Reagent matrix comprising cellulose nanofibrils and use thereof for detecting analytes

Abstract

Described herein is a reagent matrix comprising (a) a plurality of first capture components wherein the first capture component comprises a first analyte capture site; (b) a plurality of second capture components wherein the second capture component comprises a second analyte capture site and a cellulose binding domain (CBD) or carbohydrate binding module (CBM) wherein at least one of the first capture component or the second capture component comprises a detection medium; and (c) a plurality of cellulose nanofibrils. In one embodiment, the reagent matrix is combined with a porous substrate for detecting an analyte.

Description

Technical Field

[0001] The present disclosure provides a reagent matrix comprising at least two different capture components and multiple cellulose nanofibrils, which enables the detection of an analyte when contacted with the analyte of interest. Summary of the Invention

[0002] Point-of-care (POC) devices are a powerful tool for performing tests rapidly and inexpensively in clinical settings, as well as for monitoring food safety and environmental conditions. Most POC devices eliminate the need for centralized laboratory testing and provide qualitative or semi-quantitative results without the need for dedicated equipment. Due to persistent and emerging epidemics such as tuberculosis, HIV / AIDS, and COVID-19, the global demand for POC testing is increasing.

[0003] One type of POC device is the lateral flow assay. In a typical lateral flow assay, a liquid sample (or an extract thereof) is added to the device, and capillary flow of the liquid is used to move the analyte of interest to other zones of the device for detection. Because capillary flow is used to move the analyte of interest to the detection zone, these assays may require a waiting time of 10 to 30 minutes to "read" the test result.

[0004] In a vertical flow assay, the sample is applied to the device and detected at the application point. Vertical flow assays are known to have faster detection times than lateral flow assays, but are generally more complex for the end user, involving multiple reaction steps (e.g., at least two different reaction reagents used with a labeling solution), which must be performed in a certain order and require many washing steps to remove the reagents.

[0005] In one aspect, a reagent matrix is disclosed. The reagent matrix comprises:

[0006] (a) a plurality of first capture components, wherein the first capture component comprises a first analyte capture site; (b) a plurality of second capture components, wherein the second capture component comprises a second analyte capture site and a cellulose binding domain (CBD) or a carbohydrate binding module (CBM), wherein at least one of the first capture component or the second capture component comprises a detection medium; and

[0007] (c) multiple cellulose nanofibrils.

[0008] In another aspect, a kit for performing an assay to detect an analyte in a sample is disclosed. The kit comprises (i) a reagent matrix comprising: (a) a plurality of first capture components, wherein the first capture component comprises a first analyte capture site; (b) a plurality of second capture components, wherein the second capture component comprises a second analyte capture site and a cellulose binding domain (CBD) or a carbohydrate binding module (CBM), wherein at least one of the first capture component or the second capture component comprises a detection medium; and (c) a plurality of cellulose nanofibrils; and (ii) a porous substrate.

[0009] In yet another aspect, a method for detecting the presence or amount of an analyte in a sample is disclosed. The method comprises: (i) combining the sample; the reagent matrix, which comprises: (a) a plurality of first capture components, wherein the first capture component comprises a first analyte capture site; (b) a plurality of second capture components, wherein the second capture component comprises a second analyte capture site and a cellulose binding domain (CBD) or a carbohydrate binding module (CBM), wherein at least one of the first capture component or the second capture component comprises a detection medium; and (c) a plurality of cellulose nanofibrils; and a carrier solution in a container to form a test sample; (ii) contacting the test sample with the porous substrate; (iii) detecting a signal generated by aggregation of the analyte with the reagent matrix on the surface of the porous substrate.

[0010] The above summary is not intended to describe each embodiment. Details of one or more embodiments of the invention are also set forth in the following description. Other features, objects, and advantages will be apparent from the description and claims. Detailed Description

[0011] As used herein, the terms

[0012] "a," "an," and "the" are used interchangeably to mean one or more; and

[0013] "and / or" is used to mean that either or both of the stated cases may occur, e.g., A and / or B includes (A and B) and (A or B).

[0014] Also herein, ranges expressed by endpoints include all numbers included within that range (e.g., 1 to 10 includes 1.4, 1.9, 2.33, 5.75, 9.98, etc.).

[0015] Furthermore, herein, the phrase "at least one" includes one and all greater numbers (e.g., at least 2, at least 4, at least 6, at least 8, at least 10, at least 25, at least 50, at least 100, etc.).

[0016] As used herein, "comprising at least one of A, B, and C" means element A itself, element B itself, element C itself, A and B, A and C, B and C, and combinations of all three.

[0017] In the present disclosure, novel techniques for detecting a specific analyte are disclosed.

[0018] In an aggregation assay, a target analyte binds to a specific capture agent to form a detectable aggregate. In the present disclosure, in addition to the capture component, multiple cellulose nanofibrils are used for the aggregation assay to improve the detection of a specific analyte. The method of the present disclosure is a simplified method for vertical flow assays, where a single step can be used to lyse a sample, as well as capture and label the target analyte. The method is simple, rapid, and can provide a limit of detection comparable to or better than commercially available tests.

[0019] In the method of the present application, a target analyte is used to complex two different capture components, where at least one of these capture components can also bind to cellulose nanofibrils. This aggregation can occur in situ and is then applied to a porous substrate, which traps the aggregate on the surface of the substrate.

[0020] Reagent matrix

[0021] The reagent matrix of the present disclosure comprises at least two different components capable of capturing a target analyte. An analyte (or target analyte) is a compound or composition of interest to be detected. The analyte can be bio-derived or present in a bio-derived test sample fluid. Examples of such analytes can include therapeutic drugs, abused drugs, drug metabolites, hormones, peptides, polypeptides, proteins including immunoglobulins, polysaccharides, nucleic acids, and combinations thereof. In another embodiment, the analyte can be an environmental concern reagent, such as a pest control product, an environmental toxin, a halogenated substance, or dioxins and furans. In another embodiment, the analyte can be a food safety concern reagent, such as a pathogen (e.g., bacteria, virus, fungus, etc.), an allergen, a pesticide, a genetically modified organism, and a toxin.

[0022] The first capture component comprises a first site for capturing an analyte. The first capture site is capable of recognizing a specific spatial and / or chemical structure of the analyte.

[0023] The second capture component comprises a second capture site that is also capable of recognizing a specific spatial and / or chemical structure of the analyte. The second capture component further includes a cellulose binding domain (CBD) and / or a carbohydrate binding module (CBM).

[0024] In one embodiment, the capture site can be an antibody. As used herein, the term antibody encompasses not only antibodies, but also any polypeptide or protein having a binding domain (which is an antibody binding domain or homologous to an antibody binding domain). These antibodies can be derived from natural sources, or they can be produced partially or entirely synthetically. Examples of antibodies are immunoglobulin isotypes and their isotype subclasses; fragments containing antigen-binding domains, such as Fab, scFv, Fv, dAb, Fd; and bispecific antibodies. Antibodies useful in the present disclosure include those that specifically react with the target analyte. When testing a biological sample, antibody capture sites are preferred. Such antibodies are preferably IgG or IgM antibodies or mixtures thereof that are substantially not associated with antibodies capable of binding to non-analyte molecules. Antibodies can be polyclonal or monoclonal and are commercially available or can be obtained by mouse ascites, tissue culture, or other techniques known in the art. It may be necessary to use a mixture of monoclonal antibodies of different antigen specificities or a mixture of monoclonal and polyclonal antibodies. A typical description of the hybridoma process for producing monoclonal antibodies can be found, for example, in Wands et al., Gastroenterology 80, pp. 225-232 (1981); and U.S. Patent No. 4,515,893 issued to Kung et al. Fragments of antibody molecules are further envisioned to be used as specific binding reagents according to the present disclosure, including hapten molecules and Fab, Fab', or F(ab')2 fragments known in the art.

