Compositions, devices and methods for testing inanimate surfaces, populations and individuals for pathogen infection

Abstract

A novel mask insert and system for detecting an analyte in an air sample obtained from a subject is provided. Disclosed herein are compositions, devices, and methods for detecting an analyte in an air sample obtained from a subject. The compositions can be contained on an inanimate surface, such as a mask to be worn by an individual, having a test substrate for capturing the analyte.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 029,974, filed May 26, 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002]

[0002] The present disclosure relates generally to medicine, and industrial and environmental hygiene in general. More particularly, the present disclosure is directed to compositions, devices and methods for analysing air samples obtained from individuals.

[0003]

[0003] Medical monitoring and diagnosis involve sample collection and analysis. Sample collection can include invasive and non-invasive methods. Invasive sample collection methods include procedures such as surgery and blood draws, which can cause pain, discomfort, and stress to the patient. Milder forms of invasive sample collection methods can include swabbing. Devices used in invasive sample collection methods can also be expensive and require aseptic handling to prevent sample contamination. Non-invasive sample collection methods can be advantageous over invasive sample collection methods due to the reduction in pain and discomfort experienced by individuals during sample collection.

[0004]

[0004] Rapid detection and identification of pathogens is important for preventing and monitoring the presence and spread of infection, e.g., preventing and mitigating the spread of a global pandemic. Tests are administered to surfaces and individuals to detect the presence of pathogens so that the individual can be appropriately diagnosed and further tested and / or treated. Individuals can also be administered tests to detect circulating antibodies to the pathogen, indicating that the individual has previously been exposed to the pathogen and has generated an immune response to eradicate the infection.

[0005]

[0005] Detection and identification of analytes, e.g., biomarkers, can aid in the diagnosis and / or progression of disease, determine the effectiveness and / or response to treatment, and allow for correction and / or adjustment of therapeutic dosage. Detection of analytes can also be used to detect environmental exposure to potentially harmful chemicals and drug use, e.g., illicit drug use. Detection of biomarkers can also provide information regarding internal physiological conditions, e.g., disease, metabolic state, the presence of toxins, and certain chemicals.

[0006]

[0006] Current tests cannot meet societal needs during pandemics, such as the COVID-19 pandemic. For example, during the COVID-19 pandemic, there was a period when approximately 500,000 tests were conducted per day when over 20 million tests per day were required, demonstrating a testing scale gap. Current tests also suffer from drawbacks and difficulties such as scaling, sample collection and extraction, testing costs, pain associated with sample collection, risk of infection, invalid tests (e.g., temperature checks), and failure to test asymptomatic individuals. Current tests also miss a fundamental measure of those transmitting the disease.

[0007] Current tests using nasal swabs for sample collection suffer from limited swab supplies, sample collection can be painful and uncomfortable, and require collection by skilled healthcare personnel. Collection of samples, such as saliva, sputum, saliva, lavage, and other oral fluid samples, can require specialized equipment and further processing to separate the analyte from the oral fluid. Regardless of the sample collection method used, the resulting sample can begin to degrade, and sample collection reagents can incur supply chain delays. The sample must then be removed or separated from the collection device, which can be inefficient and affect the sensitivity of the test used to analyze the sample. Collection devices for collecting saliva and nasal samples add complexity and are subject to contamination of the underlying sample collection matrix; i.e., saliva and nasal swabs contain many chemicals that must be removed prior to extraction of the target analyte. Proteases and RNases can also degrade the target analyte after extraction. The ratio of the surface area of the collection material to the required elution and extraction buffer affects the concentration of the target analyte. The higher the concentration of the target analyte, the greater the sensitivity of the test. Identification of optimal materials for forming factors and performance provides maximum test sensitivity.

[0008]

[0008] Therefore, there is a need to develop compositions, devices containing the compositions, and methods of using the compositions for testing surfaces, populations, and individuals for analytes. Inexpensive exhaled bioaerosol testing worldwide is a significant unmet need in the marketplace. Summary of the Invention

[0009]

[0009] In one aspect, the present disclosure is directed to a mask insert including a first layer, a test substrate for capturing an analyte in an air sample obtained from a subject, and a second layer, wherein the first layer and the second layer form an outer layer that substantially surrounds the test substrate, an overlapping region of the first layer and the second layer are bonded, at least a portion of the test substrate is bonded to at least one of the first layer and the second layer, and the test substrate is configured to be separated from at least a portion of the outer layer.

[0010] In one aspect, the present disclosure provides a mask insert including a first layer, a test substrate for capturing an analyte in an air sample obtained from a subject, and a second layer, wherein the first layer and the second layer form an outer layer that substantially surrounds the test substrate, an overlapping region of the first layer and the second layer being bonded, at least a portion of the test substrate being bonded to at least one of the first layer and the second layer, and the test substrate being configured to be separated from at least a portion of the outer layer; and a mask to be worn by a user. system wherein the mask insert is coupled to the surface of the mask. system The target is.

[0011]

[0011] In another aspect, the present disclosure is directed to a method for detecting the presence of an analyte in an air sample obtained from a subject, the method comprising: recovering from the individual a test substrate worn by the subject; and analyzing the test substrate for the analyte.

[0012]

[0012] The present disclosure will be better understood, and other features, aspects and advantages thereof will become apparent, in consideration of the following detailed description, which refers to the following drawings: [Brief explanation of the drawings]

[0013] [Figure 1] 1 is an illustration showing a front view of one embodiment of a mask insert. [Figure 2]FIG. 1 is an illustration showing a front view of one embodiment of a mask insert showing the separation of a portion of the outer layer to expose the test substrate. A patient or healthcare provider pinches the outer layer at each end of the mask insert and pulls outward in the direction of the large arrows (pulling force indicated by the large arrows). The gripping sites on the device can be asymmetric. The gripping site on the right side of the illustration pinches the outer layer and the inner test substrate. The gripping site on the left side of the illustration pinches only the outer layer. By pinching the end and pulling outward, perforations allow removal of the outer protective layer on the left side, while the protective layer maintains hold over the inner test substrate on the right side. After removing a portion of the outer layer, the patient or healthcare provider holds the outer protective layer at the gripping site on the right side of the illustration, protecting the test substrate from manual contact, which may contain RNases, proteases, and other contaminants. The patient or healthcare provider can then place the collection substrate into a test vial. [Figure 3] 1 is an illustration showing a front view and a side view of a mask insert, the side view showing the stacking and position of microhooks for attaching the mask insert to a mask. [Figure 4] 1 is an illustration showing a mask insert with a buffer well and an integrated vertical flow assay. [Figure 5] 1 is an illustration showing a mask insert with a buffer dispensing device and an integrated vertical flow assay. [Figure 6] 1 is an illustration showing a mask insert (outer layer removed) with a buffer dispensing device and an integrated vertical flow assay. A multiplexed vertical flow assay platform is also shown. [Figure 7] 10 is an illustration showing a mask insert (outer layer removed) with three buffer dispensing devices and an integrated vertical flow multiplex assay. [Figure 8] 10 is an illustration showing a mask insert (outer layer removed) with three buffer dispensing devices and an integrated vertical flow multiplex assay. [Figure 9A]10 is an illustration showing a mask insert (outer layer removed) with three buffer dispensing plunger devices and an integrated vertical flow multiplex assay. [Figure 9B] 10 is an illustration showing a close-up view of three buffer dispensing plunger devices. [Figure 10] 1 is an illustration showing a mask insert (with outer layer in place) with a buffer well and an integrated lateral flow assay. [Figure 11] 1 is an illustration showing a mask insert (with outer layer in place) with a buffer dispensing device and with an integrated lateral flow assay. [Figure 12] 1 is an illustration showing a mask insert (outer layer removed) with a buffer dispensing device and an integrated lateral flow assay. [Figure 13A] 13A shows an exemplary embodiment of a test substrate illustrating the tabs. Figure 13A shows one exemplary embodiment of a test substrate having four tab portions and a label portion. The test substrate may have perforations along the top edge (and below the label portion) and between the individual tabs so that the tabs can be removed independently from the test substrate. [Figure 13B] 13A shows an exemplary embodiment of a test substrate illustrating the tabs. FIG. 13B shows one exemplary embodiment of a test substrate having four tab portions and a label portion. The tab portions are separated by gaps but are attached to the label portion. The test substrate may have perforations along the top edge (and below the label portion) and between the individual tabs to allow the tabs to be removed independently from the test substrate. [Figure 13C] 13A and 13B show an exemplary embodiment of a test substrate illustrating tabs. Figure 13C shows an exemplary embodiment of a test substrate having four unconnected tab portions. [Figure 14] 14 shows the embodiment of FIG. 13 as part of a face mask worn by a user. [Figure 15]1 shows an exemplary embodiment of a multi-layer test substrate having four tab portions, an inner test substrate made from a material with a high surface area that is protected on all sides and can be detached from the outer protective layer. [Figure 16] FIG. 1 shows a flow diagram for the extraction and amplification of RNA from a test substrate. DETAILED DESCRIPTION OF THE INVENTION

