Electronic assay device

Abstract

The present disclosure describes devices, systems, and methods for detecting analytes in biological, industrial, research, and environmental samples using an electrochemical assay device. Particularly described herein are vertical flow assay devices comprising a sample receiving module configured to deliver samples to an electrode chamber comprising one or more working electrodes functionalized with a capture agent specific for an analyte, and an integrated circuit electrically connected to one or more electrodes in the electrode chamber and connected to a user device via a communication module.

Description

Attny Docket No. STDU243535.601Client Ref No. 24-301ELECTRONIC ASSAY DEVICEFIELD

[0001] The present disclosure relates to detecting one or more target analytes in a sample. In particular, the disclosure related to devices, systems, and methods for detecting analytes in biological, industrial, research, and environmental samples using an electrochemical vertical flow assay device.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 690,069, filed September 3, 2024, the content of which is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0003] A variety of sample collection and assay devices for clinical or home use are available and described in the literature. These devices can be used for an assortment of purposes, including the detection of biological compounds such small molecules, drugs, metabolites, hormones, nucleic acids, proteins (e.g., antibodies), pathogens, and the like. Many of these devices are complex in design and are difficult to use. Also, these devices are often manufactured and fabricated from relatively expensive materials. Furthermore, these point of use tests are typically conducted under scenarios where resources are scarce. Instrumentation cost, size and power requirements can severely limit utilization of point of use tests in resource-limited settings. Thus, point of use devices that can rapidly detect and quantify multiple compounds of interest in biological, chemical, and environmental samples without expensive or cumbersome instrumentations are needed to enable modem diagnostics technologies to be employed more widely, providing users with real-time, actionable health and / or environmental information.SUMMARY OF THE INVENTION

[0004] Provided herein are assay devices and systems for detecting one or more analytes in a sample. In some embodiments, the devices and systems electrochemically detect the one or more analytes.

[0005] In some embodiments, the assay devices comprise one or more sensing elements. In some embodiments, each sensing element comprises a working electrode functionalized with one or more capture agents specific for a target analyte.Attny Docket No. STDU243535.601Client Ref No. 24-301

[0006] In some embodiments, the one or more sensing elements are in connection with an integrated circuit. The integrated circuit comprises elements for the operation of the sensing clcmcnt(s) and recording of output signals therefrom. In some embodiments, the assay devices further comprise a power module and a communications module. In some embodiments, the power module and a communications module are wireless.

[0007] In some embodiments, the disclosure provides vertical flow assay devices. In some embodiments, the vertical flow assay devices comprise a sample receiving module; an electrode chamber comprising one or more working electrodes functionalized with a capture agent specific for a target analyte, wherein the electrode chamber is in fluid communication with the sample receiving device; and an integrated circuit electrically connected with the one or more working electrodes, wherein the integrated circuit comprises a power module and a communication module.

[0008] In some embodiments, the sample receiving module is a puncture unit having a proximal end to receive a sample and a distal end configured to deliver samples to the electrode chamber.

[0009] In some embodiments, the vertical flow assay device comprises a solution carousel. In some embodiments, the distal end of the sample receiving module is configured to puncture sealed compartments of the solution carousel and to deliver a sample into the solution carousel. In some embodiments, the solution carousel comprises one or more chambers, each chamber comprising individual diagnostic assay solutions. In some embodiments, the electrode chamber is in fluid communication with the solution carousel.

[0010] In some embodiments, the electrode chamber comprises two or more working electrodes, each functionalized with a capture agent specific for a different target analyte.

[0011] In some embodiments, the capture agent comprises a specific binding partner to the analyte. In some embodiments, the capture agent comprises a nucleic acid, a protein, a peptide, a small molecule, or a combination thereof. In some embodiments, the target analyte and capture agent are each one half of an enzyme-substrate pair. In some embodiments, the enzyme is a redox enzyme.

[0012] In some embodiments, the capture agent comprises an electrochemical label compound. In some embodiments, the electrochemical label compound comprises a redox reporter.

[0013] In some embodiments, the capture agent comprises a bait compound. In some embodiments, the bait compound comprises the electrochemical label compound. In some embodiments, the bait compound has lower affinity for the capture agent as compared to the target analyte.Attny Docket No. STDU243535.601Client Ref No. 24-301

[0014] In some embodiments, the capture agent is configured to change confirmation upon binding to the target analyte and modify the electron transfer between the electrochemical label compound and working electrode. In some embodiments, the capture agent comprises an aptamer switch comprising an electrochemical label compound.

[0015] In some embodiments, the working electrode is part of an electrode set further comprising one or more additional working electrodes, a reference electrode and / or a counter (auxiliary) electrode for a target analyte.

[0016] In some embodiments, the electrode chamber comprises one or more non-target working electrodes functionalized with a control analyte or a capture agent specific for a control or non-target analyte.

[0017] In some embodiments, the vertical flow assay device comprises a fluid retention module. In some embodiments, the fluid retention module comprises a sponge or desiccant to retain the sample after passing through the electrode chamber.

[0018] In some embodiments, the integrated circuit comprises one or more of an electrochemical analog front end (e.g., a potentiostat), a temperature sensor, and / or a pH sensor.

[0019] In some embodiments, the power module comprises a wired power module or a wireless power module. In some embodiments, the power module comprises a battery-based power source.

[0020] In some embodiments, the communication module comprises a wireless communication component or a wired communication component. In some embodiments, the communication module comprises a short-range wireless communication. In some embodiments, the short-range wireless communication module is selected from the group consisting of a near-field magnetic induction (NFMI) module, a Bluetooth communication module, Zigbee / Z-Wave / Thread (802.15.4), Ultra Wideband (UWB), EnOcean, and a Wi-Fi communication module.

[0021] In some embodiments, the communication module and the power module are integrated into a single component. In some embodiments, the single component facilitates wireless power delivery and wireless communication. In some embodiments, the single component is a near-field magnetic induction (NFMI) module operably connected to the assay device to support wireless power delivery and wireless communication.Attny Docket No. STDU243535.601Client Ref No. 24-301

[0022] In some embodiments, the vertical flow assay devices further comprise one or more of: a delay membrane to hold samples and reagent solutions in the electrode chamber for detection, a blood filter, and a reagent zone.

[0023] Further provided are systems comprising an assay device as described herein. In some embodiments, the systems further comprise a mobile user device configured to receive and send communications from the communication module. In some embodiments, the systems comprise two or more assay devices as described herein.

[0024] In some embodiments, the mobile user device comprises a smart phone, tablet computer, smart wearable device, portable laptop computer, voice command device, or other mobile computing device. In some embodiments, the mobile user device sends and / or receives communications to the assay device using an application.

[0025] In some embodiments, the systems further comprise a sample. In some embodiments, the sample is a biological sample, an environmental sample, an industrial sample, or a research sample.

[0026] Also provided are methods of detecting one or more target analytes in a sample using the assay devices described herein. In some embodiments, the methods comprise loading the sample in the sample application region of an assay device as described herein and measuring changes in current or potential of working electrode. In some embodiments, the method further comprises determining the concentration of the target analyte(s) in the sample based on the changes in current or potential of working electrode.