[0025] In another embodiment, the capture site is a capture protein, such as an engineered protein, a peptide aptamer, or an affibody protein. Many protein engineering methods are known in the art for generating peptides and proteins with enhanced or new functions. One such method is directed evolution, which involves generating a random library that displays a wide range of protein variants. This library is then displayed using one of the various available techniques (e.g., phage, ribosome, mRNA) or cell surface display. After selection, diversity is added back to the library by DNA modification (e.g., error-prone PCR, DNA shuffling, etc.) to obtain affinity maturation. This cycle of selection is repeated until the desired binder is produced. In addition to molecular biology-based approaches, chemical combinatorial methods such as DNA-encoded libraries and single-bead single-compound libraries can be used to generate high-affinity binding peptides. Other approaches for obtaining new binding domains also include semi-rational design and computational techniques. An overview of the techniques mentioned herein can be found in Banta et al., Annu Rev. Biomed Eng., Vol. 15, pp. 93-113 (2013); and Zhao et al., Expert Opin Drug Discov., Vol. 14, pp. 735-753 (2019).

[0026] In another embodiment, the capture site is an aptamer, such as a DNA aptamer, an RNA aptamer, or a peptide aptamer. RNA aptamers and DNA aptamers bind to their targets with high selectivity and sensitivity due to their well-characterized structures. Aptamers can be generated by techniques known in the art, such as SELEX (systematic evolution of ligands by exponential enrichment). See Ellington et al., Nature, Vol. 346, pp. 818-822 (1990); and Tuerk et al., Science, Vol. 249, pp. 505-510 (1990). Peptide aptamers are alternative binding molecules in which 5 to 20 residue peptides are typically grafted onto a neutral scaffold that has undergone a selection process. Display strategies known in the art can be used to generate and select peptide aptamers. See Reverdatto et al., Curr. Top. Med. Chem., Vol. 15, No. 12, pp. 1082-1101 (2015).

[0027] The second capture component also includes a cellulose binding domain (CBD) and / or a carbohydrate binding module (CBM), which can bind to cellulose nanofibrils. CBDs and CBMs are sites that have an affinity for cellulose materials. CBD and CBM sequences and how to obtain these groups are known in the art. See, for example, U.S. Patent Publication No. 2019 / 0113512 (Sikes Johnson et al.) and U.S. Patent No. 5,496,934 (Shoseyov et al.). In some embodiments, the CBM is CBM1, CBM2, CBM3, CBM4, CBM5, CBM6, CBM9, CBM10, CBM11, CBM12, CBM14, CBM15, CBM17, CBM18, CBM19, CBM20, CBM21, CBM25, CBM27, CBM28, CBM32, CBM33, CBM48, or CBM49. The nucleic acid and amino acid sequences of CBMs contemplated herein have been described, such as those disclosed at www.cazypedia.org / index.php / Carbohydrate-binding modules, and can be readily identified by one of ordinary skill in the art using the Basic Local Alignment Search Tool (BLAST). Orthologs of CBDs have been described in a variety of bacterial species, including but not limited to Micromonospora megalomicea (GenBank ID: SCF42127.1), Mycobacterium tuberculosis (GenBank ID: CNE10097.1), Micromonospora nigra (GenBank ID: SCL15442.1), Micromonospora megalomicea (GenBank ID: SCF04121.1), Cellulomonas fimi (PDB: 1EXH_A), Mycobacterium kansasii 732 (GenBank: EUA13076.1), Ruminococcus albus 8 (GenBank: EGCO2462.1), Ralstonia aquaticus (NCBI reference sequence: WP_021763186.1), Schizosaccharomyces pombe (NCBI reference sequence: NP_593986.1), Desulfovibrio hafniensis (GenBank: CDX04743.1). CBDs expressed in other bacterial species known to one of ordinary skill in the art are also contemplated herein, such as, for example, the CBDs of Groups I, II, III, and IV disclosed in Tomme et al., J Chromatogr B Biomed Sci Appl (1998) 715(1):283-96.

[0028] The second capture component can be a bifunctional fusion protein as disclosed in U.S. Patent Publication No. 2019 / 0113512 (Sikes Johnson et al.), which is incorporated herein by reference.

[0029] The first capture component, the second capture component, or both contain a detection medium. The detection medium can be any molecule or particle that is bound or conjugated to a capture site and that enables detection. The signal can be a signal that is detected visually (e.g., by the eye) or detected with an instrument. The detection medium can be a colorant, a photoluminescent substance, a chemiluminescent substance, a radiolabel, a magnetic material, or a combination thereof.

[0030] The first capture site and the second capture site are selected to have specific binding affinities for different portions of the analyte so as to sandwich the analyte therebetween. The capture sites can be of natural derivation or synthetically produced. The first capture site specifically binds to a specific spatial and / or chemical structure of the analyte and is thus complementary thereto, while the second capture site specifically binds to a specific spatial and / or chemical structure of another portion of the analyte and is thus complementary thereto. How to select such sandwich pairs is known in the art. See, for example, the review article by Mirica et al. published in Front. Bioeng. Biotechnol. (Frontiers in Bioengineering and Biotechnology), Vol. 10, Article No. 922772 in June 2022.

[0031] In one embodiment, the capture component comprises particles, wherein the capture sites are bound or conjugated to the particles or are bound or conjugated thereon via passive adsorption or covalent attachment as known in the art. Generally, these particles can include synthetic polymers such as latex, glass, metals, metal oxides, liposomes, pollen spores, red blood cells, carbohydrates such as dextran, agarose or cellulose, microorganisms including viruses, and combinations thereof. In one embodiment, the particles include cellulose or latex. Latex is commercially available and the polymer particles therein can be derived from acrolein, acrylate, methyl acrylate, methacrylate, methyl methacrylate, glycidyl methacrylate, styrene, vinyl toluene, and tert-butyl styrene monomers and mixtures thereof. In some embodiments, the polymer particles of the latex can optionally contain crosslinking agents such as divinylbenzene and butadiene. Techniques for preparing such latexes are as well known as surface modification methods for attaching binding pair members to the particle surface. Exemplary U.S. patents describing latex particles, capture sites attachable to the particles, and / or coupling methods for attaching capture sites to the particle surface include U.S. Patent Nos. 4,064,088 (Renner); 4,210,723 (Dorman et al.); 4,264,766 (Fisher); 3,857,931 (Hager); 4,253,844 (Limet et al.); and 4,397,960 (Moussebois et al.), each of which is incorporated herein by reference. Cellulose particles can be prepared from cellulose using various techniques, such as ground cellulose, which can have a more rod-like fiber shape, as disclosed in U.S. Patent No. 5,123,962 (Komuro et al.); or prepared by dissolving cellulose and then treating it by high shear or agitation, as disclosed in Japanese H 0576496 (Shigeru et al.); prepared by coagulation, as disclosed in U.S. Patent No. 6,225,461 (Akimoto et al.); or prepared by drying a dispersion, as disclosed in U.S. Patent No. 8,629,187 (Shimomi et al.), each of which is incorporated herein by reference. Commercially available cellulose includes, for example, nanocellulose sold under the trade name "NANOACT" by Asahi Kasei.

[0032] In one embodiment, a dye is added to the particles such that when a large number of particles agglomerate at the surface of the porous substrate, a signal can be visually observed.

