moles (M) or less, or any value or range in between. For example, in various embodiments, trehalose is included at a concentration of 1 mM or more and 6 M, and optionally, 150 mM or more and 6 M. In some embodiments, the total concentration of all combined vitrifying agents is 1 mM or more and 6 M, and optionally, 1 mM or more and 6 M.

[0062] In some embodiments, the vitrification medium is intended to further contain other components, such as, but not limited to, water or other solvents, buffers, one or more salts, RNase or DNAse inhibitors, or combinations thereof. The buffer is any agent having a pKa of 6 to 8.5 at 25°C. Exemplary examples of buffers include choline, betaine, HEPES, TRIS, PIPES, MOPS, etc. In some embodiments, the buffer is a buffer containing a large organic ion (greater than 120 kDa), such as choline, betaine, or HEPES. In embodiments containing a buffer, the buffer is provided at a concentration suitable for stabilizing the pH of the vitrification medium to a desired level.

[0063] Salts may include, but are not limited to, sodium salts, potassium salts, chloride salts, or combinations thereof. When contained in a vitrification medium, salts may be provided at concentrations of 1 mM to 500 mM. For example, salts may be present at concentrations of 1 mM to 500 mM, 1 mM to 400 mM, 1 mM to 300 mM, 1 mM to 250 mM, 1 mM to 200 mM, 1 mM to 150 mM, 1 mM to 100 mM, 1 mM to 75 mM, 1 mM to 50 mM, 1 mM to 25 mM, 25 mM to 500 mM, 25 mM to 400 mM, 25 mM to 300 mM, 25 mM to 250 mM, and 25 mM to 200 mM. The ranges may be less than or equal to mM, 25 mM to 150 mM, 25 mM to 100 mM, 25 mM to 75 mM, 25 mM to 50 mM, 50 mM to 500 mM, 50 mM to 400 mM, 50 mM to 300 mM, 50 mM to 250 mM, 50 mM to 200 mM, 50 mM to 150 mM, 50 mM to 100 mM, 50 mM to 75 mM, or any range or all ranges within or included in those ranges.

[0064] A scavenger, reporter, substrate, or any other desired material may optionally be vitrified on or off a particular device in accordance with the teachings of U.S. Patent No. 10,433,540. Optionally, the molecule to be vitrified may be bound in a vitrification solution containing a sugar (e.g., trehalose) and further containing a combination of divalent metal ions and a chelating agent. Exemplary divalent metal ions include salts of Ca, Mg, Co, Fe, Zn, Mn, etc. Exemplary salts include chlorides and sulfates. Examples of chelating agents include polyols, ethylenediaminetetraacetic acid (EDTA), and ectadic acid (EGTA). The molar ratio of divalent metal salt to chelating agent in the vitrification solution is 10:90 to 90:10, and optionally 50:50.

[0065] Detection of the analyte can be performed by detecting the reporter at a test site or control site by an optional and appropriate method. Exemplary detection methods include a fluorescence reader matched with the reporter, a colorimetric assay, direct detection of a label on the reporter molecule (e.g., gold or others), chemiluminescence, or other such assay systems. In some embodiments, the fluorescence reader is light that emits a wavelength suitable for exciting a detectable label so that fluorescence emission occurs for detection.

[0066] Exemplary, analyte detection is carried out using an enzyme / substrate system, for example, horseradish peroxidase (HRP) or alkaline phosphatase (AP) conjugated with a substrate, where the substrate is 4-chloro-1-naphthol (4-CN), 3,3'-diaminobenzidine (DAB), p-nitrophenyl phosphate (PNPP), CSPD and CDP-Star substrates, DynaLight substrate with RapidGlow Enhancer, o-phenylenediamine dihydrochloride (OPD), 3,3',5,5'-tetramethylbenzidine (TMB) or its derivatives (e.g., TMBM, TMBMX), 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), SuperSignal ELISA Pico Chemiluminescent Substrate, SuperSignal ELISA Femto Maximum Sensitivity, available from ThermoFisher Scientific (Waltham, Massachusetts) and other commercial vendors. Examples include a substrate. In some embodiments, the label is a phosphor (e.g., Cy3, Cy5, alexafluor 488, alexaflour 647, or others). Illumination with an LED or a suitable wavelength source allows for the specific detection of the presence or absence of the desired molecule of interest.

[0067] Optionally, substrates used in any embodiment as described herein include TMB (3,3',5,5'-tetramethylbenzidine), ABTS (2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), OPD (o-phenylenediamine dihydrochloride), PNPP (p-nitrophenyl phosphate), ONPG (o-nitrophenyl-β-D-galactopyranoside), or any combination thereof. Such substrates are functional with HRP or alkaline phosphatase as the enzyme on the reporter, as an example.

[0068] In some embodiments, the target analyte is detected using an enzyme such as HRP, which reacts with a substrate (optionally an insoluble substrate), or a reporter containing the same. Insoluble substrates produce insoluble products, thereby providing improved localization in solid-phase assays such as those provided herein. Insoluble substrates provided herein do not necessarily require the substrate itself to be insoluble in aqueous systems, but rather mean that the product produced by the action of the enzyme is insoluble. Exemplary, non-limiting examples of insoluble substrates include TMBM, TMBMX, and derivatives of 3,3',5,5'-tetramethylbenzidine (TMB), such as 3,3'-diaminobenzidine (DAB) (available from MOSS, Inc., Pasadena, Maryland).

[0069] In other embodiments, the substrate may be a molecule that is specifically cleaved by a target analyte, such as an enzyme. For example, if the target analyte is an enzyme, the binding of a capture agent localizes the target analyte to the test site, enabling specific cleavage of the target substrate at the test site. In some exemplary cases, such a substrate may be a molecule containing a quencher and a fluorophor so that the fluorescence of the fluorophor is quenched before reaction with the target analyte. Upon contact with the enzyme target analyte, the substrate is cleaved and the fluorescent dye becomes fluorescein. Examples of such substrates include, but are not limited to, fluorescein isothiocyanate (FITC)-labeled casein (as found in protease activity kits from ABCAM, Cambridge, UK). Alternatively, the substrate may be p-nitrophenyl-α-D-glucopyranoside (which itself may be vitrified in the first matrix or other region of the instrument) that is converted to glucose and p-nitrophenol by the action of glucosidase in the presence of hydrogen peroxide. The glucoside present at the test site has the activity to cleave p-nitrophenyl-α-D-glucopyranoside after interacting with the scavenger, producing a yellow product. The reaction can be optionally stopped by adding an alkaline sodium carbonate solution if desired, although such stopping is not absolutely necessary in the lateral flow assay apparatus and method used herein.

[0070] In another embodiment, the substrate is localized to the test site by covalent or electrostatic bonding, or by other binding reagents to the test site. The enzyme target analyte acts when the enzyme is collocalized or otherwise moves across the test site. If the colorimetric portion of the reaction product remains localized, the resulting signal also remains localized to the test site, enabling reliable color localization and positive readout.

[0071] A washing solution may be added to remove nonspecifically bound immune complexes. Suitable washing solutions include water, buffer, or other materials. Washing solutions are optionally omitted. In lateral flow assay systems, washing solutions are optionally omitted.

[0072] In some embodiments, the reporter is bound to a detectable label in the form of particles such as nanoparticles. Nanoparticles are particles having nanoscale cross-sectional dimensions, e.g., 1 nm to 1000 nm. Optionally, nanoparticles may have cross-sectional dimensions of 1 to 300 nm, 5 to 80 nm, or 8 to 60 nm. Many nanoparticles are approximately spherical, and their cross-sectional dimensions can be considered the diameter of a spherical particle. Hydrodynamic radius or diameter can also be used to define nanoparticle size.

[0073] The nanoparticles are optionally metals or metalloids, latex, magnetic beads, gold colloids, gold nanoshell particles, polystyrene, carbon, or micelles with labels on their cores, optionally enzymes such as HRP. The nanoparticles may be modified to express a carboxyl or ester of N-hydroxysuccinimide (NHS) to enable strong covalent bonding with primary amines. Other examples of coupling options include modification of the nanoparticles with maleimide followed by coupling to a reporter containing exposed thiols (e.g., produced by treating the reporter with mercaptoethylamine or 2-iminothiolane (Trout's reagent)), modification of the nanoparticles with hydrazine and coupling with oxidized glycans (aldehydes), or the use of click chemistry (e.g., modifying the nanoparticles with strained alkynes and coupling to a reporter modified with azides). Additionally or alternatively, the nanoparticles may be suitable for non-covalent interactions with the reporter. An example of non-covalent bonding is the biotin-streptavidin bond, where one member of the conjugate is biotinylated and the other member is bound to streptavidin.

[0074] In some embodiments, the nanoparticles are nanoshell particles, and optionally, nanoshell particles in which the core (optionally silicon dioxide) is coated with a layer of gold. Exemplary nanoshell particles are available from NanoComposix, Inc. (San Diego, California). It has been found that the resulting color of the particles changes by adjusting the ratio of the shell thickness to the core radius. For example, increasing the ratio of the core radius to the shell thickness shifts the particle color to red. Thus, by adjusting the ratio of the core radius to the shell thickness, the particle color can be adjusted to match or substantially match the color of the product produced by the enzymatic reaction, thereby enhancing the signal detected at the test site. In some embodiments, the color of the nanoshell particles themselves has been found to be sufficient to produce a positive signal visible to the naked eye at pg / ml of the analyte. The ratio of the core radius to the shell thickness is optionally 3:1 to 1:3, optionally 1:3 to 1:12, or any value or range in between.

