CN117980744A

CN117980744A - High sensitivity analyte network detection flow assay and related methods

Info

Publication number: CN117980744A
Application number: CN202280063716.9A
Authority: CN
Inventors: 阿龙·雷哈姆伊姆; 雅各布·杰宾·约翰; 迪特·尤巴·穆罕默德·莱米纳·迪亚基特; 雅各布·拉比
Original assignee: Senzo Health Co ltd
Current assignee: Senzo Health Co ltd
Priority date: 2021-08-11
Filing date: 2022-08-11
Publication date: 2024-05-03

Abstract

Articles of manufacture (e.g., lateral flow assays) and methods for detecting analytes are generally described. These assays may involve the use of networks that block or restrict flow in the assay, for example, due to the formation of interconnected networks or grids.

Description

High sensitivity analyte network detection flow assay and related methods

Technical Field

Articles of manufacture (e.g., lateral flow assays) and methods for detecting analytes in lateral flow assays are generally described.

Background

Rapid analysis of biological and chemical targets may be important for primary or emergency medical screening, which explains the wide use of certain rapid tests to conduct the analysis. Many such rapid tests are based on chromatographic techniques using lateral flow assays on paper. Some of these lateral flow devices are typically nanoparticle-based and provide only limited sensitivity. Although these devices are limited in sensitivity, they are preferred because of the simplicity and low cost of production of these nanoparticle-based tests. However, improved lateral flow assays with increased sensitivity are needed.

Disclosure of Invention

Articles (e.g., flow assays), lateral flow assays) and methods for detecting analytes within an interconnected network (e.g., a grid) are generally described. In some cases, the subject matter of the present disclosure relates to interrelated products, alternative solutions to particular problems, and/or a plurality of different uses of one or more systems and/or articles.

In one aspect, a flow assay is described, comprising: a substrate having an upstream location and a downstream location; and a binding region comprising a plurality of capture reagents positioned at a downstream location, wherein when an analyte binds to at least a portion of the capture reagents, flow of at least a portion of the plurality of capture reagents is restricted.

In another aspect, a method of detecting multiple analytes in a sample using a flow assay is described, the method comprising: introducing the sample into an upstream location of the substrate, wherein the substrate comprises a plurality of capture reagents positioned at a binding region of the downstream location; flowing the sample from the upstream location to the downstream location; allowing at least a portion of the plurality of analytes of the sample to bind to at least a portion of the plurality of capture reagents; restricting flow along the flow assay after at least a portion of the plurality of analytes of the sample bind to at least a portion of the plurality of capture reagents; at least one analyte is detected.

In another aspect, a flow assay is described that includes: a substrate having an upstream location and a downstream location; and a binding region comprising a plurality of capture reagents positioned at a downstream location, wherein the plurality of capture reagents are configured to form an interconnected network with the plurality of analytes, the interconnected network comprising a mixture of the plurality of capture reagents and the plurality of analytes interconnected with each other.

In another aspect, a flow assay is described that includes: a substrate having an upstream location and a downstream location; a binding region comprising a plurality of capture reagents positioned at a downstream location, and an interconnecting network positioned at or between the downstream location, wherein the interconnecting network comprises a mixture of the plurality of capture reagents and the plurality of analytes interconnected with each other.

In another aspect, a method of detecting multiple analytes in a sample using a flow assay is described, the method comprising: introducing a sample at an upstream location of a substrate, wherein the substrate comprises a plurality of capture reagents positioned at a binding region at a downstream location; flowing the sample from the upstream location to the downstream location; flowing a plurality of detection reagents from an upstream location to a downstream location; allowing a plurality of analytes of the sample to bind to at least some of the plurality of capture reagents; forming an interconnection network comprising a mixture of a plurality of capture reagents and a plurality of analytes; and detecting at least one analyte.

In another aspect, a flow assay is described that includes: a substrate having an upstream location and a downstream location; a binding region comprising a plurality of capture reagents positioned at a downstream location; a plurality of detection reagents positioned upstream of the plurality of capture reagents; and a cassette enclosing at least a portion of the substrate, wherein the cassette comprises a first portion and a second portion opposite the first portion, wherein the substrate is positioned between the first portion and the second portion, wherein the second portion comprises a protrusion extending toward the first portion, and wherein the protrusion presses the substrate against the first portion.

Other advantages and novel features of the disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the drawings. In the event that the present specification and documents incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control.

Drawings

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every drawing nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the drawings:

FIGS. 1A-1C schematically illustrate the use of a flow assay to detect an analyte, according to some embodiments;

FIGS. 1D-1F schematically illustrate the use of flow assays to detect multiple analytes within an interconnected network, according to some embodiments;

FIGS. 1G-1L schematically illustrate an interconnected network including at least one analyte and at least one capture reagent, according to some embodiments;

FIGS. 2A-2B are schematic illustrations of a cassette including a tab capable of pressing a substrate against a first portion of the cassette, according to some embodiments;

FIGS. 2C-2F are schematic illustrations of a cassette and a tab included therein according to some embodiments;

FIG. 3 is a photographic image of a substrate according to one set of embodiments;

FIG. 4 illustrates a photographic image of a substrate cut into small pieces, according to some embodiments;

FIG. 5 is a photographic image of a cut substrate die within a 96-well plate, according to some embodiments;

FIG. 6 is a bar graph indicating absorbance of individual pieces of cut substrate, according to some embodiments;

FIG. 7 is a photographic image of two lateral flow assays after use, comparing the flow spectra of the two assays, according to some embodiments;

FIG. 8 illustrates photographic images of some lateral flow assays, comparing the use of different types and sizes of antibodies as capture reagents, according to some embodiments;

Figures 9-10 are images using an antigen from Respiratory Syncytial Virus (RSV) as an analyte according to some embodiments;

FIG. 11 illustrates a time lapse microscope image of a strip with a fluorescently labeled antibody during lateral flow assay operation, comparing positive and negative samples to determine the flow characteristics of the antibody, according to some embodiments;

FIG. 12 shows delayed microscope images of a marker enzyme detection reagent of a positive sample and a negative sample during flow through a membrane, according to some embodiments;

FIG. 13 shows fluorescence imaging of positive and negative sample strips before and after TMB addition, according to some embodiments; and

Fig. 14 is an image of bands of positive and negative samples after addition of TMB and color development, according to some embodiments.

Detailed Description

The present disclosure describes flow assays and methods for detecting analytes (e.g., within an interconnected network, precipitates, and/or grids). In some embodiments, for example, when an analyte binds to a reagent (e.g., capture reagent, capture antibody) within the binding region, flow in the lateral flow assay is reduced (e.g., restricted). Advantageously, reducing the flow rate (e.g., when the analyte is bound to the capture reagent) may improve detection of the analyte relative to conventional lateral flow assays where analyte flow is not reduced when the analyte is bound to the capture reagent. In some cases, the flow assay is a lateral flow assay that causes the analyte to flow on a surface of a substrate (such as a paper substrate). Many existing lateral flow assays are based on chromatographic techniques on paper substrates and can use, for example, nanoparticles to visually detect analytes (if present in the sample to be analyzed). However, these colorimetric techniques provide only limited sensitivity. In contrast, enzyme technologies, such as ELISA (enzyme linked immunosorbent assay), can provide higher sensitivity relative to most existing lateral flow assays, as ELISA uses enzymatic reactions and adds enzyme substrates, which can increase the sensitivity of the assay. However, ELISA procedures can be extremely time consuming and can require more complex processing relative to lateral flow assays.

It has been found that in the context of the present disclosure, the sensitivity and performance of a lateral flow assay can be improved by replacing nanoparticles used in a conventional lateral flow assay with an enzyme (i.e., a capture reagent) and a chromogenic substrate for the enzyme (i.e., a detection reagent). By using chromogenic enzyme substrates in combination with the features described herein, it has been unexpectedly found that the sensitivity of the lateral flow assay can be significantly improved over many existing lateral flow assays. Without wishing to be bound by any particular theory, it is believed that increasing sensitivity is achieved at least in part by forming an interconnection network (e.g., mesh, precipitate) that includes capture reagents and optionally detection reagents, and that forming such an interconnection network can increase the detection limit of the assay, thereby increasing sensitivity. It should be understood that the articles and methods described herein may be applied to any suitable flow assay, including microfluidic channel systems, and that the aspects described herein are not limited to lateral flow assays.

In an exemplary embodiment, a sample suspected of containing an analyte (or other substance of interest) is deposited onto an upstream location of a substrate (such as a nitrocellulose strip) where the strip includes a detection reagent such as an enzyme at the upstream location and a capture reagent such as a capture antibody at the downstream location. The strip may include a wicking material that flows the sample to a downstream capture reagent. In some cases, as the sample flows downstream, the analyte (if present) within the sample may bind to a detection reagent (e.g., a detection enzyme), in which case the analyte and detection reagent may flow downstream to a capture antibody. In some cases, the analyte and the capture antibody may bind and form a complex, which may further form an interconnected network (e.g., a precipitate, a mesh). Such an interconnecting network may stop the flow of at least some components of the assay (such as the detection reagent) or significantly reduce the flow rate of at least some components. Details about this interconnection network will be further described below, but briefly, and without wishing to be bound by any particular theory, it is believed that forming the interconnection network may cause the formation of a precipitate and/or a grid in which additional analytes, capture antibodies, and optionally detection reagents may bind or be captured (e.g., captured within the pores of the interconnection network). The presence of such an interconnection network within the bonding area may generate a signal (e.g., a visual signal). In some cases, the signal is visually observable. In some cases, a stain or chromogenic reagent such as TMB (3, 3', 5' -tetramethylbenzidine) is applied to the binding region to generate a signal (e.g., a colorimetric signal). In some cases, during use of such chromogenic reagents on the strip, backflow of chromogenic reagents and/or sample or other components of the assay may be caused (i.e., flow from downstream to upstream). However, as described in the present disclosure, it has also been found that the strip may be (at least partially) enclosed in a cartridge that includes a protrusion (e.g., a bridge) extending from a first portion of the cartridge to an opposing second portion of the cartridge, which may press the strip against the first portion of the cartridge, which may reduce or prevent backflow of the sample (or components within the sample, such as detection reagents).

The process of forming an interconnected network of a plurality of capture reagents (e.g., capture antibodies) and a plurality of analytes is described in more detail below, but briefly, without wishing to be bound by theory, it is believed that the interconnected network increases the sensitivity (e.g., decreases the detection limit) of a flow assay compared to some prior assays in which the analytes bind to the capture reagents but do not form an interconnected network. As described in more detail below, the interconnected network may include a precipitate and/or mesh that includes a plurality of analytes and/or capture reagents (e.g., a mixture of analytes and capture reagents) therein, wherein at least some of the analytes and/or capture reagents are linked to one another. The linkage between the various analytes and/or capture reagents may be covalent interactions (e.g., bonds) and/or non-covalent interactions (e.g., ionic interactions, hydrogen bonding, hydrophobic interactions, van der Waals interactions). In some embodiments, the linkage between the plurality of analytes and/or capture reagents may be a complementary interaction, such as an antigen-antibody pairing (e.g., one or more antigen-antibody pairing). Other connections are also possible. Details regarding the interconnection network in flow assays will be further described below.