[0033] The size of the selectable particles and the pores / pores porosity of the porous substrate can be such that the unbound particles can pass through the porous substrate, while when the analyte of interest is sandwiched between the first capture component and the second capture component, the sandwiched analyte does not pass through the porous substrate. In one embodiment, the particles have an average diameter of at least 0.1 micrometer, 0.3 micrometer, 0.5 micrometer or even 1 micrometer and at most 10 micrometers, 7.5 micrometers, 5 micrometers, 2 micrometers or even 1.5 micrometers. The particle size can be determined using techniques known in the art such as scanning electron microscopy or light scattering detection.

[0034] In addition to the first capture component and the second capture component, the reagent matrix further comprises a plurality of cellulose nanofibrils. Cellulose nanofibrils are a specific type of cellulose material. As used herein, cellulose nanofibrils include fibrillated cellulose (both nanofibrillated cellulose and microfibrillated cellulose).

[0035] Cellulose nanofibrils can be produced from cellulose materials such as wood pulp, bacteria, cellulose-containing marine animals (e.g., tunicates), or cotton, with wood pulp being the most commonly used.

[0036] Cellulose nanofibrils can be prepared using mechanical treatments such as high-pressure homogenization, high-energy ball milling, microfluidization, super low-pressure crushing, and other such methods; enzymatic treatments; and / or chemical treatments such as strong acid hydrolysis, oxidation, chemical functionalization, or combinations thereof.

[0037] Cellulose nanofibrils are composed of cellulose, which is a linear polymer of β(1 to 4)-linked D-glucose units, and the chains of these D-glucose units arrange themselves to form crystalline and amorphous domains.

[0038] The physical dimensions of cellulose nanofibrils can vary depending on the starting material and how it is processed. Generally, microfibrillated cellulose comprises long, thin fibers with a large size distribution, including individual fibers with nanometer diameters, but there are many larger fibers that form a network structure. Nanofibrillated cellulose tends to comprise individual fibrils with nanoscale diameters and a narrow size distribution. In one embodiment, the cellulose nanofibrils have an average cross-sectional dimension (the longest dimension of the cross-section of the cellulose material perpendicular to its length) of at least 2 nanometers (nm), 4 nanometers (nm), or even 5 nanometers (nm) and at most 10 nm, 20 nm, 30 nm, or even 50 nm; and an average length (the longest dimension of the cellulose nanocrystals) of at least 50 nm, 75 nm, or even 100 nm and at most 150 nm, 200 nm, 250 nm, 500 nm, 750 nm, or even 1000 nm. In another embodiment, the cellulose nanofibrils have an average cross-sectional dimension (the longest dimension of the cross-section of the cellulose material perpendicular to its length) of at least 100 nanometers (nm), 500 nanometers (nm), 1000 nanometers (nm), or even 2000 nanometers (nm) and at most 1 micrometer, 2 micrometers, 5 micrometers, 10 micrometers, 50 micrometers, 75 micrometers, or even 100 micrometers; and an average length (the longest dimension of the cellulose nanocrystals) of at least 0.5 micrometer, 1 micrometer, 2 micrometers, 5 micrometers, 10 micrometers, 50 micrometers, or even 100 micrometers and at most 150 micrometers, 200 micrometers, 250 micrometers, 500 micrometers, 750 micrometers, or even 1000 micrometers. The cross-sectional morphology of the nanofibrils is typically square, but can be rectangular or circular. Generally, cellulose nanofibrils have a high aspect ratio (the ratio of height to length). In one embodiment, the cellulose nanofibrils have an aspect ratio of 10 to 200, or even 100 - 150. The dimensions of cellulose nanofibrils can be determined based on transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy, or by other suitable means. Generally, the morphology is determined on a dry sample. In one embodiment, the cellulose nanofibrils have an average surface area of at least 30m 2 / g, 40m 2 / g, 50m 2 / g, 70m 2 / g or even 100m 2 / g. In one embodiment, the cellulose nanofibrils have an average surface area of at most 100m 2 / g, 150m 2 / g, 200m 2 / g, 300m 2 / g, 400m 2 / g or even 500m 2 / g.

[0039] The zeta potential measures the potential difference existing between the surface of a solid particle immersed in a conductive liquid (such as water) and the bulk liquid at the surface of the cellulose nanofibrils. Based on dynamic light scattering, the cellulose nanofibrils have a zeta potential higher than (i.e., a less negative value than) -50 mV, -45 mV, -40 mV, -35 mV, -30 mV or even -25 mV.

[0040] When measured under ambient conditions, the cellulose nanofibrils generally have a pH less than 7.5, 7.0, 6.5 or even 6.0 and greater than 4.5, 5.0 or even 5.5.

[0041] Cellulose nanofibrils are available from, for example: CelluForce, Montreal, Canada (CelluForce, Montreal, Canada;); Melodea Ltd., Israel (Melodea Ltd., Israel); American Process Inc., Atlanta, GA (American Process Inc., Atlanta, GA); Blue Goose Biorefineries Inc., Saskatoon, Canada (Blue Goose Biorefineries Inc., Saskatoon, Canada); the USDA Forest Products Laboratory, Madison, WI via the University of Maine (the USDA Forest Products Laboratory, Madison, WI via the University of Maine); and Weidmann Fiber Technology, Rapperswil, Switzerland (Weidmann Fiber Technology, Rapperswil, Switzerland).

[0042] In one embodiment disclosure, the cellulose nanofibrils are present in a ratio of capture component to cellulose nanofibrils of at least 0.5:1; 0.75:1; 1:1; or even 1:2. In one embodiment disclosure, the cellulose nanofibrils are present in a ratio of capture component to cellulose nanofibrils of at most 1:1; 1:2; 1:4; 1:5; 1:8; 1:10; 1:15; or even 1:20.

[0043] Advantageously, the reagent mixture can be substantially free of liquid (in other words, containing less than 5%, 3%, 2%, 1%, 0.5% or even 0.1% by weight of liquid such as water, alcohol, etc., or even undetectable liquid). The reagent mixture can be freeze-dried or dried similarly. A reagent mixture substantially free of water enables an improved shelf life.

[0044] In one embodiment, the reagent matrix further comprises a wetting agent or a lysing agent, such as a solvent (e.g., an alcohol) or a surfactant.

[0045] Porous substrate

[0046] In one embodiment, a test sample is applied to a porous substrate. Without wishing to be bound by theory, it is believed that the capture component interacts with the target analyte and multiple nanofibrils, and when applied to the porous substrate, the aggregated product is concentrated on the surface of the porous substrate while the unbound reagent is drawn away from the surface.

[0047] The porous substrate of the present disclosure is a layer comprising a series of interconnected pores from a first major surface of the porous substrate to a relative second major surface of the porous substrate.

[0048] The porous substrate is an organic material, preferably a polymeric material, which may be a porous membrane or a nonwoven fabric.

[0049] In one embodiment, the porous substrate may be a nonwoven web, which may include a nonwoven web made by any known method for producing nonwoven webs. As used herein, the term "nonwoven web" refers to a fabric having a structure of individual fibers or filaments that are randomly and / or unidirectionally interlaced in a mat-like fashion. For example, fibrous nonwoven webs can be made by carding, air-laying, spunbonding, meltblowing techniques, or combinations thereof. Spunbond fibers are typically small diameter fibers formed by extruding a molten thermoplastic polymer as filaments from a plurality of small, generally circular capillaries of a spinneret, where the diameter of the extruded fibers rapidly decreases. Meltblown fibers are typically formed by extruding a molten thermoplastic material through a plurality of small, generally circular die capillaries in the form of molten threads or filaments into a high velocity, generally heated gas (e.g., air) stream, which streamlines the filaments of the molten thermoplastic material to reduce their diameter. The meltblown fibers are then carried by the high velocity gas stream and deposited onto a collecting surface to form a web of randomly dispersed meltblown fibers. Any nonwoven web can be made from a single type of fiber or from two or more fibers that differ in type of thermoplastic polymer, its thickness, or both. Further details of available methods for manufacturing nonwoven webs are described in the content published by Wente in Indus. Eng. Chem., Vol. 48, pp. 1342 - 1346 (1956). In one embodiment, the microfibers have an effective fiber diameter of at least 0.5 microns and at most 16 microns.