[0075] Reporter / particle formation can be carried out using techniques recognized in the art. For example, the reporter agent may first be conjugated to a label (e.g., an enzyme or other), and then the labeled reporter agent may be conjugated to a particle such as a gold particle or a nanoshell particle. Alternatively, the particle may be conjugated to the label first, and then optionally, the label or particle may be conjugated to the reporter agent. The label may be conjugated to the particle or reporter agent using a conjugation kit from BioRad, Inc. (Hercules, California). The label or labeled reporter agent may be conjugated to the particle using a conjugation kit from NanoComposix, Inc. If the particle is NHS conjugated, the label and / or labeled reporter agent may be conjugated to the particle using a conjugation kit from CytoDiagnostics, Inc.

[0076] Contrary to the understanding of the art, it has been found that an improved signal can be generated by conjugating a reporter agent to nanoparticles in which part of the reporter agent is bound to a label and part of the reporter agent is not bound to a label. For example, when using an enzyme-bound reporter antibody such as HRP, the detection of the target analyte can be improved by combining both enzyme-labeled and non-enzyme-labeled antibodies in specific ratios that reduce noise in the system while simultaneously improving the signal. Exemplary, the ratio of labeled antibodies to unlabeled antibodies is 80:20 to 20:80. Optionally, the ratio of labeled antibodies to unlabeled antibodies is approximately 80:20:70:30, 60:40, 50:50:40:60, 30:70, or 20:80. Optionally, in some exemplary embodiments, the reporter agent is an antibody that specifically binds to a virus, optionally the SARS-CoV-2 viral spike protein. The particles may be coated with antibodies such that the ratio of antibodies bound to a detectable label or enzyme to antibodies not bound to a detectable label or enzyme is 80:20 to 20:80, and optionally 50:50.

[0077] In some embodiments, the apparatus described herein may further include a removable separator layer positioned between the assay layers, channels, or regions. Optionally, the separator is removed to allow contact between the analyte, reporter, and capture molecule.

[0078] In some exemplary embodiments, the capture agent may be an enzyme. The enzyme capture agent is immobilized on a membrane strip at one or more target sites. When the target analyte or a molecule bound to the target analyte interacts with the enzyme at the target site, the enzymatic conversion of the target analyte or the molecule bound to the target analyte may generate a detectable signal indicating the presence of the target analyte at the target site. In a non-limiting example, the capture agent may be an upstream component of an enzyme cascade in which the capture agent converts the target analyte into the functional molecule (e.g., the enzyme) itself. By using a substrate specific to the functional molecule, detection of the target analyte can be achieved at the target site.

[0079] In one exemplary embodiment, the scavenger is an enzyme (e.g., glucose dehydrogenase or diaphorase). The sample may be applied to a sample pad having a washing agent (e.g., dodecyltrimethylammonium bromide (DTAB)) that releases the desired molecule from the biological sample. The vitrified substrate may be contained in the conjugate pad of the apparatus, and wetting the conjugate pad rehydrates the vitrified material, so that the substrate is delivered to the site of action of the target site with the enzyme scavenger immobilized.

[0080] In some embodiments, apparatuses or methods provided herein can utilize competitive systems for detecting the presence or absence of a target analyte. In non-limiting examples, the capture agent and the target antigen may compete for the same site on the target analyte (e.g., direct competitive interaction), or the binding of the target analyte to the reporter may disrupt the reporter so that it no longer binds to the capture agent (e.g., non-competitive inhibition). Therefore, the absence of a signal at the target site, or the presence of a signal at the control site, indicates the presence of the target substance in the sample.

[0081] In other embodiments, the scavenger may be a nucleotide. As used herein, a nucleotide is a sequence of nucleic acid. The scavenger may be an unlabeled nucleic acid strand having a specific sequence, or optionally, a strand having a quencher. A labeled strand having a detectable label may be bound to the unlabeled strand at the target site. In the presence of a nucleic acid having a specific sequence, the labeled strand dissociates due to the property of the target analyte to compete with the interaction between the labeled and unlabeled strands, and the release of the labeled strand generates a detectable signal indicating the presence of the target analyte.

[0082] In the apparatus and methods provided herein, a biological sample may be applied to a sample pad. The sample pad may contain one or more vitrification assay reagents. Such vitrification assay reagents may contain buffers, detergents, salts, or any other necessary reagents. Such descriptions also apply to sample ports, as separately described herein. As an example, the sample pad may contain a detergent and / or buffer vitrified therein, thereby allowing the stored assay reagents to function to modify one or more properties of the sample being applied (e.g., pH adjustment, dissolution of one or more components, etc.) upon rehydration of the sample pad with a liquid biological sample.

[0083] Optionally, the target analyte is bacteria. As a non-limiting example, bacterial detection can be performed by adding the biological sample to a sample pad (or sample port) containing a vitrifying detergent (e.g., DTAB). Rehydration of the vitrifying detergent allows it to lyse bacterial cells in the biological sample, thereby releasing the target analyte (e.g., NAD) from within the cells. A scavenger can bind to the labeled target analyte (e.g., HRP or biotin-labeled NAD), thereby allowing the labeled target analyte to be competitively removed by the target analyte at the target site. Thus, the decrease in color upon substrate introduction is proportional to the amount of target analyte in the biological sample.

[0084] The detection limits of the assays and methods disclosed herein are advantageously superior to those of conventional ELISA and solid-phase assays. In some embodiments, the detection limits of the analyte of interest are less than about 1000 pg / ml, optionally less than about 100 pg / ml, optionally less than about 10 pg / ml, optionally less than about 5 pg / ml, optionally less than about 2 pg / ml, optionally less than about 1 pg / ml, optionally about 0.9 pg / ml, optionally less than about 0.8 pg / ml, optionally less than about 0.7 pg / ml, optionally less than about 0.50 pg / ml, optionally less than about 0.4 pg / ml, optionally less than about 0.30 pg / ml, optionally less than about 0.2 pg / ml, and optionally less than about 0.1 pg / ml.

[0085] The lateral flow assays provided herein can be completed extremely rapidly while providing excellent low detection limits for the analyte. Detection can be performed in 20 minutes or less (optionally), or in approximately 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute or less (optionally). Thus, the term “rapid” as defined herein is 20 minutes or less (optionally), or in approximately 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute or less (optionally).

[0086] The target analyte may be quantified by one of many possible methods. Exemplarially, multiple test strips may be present on a matrix containing different known amounts of capture antigen, aptamer, or antibody, and the analyte in the sample may be quantified by comparison with a known control, such as a color map, optionally. Optionally, direct quantification may be performed by the signal level in or on a single membrane strip or other test area of ​​the matrix.

[0087] In another embodiment, a kit is provided which includes one of the instruments disclosed herein, reagents necessary to carry out the methods described herein, and optionally instructions for use.

[0088] With reference to Figures 1-4, rapid diagnostic devices 100a, 100b and methods of use are provided in several embodiments. Figure 1 illustrates an exemplary multilayer device 100a comprising a membrane layer 105 containing a first matrix 110, which has a vitrified capture antigen 120 on the first matrix 110, the capture antigen 120 being optionally covalently or electrostatically coupled to the first matrix 110. In some specific embodiments, the capture antigen is a SARS-CoV-2 antigen such as a spike or nucleocapsid protein or peptide. Optionally, a screen layer 155 is positioned on or in contact with the membrane layer 105, the screen layer comprising a second matrix 160, which optionally is configured to substantially capture contaminants present in a liquid biological sample when the screen layer 155 comes into contact with the liquid biological sample. As shown in Figure 1, optionally, at least one reporter is vitrified on the first matrix 110. In some embodiments, the reporter is or includes labeled anti-human IgM130 or labeled anti-human IgG140.

[0089] Figure 2A shows the step of bringing the apparatus 100a of Figure 1 into contact with a liquid biological sample containing a target analyte 150, exemplary such as blood or saliva. Optionally, the target analyte 150 contains human IgG and / or human IgM antibodies specific to a capture antigen 120, such as a SARS-CoV-2 capture antigen or aptamer. Referring to Figure 2B, upon contact with the liquid biological sample, the membrane layer 105 is rehydrated, and reporter antibodies 130, 140 are released from the matrix 110 and become mobile within the system, allowing them to bind to the target analyte 150. Referring to Figure 2C, upon addition of a suitable substrate, the bound reporter antibodies 130, 140 are detected via signal 170. Optionally, the strength of signal 170 correlates with the concentration of the target analyte, thereby allowing quantification of the analyte in the sample by comparison with a known standard.