[0014]

[0030] The present disclosure relates generally to compositions, devices and methods for testing inanimate surfaces, groups and individuals for pathogen infection. In particular, the present disclosure is directed to devices for detecting analytes in air samples exhaled by a subject.

[0015]

[0031] In one aspect, the present disclosure is directed to a mask insert. Referring to Figures 1-3, the mask insert includes a first outer layer, a test substrate, and a second outer layer. One of the first layer or the second layer is oriented toward (close to) the subject's face, and the other layer is coupled to (attached to) the interior surface of the mask worn by the subject.

[0016]

[0032] As illustrated in Figures 1-3, the first and second layers have wider and longer dimensions than the test substrate (indicated by hash marks, the outer boundary of which is a dashed line) and together form the outer layer. The outer edges of the first and second layers, where they overlap each other but not the test substrate (indicated by a diagonal " / "), are bonded together to form an envelope or sleeve that encloses the test substrate. The side view shown in Figure 3 illustrates how the inner test substrate has a slightly smaller dimension than the outer layer. Figure 3 also illustrates the location of fasteners (e.g., microhooks in the embodiment shown in Figure 3) on the outer layer surface that are away from the subject's face and function to attach the mask insert to a mask worn by the subject. The overlapping edges of the first and second layers are also bonded together (referred to as "grab sites" in Figures 1 and 2). The bond between the edges of the first and second layers also creates a barrier that blocks or substantially reduces air movement through the first and second layers and around the test substrate. The bond between the first and second layers thus directs airflow toward the test substrate. The outer layer also functions as a protective layer for the test substrate, allowing handling and manipulation of the mask insert without direct contact with the test substrate. The outer layer contains a material for capturing bioaerosols sized less than 100 microns by providing a bioaerosol bandpass. The outer layer contains a very low pressure drop material that allows bioaerosols to easily flow and reach the test substrate. The outer layer allows bioaerosols to pass through while preventing macro-contaminants, such as RNases and proteases, that may degrade analytes from contacting the inner test substrate. The mask insert acts like a bandpass filter to allow larger bioaerosols to pass through the outer layer while the analytes are captured by the test substrate.

[0017]

[0033] The edges of the mask insert have microhooks or adhesive strips along the edges that allow the mask insert device to adhere to any face covering material and allow the device to be easily removed without damaging the face covering.

[0018]

[0034] The outer layer has perforations along the line so that when a user grasps both ends and pulls the outer layer outward, a portion of the outer layer can be torn off and discarded. This exposes the inner capture substrate while the user still holds the outer substrate on one end without contaminating the capture substrate. The user can now place the inner substrate into a test vial.

[0019]

[0035] The first and second layers may be made of the same or different materials. Suitable materials for making the first and second layers (together, the outer layers) include, for example, spunbond polypropylene, spunbond polyester, meltblown fibers, carded nonwoven fibers, etc. Particularly suitable materials for making the first and second layers (together, the outer layers) include 0.50 oz to 0.75 oz spunbond nonwoven cover web materials (commercially available from Barry Global, Evansville, Indiana, USA). The materials for the first layer, test substrate, and second layer are designed to allow air exhaled by the subject to pass through the first layer, test substrate, and second layer. The material of the first layer, which is adjacent to the subject's mouth and nose, is also designed to allow analytes contained in the air to pass through the first layer. The test substrate is designed to allow air to pass through the test substrate (top) but also capture analytes contained in the air exhaled by the subject. A second layer, away from the subject's face and adjacent to the mask surface, also allows air to flow out of the mask insert device. Suitable fiber sizes for materials forming the outer layer have average fiber diameters ranging from about 5 micrometers to about 25 micrometers, e.g., from about 10 micrometers to about 20 micrometers.

[0020]

[0036] As illustrated in Figures 1-3, the mask insert preferably includes a notch in the outer layer adjacent one end of the mask insert, allowing for easier separation of a portion of the outer layer. As also illustrated in Figure 1, the first and second layers include perforations, allowing for easier separation of a portion of the outer layer from the test substrate. Preferably, the notch is made in the outer substrate, ending at the beginning of the perforation. As further illustrated in Figure 2, separation of a portion of the outer layer exposes the test substrate. When both ends of the mask insert are held and pulled outward, a portion of the outer layer pulls away, exposing the test substrate contained within the outer layer, while still allowing a person to grasp the end of the mask insert with the remaining portion without contaminating the test substrate. The test substrate can then be processed to detect analytes captured on the test substrate. The portion of the mask insert that provided the sleeve containing the test substrate can be discarded after removal of the test substrate. This allows the user to easily tear the outer substrate layer from the inner test substrate while still holding one end.

[0021]

[0037] In another aspect, the present disclosure is directed to a mask insert including an integrated vertical flow assay (VFA). Figure 4 shows one embodiment of a mask insert with an integrated vertical flow assay. As illustrated in Figure 4, a buffer well is positioned adjacent to the notch and perforations for separating a portion of the outer layer as described herein. The well allows buffer added to the well to contact a portion of the inner test substrate. Capillary action causes the buffer to flow toward the vertical flow assay located away from the buffer well. If a charged material, such as an electret, is used as the test substrate, the buffer depletes the charge of the electret material as it flows toward the VFA, thereby transporting the analyte to the VFA. The test substrate may be made of a hydrophobic material and / or a material that is less hydrophilic than the VFA pad so that flow is toward the VFA. The test substrate is in contact with the VFA and allows the buffer with the analyte to be transported to the VFA. The buffer may also contain other biomarkers that serve as positive and / or negative controls and can also be detected by the VFA. The VFA portion of the mask insert may include a viewing "window" to allow observation of the VFA results.

[0022]

[0038] FIG. 5 shows one embodiment of a mask insert with an integrated vertical flow assay, with a buffer dispensing device positioned adjacent to the notch and perforation shown in FIG. 5. The buffer dispensing device provides an alternative or addition to the buffer well (shown in FIG. 4). The buffer dispensing device may be an ampoule embedded within the mask insert. When someone applies pressure to the mask insert in which the buffer dispensing device is positioned, all or part of the buffer dispensing device ruptures, releasing the buffer contained therein. Through capillary action, the buffer flows in a direction toward the vertical flow assay, located away from the buffer dispensing device. If a charged material, such as an electret, is used as the test substrate, the buffer depletes the charge of the electret material as it flows toward the VFA, thereby transporting the analyte to the VFA. The test substrate may be made of a hydrophobic material and / or a material that is less hydrophilic than the VFA pad, so that flow is toward the VFA. The test substrate contacts the VFA, allowing the buffer with the analyte to be transported to the VFA. The buffer may also contain other biomarkers that can serve as positive and / or negative controls and that can also be detected by the VFA. The VFA portion of the mask insert may contain a viewing "window" to allow observation of the VFA results.