[0050] In some embodiments, the porous substrate is a porous membrane, such as a thermally induced phase separation (TIPS) membrane. TIPS membranes are typically prepared by forming a homogeneous solution in a plastic compounding apparatus (e.g., an extruder) by mixing a thermoplastic material and a diluent at an elevated temperature and optionally including a nucleating agent. The solution can be formed into shape by passing through a perforated plate or an extrusion die, and after cooling, the thermoplastic material crystallizes and phase separates from the diluent. The crystallized thermoplastic material is often stretched. Optionally, the diluent is removed before or after stretching, leaving a porous polymer structure. Porous membranes are further disclosed in U.S. Patent Nos. 4,539,256 (Shipman), 4,726,989 (Mrozinski), 4,867,881 (Kinzer), 5,120,594 (Mrozinski), 5,260,360 (Mrozinski et al.), 5,962,544 (Waller), and 6,096,293 (Mrozinski et al.), all of which are assigned to 3M Company (St. Paul, MN) and are hereby incorporated by reference in their entirety.

[0051] In some embodiments, the substrate is a porous membrane, such as a solvent induced phase separation (SIPS) membrane. SIPS membranes are typically prepared by forming a homogeneous solution of a polymer in a first solvent, casting the solution into a desired shape, e.g., a flat sheet or a hollow fiber, and contacting the cast solution with another second solvent that is a non-solvent for the polymer but a solvent for the first solvent (i.e., the first solvent is miscible with the second solvent, but the polymer is not). Phase separation is induced by diffusion of the second solvent into the cast polymer solution and diffusion of the first solvent out of the polymer solution into the second solvent, thereby precipitating the polymer. The polymer lean phase is removed and the polymer is dried to produce a porous structure. SIPS is also known as phase inversion or diffusion induced phase separation or non-solvent induced phase separation, and this technique is well known in the art.

[0052] Useful porous substrates include symmetric membranes, asymmetric membranes, or multi-zone membranes, as well as multi-layer such membranes. A symmetric membrane is a membrane having substantially the same average pore size and / or porosity throughout its thickness. An asymmetric membrane is a membrane having a linear or non-linear gradient of average pore size and / or porosity extending from one major surface of the fluoroplastic substrate to the opposite major surface. In other words, the ratio of the average pore size of one major surface having larger pores to the average pore size of the opposite surface is greater than 3 or even greater than 4. A multi-zone membrane is a membrane having two or more substantially different through-thickness zones or a membrane having layers with different average pore sizes and / or different porosities. Multi-zone membranes are typically designated by the number of layers or zones (e.g., a 2-zone membrane has two substantially different zones having different average pore sizes or different porosities).

[0053] The porous substrate is an organic material, preferably a polymeric material. In one embodiment, the porous substrate can be formed from any suitable polymeric material. Suitable polymeric materials include polyolefins, poly(isoprene), poly(butadiene), fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, poly(ethersulfone), poly(sulfone), poly(vinyl acetate), polyesters (such as poly(lactic acid)), copolymers of vinyl acetate (such as poly(ethylene)-co-poly(vinyl alcohol)), poly(phosphazene), poly(vinyl ester), poly(vinyl ether), poly(vinyl alcohol), poly(carbonate), fiberglass, and the like, and combinations thereof.

[0054] Suitable polyolefins include poly(ethylene), poly(propylene), poly(1-butene), copolymers of ethylene and propylene, α-olefin copolymers (such as copolymers of ethylene or propylene with 1-butene, 1-hexene, 1-octene, and 1-decene), poly(ethylene-co-1-butene), poly(ethylene-co-1-butene-co-1-hexene), and the like, and combinations thereof.

[0055] Suitable fluorinated polymers include poly(vinyl fluoride), poly(vinylidene fluoride), copolymers of vinylidene fluoride (such as poly(vinylidene fluoride-co-hexafluoropropylene)), copolymers of chlorotrifluoroethylene (such as poly(ethylene-co-chlorotrifluoroethylene)), and the like, and combinations thereof.

[0056] Suitable polyamides include poly(iminodiacyliminohexamethylene), poly(iminodiacyliminodecamethylene), polycaprolactam, and the like, and combinations thereof. Suitable polyimides include poly(pyromellitimide), and the like, and combinations thereof.

[0057] Suitable poly(ethersulfone) includes poly(diphenyl ethersulfone), poly(diphenyl sulfone-co-diphenyl ethersulfone), and the like, and combinations thereof.

[0058] Suitable copolymers of vinyl acetate include poly(ethylene-co-vinyl acetate), copolymers in which at least some of the acetate groups in the acetate groups have been hydrolyzed to provide various poly(vinyl alcohol), and the like, and combinations thereof.

[0059] Generally, the porous substrate does not contain or substantially does not contain cellulose and groups that will specifically bind to the first binding component and / or the second binding component, because it may cause non-specific binding of the reagent to the porous substrate, resulting in background noise.

[0060] Typically, the porous substrates made from the above polymers are used in their non-functionalized state. However, the porous substrates can be functionalized with various groups such as acid groups and / or neutral hydrophilic moieties having -OCH2CH2- groups, etc., for example, for surface functionalization to enhance the signal, especially for small aggregates. Exemplary surface-functionalized porous membranes and monomers for preparing them are described in U.S. Patent No. 9,958,364 (Rasmussen et al.) and U.S. Patent Publication No. 2021 / 0031152 (Vail et al.), which are incorporated herein by reference.

[0061] In one exemplary embodiment, the porous substrate has an average pore size greater than 200 nanometers (nm), 500 nanometers (nm), 750 nanometers (nm), 1000 nanometers (nm), 2000 nanometers (nm), 3000 nanometers (nm), or even 5000 nanometers (nm). In one exemplary embodiment, the porous substrate has an average pore size less than about 100 µm (micrometers), 50 µm (micrometers), 25 µm (micrometers), 20 µm (micrometers), 10 µm (micrometers), 5 µm (micrometers), 3 µm (micrometers), or even 2 µm (micrometers). The average pore size of the porous substrate can be optimized for the specific capture component and target analyte used. The average pore size can be measured using techniques known in the art, such as optical microscopy, computed tomography (CT) scanning, or liquid porosimetry.

[0062] In some biomaterial assays, proteins are immobilized on the surface of the substrate to detect analytes. In the present application, no immobilized proteins are bound to the surface of the matrix before contact with the reagent matrix. In one embodiment, the porous substrate is substantially free (i.e., contains less than 0.5% by weight or even an undetectable amount) of immobilized proteins before contact with the reagent matrix.

[0063] In one embodiment, the porous substrate is disposed on an absorbent layer. The absorbent layer or substrate pad is typically located below the porous substrate, opposite the location where the sample mixture is dispensed, and the absorbent layer or substrate pad wicks the sample to the absorbent material below. The absorbent layer can be made of any material capable of wicking fluids by capillary action, such as paper, cellulose and cellulose derivatives (such as cellulose acetate and nitrocellulose), fiberglass, cloth, cotton, polyester, polyolefins (such as polyethylene), polyvinyl chloride membranes, and the like. In one embodiment, the absorbent layer contains a dry gel, such as silica gel, agarose, dextran, or gelatin.