[0090] Figure 3 illustrates a multilayer device 100b. This multilayer device 100b includes a membrane layer 105 comprising a first matrix 110, the membrane layer 105 having a capture agent in the form of a vitrified antigen or aptamer 120 on the first matrix 110. The capture agent 120 is optionally covalently or electrostatically coupled to the first matrix 110. Optionally, the capture agent is a SARS-CoV-2 antigen, such as a viral antigen, optionally such as a spike or nucleocapsid protein or peptide. A screen layer 155 is positioned on or in contact with the membrane layer 105, the screen layer comprising a second matrix 160, optionally configured to substantially capture contaminants present in a liquid biological sample when the screen layer 155 comes into contact with the liquid biological sample. At least one reporter agent (illustrated as labeled antibody for illustrative purposes) 130, 140 is vitrified onto a second matrix 160. Optionally, the reporter includes labeled anti-human IgM 130 or labeled anti-human IgG 140. Although Figure 3 is presented as an exploded view, it is understood that the screen layer 155 may optionally be located on or in contact with the membrane layer 105.

[0091] Figure 4 shows the step of bringing the apparatus 100b of Figure 3 into contact with a liquid biological sample containing a target analyte 150 (depicted as an antibody for illustrative purposes only), and exemplary with blood or saliva. Optionally, the target analyte 150 contains a human IgG or human IgM antibody specific to the capture antigen 120. Upon contact with the liquid biological sample, the membrane layer is rehydrated, and reporters 130 and 140 are released from the second matrix 160, becoming mobile within the system and available for binding to the target analyte. With the addition of an appropriate substrate, the bound reporter antibodies 130 and 140 are detected via signal 170, as shown in Figures 2B-2C.

[0092] Figure 5 shows exemplary embodiments of a device according to several aspects of the present disclosure, in which a removable separator layer is positioned between two channels. Channel 1 contains a capillary channel having matrix fibers inside, to which a capture molecule (exemplaryly a capture antibody) is covalently or electrostatically bound to the matrix fibers. Channel 2 contains matrix fibers, in which a reporter antibody is vitrified but not covalently bound. Serum is drawn into capillary channel 1, and when the separator is removed, channel 2 is rehydrated, and the reporter antibody is released, allowing it to freely contact the analyte in the serum. The analyte is captured by the capture molecule in channel 1. A magnified image of the fibers in channel 1 is shown in the lower left panel. The upper right panel shows the fluorescence signal of channel 2 after rehydration and release of the reporter antibody, illustrating its transition. The lower right panel shows the fluorescence signal in channel 1 that detects the presence of a target analyte (e.g., VEGF) by binding to the reporter.

[0093] Figure 6 shows the comparative advantages of the apparatus and method in this disclosure compared to standard available methods. Conventional ELISA takes more than 5 hours and has a detection limit of 6.4 pg / mL. Luminex® takes more than 3 hours and has a detection limit of approximately 10 pg / mL or less. In contrast, the apparatus and method in this disclosure can rapidly detect the target analyte within approximately 15 minutes at room temperature, without the need for cold chain transport or storage, with a detection limit of approximately 0.1 pg / mL or less.

[0094] Figures 7A–7C illustrate various apparatuses in the form of exemplary lateral flow membrane strips 200a, 200b, and 200c. In the illustrated apparatuses, the membrane strip includes a conjugate pad 210 in which a labeled trapper is vitrified, a test area 220, and an optional control area 230. Figure 7A shows the lateral flow membrane strip 200a, where the test and control areas 220 and 230 exist as strips or lines on the membrane. Figures 7B and 8C show the lateral flow membrane strips 200b and 200c, respectively, where the test and control areas 220 and 230 exist as wells on the membrane strip. It should be understood that the configuration of the test and control areas may vary (i.e., strips, line wells, circles, shapes, etc.) as long as they are detectable and / or identifiable when test and control signals are present.

[0095] Figure 8 shows a lateral flow assay apparatus 300 in which the membrane is contained within a housing. Figures 9A and 9B are exploded views of housing cassettes 300a and 300b, respectively, having a base 310, a tray 320, a membrane strip 325, a sample pad 330, a waste pad 340, and a cover 350. Figure 9A shows a membrane strip formed to contain vitrification material before being inserted into the tray 320 of the cassette. The membrane strip is optionally removable and replaceable within the housing. In Figure 9B, the membrane strip is vitrified on the tray 320 of the cassette before assembly of the cassette. Optionally, the tray containing the membrane strip is removable and replaceable. Optionally, the cover includes slots or openings for viewing control and test signals of the membrane strip beneath it.

[0096] Referring to Figure 10, an exemplary embodiment of a lateral flow membrane strip 400 is provided. The membrane strip 400 may include a sample pad 405 for receiving a liquid biological sample. The sample pad is in fluid contact with a conjugate pad 410 containing a vitrified matrix 425. Reporter agents (optionally, antibodies) 430, 440 are optionally vitrified into the matrix 425 of the conjugate pad. It is understood that the reporter agents may be bound to particles as described herein. In some embodiments, the apparatus does not include reporter agents vitrified into or on the conjugate pad, but they may be added with the sample, added after the sample, or added before the sample is brought into contact with the sample pad. Downstream of the conjugate pad, the exemplary membrane strip includes test sites or lines containing vitrified and immobilized capture agents (e.g., antibodies, light sources, or aptamers), which are optionally covalently or electrostatically bonded to them and vitrified in or on them. An optional control site or line may be located downstream of the test area and may contain a vitrified and immobilized capture agent (e.g., antigen, aptamer, or antibody) 435, 445 (exemplary, human IgG or IgM). A waste pad or absorbent pad 450 may be located at the downstream end of the membrane strip to collect excess reagent and / or facilitate the movement of reagent through the lateral flow assay.

[0097] Figure 11A shows the step of bringing the membrane strip 400 into contact with a liquid biological sample 480 containing analytes of interest 485a, 485b, and 485c. As the liquid biological sample 480 moistens the sample pad 405, the liquid and analytes in the liquid sample move through the membrane strip to the vitrification matrix 425 by capillary action. The partial liquid of the biological sample reconstitutes the vitrification matrix 425 by releasing reporter agents 430 and 440 that are mobile in the system. As shown in Figure 11B, the target analyte 485 is captured by the capture agent 420 in the test line. Reporter antibodies 430 and 440 bind to the capture antibodies 435 and 445 in the target analyte and / or control line. Excess reagent is absorbed into the waste pad 450. Once the reporter is localized and optionally a substrate is added, the signal can optionally be detected using a light source 470, or by visual inspection for color change, or by other means for detecting the signal.

[0098] Figure 12 shows a lateral flow assay containment cassette 500 containing multiple membrane strips. Figures 13A and 13B are exploded views of cassettes 500a and 500b, respectively, having a base 510, a tray 520, multiple membrane strips 525, a sample pad 530, a waste pad 540, and a cover 550. In Figure 13A, the membrane strips are vitrified before being inserted into the cassette tray 520. The membrane strips are optionally removable and replaceable independently. In Figure 13B, the membrane strips are vitrified on the cassette tray 520 before the cassette is assembled. The tray 520 is optionally removable and replaceable. While test and control lines are exemplified on the membrane strip 525, it should be understood that the test and control areas may be in the form of lines, wells, or any other configuration that allows for rapid detection of signals. Optionally, the cover includes slots or openings for viewing the control and test signals of the membrane strips beneath it.

[0099] Figures 14 and 15 show exemplary multistrip lateral flow assays 600 according to several aspects of the present disclosure, both in a housing (15A) and without a housing (15B). The assay includes a sample pad 605 for receiving a liquid biological sample. Although three membrane strips are illustrated, it will be understood that any number of membrane strips, including two, three, four, five, or more membrane strips, may be included in the multistrip assay. Each membrane strip has a conjugate pad 615 containing a vitrification matrix, one or more vitrification reporters 630, 640, and optionally one or more substrates 645. A test line having a capture agent 620 (optionally coupled covalently or electrostatically) is positioned downstream of the conjugate pad 615. Optionally, individual membrane strips may contain the same or different capture agents 620. A control strip is positioned downstream of the test strip and contains one or more covalently or electrostatically coupled control capture agents 655, 660. A waste pad 650 is placed downstream of the membrane strip to receive excess sample fluid. Optionally, the waste pad contains an indicator 675, such as a dye, which provides a color change in the presence of a protein, liquid, or other indicator to indicate the completion of the test. Exemplarily, the dye includes Coomassie G-250.

[0100] When the multi-strip lateral flow assay 600 comes into contact with one or more liquid biological samples, a portion of the sample liquid moistens the sample pad 605 and flows downstream into the membrane strip. As the sample liquid reconstitutes the conjugate pad 615, one or more vitrified reporters 630, 640 and substrates 645 are released from the matrix of the conjugate pad 615. The reporters are then free to bind to the target analyte in the sample. The fluid and mobile components move downstream, where the “sandwich” binding is detectable in the test and control lines. To stop the interaction between the reporter and substrate, a stop solution may be added to the sample pad.

[0101] Optionally, in any of the embodiments disclosed herein, the control area (e.g., a strip or well) may contain, in addition to or instead of the capture agent, an indicator dye such as Coomassie G-250. In such embodiments, the indicator dye detects the presence of any protein in the liquid sample and thereby indicates the completion of the test via a signal, e.g., a change in color.