[0027] In some embodiments, the sample is a biological sample, an environmental sample, an industrial sample, or a research sample.

[0028] In some embodiments, the analyte is selected from the group consisting of: a small molecule, a lipid, an amino acid, a peptide, a protein, a nucleic acid, and a carbohydrate.

[0029] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.BRIEF DESCRIPTIONS OF THE DRAWINGS

[0030] FIG. 1 is an expanded view of an exemplary vertical assay device comprising: a puncture straw sample receiving module, a solution carousel, a blood filter, a reagent zone comprising a binding partner, an electrode chamber comprising five electrode sets, a delay membrane, a fluid retentionAttny Docket No. STDU243535.601Client Ref No. 24-301 module (referred to in the drawings as a sample retention module) and an integrated circuit contained within a vertical flow cylinder.

[0031] FIGS. 2A-2E show the exemplary vertical assay device and the process for the delivery of the sample and reagent solutions to the electrode chamber for detection of the target analyte(s).

[0032] FIGS. 3 A and 3B are schematics of the electrode preparation (FIG. 3 A) and test scheme (FIG. 3B) for a procalcitonin (PCT) assay.

[0033] FIG. 4 is a graph of indirect ELISA screening of anti-PCT antibody panel.

[0034] FIG. 5 is interpolated indirect ELISA curves to characterize antibody candidates.

[0035] FIG. 6 is cyclic voltammetry (CV) of EDC / S-NHS concentrations, as indicated, for surface activation.

[0036] FIG. 7 is square wave voltammetry (SWV) peaks showing decreasing current in response to increasing PCT concentrations (0, 10, 100 ng / mL).

[0037] FIGS. 8A and 8B show expanded views of exemplary vertical flow assay (VFA) devices and associated system having a mobile user device. The exemplary device shown in FIG. 8A includes a solution carousel, blood filter, delay membrane. The exemplary device shown in FIG. 8B does not include a solution carousel, blood filter, delay membrane. Either exemplary device may associate to support wireless power delivery and wireless communication of instructions and / or test results with a mobile user device as shown in FIG. 8A.

[0038] It is to be understood that the figures are not necessarily drawn to scale, nor are the objects in the figures necessarily drawn to scale in relationship to one another. The figures are depictions that are intended to bring clarity and understanding to various embodiments of apparatuses, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, it should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way.DETAILED DESCRIPTION OF THE INVENTION

[0039] The present disclosure provides technology related to sample collection and detection of one or more analytes in a sample. The disclosed device has the capability to measure multiple analytes simultaneously, providing quantitative measurements of the analytes in the sample. The devices also enable the use of wireless power and wireless communications, eliminating the requirement forAttny Docket No. STDU243535.601Client Ref No. 24-301 expensive and externally powered test analyzers or reading instrumentation and facilitating the use in under-resourced point of use settings.

[0040] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.1. Definitions

[0041] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0042] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0043] As used herein, the term “analyte” refers to any molecule detected by the device and methods described herein. In some non-limiting example, the analyte is a biological molecule (e.g., protein, nucleic acid, carbohydrate, hormone, etc.).

[0044] “Polynucleotide” or “oligonucleotide” or “nucleic acid,” as used herein, means at least two nucleotides covalently linked together. The polynucleotide may be DNA, both genomic and cDNA, RNA, or a hybrid, where the polynucleotide may contain combinations of deoxyribo- and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. The nucleic acid, whether DNA or RNA may comprise non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”). Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. Polynucleotides may be single- or double-stranded or may contain portions of both double stranded and single stranded sequence. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acidAttny Docket No. STDU243535.601Client Ref No. 24-301 may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.

[0045] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms “polypeptide” and “protein” are used interchangeably herein.

[0046] The term “capture agent” refers to i) an unlabeled specific binding partner that is specific for a target analyte, as in a sandwich assay, ii) a label compound and specific binding partner, as in a competitive assay, or iii) an ancillary specific binding partner, which is a specific binding partner that binds to the specific binding partner of an analyte. For example, an ancillary specific binding partner may include an antibody specific for another antibody, for example, goat anti-human antibody.

[0047] The term “specific binding partner” or “binding partner” refers to a member of a pair of molecules that interact by means of specific, noncovalent interactions that depend on the three- dimensional structures of the molecules involved. Typical pairs of specific binding partners include antigen / antibody, hapten / antibody, hormone / receptor, nucleic acid strand / complementary nucleic acid strand, substrate / enzyme, inhibitor / enzyme, carbohydrate / lectin, polypeptide / metal, biotin / (strept)avidin, and virus / cellular receptor.

[0048] “Antibody” and “antibodies” as used herein refers to monoclonal antibodies, monospecific antibodies (e.g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multi- specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, disulfide-linked Fvs (“sdFv”), and anti- idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology , 25(11): 1290- 1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by reference), or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology 21:484-490Attny Docket No. STDU243535.601Client Ref No. 24-301(2014)), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camclid, or which arc synthetic, c.g., nanobodics, VHH, or other domain structure), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. For simplicity sake, an antibody against an analyte is frequently referred to herein as being either an “anti-analyte antibody” or merely an “analyte antibody”.

[0049] “Antibody fragment” as used herein refers to a portion of an intact antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)) (e.g., comprises the antigen-binding site or variable region). Any antigen-binding fragment of the antibody described herein is within the scope of the present disclosure. The antibody may not include the constant heavy chain domains (e.g., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab’ fragments, Fab’-SH fragments, F(ab’)2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

[0050] As used herein, when a first entity (e.g., antigen binding domain) “specifically recognizes” or “specifically binds” another entity, it preferentially recognizes the other entity in a complex mixture of proteins and / or macromolecules, and binds with an affinity which is substantially higher than to other entities. In this regard, “affinity which is substantially higher” means affinity that is high enough to enable detection of an entity which is distinguished from entities using a desired assay or measurement apparatus. Typically, it means binding affinity having a binding constant (Ka) of at least 107M'1(e.g., >107M’1, >108M'1, >109M'1, >1010M'1, >10nM'1, >1012M’1, >1013M’1, etc.).