[0023]

[0039] Figure 6 shows another embodiment of the mask insert as illustrated in Figure 5 but with a removable outer layer. Figure 6 shows a VFA for single analyte detection and a VFA for performing multiplex testing. Removal of the outer layer of the mask insert between the buffer dispensing device and the VFA allows the buffer to flow through the test substrate without further flowing into the material forming the outer layer.

[0024]

[0040] In another embodiment, the mask insert can include two or more buffer dispensing devices. Figure 7 shows an exemplary embodiment of a mask insert including three buffer dispensing devices. The embodiment illustrated in Figure 7 also includes a removable outer layer. Buffer from the buffer dispensing devices flows toward the VFA, while barriers and / or occlusive nonporous spacing (or separations) in the test substrate allow independent elution and extraction of analytes from the test substrate "lanes" and prevent crossflow of each buffer into other "lanes" of the test substrate intended to detect different analytes. Via capillary action, the buffer flows toward the vertical flow assay located away from the buffer dispensing device. If a charged material, e.g., an electret, is used as the test substrate, the buffer depletes the charge of the electret material as it flows toward the VFA, thereby transporting the analytes to the VFA. The test substrate may be made of a hydrophobic material and / or a material that is less hydrophilic than the VFA pad so that flow is toward the VFA. Removal of the outer layer of the mask insert between the buffer dispensing device and the VFA allows the buffer to flow through the test substrate without further flowing into the material forming the outer layer. The test substrate contacts the VFA, allowing the buffer with the analyte to be transported to the VFA. The buffer may also contain other biomarkers that serve as positive and / or negative controls and can also be detected by the VFA. The VFA portion of the mask insert may include a display "window" to allow observation of the VFA results. Figure 7 also shows an exemplary embodiment for multiplex testing.

[0025]

[0041] FIG. 8 shows another exemplary embodiment of a mask insert including three buffer dispensing devices. The embodiment shown in FIG. 8 also includes perforations adjacent to the buffer dispensing devices and adjacent to the VFA to allow removal of only the outer layer surrounding the central portion of the test substrate. Buffer from the buffer dispensing devices flows toward the VFA, but barriers and / or spacing (or separations) in the test substrate prevent crossflow of each buffer into the test substrate. Through capillary action, the buffer flows toward the vertical flow assay located away from the buffer dispensing devices. If a charged material, such as an electret, is used as the test substrate, the buffer depletes the charge of the electret material as it flows toward the VFA, thereby transporting the analyte to the VFA. Removal of the outer layer of the mask insert between the buffer dispensing devices and the VFA allows the buffer to flow through the test substrate without further flowing into the material forming the outer layer. The test substrate may be made of a hydrophobic material and / or a material that is less hydrophilic than the VFA pad so that flow is toward the VFA. The test substrate contacts the VFA and allows the buffer with the analyte to be transported to the VFA. The buffer may also contain other biomarkers that serve as positive and / or negative controls and that may also be detected by the VFA. The VFA portion of the mask insert may include a display "window" to allow observation of the VFA results. Figure 8 also shows an exemplary embodiment for a multiplex test.

[0026]

[0042] In another embodiment, the mask insert includes an integrated vertical flow assay and at least one integrated delivery plunger for introducing buffer into the test substrate. Figure 9A shows an exemplary embodiment with three integrated delivery plungers. Figure 9B shows the delivery plungers. The vertical bar on the right is a locking mechanism that can be flipped up to allow depression of the plunger on the left, expelling buffer from the plunger cavity onto the test substrate.

[0027]

[0043] In another embodiment, the mask insert includes an integrated lateral flow assay. Figure 10 shows an exemplary embodiment of a mask insert with an integrated lateral flow assay (LFA). The embodiment of Figure 10 includes wells for introducing buffer to extract and / or elute analytes and transport the analytes from the test substrate toward the lateral flow assay sample pad. As shown in Figure 10, the test substrate is in contact with the sample pad of the LFA. The LFA may be integrated with the mask insert in the form of a standard LFA cassette or as a laminated LFA. The embodiment shown in Figure 10 may also be used for multiplex testing.

[0028]

[0044] In another embodiment, shown in FIG. 11, the mask insert includes an integrated lateral flow assay device that further includes a buffer dispensing device (ampule) for introducing buffer to extract and / or elute analytes and transport the analytes from the test substrate toward the lateral flow assay sample pad. As discussed herein, pressure applied to the buffer dispensing device releases buffer, which flows via capillary action through the test substrate toward the LFA. As shown in FIG. 11, the test substrate is in contact with the sample pad of the LFA. The LFA may be integrated with the mask insert in the form of a standard LFA cassette or as a laminated LFA. The embodiment shown in FIG. 11 may also utilize multiplex testing.

[0029]

[0045] In another exemplary embodiment, shown in FIG. 12, the mask insert includes an integrated lateral flow assay device that further includes a buffer dispensing device (ampule) for introducing buffer to extract and / or elute analytes and transport them from the test substrate toward the lateral flow assay sample pad. As discussed herein, pressure applied to the buffer dispensing device releases the buffer, which flows through the test substrate and toward the LFA via capillary action. Removal of the outer layer of the mask insert between the buffer dispensing device and the LFA allows the buffer to flow through the test substrate without further flowing into the material forming the outer layer. As shown in FIG. 12, the test substrate is in contact with the sample pad of the LFA. The LFA may be integrated with the mask insert in the form of a standard LFA cassette or as a laminated LFA. The embodiment shown in FIG. 12 may also be used for multiplex testing.

[0030]

[0046] The test substrate is designed to capture an analyte in an air sample from a subject. In some embodiments, the test substrate has a pore size smaller than the size of the analyte. In other embodiments, the test substrate is made of a material that attracts the analyte. In some embodiments, the test substrate has a pore size smaller than the size of the analyte and is made of a material that also attracts the analyte. Pore sizes for capturing analytes are less than 100 microns. Particularly suitable pore sizes for capturing analytes, such as pathogens, are less than 75 microns, less than 50 microns, and less than 25 microns. Materials can be modified to target specific analytes while minimizing pressure drop. For example, capturing COVID may require a higher pressure drop because COVID is smaller, requiring a higher-density material. TB can be captured with similar efficiency using a lower-density material, thus having a lower pressure drop and higher throughput. Exemplary analytes contemplated for capture by the test substrate of a mask insert include COVID-19 (approximately 100 nm), influenza (approximately 80-120 nm); Mycobacterium (approximately 7 microns), and other bioaerosols containing respiratory pathogens (less than 10 microns). As described herein, the test substrate material can be treated (e.g., by imparting an electrical charge to the material) to "attract" the analyte to the test substrate. Thus, for example, a lower density material can be treated to attract COVID to the test substrate compared to an untreated material.

[0031]

[0047] The mask insert is connected (adhered) to the inner surface of the mask. The mask insert is connected to the mask surface by adhesives and other fasteners, such as hook and loop fasteners. Preferably, the mask insert includes microhooks that allow the mask insert to be attached to the inner surface of the mask worn by the subject. Other attachment mechanisms are also suitable. For example, the mask insert can be attached to the mask surface using an adhesive. Generally, a non-permanent adhesive is preferred so that the mask insert can be removed from the mask surface.

[0032]

[0048] Air is exhaled by a subject through breathing, coughing, sneezing, speaking, and combinations thereof.

[0033]

[0049] In one embodiment, the mask insert is reversibly coupled to the mask worn by the user. As used herein, "reversibly coupled" refers to the ability to attach the mask insert to the mask, then remove the mask insert from the mask, and then reattach the mask insert to the mask. In some embodiments, the mask is reusable, and a new mask insert is attached to the mask.

[0034]

[0050] In another embodiment, the mask insert is semi-permanently connected to the mask worn by the user, in which the mask insert can be separated from the mask after being attached to the mask surface, but is not intended to be reattached to the mask insert after removal from the mask surface.