Turning to the drawings, specific non-limiting embodiments are described in more detail. It should be understood that the various components, features, systems, assays, and methods described with respect to these embodiments can be used alone and/or in any desired combination, as the present disclosure is not limited to the specific embodiments described herein.

FIG. 1A depicts a schematic diagram of a flow assay 100 for detecting an analyte. In this figure, flow assay 100 includes a substrate 110, the surface of substrate 110 having an upstream location 112 and a downstream location 114. Sample region 116 and detection reagent region 118 are located on substrate 110 near upstream location 112, where sample and detection reagent may be deposited, respectively. When a sample is introduced onto a substrate (e.g., in a sample region), a sample (or analyte within the sample) stream flows from an upstream location to a downstream location (e.g., via capillary action, wicking).

The flow assay 100 may also include a detection reagent 130 deposited in the detection reagent zone 118. The capture reagent 120 may be positioned at the binding region 122, and the capture reagent 120 may be configured to bind to the detection reagent 130 and/or an analyte within the sample. Thus, in some cases, capture reagent 120 is configured to bind to a particular analyte of interest, and may be of a size, shape, and/or composition that is complementary to the detection reagent so as to bind to both the detection reagent and the analyte of interest.

In fig. 1B, the sample containing analyte 140 has been deposited at the sample region 116, i.e., at a location upstream of the binding region 122. The detection reagent 130 and analyte 140 may flow toward the binding region 122 (e.g., via wicking), shown in this figure as flow 142, toward the downstream location 114. First, the detection reagent 130 may bind to the capture reagent 120. Next, after analyte 140 flows to binding area 122, analyte 140 may bind to capture reagent 120, as schematically shown in fig. 1C. After binding, the analyte-capture reagent-detection reagent complex can be detected (e.g., visually by an external user). The capture reagent 120 may be configured to remain in the binding region 122 once the capture reagent 120 binds to the analyte 140 (e.g., not removed from the binding region) (e.g., even if flow from the upstream location to the downstream location continues) as schematically illustrated in the figure.

In some embodiments, a plurality of analytes are present in the sample, and the flow assay can detect one or more of the plurality of analytes. For example, as schematically illustrated in fig. 1D, a plurality of analytes 140 are present within the sample region 116 and a plurality of capture reagents 120 are present within the binding region 122. A flow (e.g., flow 142) may be initiated and then the plurality of analytes 140 and detection reagents 130 may flow to the plurality of capture reagents 120 (e.g., from an upstream location to a downstream location), as shown in the figure.

The plurality of analytes and/or capture reagents may form an interconnected network (or be configured to form an interconnected network) that includes at least some of the plurality of analytes, capture reagents, and/or detection reagents. For example, as shown in FIG. 1E, an interconnection network 150 is shown that includes multiple analytes and multiple capture reagents. Advantageously, forming the interconnection network may increase the sensitivity of the flow assay compared to some existing flow assays. Details about this interconnection network will be described further below.

After the formation of the interconnection network, the analyte may be detected. For example, as shown in fig. 1F, staining reagent 160 is dispensed from pipette 162 onto binding area 122, which binding area 122 includes interconnection network 150. The staining reagent may alter the detection reagent and/or the analyte such that the detection reagent generates a signal in the presence of the analyte. For example, in this figure, detection reagent 130B may bind to or simply be blocked or captured by the interconnection network, and may generate a signal indicative of the presence of at least one analyte 140 and exposure to staining reagent 160.

The interconnected network (e.g., precipitates, grids) may have a variety of configurations. Fig. 1G-1K schematically depict some example configurations. For example, in fig. 1G, the interconnection network 150 includes a plurality of capture reagents 120 and a plurality of analytes 140, and is connected by non-covalent interactions between the plurality of analytes 140. In contrast, fig. 1H depicts an interconnected network joined by at least one bond 152 between two analytes 140. Fig. 1I schematically depicts a configuration of an interconnection network 150, wherein a bond 152 is located between two capture reagents 120. In fig. 1J, capture reagent 120 bridges the two analytes, forming a cement for the interconnection network 150. Fig. 1K schematically depicts a configuration of an interconnection network 150 in which an analyte 140 bridges two capture reagents 120, thereby forming a cement for the interconnection network 150. In some embodiments, the capture reagent is configured to bind two or more analytes. For example, fig. 1L schematically illustrates capture reagent 120 configured to bind two analytes 140 such that interconnection network 150 includes capture reagent 120 that binds two analytes. In some embodiments, at least some analytes may bind to more than one capture reagent, and at least some capture reagents may also bind to more than one analyte and form chains or cements of capture reagents and analytes. That is, in some embodiments, the interconnected network includes chains or cements comprising multiple capture reagents and/or analytes. Of course, other configurations of the interconnection network are possible, and these configurations are not limited to those schematically depicted in the drawings. Further details regarding the interconnection network will be described below and elsewhere herein.

The formation of an interconnected network (e.g., a mesh) in a flow assay may affect the flow characteristics of the assay. For example, the interconnection network may block the pores of the substrate such that flow is reduced, restricted, and/or stopped over at least some portions of the substrate. In some embodiments, backflow (i.e., flow from a downstream location to an upstream location) is possible. To mitigate or eliminate reflow, it has been found that the substrate can be encapsulated (e.g., at least partially encapsulated) in a cassette that includes a protrusion that presses the substrate against the cassette. By way of illustration, and not limitation, fig. 2A and 2B depict schematic diagrams of such a box. Fig. 2A shows a top view of a cartridge 200, the cartridge 200 having a sample window 210 for depositing a sample, and a viewing window 220 for viewing a substrate binding area. Fig. 2B depicts a cross-sectional view of the cartridge 200, showing a first portion 230 and a second portion 240 of the cartridge 200, the second portion 240 having a tab 250, the tab 250 extending toward the opposite first portion 230 of the cartridge. The protrusion may press the substrate against the first portion of the cartridge, which may advantageously reduce or eliminate backflow of one or more components of the flow assay (e.g., detection reagent, sample solvent). While fig. 2A-2B depict one tab of the cassette, it should be understood that one or more tabs may be present. Additional details regarding the cartridge and its protrusions will be further described below and elsewhere herein.

As mentioned above, the articles and methods described herein may be used in flow assays. For example, in some embodiments, the flow assay is a lateral flow assay. As understood by those of skill in the art, lateral flow assays are a diagnostic technique for confirming the presence (or absence) of an analyte. Lateral flow assays typically include a planar substrate, such as a sheet or strip of material (e.g., paper) that can absorb a liquid sample, and may include test lines where a user can view the assay results and control lines where a user can ensure that the assay is functioning properly. The sample may be deposited at a sample deposition area at a location upstream of the lateral flow assay, and then the sample may flow from the location upstream of the substrate to a location downstream of the substrate (e.g., to a test line and/or control line of the substrate). However, it should be noted that while the articles and methods described herein may be applicable to lateral flow assays, other types of flow assays are also possible. For example, the flow assay may also include chromatographic techniques (e.g., thin layer chromatography, liquid chromatography, reverse phase chromatography), and the sample (or analyte within the sample) may flow through one or more stationary phases of the chromatography-based assay with the assistance of one or more mobile phases (e.g., solvents), as the disclosure is not so limited. Gel assays or microfluidic channel assays are also possible.

For some embodiments, the flow-assayed substrate has an upstream location and a downstream location. In some embodiments, the substrate includes a detection reagent (or reagents) positioned at a location upstream of the substrate. In some embodiments, the sample is introduced into a location upstream of the substrate (e.g., within the sample loading region or within the sample deposition region).

The substrate may comprise any suitable material. In exemplary embodiments, the substrate is or includes cellulose (e.g., nitrocellulose). However, other materials are also possible. For example, in some embodiments, the substrate comprises or is formed from a polymer or polymeric material (e.g., cellulose fibers, nylon, PVDF, polymer gel), wherein the polymer or polymeric material is not limited thereto. Other non-limiting examples of substrate materials include paper, glass, quartz, capillaries, gels, filled beads (e.g., silica beads), and woven webs. In some embodiments, the substrate comprises an absorbent material (e.g., cotton, cellulosic fibers, absorbent pads). In some embodiments, the substrate is or includes one or more membranes or membrane materials that can selectively pass certain substances while rejecting others (e.g., do not allow passage of an interconnected network containing one or more analytes, but allow passage of other components). In some embodiments, the substrate comprises a flexible or hard material to facilitate convenient gripping of the substrate. In some embodiments, the substrate comprises one or more portions of a plurality of layers. In some such embodiments, each portion and/or each layer may independently comprise or be formed from a suitable material.

In some embodiments, the substrate is a porous substrate. For example, in some embodiments, the porosity of the substrate is greater than or equal to 20%, greater than or equal to 25%, greater than or equal to 30%, greater than or equal to 40%, or greater than or equal to 50%. In some embodiments, the porosity of the substrate is less than or equal to 70%, less than or equal to 60%, less than or equal to 50%, less than or equal to 40%, less than or equal to 30%, less than or equal to 25%, or less than or equal to 20%. Combinations of the above-mentioned ranges are also possible (e.g., greater than or equal to 20% and less than or equal to 40%). Other ranges are also possible.

In some embodiments, the porous substrate may have pores of a particular pore size. For example, in some embodiments, the pores of the substrate have an average pore size greater than or equal to 1 μm, greater than or equal to 2 μm, greater than or equal to 5 μm, greater than or equal to 10 μm, greater than or equal to 15 μm, greater than or equal to 20 μm, greater than or equal to 25 μm, greater than or equal to 30 μm, greater than or equal to 40 μm, or greater than or equal to 50 μm. In some embodiments, the average pore size of the pores of the substrate is less than or equal to 50 μm, less than or equal to 40 μm, less than or equal to 30 μm, less than or equal to 25 μm, less than or equal to 20 μm, less than or equal to 15 μm, less than or equal to 10 μm, less than or equal to 5 μm, less than or equal to 2 μm, or less than or equal to 1 μm. Combinations of the above-mentioned ranges are also possible (e.g., greater than or equal to 1 μm or less than or equal to 50 μm). Other ranges are also possible.

In some embodiments, the substrate includes one or more layers of material (e.g., pads of material) that may be stacked one on top of the other, and/or one or more layers that are placed adjacent to each other to form the substrate. Retention and/or rejection may be selected based on a variety of factors including, but not limited to, size, charge, and/or mass of the substance (e.g., analyte-capture antibody complex, interconnected network comprising one or more analytes). For example, in some embodiments, the substrate includes a membrane that can allow some substances to pass through while retaining other substances based on size exclusion. In some embodiments, specific pore sizes, charges, or other characteristics of the substrate may be important for determining the flow difference between the free analyte and the analytes within the interconnected network.