[0051] As used herein, the term “sample” is used in its broadest sense. A “sample” refers to any sample comprising an analyte or a part or component thereof or that potentially comprises an analyte or a pail or component thereof. Accordingly, the term “sample” refers to any material to be tested for theAttny Docket No. STDU243535.601Client Ref No. 24-301 presence or amount of an analyte. “Sample” includes a specimen obtained from any source, including biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples can be obtained from a subject using routine techniques known to those skilled in the art. Such examples are not however to be construed as limiting the sample types. Preferably, a sample is a fluid sample such as a liquid sample. Examples of liquid samples that may be assayed include bodily fluids (e.g., blood, serum, plasma, saliva, urine, ocular fluid, semen, sputum, sweat, tears, and spinal fluid), water samples (e.g., samples of water from oceans, seas, lakes, rivers, and the like), samples from home, municipal, or industrial water sources, runoff water, or sewage samples; and food samples (e.g., milk, beer, juice, or wine). Viscous liquid, semisolid, or solid specimens may be used to create liquid solutions, eluates, suspensions, or extracts that can be samples. For example, throat or genital swabs may be suspended in a liquid solution to make a sample. Samples can include a combination of liquids, solids, gasses, or any combination thereof (e.g., a suspension of lysed or unlysed cells in a buffer or solution). Samples can comprise biological materials, such as cells, microbes, organelles, and biochemical complexes. Liquid samples can be made from solid, semisolid, or highly viscous materials, such as soils, fecal matter, tissues, organs, biological fluids, or other samples that are not fluid in nature. For example, solid or semisolid samples can be mixed with an appropriate solution, such as a buffer, a diluent, and / or extraction buffer. The sample can be macerated, frozen and thawed, or otherwise extracted to form a fluid sample. Samples may be used directly as obtained from the source or following a pretreatment to modify the character of the sample. Such pretreatment may include, for example, preparing plasma from blood, diluting viscous fluids, filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, and the like.

[0052] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, guinea pigs, andAttny Docket No. STDU243535.601Client Ref No. 24-301 the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.

[0053] As used herein, the terms “detection” or “detecting” refer to the qualitative determination of the presence or absence of an analyte in a sample or quantification of an analyte in a sample, e.g., the amount and / or concentration of an analyte in a sample.

[0054] As used herein, the word “presence” or “absence” (or, alternatively, “present or “absent”) is used in a relative sense to describe the amount or level of a particular entity (e.g., an analyte). For example, when an analyte is said to be “present” in a sample, it means the level or amount of this analyte is above a pre-determined threshold; conversely, when an analyte is said to be “absent” in a sample, it means the level or amount of this analyte is below a pre-determined threshold. The pre-determined threshold may be the threshold for detectability associated with the particular assay used to detect the analyte or any other threshold. When an analyte is “detected” in a sample it is “present” in the sample; when an analyte is “not detected” it is “absent” from the sample. Further, a sample in which an analyte is “detected” or in which the analyte is “present” is a sample that is “positive” for the analyte. A sample in which an analyte is “not detected” or in which the analyte is “absent” is a sample that is “negative” for the analyte.

[0055] As used herein, an element of the present device is “integral” to another element of the present device when the two elements are manufactured or assembled as a single piece.

[0056] As used herein, an element of the present device is “separate” from another element of the present device when the two elements are manufactured or provided as separate pieces.

[0057] The terms “fluidically connected” or “in fluid communication” refer to any connection in an appropriate manner such that a fluid or a least a portion of a fluid can directly or indirectly pass or flow between the named components.

[0058] The term “electrically connected” refers to the ability of two or more materials and / or components, e.g., of the device disclosed herein, to transfer charge between them, such as in the form of the transfer of electrons or ions. The term “electrically connected” may refer to a configuration of two or more components such that an electronic signal or charge carrier can be directly or indirectly transferred from one component to another. The connection may include one-way and two-way electrical communication. In some embodiments, components in electrical contact or electronic contact are in indirect electrical communication wherein an electronic signal or charge carrier is indirectly transferredAttny Docket No. STDU243535.601Client Ref No. 24-301 from one component to another via one or more intermediate structures, such as circuit elements, separating the components.

[0059] The term “electrochemical detection” refers to methods which operate on principles of electricity and chemistry, or electrochemistry, e.g., on principles of relating the magnitude of a current flowing through a substance, the resistance of a substance, or a voltage across the substance given a known current, to the presence of a chemical species within the substance. Some of these methods can be referred to as potentiometric, chronoamperometric, or impedance, depending on how they are practiced, e.g., whether potential difference or electric current is controlled or measured. The systems, methods, and devices disclosed herein, can measure current flowing through a substance due directly or indirectly to the presence of a particular analyte or an electroactive compound. Electrochemical detection includes detection with amplification or cycling. For examples, the current can be amplified by diffusional recycling under steady state conditions and / or enzyme recycling.

[0060] An “electrochemical label” as used herein refers to a chemical species capable of reversible oxidation and reduction in a liquid sample. Electrochemical labels can include complexes of transition metal ions, for example iron (ferrocene and ferrocene derivatives), ruthenium, and osmium.

[0061] The term “working electrode” as used herein refers to an electrode where measured events (e.g., oxidation, reduction, binding) take place and the resultant current flow can be measured as an indicator of analyte concentration.

[0062] The term “functionalized” may refer to modification of a material or layer surface to add chemical, physical, electrical, optical or electrochemical functionality. In an embodiment, biological molecules or reagents may be deposited onto a working electrode, such that the electrode is functionalized. A variety of methods may be used to deposit biological molecules and reagents on the surface of an electrode and the method of functionalization depends on the nature of the electrode and the biological molecules or reagents with which the electrode is being functionalized. For example, carbon surfaces can be modified for attachment of a linker, biological molecule, or reagent by electroreduction of a diazonium salt, whereas gold surfaces can be functionalized by an amine or thiol (Au-S bond) group of a linker, biological molecule, or reagent. The electrode may be coated with linker molecules configured to interact with the electrode surface and have a specif ic interaction or aff inity with the desired capture agent or provide a functional group with which to react the capture agent, e.g., a reactive moiety for crosslinking, including but not limited to this, amino, hydroxylamino, diazanyl,Attny Docket No. STDU243535.601Client Ref No. 24-301 azide, phenoxide, carboxylic acid, aldehyde, acetylene, dithiocarbamate, and dithiocarboxylate. Linker molecules encompass any group, moiety, or compound which is typically derived from a bi- or trifunctional compound which is or configured to be connected to at least two groups, moieties, or compounds.

[0063] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0064] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.2. Assay Device

[0065] The technology relates to assay devices. As used herein, the term “assay device” refers to a device for detecting the presence, absence, concentration, and / or amount of one or more target analytes in a sample. In some embodiments, an “assay device” detects the presence, absence, concentration, and / or amount of two or more analytes in a sample.

[0066] In some embodiments, the technology relates to assay devices that are suitable for use in the home, clinic, hospital, or on-site or in the field for environmental or industrial analytes, and that are intended to give an analytical result that is rapid with minimum degree of skill and involvement from the user. In some embodiments, the assay devices are fully or partially disposable. In some embodiments, the assay devices are fully or partially single use. In some embodiments, one or more of the components of the assay devices may be multiuse.

[0067] In some embodiments, the assay devices are vertical flow assay devices. With reference to FIG. 1, the vertical flow assay device comprises a sample receiving module configured to receive, optionally store, and deliver samples to an electrode chamber comprising one or more working electrodes functionalized with a capture agent specific for an analyte, and an integrated circuitAttny Docket No. STDU243535.601Client Ref No. 24-301 electrically connected with the one or more working electrodes comprising a power module and a communication module.