[0102] Optionally, the capture agent is an aptamer, such as an aptamer that binds to an intact pathogen. Optionally, the aptamer is an ACE2 protein or peptide capable of binding to a viral surface protein, and the viral surface protein is optionally a SARS-CoV-2 surface protein. In some embodiments, the membrane may include a vitrification matrix and a vitrification chromogen that complex with the bound virion / aptamer complex. For example, when a liquid biological sample comes into contact with the matrix, the matrix is ​​reconstituted and the chromogen is released. The target virion binds to the capture aptamer, and the chromogen binds to the complex to generate a detection signal.

[0103] Optionally, the vitrification scavenger is a biotin-labeled aptamer that binds to an intact pathogen. In this embodiment, the membrane may include a vitrification matrix, a vitrification reporter, and a vitrification biotin-labeled aptamer. The biotin-labeled aptamer binds to a virion (or other target), and the complex is captured in the test region by covalently bound avidin or streptavidin. The reporter binds to this complex and generates a detection signal.

[0104] Optionally, the vitrification scavenger is an oligonucleotide (e.g., an aptamer) that binds to an intact pathogen, optionally to a virus, and optionally to SARS-CoV-2. The membrane may also contain a vitrification matrix and a vitrification beacon aptamer, the beacon aptamer having a phosphor and a quencher. If the beacon aptamer is not bound to the target analyte, the phosphor is quenched by the quencher and no signal is generated. However, when the beacon aptamer binds to SARS-virion, the complex is captured in the test region by covalently or electrostatically bonded oligonucleotides. At this point, the formation of the aptamer complex prevents the quenching of the beacon aptamer's fluorescent dye, and a signal is generated.

[0105] Optionally, the vitrification capture agent is an antibody that binds to an intact pathogen, the intact pathogen being a virus or a part thereof, such as the S or N protein of SARS-CoV-2, as one exemplary example. The membrane may also contain a vitrification matrix and the vitrification capture antibody. Once the liquid sample reconstitutes the capture antibody, the capture antibody can freely bind to any analyte in the system. In some embodiments, if a reporter agent is also bound to the analyte, the reporter is concentrated in the region where the capture agent is localized, optionally covalently or electrostatically. Detection of the presence or absence of the analyte may be achieved by a change in color or concentration, optionally by a reaction between an enzyme bound to the reporter agent and a suitable substrate to produce a detectable product.

[0106] Figure 16A is a diagram showing an exemplary diagnostic assay apparatus 600, which optionally includes multiple membrane strips 610 within a housing. As shown in Figures 16A and 16B, each membrane strip 610 includes a conjugate pad 615 having a vitrified reporter in a matrix, and multiple test regions 622 containing vitrified covalently or electrostatically coupled capture agents for capturing a target analyte. As the target analyte moves downstream from the conjugate pad, the capture concentration is successively diluted across the multiple test regions 622, thereby providing multiple signals that can be compared to a color map or other reference standard to determine the concentration of the target analyte.

[0107] Figure 16 shows an exemplary diagnostic assay apparatus 700. The assay apparatus 700 optionally includes a sample pad 705 and optionally includes a substrate pad 702 positioned adjacent to and / or overlapping and / or in contact with the sample pad 705. Optionally, the sample pad may receive a liquid biological sample, exemplary a blood sample or saliva sample, and may isolate, remove, or destroy red blood cells in the sample (if present) so that the remaining components of the sample and the target analyte can flow into the conjugate pad 715. The conjugate pad 715 comprises a matrix 725 and at least one reporter 730a, 730b, the at least one reporter 730a, 730b being optionally vitrified in the matrix and optionally conjugated to a particle. Optionally, the conjugate pad 715 comprises at least one labeled reporter and optionally an antigen 730b being vitrified in the matrix. Optionally, the conjugate pad 715 contains at least one labeled control antibody 730a vitrified in a matrix. Downstream of the conjugate pad 715, and optionally integral therewith, is a membrane strip containing one or more test regions 722, each test region containing a vitrification-immobilized capture agent 720, the capture agent being specific to the type of test analyte (e.g., human IgG, SARS-CoV-2 S protein, etc.) or control. Optionally, each test region may contain a different vitrification-immobilized capture agent 720, such as a capture antigen, antibody, or aptamer. The control region 745 is optionally placed on the membrane strip, optionally downstream of the test regions. In some embodiments, the assay further includes a waste fluid pad 750 for absorbing excess fluid from the sample.

[0108] In some embodiments, the substrate pad 702 includes an embedded or vitrified substrate to which a reactant suitable for color development upon reaction with the label or label molecule is bound. In a non-limiting example, the capture agent may be bound to HRP. The substrate pad may contain one or more substrates for HRP (or other enzyme labels) such that the substrate comes into contact with HRP and a color indicating a positive result is produced at the location of HRP on the membrane strip. Optionally, when a biological sample is added to the sample pad, a buffer may be added to the substrate pad to reconstitute and release the substrate so that the substrate flows through the membrane strip and binds to the appropriate reporter molecule.

[0109] In some embodiments, the sample pad 705 may further contain an agent for isolating and / or lysing red blood cells, thereby allowing plasma and target analytes to move across the membrane strip. Various erythrolytic agents are known in the art. Exemplary agents include, but are not limited to, erythrolytic buffers (available from various suppliers) or embedding solutions containing one or more of ammonium chloride, potassium bicarbonate, and disodium EDTA. Those skilled in the art will understand that erythrolytic agents are well known in the art.

[0110] When a biological sample (exemplified, a blood sample) containing a target analyte (exemplified, an antibody against an infectious agent, the virus itself, or a part thereof such as the viral S protein) is brought into contact with the apparatus 700, the blood is deposited onto the sample pad. Optionally, a buffer is added to the substrate pad to release the substrate molecules. Optionally, the sample pad contains an agent that isolates and / or lyses red blood cells in the sample, thereby allowing the plasma and target analyte to move across the membrane strip.

[0111] A vitrified reporter, such as a labeled viral antigen 730b, and a vitrified control antibody 730a are released from the conjugate pad 715 as the liquid from the biological sample reconstitutes the matrix. The sample, analyte, reporter molecule, and control molecule flow downstream toward the test area. In the test area, individual target analytes are captured at the desired location depending on the identity of the captured molecule in each test area. The control antibody binds to the capture antibody in the control area, confirming that the test is complete and / or successful. In some embodiments, a buffer may be added to the substrate and optionally further include a detection substrate for generating a signal in the presence of a “sandwich” complex in the test area.

[0112] Optionally, a biological sample is taken from a subject and combined with an assay buffer suitable for adjusting one or more properties of the biological sample and / or for releasing one or more analytes within the biological sample. In an exemplary embodiment, the test sample may be saliva. The saliva may be combined with an assay buffer containing a washing agent to release a desired protein or nucleic acid analyte from a target viral system. The resulting sample may then be applied to the sample pad of the instrument, thereby the fluid may optionally reconstitute a reporter agent at or downstream of the sample pad (e.g., in a conjugate pad) so that the reporter agent can freely bind to the analyte of interest in the sample. The lateral flow of the sample moves toward a test area 722 which may contain one or more capture agents that specifically bind to the analyte of interest, thereby capturing the analyte and reporter agent and localizing them at the test site. Optionally, to detect an analyte in the sample, a substrate is applied to or upstream of the test site to produce a colored product of a reaction catalyzed by an enzyme bound to the reporter agent.

[0113] In some embodiments, the sample is brought into contact with a detergent before or during contact with the drug on the sample pad. The detergent is any detergent suitable for releasing proteins from viral particles. In some embodiments, an exemplary detergent is Zwittergent 3-14 (CAS 14933-09-6) (optionally, 1-5% by weight in the fluid). Optionally, the detergent is combined with dithiothreitol (DTT), which may help improve signal results in the assay system.

[0114] Alternatively, the sample can be dissolved to release one or more analytes on the sample pad. Optionally, the sample pad contains a detergent combined with DTT, DTAB, or other vitrified detergent on the sample pad, and the sample is applied directly to the sample pad to reconstitute the detergent and directly release analytes from any desired sources contained in the sample.

[0115] Simultaneously with or following the release of the target analyte, the sample is brought into contact with a reporter agent. The reporter agent may be contained in the same solution as the washing agent or in a separate solution and conjugate to the sample, or the reporter agent may be contained in or on the conjugate pad in a vitrified state. The reporter agent (optionally an antibody, optionally conjugated to a particle) is then free to come into contact with the analyte in the sample. After the sample has passed through the conjugate pad, a capture agent at the test site concentrates and collects the analyte at the test site, thereby concentrating the label in the reporter agent and providing a detectable signal at the test site.

[0116] In some embodiments, it has been found that delivering a substrate to a target site for enhancing or developing a positive indication of the presence of a target analyte can be improved by altering the flow characteristics of the substrate through the instrument. Readout in lateral flow assay instruments is typically performed visually, which is less sensitive than machine readout. Therefore, using highly sensitive color, fluorescent, luminescent, or other readout devices suitable for high-sensitivity detection in situ or outside the laboratory tends to improve the detection limits of the instrument. Enzymatic amplification, such as the use of enzyme-conjugated reporters (e.g., HRP), improves sensitivity but introduces other problems in effectively localizing signal generation to the target site.