[0064] The choice of material for the absorbent layer is not critical and a variety of fibrous filter materials can be used, including one or more layers of the same or different materials, provided that the materials selected are compatible with the target analyte and assay reagents. Any conventionally used absorbent material that can, for example, draw or wick fluid through a porous membrane by capillary action can be used in the present invention. The absorbent material should be able to absorb a volume of fluid test sample equal to or greater than the total volume capacity of the material itself. Known materials that can be used include cotton, cotton wool, cellulose acetate fibers, polyester, polyolefin, or other such materials. The absorbent material provides a means of collecting the sample by providing a uniform "suction" to deliver the sample from the well through the reaction zone and down into the absorbent material. Thus, the absorbent also serves as a reservoir to hold the sample as well as the various reagents used in performing the assay. Therefore, when used in assays that use a relatively large volume of fluid, the absorbent material should have a high absorption capacity to prevent or minimize the likelihood of the sample and reagents flowing back from the absorbent into the reaction membrane.

[0065] The absorbent layer is placed on the opposite side of the location where the porous substrate is added with the sample matrix. In one embodiment, the absorbent layer is fixedly attached to the porous substrate, for example, by using an adhesive.

[0066] Method

[0067] In one embodiment, the multi-layer substrate of the present disclosure is used in a downward or vertical flow assay, where the multi-layer substrate is placed in a test device that includes a test area. In the test area, the porous substrate faces outward, and the absorbent layer is located below the porous substrate and is in vertical communication with the porous substrate.

[0068] Exemplary biological samples that can be sampled include: body fluids and tissue samples. Body fluids can include amniotic fluid, aqueous humor, vitreous humor, bile, blood, cerebrospinal fluid, chyle, endolymph, perilymph, female ejaculate, lymph fluid, mucus (including nasal mucus and phlegm), pericardial fluid, peritoneal fluid, pleural effusion, pus, inflammatory exudate, saliva, sputum, synovial fluid, vaginal discharge, semen, blood, serum, or plasma. Tissue samples can include organ or tissue extracts, such as organ or tissue extracts taken from: placenta, brain, eye, pineal gland, pituitary gland, thyroid gland, parathyroid gland, chest, heart, lung, esophagus, thymus, pleura, adrenal gland, appendix, gallbladder, bladder, large intestine, small intestine, kidney, liver, pancreas, spleen, stomach, ovary, uterus, testis, skin, blood, or buffy coat samples of blood. Additional examples of organs and tissues from any biological source are well known to those of ordinary skill in the art, and such embodiments are within the scope of the methods provided herein. In one example, swabs from the nasal cavity, throat, or other body sites are used for point-of-care testing.

[0069] In environmental testing and food safety testing, the test sample can be or can not be a liquid. For example, a water sample or a liquid food sample can be the sample, or a swab can be used to wipe the surface of a food preparation surface, a container, or a surface in a high-traffic area.

[0070] Disperse the reagent matrix in a liquid (if not already dispersed), and add the sample to the reagent matrix to form a test sample. In one embodiment, the reagent matrix is in a dry form, and a liquid such as a buffer and / or a surfactant can be used to disperse the capture component reagent and the cellulose nanofibrils. The buffer can include those buffers known in the art, especially biological buffers. Surfactants can be used to assist in the denaturation of the analyte and / or the sample. Such surfactants include those known in the art. Suitable buffer solutions for use in the present disclosure include, but are not limited to, acetate buffer, buffered saline, citrate buffer, barbital buffer, borate buffer, phosphate buffer, or buffers prepared using: tris(hydroxymethyl)aminomethane (TRIS), N-(2-acetamido)iminodiacetic acid (ADA), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), 2,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3,4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), N-tris(hydroxymethyl)methylglycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), and / or buffers containing one or more buffer salts selected from K2HPO4, KH2PO4, Na2HPO4, NaH2PO4, NaHCOs, NaBO4, (NH4)2CO3. In one embodiment, the buffer is a multifunctional buffer as disclosed in U.S. Patent No. 7,531,362 (Chan, incorporated herein by reference), which buffer contains (1) a biological buffer for maintaining the pH between about 7.0 and 10.0; (2) at least one surfactant for reducing non-specific binding of the assay reagent while avoiding inhibition of specific binding interactions; (3) a molecular weight between about 2×10 2 D to about 2×10 6A high molecular weight polymer in the range of D, as a dispersion suspension reagent; (4) a pH stabilizer for maintaining the pH of the multi-functional buffer between about pH 7.0 and 10.0; (5) an ionic salt for reducing non-specific binding of antibodies; (6) at least one preservative for reducing bacterial and microbial growth; and (7) a calcium chelator for preventing clotting of whole blood test samples.

[0071] Mix a test sample comprising a sample and a reagent matrix in a liquid medium with an optional surfactant, so that the analyte can agglomerate with the first capture component, the second capture component, and cellulose nanofibrils. Then the test sample is placed on the test area of the test device. Although not wishing to be bound by theory, it is believed that the agglomerated sample is captured on the surface of the porous substrate layer while the reagent flows vertically or downward. After applying the test sample, it may be advantageous to include one or more washing steps, for example by adding a washing solution such as a buffer dropwise onto the test area. It is believed that the capture of the analyte and its concentration on the surface of the porous substrate enables good detection of the analyte. Visual detection (in other words, by the human eye) or detection using an instrument (such as a spectrophotometer, a fluorescence detector, a Geiger counter, etc.) can be used to determine whether an analyte is present in the sample and / or the amount of the analyte present.

[0072] In one embodiment, the test device is a device capable of simultaneously detecting multiple analytes (for example, 2, 3, or even more different analytes). For example, a first capture component with detection medium 1 can be used to detect analyte 1, while another first capture component with detection medium 2 can be used to detect analyte 2. In one embodiment, both detection medium 1 and detection medium 2 are colorimetric, and the observed colors are both additive. In another embodiment, detection medium 1 and detection medium 2 use different detection mechanisms (for example, colorimetry and radiolabels).

[0073] Example

[0074] Unless otherwise specified, all parts, percentages, ratios, etc. in this example and the rest of the specification are by weight, and all reagents used in the examples are obtained from or can be purchased from common chemical suppliers, such as Sigma-Aldrich Company, Saint Louis, Missouri, or can be synthesized by conventional methods.

[0075] Materials and methods

[0076] 4-Morpholineethanesulfonic acid (MES) was obtained from Sigma-Aldrich (Saint Louis, Missouri).

[0077] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), Blocker Casein in PBS (Phosphate Buffered Saline, 1% weight / volume), sucrose, TRITON X-100 surfactant, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and N-hydroxysulfosuccinimide (sulfo-NHS) were obtained from Thermo Fisher Scientific (Waltham, MA).

[0078] Boric acid was obtained from VWR International (Radnor, PA).

[0079] Antigen 1: SARS-CoV-2 nucleoprotein (N-protein) was obtained from Professor Hadley Sikes at the Massachusetts Institute of Technology in Cambridge, MA. The preparation of the SARS-CoV-2 nucleoprotein is described in Kim, S. et al., "Developing a SARS-CoV-2 Antigen Test Using Engineered Affinity Proteins", Applied Materials & Interfaces, 2021, 13, 38990 - 39002, and Supporting Information S1 - S23.