[0068] In some embodiments, the vertical flow assay device comprises a solution carousel. Accordingly, the vertical flow assay device may comprise a sample receiving module, a solution carousel, an electrode chamber, and an integrated circuit. The sample receiving module may deliver samples into the solution carousel (e.g., into a running buffer solution chamber of the solution carousel). The sample receiving module may be configured for direct receipt of a sample, e.g., as a collection vessel from the source of the sample, or may be configured for receipt of a sample following collection, processing, and / or transportation.

[0069] The sample receiving module may be configured to puncture various seals or compartments of the vertical flow assay device to facilitate delivery of samples to the electrode chamber. For example, the sample receiving module may be configured to puncture sealed compartments of the solution carousel such that the fluid contents of each compartment are delivered downwards towards the electrode chamber for analysis. In some embodiments, the sample receiving device is a puncture unit having a proximal end to receive a sample and a distal end configured to puncture sealed compartments in the solution carousel. The puncture unit may be designed to puncture both the top and bottom of the sealed compartments in the solution carousel resulting in delivery of samples to the solution carousel and transfer of the sample / solution mixture downwards towards the electrode chamber for analysis.

[0070] With reference to FIGS. 2A-2E, the sample (e.g., a blood sample) is delivered to the proximal end of the puncture unit (e.g., a puncture straw as shown in FIG. 2A). Once the puncture unit punctures the vertical flow assay device and / or the top of the sealed compartments in the solution carousel, the sample mixes with the solution inside the solution carousel (e.g., a running buffer solution as shown in FIG. 2B). Following puncture of the bottom of the sealed compartments in the solution carousel, the sample in the assay solution(s) is applied to a blood filter (FIG. 2C). After the sample in the running buffer solution passes through the blood filed (FIG. 2D) it can enter the reagent zone before entering the electrode chamber (FIG. 2E).

[0071] The sample receiving module may be composed of a variety of materials. In some embodiments, the sample receiving module is composed of one or more bio-based, biodegradable, compostable, or recyclable materials. For example, the sample receiving module may be comprised of materials such as paper, bamboo, PLA (polylactic acid) or PHA (polyhydroxy alkanoate).Attny Docket No. STDU243535.601Client Ref No. 24-301

[0072] The sample receiving module and the device may be configured to accommodate sample volumes greater than approximately 0.01 mL, 0.05 mL, O.lmL, 0.25 mL, 0.5 mL, 1 mL, 2, mL, 3 rnL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, or more. The sample receiving module and the device may be configured to accommodate sample volumes of less than about 10 mL, less than about 9 mL, less than about 8 mL, less than about 7 mL, less than about 6 mL, less than about 5 mL, less than about 4 mL, less than about 3 mL, less than about 2 mL, less than about 1 mL, less than about 0.5 mL, less than about 0.25 mL, less than about 0.1 mL, less than about 0.05 mL, less than about 0.01 mL, less than about 0.005 mL, or less.

[0073] In some embodiments, the sample receiving module may include volume indicators. In some embodiments, these indicators are provided on the outer surface of the sample receiving module as protrusions or indentations in the material from which they are manufactured. In some embodiments, these indicators are provided as inked labels on the outer surface of the sample receiving module.

[0074] The solution carousel houses and segregates various solutions required to perform one or more assays within the diagnostics device. In instances in which more than one solution is required for an assay, the solution carousel may be divided into separate chambers or compartments, each housing a separate assay solution. Exemplary assay solutions include but are not limited to running buffers, wash solutions, redox solutions (e.g., any solution comprising components that enable taking up or releasing said ions in solution during detection), and amplification solutions (e.g., any solution comprising components, that enable redox cycling reactions as described elsewhere herein). Accordingly, assay solutions include ferro / ferricyanide, trimethylene blue, peroxidases, and the like. In embodiments of the vertical flow assay device lacking the solution carousel, assay solutions (e.g., solutions comprising assay buffers and reagents) may be independently delivered to the electrode chamber by the sample receiving module.

[0075] The solution carousel delivers solutions downwards towards to the electrode chamber for analysis once a seal on the solution carousel is punctured or released. Seals on the top and / or bottom of the solution carousel can be of various types including, but not limited to, foil seals, tape seals, a wax seals, and a plastic heat shrunk seal. The body or chambers of the solution carousel can be comprised of plastic, metal or glass.

[0076] The electrode chamber is the location of the electrochemical detection of the analyte. The electrode chamber is in fluid communication with the sample receiving device and, optionally theAttny Docket No. STDU243535.601Client Ref No. 24-301 solution carousel. For example, once the sample and assay solution from the solution carousel are released or independently delivered the electrode chamber will fill and detection of the analyte will commence.

[0077] The electrode chamber comprises one or more electrode sets, each comprising at least one working electrode functionalized with a capture agent specific for an analyte. Each electrode set may further comprise a reference electrode and / or a counter electrode. In some embodiments, the electrode set comprises a working electrode, a reference electrode, and a counter electrode. In some embodiments, the electrode set comprises two or more working electrodes, a reference electrode, and / or a counter electrode.

[0078] In some embodiments, each of the one or more electrode sets are configured for detection of a different analyte, e.g., are functionalized with a different capture agent specific to a different analyte. Electrodes may be printed / deposited on flexible substrate such as polyimide, or can be printed directly onto an inner surface of the vertical flow cylinder comprising one or more components of the device. Metallurgy of the electrodes can vary based on application, and can include gold, platinum, silver, carbon, and silver chloride.

[0079] The capture agent comprises a specific binding partner to the analyte. The nature and identity of the specific binding partner will depend on the analyte of interest. In some embodiments, the capture agent comprises a nucleic acid, a protein, a peptide, a small molecule, or a combination thereof. In select embodiments, the capture agent comprises an antibody, or fragment thereof, for the target analyte.

[0080] In some embodiments, the target analyte and capture agent are each one half of an enzymesubstrate pair. For example, the target analyte may be a substrate, and the capture agent may be its cognate enzyme. The electrode may be functionalized with either linker molecules specific to an enzyme or the enzyme itself. Alternatively, the target analyte may be an enzyme and the capture agent is its substrate. When the target analyte and capture agent are each one half of an enzyme-substrate pair, the interaction of the substrate / enzyme or the catalysis of the enzymatic reaction can result in a change which is observable by the electrode, e.g., an observable change in the peak frequency and / or interfacial capacitance. Thus, allowing detection of the target analyte.

[0081] In select embodiments, the target analyte or capture agent is a redox enzyme. Redox enzyme is a general term for an enzyme that catalyzes the reduction-oxidation between two molecules.Exemplary redox enzymes include, but are not limited to, protein complexes of the respiratory chains ofAttny Docket No. STDU243535.601Client Ref No. 24-301 mitochondria and bacteria, enzymes involved in photosynthetic metabolism, ROS scavenging enzymes including superoxide dismutases, peroxidases, oxidases, and catalases, and the like.