[0035]

[0051] In another embodiment, the mask insert is permanently connected to the mask worn by the user, in this embodiment, the mask insert is not intended to be separated from the mask once the mask insert is attached to the mask surface.

[0036]

[0052] Generally, the mask insert (whether reversibly or permanently connected) is positioned on the inner surface of the mask such that air exhaled by the user through normal breathing, labored breathing, coughing, sneezing, and any combination thereof, causes air to flow into the mask insert.

[0037]

[0053] As used herein, the substrate to be tested (also referred to interchangeably herein as a "test substrate" and a "collection substrate") can include any inanimate surface that may come into contact with an individual or population of individuals that may contain pathogens on their exterior surface. For example, in one embodiment, the substrate to be tested can be a complete mask or a mask from which a portion of the mask (i.e., the test substrate) has been removed. The substrate can also include a removable mask insert. In some embodiments, the mask insert can be cleaned and reused by placing it back into the mask.

[0038]

[0054] In some embodiments, the test substrate is treated to extract and / or remove pathogen material from the test substrate. In other embodiments, the test does not require removal or extraction of pathogen material from the test material. For example, the test substrate can be analyzed by placing it in a reaction solution (e.g., buffer and / or water) that produces a colorimetric reaction indicating the presence or absence of the pathogen. In another embodiment, the test substrate is placed in a reaction solution (e.g., buffer and / or water) in which the test substrate dissolves. In another embodiment, a reagent is applied to the test substrate. In another embodiment, the reagent is contacted with the test substrate, which draws the reagent into the test substrate by capillary action.

[0039]

[0055] The test substrate may be suitably made of synthetic fibers, natural fibers, and combinations thereof. The fibers used to form the layers may be hydrophobic fibers, hydrophilic fibers, and combinations thereof. Hydrophobic fibers include, for example, polylactone, poly(caprolactone), poly(L-lactic acid), poly(glycolic acid), similar copolymers, poly(alkyl acrylate), polybutadiene, polyethylene, polystyrene, polyacrylonitrile, polyethylene(terephthalate), polysulfone, polycarbonate, poly(vinyl chloride), and combinations thereof. Hydrophilic fibers include, for example, linear poly(ethyleneimine), cellulose, cellulose acetate and other grafted cellulose derivatives, poly(hydroxyethyl methacrylate), poly(ethylene oxide), polyvinylpyrrolidone, poly(acrylic acid), poly(ethylene glycol), poly(vinyl alcohol), poly(vinyl acetate), poly(acrylamide), proteins, poly(vinylpyrrolidone), poly(styrene sulfonate), and combinations thereof.Other suitable fiber materials include, for example, acrylonitrile / butadiene copolymer, cellulose, cellulose acetate, chitosan, collagen, DNA, fibrinogen, fibronectin, nylon, poly(acrylic acid), poly(chlorostyrene), poly(dimethylsiloxane), poly(etherimide), poly(ethersulfone), poly(ethyl acrylate), poly(ethyl vinyl acetate), poly(ethyl-co-vinyl acetate), poly(ethylene oxide), poly(ethylene terephthalate), poly(lactic acid-co-glycolic acid), poly(methacrylates), poly(methyl methacrylate), poly(methylstyrene), poly(styrenesulfonates), poly(styrenesulfonyl fluoride), poly(styrene-co-acrylonitrile), Examples include poly(styrene-co-butadiene), poly(styrene-co-divinylbenzene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene fluoride), polyacrylamide, polyacrylonitrile, polyamic acid (PAA), polyamide, polyaniline, polybenzimidazole, polycaprolactone, polycarbonate, polydimethylsiloxane-co-polyethylene oxide, polyether ether ketone, polyethylene, polyethyleneimine, polyimide, polyisoprene, polylactide, polypropylene, polystyrene, polysulfone, polyurethane, polyvinylpyrrolidone, proteins, SEBS copolymer, raw silk, styrene / isoprene copolymer, and combinations thereof.Polymer blends, such as poly(vinylidene fluoride)-blends-poly(methyl methacrylate), polystyrene-blends-poly(vinyl methyl ether), poly(methyl methacrylate)-blends-poly(ethylene oxide), poly(hydroxypropyl methacrylate)-blends-poly(vinylpyrrolidone), poly(hydroxybutyrate)-blends-poly(ethylene oxide), protein blends-polyethylene oxide, polylactide-blends-polyvinylpyrrolidone, polystyrene-blends-polyester, polyester-blends-poly(hyroxyethyl methacrylate), poly(ethylene oxide)-blends-poly(methyl methacrylate), poly(hydroxystyrene)-blends-poly(ethylene oxide), and combinations thereof.

[0040]

[0056] Another suitable layer of the test substrate can be an electret (e.g., a thermal electret and a fibrillated electret film). Electrets are dielectric materials with a pseudo-permanent charge or dipole polarization. Electrets are available from commercial sources. Electrets can be prepared by heating a material and simultaneously exposing it to an electric field, which orients many dipoles in the material in a preferred direction. After heating, the material "frozen" and can retain the position of its electric dipoles for an extended period of time. Suitable materials for preparing electrets include, for example, materials currently available for fabricating thermal electrets, such as organic materials, e.g., ebonite, naphthalene, polymethylmethacrylate, and many polymers, as well as inorganic materials, e.g., sulfur, quartz, glass, steatite, and some ceramics. Electret fibrous membranes are particularly suitable. Polyvinylidene fluoride (PVDF) / polytetrafluoroethylene (PTFE) NP electret nanofibrous membranes can be formed by electrospinning. Also suitable are the fibrillated electret films described in Van Turnhout (US Pat. No. 3,998,916).

[0041]

[0057] Particularly suitable test substrates are charged materials (e.g., polypropylene, polylactic acid, and electrets) that attract analytes contained in the air sample. Thus, analytes contained in the air of interest are attracted to the test substrate, although they are not necessarily captured by the test substrate due to the size difference between the test substrate and the analyte.

[0042]

[0058] Embodiments using polypropylene as the test substrate advantageously provide an inert substrate that allows biological materials, e.g., pathogens, to remain stable for extended periods of time, e.g., when the mask insert and / or test substrate require transportation for testing. The test substrate may also include coatings and chemical treatments that allow for easier elution of analytes. A low surface energy material can be used to increase the wettability of the test substrate material, thus requiring less buffer to impregnate the material and thus concentrate the analyte in the elution buffer. Hydrophobic polypropylene has the advantage of being easily discharged, allowing elution of virions from the nonwoven with a simple application of a buffer, e.g., 0.1% Triton X-100, which performs crude extraction for certain pathogens, e.g., SARS-CoV-2.

[0043]

[0059] The test substrate material can also be treated to impart an electrical charge to make the material more or less hydrophilic, to make the material more or less hydrophobic, and combinations thereof.

[0044]

[0060] Suitable test substrate materials also include materials that are soluble and / or dissolvable in liquid. For example, cellulose acetate nanofibers can dissolve upon contact with liquid. It should be understood that the entire test substrate and / or a portion thereof (e.g., the collection substrate) can be soluble or dissolvable. Advantageously, the substrate itself can completely dissolve, liberating all analyte material into the eluent without requiring a removal process. Preferably, the substrate can be fabricated such that the substrate dissolves and reacts, stabilizes RNA, or performs some other service detection. Preferably, the substrate can be inert before it is dissolved. Additionally or alternatively, a protective inert layer on the inner substrate dissolves, exposing a less inert, active inner substrate with active diagnostic properties. For example, the test substrate (e.g., a rod) can be placed in an eluent that dissolves the outer coating, releasing all of the viral material. The test reagent then dissolves and interacts with the analyte material.