[0080] Capture Component 1: The preparation of the SARS CoV-2 antigen-binding protein-cellulose binding domain capture affinity reagent (abbreviation: SsoNP.E2-CBD) is described in Kim, S. et al., Applied Materials & Interfaces, 2021, 13, 38990 - 39002, and Supporting Information S1 - S23. A 60 μM stock solution of SsoNP.E2-CBD was prepared in 1X PBS (pH 7.4) containing 2% weight / volume trehalose.

[0081] Capture Component 2: See preparation below

[0082] Capture Component 3: For Comparative Example C (CE-C), the CBD sequence of SsoNP.E2-CBD was replaced with a cysteine (Cys) group to provide a non-CBD-binding mutant SsoNP.E2-Cys. SsoNP.E2-Cys can be prepared according to the general protocol described in Kim, S. et al., Applied Materials & Interfaces 2021, 13, 38990-39002 and Supporting Information S1-S23. A 60 μM stock solution of SsoNP.E2-Cys was prepared in 1X PBS (pH 7.4) containing 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP).

[0083] Microfibrillated cellulose (MFC) was obtained from Weidmann Fiber Technology (Rapperswil, Switzerland) under the trade name "CELOVA M250R-G" as a white gel with the specifications reported by the manufacturer of 2.9% MFC content, pH 8, a specific surface area of 234 m 2 / g and a particle length D of 9 μm. 50 A 0.3% (v / v) stock solution of MFC was prepared in 1X PBS (pH 7.4) containing 1% (v / v) TRITON X-100 surfactant.

[0084] AVICEL PH-101 microcrystalline cellulose (50 μm particle size) was obtained from Sigma-Aldrich (Catalog #11363). A 0.3% (w / v) stock solution of AVICEL PH-101 microcrystalline cellulose was prepared in 1X PBS (pH 7.4) containing 1% (v / v) TRITON X-100 surfactant.

[0085] Sigmacell cellulose powder (20 μm particle size) was obtained from Sigma-Aldrich (Catalog #S3504). A 0.3% (w / v) stock solution of Sigmacell cellulose was prepared in 1X PBS (pH 7.4) containing 1% (v / v) TRITON X-100 surfactant.

[0086] Pooled human saliva (Catalog #IRSHUSML) was obtained from Innovative Research, Novi, MI.

[0087] Unless otherwise specified, 18 MΩ deionized water from a MILLI-Q water purification system (EMD Millipore, Billerica, MA) was used.

[0088] Nonwoven substrate

[0089] Nonwoven substrate A (NWS-A) is a meltblown polypropylene (PP) nonwoven web that is white in color and has an effective fiber diameter of approximately 12 μm, a basis weight of approximately 200 grams per square meter (gsm), a density of approximately 10%, and a calculated average pore diameter of 36 μm.

[0090] Nonwoven substrate B (NWS-B) is a meltblown polypropylene (PP) nonwoven web that is white in color and has an effective fiber diameter of approximately 8 μm, a basis weight of approximately 200 grams per square meter (gsm), a density of approximately 10%, and a calculated average pore diameter of 24 μm.

[0091] Nonwoven substrate C (NWS-C) is a meltblown polypropylene (PP) nonwoven web that is white in color and has an effective fiber diameter of approximately 6 μm, a basis weight of approximately 200 grams per square meter (gsm), a density of approximately 10%, and a calculated average pore diameter of 18 μm.

[0092] Nonwoven substrate D (NWS-D) is a meltblown polypropylene (PP) nonwoven web that is white in color and has an effective fiber diameter of approximately 4 μm, a basis weight of approximately 106 grams per square meter (gsm), a density of approximately 10%, and a calculated average pore diameter of 12 μm.

[0093] Nonwoven substrate E (NWS-E) is a meltblown nylon 6,6 nonwoven web that is white in color and has an effective fiber diameter of approximately 4 μm, a basis weight of approximately 130 grams per square meter (gsm), a density of approximately 10%, and a calculated average pore diameter of 13 μm.

[0094] "Average pore diameter" (also known as average pore size) was calculated according to Equation 1.

[0095] Equation 1: Average pore diameter = d f ((2α / π) (-1 / 2) -1)

[0096] where "d f " is the arithmetic mean fiber diameter and "α" is the web density.

[0097] Preparation of capture component 2: Nanocellulose beads functionalized with SARS-CoV-2 monoclonal antibody (mAb)

[0098] NANOACT blue nanocellulose beads (Catalog #BL1BCA005, carboxyl-functionalized, diameter 318 nm, 1.05 wt% suspension in water) were obtained from Asahi Kasei Corporation, Tokyo, Japan. An aliquot (60 μL) of the NANOACT suspension was added to a pre-weighed 15 mL conical centrifuge tube, followed by the sequential addition of 540 μL of MES (100 mM aqueous solution, pH 6), 7.5 μL of EDC (4 wt% aqueous solution), and 15 μL of sulfo-NHS (4 wt% aqueous solution). The tube was kept at room temperature for 15 minutes and then centrifuged at 20 °C (5000 g) for 20 minutes using a tabletop centrifuge. The resulting supernatant was removed from the tube using a micropipette. An aliquot of MES (600 μL of 100 mM aqueous solution, pH 6) was added to the tube and the suspension was sonicated for 10 seconds using a probe sonicator in pulse mode (set to 3.2 seconds on and 0.5 seconds off). An antibody (60 μg of monoclonal antibody (mAb) against SARS-CoV-2 nucleocapsid protein, product number 9547, obtained from Meridian Bioscience, Memphis, TN) was added to the resuspended beads and the tube was vortexed for 5 to 10 seconds using a Vortex Genie-2 (Scientific Industries, Inc., Bohemia, NY) at maximum speed. The suspension was then incubated at 37 °C for 120 minutes in a temperature-controlled chamber. Blocker casein in PBS (7200 μL) was added, the tube was vortexed for 5 to 10 seconds, and then incubated at 37 °C for 60 minutes. The tube was removed from the temperature-controlled chamber and centrifuged at 20 °C (5000 g) for 20 minutes using a tabletop centrifuge. The resulting supernatant was removed by decantation, and then 7200 μL of boric acid (50 mM aqueous solution, pH 10) was added to the tube. The suspension was sonicated for 10 seconds using a probe sonicator in pulse mode (set to 3.2 seconds on and 0.5 seconds off), followed by centrifugation of the tube at 20 °C (5000 g) for 20 minutes. The supernatant was removed by decantation, and the resulting conjugated beads were resuspended in 33 mM aqueous boric acid solution (pH 9.2) also containing blocker casein (0.2 wt%) and sucrose (15 wt%). Enough solution was added such that the total weight of the suspension was 1579 mg. This yielded a 0.038 wt% stock suspension of antibody-conjugated beads. The suspension was sonicated for 10 seconds using a probe sonicator in pulse mode (set to 3.2 seconds on and 0.5 seconds off). The tube was stored at 4 °C and used within 2 to 3 days.Alternatively, a suspension of conjugated beads is aliquoted into 1.5 mL Eppendorf tubes and lyophilized. The lyophilized samples are sealed in foil bags. Each bag contains two 1 g silica gel desiccant packets (product number S-3902, obtained from ULINE Company, Pleasant Prairie, WI) and is stored at room temperature.