It should also be noted that when a portion or layer of a substrate is referred to as being "adjacent" to another portion or layer, it can be directly adjacent to the portion or layer, or one or more intervening components (e.g., portions or layers including, but not limited to, films, pads, polymer layers, glass layers, coatings, and/or fluids) may also be present. One part or layer is "directly adjacent" to another part or layer, meaning that there are no intervening components present.

The substrate or base layer may have any suitable thickness. In some embodiments, the thickness of the substrate (or substrate layer) is greater than or equal to 100 μm, greater than or equal to 250 μm, greater than or equal to 500 μm, greater than or equal to 750 μm, greater than or equal to 1mm, greater than or equal to 2mm, greater than or equal to 3mm, greater than or equal to 4mm, greater than or equal to 5mm, or greater than or equal to 10mm. In some embodiments, the thickness of the substrate (or substrate layer) is less than or equal to 10mm, less than or equal to 5mm, less than or equal to 4mm, less than or equal to 3mm, less than or equal to 2mm, less than or equal to 1mm, less than or equal to 750 μm, less than or equal to 500 μm, less than or equal to 250 μm, or less than or equal to 100 μm. Combinations of the above-mentioned ranges are also possible (e.g., greater than or equal to 100 μm and less than or equal to 5 mm). Other ranges are also possible.

In some embodiments, the binding region is positioned on the substrate. The binding region may include a test line where a capture reagent (e.g., a capture antibody) may be deposited. In some embodiments, the binding region further comprises a control line, wherein a control reagent (e.g., another capture antibody) may be used in order to ensure that the flow assay adequately signals the user. In some embodiments, the binding region comprises a plurality of capture reagents positioned at a downstream location. In some embodiments, an interconnected network comprising one or more analytes is deposited before or within the binding region.

In some embodiments, the binding region comprises one or more capture reagents, wherein the capture reagents are a single type of capture antibody that is localized at the binding region at or on the substrate surface. That is, a single type of capture antibody may bind a single specific antigen, and may be the only type of antibody present within a lateral flow assay (i.e., within the binding region of a flow assay, or within all reagents used for an assay for a particular analyte of interest). In some embodiments, there may be more than one capture reagent, but these capture reagents may be a single type of capture reagent of the same type, wherein each capture reagent targets the same analyte. In some such embodiments, the binding region comprises only a single type of capture antibody, such that all capture antibodies each can bind an analyte, each analyte being located at the same location of the antibody. However, in other embodiments, there may be more than one capture reagent, but each capture reagent may target a different analyte, or a different portion of the same analyte (e.g., where the capture reagent comprises polyclonal antibodies, each of which may target a different epitope of the analyte or antigen).

The articles and methods described herein may be used to detect an analyte within a sample. The sample may be any suitable sample containing or suspected of containing the analyte of interest. Thus, in some cases, the sample (or at least a portion of the sample) does not contain an analyte, but may be suspected of containing an analyte of interest.

The sample may be obtained from any suitable subject (such as a human or animal), or may be obtained from the environment in order to test for analytes in the environment (e.g., to detect analytes in sewage or river water). In some embodiments, the sample is obtained from a cell (e.g., a human cell). In some embodiments, the sample may be obtained from a nasal swab of a patient in order to determine whether the patient has a disease. In some embodiments, the sample is (or is obtained from) a blood sample, a saliva sample, or a urine sample. In some embodiments, the sample may be subjected to a treatment (e.g., physical treatment, chemical treatment) prior to introduction of the sample into the sample region, in order to (at least partially) purify the sample and/or the analyte. For example, the analyte may be contained in a solid sample (e.g., a fecal sample), and the solid sample may be further processed (e.g., suspended in a solvent, filtered) to produce a liquid sample.

In some embodiments, the sample may include a solvent for dissolving and/or suspending sample components (e.g., analytes contained within the sample). In some embodiments, the sample solvent may also facilitate the flow of the sample along the flow assay (e.g., via capillary action, via wicking). In some embodiments, the solvent may also be used to wash the flow assay, e.g., to wash unbound detection reagent and/or capture reagent from the assay. In some embodiments, the solvent is an aqueous solvent (e.g., water). However, other solvents are also possible. The solvent may be selected based on a variety of factors including, but not limited to, the ability to dissolve the sample components, and compatibility with the substrate material of the substrate. Non-limiting examples of other solvents include acetone, acetonitrile (MeCN), benzene, butanol, carbon tetrachloride, chloroform, dichloromethane (DCM), dimethylformamide (DMF), dimethylsulfoxide (DMSO), dioxane, ethyl acetate, diethyl ether, isopropanol, ethanol, methanol, tetrahydrofuran (THF), toluene, or water. Other solvents are also possible.

In some embodiments, the sample (or solvent of the sample) flows via wicking, such as in a lateral flow assay. However, the sample may also flow via other mechanisms, such as via capillary forces, or via an applied negative or positive pressure (e.g., via vacuum, via a pump, using compressed gas). In some embodiments, the flow assay is operably associated with an external pump to provide flow.

The articles and methods described herein may be suitable for detecting a variety of analytes. In exemplary embodiments, the sample comprises an analyte that is or is derived from a pathogen, such as an antigen (e.g., a viral protein or nucleic acid). In some such embodiments, the analyte comprises a nucleic acid from or related to SARS-CoV-2. However, other analytes or antigens are also possible. In some embodiments, the analyte comprises a protein, peptide, hormone, toxin, nucleic acid (e.g., a nucleic acid fragment), and/or gene fragment, or other suitable biomarker. In some embodiments, the analyte may be derived from a virus, a bacterium, a fungus, a plant cell, or an animal cell (e.g., a human cell). In some embodiments, the sample is obtained from a fluid sample (e.g., blood, urine, or saliva) of a user or patient.

In some embodiments, analytes can be detected at a relatively low limit of detection (LOD) compared to certain existing lateral flow assays. In the case where the analyte is derived from a pathogen, LOD may be determined by measuring the median tissue culture infection dose (TCID 50) assay. As will be appreciated by those skilled in the art, the assay is performed by adding serial dilutions of a sample containing a pathogen of interest (e.g., a virus) to a plurality of cells in a 96-well format plate. The cell type is specifically selected to exhibit cytopathic effects, i.e., morphological changes when the cell is infected with a pathogen of interest or alternatively when the cell dies. After the incubation period, cells were checked for CPE or cell death, and each well was then classified as infected or uninfected. In some cases, colorimetric or fluorescent readings may aid in this classification, which may improve assay sensitivity. TCID ₅₀ was calculated using 50% of the wells to show CPE or dilution at cell death. In some embodiments, the pathogen concentration is less than or equal to 1,000TCID ₅₀/mL, less than or equal to 200TCID ₅₀/mL, less than or equal to 32TCID ₅₀/mL, less than or equal to 24TCID ₅₀/mL, less than or equal to 20TCID ₅₀/mL, less than or equal to 15TCID ₅₀/mL, less than or equal to 10TCID ₅₀/mL, less than or equal to 5TCID ₅₀/mL, less than or equal to 3TCID ₅₀/mL, less than or equal to 1TCID ₅₀/mL, less than or equal to 0.5TCID ₅₀/mL, less than or equal to 0.4TCID ₅₀/mL, less than or equal to 0.3TCID ₅₀/mL, less than or equal to 0.2TCID ₅₀/mL, or less than or equal to 0.1TCID ₅₀/mL. In some embodiments, TC50 is greater than or equal to 0.1TCID ₅₀/mL, greater than or equal to 0.5TCID ₅₀/mL, greater than or equal to 1TCID ₅₀/mL, greater than or equal to 3TCID ₅₀/mL, greater than or equal to 5TCID ₅₀/mL, greater than or equal to 10TCID ₅₀/mL, greater than or equal to 15TCID ₅₀/mL, greater than or equal to 20TCID ₅₀/mL, greater than or equal to 24TCID ₅₀/mL, greater than or equal to 32TCID ₅₀/mL, greater than or equal to 200TCID ₅₀/mL, or greater than or equal to 1,000TCID ₅₀/mL. Combinations of the above-mentioned ranges are also possible (e.g., greater than or equal to 0.1TCID ₅₀/mL and less than or equal to 32TCID ₅₀/mL). Other ranges are also possible.

As mentioned above, the flow assays described herein may include one or more capture reagents (e.g., a plurality of capture reagents). The capture reagent may be configured to bind to the analyte, but may also be configured to bind to one or more detection reagents. In some embodiments, each of the plurality of capture reagents may be independently bound to one or more analytes (e.g., two analytes, three analytes). In some embodiments, the plurality of capture reagents are positioned at a downstream location on the substrate surface. In some embodiments, the method comprises allowing the plurality of analytes (or at least a portion of the plurality of analytes) of the sample to bind to at least some of the plurality of capture reagents. In some embodiments, the plurality of capture reagents is configured to form an interconnected network with the plurality of analytes and/or the plurality of detection reagents or other capture reagents.

In some embodiments, the capture reagent (e.g., plurality of capture reagents) is positioned (e.g., immobilized, bound, bonded) on the substrate, e.g., within a binding region of the substrate, and/or positioned on a test line of the substrate (e.g., within a binding region). The capture reagent can be positioned on the substrate in a variety of ways, such as via covalent interactions or via non-covalent interactions (e.g., absorption, adsorption, electrostatic interactions, dispersal forces, or a combination thereof). In exemplary embodiments, the capture reagent is a capture antibody (e.g., igG, igM, igA, igD or IgE antibody) configured to complementarily bind to one or more analytes (e.g., antigens or viral-derived nucleic acids). In exemplary embodiments, the capture reagent is further configured to form an interconnected network with other capture reagents and/or with the analyte and/or detection reagent. However, other capture reagents are also possible, as the disclosure is not so limited. In some embodiments, the capture reagent includes an aptamer (e.g., protein, oligonucleotide) configured to bind to a particular target molecule.

In some embodiments, the capture reagent may be disposed, deposited, immobilized, or positioned (e.g., prior to first use) at a downstream location relative to the detection reagent and/or the sample introduction region (e.g., in a binding region positioned downstream of the sample introduction region). However, in other embodiments, the capture reagent may alternatively or additionally be provided with the detection reagent (e.g., prior to first use).

In some embodiments, the capture reagent may be configured to complementarily bind to both the detection reagent and the analyte. In some such embodiments, binding to both the detection reagent and the analyte may allow the capture reagent to remain attached to the binding region of the substrate. However, in some embodiments, when the capture reagent binds to the detection reagent but does not bind to the analyte, the capture reagent may be configured to be removed (e.g., separated, released) from the binding region of the substrate as liquid flows through the binding region during use. In some embodiments, when the capture reagent binds to the detection reagent but not to the analyte, the capture reagent does not form an interconnected network and thus can be washed or flowed to different locations on the substrate (e.g., through the binding region to the waste region).