[0082] In some embodiments, the capture agent comprises an electrochemical label compound. The electrochemical label compound can be directly or indirectly (e.g., by means of a linker moiety) covalently linked to the capture agent. The electrochemical label compound facilitates measurement of the capture agent- analyte interactions. For example, upon analyte binding to the capture agent, the electrode may measure a change (e.g., signal decrease) from the electrochemical label compound.

[0083] An electrochemical label compound is a redox active species, also referred to as electrochemical redox labels, electron transfer agents, redox reporter label, or electrochemical mediator labels. The redox moiety can be a pheno thiazine, a phenoxazine, a ferrocene, ruthenium (II), osmium (II), an anthraquinone, a phenazine, or derivatives thereof. For example, the redox moiety can be a phenothiazine derivative such as methylene blue, PZ1, methylene green, toluidine Blue O, Azure A and PZ9 or a phenoxazine such as PO1, gallocyanine, nile blue, and Meldola’s blue. Metal complexes which are defined by the identity of the metal and its associated ligands, including ruthenium (II) complexes, Osmium (II) complexes, and Iron (II) complexes, are exemplary redox moieties suitable for use as an electrochemical label in the device herein.

[0084] In some embodiments, the redox active species is a molecule that is capable of cycling through states of oxidation and or reduction without decomposing or reacting irreversibly with other molecules in solution. Thus, allowing redox cycling reactions, wherein the current flow / voltage signal that is measured from the cavity is amplified. Redox cycling is a technique in which multiple electrodes are used to repeatedly flip the charge state of the redox active molecules allowing each redox active molecule to participate in multiple redox reactions and thereby contribute multiple electrons to the measured current value (leading to signal amplification). Redox-active molecules diffuse in the cavity between the two electrodes and shuttle multiple electrons between the electrodes, leading to amplification of the measured electrochemical current. Signals from the redox active species are potentially amplified greater than 100 times, depending on factors, such as the stability of the redox species and the diffusion of the redox species out of the sensing region.

[0085] The disclosed device is suitable for use with both sandwich assays and competition assays. In the sandwich assay configuration, electron transfer increases when the sandwich is formed by an analyte of interest coming into contact with both of its binding partners, the first binding partner having comeAttny Docket No. STDU243535.601Client Ref No. 24-301 into contact with the target analyte in the reagent zone to which the sample has been introduced, and the second binding partner being bound to the surface of an electrode in the electrode chamber. Electron transfer (voltammetry) increases with concentration of the analyte(s) in a sandwich assay.

[0086] In a competition assay format, electron transfer decreases when a redox reporter labeled bait molecule is displaced from the single binding partner bound to the electrode. The single binding partner has greater affinity for the analyte of interest in the sample than it does for the redox reporter labeled bait molecule. Therefore, when the binding partner comes in contact with the target analyte, the redox reporter labeled molecule is displaced by the target analyte and electron transfer (voltammetry) decreases with increasing concentration of the analyte(s) in a competition assay. Thus, in some embodiments, the capture agent comprises a bait compound. The bait compound may have lower affinity for the capture agent as compared to the analyte. The bait compound may comprise a variant of the analyte that has been engineered to have a lower affinity for the capture agent. Alternatively, the bait compound may be a compound entirely separate and distinct from the target analyte. In some embodiments, the bait compound comprises the electrochemical label compound.

[0087] In some embodiments, the capture agent is configured to change confirmation upon binding to the analyte. Such a confirmation change may modify the electron transfer between the electrochemical label compound and working electrode, thus resulting in a change in the observed signal. In select embodiments, the capture agent comprises an aptamer switch comprising an electrochemical label compound. For example, an aptamer switch with a methylene blue (MB) redox reporter when in the absence of a target analyte is in an unfolded conformation which limits the electron transfer between the reporter and the electrode. Upon binding to the target analyte, the aptamer transitions to a folded conformation, increasing the proximity of the MB tag to the electrode surface and enhancing the rate of electron transfer. This change in electron transfer rate is indicative of the presence and concentration of the target metabolite and can be measured using voltammetry.

[0088] In some embodiments, the electrode chamber further comprises one or more non-target working electrodes functionalized with a control analyte or a capture agent specific for a control or nontarget analyte. The control or non-target analyte may include any analyte known to be present in the sample which provides confirmation or positive control for the assay. The control or non-target analyte may include analytes known to be associated with certain types of samples, providing additional verification of the sample source, type, disease status, and the like. The control or non-target analyteAttny Docket No. STDU243535.601Client Ref No. 24-301 may be an analyte of similar characteristics as the target analyte, e.g., similar size. Alternatively or in addition, the control or non-targct analyte may be an analyte other than the target analyte of interest. In some embodiments, the electrode chamber comprises at least one target working electrode functionalized with a capture agent specific for the target analyte and at least one non-target working electrode functionalized with capture agent specific for a non-target analyte.

[0089] The device may further comprise one or more components to process the sample. For example, the sample may comprise a filter to retain cells (e.g., blood cells) and other insoluble components of samples which may interfere with detection. Such components may be provided prior to the electrode chamber, e.g., in the sample receiving module, after the sample receiving module, after the solution carousel.

[0090] In some embodiments, the sample receiving module or solution carousel includes reagents for use in processing the sample. In some embodiments, a reagent is used to process a sample, e.g., to digest a solid sample with appropriate reagents (e.g., chemicals, acids, bases, and / or enzymes (e.g., proteases)). In some embodiments, reagents are used to extract or partially purify an analyte from a sample. In some embodiments, reagents are used to stabilize an analyte in a sample.

[0091] In some embodiments, the device may further comprise a blood filter. Blood separation membrane / filter can be made from variety of materials, such as glass fiber, polysulfone, nitrocellulose, hydrophilic poly ethersulfone (PES), or hydrophilic polytetrafluoroethylene (PTFE) bonded to polypropylene support.

[0092] Additional reagents for the assay may be provided to the sample solution prior to entry into the electrode chamber in the reagent zone. As used herein, the term “reagent zone” refers to a region or module where a reagent is provided. Generally, the reagent is provided in a form that is immobile in the dry state and mobile in the moist state. A reagent can be a specific binding partner, an enzyme, a substrate, indicators, components of a signal producing system, reducing agents, chelators, surfactants, etc., that contribute to the function of the assay. In some embodiments, the binding partner zone contains one or more binding partners that bind to one or more analytes of interest within a sample. The reagent zone may be made from any material which immobilizes the desired reagents while also allowing passage of the sample solution through to the electrode chamber. The reagent zone may comprise materials such as glass fiber filters, cellulose filters, and surface-treated (hydrophilic) polyester or polypropylene filters.Attny Docket No. STDU243535.601Client Ref No. 24-301

[0093] The electrode chamber may be followed by or may include a delay membrane or fluidic arrangement that focuses the sample and / or assay reagents to the surface of the electrodes for detection. In some embodiments, the delay membrane or fluidic arrangement prevents or delays solutions from passing through the electrode chamber or specifically channels the solutions to the surface of the electrodes, allowing sufficient amount of time for chemical and / or biological processes and reactions to occur between / among the various components of the diagnostics device and at the surface of the electrodes, including but not limited to: binding events between analytes of interest and their binding partner(s); redox cycling between / among electrodes, binding partners, and various chemical and biological compounds within sample and reagent solutions; and amplification of biological, chemical and / or electrochemical signals originating from binding events between analytes of interest and their binding partners. Delay membranes are generally membrane or filters with varying thickness or decreased pore sizes which allow adjustment for the delay time based on assay type and / or sample volumes. Fluidic arrangements can include any channels, capillaries, or other structures which direct fluid flow.