[0099] Preparation of vertical flow (VF) detection device

[0100] Vertical flow assay devices are prepared using different types of nonwoven substrates. Each device consists of a sealed plastic housing having an internal cavity (external device dimensions: 10 cm (length) × 7.5 cm (width) × 14 mm (depth)), which is prepared by 3D printing using ACCURA 25 plastic and a PROJET 7000 printer from 3D Systems (3DSystems, Rock Hill, SC). The device housing is prepared from two halves (i.e., an upper housing portion and a lower housing portion that are latched together). The lower housing portion of the device contains the internal cavity (dimensions 51 mm (length) × 13 mm (width) × 1.5 mm (depth)). The upper housing portion of the device contains two circular openings (each 4 mm in diameter). The openings are positioned to align with the cavity portion of the lower housing and are spaced 20 mm apart in the length direction. The absorbent pads for the device are 50 mm × 13 mm sections cut from WHATMAN grade GB003 cellulose blotting paper (0.8 mm thick) (obtained from Cytvia, Marlborough, MA). The porous substrate layer of the device is a 50 mm × 13 mm section cut from a single nonwoven substrate selected from nonwoven substrates A to E. A stack of three absorbent pads is placed in the cavity of the lower housing, and a single section of the selected nonwoven substrate is placed on top of the stack of absorbent pads. The upper housing portion and the lower housing portion are then mated and secured using a magnetic closure to form the completed device. In the completed device, the inward-facing surface of the upper housing portion presses against the surface of the nonwoven layer in the stack. The two openings in the upper housing and the nonwoven surface form two holes in the device for sample delivery and assay result detection.

[0101] Example 1: Detection of antigen 1

[0102] Two different test samples and a control sample were prepared using human oral swab samples. For each sample, a fresh cheek swab from a human volunteer was obtained using a PURITAN 25-8061PR Pur-wraps sterile rayon tipped swab (PURITAN Medical Products, Guilford, ME). Test sample 1 was prepared by immersing the absorbent material end of the swab in 1 mL aliquots of buffer solution spiked with antigen 1 (20 nM) (50 mM borate buffer (pH 10) containing NaCl (300 mM) and 1% by volume TRITON X-100 surfactant). The swab was rotated in the spiking solution (10 cycle rotation) by manually rotating the shaft of the swabbing applicator. The spiked sample was held at room temperature for one minute and then 300 μL aliquots of the spiked sample were added to microcentrifuge tubes containing 0.00475 mg of capture component 2. Capture component 1 (10 μL of the above 60 μM stock solution) and MFC (40 μL of the above 0.3% stock solution) were mixed together and 25 μL of the resulting mixture was added to the tubes. The samples were held at room temperature for 2 minutes and then added to the VF detection device.

[0103] Test sample 2 was prepared using the same procedure described for test sample 1, except that the swab was immersed in 1 mL aliquots of buffer solution spiked with antigen 1 (2 nM).

[0104] The control sample was prepared using the same procedure described for test example 1, except that the swab was immersed in 1 mL aliquots of buffer solution not spiked with antigen 1.

[0105] A VF detection device containing NWS-A was used and a separate device was used for each test sample. Test sample 1 or 2 (300 μL) was added to the first well opening of the device by pipette and the corresponding control sample (300 μL) was added to the second well opening of the selected device by pipette. Approximately 30 seconds after sample application, 100 μL aliquots of 0.1 M sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 1% by volume TRITON X-100 surfactant were added to each well.

[0106] Images of each device were taken using a Canon EOS Rebel T3i digital camera (Canon USA, Melville, NY). Pixel intensities observed in each well of the device were quantified using the ImageJ software program (National Institutes of Health, Bethesda, MD). Images were converted to 8-bit and inverted prior to quantification. The normalized pixel intensity of the first well was obtained by subtracting the pixel intensity value of the second well (i.e., the pixel intensity of the control sample) from the pixel intensity value of the first well (i.e., the sample spiked with antigen 1). The pixel intensity of the normalized value is related to the amount of antigen 1 in the test sample. The results are reported in Table 1.

[0107] Comparative examples A and B (CE-A and CE-B): Test samples without MFC component

[0108] Two different comparative test samples and a control sample were prepared according to the procedure of Example 1, except that the samples did not contain MFC. Comparative test sample CE-A was prepared by first immersing a swab in a 1 mL aliquot of a buffer solution spiked with antigen 1 (20 nM) (50 mM borate buffer (pH 10) containing NaCl (300 mM) and 1% (v / v) TRITON X-100 surfactant). The swab was rotated in the spiking solution (10 cycle rotations) by manually rotating the shaft of the swab. The spiked sample was held at room temperature for one minute and then a 300 μL aliquot of the spiked sample was added to a microcentrifuge tube containing 0.00475 mg of capture component 2. Capture component 1 (10 μL of the above 60 μM stock solution) and buffer solution (40 μL of 1X PBS (pH 7.4) containing 1% (v / v) TRITON X-100 surfactant) were mixed together and 25 μL of the resulting mixture was added to the tube. The sample was held at room temperature for 2 minutes and then added to the VF detection device.

[0109] Comparative test sample CE-B was prepared using the same procedure described for comparative test sample CE-A, except that the swab was immersed in a 1 mL aliquot of a buffer solution spiked with antigen 1 (2 nM).

[0110] The control sample was prepared using the same procedure described for comparative test sample CE-A, except that the swab was immersed in a 1 mL aliquot of a buffer solution not spiked with antigen 1. The pixel intensities of the comparative test samples were detected using the VF device using the same procedure described in Example 1. The results are reported in Table 1.

[0111] Table 1. Normalized Pixel Intensity of the First Well of the Device after Adding Test Samples

[0112]

[0113] Example 2: Detection of antigen 1

[0114] Pooled human saliva containing 1% (v / v) Triton X-100 was diluted 1:1 (v / v) with 50 mM borate buffer (pH 10) containing NaCl (300 mM) and 1% (v / v) Triton X-100 surfactant. The diluted saliva was spiked with 20 nM, 2 nM, 0.5 nM, 0.2 nM, 0.05 nM, or 0 nM antigen 1. A solution without antigen 1 (0 nM) was used to prepare the control sample. Each solution (300 μL) was added to a separate microcentrifuge tube containing 0.00475 mg of capture component 2. Capture component 1 (10 μL of the above 60 μM stock solution) and MFC (50 μL of the above 0.3% stock solution) were mixed together, and 30 μL of the resulting mixture was added to each tube. Each resulting test sample was held at room temperature for 5 minutes, and then the entire volume from the test sample tube was added to the first well opening of the VF detection device by pipette. Each VF detection device was configured to comprise a single part of NWS-B (polypropylene, EFD = 8 μm), and a single test sample was added to the device. The control sample was added to the second well opening of each device by pipette. Approximately 30 seconds after sample application, 100 μL aliquots of 0.1 M sodium phosphate buffer solution (pH 8.0) containing 300 mM NaCl and 1% (v / v) Triton X-100 surfactant were added to each well by pipette. Images of the device were taken and pixel intensities were analyzed according to the procedure described in Example 1. The normalized pixel intensities of the first well (i.e., the sample spiked with antigen 1) of each device are reported in Table 2.

[0115] Comparative example C (CE-C): Test sample without capture component 1

[0116] The same procedure reported in Example 2 was followed, except that capture component 1 of the test sample was replaced with a variant (capture component 3) that does not have a cellulose binding domain. The results are reported in Table 2.

[0117] Table 2.

[0118]

[0119] Example 3: Detection of antigen 1 using devices with different nonwoven substrates

[0120] Following the procedure described in Example 1, multiple test samples were prepared by immersing swabs in 1 mL aliquots of buffer solution spiked with 20 nM, 2 nM, 0.5 nM, 0.2 nM, or 0.05 nM of Antigen 1 (50 mM borate buffer (pH 10) containing NaCl (300 mM) and 1% (v / v) TRITON X-100 surfactant). Additionally, the VF device was modified to incorporate a single polypropylene nonwoven substrate selected from NWS-A, NWS-B, NWS-C, NWS-D. The normalized pixel intensity of the first well of each device (i.e., the sample spiked with SARS-CoV-2 N-protein) is reported in Table 3.