[0045]

[0061] Fibers can be subnanofibers, nanofibers, microfibers, and combinations thereof, with diameters ranging from subnanometers to micrometers. Subnanofibers can be prepared using template-assisted growth, orientation-assisted growth, ligand-controlled growth, and catalyst-guided growth. Subnanometer fibers (also referred to herein as "subnanofibers") refer to single-dimensional structures with extremely high aspect ratios, with diameters ranging from a few angstroms to tens of angstroms. Ultrathin nanofibers with diameters less than 100 nm can be produced by phase separation, self-assembly, the Sea Island process, template synthesis, electrospinning, and bubble electrospinning. Nanofiber diameters range, for example, from about 3 nm to less than 1 μm. Microfiber diameters range, for example, from 1 μm to about 10 μm. Nanofibers and microfibers can be produced using phase separation, self-assembly, the Sea Island process, template synthesis, electrospinning, bubble electrospinning, meltblowing, spinblowing, and other methods. Fiber diameter and morphology can be determined using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). Test substrate fibers can be woven, knitted, crocheted, knotted, co-pressed, interlaced, bonded, co-stapled in the form of a sheet web, and combinations thereof. Layers can be formed using spunbond materials prepared with continuous fibers formed by continuously extruding a polymer through a spinneret to form distinct filaments. The filaments can then be mechanically or pneumatically stretched without disrupting the orientation of the polymer filaments. Continuous filaments can be deposited substantially randomly on a carrier belt to form a web. Layers can also be formed using meltblown fibers. Layers can also be formed using electrospun fibers. As known to those skilled in the art, electrospinning involves drawing individual polymer chains in a polymer solution under high voltage through a nozzle / orifice, typically having an extremely small diameter, into nano- or sub-microscale structures in the form of fibers.

[0046]

[0062] Additives can be included with the test substrate layer. Suitable additives include, for example, antimicrobial additives, such as silver-containing antimicrobial agents and antimicrobial polypeptides, analgesic compounds, such as lidocaine, antibiotics, such as neomycin, thrombogenic compounds, nitric oxide-releasing compounds, such as sydnonimine and NO complexes, bactericidal compounds, fungicidal compounds, bacteriostatic compounds, other pharmaceutical compounds, adhesives, fragrances, deodorizing compounds, preservatives, RNase inhibitors, protease inhibitors, and nucleic acids, such as deoxyribonucleic acid, ribonucleic acid, and nucleotide analogs.

[0047]

[0063] Suitable analytes are contained in the airborne gases and aerosol droplets exhaled by the user. Suitable analytes include microorganisms, chemicals, proteins, nucleic acids, and combinations thereof. Suitable microorganisms include bacteria and viruses.

[0048]

[0064] Particularly suitable microorganisms include pathogens. The term "pathogen" is used according to its ordinary meaning to refer to bacteria, viruses, and other microorganisms that directly or indirectly cause disease. Exemplary pathogens include, for example, Yersinia, Klebsiella, Providencia, Erwinia, Enterobacter, Salmonella, Serratia, Aerobacter, Escherichia, Pseudomonas, Shigella, and the like. ella, Vibrio, Aeromonas, Streptococcus, Staphylococcus, Micrococcus, Moraxella, Bacillus, Clostridium, Corynebacterium, Eberthella, Francisella sella, Haemophilus, Bacteroides, Listeria, Erysipelothrix, Acinetobacter, Brucella, Pasteurella, Flavobacterium, Fusobacterium, Streptobacillus Ocacillus, Calymmatobacterium, Legionella, Treponema, Borrelia, Leptospira, Actinomyces, Nocardia, Rickettsia, Micrococcus, Mycobacterium,Neisseria, Campylobacter, pathogenic viruses such as papillomaviruses, parvoviruses, adenoviruses, herpesviruses, vaccine viruses, arenaviruses, coronaviruses, rhinoviruses, respiratory syncytial viruses, influenza viruses, picornaviruses, paramyxoviruses, reoviruses, retroviruses, rhabdoviruses, human immunodeficiency virus (HIV), etc., Taenia, Hymenolepsis, Diphyllobothria, etc. lobothrium), Echinococcus, Fasciolopsis, Heterophyes, Metagonimus, Clonorchis, Fascio la), Paragonimus, Schistosoma, Enterobius, Trichuris, Ascaris, Ancylostoma, Necator, Wuc hereria, Brugi, Loa, Onchocerca, Dracunculus, Naegleria, Acanthamoeba, Plasmodium, Trypanosoma, Leishmania, Toxoplasma, Entamoeba, Giardia, Isospora pora, Cryptosporidium, Enterocytozoa, Strongyloides, Trichinella, e.g., ringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis, coccidiodomycosis, paracoccidioidomycosis, mucomycosis, candidiasis, dermatophytosis, protothecia, pityriasis, mycetoma,Fungi causing Paracoccidiodomycosis, Phaeohphomycosis, Pseudoalescheriasis, Trichosporonosis, Pneumocystis, and combinations thereof.

[0049]

[0065] Particularly suitable chemicals include ketones, nicotine, cocaine, opioids, marijuana, benzodiazepines, amphetamines, barbiturates, and combinations thereof.

[0050]

[0066] Proteins, DNA, and RNA can also be detected.

[0051]

[0067] Suitable masks include any face covering worn over or in front of a user's mouth and nasal passages through which air is expelled from the user, for example, during speaking, coughing, and sneezing. Masks can be secured to the user's face using ties, straps, bands, and combinations thereof. Masks also include face shields. Masks also include bandanas, neck gaiters, scarves, towels, and cloth covers placed over the user's nose and mouth. Thus, the mask insert of the present disclosure can be coupled with any type of face covering such that at least a portion of the exhaled air (due to breathing, coughing, sneezing, speaking) passes through the device, where analytes contained in the air sample can be collected by a test substrate.

[0052]

[0068] When the analyte is a pathogen, the amount of pathogen particles captured can be expressed as a filtration efficiency, a minimum efficiency report value rating, and a particulate performance rating. Preferably, the filtration efficiency of the test substrate ranges from about 60% to about 95%. Preferably, the minimum efficiency report value rating is at least 12. Preferably, the particulate performance rating is at least 1900.

[0053]

[0069] Any suitable method can be used to clean the mask and / or mask insert, such as cleaning and / or sterilization by heat treatment, ultraviolet light exposure, and other sterilization methods. In other embodiments, the mask and / or mask insert is a disposable mask or mask insert. In these embodiments, the mask and / or mask insert is destroyed after removal.

[0054]

[0070] In one aspect, the present disclosure is directed to a mask for testing for pathogen infection. The mask includes a test substrate that includes a capture agent for capturing the pathogen.

[0055]

[0071] Suitable capture agents include, for example, antibodies that specifically bind to pathogens, ligands that specifically bind to pathogens, such as surface molecules, e.g., sugars, glycoproteins, etc., to which a pathogen must bind in order to infect its host. The capture reagent may be covalently or non-covalently linked to the test substrate by a linker. Any suitable linker can be used, such as organic molecules, e.g., polymers or copolymers (e.g., substituted or unsubstituted polyalkylene glycols, e.g., polyethylene glycol), and / or biological molecules, such as bovine serum albumin.

[0056]

[0072] In one embodiment, a mask worn by an individual covers the subject's mouth and nose, such that pathogens are captured and / or adsorbed by the mask material when the individual breathes, coughs, and / or sneezes into the mask. The mask may include ear loops and / or ties for securing the mask to the individual wearer's face. The mask may include strips of material attached to the mask and extending along each side thereof for use in attaching the mask to the wearer's face to provide an improved fluid seal between the perimeter of the mask and the wearer's face.

[0057]

[0073] The test substrate may be coated and / or treated to absorb and / or capture pathogens.

[0058]

[0074] The mask and / or the mask insert portion of the mask can be constructed to maximize the surface area of the test substrate. For example, the test substrate can include particles, such as microparticles, nanoparticles, beads, and the like. The particles can be coated with a capture agent.