In some embodiments, the capture reagent may not be bound to the substrate (e.g., prior to first use), but may be subsequently bound to the substrate or positioned on the substrate (e.g., at the binding region) when it binds the detection reagent and/or analyte. In some such embodiments, the capture reagent may form an interconnected network with the detection reagent and/or analyte. In some such embodiments, the capture reagent is configured to not flow (or to flow at a slower rate) on or in the substrate (e.g., away from the binding region of the substrate during the first use flow assay) in the event that the capture reagent binds to the analyte. That is, in some such embodiments, the analyte (and/or the liquid carrying the analyte) may cease to flow or have a reduced flow rate when bound to the capture reagent and/or may cause another component of the assay to cease to flow or have a reduced flow rate (e.g., the flow of upstream capture reagent and/or detection reagent may be reduced or stopped when the analyte binds to the capture reagent and/or forms a network containing the analyte along with the capture reagent and/or detection reagent). In some embodiments, the flow of capture reagent and/or detection reagent is restricted (e.g., due to capture reagent-analyte interactions) such that more capture reagent remains on the substrate. However, for embodiments in which the sample contains no analyte (e.g., target analyte) or relatively little analyte, the flow of capture reagent and/or detection reagent is less restricted or completely unrestricted. Thus, when a chromogenic reagent (e.g., TMB) is applied to a substrate (e.g., a binding reagent of the substrate), more color development of the capture reagent and/or detection reagent occurs relative to when less analyte is present (or no analyte is present) within the sample. Without wishing to be bound by any particular theory, it is believed that when more analyte is present, the flow restriction due to analyte-capture reagent interactions may be stronger (and thus more restrictive to flow rate relative to a sample containing no or less analyte), and that such differences may enable quantitative or semi-quantitative measurement of the amount (e.g., concentration) of analyte within the sample. It is believed that this is possible when more analyte is present within the sample, because more analyte results in more restriction to flow, and thus detection of analyte (e.g., visual detection of analyte) becomes easier, because more analyte can bind to more capture reagent on the substrate than less analyte (or no analyte) is present in the sample, and can also bind to more detection reagent. In some embodiments, this difference in flow restriction may occur due to the formation of an analyte-capture reagent network (e.g., a grid) when there is more analyte in the sample relative to the sample with less analyte (or no analyte). However, it should be appreciated that such a difference in flow restriction may occur even without the formation of an analyte-capture reagent network.

In some embodiments, when a sample comprising an analyte (e.g., at least one analyte, multiple analytes) flows (e.g., flows through and/or binds to one or more capture reagents) in a lateral flow assay, the flow is restricted, wherein when the sample is free of analytes, the flow is unrestricted. These events may occur before the sample and/or liquid has reached the end of the substrate (e.g., the end of the substrate furthest downstream).

In some embodiments, the sample flow and/or liquid (e.g., solvent) flow over the assay may be limited, reduced, or otherwise slowed relative to the initial flow rate. By way of illustration and not limitation, the sample can flow on the assay at a flow rate of 2mm/s, e.g., after at least one of the plurality of analytes binds to a portion of the plurality of capture reagents, the flow rate of the sample can be limited to 1mm/s or less. Without wishing to be bound by any particular theory, it is believed that forming a network or network comprising analytes and/or capture reagents may reduce the flow rate of the sample, for example, by filling pores (i.e., size exclusion) on the substrate. Alternatively, without wishing to be bound by any theory, analyte binding to capture reagent may block pores in nitrocellulose membrane (or other substrate) due to physical obstruction, charge-based obstruction, or other effects. In some embodiments, the flow restriction is due to the formation of an interconnecting network between the analyte, the capture reagent (and in some embodiments, other components of the sample liquid, such as salts and surfactants). In other embodiments, simply stated, individual analyte-capture reagents may block flow only, thereby forming a non-interconnected blocking network. Without wishing to be bound by any particular theory, this may be a result of pores of the nitrocellulose membrane or other substrate being blocked due to size, charge, or other limitations. In some embodiments, the sample flow rate and/or liquid flow (e.g., as compared to the flow rate of the sample/liquid at the upstream portion of the substrate (such as at any of the regions 112, 116, 118, and/or 110 shown in fig. 1A)) on/in the assay is reduced to at least 0.9, 0.8, 0.6, 0.5, 0.4, 0.2, or 0.1 times the sample or liquid flow rate before flow restriction. In some embodiments, the sample flow rate and/or liquid flow (e.g., as compared to the flow rate of the sample/liquid at the upstream portion of the substrate (such as at any of the regions 112, 116, 118, and/or 110 shown in fig. 1A)) on/in the assay is reduced to less than or equal to 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, or 0.9 times the sample or liquid flow rate prior to the flow restriction. Combinations of the above mentioned ranges are also possible. These events may occur before the sample and/or liquid has reached the end of the substrate (e.g., the end of the substrate furthest downstream).

In some embodiments, the sample flow rate and/or the liquid (e.g., solvent) flow over/in the assay is reduced by greater than or equal to 0.1mm/s, greater than or equal to 0.2mm/s, greater than or equal to 0.3mm/s, greater than or equal to 0.4mm/s, greater than or equal to 0.5mm/s, greater than or equal to 1mm/s, greater than or equal to 2mm/s, greater than or equal to 3mm/s, or greater than or equal to 5mm/s relative to the initial flow rate of the sample (or liquid) over the lateral flow assay (e.g., prior to limiting relative to the sample flow rate). In some embodiments, the sample flow rate and/or the liquid (e.g., solvent) flow over/under the assay is reduced by less than or equal to 5mm/s, less than or equal to 3mm/s, less than or equal to 2mm/s, less than or equal to 1mm/s, less than or equal to 0.5mm/s, less than or equal to 0.4mm/s, less than or equal to 0.3mm/s, less than or equal to 0.2mm/s, or less than or equal to 0.1mm/s relative to the initial flow rate of the sample (or liquid) over the lateral flow assay (e.g., relative to the flow rate of the sample/liquid at the upstream portion of the substrate, such as at any of regions 112, 116, 118, and/or 110 shown in fig. 1A, prior to the sample flow rate being limited). Combinations of the above-mentioned ranges are also possible (e.g., greater than or equal to 0.1mm/s and less than or equal to 5 mm/s). Other ranges are also possible. For some embodiments, the sample/liquid flow rate is slowed, but not stopped (e.g., the flow rate is not 0 mm/s). For other embodiments, the flow rate of the sample is stopped. These events may occur before the sample and/or liquid has reached the end of the substrate (e.g., the end of the substrate furthest downstream).

In some embodiments, the restriction and/or reduction in the flow rate of the sample/liquid may occur in any of the flow assays described herein. In some embodiments, the flow assay comprises: a substrate having an upstream location and a downstream location; and a binding region comprising a plurality of capture reagents positioned at a downstream location, wherein the plurality of capture reagents are configured to form an interconnected network with the plurality of analytes, the interconnected network comprising a mixture of the plurality of capture reagents and the plurality of analytes interconnected with each other. Other configurations are also possible. The method may involve, for example, introducing a sample into an upstream location of a substrate, wherein the substrate includes a plurality of capture reagents positioned at a binding region at a downstream location; flowing the sample from the upstream location to the downstream location; allowing at least a portion of the plurality of analytes of the sample to bind to at least a portion of the plurality of capture reagents; restricting flow along the flow assay after at least a portion of the plurality of analytes of the sample bind to at least a portion of the plurality of capture reagents; and detecting at least one analyte. Other steps are also possible.

In some embodiments, the capture reagent is positioned on the substrate but not bound (or only slightly bound) to the substrate such that the capture reagent can flow along the substrate when a liquid (e.g., solvent) is applied. However, when the capture reagent binds to the analyte and/or detection reagent, the resulting complex may not move (or move slower) along the substrate. In some embodiments, the complex forms or is located within an interconnecting network, and the interconnecting network may not move or may stop within the bonding region of the substrate. Further details regarding the interconnection network will be described below.

In some embodiments, a chemical substance (such as a binding entity) may be attached (e.g., bonded) to the capture reagent, e.g., to facilitate its attachment to the substrate. In some embodiments, the chemical is or includes biotin, a biotinylated derivative, or other suitable binding entity. That is, the capture reagent may be biotinylated, which may facilitate binding of the capture reagent to the substrate (e.g., a binding region on the substrate). In some embodiments, the capture reagent comprises, is attached to, or is bound to a detection reagent, e.g., via biotin attached to the capture reagent. However, other binding entities besides biotin are also possible, as the disclosure is not so limited.

As mentioned above, various embodiments may also include one or more detection reagents (e.g., a plurality of detection reagents). For example, in some embodiments, multiple detection reagents are positioned at upstream locations on the substrate surface. The detection reagent (when present) is configured to facilitate detection and/or identification of the analyte. For example, the detection reagent may be configured to allow detection of the analyte via a color change of the detection reagent itself, by facilitating an enzymatic reaction that may be detected, and/or by allowing binding of additional reporter molecules. The detection reagent may be positioned on any suitable portion of the substrate to facilitate detection of the analyte (if present) within the sample. In some embodiments, the plurality of detection reagents is positioned upstream of the plurality of capture reagents. In some embodiments, the method comprises allowing the plurality of analytes (or at least a portion of the plurality of analytes) of the sample to bind to at least a portion of the plurality of detection reagents. However, in other embodiments, one or more detection reagents are not positioned on the substrate (e.g., prior to first use of the flow assay), but may be added to the flow assay later (e.g., after sample deposition on the flow assay).

The detection reagent may be any suitable reagent for generating a signal to determine the presence (or absence) of an analyte. In some embodiments, the detection reagent includes a nanoenzyme (i.e., a metal nanoparticle catalyst), such as Fe ₃O₄ nanoparticles. In some embodiments, the detection reagent comprises gold nanoparticles and/or latex nanoparticles, or other nanoparticles. In some embodiments, the detection reagent comprises an enzyme, such as glucose oxidase, cholesterol esterase, cholesterol oxidase, or horseradish peroxidase, or a conjugate, molecule, or polymer thereof. Other detection reagents are also possible.