[0121] Table 3.

[0122]

[0123] Example 4: Detection of antigen 1 using nylon substrate 。

[0124] Separate solutions of Antigen 1 (20 nM, 2 nM, 0.5 nM, 0.2 nM, 0.05 nM, and 0 nM) were prepared in 1X PBS (pH 7.4) containing 1% (v / v) TRITON X-100 surfactant. The solution without Antigen 1 (0 nM) was used to prepare control samples. Each solution (300 μL) was added to a separate microcentrifuge tube containing 0.00475 mg of Capture Component 2. Capture Component 1 (10 μL of the above 60 μM stock solution) and MFC (50 μL of the above 0.3% stock solution) were mixed together, and 30 μL of the combined solution was added to each tube. Each resulting test sample was held at room temperature for 5 minutes, and then the entire volume from the test sample tube was added to the first well opening of the VF detection device by pipette. Each VF detection device consisted of a single piece of NWS-E (nylon, EFD = 4 μm). A single test sample was added to the device. The control sample was added to the second opening of each device by pipette. Approximately 30 seconds after sample application, 100 μL aliquots of 0.1 M sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 1% (v / v) TRITON X-100 surfactant were added to each well by pipette. Images of the device were taken and pixel intensities were analyzed according to the procedure described in Example 1. The results are reported in Table 4.

[0125] Table 4.

[0126]

[0127] Comparative examples D-K (CE-D to CE-K): Comparative test samples replacing microfibrillated cellulose (MFC) with cellulose particles

[0128] Following the procedure described in Example 1, except that 30 μL of an AVICEL PH-101 microcrystalline cellulose stock solution (as described above) or 30 μL of a Sigmacell cellulose stock solution (as described above) was used to replace the MFC in the sample. As used in the Examples section, the term solution can include clear mixtures as well as suspensions (e.g., suspended particles, emulsions, etc.). Each resulting comparative test sample was held at room temperature for 5 minutes and then the entire volume from the sample tube was added by pipette to the first well opening of a single section of the VF detection device containing NWS-B (polypropylene, EFD = 8 μm) or NWS-E (nylon, EFD = 4). A single comparative test sample was added to the device. The control sample was added to the second well opening of each device by pipette. Approximately 30 seconds after sample application, 100 μL aliquots of a 0.1 M sodium phosphate buffer solution containing 300 mM NaCl and 1% by volume TRITON X-100 surfactant were added to each well by pipette. Images of the device were taken and pixel intensities were analyzed according to the procedure described in Example 1. The results are reported in Table 5.

[0129] Table 5.

[0130]

[0131] To those skilled in the art, predictable modifications and changes can be made to the present invention without departing from the scope and spirit of the invention. The present invention should not be limited to the embodiments presented for illustrative purposes in this application. If any conflict or contradiction exists between the disclosure in the written specification and any document mentioned or incorporated herein by reference, the written specification shall prevail.

Claims

1. A reagent matrix, the reagent matrix comprising: (a) a plurality of first capture components, wherein the first capture components comprise first analyte capture sites; (b) a plurality of second capture components, wherein the second capture components comprise second analyte capture sites and cellulose binding domains (CBDs) or carbohydrate binding modules (CBMs), wherein at least one of the first capture components or the second capture components comprises a detection medium; and (c) a plurality of cellulose nanofibrils.

2. The reagent matrix according to claim 1, wherein the plurality of cellulose nanofibrils comprise fibrillated cellulose, cellulose nanocrystals, bacterial cellulose, or a combination thereof.

3. The reagent matrix according to any one of the preceding claims, wherein the plurality of cellulose nanofibrils have an average surface area of at least 30 m 2 / g.

4. The reagent matrix according to any one of the preceding claims, wherein the detection medium comprises a colorimetric material, a fluorescent material, a chemiluminescent material, a phosphorescent material, a radiolabel, a magnetic material, or a combination thereof.

5. The reagent matrix according to any one of the preceding claims, wherein at least one of the first capture components or the second capture components comprises particles, optionally, wherein the particles comprise at least one of the following: synthetic polymers, cellulose, glass, ceramics, metals, or a combination thereof.

6. The reagent matrix according to claim 5, wherein the particles comprise cellulose.

7. The reagent matrix according to any one of claims 5 to 6, wherein the plurality of particles have an average particle size of at least 0.1 micrometer to at most 10 micrometers.

8. The reagent matrix according to any one of the preceding claims, wherein the first capture component comprises a protein or particles.

9. The reagent matrix according to any one of the preceding claims, wherein the first analyte capture site comprises at least one of the following: a monoclonal antibody, a polyclonal antibody, its F(ab') or F(ab')2 fragment, a capture protein, or an aptamer.

10. The reagent matrix according to any one of the preceding claims, wherein the second capture component comprises a protein or particles.

11. The reagent matrix according to any one of the preceding claims, wherein the second analyte capture site comprises at least one of the following: a monoclonal antibody, a polyclonal antibody, its F(ab') or F(ab')2 fragment, a capture protein, or an aptamer.

12. The reagent matrix according to any one of the preceding claims, wherein the second capture component is a bifunctional fusion protein comprising a cellulose binding domain and a second analyte capture site.

13. The reagent matrix according to any one of the preceding claims, wherein the reagent matrix is substantially free of water.

14. The reagent matrix according to any one of claims 1 to 13, wherein the reagent matrix further comprises a carrier solution, optionally, wherein the carrier solution is an aqueous buffer.

15. A kit for performing an assay to detect an analyte in a sample, the kit comprising: (i) the reagent matrix according to any one of claims 1 to 14; and (ii) a porous substrate.

16. The kit according to claim 15, wherein the porous substrate is a nonwoven substrate, optionally wherein the nonwoven substrate comprises a plurality of polyolefin fibers, polyamide fibers, polyester fibers, glass fibers, or a combination thereof.

17. The kit according to any one of claims 15 to 16, wherein the nonwoven substrate comprises a plurality of microfibers having an effective fiber diameter of at least 0.5 microns and at most 16 microns.

18. The kit according to claim 15, wherein the porous substrate is a microporous membrane, optionally wherein the microporous membrane comprises a thermoplastic polymer material.

19. The kit according to claim 18, wherein the microporous membrane has an average flow pore size of at least 0.1 microns and at most 100 microns.

20. The kit according to any one of claims 15 to 19, wherein the analyte comprises at least one of the following: an antigen, a protein, a biological contaminant, or a chemical of environmental concern.

21. A method for detecting an analyte in a sample, the method comprising: (i) combining the sample, a carrier solution, and a reagent matrix according to any one of claims 1 to 16 in a container to form a test sample; (ii) contacting the test sample with a porous substrate; and (iii) detecting a signal generated by the aggregation of the analyte and the reagent matrix on the surface of the porous substrate, optionally further comprising: a washing step after contacting the test sample with the porous substrate.

22. The method according to claim 21, wherein the detection is performed by visual inspection.

23. The method according to any one of claims 21 to 22, wherein the detection is performed by an instrument.

24. The method according to any one of claims 21 to 23, wherein the method further comprises a positive control.

25. The method according to any one of claims 21 to 24, the method further comprising contacting the reagent matrix with the carrier solution before combining with the sample.

26. The method according to any one of claims 21 to 26, wherein the analyte is an antigen, a protein, a biological contaminant, or a chemical of environmental concern.

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