[0117] The rate at which the liquid (liquid biological sample or a portion thereof) passes through the membrane has been found to affect assay performance. Lowering the fluid flow rate improves the interaction between the capture agent and the target analyte, but increases the probability of reporter retention outside the target site. This is particularly problematic when using nanoparticles such as gold as the reporter visualizer. When using enzyme / fluorescence / luminescence amplification, reporter retention outside the target site can lead to high background and / or false negatives due to non-specific retention of the reporter, which is later amplified by substrate addition. When using a lateral flow apparatus including a membrane strip and associated conjugate pads and / or substrate pads, as substantially described herein and optionally depicted in Figure 17A, the liquid flow rate is modified depending on the total strip volume and the amount of liquid sample placed in the system relative to the time since liquid application. As shown in Figure 17B, the dotted line indicates the total wet volume of the strip. The strip house increases as the flow duration progresses to the total volume. However, as shown by the solid line, the rate at which the liquid flows through the membrane strip changes over time, depending on the time since sample application. This can be explained by dividing the total flow rate into four different flow modes, separated by vertical dashed lines in Figure 17B. Region 1 shows the initial wet-out of the membrane strip. Region 2 illustrates the wetting of the membrane strip and waste pad interface. Region 3 illustrates the wet-out of the waste pad. Finally, Region 4 shows the flow rate due to evaporation from the waste pad (if present). The maximum flow rate is reached in Region 1, which represents the initial wet-out where the membrane strip material fills the pores and moves the liquid from the conjugate pad through the membrane. When the membrane strip is saturated with fluid to about 75% of its volume, the flow rate becomes substantially false. In apparatuses such as those provided herein, the length and width of the channels can be adjusted to improve the fluid flow characteristics and improve the specific binding of the target analyte to the test site, while sensitivity can be improved by moving the unbound reporter away from the site to reduce background. Optionally, at the end of Region 3, illustrated in Figure 17B at about 8–10 minutes, the substrate may be added or a display may be obtained.

[0118] Thus, it was found that by specifically creating a surface flow of the substrate, the enzyme / substrate reaction can be more effectively localized to the target site, lowering the detection limit, reducing false negatives, and improving the signal-to-noise ratio. Further challenges remain regarding substrate addition for enzyme amplification. Adding a liquid substrate alters the overall flow characteristics of the liquid on the membrane strip. Therefore, the use of a liquid substrate causes the membrane strip to overlay, which is especially true from region 3 (optimal substrate addition time) onward. When a liquid substrate is used, the additional liquid pool takes a very long time to clear up the test line region and may leave nonspecific signals that lead to false positives.

[0119] These problems can be solved, according to some embodiments provided herein, by optionally bringing a dry form of the substrate into contact with the membrane layer after the membrane layer has been wetted with a biological sample or the like. The flow of the substrate on / in the membrane layer becomes a surface flow, with a reduced amount of flow through the membrane itself. Thus, according to some embodiments, the substrate is vitrified into a substantially dry substrate film, which is then brought into contact with the membrane layer after wetting. The fluid in the wet membrane layer used to rehydrate the vitrified material in the membrane layer is also used to rehydrate the substrate film, thereby enabling the flow of the substrate from the substrate film to the desired target site and improving signal generation. The substrate may be introduced at a site considerably upstream of the test site so that a substantially linear flow of the substrate is introduced into the membrane to improve signal and false positives, as substantially shown in Figure 17C.

[0120] Figures 18A-E show various embodiments of exemplary assay apparatus 800 for delayed application of substrate to a membrane layer to achieve these unique substrate flow characteristics. The membrane strip includes a sample pad 805 and a conjugate pad 815 positioned adjacent to and / or overlapping the sample pad 805. The conjugate pad may contain one or more reporters vitrified in or on it, which flow onto the membrane strip containing one or more test sites 822 on it. Downstream of the conjugate pad 815 is a membrane strip containing one or more test areas 822, which are fluidically connected thereto and optionally integral thereto, each test area containing a vitrification-immobilized capture agent, the capture agent being specific to the type of test analyte (e.g., human IgG, SARS-CoV-2 S protein, etc.) or control. Optionally, each test area may contain different vitrification-immobilized capture agents such as capture antigens, antibodies, aptamers, etc. A control region 845 is optionally positioned on the membrane strip, optionally downstream of the test region. In some embodiments, the assay further includes a waste fluid pad 850 for absorbing excess fluid from the sample. The housing 860 may be formed from an upper housing 862 and a lower housing 863 mounted together to accommodate the membrane strip, although it is understood that the upper and lower housings may be integral and formed from a single piece. The housing may further include a substrate delivery lever 870 containing a substrate pad 875 attached to or integrated with it. The substrate pad may be attached to the substrate delivery lever by adhesive, optionally by a peelable adhesive, or by any desired means such as directly incorporating a membrane suitable for the vitrification of the substrate within into the lever. The substrate pad 875 is positioned on the lever 870 such that when the lever is actuated, the substrate pad comes into contact with a portion of the membrane to rehydrate the substrate and is movable for reaction with the reporter.

[0121] Continuing to refer to Figure 18, the lever 870 can be fitted into the lever slot 881 in the housing 862. The mounting between the lever 870 and the lever slot 881 allows the lever to be actuated, enabling the substrate pad to contact the membrane strip. In some embodiments, the lever includes a threaded element or shape complementary to the threaded element or shape in the lever slot, such that rotating the lever moves the substrate pad from a first position to a second position in contact with the membrane strip, as illustrated in Figure 18C. In other embodiments, the lever 780 may be frictionally fitted into the lever slot 881 so that by applying a desired amount of force, the substrate pad 875 moves to contact the membrane strip. In some embodiments, it is desired to include a system that can be actuated by a user to move the membrane strip from a first position to a second position and to contact the membrane strip at a desired time, while being resistant to accidental movement of the substrate membrane. Note that the position of the substrate pad in the housing can be adjusted to contact the membrane strip at any desired position. Optionally, the lever is positioned to bring the substrate pad 875 into contact with the membrane strip at or near the conjugate pad 815, as illustrated in Figure 18B. Alternatively or additionally, the lever is positioned to bring the substrate pad and the membrane strip into contact at or near the sample pad 805. Alternatively or additionally, the lever is positioned at or near the inspection site of the sample pad, or at any position upstream of the inspection site, so that the substrate pad is in contact with the membrane strip, allowing the substrate to flow from the substrate pad to the inspection site by surface capillary action during rehydration.

[0122] The housing 862 optionally further includes a sample port 880 positioned substantially close to the sample pad 805 to allow the user to easily localize a biological sample onto the sample pad. The sample port 880 may further include an assay reagent delivery port 885. The assay reagent delivery port may contain one or more assay reagents, vitrified therein, which are used to replenish the biological sample or to deliver one or more additives to the membrane strip. Exemplary examples of assay reagents include, but are not limited to, buffers (such as potassium phosphate, HEPES, TRIS) and washing agents (such as tween). The user may take a biological sample or a portion thereof, add it to the delivery port 885 to rehydrate an additive, and conjugate the additive with the biological sample to improve the overall assay or instrument performance. Optionally, the instrument includes an assay reagent delivery port. Optionally, the instrument omits an assay reagent delivery port.

[0123] Continuing to refer to Figure 18, the apparatus housing may further include a lower housing 863 which includes one or more membrane strip slots 890, 892 for housing and maintaining the orientation of a membrane strip within the housing. A readout shelf 895 for maintaining the position of the membrane relative to the lower housing may be optionally present, which prevents distortion of the membrane when the lever 870 is operated and forces the membrane strip into contact with the substrate pad 875.

[0124] Referring to Figure 18E, the lever 870 may include an enlarged area 871 that can be stationary over one or more inspection areas 822 when the lever 870 is in operation, allowing the user to more easily read out the inspection areas. As is evident from Figures 17C and 17E, the amount of rotation of the other movement of the lever may be designed so that contact of the sample pad with respect to the membrane strip is achieved at a desired rotation level. As shown in Figure 17C, the contact between the sample pad and the membrane strip is about 90 degrees. As shown in Figure 17E, the contact between the sample pad and the membrane strip is about 180 degrees. Thus, the rotation of the lever may be any amount between 0 and 180 degrees to affect the contact between the substrate pad and the membrane strip.

[0125] Apparatus or processes provided herein can rapidly detect the presence or absence of a target analyte in a liquid biological sample. Optionally, the target analyte can be detected within 30 minutes, optionally within 25 minutes, optionally within 10 minutes, optionally within 15 minutes, or optionally within 10 minutes of application of the liquid biological sample to the apparatus. In some embodiments, such as when a substrate is applied to a membrane layer after the application of the liquid biological sample, the liquid biological sample can be moved through the membrane layer for approximately 10 to 20 minutes, optionally within 15 minutes, before the application of the substrate. The substrate may then be applied in contact with the membrane layer, and the detection of the presence or absence of the target analyte may be performed by development for approximately 1 to 10 minutes, optionally within 5 minutes, after which a detectable label may be detected visually or by a detection instrument. Thus, the apparatus and methods provided herein are substantially faster than prior methods such as ELISA-based systems or chemiluminescence-based systems.

