Lateral flow assay for quantitative ultrasensitive detection of analytes

Abstract

Lateral flow devices, systems and methods can be used to detect analytes by monitoring changes in electronic properties. In one embodiment, a device for detecting an analyte can include a porous substrate and a sensing region that includes a semiconductor material on the surface of the porous substrate that can transport a sample through capillary action. The sensing region can have electronic properties that change in response to the analyte. In another embodiment, a system for detecting an analyte can include a device as described herein and a reader configured to measure the electronic properties of the sensing region. For example, the reader can include a wireless reader (e.g., a smartphone) or any circuit that can measure changes in resistance.

Description

[Technical field]

[0001] (Priority Claim) This application claims priority to U.S. Provisional Patent Application No. 63 / 308,630, filed February 10, 2022, and U.S. Provisional Patent Application No. 63 / 339,728, filed May 9, 2022, each of which is incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to systems and methods for lateral flow assays. [Background technology]

[0003] (background) Many biosensing methods rely on signals generated by enzyme-catalyzed reactions and efficient methods to detect and record this activity. Other chemical sensing methods rely on other chemical interactions and reactions to provide a response. Summary of the Invention [Means for solving the problem]

[0004] (overview) Overall, a new type of lateral flow device can detect analytes by monitoring changes in electronic properties.

[0005] In one aspect, a device for detecting an analyte may comprise a porous substrate and a sensing region comprising a semiconductor material on a surface of the porous substrate capable of transporting a sample via capillary action, the sensing region having electronic properties that change in response to the analyte.

[0006] In another embodiment, a system for detecting an analyte may comprise a device described herein and a reader configured to measure an electronic property of the sensing region. For example, the reader may include a wireless reader (e.g., a smartphone) or any circuit capable of measuring a change in resistance.

[0007] In another aspect, the method of making a device can include depositing a colloidal dispersion on a surface of a substrate to form a semiconductor material. In certain embodiments, the substrate can be a porous substrate. In certain embodiments, the method can be used to create a coating from a material that is a nominally insoluble substance, but small fluidized particles can be trapped at the liquid-liquid interface of the colloidal dispersion and then used to create a conformal coating on the surface. In certain circumstances, the method can include reductive functionalization of the semiconductor material to create a film that is optimal for analyte detection by oxidative doping. In some circumstances, the semiconductor material can be deposited by polymerization on the substrate.

[0008] In another embodiment, a method of detecting an analyte may include exposing a device as described herein and detecting the analyte in the sample. In certain circumstances, detecting the analyte in the sample may be quantitative.

[0009] In certain circumstances, the semiconductor material may include a semiconducting polymer film, carbon nanotubes, or MXene film (e.g., metal carbide or metal nitride) on the surface of the porous substrate. For example, the semiconducting polymer film may include polypyrrole, polyaniline, polythiophene, polyacetylene, polyphenylene, polyarylene, polyarylenevinylene, or polyphenylenevinylene, or combinations thereof. The polymer deposited on the surface may be initially prepared in a form conducive to analyte detection. In some embodiments, the polymers are in a more conductive form caused by oxidative doping, and an analyte-induced event reduces the conductivity by pinning or expelling charge carriers. In some embodiments, the polymers are in a lower conductive state, and an analyte-induced event injects new charge carriers or liberates pinned carriers, increasing the conductivity. In certain circumstances, the semiconductor material may initially be in a lower conductive state than it is after analyte detection. In other circumstances, the semiconductor material may initially be in a higher conductive state than it is after analyte detection.

[0010] In certain circumstances, the analyte detection results in a change in conductivity of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100%.

[0011] In certain circumstances, the semiconductor material may comprise a recognition moiety configured to respond to the analyte. The recognition moiety may include an enzyme, a protein, a synthetic receptor, an antibody, a nanobody, a nucleic acid, a molecular catalyst, a metal binding site, a Lewis base, a Lewis acid, or a combination thereof. In certain circumstances, the recognition moiety may be attached to the material on which the semiconductor material is deposited.

[0012] In certain circumstances, the device may include a catalyst in proximity to the semiconductor material, which may be immobilized in proximity to the semiconductor material by the action of an analyte.

[0013] In certain circumstances, the device may include a redox catalyst initially present in the analyte solution that can react with the semiconductor material.

[0014] In certain circumstances, the sensing region may comprise an enzyme. The sensing region may include adjacent functional groups. For example, an enzyme may be entrapped upstream of the semiconductor material. In another example, a flow regulation element may be present downstream of the semiconductor material.

[0015] In certain circumstances, the analyte may include bacteria, proteins, viruses, nucleic acids, cells, biomolecules, drugs, toxins, biomarkers, reactive biomarkers, enzyme substrates, carbohydrates, metals (e.g., toxic metals), toxins, toxic molecules, metal ions, heavy metals (e.g., mercury or lead), ions, inorganic ions, organic molecules, highly fluorinated molecules, oxidizing agents, Bronsted acids or bases, or combinations thereof.

[0016] In certain circumstances, the analyte may be present at a concentration of less than 1 ng / L, less than 100 pg / L, or less than 10 pg / L.

[0017] In certain circumstances, the analyte may be capable of binding to or reacting with an enzyme, or may be capable of binding to two or more recognition moieties simultaneously.

[0018] In certain circumstances, the analyte may participate in or initiate one or more reactions that result in the oxidation or reduction of the semiconductor material.

[0019] In certain circumstances, the porous substrate may include a control region adjacent to the sensing region.

[0020] In certain circumstances, the porous substrate may include a sampling region that is adjacent to the control region or the sensing region.

[0021] In certain circumstances, the control signal may be a change in electrical resistance.

[0022] In certain circumstances, the device may include a redox catalyst that may enhance or facilitate an analyte-induced change in the resistive state of the semiconductor. The redox catalyst may enhance or facilitate an analyte-induced change in the resistive state of the semiconductor.

[0023] In certain circumstances, the electronic property may be resistivity.

[0024] In certain circumstances, the device may include a radio frequency identification tag electrically connected to the sensing region. For example, the radio frequency identification tag may include an integrated circuit in parallel with the sensing region. In other embodiments, the radio frequency identification tag may include an integrated circuit in series with the sensing region.

[0025] Other aspects, embodiments, and features will be apparent from the following description, drawings, and claims. [Brief description of the drawings]

[0026] [Figure 1A] FIG. 1A shows a schematic diagram illustrating a device for detecting an analyte.

[0027] [Figure 1B] FIG. 1B shows a schematic diagram illustrating a system for detecting an analyte.

[0028] [Figure 2A] Figures 2A-B show the preparation of carboxylate-functionalized pPy core-shell particles: Figure 2A is a schematic of the preparation of carboxylate-functionalized pPy core-shell particles; Figure 2B is a diagram of the conjugation and reduction of pPy core-shell particles. [Figure 2B] Figures 2A-B show the preparation of carboxylate-functionalized pPy core-shell particles: Figure 2A is a schematic of the preparation of carboxylate-functionalized pPy core-shell particles; Figure 2B is a diagram of the conjugation and reduction of pPy core-shell particles.

[0029] [Diagram 3] Figures 3A-B show schematic diagrams of the preparation and rupture process of pPy core-shell particles: Figure 3A shows the preparation of pPy core-shell particles; Figure 3B shows the spontaneous rupture process of carboxylate-functionalized pPy core-shell particles on a glass surface.

[0030] [Figure 4] Figures 4A-4B show optical images of pPy core-shell particles and carboxylate-functionalized pPy core-shell particles. Figure 4A shows an optical image of a pPy core-shell particle. Figure 4B shows an optical image of a pPy core-shell particle after carboxylate-functionalization.