[0059]

[0075] Preferably, the test substrate portion of the mask and / or mask insert can be an immunochromatographic test substrate, a colloidal gold test substrate, or a combination thereof, including, for example, a quantum dot-labeled test substrate, a colloidal gold-labeled test substrate, a colloidal selenium-labeled test substrate, an upconversion phosphorescent-labeled test substrate, a nano-rare earth fluorescent complex-labeled test substrate, a time-resolved chromatography test substrate, a chemiluminescent test substrate, and other test substrates.

[0060]

[0076] In embodiments of the mask intended to include a test substrate, the mask is configured to receive the test substrate. The mask may have perforations, for example, so that the test substrate can be inserted into the mask at the perforations. In other embodiments, the test substrate can be attached to the mask by, for example, hook and loop fasteners, adhesive glue, and the like.

[0061]

[0077] The mask insert may also include tabs for labeling the mask insert and for handling the mask without contaminating the test substrate.

[0062]

[0078] As illustrated in FIG. 1 , the perforations in the mask insert, particularly the test substrate, can be formed to separate the test substrate into different portions (or tabs). The perforations can be between each tab. Additionally, or alternatively, the perforations can be parallel to the top (or bottom) edge of the test substrate. As shown in FIGS. 1A and 1B , in addition to the perforations between each tab, the perforations can be oriented to separate individual tabs from the label portion, such that individual tabs can be separated from the test substrate independently of other tabs on the test substrate, while the remaining tabs remain attached. Additionally, or alternatively, the test substrate can have perforations along the top (or bottom) edge of the test substrate, and a space (or gap) can separate each tab so that each tab is independently connected to the top or bottom edge of the test substrate, as shown in FIG. 1B . In another embodiment, illustrated in FIG. 1C , the tabs can be “floating” within the test substrate. For example, rather than a single strip with perforations creating four tabs, the test substrate may have four individual tabs that are unconnected but contained on the same plane of the test substrate. The exemplary embodiment illustrated in Figures 1A-1C shows four tab portions that can be individually tested. For example, tab 1 can be pooled with other tabs from multiple patients. Tab 2 can then be individually tested. Tab 3 can be used as a control. Tab 4 can be stored for future testing. While Figure 1 illustrates an exemplary embodiment with four tabs, it should be understood that the test substrate can have fewer than four tabs and five or more tabs. Figure 2 illustrates a mask on a user with a test substrate having four test tabs.

[0063]

[0079] It should be understood that "tab" can refer to the collection substrate alone as well as to a separable portion of a test substrate. For example, a test substrate tab can include one or more protective layers with the collection substrate sandwiched between the protective layers (and other layers). In this embodiment, the test substrate tab (i.e., a portion of the test substrate) can be separated from the remainder of the test substrate while leaving the other tab portions unbroken. In another embodiment, the collection substrate tab can be sandwiched between protective layers. In this embodiment, the collection substrate tab can be independently removed from between the protective layers without the protective layer(s) and / or without contacting or damaging the remaining collection substrate tab. An advantage of the "tooth" embodiment is that a clinician can simply hold the tab and pull apart the perforation along the top, which detaches both the other and inner layers from the common back, allowing the inner layer to be easily placed in a container for further testing without contamination, and the outer layer can be discarded, etc. Tabs can also include multiple layers as described herein with respect to multi-layer test substrates. The tab(s) can share a common back but be separate (e.g., as shown in FIG. 1C). The tab may have perforations for easy removal and non-contamination.

[0064]

[0080] The test substrate may be a multi-layer test substrate. A multi-layer test substrate has an inner collection substrate protected on all sides by additional layers. The inner collection layer is made of a material with a high surface area for collection of the pathogen to be detected. The inner collection substrate can also be detachable from the protective layer. The protective layer and inner collection substrate can be made of different materials designed for the layer's specified purpose (e.g., protection and sample collection). For example, the inner collection substrate can be made of a corrugated double-sided polyester swab material. The inner collection substrate can be coated with a reagent to retain the pathogen load collection. The inner collection substrate can also be coated with a reagent to stabilize the pathogen material. Figure 3 shows a multi-layer test substrate. The protective layer can also function as a porous, flexible transport layer capable of receiving and temporarily storing exhaled droplets before they are transferred and absorbed by the underlying collection layer(s). The protective layer can be an air-permeable contact protective coating. The protective layer positioned furthest from the user's face may block all airflow, while the protective layer positioned closest to the user's face may allow airflow.

[0065]

[0081] In some embodiments, the inner retrieval substrate(s) may be separated from the protective layer. In some embodiments, the protective layer may be opened to remove or expose the inner retrieval substrate. For example, for one exemplary embodiment having adjacent and distant protective layers, with the inner retrieval substrate positioned between them, the protective layers may be larger in size than the inner retrieval substrate. When sandwiched together, the surfaces of the protective layers may contact each other and may be held together using adhesive, press-fit locking zippers, tape, or hinges. Separation of the protective layers allows access to the inner retrieval substrate, which can be removed for testing. Separation of the protective layers also allows for insertion or replacement of an unused inner retrieval substrate.

[0066]

[0082] Features of the multi-layer test substrate and multi-layer tab include, for example, that the outer layer(s) protect the inner layer(s) from contamination, the inner layer(s) may be double-sided, the inner layer(s) may include an adhesive, the inner layer(s) may include a stabilizer, the inner layer(s) may include a test layer, and the inner layer(s) may be multi-layered.

[0067]

[0083] In one embodiment, the multi-layer test substrate can include one or more reagent layers (also referred to herein as "reagent material" that includes analytical reagents). As used herein, "analytical reagents" refers to components used in the detection of pathogens. For example, analytical reagents can include, for example, buffer components, salts, dNTPs, oligonucleotide primers, polymerase, reverse transcriptase, and combinations thereof. The analytical reagents can preferably be lyophilized, in liquid form, gel, or combinations thereof. The reagent layer containing the analytical reagents can be separated from other layers of the test substrate by a coating that prevents the test reagents from contacting and / or activating the recovery layer until the test substrate is to be processed for analysis. The analytical reagents can be activated, for example, by placing the test substrate (and / or the recovery layer with the dissolvable layer containing the test reagents) in a liquid medium, such as a buffer solution, e.g., water, whereby the coating dissolves and releases the analytical reagents, which can then also dissolve in the buffer solution. The coating can be meltable, allowing the temperature to be adjusted so that the coating melts and releases the analytical reagents, forming a mixture capable of detecting pathogens. The reagent layer may include microparticles and / or beads containing analytical reagents.

[0068]

[0084] In some embodiments, the test substrate and inner collection substrate(s) can be packaged in sterile packaging to prevent contamination during storage and handling, and the packaging can then be opened to allow the test substrate or inner collection substrate to be placed in a mask and / or with a protective layer.

[0069]

[0085] The multi-layer test substrate may include spacers between the individual layers. The spacers may be located at the ends of the collection area to prevent air from flowing through the spacer material. Additionally or alternatively, the spacers may be of a material that allows air and pathogen particles to pass through.

[0070]

[0086] The multi-layer test substrate may include a label (see, e.g., FIG. 1). The label may be a blank area onto which information can be printed. The label may be pre-printed or may have a machine-readable barcode. Information contained on the label can be used to identify the test substrate (including the individual test substrate tabs) and to track and associate collected test substrates with users.

[0071]

[0087] In a multi-layer test substrate, individual layers may have different pore sizes than other layers. In one exemplary embodiment, a first protective layer may have a pore size with low filtration efficiency such that pathogen-sized particles may pass freely through the protective layer to reach the collection layer.

[0072]

[0088] In one exemplary embodiment, the test substrate may have a first protective layer made of one fiber material with a pore size range that allows pathogen-sized particles to pass freely through the first protective layer, and at least one collection layer that is a different fiber material with a pore size smaller than the pathogen-sized particles so that the pathogen-sized particles are captured by the collection layer. Preferably, the collection layer may be a multi-layer laminate itself to increase the virus capture load of each test. If the inner substrate tab can capture a virus density of X, then there should be a function f(Ln) = Ln × virus load of a single layer, where Ln is the number of layers. It should be understood that the reduction in efficiency due to the addition of layers.