In some embodiments, the articles and methods described herein may further comprise additional reagents for detecting an analyte. For example, some embodiments may include the following reagents (e.g., staining reagents): configured to react with a detection reagent and/or an analyte to generate a signal for detecting the analyte, for example, by staining the analyte, the detection reagent, and/or a product produced by the analyte and/or the detection reagent (e.g., the detection reagent may react with the analyte to produce a product that generates a signal for detection). The stain may be used to qualitatively detect the presence of an analyte. In an exemplary embodiment, 3', 5' -Tetramethylbenzidine (TMB) is used as a staining agent after the analyte interacts with the detection reagent. However, other reagents are also possible. Non-limiting examples of other agents include OPD (o-phenylenediamine dihydrochloride), ABTS (2, 2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) and/or NPP (disodium p-nitrophenylphosphate). In some embodiments, additional reagents for analyte detection include nanoparticles (e.g., gold nanoparticles, latex nanoparticles) and/or dyes (e.g., luminol, CPD-Star, ABEI). While some embodiments may facilitate visual detection, in other embodiments, any suitable detection device may be used to detect the analyte, including spectroscopic methods using absorbance or luminosity, or other spectroscopic detection methods, which may be used to qualitatively and/or quantitatively determine the presence of the analyte. For example, some embodiments may include a detector (e.g., PMT detector, PDA detector) to detect the analyte. In some embodiments, a digital reader may be attached to the substrate to facilitate detection of the analyte.

The time for detecting the analyte may be any suitable time. In some embodiments, the detection time is less than or equal to 30 minutes, less than or equal to 20 minutes, less than or equal to 15 minutes, less than or equal to 10 minutes, less than or equal to 6 minutes, less than or equal to 5 minutes, less than or equal to 3 minutes, or less than or equal to 1 minute. In some embodiments, the detection time is greater than or equal to 1 minute, greater than or equal to 5 minutes, greater than or equal to 10 minutes, greater than or equal to 15 minutes, greater than or equal to 20 minutes, or greater than or equal to 30 minutes. Combinations of the above-mentioned ranges are also possible (e.g., greater than or equal to 1 minute and less than or equal to 30 minutes). Other ranges are also possible.

As described above, the analyte and capture reagent (e.g., capture antibody) may form (or be configured to form) an interconnected network. The interconnected network may be formed by or comprise a precipitate (e.g., a precipitate comprising a plurality of analytes and a plurality of capture reagents) or a mesh comprising a plurality of analytes and a plurality of capture reagents. The interconnected network may optionally include other reagents, such as a plurality of detection reagents and/or other binding entities. In some embodiments, the interconnection network comprises a plurality of capture reagents and analytes, wherein at least some of the capture reagents and/or analytes are interconnected via one or more bridges comprising capture reagents and/or analytes linking two or more other capture reagents and/or analytes.

Without wishing to be bound by any particular theory, it is believed that the combination of a soluble analyte and a soluble capture reagent (i.e., one or more components that are soluble in the sample, such as a solvent sample and/or sample buffer) may cause a grid to be formed that includes both components that is visible to the user. In some cases, a region of precipitates (e.g., a precipitation ring) is formed when a soluble analyte and a soluble capture reagent meet on a substrate, and the region may be formed at an equivalence point between the capture reagent and the analyte (e.g., capture antibody and antigen), with an interconnecting network formed (or beginning to form) at the equivalence of the capture reagent and the analyte. In some embodiments, the mesh is soluble (e.g., at least slightly soluble, at least partially soluble) when within the flow assay. However, in other embodiments, the mesh is insoluble on or within the flow assay. As will be appreciated by those skilled in the art, the solubility (or insolubility) may vary depending on the sample solvent, sample buffer, and may also vary with other factors such as temperature. Without wishing to be bound by any particular theory, a substance is insoluble if its solubility product (K _sp) in a particular solvent at 25 ℃ is less than 10 ^-3 (e.g., less than or equal to 10 ^-4, less than or equal to 10 ^-5, less than or equal to 10 ^-6, less than or equal to 10 ^-9). A substance may be slightly or partially soluble/insoluble if K _sp at 25 ℃ in a particular solvent is between 10 ^-3 and 10 ³ (e.g., between 10 ^-3 and 1, between 10 ^-2 and 1, between 0.1 and 1, between 1 and 10, between 1 and 100, between 1 and 10 ³). Generally, a substance is soluble if the K _sp at 25 ℃ is greater than 10 ³ (e.g., greater than 10 ⁴, greater than 10 ⁵, greater than 10 ⁶, greater than 10 ⁹). Combinations of these ranges are also possible. However, those skilled in the art will appreciate that these boundaries may vary depending on the factors described above, and that those skilled in the art will be able to select analytes and/or capture reagents whose complexes have the appropriate solubility (i.e., solubility that forms a grid between an upstream location and a downstream location or within a binding region of a substrate) in light of the present disclosure. In some embodiments, the solvent used to determine K _sp is water. However, other solvents are also possible, such as, but not limited to, the sample solvents described elsewhere herein.

In some embodiments, a complex is formed (or configured to form) between the capture reagent and the analyte, which complex can flow downstream to the binding region. In some embodiments, the size of the complex may increase (e.g., the number of capture reagents and/or analyte reagents in the complex may increase) as the complex flows from the upstream location to the downstream location of the assay. In some embodiments, the complex forms an interconnected network that includes a plurality of capture reagents and a plurality of analyte reagents. As the interconnected network (e.g., including precipitates and/or grids) migrates or flows toward the bonding area, the size of the interconnected network may increase. Thus, in some embodiments, the size of the composite or interconnecting network may increase (or decrease) as it moves from an upstream location toward a downstream location, or the size may increase within the bonding area of the substrate. In other embodiments, the reagents and/or complexes flow downstream, but do not form an interconnecting network until they reach the binding region.

Without wishing to be bound by any particular theory, it is believed that the analyte-capture reagent complexes (e.g., antigen-capture antibody complexes) may bind additional analytes and/or capture reagents (e.g., covalently bound, bound via non-covalent interactions) upon flow-through assay, or the analyte-capture reagent complexes may be immobilized within the binding region and bind with additional analytes and/or capture reagents as they move toward the binding region to form an interconnected network. The size of the interconnected network (including the analyte-capture reagent complex) may increase as the analyte-capture reagent complex binds additional analyte and/or capture reagent. In some embodiments, the analyte-capture reagent complex or interconnected network may also bind (or be configured to bind) one or more detection reagents, which may also increase the size of the complex or interconnected network. In some embodiments, increasing the size of the network includes adding a salt or buffer to the flow assay.

In some embodiments, a method describes forming an interconnected network that includes a plurality of capture reagents, a plurality of detection reagents, and/or a plurality of analytes. In some embodiments, the interconnected network comprises a precipitate or mesh comprising a plurality of capture reagents, detection reagents, and/or analytes. In some embodiments, the interconnected network includes agglomerates of analyte and capture reagent. In some embodiments, the interconnecting network forms precipitates within the substrate (e.g., within the bonding region of the substrate). In some embodiments, the plurality of capture reagents and/or the plurality of detection reagents are configured to form an interconnected network with the plurality of analytes at a location downstream of the substrate. In some embodiments, the interconnected network includes a mixture of a plurality of capture reagents and a plurality of analytes interconnected with each other. In some embodiments, the plurality of capture reagents and the plurality of detection reagents are configured to form an interconnected network with at least some of the plurality of analytes. In some embodiments, the interconnected network includes a mixture of a plurality of capture reagents, a plurality of detection reagents, and/or a plurality of analytes interconnected with one another.

In some embodiments, the interconnection network includes one or more linkages (e.g., bonds) that connect components of the interconnection network (e.g., analyte, capture reagent, and/or detection reagent) to each other. In some embodiments, the interconnected network includes one or more covalent bonds between the analyte, capture reagent, and/or detection reagent (e.g., at least some of the plurality of analytes, at least some of the plurality of capture reagents, and/or at least some of the plurality of detection reagents). In some embodiments, the interconnected network includes one or more non-covalent interactions between the analyte, capture reagent, and/or detection reagent. Non-limiting examples of non-covalent interactions include ionic interactions (e.g., salt bridges), hydrogen bonds, van der Waals interactions, and/or hydrophobic interactions. For example, in some embodiments, the interconnected network includes hydrogen bonds between the plurality of analytes, the plurality of detection reagents, and/or the plurality of capture reagents.

In some embodiments, one or more salts (or ionic species of one or more salts) may contribute to substrate flow obstruction. In some embodiments, these one or more salts (or ionic species of one or more salts) may contribute to the formation of an interconnecting network structure. The different types of salts and ions may come from a variety of sources, e.g., sample buffer, sample solvent, substrate (e.g., metal components of nitrocellulose membranes). Salts (or ionic species of salts) may affect fragmentation and/or activation of the side chains of an analyte (e.g., an analyte comprising an amino acid, such as a protein) during movement on a substrate. In some embodiments, the salt may also form at least a portion of an interconnection network; or may surround at least a portion of the interconnected network, which occurs in conjunction with analyte binding to the capture reagent. In some embodiments, the analyte (e.g., antigen) and capture reagent (e.g., capture antibody) bind and may form a single "seed" or nucleation site of the interconnected network (e.g., due to antigen-antibody chain formation or agglomeration), such seed may form the basis for salt accumulation or precipitation around the interconnected network, thereby enhancing the network and may further impede flow on the substrate after the interconnected network has been formed. Advantageously, this effect may cause a positive (target analyte-containing) sample to generate an even greater signal than a negative sample that does not contain the target analyte. Without wishing to be bound by any particular theory, when an analyte binds to a capture reagent, a conformational change may occur, which may cause the pores of the substrate to clog, for example, due to a negative or positive charge from a salt (e.g., a salt of a buffer system).

In some embodiments, a change in the percentage or concentration of salts and/or other ions may cause the formation of an interconnected network structure. In some such embodiments, the interconnected network may form a solid precipitate, which optionally includes one or more salts (e.g., salts of a buffer), which may block the pores of the substrate. In some such embodiments, the interconnecting network may accelerate the formation of salt bridges. In some such embodiments, salt blockage may be "seeded" for the formation of an interconnected network, and without wishing to be bound by any particular theory, salt blockage may be formed by several pathways: (A) Alternate ion charge agglomeration occurs (e.g., in sample buffer, antibody solution, HRP conjugate solution) such that a network of different types of ions is formed. The ions may be positively charged or negatively charged. Without wishing to be bound by any particular theory, these charges may play a role in exposing, breaking and/or activating side chains of the analyte as it moves on the substrate. Binding of the analyte to the capture reagent may result in blocking of the well. Additionally, accumulation of salts at or within the binding region can alter the charge distribution of the capture reagent and/or analyte within the binding region, and agglomeration may exist; (B) The counter ion causes salt formation, wherein one or more components of the buffer and the substrate contribute anions and cations, respectively. Some substrates may include cationic components, such as Ca ²⁺、Mg²⁺, silicon-containing cations, which may interact with ions of the sample buffer (if present) to cause formation of an interconnecting network or salt bridge (upon which the interconnecting network may be formed or evolved); (C) A calcium-phosphate is formed wherein the substrate comprises components such as Ca ²⁺、Mg²⁺, silicon-containing cations, and the like. At higher pH values (i.e., > 7), calcium-phosphate has a tendency to interact, which causes CaHPO ₄ to form. The presence of NaCl may also assist in the supply of carbonate, thereby promoting calcium-phosphate formation of precipitates.