[0031] [Figure 5A-B] Figures 5A-5E show SEM images of pPy core-shell particles with various volume ratios of 3-mercaptopropionic acid. Figure 5A shows no post-functionalization. Figures 5B-5E show pyrrole:3-mercaptopropionic acid with volume ratios equal to 5:1, 5:2, 5:3, and 5:4, respectively. [Figure 5C-D] Figures 5A-5E show SEM images of pPy core-shell particles with various volume ratios of 3-mercaptopropionic acid. Figure 5A shows no post-functionalization. Figures 5B-5E show pyrrole:3-mercaptopropionic acid with volume ratios equal to 5:1, 5:2, 5:3, and 5:4, respectively. [Figure 5E]Figures 5A-5E show SEM images of pPy core-shell particles with various volume ratios of 3-mercaptopropionic acid. Figure 5A shows no post-functionalization. Figures 5B-5E show pyrrole:3-mercaptopropionic acid with volume ratios equal to 5:1, 5:2, 5:3, and 5:4, respectively.

[0032] [Figure 6A-B] Figures 6A-C show schematic illustrations of pPy particle bioconjugation. Figure 6A shows the bioconjugation of streptavidin to carboxylate-functionalized pPy particles. Figure 6B shows a fluorescence microscopy image of pPy particles after streptavidin bioconjugation and AFDye 594 biotin addition for 2 hours. Red fluorescence indicates successful bioconjugation in the particles. Scale bar = 50 μm. Figure 6C shows a schematic illustration of bioconjugation of biotinylated glucose oxidase to pPy core-shell particles. Only one of the four possible biotin-streptavidin sites is shown as occupied for clarity. [Figure 6C] Figures 6A-C show schematic illustrations of pPy particle bioconjugation. Figure 6A shows the bioconjugation of streptavidin to carboxylate-functionalized pPy particles. Figure 6B shows a fluorescence microscopy image of pPy particles after streptavidin bioconjugation and AFDye 594 biotin addition for 2 hours. Red fluorescence indicates successful bioconjugation in the particles. Scale bar = 50 μm. Figure 6C shows a schematic illustration of bioconjugation of biotinylated glucose oxidase to pPy core-shell particles. Only one of the four possible biotin-streptavidin sites is shown as occupied for clarity.

[0033] [Figure 7]7A-7B show fluorescence microscopy images of pPy core-shell particles after NHS activation. Figure 7A shows a fluorescence microscopy image. Figure 7B shows a bright field microscopy image. Scale bar = 50 μm.

[0034] [Figure 8] Figure 8 shows a schematic illustration of the bioconjugation of pyruvate oxidase to pPy core-shell particles, with only one of the four possible biotin-streptavidin sites shown as occupied for clarity.

[0035] [Figure 9A] Figures 9A-9C show the preparation of a lateral flow device. Figure 9A shows a schematic illustration of the conformal coating process. Figure 9B shows a demonstration of the construction of the lateral flow device. Figure 9C shows an optical image of the lateral flow device. [Figure 9B-C] Figures 9A-9C show the preparation of a lateral flow device. Figure 9A shows a schematic illustration of the conformal coating process. Figure 9B shows a demonstration of the construction of the lateral flow device. Figure 9C shows an optical image of the lateral flow device.

[0036] [Figure 10A-B] Figures 10A-10C show SEM images of nitrocellulose membranes. Figure 10A shows an SEM image of the nitrocellulose membrane before coating with pPy core-shell particles. Figure 10B shows an SEM image of the nitrocellulose membrane after coating with bioconjugated pPy core-shell particles. The white circles on the images indicate pores on the coated nitrocellulose membrane. Figure 10C shows an SEM image of the nitrocellulose membrane after coating with bioconjugated pPy core-shell particles at a higher magnification. [Figure 10C]Figures 10A-10C show SEM images of nitrocellulose membranes. Figure 10A shows an SEM image of the nitrocellulose membrane before coating with pPy core-shell particles. Figure 10B shows an SEM image of the nitrocellulose membrane after coating with bioconjugated pPy core-shell particles. The white circles on the images indicate pores on the coated nitrocellulose membrane. Figure 10C shows an SEM image of the nitrocellulose membrane after coating with bioconjugated pPy core-shell particles at a higher magnification.

[0037] [Figure 11] 11 shows a schematic illustration of the increase in conductivity of glucose oxidase- or pyruvate oxidase-conjugated pPy films in an LFA upon addition of glucose- or sodium pyruvate-containing samples. Doping of the pPy films was induced by the generation of H2O2 by glucose oxidase or pyruvate oxidase.

[0038] [Figure 12A-B] 12A-12E show the conversion of a commercially available NFC tag to a p-CARD for wireless detection of enzyme responses. FIG. 12A shows the wired integration of a test line of a lateral flow device in parallel with an NFC integrated circuit (IC). FIG. 12B shows the circuit design of the p-CARD. FIG. 12C shows the resonant frequency traces for a p-CARD with various concentrations of glucose solutions and the addition of 0.16 wt% catalyst. FIG. 12D shows the correlation between the magnitude of the response (Δ gain) at 13.56 MHz and the glucose concentration. All error bars are standard deviations calculated from three independent experiments (n=3). FIG. 12E shows the magnitude of the response (Δ gain) at 13.56 MHz upon the addition of water or 1 wt% sodium pyruvate (with 0.16% catalyst) to the lateral flow device. All error bars are standard deviations calculated from three independent experiments (n=3). [Fig. 12C-D]12A-12E show the conversion of a commercially available NFC tag to a p-CARD for wireless detection of enzyme responses. FIG. 12A shows the wired integration of a test line of a lateral flow device in parallel with an NFC integrated circuit (IC). FIG. 12B shows the circuit design of the p-CARD. FIG. 12C shows the resonant frequency traces for a p-CARD with various concentrations of glucose solutions and the addition of 0.16 wt% catalyst. FIG. 12D shows the correlation between the magnitude of the response (Δ gain) at 13.56 MHz and the glucose concentration. All error bars are standard deviations calculated from three independent experiments (n=3). FIG. 12E shows the magnitude of the response (Δ gain) at 13.56 MHz upon the addition of water or 1 wt% sodium pyruvate (with 0.16% catalyst) to the lateral flow device. All error bars are standard deviations calculated from three independent experiments (n=3). [Figure 12E] 12A-12E show the conversion of a commercially available NFC tag to a p-CARD for wireless detection of enzyme responses. FIG. 12A shows the wired integration of a test line of a lateral flow device in parallel with an NFC integrated circuit (IC). FIG. 12B shows the circuit design of the p-CARD. FIG. 12C shows the resonant frequency traces for a p-CARD with various concentrations of glucose solutions and the addition of 0.16 wt% catalyst. FIG. 12D shows the correlation between the magnitude of the response (Δ gain) at 13.56 MHz and the glucose concentration. All error bars are standard deviations calculated from three independent experiments (n=3). FIG. 12E shows the magnitude of the response (Δ gain) at 13.56 MHz upon the addition of water or 1 wt% sodium pyruvate (with 0.16% catalyst) to the lateral flow device. All error bars are standard deviations calculated from three independent experiments (n=3).

[0039] [Figure 13] FIG. 13 shows the resonant frequency traces for the p-CARD before and after adding deionized water with 0.16 wt % of the catalyst.

[0040] [Figure 14]FIG. 14 shows the resonant frequency traces for p-CARD (coated with pyruvate oxidase-conjugated pPy particles) before and after the addition of 1% sodium pyruvate and 0.16 wt % catalyst.

[0041] [Figure 15] FIG. 15 shows the thickness of the pPy film on the nitrocellulose membrane as measured by a profilometer.