[0073]

[0089] The capture layer(s) can be treated to enhance capture of pathogen particles. In one embodiment, the capture layer is treated with an adhesive to which pathogen particles adhere. In another embodiment, the capture material is treated with a capture ligand. In another exemplary embodiment, the capture material is treated with a combination of an adhesive and a capture ligand. The capture ligand can be an antibody, including an antibody fragment, an aptamer, a magnetic particle, or other ligand type that can bind to the pathogen of interest. The capture ligand is intended to attract and retain the pathogen so that subsequent analysis can be performed to detect and / or identify the pathogen.

[0074]

[0090] An exemplary multi-layer test substrate includes, for example, only a recovery layer without a capture agent and without a protective layer. Another exemplary multi-layer test substrate includes a protective layer and a recovery layer. Another exemplary multi-layer test substrate includes a protective layer and a recovery layer, where the recovery substrate includes a capture agent (e.g., an adhesive, a capture ligand, and combinations thereof). Another exemplary multi-layer test substrate includes a protective layer and multiple recovery layers.

[0075]

[0091] The protective layer can be of a material that allows for airflow and protection of the inner collection layer. The protective layer can be treated or coated with reagents, such as antimicrobials, RNase inhibitors, protease inhibitors, preservatives, and combinations thereof. The protective layer also provides physical protection for the inner collection substrate, for example, from contact. Each layer can have perforations to allow for separation of the tabs.

[0076]

[0092] The orientation of the test substrate can be flat (similar to a paper-like sheet), coiled, rod-shaped, hollow cylindrical, honeycomb, and combinations thereof. Coiled, rod-shaped, and hollow cylindrical substrates can be utilized in an axial or perpendicular orientation relative to the direction of respiration. Coiled, rod-shaped, and hollow cylindrical substrates can also be easily placed into tubes for storage and / or processing. It should be understood that each of these embodiments provides a three-dimensional structure to enhance collection and allow airflow.

[0077]

[0093] In one aspect, the present disclosure is directed to a method for detecting the presence of a pathogen. In one embodiment, the method detects the presence of a pathogen on an inanimate surface that may come into contact with an individual or a group of individuals. In another embodiment, the method detects the presence of a pathogen infection in a population where at least one individual in the population has or is suspected of having a pathogen infection. The method includes collecting, from the group of individuals, a test substrate worn by each individual in the population; combining the test substrates collected from each individual to form a pooled sample of test substrates; and analyzing the pooled sample of test substrates for the pathogen, wherein detecting the presence of the pathogen in the pooled sample of test substrates indicates that at least one individual in the population has had close exposure to the pathogen, is infected with the pathogen, or may require further diagnostic testing.

[0078]

[0094] The method may further comprise independently administering a second test to each individual in the group to identify those individuals infected with the pathogen.

[0079]

[0095] In some embodiments, the method is repeated for a subgroup or subpool of the first population. By way of example only, the method of this embodiment may include: collecting, from the first population, a test substrate worn by each individual in the population; combining the test substrates collected from each individual to form a first pooled sample of test substrates; analyzing the first pooled sample of test substrates for a pathogen, wherein detecting the presence of a pathogen in the pooled sample of test substrates indicates that at least one individual in the population has had close exposure to the pathogen, is infected with the pathogen, or may require further diagnostic testing; and analyzing a second population. from the first population, where the second population is a subgroup of the first population; combining the test substrates collected from each individual to form a pooled second sample of test substrates; and analyzing the pooled second sample of test substrates for a pathogen, where detecting the presence of a pathogen in the pooled sample of test substrates indicates that at least one individual in the population has had close exposure to the pathogen, is infected with the pathogen, or may require further diagnostic testing. The method can be repeated any number of times to identify individuals or subgroups of individuals or material substrates in contact with the individuals or subgroups of individuals that are infected with the pathogen.

[0080]

[0096] Suitable test substrates are described herein. Preferably, the test substrate is a mask or part of a mask (e.g., a mask insert) as described herein.

[0081]

[0097] Suitable pathogens to test for are described herein.

[0082]

[0098] In one aspect, the present disclosure is directed to a method of detecting the presence of a pathogen infection in an individual having or suspected of having a pathogen infection, the method comprising: recovering from the individual a test substrate worn by the individual; and analyzing the test substrate for the pathogen, wherein detection of the presence of the pathogen indicates that the individual has had close exposure to the pathogen, is infected with the pathogen, or may require further diagnostic testing.

[0083]

[0099] Suitable test substrates are described herein. Preferably, the test substrate is a mask or part of a mask (e.g., mask insert, test substrate, tab) described herein.

[0084]

[0100] In some embodiments, the pathogen(s) and / or pathogen material to be tested are extracted (eluted or removed) from the test substrate. In some embodiments, the test substrate can be analyzed directly, such as, for example, embodiments in which the test substrate used (including tabs, inner test substrate layers, etc.) is a soluble test substrate, a volatile test substrate, etc.

[0085]

[0101] Suitable pathogens to test for are described herein.

[0086]

[0102] The test substrates used in the test substrates and methods of the present disclosure can be tested directly or can be removed from the mask, for example, by cutting the mask to remove the portion of the mask to be tested, or the test substrate in the form of a mask insert can be configured for use in an analyzer. Any analyzer used to test a substrate for the presence of an analyte (e.g., a pathogen in the present disclosure) is suitable for use in the present disclosure. In one embodiment, the test substrate can be inserted into the analyzer, and the pathogen can be detected by the analyzer. It should be understood that in other embodiments, the analyzer can be a point-of-care analyzer in which the detection reagent is directly contacted with the test substrate, for example, by dropping a solution containing the detection reagent directly onto the test substrate. These point-of-care analyzers can be for personal use at home, in the workplace, etc. The signal output of the analyzer can provide whether a pathogen is present in the sample. For example, the signal output by the analyzer's detector can be transmitted to a data processor for storage, processing, and analysis. Preferably, the test substrate (including the tab) can be placed in an ion mobility spectrometer and tested for pathogens.

[0087]

[0103] As illustrated in Figure 4, the test substrate is compatible with additional sample preparation methods, such as mechanical, chemical, and application of force (eg, magnetic force).

[0088]

[0104] Figure 5 shows a flow diagram for sample detection using amplification. After recovery, RNA is extracted from the sample and subjected to reverse transcription to create a pool of cDNA. The cDNA is then amplified (e.g., using polymerase chain reaction ("PCR") using primers specific for the pathogen of interest. After amplification, the PCR products can be further analyzed. Analysis can include simple product / no product results and / or the PCR products can be sequenced. The amount of product can also be used to determine the concentration of the pathogen.

[0089]

[0105] After sample collection through the mask and / or test substrate, the collected sample can be analyzed using commercially available equipment and methods.

[0090]

[0106] The viral density and load capacity ranges for the above device embodiments can be determined by utilizing ratios based on internal control spike tests on the mask.

[0091]

[0107] The approximate range of viral loads recovered by embodiments of the test substrate device is given by the following assumptions: VD d1 / VD m1 :VD d2 / VD m2 (Wherein, VD = 1 square millimeter (mm 2 The viral density of the inner substrate normalized based on the viral load per mask; d1 = pathogen-spiked device in the mask; m1 = pathogen-spiked mask with device in the mask; d2 = estimated performance of the device embodiment; m2 = mask retrieved from the patient after removal can be estimated for infected humans wearing the device under different conditions. Once the range of results from the pathogen-spiked test and retrieved masks is determined, an approximate range of recovery potential for the device embodiment can be estimated. In another example, a test substrate device can be spiked with a range of viral loads to simulate low, average, and high densities to estimate the viral load that can be recovered by the test substrate device in use. The predicted device sensitivity can be determined based on a range of pool sizes and a range of densities.