In some embodiments, the sample comprises a salt and/or a buffer (e.g., phosphate buffer, naH ₂PO₄/Na₂HPO₄). In some such embodiments, the salt may assist in forming the interconnection network, or enhance the interconnection network by forming such structures around the interconnection network. Non-limiting examples of salts include NaCl、KCl、NH₄Cl、KNO₃、Al(NO₃)₃、(NH₄)₂SO₄、Mg₂SO₄、FeCO₃、CaCO₃、FePO₄、KH₂PO₄、Na₂HPO_4、(NH₄)₃PO₄、Pb(CH₃COO)₂、Cu(CH₃COO)₂. other salts are also possible.

In some embodiments, the sample (e.g., sample solvent, sample buffer) comprises a surfactant. In some such embodiments, the surfactant may also be attached to the capture reagent and/or analyte in a manner that helps to limit flow in the substrate. In some embodiments, the surfactant may form at least a portion of the interconnected network, or may assist in forming the interconnected network. Without wishing to be bound by any particular theory, the analyte (e.g., antigen) and capture reagent (e.g., capture antibody) bind, which may form a single "seed," which may form the basis for the accumulation or precipitation of surfactant around the interconnected network, thereby enhancing the network and further impeding flow on the substrate. In some embodiments, the surfactant may act to expose, cleave, and/or activate side chains of the analyte as the analyte moves on the substrate. In some embodiments, the surfactant may act to increase or decrease the hydrophilicity of the capture reagent and/or analyte or other portions of the interconnected network, which may cause or enhance network formation.

In some embodiments, this effect may cause a positive (i.e., analyte-containing) sample to generate an even greater signal than a negative sample that does not contain the analyte of interest.

In some embodiments, the surfactant includes anionic surfactants such as, but not limited to, ammonium lauryl sulfate, sodium laureth sulfate, sodium lauryl sarcosinate, sodium laureth sulfate, sodium alkanolamine sulfate, sodium dodecyl sulfate, sodium stearate, sodium deoxycholate, alpha-alkenyl sulfonate, and/or ammonium laureth sulfate.

In some embodiments, the surfactant includes a cationic surfactant such as, but not limited to, C8-10 alkyl hydroxyethyl dimethyl ammonium chloride, C8-10 alkyl amino dimethyl propylamine, and/or ditalloyl dimethyl ammonium chloride.

In some embodiments, the surfactant includes a nonionic surfactant, such as, but not limited to, polysorbates (e.g., tween 20), sorbitan esters, alkyl-phenol polyethylenes (e.g., triton), oligo-alkyl-ethyleneoxides, poly (alkylene oxide) block copolymers. In some embodiments, the surfactant is an amphoteric surfactant.

In some embodiments, a change in the percentage or concentration of surfactant may cause the formation of an interconnected network structure. In some such embodiments, the interconnecting network includes a surfactant and/or the surfactant surrounds at least a portion of the interconnecting network, which may block at least some of the pores of the substrate if the substrate is porous.

In some embodiments, surfactants (such as those described without limitation above) may cause a change in the configuration of the analyte (e.g., cause a change in the protein-containing analyte from a "closed" configuration to an "open" configuration), which may also enable or enhance binding to the capture reagent.

As mentioned above, various embodiments describe flow assays for detecting analytes. Thus, the sample (or sample solvent) can flow. Generally, flow is from an upstream location to a downstream location. For example, in some embodiments, the method includes flowing the interconnection network to a bonding region (e.g., a bonding region at a downstream location). In some embodiments, the method includes stopping flow of the interconnection network within the union region. In some embodiments, the method includes reducing or stopping flow on at least a portion of the substrate after forming the interconnection network. In some embodiments, at least some of the plurality of detection reagents do not flow from the flow assay during any one of the flow steps, such as after formation of an interconnection network in the flow assay. However, as noted above, in some embodiments, the formation of an interconnected network (e.g., a precipitate, a grid) in a flow assay may affect the flow characteristics of the assay such that flow from a downstream location to an upstream location (i.e., back flow) may occur. For example, in some embodiments, the method includes reflowing the interconnection network away from the bonding region.

In some embodiments, a control indicator may be used to indicate when a sample has passed through a portion or all of the substrate. In some embodiments, the control indicator may be a dye disposed in a downstream region of or on the substrate (e.g., in an absorbent pad or other pad at a downstream location on or near the substrate) such that as the sample liquid passes through the dye, the dye diffuses downstream and occurs in a region downstream of its initial location. The indicator may be any suitable color, such as a red or other color dye disposed at a location downstream of the substrate (e.g., the bottom of the absorbent pad). In the case of a red indicator, the indicator may diffuse downstream as the sample passes, such that red appears in the window of the cartridge downstream of the initial position of the dye. In some embodiments, the operator may use the red appearance in the cartridge to determine when to add detection reagents and/or chromogenic reagents to the assay. The indicator may be any flowing colorimetric reagent and may be read by eye or a reading device. In other embodiments, the color indicator comprises a liquid or solid containing fluorescent particles or charged or magnetic particles. Other color indicators are also possible. The use of an indicator may indicate when a sample (e.g., a liquid that may contain a sample analyte) has passed through the substrate. This may indicate that an interconnecting network has formed when one or more analytes are present in the sample, and/or that the detection reagent has flowed out of the substrate when no target analytes are present in the sample. Thus, when an indicator is present, detection reagents and/or chromogenic reagents (e.g., TMB) can be added later, which can distinguish positive (analyte-containing) samples from negative samples (analyte-free). The indicator may be a preferred alternative to sample flow timing because in many existing lateral flow assays or other systems, the sample may flow at different rates depending on temperature, humidity and other factors, but in all cases the indicator indicates the time at which the sample has flowed through the substrate at that location.

As mentioned above, the cartridge may be used to change the flow characteristics of a flow assay (e.g., a lateral flow assay). Thus, some embodiments include a cassette that at least partially encapsulates a substrate. As described above with reference to the figures, the cassette includes a first portion and a second portion opposite the first portion, and the flow assay substrate may be positioned between the first portion and the second portion. In some embodiments, the second portion includes a protrusion extending toward the first portion, and wherein the protrusion presses the substrate against the first portion. Advantageously, the protrusion may prevent excess sample from flowing over the substrate or the first portion of the cartridge without flowing through the substrate, and also reduce or prevent backflow of flowing assay components (e.g., sample solvent, at least a portion of the plurality of detection reagents, at least a portion of the chromogenic reagent). The protrusion may be pressed against the substrate so as to contact the first portion, but not cause any liquid (e.g., sample solvent) to be forced out of the substrate. The protrusions should contact the substrate with sufficient force to prevent liquid flow through the substrate or liquid backflow through the substrate; however, the protrusion should not be pressed into the substrate with too much force, as this may force the liquid out of the substrate and/or create indentations or grooves in the substrate where the liquid may accumulate. For example, such a groove may be used as a place where the color-developing agent is accumulated, and thus, such an excessive amount of the color-developing agent is not discharged from the substrate, but may remain in the groove. This may lead to false positive assay results because if given long enough, some chromogenic reagents may develop even in the absence of chromogenic reagent, which may lead to a signal that may appear positive, even in the absence of the target analyte in the sample. In other embodiments, false negative assay results may be generated due to dents. Thus, control of the specific features and manufacturing process of the tab is critical to proper operation. The cartridge may also include one or more windows, for example, for sample deposition and/or observation of analyte detection signals. For example, in fig. 2C to 2D, the observation window 251 is included in the box 200. Fig. 2E-2F show schematic views of the projection (e.g., bridge) 250 schematically illustrated in fig. 2C-2D.

In some embodiments, the protrusions are positioned to align with a particular location of the substrate. For example, in some embodiments, the protrusion is positioned at a midpoint between an upstream location and a downstream location (e.g., a union region of the downstream location). In some embodiments, the protrusion is less than or equal to 10% of the distance between the upstream location and the downstream location, less than or equal to 20% of the distance, less than or equal to 30% of the distance, less than or equal to 40% of the distance, less than or equal to 50% of the distance, less than or equal to 60% of the distance, less than or equal to 70% of the distance, less than or equal to 80% of the distance, or less than or equal to 90% of the distance. By way of illustration and not limitation, if the distance between the upstream and downstream locations is 3cm, the protrusion may be positioned at 50% of the distance, i.e., 1.5cm from the upstream location. In some embodiments, the protrusion is greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, or greater than or equal to 90% of the distance between the upstream and downstream positions. Combinations of the above-mentioned ranges are also possible (e.g., the protrusion is positioned at greater than 10% and less than or equal to 90% of the distance between the upstream and downstream positions). Other ranges are also possible.

The protrusion may extend from a surface of the second portion towards the first portion. The extent of the protrusion may influence the extent of the force (when present) applied to the substrate when the protrusion is pressed against the first portion. In some embodiments, the protrusion extends from the surface of the second portion a distance of greater than or equal to 0.1mm, greater than or equal to 0.2mm, greater than or equal to 0.3mm, greater than or equal to 0.5mm, greater than or equal to 0.7mm, or greater than or equal to 1mm. In some embodiments, the protrusion extends from the surface of the second portion a distance of less than or equal to 1mm, less than or equal to 0.7mm, less than or equal to 0.5mm, less than or equal to 0.3mm, less than or equal to 0.2mm, or less than or equal to 0.1mm. Combinations of the above-mentioned ranges are also possible (e.g., the protrusion extends from the surface of the second portion a distance greater than or equal to 0.1mm and less than or equal to 1 mm). Other ranges are also possible.

In some embodiments, the tab is formed of the same material as another portion of the cassette, such as plastic, including but not limited to polyethylene, polypropylene, ABS, HIPS. In other embodiments, the tab is formed of a different material than another portion of the cassette.

In some embodiments, the tab may be formed of a different material than the rest of the cassette. For example, the protrusions may be formed of a softer material relative to other components of the flow assay. In some embodiments, the tab comprises a soft material, such as rubber (e.g., silicone rubber) or a thermoplastic elastomer (e.g., polyurethane), that forms the entire tab or is over-molded onto another material that is the base of the tab. In some embodiments, the softer material may have a low shore hardness of 25A to 40A or less. Advantageously, it may be difficult to accurately achieve the depth required for the hard protrusions to penetrate the substrate (e.g., nitrocellulose membrane) using a low shore hardness material in order to accurately provide the correct force (i.e., the force is not so great as to squeeze the liquid from the substrate, but the force is not so small as to provide an improperly sized force to squeeze the substrate). Such tight tolerances may be difficult to achieve in large-scale manufacturing. However, it has been found by the present disclosure that the use of a softer protrusion having a material with a shore hardness of 25A to 40A may instead improve the manufacture of the protrusion.