[0042] [Figure 16A-B] 16A-C show wireless detection of C-reactive protein (CRP) using a lateral flow device. FIG. 16A shows a schematic diagram of the lateral flow device configuration. FIG. 16B shows a graph of the resonant frequency trace for p-CARD pre-treated with various concentrations of CRP. 0.2 wt% glucose and 0.16 wt% catalyst were finally added to the device to generate a signal. FIG. 16C shows a graph of the magnitude of response (Δ gain) at 13.56 MHz versus CRP concentration. All error bars are standard deviations calculated from three independent experiments (n=3). [Figure 16C] 16A-C show wireless detection of C-reactive protein (CRP) using a lateral flow device. FIG. 16A shows a schematic diagram of the lateral flow device configuration. FIG. 16B shows a graph of the resonant frequency trace for p-CARD pre-treated with various concentrations of CRP. 0.2 wt% glucose and 0.16 wt% catalyst were finally added to the device to generate a signal. FIG. 16C shows a graph of the magnitude of response (Δ gain) at 13.56 MHz versus CRP concentration. All error bars are standard deviations calculated from three independent experiments (n=3).

[0043] [Figure 17] FIG. 17 is a schematic diagram of a lateral flow device. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] (Detailed Description) Described herein is the formation of a system and method for converting a conventional lateral flow assay (LFA) process into an electronically readable system. The conversion material used to detect the analyte is deposited using a colloidal dispersion, printed from a solution, or polymerized on a support. In an operable lateral flow device, the sample is drawn through a test strip to provide a measurement. The method does not use conventional optical methods for detection, but uses the change in electrical resistivity of a band of material containing a π-conjugated or other semiconductor system capable of a conductivity change as a result of oxidation or reduction by an enzyme or its product. In an illustrative example, poly(pyrrole) is functionalized with an oxidase enzyme that generates a localized hydrogen peroxide signal in response to an analyte. An oxidation (redox) catalyst can be used to efficiently affect oxidative doping of the polymer in the presence of the hydrogen peroxide. The conversion scheme is general, and enzymes that generate other oxidizing or reducing molecules can be localized to the conductive material to produce a conductivity change in response to a biomolecular recognition event. The ideal pairing of the molecules responsible for the conductivity change depends on the type of semiconductor and its functionalization. An n-type semiconductor, i.e., a semiconductor that increases its conductance, when reduced, pairs better with a reduced molecule generated as a result of a biomolecular recognition event. A p-type semiconductor that has already been oxidatively doped to a conductive state can similarly be induced to decrease its conductivity by a reduced molecule that reduces its doping level. A p-type semiconductor in a low conductivity (low doping level) state can, in some embodiments, be paired with an oxidized molecule generated by an enzyme that can cause its conductivity to increase. Semiconductors can also exhibit a change in conductivity with other molecules or ions, and in some cases, the binding of anions or cations changes their conductivity.

[0045] As a first demonstration of this LFA concept, quantitative detection of glucose based on electrical resistance changes and / or shifts in the resonant frequency of a passive radio frequency identification (RFID) tag is illustrated below. The system may be equipped with an inexpensive reader, which may allow the device to be read by other wireless methods. For example, wireless methods are interesting for connecting to a smartphone for at-home diagnostics, which may also be centrally monitored if necessary. Wireless methods may include methods such as Bluetooth, local wireless networks, or other radio frequencies. In some embodiments, the radio frequency is 13.56 mHz for pairing with widely available readers, including smartphones. In the example below, the signal is large, and the resistance of the poly(pyrrole) dropped by 700,000% in response to physiological concentrations of glucose. A large change (decrease) in resistance was used to generate a response to pyruvate. The large resistivity changes observed may provide sensitivity beyond conventional LFA techniques. In some embodiments, the analyte may be detected at concentrations lower than 1 ng / L. In some embodiments, the analyte may be detected at concentrations below 100 pg / L. In some embodiments, the analyte may be detected at concentrations below 10 pg / L.

[0046] The initial demonstration begins with an enzyme immobilized on a semiconducting polymer by the action of an analyte, but the conversion scheme can be much broader. For example, the enzyme can be configured in close proximity to the semiconducting polymer and can be effective as long as the action of the enzyme causes a resistivity change in the semiconducting polymer. In some embodiments, the sample can be added to a reagent pad that includes a receptor for the analyte bioconjugated with one or more enzymes, and other reagents that cause oxidation of the reduction product can be added to the reagent pad or can also be present in the reagent pad. The reagent pad can be configured to release these materials simultaneously or to release one material in a stepwise manner before the other in a flow with the assay. An assay can include two or more separate reagent pads, in some embodiments, one pad to which a sample containing the analyte is added and a second reagent pad to which a solution that delivers a second reagent for the assay is added. Additional reagent pads can deliver materials as needed. The times at which the various reagents are released can be controlled in some embodiments by the fluid flow path length, the configuration of the reagent pad, or the timing of fluid addition to the reagent pad. A multivalent analyte complementary to the receptor conjugated to the enzyme can be functionalized, and then a second receptor attached to the semiconductive conversion material or support structure can capture the analyte-enzyme complex as it flows along the test strip. An enzyme substrate (including, but not limited to, a substrate for an oxidase enzyme, including glucose or pyruvate, or a substrate for a reductase enzyme) can provide a local concentration of an oxidizing or reducing agent that causes a resistance change in the control and test bands of the assay.

[0047] This scheme is general and can be applied to the detection of bacteria, proteins, viruses, nucleic acids, cells, reactive biomarkers, enzyme substrates, carbohydrates, toxins, heavy metals, organic molecules, combinations thereof, or any analyte that can bind to or react with an enzyme, and any analyte that can bind to two or more receptors simultaneously. For example, tetrameric C-reactive protein (CRP) can be detected by using a test line with a semiconductor functionalized with a CRP-specific antibody and a reagent pad containing glucose oxidase conjugated to a CRP-specific antibody. Dimeric or monomeric proteins can be detected, provided that two binding elements can interact with the protein. Multiple binding proteins can be used to attach to two separate epitopes of a protein analyte. In some embodiments, there may be advantages to using multiple binding proteins that recognize different epitopes. For example, when all of the target analytes are bound to one binding protein in the reagent pad, there may still be an open binding site (second epitope). Binding to this second epitope by a second binding protein can be used to localize the enzyme on or in proximity to the semiconductor material.

[0048] By way of example, semiconductor materials that may be used with the device include, but are not limited to, semiconducting polymers, conducting polymers, carbon nanotubes, graphene, carbon nitride nanomaterials, metal oxides, MXenes (e.g., transition metal carbides or transition metal nitrides), or metal sulfides. In some embodiments, a first semiconductor may be doped, which may then change the conductivity of a second semiconductor. In some embodiments, the second semiconductor may be silicon, and in some embodiments, the resistivity change of the silicon may be used to determine the presence of an analyte. In other embodiments, the first semiconductor is not required to have high conductivity when doped. In some embodiments, the first semiconductor may be an isolated molecular form in which bulk semiconductor behavior is not possible.

[0049] In some circumstances, LFAs may exploit resistivity changes in semiconductor materials for the quantitative detection of analytes.

[0050] In other situations, the LFA may utilize resonance changes in the RFID circuit for the detection of analytes.

[0051] In other situations, methods for the deposition of semiconducting polymer films can include the use of colloidal dispersions, which provide a method for producing high quality conformal coatings. In some embodiments, the colloids can be reductively functionalized with thiols or reagents capable of carrying out reactions equivalent to the thiol-Michael reaction to produce films that are optimally conditioned for oxidative doping. This coating and functionalization method can be inexpensive and scalable for commercial production of LFAs as well as other coated materials.

[0052] In other situations, the method for depositing the semiconductor material may include a solution that may be printed. In some embodiments, the printed material may be chemically or thermally activated after deposition to create a converted material. In other situations, the semiconductor material may be synthesized directly on a support material from molecular precursors. In some embodiments, multiple materials may be deposited either together or in close proximity to each other.

[0053] In other situations, methods that use catalysts to promote the reaction of oxidizing or reducing reagents with semiconductor materials can produce optimal conductivity changes. The redox catalyst can enhance or promote the analyte-induced change in the resistive state of the semiconductor.

[0054] In other situations, the quantitative LFA may be wirelessly readable using a smartphone. In other situations, an inexpensive device capable of detecting resistivity changes may read the LFA and transmit the information to a smartphone, local area network, cellular network, or computer.