[0094] In some embodiments, the vertical flow assay device comprises a fluid retention module. The fluid retention module absorbs, retains, and sequesters the sample with the various assay solutions once they have passed through the electrode chamber for analysis. The fluid retention module may comprise a void volume necessary to retain the sample and the various assay solutions. In some embodiments, the fluid retention module protects device users from coming into contact with potentially hazardous biological agents and / or chemicals introduced into or present within sample and reagent solutions. The sequestration of solutions within the fluid retention module also serves to promote unimpeded communication and power supply to the integrated circuit.

[0095] The fluid retention module may comprise an adsorbent or absorbent material. The quantity of the adsorbent or absorbent material scales to the volume of the sample and the various assay solutions. Exemplary adsorbent or absorbent material include, but are not limited to, a sponge, fibrous material, polymeric materials, desiccants, or other liquid-expandable materials. The absorbent material may comprise a polysaccharide such as, but not limited to, cellulose, starch, chitin or chitosan; cotton; and / or glass fibers. Exemplary desiccants include natural or synthetic desiccants including anhydrous calcium sulfate (e.g., gypsum), aluminas, including activated aluminas (e.g., aluminum oxide or AI2O3), glass, silicas (e.g., SiCE, silica gels, Ascarite II® absorbents (e.g., carbon dioxide adsorbents including sodiumAttny Docket No. STDU243535.601Client Ref No. 24-301 hydroxide-coated silica), or diatomaceous silicas (e.g., Celite®, Celatom®, CAFA (Celite® Analytical Filter Aid))), a hygroscopic polymer and / or salt (e.g., CaCh, CaO, ZnCh, KOH, NaOH, CaH , CaSO4, and Na2SO4), molecular sieves or crystalline metal aluminosilicates, activated carbon, montmorillonites or montmorillonite clays (e.g., (Al2O3.4SiO2.xH2O)), and drying agents (e.g., barium oxide, boron oxide, calcium salts (e.g., calcium chloride, calcium oxide, calcium hydride), copper(II) sulfate, lithium aluminum hydride, lithium chloride, magnesium oxide, magnesium perchlorate, magnesium sulfate, phosphorus pentoxide, potassium hydroxide, sodium, sodium hydroxide, or sodium-potassium alloy).

[0096] The vertical flow assay device may comprise a sample receiving module, an electrode chamber, an integrated circuit, and one or more or all of: a solution carousel, a sample processing component (e.g., a blood filter), a reagent zone, a delay membrane or fluidic arrangement, and a fluid retention module. In some embodiments, the vertical flow assay device comprises a sample receiving module, a solution carousel, an electrode chamber, an integrated circuit, and one or more or all of: a sample processing component (e.g., a blood filter), a reagent zone, a delay membrane or fluidic arrangement, and a fluid retention module. In some embodiments, the vertical flow assay device comprises a sample receiving module, a solution carousel, an electrode chamber, an integrated circuit, a sample processing component (e.g., a blood filter), a reagent zone, a delay membrane or fluidic arrangement, and a fluid retention module.

[0097] The integrated circuit (IC) is intended to enable multiplexed detection of several analytes in the electrode chamber via direct electrical connection to the one or more electrode sets. The custom IC can be in form of bare-die, package or module (with embedded passive components or multiple die), comprising an electrochemical detection front-end (e.g., a potentiostat) that can be configured to perform any of established electrochemical methods of analysis. The electrode connections to the custom IC may be rigid or flexible, for example, including wires, leads, or printed circuit board (PCB) elements. The electrodes can be either part of the PCB (one-piece design) or can be made modular via use of FPC connectors. The IC may further comprise a temperature sensor and a pH sensor.

[0098] The integrated circuit or integrated circuit mounted on a printed circuit board (PCB) will comprise components suitable for operation of the sensing elements. The integrated circuit or integrated circuit mounted on a PCB may comprise, for example, one or more processors, controllers, voltage sources, or potentiostats for the application of controlled potentials to the electrode sets in selected waveforms. Electrochemical sensors may be interrogated by methods known in the art and asAttny Docket No. STDU243535.601Client Ref No. 24-301 appropriate for the selected sensor configuration. In one embodiment, the sensors are configured for pulsed voltammetric measurements wherein a scries of potentials is applied in a pulsed waveform to the functionalized sensing electrode of the sensor, and wherein one or more measurements of faradic current are assessed in each cycle. In one embodiment, the sensor is interrogated by square-wave voltammetry (SWV). The integrated circuit, by its electrical connection to the electrode sets, will further comprise elements for measuring and recording voltages, faradaic currents, or other outputs from the electrode sets and memory and / or data storage for recording sensor outputs.

[0099] The integrated circuit or integrated circuit mounted on a PCB further comprises a power molecule. In some embodiments the power module is a wired power module. In some embodiments, the power module is a wireless power module. The wireless power module enables the custom chip to be powered with a user device in proximity to the test device. In some embodiments the power module utilizes or comprises a battery-based power source.

[0100] The integrated circuit or integrated circuit mounted on a PCB further comprises a communication module. The communication module may be a wireless communication component or a wired communication component. In some embodiments, the communication module comprises a short- range wireless communication. Exemplary short-range wireless communication modules include nearfield magnetic induction (NFMI) modules (e.g. NFC), Bluetooth communication modules, Zigbee / Z- Wave / Thread (802.15.4), Ultra Wideband (UWB), EnOcean, and Wi-Fi communication modules. The communication module may further comprise an antenna and / or an antenna interface.

[0101] In some embodiments, the power module and the communication module are integrated into a single component. In some embodiments, the single component is a near-field magnetic induction (NFMI) module, e.g., near-field communication (NFC) module, operably connected to the assay device to support wireless power delivery and wireless communication. The wireless data-communication module (e.g., NFMI module) provides the capability for data communication or transfer between the chip and a user device (e.g., a smart phone, tablet computer, smart wearable device, portable laptop computer, voice command device, or other mobile computing device).

[0102] Integrated circuits or integrated circuits mounted on a PCB may be manufactured using standard CMOS semiconductor technology. Printed circuit boards can be manufactured on rigid (FR4) or flexible (polyimide) substrates.Attny Docket No. STDU243535.601Client Ref No. 24-301

[0103] Any or all of the components of the device may be contained within a vertical flow cylinder. For example, any or all of the components may be integral to the vertical flow cylinder. In some embodiments any two or more of: the solution carousel, reagent zone, electrode chamber, blood filter, delay membrane / fluidic arrangement, and the fluid retention module are provided as a single unit. In some embodiments, the integrated circuit is separate from the device. In some embodiments, the integrated circuit is integral to the device. See FIG. 1 for example.