The articles and methods disclosed herein are suitable for use in a variety of applications. In some embodiments, the article is a flow assay (e.g., a lateral flow assay). As mentioned above, for example, the presently disclosed articles and methods can be used to detect pathogens, such as SARS-CoV-2. As a non-limiting example, the presently disclosed articles and methods can be used to detect other pathogens, such as SARS-CoV-2 or variants of influenza virus. However, other applications are also possible. For example, the articles and methods described herein may be used to test for the presence of environmental contaminants, such as contaminants or toxins suspected to be present in a particular environment.

Example 1

The following examples show the movement and position of capture reagents (antibodies) and detection reagents (HRP conjugates) when run with positive samples (i.e., containing analyte) and negative samples (i.e., without analyte). HRP here means horseradish peroxidase, HRP conjugate means HRP is conjugated covalently or non-covalently with other molecules or substances.

In order to test the concept that in a negative sample, HRP conjugate and biotinylated antibody detection reagent were washed out into the absorbent pad of the negative sample substrate and then remained in the nitrocellulose membrane substrate with the target protein contained in the sample, 6 devices were used, 3 of which were confirmed positive by PCR and the other 3 were confirmed negative by PCR.

The sample is added to the sample pad. Once the sample has flowed through the strip and into the absorbent pad of the substrate. The absorbent pad is removed and placed into the well of a 96-well plate. Mu.l of TMB staining reagent was added. At room temperature, no color development was observed within 20 minutes.

Since the detection reagent HRP conjugate is known to be present on the absorbent pad, it is believed that the absorbent pad is highly likely to prevent the staining reagent TMB from contacting the detection reagent HRP conjugate and/or that a majority of the HRP conjugate is located on the back pad of the strip, considering sample flow. A second example, example 2, was designed to provide a different method to determine the location of detection reagent HRP conjugate.

Example 2

The following examples describe the separation of each segment of the lateral flow assay to determine the presence and amount of detection reagent HRP conjugate.

12 Flow assays were used, 6 with absorbent pads (fig. 3), and the other 6 without absorbent pads. For each device type, 6 samples were run, 3 of which were positive samples confirmed by PCR, and the other 3 were negative samples confirmed by PCR. The sample is added to the sample pad. Once the flow reaches the absorbent pad, having flowed through the entire film substrate, the lateral flow assay is split into its components and the 2.5cm film substrate is cut into 0.5cm sections, creating 8 discrete assay regions, as shown in fig. 4. As shown in fig. 5, these sections were placed into individual wells of a 96-well plate. mu.L of the staining reagent TMB was added. After 17 minutes, the reaction was terminated by adding 150. Mu.l of a termination solution. The assay components were removed from the wells and the plates were read at 450 nm.

Results

The results are shown in fig. 6. In all lateral flow assays, the conjugate pad of the detection reagent zone still contained a significant amount of HRP conjugate. For assays on which positive samples are run, the enzyme level is maintained at a constant level until the next section of the membrane substrate, section 2, which contains a lower level of antibody but passes the test line. In contrast, the level of enzyme remaining in the conjugate pad was much lower for the negative samples, extremely low in the following 2 membrane sections, and negligible in membrane sections 1 to 3. The enzyme level in the conjugate pad was lower for the negative samples and also for the membrane, indicating that in the case of negative samples (no antigen/protein of interest), HRP conjugate had mostly washed out of the membrane by the sample. This suggests that for positive samples, flow was reduced or stopped, and for negative samples, flow was neither reduced nor stopped, and HRP conjugate could be largely washed off the assay substrate (membrane).

There is a high level of enzyme in each section of the device running a positive sample that descends after the test line, indicating that binding of the target analyte antigen to the biotinylated antibody capture reagent restricts sample flow, thereby preventing enzyme flow.

The antigen-HRP conjugate complex (network or mesh) appears to block flow by physically blocking pores of the substrate or by causing a change in properties (e.g., pH or charge) of the membrane substrate or any other mechanism, which limits complex formation by a cascade or chain reaction between antigen, antibody and/or HRP conjugate. In the absence of antigen (negative sample), no limiting complex (network) was formed, so HRP conjugate could be washed away.

Example 3

The following examples describe the flow patterns of samples analyzed on lateral flow assays.

To further confirm the observations discussed in example 2, a third example was designed to track the flow profile of the sample for further analysis of HRP conjugate detection reagent movement along the strip (substrate) in the presence and absence of antigen (positive and negative samples).

A lateral flow assay was made using a longer nitrocellulose membrane substrate than the assay of example 2. An additional 1cm was added from the test line, making the distance between the sample pad and the test line the same as in the standard device (the strip used in experiment 2), while the absorbent pad was removed from the test line by 1cm.

Results

These assays were run as described previously above, and TMB staining reagent was added to the test area once the sample had stopped flowing along the membrane. The addition of TMB staining reagent generates TMB staining along the entire band to track the flow profile. As shown in fig. 7, depending on the sample, two flow spectra can be distinguished in the test line area.

For the negative samples, a poiseuille flow profile was observed, suggesting that the negative samples completely formed velocity flow. Whereas for positive samples the hydrodynamic inlet region may be distinguished from the non-swirling region.

This result is consistent with the previous observations in example 2, demonstrating that in a negative sample, the complex will flow along with the sample fluid and reach the absorbent pad. In contrast, in a positive sample, the antibody binds to the target antigen analyte, forming a complex through a cascade reaction or chain reaction between the antigen, antibody capture reagent, and/or HRP conjugate detection reagent. The captured composite creates a blocked region, thereby creating a non-swirling region.

Example 4

The following examples use IgG as the detection reagent.

The anti-S1 IgM was biotinylated and placed in the device for use in place of the biotinylated IgG detection reagent. These samples were used to run positive and negative samples.

The positive sample developed well and the negative sample remained blank. The size of IgG is 150kDa. The results are shown in fig. 8. In contrast, igM was 970kDa. In view of the large size difference between these two types of antibodies, the results suggest that not only does the size of the antibody-antigen complex prevent movement along the membrane substrate, but that there is further interaction between a single complex and the membrane substrate, or between two or more antibody-antigen complexes and/or salts or other components of the sample buffer. The latter suggests that an interconnected network is formed as the flow assay runs.

Example 5

The following examples demonstrate that other analytes besides the SARS-CoV-2 spike glycoprotein S1 subunit can also be used. For this example, respiratory Syncytial Virus (RSV) was selected as the source of analyte.

The mouse anti-RSV antibody IgG targeting the F protein, MAB12398-100, was used as detection reagent. Respiratory syncytial virus a lysate was used as the target analyte. Initially, the dilution was 1:10, decreasing from 100. Mu.g/mL to 1pg/mL. For positive samples, the signal reached 1pg/mL, and a 1:2 serial dilution was performed in the extraction buffer, decreasing from 0.5pg/mL to 3.91fg/mL.

Results

The results of each assay are shown in fig. 9-10. As can be seen, RSV lysate gave a positive signal as low as 62.5 fg/mL. These results indicate that the mechanism described in the above examples can be used to detect other pathogens and biomarkers and is not limited to SARS-CoV-2 spike glycoprotein S1.

Similarly, PSA was also run and tested in the same way, showing detection as low as pg/mL levels, and no signal in the case of negative samples (no antigen of interest). Since there may be a large difference in size between these different antigens (e.g., PSA is very small), the results suggest that not only does the size of the antibody-antigen complex prevent movement along the membrane substrate, but there is further interaction between a single complex and the membrane substrate, or between two or more antibody-antigen complexes and/or salts or other components of the sample buffer.

Example 6

The following examples describe the determination of the location of the capture antibodies after running positive and negative samples.

To verify the concept that the capture antibodies were shed from the test line in the case of a negative sample and left in place in the case of a positive sample, the antibodies were labeled with fluorophores to enable imaging of the strip via microscopy.

Anti-S1 IgG was conjugated to the uv tag using a commercially available conjugation kit.

Antibodies were deposited at the test lines on nitrocellulose membranes and test strips were then fabricated following a procedure similar to the previous examples.

SARS-CoV-2 positive nasal swabs were run on one device and negative nasal swabs were run on the other device. From 6 minutes after sample application, the positive and negative test strips were imaged using a fluorescence microscope, with 50 second intervals within 10 minutes.

Results

The results are shown in fig. 11. The antibody lines in both the positive and negative samples remained in the same position throughout the 10 minute imaging period. This indicates that during manufacture of the test strip, the antibodies bind strongly to the nitrocellulose membrane and do not flow out of the test line in the case of positive and negative samples.

Example 7

The following examples describe labeling of streptavidin-linked HRP conjugates to visualize the process of enzyme flow through the test strip during running positive and negative nasal swab samples.

To test the concept that the flow of an enzyme as a detection reagent is limited in the case of a positive sample and not in the case of a negative sample, the enzyme is labeled so that a delayed imaging of the labeled enzyme can be performed during flow through the membrane.

ATTo 488 is attached to the enzyme as a fluorophore. Streptavidin-linked HRP conjugate was incubated with biotinylated fluorophore at a ratio of 1:4 to bind all binding sites on streptavidin.

The fluorophore conjugated streptavidin-linked HRP conjugate was deposited onto conjugate pads and test strips manufactured as described elsewhere herein.

SARS-CoV-2 positive nasal swabs were run on one device and negative nasal swabs were run on the other device.

From 6 minutes after sample application, the positive and negative test strips were imaged using a fluorescence microscope, with 30 second intervals within 10 minutes.

Results

The flow of enzyme in the positive swab sample was significantly delayed compared to the negative sample, indicating that flow was restricted. The enzyme in the positive sample concentrated toward the test line and the front of the absorbent pad during the imaging period of 10 minutes, whereas in the negative sample, the flow dispersed into the absorbent pad during the imaging period of 10 minutes. These results are shown in fig. 12.

Example 8

The following example describes the use of HRP substrate TMB to examine the relationship between enzyme development and imaging of enzyme movement achieved by the fluorophore and to determine whether the addition of TMB caused further movement of the enzyme within the test strip.

Streptavidin-linked HRP conjugate was incubated with biotinylated fluorophore at a ratio of 1:4 to bind all binding sites on streptavidin.

SARS-CoV-2 positive nasal swabs were run on one lateral flow assay device and negative nasal swabs were run on the other device.

At the end of the 10 minute imaging period, the test strips were removed from the microscope. TMB was deposited onto the test line and allowed to develop for 5 minutes. The color development is imaged and the test strip is then returned to the microscope to image the final location of the enzyme.

Results

The results are shown in fig. 13 and 14, and in the case of the negative sample, after TMB was deposited on the test line, as TMB was reflowed along the nitrocellulose membrane, a single chromogenic region appeared upstream of the test line (i.e., region 1 in fig. 13), demonstrating the presence of the enzyme at this location.