[0055] Referring to FIG. 1A, the device 10 may include a porous substrate 20. The porous substrate may be capable of transporting a sample through capillary action. The porous substrate 20 is arranged to receive a fluid sample, for example, at a sampling region 50. The fluid sample may be delivered to the sampling region 50 as a droplet or as a flow of fluid. The sampling region may include reagents and recognition moieties necessary for the assay. A control region 30 may be present in the porous substrate 20. The control region may provide a visual or electronic signal that confirms the presence of a fluid sample in the device. A sensing region 40 may be present in the porous substrate 20 and may be adjacent to the control region 30. The sensing region 40 may include a semiconductor material having electronic properties that change in response to the analyte. The electronic properties may be monitored by a circuit 60. The electronic properties in the control region 30 may also be measured by a comparable circuit 60 (not shown). The control region 3 and the sensing region 40 need not be in the order shown, and in some embodiments, the control region may precede the sensing region. The device is also not limited to one sensing region; multiple sensing regions can be used to create an LFA capable of detecting multiple analytes from a single sample. Again, multiple circuits 60 can be connected to each sensing region. The sensing regions can be wired separately in parallel depending on the intent of the assay. The electronic property can be the resistivity of the sensing region.

[0056] The fluid sample may have a volume of 5 nL, 10 nL, 50 nL, 100 nL, 500 nL, 1 μL, 5 μL, 10 μL, 20 μL, 50 μL, 100 μL, 500 μL, or 1 mL. The sample may be in a liquid (e.g., water, an aqueous solution containing molecules and / or ions), or in an organic solvent (e.g., an alcohol, ether, or ester), or a combination thereof.

[0057] The semiconductor material may include a semiconducting polymer film on the surface of the porous substrate. The semiconducting polymer film may include polypyrrole, polythiophene, polyacetylene, polyphenylene, polyphenylenevinylene, or another semiconducting polymer. In certain embodiments, the semiconducting polymer film is initially in a low conductive state. In certain embodiments, the semiconducting polymer film is initially in a high conductive state.

[0058] The porous substrate may be a fibrous, foamed, matted, or non-matted structure. The porous substrate allows the fluid sample to flow to the sensing region. In other embodiments, the porous substrate is a filter through which the fluid can pass. The porous substrate may include paper, cotton, polyester, glass, nylon, cellulose mixed esters, spun polyethylene, polysulfone, nitrocellulose, nylon, poly(arylene ether), or cellulose mixed esters. The porous substrate may have an average pore size of 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100 microns, 110 microns, 120 microns, 130 microns, 140 microns, 150 microns, 160 microns, 170 microns, 180 microns, 190 microns, 200 microns, 210 microns, 220 microns, 230 microns, 240 microns, or 250 microns. The porous structures can have thicknesses of 0.01 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, or 2.5 mm. When coated, the average pore size can be reduced by 20%, 30%, 40%, 50%, 60%, 70%, or 80%. The porous substrates can be designed to transport analytes and key reagents in LFAs, and other structured materials based on polymers, biomolecules, or inorganic materials can have comparable performance and can be used in these devices.

[0059] In some embodiments, the porous material can be designed to reduce the rate at which the solution flows through the semiconductor material, allowing more time for the redox reaction. The additional time can result in greater sensitivity. This characteristic can be a direct effect of the change in the characteristics of the porous material as a result of the deposition of the semiconductor material. For example, if the semiconductor material reduces its interaction with water or reduces the size of the pores, it can slow the passage of water. Alternatively, the porous substrate can be deposited in close proximity to the semiconductor material to slow the fluid flow through the semiconductor material.

[0060] The semiconductor material, or a material proximal to the semiconductor material, may comprise a recognition moiety configured to respond to the analyte. The recognition moiety may selectively bind to the analyte or otherwise identify the analyte. For example, a probe may bind to a recognition moiety that oxidizes or reduces the analyte. In other examples, the analyte may itself be a catalyst for the reduction process. In certain circumstances, a catalyst may be present to oxidize or reduce the analyte. The catalyst may, in some embodiments, facilitate the reaction of an oxidation reagent with the semiconductor material to cause an optimal conductivity change. Other specific interactions may induce a conductivity change, including metal binding, protonation, or other reactions. The catalyst may be a metal catalyst, such as a metal oxide, such as a molybdate. Alternatively, the catalyst may include an enzyme for which the analyte is a substrate. In certain circumstances, both a catalyst and an enzyme may be present in the sensing region. For example, the analyte may be capable of binding or reacting with an enzyme, or may be capable of binding to two or more receptors simultaneously. In some embodiments, the analyte may bind to one recognition moiety in the reagent pad and a second recognition moiety in the sensing region. In some embodiments, the first recognition moiety comprises an enzyme. The recognition moiety may be an antibody-enzyme conjugate, a protein-enzyme conjugate, a conductive polymer-protein conjugate, a conductive polymer-antibody conjugate. These are non-limiting examples intended to show how an analyte that binds to two or more recognition moieties may be used to co-localize materials in the sensing region. In some embodiments, one or two additional reagents are added that do not directly interact with the analyte, but react with the analyte-assembled complex localized in the sensing region to produce a change in the electronic properties of the semiconductor material. The recognition moiety may comprise an enzyme, a protein, a synthetic receptor, an antibody, a nanobody, a nucleic acid, a molecular catalyst, a metal binding site, or a combination thereof.

[0061] The method of manufacturing the device described herein may include depositing a colloidal dispersion on the surface of a substrate to form a semiconductor material. The colloidal dispersion may include particles capable of having flowable properties, which subsequently burst at the surface of the substrate to form a substantially uniform film. The colloidal dispersion may be functionalized such that the resulting film has chemical or biological functionality. For example, the colloids may be connected to recognition moieties prior to forming the film, and the film may present these moieties in a manner that can recognize an analyte. Alternatively, in other embodiments, the colloidal dispersion is functionalized such that the film formed has surface functionality that can be used for subsequent functionalization. The semiconductor material may be deposited from solution by spray coating, silk screen printing, inkjet printing, blade coating, or other physical deposition methods, or combinations thereof, to create structures on the substrate. In some embodiments, the semiconductor material may be soluble and may be deposited from solution. In some embodiments, the deposited material is a precursor of the semiconductor material, which is then chemically or photochemically activated. In other embodiments, the semiconductor material is directly assembled or synthesized on the substrate. For example, a polymer can be produced on the substrate by polymerization of monomers.

[0062] In certain embodiments, the surface may be a rough surface.

[0063] In certain embodiments, the surface may be porous.

[0064] In certain embodiments, the surface may be fibrous.

[0065] In certain embodiments, the surface may be a fabric.

[0066] In certain embodiments, the surface may be metallic.

[0067] In certain embodiments, the surface may be a metal oxide.

[0068] In certain embodiments, the surface can be a filter.

[0069] In certain embodiments, the substrate may include a collection of particles.

[0070] In certain embodiments, the substrate may be a composite, for example, the substrate may be a composite of one or more of the materials mentioned herein.

[0071] Analyte detection refers to the ability to confirm the presence of an analyte at a concentration of interest for a given application. In some embodiments, the concentration can be millimolar. In some embodiments, the concentration can be micromolar. In some embodiments, the concentration can be nanomolar. In some embodiments, the concentration can be picomolar. In some embodiments, the concentration can be femtomolar. The detection signal can be greater than background noise. The greater the signal relative to noise, the greater the accuracy with which the concentration of the analyte can be determined in the analyte detection process. As a result, a larger resistance change in the analyte detection process, in some embodiments, provides greater accuracy in determining the concentration of the analyte.

[0072] Each of the substrates and semiconductor materials can be independently modified by surface chemical reactions applied at various stages. For example, reductive functionalization of semiconductor materials assembled in colloidal dispersions with thiols or reagents capable of carrying out the equivalent of the thiol-Michael reaction can produce materials capable of forming films that are optimal for analyte detection by oxidative doping. This method can be inexpensive and scalable for commercial production of LFAs. In other embodiments, the thiol-Michael reaction can also be formed on the material after forming the film. The thiol-Michael reaction can be used to directly attach a recognition moiety or can be used to provide other functional groups that can be used to attach the recognition moiety in a later step.