[0104] In such embodiments when the integrated circuit or integrated circuit mounted on a PCB is provided separate to the device, the integrated circuit may include a means of attachment to the vertical flow cylinder or the rest of the components of the device, which optionally comprise an opposing attachment means. Exemplary means of attachment include, but are not limited to, compressive gaskets, screw mechanisms, snap couplings, compressive latching mechanisms, compressive spring mechanisms, bayonet or pin / slot couplings, ring-lock couplings, sliding mechanisms, compressive clips, epoxy, and combinations thereof. Assembly may be performed by the manufacturer or at the point of care or prior to use by aligning the integrated circuit with the vertical flow cylinder or the rest of the components of the device and may involve reversibly affixing the integrated circuit to the vertical flow cylinder or the rest of the components of the device using interlocking surfaces (e.g., snaps), and the like.

[0105] In some embodiments, the assay device and its component parts described herein are constructed using methods of construction known in the mechanical arts or medical device construction arts. The materials from which the assay device or components thereof are manufactured are varied, in some instances as indicated herein. In some embodiments, the assay device or a component thereof comprises a material that is metal, silicon, glass, ceramic, plastic, and synthetic and natural polymers and combinations and mixtures thereof. In some embodiments, the assay device or a component thereof comprises a polypropylene and / or high impact polystyrene composition using an appropriate manufacturing method (e.g., pressure injection molding, machining, three-dimensional printing, etc.). In some embodiments, the assay device or a component thereof is constructed using other suitable methods of manufacturing such as milling, casting, blowing, spinning, and other methods known in the mechanical ails and medical device construction.3. Systems or Kits

[0106] In some embodiments, a system comprises one or more assay devices as described herein.Attny Docket No. STDU243535.601Client Ref No. 24-301

[0107] In some embodiments, the system further comprise a mobile user device configured to receive and send communications from the communication module of the assay device. The mobile user device may further be configured to provide power to the assay device. Thus, the mobile user device comprises any computing device which has an integrated power and communication components which are compatible with the communication module and power module of the disclosed assay device. In some embodiments, the mobile user device comprises a small phone, tablet computer, smart wearable device, portable laptop computer, voice command device, or other mobile computing device.

[0108] In some embodiments, the mobile computing device comprises software to send and receive communications from the device. For example, the software components may be provided via an application service provider (ASP) (e.g., are accessed by users within a web-based platform via a web browser across the internet; is bundled into a network-type appliance and run within an institution or an intranet; is provided as a software package and used as a stand-alone system; or is provided as downloadable software to a user device). The software components may be built on a system that comprises appropriate privacy and security features to comply with legal regulations regarding sharing and transferring of patient data and medical information. The software may allow receiving, processing, and transmitting the result or indicator to and from laboratories conducting the assays, information providers, medical personal, and subjects. The result and / or indicator may be displayed by any suitable method.

[0109] The system may further comprise a sample, e.g., a biological sample, an environmental sample, an industrial sample, or a research sample. The system may further comprise one or more reagents for processing the sample prior to analysis, e.g., prior to delivery of the sample to the device via the sample receiving module .

[0110] The disclosure also provides kits comprising the devices or systems disclosed herein. Individual member components of the kits may be physically packaged together or separately.

[0111] The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kit. The materials may include any combination of the following: background information, list of components, brief or detailed methods for using the system, trouble-shooting, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic formAttny Docket No. STDU243535.601Client Ref No. 24-301 which may be supplied on a computer readable memory device or downloaded from an internet website, or as a recorded presentation.4. Methods of Use

[0112] The disclosure also provides methods for detecting one or more target analytes in a sample using the assay devices or systems as described herein. Detecting may comprise determining the presence, absence, concentration, and / or amount of one or more analytes in the sample.

[0113] Analytes include, but are not limited to, small molecules, amino acids, peptides, proteins, nucleic acids, lipids, carbohydrates, and minerals. In some embodiments, the analyte is a prognostic or diagnostic analyte for a patient’s health and / or well-being. Analytes include biomarkers, which are measurable indicators of some biological state or condition. The analyte(s) can be any molecule of interest for diagnosis, screening, disease staging, forensic analysis, pregnancy testing, drug testing, and other reasons. The analyte can include a protein, a peptide, a polypeptide, an amino acid, a hormone, a steroid, a vitamin, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, a bacterium, a virus, and metabolites of or antibodies to any of the above substances.

[0114] The methods comprise providing a sample to the device through the sample receiving module, optionally, delivering and or puncturing the sealed solution carousel, to deliver the sample and any reagents or solutions to the electrode chamber, and monitoring a change in the electrochemical detection at the one or more electrodes or electrode sets. In some embodiments, the methods comprise simultaneously or sequentially in any order providing a sample to the device through the sample receiving module and providing one or more assay solutions and / or reagents to the sample receiving module, thereby delivering the sample and any reagents or solutions to the electrode chamber, and monitoring an electrochemical change one or more electrodes or electrode sets. In some embodiments, the methods comprise measuring changes in current or potential of the working electrode(s) in the electrode chamber. For example, the changes in current may be monitored with a user device in communication with the assay device. In some embodiments, the methods further comprise determining the concentration of the analyte in the sample based on the changes in current or potential of working electrode.

[0115] The assay device and related methods find use in a variety of settings. In some embodiments, the technology finds use in a home, medical clinic, emergency room, or doctor’s office. In someAttny Docket No. STDU243535.601Client Ref No. 24-301 embodiments, the assay device finds use in remote locations or in areas of limited resources or technology. In some embodiments, the technology finds use in an environmental setting, testing air, water, or soil samples. In some embodiments, the technology finds use in an industrial setting (e.g., for detection of contaminants or by-products in the desired product). In some embodiments, the technology finds use in a research or laboratory setting.

[0116] In some embodiments, the assay device is configured for a person who is a sample collector to collect a sample. In some embodiments, the sample is collected from a person or subject who is a sample donor. In some embodiments, the sample is collected from an environmental or other nonsubject source (e.g., industrial or research sample). Thus, in some embodiments, the user of the device is a sample collector. In some embodiments, the sample collector is the same individual as the sample donor.5. Examples

[0117] The following are examples of the present invention and are not to be construed as limiting.Example 1

[0118] Electrodes were functionalized with antibodies to procalcitonin (PCT) as shown in FIG. 3A. A self-assembling monolayer comprised of thiols was formed on the electrode surface by Au-S covalent bonding followed by activation and attachment of the antibodies.

[0119] Antibody Assessment and Screening

[0120] Screening began with indirect ELISA to quantitatively rank available anti-PCT antibodies by their binding efficiency to the PCT antigen. In this comparative analysis, antibodies labeled as #6 (BM- PCT-02) and #3 (Biospacific A91066508P) demonstrated the highest signal intensity, indicative of superior binding strength (FIG. 4). These antibodies advanced to integration into the electrochemical PCT assay for further evaluation.