In the case of a positive sample, after TMB deposition on the test line, a distinct strongly colored region appears near the test line (i.e., region 2 in fig. 13), followed by color development in region 1, as seen in the case of a negative sample. This indicates that in the case of a positive sample, the enzyme is present in two different areas, where a larger amount of enzyme remains near the test line.

Fluorescence imaging of the test strip after TMB development revealed a non-specific signal or background (i.e., enzyme independent) due to the presence of unbound fluorophores on the nitrocellulose membrane at all times. The figure shows dark areas, which correspond to the areas where TMB development was observed (FIG. 14). This may be due to the fact that the optical density of the chromogenic TMB limits the excitation and/or emission of the fluorophore.

Although several embodiments of the invention have been described and illustrated herein, a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more advantages described herein will be apparent to those of ordinary skill in the art, and each of such variations and/or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application for which the teachings of the present disclosure is used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, and/or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and/or methods (if such features, systems, articles, materials, and/or methods are not mutually inconsistent) is included within the scope of the present disclosure.

The indefinite articles "a" and "an" as used herein in the specification and claims should be understood to mean "at least one" unless explicitly indicated to the contrary.

The phrase "and/or" as used herein in the specification and claims should be understood to mean "either or both" of the elements so combined, i.e., the elements are in some cases combined and in other cases separated. Other elements than those specifically identified by the "and/or" clause may optionally be present, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, references to "a and/or B" when used in conjunction with an open-ended term such as "comprising" may refer to a in one embodiment rather than B (optionally including elements other than B); in another embodiment, B may be referred to instead of a (optionally including elements other than a); in yet another embodiment, both a and B (optionally including other elements) may be referred to; etc.

As used in the specification and claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, an "or" and/or "item in a separate list should be construed as inclusive, i.e., including at least one item in a plurality of elements or element lists, but also including more than one item therein, and optionally, additional unlisted items. Only terms explicitly indicated as the contrary, such as "only one" or "exactly one", or "consisting of … …" when used in the claims, will refer to an element that exactly contains a plurality of elements or one element of a list of elements. Generally, the term "or" as used herein before an exclusive term such as "any", "one", "only one", or "exactly one" should be interpreted to mean only an exclusive alternative (i.e., "one or the other, but not both"). "consisting essentially of … …" when used in the claims should have its ordinary meaning as used in the patent statutes.

As used herein in the specification and claims, the phrase "at least one" referring to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including each and every element specifically listed within the list of elements, and not excluding any combination of elements in the list of elements. The definition also allows that, optionally, additional elements may be present other than the specifically identified elements within the list of elements referred to by the phrase "at least one," whether related or unrelated to those elements specifically identified. Thus, as one non-limiting example, "at least one of a and B" (or equivalently, "at least one of a or B," or equivalently, "at least one of a and/or B") may refer in one embodiment to at least one a, optionally including more than one a, without the presence of B (and optionally including elements other than B); in another embodiment may refer to at least one B, optionally including more than one B, without a being present (and optionally including elements other than a); in yet another embodiment may refer to at least one a, optionally comprising more than one a, and at least one B, optionally comprising more than one B (and optionally comprising other elements); etc.

Some embodiments may be embodied as a method, various examples of which have been described. Acts performed as part of the method may be ordered in any suitable manner. Thus, embodiments may be constructed in which acts are performed in a different order than illustrated, which may include acts different from those described (e.g., more or less acts), and/or may involve the performance of some acts simultaneously, even though they are shown as being performed in order in the embodiments explicitly described above.

Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

In the claims and the above description, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like are to be understood to be open-ended, i.e., to mean including, but not limited to. As set forth in section 2111.03 of the U.S. patent office patent review program manual, only the transitional phrases "consisting of … …" and "consisting essentially of … …" should be closed or semi-closed transitional phrases, respectively.

Claims

1. A flow assay, comprising:

a substrate having an upstream location and a downstream location; and

A binding region comprising a plurality of capture reagents positioned at the downstream location,

Wherein the plurality of capture reagents is configured to form an interconnected network with a plurality of analytes, the interconnected network comprising a mixture of the plurality of capture reagents and the plurality of analytes interconnected with each other.

2. A flow assay, comprising:

a substrate having an upstream location and a downstream location;

A binding region comprising a plurality of capture reagents positioned at the downstream location; and

An interconnection network located at the downstream location or between the upstream location and the downstream location, wherein the interconnection network comprises a mixture of the plurality of capture reagents and the plurality of analytes interconnected with each other.

3. The flow assay of claim 2, further comprising a plurality of analytes positioned upstream of the plurality of capture reagents and configured to bind to the plurality of capture reagents.

4. A flow assay, comprising:

a substrate having an upstream location and a downstream location; and

A bonding region including a bonding region positioned at the downstream location

A plurality of capture reagents are used to capture the sample,

Wherein the flow of at least a portion of the plurality of capture reagents is restricted when the analyte binds to at least a portion of the capture reagents.

5. The flow assay of any one of claims 1 to 4, further comprising a plurality of detection reagents positioned upstream of the plurality of capture reagents and configured to bind to the plurality of capture reagents.

6. The flow assay of any one of claims 1 to 5, wherein the plurality of capture reagents and the plurality of detection reagents are configured to form the interconnected network with a plurality of analytes.

7. The flow assay of any one of claims 1 to 6, wherein the flow of the detection reagent is restricted such that when more analyte is present in the sample, less of the detection reagent flows out of the assay than when less or no analyte is present.

8. A method of detecting a plurality of analytes in a sample using a flow assay, the method comprising:

Introducing the sample at a location upstream of a substrate, wherein the substrate comprises a plurality of capture reagents positioned at a binding region at a downstream location;

Flowing the sample from the upstream location to the downstream location;

Flowing a plurality of detection reagents from the upstream location to the downstream location;

Allowing the plurality of analytes of the sample to bind to at least some of the plurality of capture reagents;

forming an interconnected network comprising a mixture of the plurality of capture reagents and the plurality of analytes; and

Detecting at least one of the analytes.

9. A method of detecting a plurality of analytes in a sample using a flow assay, the method comprising:

Introducing the sample into an upstream location of a substrate, wherein the substrate comprises a plurality of capture reagents positioned at a binding region at a downstream location;

Flowing the sample from the upstream location to the downstream location,

Allowing at least a portion of the plurality of analytes of the sample to bind with at least a portion of the plurality of capture reagents;

restricting flow along the flow assay after at least a portion of the plurality of analytes of the sample bind with at least a portion of the plurality of capture reagents;

Detecting at least one of the analytes.

10. The method of claim 9, further comprising forming an interconnected network comprising a mixture of the plurality of capture reagents and the plurality of analytes.

11. The method of the preceding claim, further comprising allowing the plurality of analytes of the sample to bind to the plurality of detection reagents.

12. The method of any of the preceding claims, further comprising reflowing the interconnection network away from the bonding region.

13. The method of any of the preceding claims, further comprising flowing the interconnection network to the bonding region.

14. The method of any of the preceding claims, further comprising stopping flow of the interconnection network within the bonding region.

15. The method of any of the preceding claims, further comprising increasing a size of the interconnection network.

16. The method of any of the preceding claims, further comprising reducing or stopping flow on at least a portion of the substrate after forming the interconnection network.

17. The method of any one of the preceding claims, wherein during any one of the flowing steps, one or more detection reagents do not flow out of the substrate.

18. The method of any one of the preceding claims, wherein one or more detection reagents flow from the substrate during the flowing step when one or more analytes are not bound to the capture reagent and do not flow when one or more analytes are bound to the capture reagent.

19. The method of any one of the preceding claims, wherein the reduction in flow rate of the sample is greater than or equal to 0.1mm/s and less than or equal to 5mm/s.

20. The flow assay or method of any one of the preceding claims, wherein the interconnected network comprises a mixture of the plurality of capture reagents, the plurality of detection reagents, and/or the plurality of analytes interconnected with one another.

21. The flow assay or method of any of the preceding claims, wherein the interconnected network comprises a precipitate or mesh comprising a plurality of capture reagents, detection reagents and/or analytes.

22. The flow assay or method of any of the preceding claims, further comprising a sample comprising the analyte.

23. The flow assay or method of any of the preceding claims, wherein the plurality of capture reagents and the plurality of detection reagents are configured to form the interconnected network with the plurality of analytes at the downstream location.

24. The flow assay or method of any of the preceding claims, further comprising a cassette at least partially enclosing the substrate.

25. The flow assay or method of any of the preceding claims, wherein the interconnected network comprises non-covalent interactions between the plurality of analytes, the plurality of detection reagents, and/or the plurality of capture reagents.

26. The flow assay or method of any of the preceding claims, wherein the plurality of capture reagents are positioned at the downstream location on the surface of the substrate.

27. The flow assay or method of any of the preceding claims, wherein the plurality of detection reagents are positioned at the upstream location on the surface of the substrate.

28. The flow assay or method of any one of the preceding claims, wherein the substrate comprises a detection reagent positioned at a location upstream of the substrate.

29. The flow assay or method of any of the preceding claims, wherein the interconnected network is sparingly soluble or insoluble in the sample or a solvent for the sample.

30. The flow assay or method of any of the preceding claims, wherein the interconnected network forms a precipitate within the substrate.

31. The flow assay or method of any of the preceding claims, wherein the interconnected network comprises agglomerates of the analyte and the capture reagent.

32. The flow assay or method of any of the preceding claims, wherein the sample further comprises a salt, buffer, and/or surfactant.

33. The flow assay or method of any of the preceding claims, wherein the plurality of capture reagents is a plurality of capture antibodies and the analyte is an antigen of the plurality of capture antibodies.

34. The flow assay or method of any one of the preceding claims, wherein the flow of the detection reagent is restricted such that when more analyte is present in the sample, less of the detection reagent flows out of the assay than when less or no analyte is present.

35. The flow assay or method of any of the preceding claims, wherein the detection reagent is one or more HRP conjugates.

36. The flow assay or method of any one of the preceding claims, wherein the detection reagent is one or more nanoenzymes.

37. A flow assay, comprising:

a substrate having an upstream location and a downstream location;

a binding region comprising a plurality of capture reagents positioned at the downstream location;

a plurality of detection reagents positioned upstream of the plurality of capture reagents; and

A box enclosing at least a portion of the substrate, wherein the box comprises a first portion and a second portion opposite the first portion, wherein the substrate is positioned between the first portion and the second portion, wherein the second portion comprises a protrusion extending toward the first portion, and

Wherein the protrusion presses the substrate against the first portion.

38. The flow assay of claim 37, wherein the protrusion is greater than or equal to 10% of the distance between the upstream location and the downstream location of the substrate.

39. The flow assay of claim 37, wherein the protrusion is less than or equal to 90% of the distance between the upstream location and the downstream location of the substrate.