[0126] Various aspects of this disclosure are illustrated by the following non-limiting examples. The examples are for illustrative purposes only and do not limit any implementation of this disclosure. It will be understood that modifications and alterations can be made without departing from the spirit and scope of this disclosure. Similar and other technologies known in the art readily translate to the examples. The reagents illustrated herein are commercially available, and it will be readily apparent to those skilled in the art where such reagents are available.

[0127] <Example> The sample apparatus is formed substantially as illustrated in Figure 17A and housed in a polymer housing. This polymer housing includes a sample application port localized on the sample pad, a second reagent port located upstream of the sample pad, and a test site port that allows access to the test site on the membrane strip. The conjugate pad and membrane are made of cellulose nitrate (CN95) from Sartorius GmbH (Göttingen, Germany). The sample pad is made of binder-bound chopped glass and is sold as material 8951 by AHLSTROM-MUNKSJO GmbH (Helsinki, Finland). The absorbent pad, or waste pad, is made of cotton fiber and is sold as material 222 by AHLSTROM-MUNKSJO GmbH (Helsinki, Finland).

[0128] The conjugate pads (25mm x 5mm) are formed using an Automated Lateral Flow Reagent Dispenser (ALFRD) from ClaremontBio (Upland, California). The amount of coating solution dispensed is precisely controlled by a syringe pump while the dispenser translates across the nitrocellulose surface. A dispensing rate ratio of 0.291 μL / mm to 0.401 μL / mm allows for the formation of a test line approximately 1 mm wide. To form a test strip line, an anti-Fc antibody is first coated onto the desired location on the nitrocellulose. After the first coating layer dries, a second anti-nucleocapsid antibody is coated onto the same location. For example, first, 2 mg / mL of goat anti-mouse IgG (Fc-specific) antibody (Sigma Aldrich M4280-1ML) is striped onto CN95 at a dispensing rate / movement speed of 0.291 μL / mm. After the first layer has dried, 1 mg / mL of mouse MAb SARS-CoV / SARS-CoV-2 Nucleocapsid Antibody (SinoBio 40143-MM05) containing 5% disaccharides is coated onto the first layer and vitrified according to the instructions of U.S. Patent No. 10,433,540.

[0129] The sample pad (18mm x 5mm) is pre-treated with 2% Casein and 0.5% Tween-20 in PBS and dried before assembling the device. The waste pad is 21mm x 5mm and is used as received from the manufacturer. The device is formed on a clear polycarbonate support card coated with double-sided tape. The sample pad, conjugate pad, and absorbent pad are placed on the tape and covered with a cover that exposes the desired area of ​​the device.

[0130] To detect the analyte, prepare a sample solution containing SARS-CoV-2 (2019-nCoV) S protein (Sino Biological, Chesterbrook, Pennsylvania) in 1 wt% BSA and 0.1 wt% Tween-20 in phosphate-buffered saline. Alternatively, use thermoactivated SARS-CoV-2 (2019-nCoV) (VR1986HK, ATCC) in 0.1 wt% BSA in PBS. If using inactive virus, digest a TCID50 0.625 x 10² / ml virus load in artificial saliva (960 μl) (Cat.#1700-0304, Pickering Laboratories) containing 2 wt% zwittergent3-14 (0.2 g) and 20 mM DTT (20 μl) for 5 minutes, then apply to the sample pad.

[0131] Gold-Ab-HRP conjugates are formed using Anti-Human SARS Coronavirus Nucleoprotein Mouse MAb (40143-MM05, Sino Biologicals) or Anti-Human SARS Coronavirus Nucleoprotein Rabbit MAb (40143-R001, Sino Biologicals). The antibody is first purified to remove any protein additives for stabilization (e.g., BSA), salts as preservatives (e.g., sodium azide), or salts in the storage buffer (e.g., PBS). The antibody is purified by centrifugation, and the buffer is replaced with 10 mM potassium phosphate, pH 7.4, with a concentration of 1 mg / mL or higher. The HRP-bound antibody sample is mixed with an equal volume of unused antibody for conjugation into particles. The antibody for conjugation is mixed with gold at a ratio of 100 μg Ab / 500 μL OD=20 gold, and the mixture is incubated at room temperature for 30 minutes with shaking or rotation. For example, in the case of an Ab-HRP 50% / Ab50% mixture, 50 μg of Ab-HRP and 50 μg of Ab (unlabeled) are mixed in 500 μL of OD=20 gold. Blocking solution (25 mM borate, 10% BSA, pH 8.0) is added in 200 μL blocking buffer / 500 μL conjugate solution and incubated at room temperature for 30 minutes with shaking or rotation. The conjugate material is rotated in a centrifuge at 3600 RCF for 10 minutes and the supernatant is removed. The pellet may be resuspended in a vitrification matrix containing 25 mM borate, pH 8.0, 50:50 mM FeSO4-EDTA and 600 mM trehalose disaccharide with 1% BSA, and then vitrified on a sample pad according to the teachings of U.S. Patent No. 10,433,540.

[0132] In the test study, load 30 μL of sample into the cassette's sample port and add 70 μL of running buffer (1 wt% BSA, 1 wt% Tween-20 in PBS) onto the sample pad in the running buffer port upstream of the sample port. Allow the assay to proceed for 15 minutes. In some cases, add 10 μL of substrate TMBM or TMBMX (Moss Bio) onto the nitrocellulose test line and allow to develop for 5 minutes.

[0133] Optionally, the substrate is applied by bringing a dry substrate membrane into contact with a membrane strip. The dry substrate pad may be formed by combining 250 mM trehalose, 2.5 mM EDTA, 0.5 wt% PEG, 62.5 μl TMB (substrate) stock, 399.4 μl reconstitution buffer, and 5 μl 30 wt% H2O2. The reconstitution buffer is 0.03 M acteat containing 0.4 wt% sodium nitroferricyanide at pH 3.3. The material is dissolved by vortexing and vitrified into a nitrocellulose membrane according to the instructions of U.S. Patent No. 10,433,540. To apply the substrate, the vitrified substrate membrane is brought into contact with the membrane strip upstream of the inspection site.

[0134] The results using the isolated sample are shown in Figure 19, and the results using the TMBM substrate are shown in Figure 20. It is demonstrated that the SARS-CoV-2 S protein can be detected in the system down to less than 1 pg / ml. The results using inactivated SARS-CoV-2 as the target analyte are shown in Figure 21. It is demonstrated that dilutions of 0.625 x 10² / ml and 0.625 x 100 / ml, as well as the negative control, are detectable.

[0135] In further studies, 30 μL of sample is loaded into the cassette's sample port. The sample pad is vitrified with running buffer (BSA, Tween-20 in phosphate buffer, and sodium salt at concentrations that constitute the running buffer). The running buffer material is rehydrated upon addition of the sample. The assay is allowed to proceed for 15 minutes. In some cases, 10 μL of substrate TMBM or TMBMX (Moss Bio) is added to a nitrocellulose test line and developed for 5 minutes. Similar results are expected.

[0136] The same study was repeated, comparing binding of the capture agent alone or on a pre-coated anti-IgG antibody at the test site. Nitrocellulose membrane strips were prepared using 1.5 mg / ml Human SARS Coronavirus Nucleoprotein / NP Ab (40144-MM05, Sino Biological) with 5% sucrose added to the target site. SARS-CoV-2 nucleoprotein was diluted 10-fold in serial dilutions from 1000 ng / ml to zero 1 wt% BSA / 0.1 wt% PBST. 30 μl of each dilution was added to the sample pad along with 0.25 μl of 50 wt% R001 + 50 wt% R001-HRP conjugated to 40 nm GNP (passive conjugation). 70 μL of 1 wt% BSA / 1 wt% PBST was added to the sample port as running buffer. The test was run for 20 minutes. Subsequently, 15 μL of TMBMX(Moss) was added to the matrix upstream of the test site. After 5 minutes, a photograph of the resulting test apparatus was taken, yielding the results shown in Figure 22A.