[0092]

[0108] The viral load and number of layers of the test substrate can be determined using the formula: f(Ln) = VD x Ln for a single layer. Without being bound by theory, the more layers that need to be incorporated into the function, the smaller the return value may be because exhaled air cannot efficiently reach the inner layers. Some devices may require only one to five layers to screen smaller pools, while other devices with a greater number of layers are useful for screening larger pools.

[0093]

[0109] The viral load recovery capacity range of the test substrate can also be determined as a function of wear time. Device embodiments can be tested at two or more time points. Viral load can be quantified based on the amount of time each embodiment is worn.

[0094]

[0110] [Example]

[0111] In this example, detection of COVID-19 using swabs to collect samples was compared with detection using mask inserts. Patients suspected of being COVID-19 positive wore masks containing mask inserts for 30 to 60 minutes. After removing the device from the mask, the test substrate was removed and analyzed by RT-PCR. Simultaneously, patients were swabbed with nasal swabs, which were also tested by RT-PCR. The results for the nasal swabs and mask inserts were compared, as summarized in Table 1.

[0095]

[0112] [Table 1]

[0096]

[0113] The disclosed compositions, devices, and methods allow for rapid, large-scale identification of pathogen infections in populations or in inanimate surfaces that have come into contact with individuals and / or populations with a single test. If a positive test result is obtained in a pooled sample, individuals or subgroups of individuals from the pooled sample can be separately retested to identify the infected individual and / or individuals. The method may also identify individuals exposed through contact with the infected individual(s) identified as positive. Subsequent steps could then be taken to isolate both the positively infected individual and those exposed to the positively infected individual to prevent the spread of infection. The disclosed compositions, devices, and methods also reduce the costs associated with individual testing of populations because 1) a single test can be administered to a population and 2) relatively inexpensive inserts can be used to allow for the reuse of masks. Administration of a single test to individuals, groups, and / or subgroups of individuals reduces the time required to administer tests to multiple individuals separately. The disclosed methods also reduce the amount of reagents required to administer a single test compared to the amount of reagents required to test multiple individuals separately. Furthermore, the disclosed method allows for limited invasive testing and eliminates the discomfort associated with conventional serological or nasal testing methods.

Claims

1. 1. A mask insert comprising: a first layer; a test substrate for capturing an analyte in an air sample obtained from a subject; and a second layer, wherein the first layer and the second layer form an outer layer that substantially surrounds the test substrate, an overlapping region of the first layer and the second layer is bonded, at least a portion of the test substrate is bonded to at least one of the first layer and the second layer, the test substrate is configured to be separate from at least a portion of the outer layer, and the mask insert is configured to couple to a surface of a mask.

2. 10. The mask insert of claim 1, further comprising a fastener present on a surface of at least one of the first layer or the second layer and configured to connect the mask insert to a surface of a mask.

3. 10. The mask insert of claim 1, further comprising perforations in the outer layer proximate an end of the mask insert.

4. A mask insert as described in claim 1, further comprising a notch coextensive with the perforation.

5. 10. The mask insert of claim 1, wherein the test substrate comprises a material selected from the group consisting of synthetic fibers, natural fibers, and combinations thereof.

6. 6. The mask insert of claim 5, wherein the test substrate comprises a material selected from the group consisting of hydrophobic fibers, hydrophilic fibers, and combinations thereof.

7. 7. The mask insert of claim 6, wherein the hydrophobic fiber is selected from the group consisting of polypropylene, polylactone, poly(caprolactone), poly(L-lactic acid), poly(glycolic acid), co-poly(alkyl acrylate), polybutadiene, polyethylene, polystyrene, polyacrylonitrile, polyethylene(terephthalate), polysulfone, polycarbonate, poly(vinyl chloride), and combinations thereof.

8. 7. The mask insert of claim 6, wherein the hydrophilic fibers are selected from the group consisting of linear poly(ethyleneimine), cellulose, cellulose acetate and other grafted cellulose derivatives, poly(hydroxyethyl methacrylate), poly(ethylene oxide), polyvinylpyrrolidone, poly(acrylic acid), poly(ethylene glycol), poly(vinyl alcohol), poly(vinyl acetate), poly(acrylamide), proteins, poly(vinylpyrrolidone), poly(styrene sulfonate), and combinations thereof.

9. The test substrate may be any of acrylonitrile / butadiene copolymers, cellulose, cellulose acetate, chitosan, collagen, DNA, fibrinogen, fibronectin, nylon, poly(acrylic acid), poly(chlorostyrene), poly(dimethylsiloxane), poly(etherimide), poly(ethersulfone), poly(ethyl acrylate), poly(ethyl vinyl acetate), poly(ethyl-co-vinyl acetate), poly(ethylene oxide), poly(ethylene terephthalate), poly(lactic-co-glycolic acid), poly(methacrylic acid), poly(methyl methacrylate ... methacrylate), poly(methylstyrene), poly(styrenesulfonate), poly(styrenesulfonyl fluoride), poly(styrene-co-acrylonitrile), poly(styrene-co-butadiene), poly(styrene-co-divinylbenzene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene fluoride), polyacrylamide, polyacrylonitrile, polyamic acid (PAA), polyamide, polyaniline, polybenzimidazole, polycaprolactone, polycarbonate, polydimethylsiloxane the polymer blend comprises a material selected from the group consisting of ethylene-co-polyethylene oxide, polyetheretherketone, polyethylene, polyethyleneimine, polyimide, polyisoprene, polylactide, polypropylene, polystyrene, polysulfone, polyurethane, polyvinylpyrrolidone, protein, SEBS copolymer, raw silk, styrene / isoprene copolymer, poly(vinylidene fluoride)-blend-poly(methyl methacrylate), polystyrene-blend-poly(vinyl methyl ether), poly(methyl methacrylate)-blend-poly(ethylene oxide), poly(hydroxypropyl methacrylate)-blend-poly(vinylpyrrolidone), poly(hydroxybutyrate)-blend-poly(ethylene oxide), protein blend-polyethylene oxide, polylactide-blend-polyvinylpyrrolidone, polystyrene-blend-polyester, polyester-blend-poly(hydroxyethyl methacrylate), poly(ethylene oxide)-blend-poly(methyl methacrylate), poly(hydroxystyrene)-blend-poly(ethylene oxide), and combinations thereof;The mask insert of claim 5.

10. The mask insert of claim 1 , wherein the test substrate comprises an electret.

11. 10. The mask insert of claim 1, wherein the first layer and the second layer comprise synthetic fibers, natural fibers, and combinations thereof.

12. 10. The mask insert of claim 1, wherein the bond substantially reduces airflow through the bonded area of the mask insert.

13. 10. The mask insert of claim 1, further comprising an assay selected from the group consisting of a vertical flow assay and a lateral flow assay.

14. The mask insert of claim 1, further comprising a buffer dispensing device having an integrated vertical flow assay.

15. 1. A system for detecting an analyte in an air sample obtained from a subject, comprising: a mask insert including a first layer, a test substrate for capturing an analyte in an air sample obtained from a subject, and a second layer, wherein the first layer and the second layer form an outer layer that substantially surrounds the test substrate, an overlapping area of the first layer and the second layer are bonded, at least a portion of the test substrate is bonded to at least one of the first layer and the second layer, and the test substrate is configured to be separated from at least a portion of the outer layer; a mask suitable for being worn by said subject; Including, The mask insert is coupled to a surface of the mask.

16. 16. The system of claim 15, wherein the mask insert is coupled to one of a vertical flow assay and a lateral flow assay.

17. 16. The system of claim 15, wherein the mask insert further comprises a buffer dispensing device with an integrated vertical flow assay.

18. 16. The system of claim 15, wherein the test substrate comprises synthetic fibers, natural fibers, and combinations thereof.

19. The system of claim 16 , wherein the test substrate comprises an electret.

20. 16. The system of claim 15, wherein the first layer and the second layer comprise synthetic fibers, natural fibers, and combinations thereof.

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