[0073] In other situations, the substrate on which the semiconductor material is placed can be functionalized. This functionalization can occur before or after the deposition of the semiconductor material. The functionalization of the substrate can be performed in the vicinity of the semiconductor material. In some embodiments, both the substrate and the semiconductor material can be reacted to produce and contain the same or separate functional groups. The functionalization chemistry can be selected to be appropriate for the functionality and substrate reactivity. In some embodiments, multiple functionalization steps can be used. For example, in one step, a functional group can be added that allows for easy attachment of another group. In some embodiments, the first functionalization can attach a reactive ester, which can then be reacted with a biomolecule, which in some cases can be further reacted with another biological molecule to produce the final structure.

[0074] The sensing region may have multiple sub-regions, including regions that may have recognition moieties or elements, regions that may include semiconductor material, and regions that may have functionality designed to control the rate of fluid flow, optionally or in combination. The sensing region components may be co-localized or adjacent to each other, as appropriate. The order of the regions may reflect the direction of fluid flow. For example, adjacent groups that slow fluid flow may be located downstream of the semiconductor material, while recognition moieties that bind to analytes and cause changes in the semiconductor material may be at the same location or upstream from the semiconductor material. Referring to FIG. 17, a device 202 with a directional fluid flow 200 may include a recognition zone 205, a semiconductor material zone 210, and a flow modifier zone 220. The recognition zone 205 may bind to an analyte, and catalysis may produce a reactant that changes the electrical conductivity. The semiconductor material zone 210 may include features that may bind to an analyte. The flow modifier zone 220 may slow the flow of liquid medium past the semiconductor material zone.

[0075] In some embodiments, the sensing area can be relatively narrow with respect to the flow of the solution in the lateral flow assay.The sensing area can have a width of 0.01mm, 0.1mm, 0.2mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1.0mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2.0mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm, or 2.5mm.In some embodiments, the sensing area can be wider than 2.5mm.The desired specific width of the sensing area can be selected based on one or more of the following characteristics: the manufacturing method; the desired base resistivity of the sensing material before and after the sensing event; the need to make electrical contact; the size of the support; the nature of the reaction that causes the change in the semiconductor material; and the ease with which electrical measurement can be made.

[0076] The circuit 60 can be a resistivity sensor. In other embodiments, the circuit 60 can be a radio frequency identification tag electrically connected to the sensing region 40. For example, the radio frequency identification (RFID) tag can include an integrated circuit in parallel or series with the sensing region. The electronic properties of the sensing region can be measured, for example, by the frequency of the RFID tag or monitored by a reader (e.g., a wireless reader). The wireless reader can be, for example, a loop antenna or a smartphone, or any circuit capable of monitoring the physical properties of the sensing region, which can be calibrated to determine the analyte concentration in a sample.

[0077] The analytes may include bacteria, proteins, viruses, nucleic acids, cells, reactive biomarkers, enzyme substrates, carbohydrates, toxins, heavy metals, organic molecules, or combinations thereof.

[0078] An example of a system including the device and a detector is shown in FIG. 1B. The system may perform a method of detecting an analyte, which may include exposing the device to a sample and detecting the analyte in the sample. The detecting an analyte in the sample may be quantitative. For example, the reader may be calibrated to provide a quantitative measure of the analyte. Quantitative assays may be used to determine biomarkers that are always present, but may indicate a health problem if the concentrations of the biomarkers are outside of the normal range.

[0079] Herein, the design of a wireless lateral flow device is reported that shows the conversion of an oxidase reaction into a change in the resonance of a radio frequency identification (RFID) circuit. The detection is triggered by a large conductivity change induced by polyoxometalate-catalyzed oxidative doping of polypyrrole (pPy) when exposed to HO generated by oxidase. This conversion and RFID capabilities are incorporated into a lateral flow device to create a low-cost, rapid, portable and mobile method for quantitative detection of biological signals. Furthermore, a novel method is reported for making functional coatings from pPy core-shell colloidal particles bioconjugated with glucose oxidase or pyruvate oxidase using streptavidin-biotin recognition. The biofunctionalized pPy particles coalesce on a nitrocellulose membrane to generate a chemoresistive band. The addition of glucose or pyruvate solutions results in the formation of HO at the pPy bands functionalized with the respective oxidases, resulting in a conductivity enhancement of over 700,000%. The pPy band, when placed in an RFID circuit, converts the change in resistivity into a change in resonance. Using this lateral flow device, detection of the glucose oxidase enzyme response within 30 minutes with as little as 0.01 wt% glucose has been achieved. Pyruvate has also been shown to produce a significant response. The ability to use oxidase enzymes as the transduction element establishes this method as a platform for the construction of a family of novel lateral flow devices capable of detecting and quantifying biological targets. The oxidase enzyme transduction is a non-limiting example of the methods and sensors disclosed herein.

[0080] The generation and detection of biochemical signals is essential for all biological systems (Refs. 1-3) and often involves enzyme-catalyzed reactions (Refs. 4-8). Understanding of this biochemistry has revealed many conjugated systems (Refs. 7, 9-11). Glucose oxidase is a highly robust enzyme that is widely distributed in living organisms and produces hydrogen peroxide (H2O2) by oxidation of glucose (Refs. 12-14). In addition to its utility in glucose detection, this enzyme also finds applications in wound healing, food preservation, and the pharmaceutical industry (Refs. 15-17). Glucose oxidase can be used in colorimetric glucose assays by converting the production of H2O2 into an absorbance (color) change (Refs. 18-19). The produced H2O2 can also be detected electrochemically, which is the basis of conventional glucose monitoring devices (Refs. 20-22). This robust and versatile nature of glucose oxidase qualifies it as an attractive platform for the creation of additional biosensor platforms. In some embodiments, the analyte may itself be the catalyst of the reagent for the transduction event that provides the sensing response. In other embodiments, a reductase enzyme may be used to generate the transduction event.

[0081] Lateral flow assays (LFAs) have emerged as an economical, rapid, and easy-to-use platform for laboratory medical diagnostics and at-home testing (References 23-26). In LFAs, liquid flow driven by capillary forces moves sample and reagents along a nitrocellulose test strip (References 27-28). The sample pad is loaded with capture reagents that immobilize specific biological capture agents designed to generate signals at the test and control lines. A limitation of traditional colorimetric LFAs is that most provide binary (yes / no) readouts rather than quantitative data (References 26, 29). As a result, sensitive LFA platforms that can be easily used to generate quantitative data have been created. As an example, C-reactive protein (CRP) is an inflammatory biomarker tetrameric protein that has a normal range in humans and increases the response generated in response to an immune response. 16A-16C show that the test line has an organic semiconducting polymer (polypyrrole) functionalized with an antibody that specifically binds to CRP, the reagent pad has a CRP-specific antibody conjugated to glucose oxidase, and when the solution contains glucose, this biomarker can be quantitatively detected. Such a device can be used, for example, to monitor the health of individuals undergoing immunotherapy for cancer treatment.

[0082] As described herein, carboxylate-functionalized pPy core-shell particles can be used to create oxidase-conjugated films for signal transduction. The pPy functionalization creates a reduced low-conductivity state that can be coated to produce a resistive band on an LFA test strip. The pPy band can be oxidatively doped with H2O2, resulting in a >700,000% increase in conductivity. These large resistance changes can be determined by incorporation into a resonant radio frequency identification (RFID) circuit or measured directly by four-probe or two-probe methods, thereby determining glucose or pyruvate concentrations (References 30-35). RFID detection methods using passive near-field communication (NFC) tags operating at 13.56 MHz have been demonstrated herein. These devices can be powered and read by a conventional smartphone for convenient at-home testing (References 36-39).