[0137] For comparison, nitrocellulose CN95 was first coated with 2 mg / ml anti-mouse IgG (Fc Specific) Ab (M4280, Sigma), and then coated with 1 mg / ml Human SARS Coronavirus Nucleoprotein / NP Ab (40144-MM05, Sino Biological). 5% sucrose was added to each coating layer to prepare test specimens. The remaining tests were performed in the same manner as above. Figure 22B shows the results. Neither test showed a positive signal without substrate addition. Using pre-coated antibodies before adding the antibody capture agent reduced the detection limit by nearly 10 times. <Further examples> 1. The rapid diagnostic device, Housing and The housing comprises a substantially dry film layer, The aforementioned film layer The first matrix and, In the first inspection site, a vitrified scavenging agent is found within or on the first matrix, A rapid diagnostic device comprising, optionally, at least one vitrified reporter in the first matrix. 2. The apparatus according to Embodiment 1, further comprising a substantially dry substrate film, wherein the substrate film comprises a vitrified substrate in or on the substrate film, and the substrate film is associated with a housing and optionally located on a lever or other suitable device, and configured to be in contact with the substrate comprising the film layer. 3. The apparatus according to embodiment 1 or 2 further includes a screen layer disposed near the film layer and optionally in contact with the film layer, wherein the screen layer Apparatus comprising: a second matrix configured to substantially capture contaminants present in a liquid biological sample when the screen layer comes into contact with the liquid biological sample. 4. The apparatus according to any one of embodiments 1 to 3, wherein the reporter is present and vitrified in or on a first matrix between the sample pad and the first inspection site. 5. The apparatus according to any one of embodiments 1 to 4, wherein the capturing agent is covalently bonded to the first matrix at the first inspection site. 6. An apparatus in any embodiment provided herein, wherein the first matrix of the film layer comprises nitrocellulose, collagen, polycaprolactone, lactic acid, lactic acid-glycolic acid copolymer, or a combination thereof. 7. An apparatus according to any one embodiment of embodiments 1 to 6, wherein the scavenger is an antigen comprising nucleic acid, protein, peptide, or fragment thereof. 8. The apparatus according to Embodiment 7, wherein the capture agent comprises a virus coat protein, optionally an influenza coat protein, optionally a coronavirus coat protein, optionally a SARS-CoV-2 spike (S) protein, an SARS-CoV-2 nucleocapsid (N) protein, or fragments thereof. 9. The apparatus according to any one embodiment of embodiments 1 to 6, wherein the capture agent is an antibody that selectively binds to the coat protein of the virus, or optionally to the SARS-CoV-2 spike (S) protein, the SARS-CoV-2 nucleocapsid (N) protein, or a fragment thereof. 10. An apparatus according to any one embodiment of embodiments 1 to 9, wherein the at least one reporter comprises an antibody, or optionally IgG, IgM, or a combination thereof. 11. An apparatus according to any one embodiment of embodiments 1 to 10, wherein the at least one reporter includes a detectable label, an enzyme, or both. 12. An apparatus according to any one embodiment of embodiments 1 to 11, wherein the reporter is bound to a particle. 13. The apparatus according to embodiment 12, wherein the particles are nanoparticles, or optionally nanoshell particles, gold colloid particles, or a combination thereof. 14. The apparatus according to embodiment 13, wherein the particles are nanoshell particles comprising a silicon oxide core and a gold shell layer covering the core, the core having a radius, the shell layer having a thickness, and optionally the ratio of the radius to the thickness being 3:1 to 12:1. 15. The apparatus according to embodiment 12, wherein the reporter is coupled with the particles and enzyme, a luminescence-detectable label, a radioisotope-detectable label, or a combination thereof. 16. The apparatus according to embodiment 12, wherein the reporter is covalently or electrostatically bonded to an enzyme and optionally to horseradish peroxidase or alkaline phosphatase. 17. The apparatus according to embodiments 1 to 16, wherein the reporter further comprises a second antibody that is not bound to an enzyme or a detectable label. 18. The apparatus according to embodiment 17, wherein the ratio of the number of reporters to the number of second reporter antibodies is 80:20 to 20:80, or optionally approximately 50:50. 19. Apparatus according to any one of embodiments 1 to 18, wherein the housing includes a sample port. 20. The apparatus according to embodiment 19, wherein the sample port further contains a vitrified assay reagent. 21. The apparatus according to embodiment 19, wherein the sample port is located close to the first end of the film layer, and the first target area is located close to the second end of the film layer. 22. Apparatus according to any one of embodiments 1 to 21, further comprising the substantially dried substrate film, wherein the substantially dried substrate film is located within the housing between the sample port and the first test site. 23. The apparatus according to embodiment 22, wherein the substrate film is associated with a lever configured to bring the substrate film into contact with the film layer by adjusting its rotational position. 24. The apparatus according to any one of embodiments 1 to 23, wherein the capture agent is an antibody, antigen, or aptamer. 25. An apparatus according to any one of embodiments 1 to 24, further comprising a removable separator layer disposed between a film layer and a screen layer. 26. An apparatus according to any one of embodiments 1 to 25, wherein the capture agent is an aptamer specific to the target analyte in the biological sample. 27. The apparatus according to embodiment 26, wherein the target analyte contains a pathogen. 28. The apparatus according to embodiment 27, wherein the pathogen includes a virus, bacteria, or toxin. 29. The apparatus according to embodiment 27, wherein the pathogen is a virus, optionally SARS-CoV-2, or influenza virus. 30. The apparatus according to embodiment 26, wherein the scavenger is an aptamer containing a human ACE2 protein, a peptide, or a fragment thereof. 31. A method for determining the presence or absence of a target analyte in a liquid biological sample, To provide an apparatus comprising a housing and a vitrified film layer associated with the housing, wherein the vitrified film layer comprises a first matrix and a scavenger covalently or electrostatically bonded to the first matrix at a first target site, The device is brought into contact with a liquid biological sample, and the liquid biological sample rehydrates the vitrified film layer or a portion thereof, thereby causing the target analyte to be present in the liquid biological sample, and the target analyte to bind to the capture agent and the reporter through contact. A method comprising detecting the presence or absence of a target analyte based on the presence or absence of a detectable signal at the first target site. 32. A method according to embodiment 31, further comprising bringing the rehydrated vitrified film layer into contact with a substrate film that has been vitrified in or on a substrate film, such that the liquid in the rehydrated vitrified film layer rehydrates the substrate film. 33. A method according to embodiment 31 or 32, further comprising moving the substrate from the substrate film toward the first target site by surface capillary action. 34. A method according to any one of embodiments 31 to 33, wherein the reporter is vitrified in or on the vitrified film layer, thereby the contact step releases the at least one reporter agent from the first matrix so that the reporter agent can bind the analyte. 35. A method according to any one of embodiments 31 to 34, wherein the reporter is an antibody. 36. A method according to the method of embodiment 35, wherein the antibody comprises a detectable label and optionally an enzyme. 37. The method according to embodiment 35, wherein the reporter further comprises a second antibody that is not bound to a detectable label. 38. The method according to embodiment 37, wherein the ratio of the number of reporters to the number of second reporter antibodies is 80:20 to 20:80, and optionally approximately 50:50. 39. A method according to any one of embodiments 31 to 34, wherein the reporter is bound to a particle. 40. The method according to embodiment 39, wherein the particles are nanoparticles, optionally nanoshell particles, gold colloid particles, or a combination thereof. 41. The method according to embodiment 40, wherein the particles are nanoshell particles comprising a silicon oxide core and a gold shell layer covering the core, wherein the core has a radius and the shell layer has a thickness, and optionally the ratio of the radius to the thickness is 3:1 to 12:1. 42. A method according to any one embodiment of aspects 39 to 41, wherein the reporter is bound to the particle and an enzyme, a detectable label, or a combination thereof. 43. A method according to any one embodiment of aspects 31 to 42, wherein the reporter is covalently bound to a detectable label selected from the group consisting of an enzyme, a phosphor, or both. 44. A method according to any one of embodiments 31 to 43, wherein the liquid biological sample includes saliva, blood, serum, plasma, bronchoalveolar lavage fluid, sputum, or nasal fluid. 45. A method in any one embodiment of aspects 31 to 44 or any other embodiment provided herein, wherein the first matrix of the film layer comprises nitrocellulose, collagen, polycaprolactone, polylactic acid, lactic acid-glycolic acid copolymer, or a combination thereof. 46. ​​A method according to any one embodiment of aspects 31 to 45, wherein the scavenger comprises nucleic acids, proteins, peptides, or fragments thereof. 47. A method according to any one embodiment of aspects 31 to 46, wherein the scavenger comprises a viral coat protein, or optionally, a SARS-CoV-2 spike (S) protein, a SARS-CoV-2 nucleocapsid (N) protein, or a fragment thereof. 48. A method according to any one embodiment of aspects 31 to 47, wherein the method comprises at least one reporter-detectable label. 49. In the method described in any one embodiment of aspects 31 to 48, the presence or absence of the target analyte is detected. Bringing the substrate for the detectable label into contact with the apparatus; and A method comprising detecting a signal generated when the substrate binds to the detectable label. 50. A method according to any one of embodiments 31 to 34, wherein the signal includes a fluorescent signal or a change in color. 51. A method according to any one embodiment of aspects 31 to 34, wherein the apparatus further comprises a screen layer disposed on the film layer, the screen layer being configured to substantially capture contaminants present in the liquid biological sample. 52. A method according to any one embodiment of embodiments 31 to 34, wherein the presence or absence of the target analyte is detected within approximately 20 minutes.

[0138] The apparatus and methods described herein are representative of exemplary embodiments and are not intended to limit the scope of the invention. Modifications and other uses therein will arise for those skilled in the art. Such modifications and other uses can be made without departing from the scope of the invention.