[0083] Efficient manufacturing methods compatible with large-scale production of LFA test strips are necessary for real-world applications. To this end, a novel method has been developed that utilizes colloidal dispersions containing insoluble conductive polymers generated at liquid-liquid interfaces. These materials have fluid-like properties and can be used to create high-quality conformal coatings on a variety of substrates, including porous materials. The method is also ideally suited for easy functionalization, where bioconjugated pPy particles or other semiconductor particles can be generated as activated colloids to conformally coat nitrocellulose membranes. As detailed in Figures 2A-B, 3A-B, and 4A-B, the method utilizes core-shell particles generated by interfacial emulsion polymerization of organo-pyrrole droplets in water. The initial core-shell particles contain oxidized (doped) pPy, which when directly deposited on a surface, generate or create a conductive coating. The thin pPy shell and its organic core solvent create a reproducible method to deposit uniform coatings. However, in our application to create the necessary conversion lines for our LFA devices, the pPy needs to be in a highly resistive (undoped) state that can optimally provide the maximum conductance response to an oxidation (doping) event. To meet this design requirement, a method (FIGS. 2A-2B) was developed to achieve both conjugation and reduction in one step. Specifically, the free base form of doped pPy is generated in situ and easily functionalized by a thiol-Michael reaction with a functional thiol. After functionalization, the resulting state shown in FIG. 2B is an undoped polymer. 3-mercaptopropionic acid is used as the functional thiol for addition, and its carboxylate group can be used for bioconjugation (FIGS. 5A-5E). These materials can be coated at higher concentrations on glass as well as nitrocellulose. This and similar methods can be used to efficiently synthesize, functionalize, and coat a variety of semiconducting materials, including polythiophenes and polyanilines.Other fabrication methods using soluble semiconductor materials or precursors can be used.

[0084] The carboxylate-functionalized pPy core-shell particles were further bioconjugated by NHS-ester activated condensation reactions (Figures 6A-3C and 7A-7B, and Figure 8). In this process, the carboxyl groups were treated with N-hydroxysuccinimide and carbodiimide to generate NHS esters, which reacted with the amines of streptavidin. The streptavidin-pPy core-shell particles were then conjugated to biotinylated glucose oxidase or biotinylated pyruvate oxidase. The advantage of this streptavidin / biotin conjugation scheme is that the tetravalent valency of streptavidin could, in principle, generate a high density of enzyme on the surface of the pPy core-shell particles. Fluorescence microscopy (Figure 6C) confirmed the streptavidin conjugation by staining with a red fluorescent biotin-dye conjugate.

[0085] The bioconjugated pPy core-shell particles were coated directly onto nitrocellulose test strips for use in LFAs (Figures 9A-C). Scanning electron microscope images shown in Figures 10A-C confirm that the pPy-coated nitrocellulose test strips retain their porosity, including 7 μm pores. For an LFA to be effective, fluid must flow over both the test and control lines, which are functionalized with pPy films containing glucose oxidase and pyruvate oxidase enzymes on their surfaces, respectively. The nitrocellulose is typically functionalized with surfactants to allow the hydrophilic surface to promote lateral capillary flow of the aqueous sample solution (40). The hydrophilic nature of the deposited bioconjugated pPy films appears to function similarly to the surfactants and does not impede this necessary fluid flow. The device was constructed with a glucose oxidase-pPy test line and a pyruvate oxidase-pPy control line. These enzymes generate H2O2 that can be used to oxidatively dope the pPy, resulting in a change in conductivity determined by the amount of glucose and / or pyruvate present (Figure 11). In such devices, a known amount of pyruvate is added as a control to ensure the authenticity of the LFA. [Table 1]

[0086] Using a nitrocellulose strip containing test and control lines of bioconjugated pPy, a lateral flow device was constructed as shown in Figure 9B and Figure 9C. Application of a liquid sample containing glucose, a redox catalyst, and sodium pyruvate resulted in transport of the solution across the test and control lines. Oxidation catalyzed by glucose oxidase and pyruvate oxidase resulted in the formation of H2O2, doping the pPy film and causing an increase in conductivity. The change in conductivity in the pPy film coated on the nitrocellulose membrane was first quantified by a four-probe technique. It was determined that the intrinsic reactivity of H2O2 with pPy was too slow, and therefore, phosphovanadomolybdate (H5[PV2Mo), which has been shown to be compatible with oxidases (Refs. 41-42), was used. 10 O 40]) was used as a water-soluble oxidation (redox) catalyst. Tables 1 and 3 detail the conductivity changes observed with various concentrations of glucose or sodium pyruvate. Optimized experimental conditions revealed that 0.16 wt% of catalyst provided the best conductivity enhancement (Table 2). LFA analysis of solutions containing 0.2 wt% glucose and 0.16 wt% of catalyst produced a 7000-fold increase in conductivity. Using this optimized catalyst, solutions containing 0.05 wt% glucose and 0.01 wt% glucose were also tested, and 204- and 8-fold conductivity enhancements, respectively, were observed. These results confirm that a dramatic increase in the conductivity of pPy films could be observed due to glucose triggering. These studies were performed at glucose concentrations below physiological levels. Even greater conductivity enhancements can be expected at higher glucose concentrations. It is noteworthy that highly oxidized pPy often exhibits conductivities above 200 S / cm, with the highest values ​​in Table 1 being approximately 100 times lower than this limit. Control tests in which solutions are applied in this system without the addition of glucose did not result in any increase in conductivity (Table 4), indicating that the conductivity of pPy films is stable to background conditions. A two-probe method (Table 5) was used, which is a more convenient method for measuring conductivity changes for lateral flow devices. [Table 2] [Table 3] [Table 4] [Table 5]

[0087] RFID devices have been widely used to track and identify goods using wireless communication. When the one-step conversion of a short-range RFID tag to a chemically actuated resonant device (CARD) and the chemiresistive material are wired in parallel to an integrated circuit, the device is called a p-CARD (Reference 37). See also U.S. Patent No. 11,200,474, which is incorporated by reference in its entirety. A pPy-coated film was incorporated as a chemiresistive to create a p-CARD device capable of measuring glucose concentration using wireless communication (FIGS. 12A and 12B). The resonance (reflection coefficient) frequency trace of the lateral flow-based p-CARD was first measured without the addition of glucose solution using a vector network analyzer and a homemade loop antenna. Solutions containing 0 wt% glucose, 0.01 wt% glucose, 0.05 wt% glucose, 0.1 wt% glucose, 0.2 wt% glucose, 0.3 wt% glucose with 0.16 wt% catalyst were added to the pads of separate LFA devices and held for 30 minutes, whereupon all of the solutions left the pads and migrated along the test strip. The resonant frequency traces of the treated p-CARDs were measured (FIG. 12C). A plot of the magnitude of the response (Δ gain) versus glucose concentration is shown in (FIG. 12D). An increase in the magnitude of the response was observed when the concentration of the glucose solution was 0.01 wt%. The magnitude of the response (Δ gain) increased significantly to 4.3 dB after the addition of 0.3 wt% glucose. The trend in FIG. 12D indicates that our lateral flow device is highly sensitive and capable of accurate detection of glucose concentrations at 0.01 wt% and above. These results are also consistent with predictions based on the four-probe and two-probe conductance measurements. A control line coated with pyruvate oxidase-conjugated pPy particles was also tested, and the magnitude of response (Δgain) was also well behaved (FIG. 12E and FIG. 13 and FIG. 14). In certain embodiments, the control line does not need to be functionalized.In some embodiments, it may only be necessary to verify that the aqueous fluid has reached the control line, and the resulting resistivity change of that water may be used as the control signal.