[0139] The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural form, which includes “at least one,” unless the context explicitly indicates otherwise. “Or” means “and / or.” Where used herein, the term “and / or” includes any and all combinations of one or more of the enumerated items relating to the subject. Where used herein, the terms “comprise” and / or “comprising,” or “includes” and / or “including” specify the presence of the described features, regions, integers, steps, operations, elements, and / or components, but it will be further understood that they do not exclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and groups thereof. The term “or combination thereof” means a combination that includes at least one of the aforementioned elements.

[0140] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as they would be generally understood by a person skilled in the art in which this disclosure pertains. Terms as defined in commonly used dictionaries should be interpreted in a way that is consistent with their meaning in the context of the relevant art and this disclosure, and it will be further understood that they should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0141] In addition to those shown and described herein, various modifications of the present invention will be apparent to those skilled in the art of the above description. Such modifications are also intended to be included within the scope of the appended claims.

[0142] The patents, publications, and applications referenced herein represent the level of skill of those skilled in the art to which the present invention pertains. These patents, publications, and applications are incorporated herein by reference to the same extent that individual patents, publications, or applications are incorporated herein by reference specifically and individually.

[0143] The foregoing description illustrates specific embodiments of the present invention and is not intended to limit its implementation. The following claims (including all equivalents thereof) are intended to define the scope of the present invention.

Claims

1. A rapid diagnostic device comprising a housing and a substantially dry film layer within the housing, wherein the film layer comprises a first matrix containing nitrocellulose, collagen, polycaprolactone, polylactic acid, poly(lactic acid-glycolic acid copolymer), or a combination thereof, and a capture agent covalently or electrostatically bonded to the first matrix at a first inspection site and vitrified in the form of an amorphous material substantially without a crystalline structure within or on the first matrix at the first inspection site.

2. The apparatus according to claim 1, further comprising a substantially dry substrate film, wherein the substrate film comprises a vitrified substrate in or on the substrate film, and the substrate film is associated with a housing and configured to be in contact with the film layer.

3. The apparatus according to claim 1, further comprising a screen layer disposed in the vicinity of the membrane layer, the screen layer comprising a second matrix configured to substantially capture contaminants present in the liquid biological sample when the screen layer comes into contact with the liquid biological sample.

4. The apparatus according to any one of claims 1 to 3, further comprising a vitrified reporter in or on the first matrix between the sample pad and the first inspection area.

5. The apparatus according to any one of claims 1 to 3, wherein the capturing agent is electrostatically coupled to the first matrix at the first inspection site.

6. The apparatus according to any one of claims 1 to 3, wherein the first matrix of the film layer comprises nitrocellulose.

7. The apparatus according to any one of claims 1 to 3, wherein the capture agent is an antigen comprising nucleic acid, protein, peptide, or fragment thereof, or the capture agent is an anti-Fc antibody.

8. The apparatus according to claim 7, wherein the scavenger comprises a viral coat protein, a SARS-CoV-2 spike (S) protein, a SARS-CoV-2 nucleocapsid (N) protein, or a fragment thereof.

9. The apparatus according to any one of claims 1 to 3, wherein the scavenger is an antibody, and the antibody selectively binds to any of the following: a viral coat protein, a SARS-CoV-2 spike (S) protein, a SARS-CoV-2 nucleocapsid (N) protein, or a fragment thereof.

10. The apparatus according to claim 4, wherein the reporter includes an antibody.

11. The apparatus according to claim 4, wherein the reporter comprises a detectable label, an enzyme, or both.

12. The apparatus according to claim 4, wherein the reporter is bound to the particle.

13. The apparatus according to claim 12, wherein the particles are nanoparticles.

14. In the apparatus according to claim 13, the nanoparticles are nanoshell particles comprising a silicon oxide core and a gold shell layer covering the core, wherein the core has a radius and the shell layer has a thickness. ,Device.

15. The apparatus according to claim 12, wherein the reporter is bound to the particles and enzyme, a detectable label, or a combination thereof.

16. The apparatus according to claim 12, wherein the reporter is covalently bonded to the enzyme.

17. The apparatus according to claim 12, wherein the reporter further comprises a second antibody that is not bound to an enzyme or a detectable label.

18. The apparatus according to claim 17, wherein the ratio of the number of reporters to the number of second antibodies is 80:20 to 20:

80.

19. The apparatus according to any one of claims 1 to 3, wherein the housing includes a sample port.

20. The apparatus according to claim 19, wherein the sample port further comprises a vitrified assay reagent.

21. The apparatus according to claim 19, wherein the sample port is located close to the first end of the film layer, and the first target area is located close to the second end of the film layer.

22. The apparatus according to claim 21, further comprising the substantially dried substrate film, wherein the substantially dried substrate film is located within the housing between the sample port and the first inspection area.

23. The apparatus according to claim 22, wherein the substrate film is associated with a lever configured to bring the substrate film into contact with the film layer by adjusting its rotational position.

24. The apparatus according to any one of claims 1 to 3, wherein the scavenger is an antibody, an antigen, or an aptamer.

25. The apparatus according to claim 3, further comprising a removable separator layer disposed between the film layer and the screen layer.

26. The apparatus according to any one of claims 1 to 3, wherein the capture agent is an aptamer specific to the target analyte in the biological sample.

27. The apparatus according to claim 26, wherein the target analyte includes a pathogen.

28. The apparatus according to claim 27, wherein the pathogen includes a virus, bacteria, or toxin.

29. The apparatus according to claim 27, wherein the pathogen is a virus, SARS-CoV-2 virus, or influenza virus.

30. The apparatus according to claim 26, wherein the scavenger is an aptamer containing human ACE2 protein, a peptide, or a fragment thereof.

31. A method for determining the presence or absence of a target analyte in a liquid biological sample, comprising: an apparatus comprising a housing and a vitrified film layer associated with the housing, wherein the vitrified film layer comprises a first matrix and a capture agent covalently or electrostatically bonded to the first matrix at a first target site, the capture agent being in the form of a substantially amorphous material lacking a crystalline structure, and the first matrix comprising nitrocellulose, collagen, polycaprolactone, polylactic acid, poly(lactic acid-glycolic acid), or a combination thereof; and a method for contacting the apparatus with a liquid biological sample, wherein the liquid biological sample rehydrates the vitrified film layer or a portion thereof, thereby binding the target analyte with the capture agent and reporter if the target analyte is present in the liquid biological sample; and detecting the presence or absence of the target analyte by the presence or absence of a detectable signal at the first target site.

32. A method according to claim 31, further comprising bringing the rehydrated vitrified film layer into contact with a substrate film containing a vitrified substrate in or on the substrate film, such that the liquid in the rehydrated vitrified film layer rehydrates the substrate film.

33. A method according to claim 32, further comprising moving the substrate from the substrate film toward the first target site by surface capillary action.

34. A method according to claim 31, wherein the reporter is vitrified in or on the vitrified film layer, thereby the contact step releases the at least one reporter agent from the first matrix so that the reporter agent can bind the analyte.

35. A method according to any one of claims 31 to 34, wherein the reporter is an antibody.

36. The method according to claim 35, wherein the antibody comprises a detectable label.

37. The method according to claim 35, wherein the reporter further comprises a second antibody that is not bound to a detectable label.

38. The method according to claim 37, wherein the ratio of the number of reporters to the number of second reporter antibodies is 80:20 to 20:

80.

39. A method according to any one of claims 31 to 34, wherein the reporter is bound to a particle.

40. The method according to claim 39, wherein the particles are nanoparticles, nanoshell particles, gold colloidal gold particles, or a combination thereof.

41. The method according to claim 40, wherein the particles are nanoshell particles comprising a silicon oxide core and a gold shell layer covering the core, wherein the core has a radius and the shell layer has a thickness.

42. The method according to claim 39, wherein the reporter is bound to the particles and an enzyme, a detectable label, or a combination thereof.

43. A method according to any one of claims 31 to 34, wherein the reporter is covalently bound to a detectable label selected from the group consisting of an enzyme, a phosphor, or both.

44. A method according to any one of claims 31 to 34, wherein the liquid biological sample includes saliva, blood, serum, plasma, bronchoalveolar lavage fluid, sputum, or nasal fluid.

45. A method according to any one of claims 31 to 34, wherein the first matrix of the film layer comprises nitrocellulose.

46. A method according to any one of claims 31 to 34, wherein the scavenger comprises a nucleic acid, a protein, a peptide, or a fragment thereof.

47. A method according to any one of claims 31 to 34, wherein the scavenger comprises a viral coat protein, a SARS-CoV-2 spike (S) protein, a SARS-CoV-2 nucleocapsid (N) protein, or a fragment thereof.

48. A method according to any one of claims 31 to 34, wherein the reporter includes a detectable marker.

49. A method according to any one of claims 31 to 34, wherein detecting the presence or absence of a target analyte includes bringing a substrate for the detectable label into contact with the apparatus, and detecting a signal generated when the substrate binds to the detectable label.

50. A method according to any one of claims 31 to 34, wherein the signal includes a fluorescent signal or a change in color.

51. A method according to any one of claims 31 to 34, wherein the apparatus further comprises a screen layer disposed on the film layer, the screen layer being configured to substantially capture contaminants present in the liquid biological sample.

52. A method according to any one of claims 31 to 34, wherein the presence or absence of the target analyte is detected within 20 minutes.

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