[0088] In summary, the design and fabrication of a wireless LFA device that can be used for the detection of oxidase enzyme responses is described herein. A method was developed to create a conductive polymer-enzyme film using bioconjugated pPy core-shell particles. The method conformally coats nitrocellulose test strips with the polymer in an optimal undoped state. These methods are general and can be used to immobilize other biomolecular elements. The designed pPy core-shell particles show significant enhancement of conductivity when exposed to H2O2 in the presence of a catalyst. Conductivity and resonant radio frequency measurements confirm that this pPy-based LFA can quantify glucose concentration in a test solution. Wireless detection using p-CARD can detect glucose at 0.01 wt% or higher. This device provides a novel mechanism for the operation of LFAs that can be used in the laboratory or for home testing. LFAs can be made for a variety of assays that are interesting for health care.

[0089] (material) Unless otherwise indicated, all reagents and solvents were used as received without further purification. Glucose oxidase from Aspergillus niger, microbial pyruvate oxidase, (+)-biotin N-hydroxysuccinimide ester, pyrrole, 1,2-dichlorobenzene (oDCB), sulfolane, cetrimonium bromide (CTAB), phosphomolybdic acid (PMA), magnesium sulfate, triethylamine, and 3-mercaptopropionic acid were purchased from Sigma-Aldrich. N-hydroxysuccinimide was purchased from Alfa Aesar. Flavin adenine dinucleotide (FAD) and thiamine pyrophosphate were purchased from Acros Organics. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was purchased from Fluka. AFDye 594 biotin was purchased from Click Chemistry Tools. Phosphovanadomolybdate catalyst (H5[PV2Mo 10 O 40 ]) was prepared according to literature procedures (see, e.g., Tsigdinos, GA; Hallada, CJ, Molybdovanadophosphoric acids and their salts. I. Investigation of methods of preparation and characterization., Inorg. Chem., 1968, 7, 437-441, which is incorporated by reference in its entirety).

[0090] All Near Field Communication (NFC) tags were HF-I Tag-It 13.56MHz RFID transponder square inlays manufactured by Texas Instruments and purchased from DigiKey. Nitrocellulose membranes were purchased from GE Healthcare Life Sciences. 0.020 inch thick backing cards and 22mm x 300mm sample pads were purchased from DCN Diagnostics.

[0091] (device) Optical images of pPy core-shell particles were acquired by an AmScope Trinocular Inverted Microscope equipped with an 18MP USB 3.0 camera. SEM images of the films and particles were acquired by Merlin and Crossbeam 540 Zeiss scanning electron microscopes. Four-point and two-point probe measurements were performed by a Keithley 2400 and a Signatone Four Point Resistivity System. The thickness of the pPy film on the nitrocellulose membrane was acquired using a Dektak 6M stylus profilometer. Fluorescence microscopy images were acquired by a ZEISS AXIO Observer equipped with a ZEISS Axiocam 702 mono Megapixel Microscope Camera. Resonant frequency traces of the p-CARD were recorded using an Agilent E5061B 5Hz-3GHz Network Analyzer.

[0092] Colloidal methods for the synthesis of semiconducting polymers provide an inexpensive, scalable method that can generate materials for coating a variety of surfaces. Many applications exist besides the formation of test lines in lateral flow assays. Coatings can be used for antistatic applications, printed electronics, stabilization of metal surfaces, coating of fabrics, and deposition of chemiresistive sensors.

[0093] The ability of the colloidal platform to allow controlled functionalization of polymerized materials is also important. The synthesis of functional monomers is expensive and the polymerization may not be efficient for the material being functionalized. The functionalization of the colloids as they are formed is cost-effective and the fluidity of the interface allows for easy and uniform functionalization of semiconductor materials. Many types of functional groups can be envisioned. Groups that provide attachment to other materials can be included. The functional groups can be used to provide different surface properties of the deposited semiconductor film. In this way, the film can be made hydrophobic or hydrophilic, for example. The materials can bind toxic metal ions (e.g., mercury or lead) to create specific interactions with the semiconductor material for selective detection of these ions. Alternatively, other ions (e.g., arsenate ions) can be targeted. Lateral flow assays or related devices for detection of toxic components in water can be developed. Fluorocarbon functionalization that promotes the binding of fluorocarbons in water samples can also be achieved. For example, perfluorooctanoic acid can be detected in a lateral flow assay using a semiconductor functionalized to have a fluorophilic character. Other inorganic ions can be detected by selective binding to receptors, selective binding to ligands, or hydrogen bonding. Small organic molecules can also be selectively bound to functionalized semiconductor materials to create lateral flow assays.

[0094] (Experimental Procedure) (Preparation of PPy core-shell particles) The synthesis approach involved first mixing pyrrole, 1,2-dichlorobenzene (oDCB), and sulfolane together as the dispersed phase. Then, an aqueous solution containing 0.1 wt% cetrimonium bromide (CTAB) was added to the dispersed organic phase at a volume ratio of organic phase:aqueous phase equal to 1:6. Furthermore, phosphomolybdic acid (PMA) was introduced into the solution mixture to polymerize pyrrole. After the introduction of PMA, sodium sulfite powder, a weak reducing agent, was also dispersed in the solution to quench the excess amount of PMA to prevent the pyrrole from being doped to a higher oxidation state. The entire mixture was then emulsified for 10 seconds to form stable pPy core-shell particles.

[0095] (Post-functionalization methods for incorporating carboxyl groups) The pPy particles were treated with triethylamine to ensure that they were fully deprotonated, followed by thiol addition with 3-mercaptopropionic acid to introduce carboxyl groups. Figures 5B-5E show SEM images of the same amount of pyrrole but various volume ratios of 3-mercaptopropionic acid, while Figure 5A shows SEM images of pPy core-shell particles without post-functionalization. It can be concluded from Figures 5A-5E that with the minimum amount of 3-mercaptopropionic acid, the pPy particles mostly retained a stable shell structure compared to that shown in Figure 5A. However, when it reached the maximum amount of carboxylate content shown in Figure 5E, the pPy behaved to completely explode onto the silicon wafer substrate of the SEM.

[0096] (Bioconjugation on PPy core-shell particles) Carboxyl groups on the particles were activated by formation of NHS ester. Two equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and two equivalents of N-hydroxysuccinimide were added to the continuous phase of the pPy particles and allowed to react overnight. The pPy particles were then washed three times with deionized water. Streptavidin was bound to the pPy particles by adding one equivalent of streptavidin to the continuous phase and allowed to react for 2 hours.

[0097] The particles were washed three times with deionized water to remove unreacted streptavidin.

[0098] Biotin was chemically bound to glucose oxidase and pyruvate oxidase. We mixed 10 mg of glucose oxidase or pyruvate oxidase with 5 mg of biotin N-hydroxysuccinimide ester in 1 mL of PBS buffer and reacted overnight. The resulting mixture was purified by a desalting column. 100 μL of the biotin-glucose oxidase solution or biotin-pyruvate oxidase solution was added to the continuous phase of the streptavidin-conjugated pPy particles (100 μL particles in 1 mL of continuous phase) overnight. After washing the particles three times with deionized water, glucose oxidase-functionalized pPy particles or pyruvate oxidase-functionalized pPy particles were obtained.

[0099] (Preparation of Lateral Flow Devices Coated with PPy Core-Shell Particles) 100 μL of pPy core-shell particles bioconjugated with glucose oxidase and 100 μL of pPy core-shell particles bioconjugated with pyruvate oxidase were added to a nitrocellulose membrane and allowed to dry overnight. The emulsions were automatically coated onto the membrane. When the films were dry, 0.01 mM flavin adenine dinucleotide (FAD), 10 mM magnesium sulfate, and 0.2 mM thiamine pyrophosphate (which were cofactors) were added to the films coated with pPy particles bioconjugated with pyruvate oxidase. After the films were dry, the nitrocellulose membrane was attached to a backing card by attaching the plastic backing of the nitrocellulose to the adhesive on the card. Sample pads and absorbent pads were cut to size and added to the backing card with a 1 mm overlap with the nitrocellulose membrane.

[0100] (Preparation of Poly(ethylenedioxythiophene) Core-Shell Particles) The formation of PEDOT core-shell particles is initiated by thoroughly mixing the surfactant (polyvinyl alcohol) and reactant (FeCl3) for 30 minutes. 3,4-Ethylenedioxythiophene is added to the reaction system and mixed and reacted under the surfactant conditions at room temperature for 6 hours. The temperature is then increased to 90°C and reacted for 30 minutes. Finally, the entire system is stirred and reacted at room temperature for another 30 minutes to 1 hour. This reaction process produces micron-sized core-shell emulsion particles with diameters of 20 μm to 40 μm. In this case, the core is an ethylenedioxythiophene monomer. The addition of polystyrene sulfonate to the system results in the formation of slightly larger sized PEDOT core-shell particles.

[0101] (Preparation of polyaniline core-shell particles) Polyaniline (PANI) is polymerized with a mixture of freshly distilled aniline, orthodichlorobenzene, and sulfolane in a 1:1:1 ratio as the oil phase, and an aqueous solution of CTAB mixed with 0.1 wt% reduced PMA as the continuous phase, and a surfactant ammonium persulfate (APS) treatment. The introduction of APS and vortexing for 10 times (30 seconds each time) produced stable PANI core-shell emulsions. The thiol addition reaction on these core-shell particles increases the stability of the emulsion. Under conditions where thiolates are formed and 4.5 molar equivalents of 3-mercaptopropionic acid are added, PANI core-shell particles are obtained. These particles can be used for coating or biofunctionalization.

[0102] (Calculation of Conductivity) The conductivity was calculated by using the resistance measured by four-point probe and the thickness of the coated pPy film, one of which is shown below as an example: D=11mm; before: ρ s =R*C=4.2548*138.0484MΩ*1000=587368.332 Bulk resistivity: 569903.321Å*587368.332*10 -10 =33.474 Conductivity = 1 / bulk resistivity = 2.987*10 -4 S / cm rear: ρ s =R*C=4.2548*18.668KΩ=79.429 Bulk resistivity: 569903.321Å*79.429*10 -10 =4.527*10 -3 S / cm Conductivity = 1 / bulk resistivity = 2.209 S / cm

[0103] (RFID measurement) The sample solutions were added to the sample pad of the lateral flow device for 30 minutes. When the films were dry, a vector network analyzer and loop were used to record the resonance (reflection coefficient, or S11 parameter)-frequency trace. The probe was placed above the lateral flow device at a consistent distance to keep all the parameters constant.

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[0105] The details of one or more embodiments are set forth in the accompanying drawings and description. Other features, objects, and advantages are apparent from the specification, drawings, and claims. Although a number of embodiments of the invention have been described, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. It should also be understood that the accompanying drawings are not necessarily to scale, but rather depict somewhat simplified representations of various features and underlying principles of the invention.

Claims

1. 1. A device for detecting an analyte, comprising: a porous substrate; and a sensing region comprising a semiconductor material on a surface of the porous substrate capable of transporting a sample via capillary action and a recognition moiety configured to respond to the analyte; Equipped with The device wherein the sensing region has an electronic property that changes in response to the analyte.

2. 10. The device of claim 1, wherein the semiconductor material comprises a semiconducting polymer film, carbon nanotubes, or an MXene film on a surface of the porous substrate.

3. The device of claim 2 wherein the semiconducting polymer film comprises polypyrrole, polythiophene, polyaniline, polyacetylene, polyphenylene, polyarylene, polyphenylenevinylene, or polyarylenevinylene.

4. A device according to any one of claims 1 to 3, wherein the semiconducting material is initially in a less conductive state than after analyte detection.

5. A device according to any one of claims 1 to 3, wherein the semiconducting material is initially in a more conductive state than after analyte detection.

6. The device of any one of claims 1 to 3, wherein the analyte detection results in a change in conductivity of at least 10%.

7. The device of any one of claims 1 to 3, wherein the analyte detection produces a conductivity change of about 100%.

8. The device of any one of claims 1 to 3, wherein the recognition moiety comprises an enzyme, a protein, a synthetic receptor, an antibody, a nanobody, a nucleic acid, a molecular catalyst, a metal binding site, or a combination thereof.

9. The device of claim 8 further comprising a redox catalyst proximate to the semiconductor material.

10. 10. The device of claim 8, further comprising a redox catalyst initially present in the analyte solution capable of reacting with the semiconductor material.

11. The device of any one of claims 1 to 3, wherein the sensing region comprises an enzyme.

12. 4. The device of any one of claims 1 to 3, wherein the analyte comprises bacteria, proteins, viruses, nucleic acids, cells, reactive biomarkers, enzyme substrates, carbohydrates, highly fluorinated molecules, toxic molecules, metal ions, or combinations thereof.

13. A device according to any one of claims 1 to 3, wherein the analyte is capable of binding to or reacting with an enzyme or of binding simultaneously to two or more recognition moieties.

14. The device of any one of claims 1 to 3, wherein the porous substrate further comprises a control region adjacent to the sensing region.

15. 15. The device of claim 14, wherein the porous substrate comprises a sampling region, the sampling region being adjacent to the control region or the sensing region.

16. The device of any one of claims 1 to 3, further comprising a catalyst.

17. The device of any one of claims 1 to 3, wherein the electronic property is resistivity.

18. The device of any one of claims 1 to 3, further comprising a radio frequency identification tag electrically connected to the sensing region.

19. 20. The device of claim 18, wherein the radio frequency identification tag comprises an integrated circuit in parallel with the sensing area.

20. 20. The device of claim 18, wherein the radio frequency identification tag comprises an integrated circuit in series with the sensing region.

21. A device according to any one of claims 1 to 3, and a reader configured to measure an electronic property of the sensing region; 1. A system for detecting an analyte, comprising:

22. The device of claim 21 , wherein the reader comprises a wireless reader.

23. 22. The device of claim 21, wherein the wireless reader is a smartphone.

24. Depositing the colloidal dispersion onto the surface of a substrate to form a semiconductor material. A method for manufacturing a device, comprising:

25. 25. The method of claim 24, wherein the surface is a rough surface.

26. 25. The method of claim 24, wherein the surface is porous.

27. 25. The method of claim 24, wherein the substrate is fibrous.

28. The method of claim 24, wherein the substrate is a fabric.

29. The method of claim 24 wherein the substrate is a metal.

30. 25. The method of claim 24, wherein the substrate is a metal oxide.

31. 25. The method of claim 24, wherein the substrate is a filter.

32. 25. The method of claim 24, wherein the substrate comprises a collection of particles.

33. 25. The method of claim 24, wherein the substrate is a composite.

34. reductive functionalization of said semiconductor material to produce a film that is optimally conditioned for the detection of an analyte by oxidative doping.

25. The method of claim 24, further comprising:

35. 1. A method for detecting an analyte, comprising: exposing the device of any one of claims 1 to 3 to a sample; and detecting the analyte in the sample. A method comprising:

36. 36. The method of claim 35, wherein detecting the analyte in the sample is quantitative.

37. 36. The method of claim 35, wherein the analyte is a biomolecule.

38. 36. The method of claim 35, wherein the analyte is a biomarker.

39. 36. The method of claim 35, wherein the analyte is a drug.

40. 36. The method of claim 35, wherein the analyte is a toxicity.

41. 36. The method of claim 35, wherein the analyte is a heavy metal such as mercury or lead.

42. 36. The method of claim 35, wherein the analyte is a virus.

43. 36. The method of claim 35, wherein the analyte is an oxidizing agent.

44. 36. The method of claim 35, wherein the analyte is a Bronsted acid or base.

45. 36. The method of claim 35, wherein the analyte is an ion.

46. 36. The method of claim 35, wherein the analyte is an organic molecule.

47. 36. The method of claim 35, wherein the analyte is present at a concentration of less than 1 ng / L.

48. 36. The method of claim 35, wherein the analyte is present at a concentration of less than 100 pg / L.

49. 36. The method of claim 35, wherein the analyte is present at a concentration of less than 10 pg / L.

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