[0121] EC50 Determination and Application-Relevant Validation

[0122] ELISA experiments focused on quantifying each antibody’s EC50, the effective concentration of antibody necessary to generate half-maximal binding. Antibody #6 (BM-PCT-02) and antibody #3 (Biospacific A91066508P) yielded EC50 values of 160pM (24ng / mL) and 572pM (85.8ng / mL), respectively (FIG. 5).

[0123] EDC / S-NHS Coupling for Antibody ImmobilizationAttny Docket No. STDU243535.601Client Ref No. 24-301

[0124] Covalent immobilization of antibodies onto the SAM was achieved via carbodiimide chemistry. The combination of EDC (N-cthyl-N’-(3-dimcthylaminopropyl)carbodiimidc) and S-NHS (Sulfo-N-hydroxysulfosuccinimide) enables activation of the carboxyl groups on the SAM for subsequent amide bond formation with antibody amines.

[0125] Recognizing that the O-acylisourea intermediate formed when EDC reacts with a carboxylic acid is unstable in aqueous environments, undergoing rapid hydrolysis, S-NHS was added in excess to convert this transient species into a more hydrolytically stable NHS ester.

[0126] A range of EDC / Sulfo-NHS concentrations were tested to identify a saturation point beyond which no further increase in surface activation was observed. This was assessed electrochemically by comparing the CV response of surfaces post-activation to the baseline SAM (pre-activation). The CV curves plateaued at activation towards 200 mM EDC and 400 mM S-NHS (FIG. 6), which was selected as the condition for consistent antibody immobilization.

[0127] Square Wave Voltammetry (SWV) PCT Assay

[0128] The PCT assay was conducted as in FIG. 3B. PCT molecules in the diluted plasma bind specifically to the immobilized antibodies on the sensor surface (FIG. 3B, step 2). After an incubation period, the sensor is washed (FIG. 3B, step 3) and a redox solution containing ferrocyanide / ferricyanide is added (FIG. 3B, step 4). This redox mediator enables sensitive electrochemical measurement by facilitating electron exchange between the electrode and the solution. When PCT is captured by surfacebound antibodies, it partially occludes the electrode interface, thereby impeding electron transfer from the redox couple. This results in a reduction of measured current, which is inversely proportional to the analyte’s concentration: higher levels of PCT generate a lower electrochemical signal (“signal-off’ response).

[0129] Square Wave Voltammetry results indicated dose-dependent changes in peak current (FIG. 7). The SWV signal decreases (signal-off) with increasing PCT concentration on electrodes functionalized with 20 pg of antibody #6 (BM-PCT-02) and blocked with 1% BSA.

[0130] It is understood that the foregoing detailed description are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.Attny Docket No. STDU243535.601Client Ref No. 24-301

[0131] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit and scope thereof.

Claims

Attny Docket No. STDU243535.601Client Ref No. 24-301CLAIMSWhat is claimed is:

1. A vertical flow assay device for detection of one or more target analytes in a sample, comprising: a sample receiving module; an electrode chamber comprising one or more working electrodes functionalized with a capture agent specific for a target analyte, wherein the electrode chamber is in fluid communication with the sample receiving device; and an integrated circuit electrically connected with the one or more working electrodes, wherein the integrated circuit comprises a power module and a communication module.2 The vertical flow assay device of claim 1, wherein the sample receiving module has a proximal end to receive a sample and a distal end configured to deliver samples to the electrode chamber.3 The vertical flow assay device of claim 1 or 2, wherein the vertical flow assay device comprises a solution carousel.4 The vertical flow assay device of claim 3, wherein the distal end of the sample receiving module is configured to puncture sealed compartments of the solution carousel.5 The vertical flow assay device of claim 3 or 4, wherein the electrode chamber is in fluid communication with the solution carousel.6 The vertical flow assay device of any of claims 3-5, wherein the solution carousel comprises one or more chambers, each chamber comprising individual diagnostic assay solutions.7 The vertical flow assay device of any of claims 1-6, wherein the electrode chamber comprises two or more working electrodes, each functionalized with a capture agent specific for a different target analyte.8 The vertical flow assay device of any of claims 1-7, wherein the capture agent comprises a specific inding partner to the target analyte.9 The vertical flow assay device of any of claims 1-8, wherein the capture agent comprises a nucleic acid, a protein, a peptide, a small molecule, or a combination thereof.10 The vertical flow assay device of any of claims 1-9, wherein the target analyte and capture agent are each one half of an enzyme-substrate pair.Attny Docket No. STDU243535.601Client Ref No. 24-30111 . The vertical flow assay device of claim 10, wherein the enzyme is a redox enzyme.

12. The vertical flow assay device of any of claims 1-11, wherein the capture agent comprises an electrochemical label compound.

13. The vertical flow assay device of any of claims 1-12, wherein the capture agent comprises a bait compound.

14. The vertical flow assay device of claim 13, wherein the bait compound comprises the electrochemical label compound.

15. The vertical flow assay device of claim 13 or 14, wherein the bait compound has lower affinity for the capture agent as compared to the target analyte.

16. The vertical flow assay device of any of claims 1-5, wherein the capture agent is configured to change confirmation upon binding to the target analyte and modify the electron transfer between the electrochemical label compound and working electrode.

17. The vertical flow assay device of claim 16, wherein the capture agent comprises an aptamer switch comprising an electrochemical label compound.

18. The vertical flow assay device of any of claims 12-17, wherein the electrochemical label compound comprises a redox reporter.

19. The vertical flow assay device of any of claims 1-18, wherein the working electrode is part of an electrode set further comprises one or more additional working electrodes, a reference electrode and / or a counter (auxiliary) electrode for a target analyte.

20. The vertical flow assay device of any of claims 1-19, wherein the electrode chamber comprises one or more non-target working electrodes functionalized with a control analyte or a capture agent specific for a control or non-target analyte.

21. The vertical flow assay device of any of claims 1-20, wherein the vertical flow assay device comprises a fluid retention module.

22. The vertical flow assay device of any of claims 1-21, wherein the fluid retention module comprises a sponge or desiccant to retain the sample and reagent solutions after passing through the electrode chamber.Attny Docket No. STDU243535.601Client Ref No. 24-30123. The vertical flow assay device of any of claims 1-22, wherein the integrated circuit comprises one or more of an electrochemical analog front end, a temperature sensor, and / or a pH sensor.

24. The vertical flow assay device of any of claims 1-23, wherein the power module comprises a wired power module or a wireless power module.

25. The vertical flow assay device of any of claims 1-24, wherein the power module comprises a battery-based power source.

26. The vertical flow assay device of any of claims 1-25, wherein the communication module comprises a wireless communication component or a wired communication component.

27. The vertical flow assay device of any of claims 1-26, wherein the communication module comprises a short-range wireless communication.

28. The vertical flow assay device of claim 27, wherein the short-range wireless communication module is selected from the group consisting of a near-field magnetic induction (NFMI) module, a Bluetooth communication module, Zigbee / Z-Wave / Thread (802.15.4), Ultra Wideband (UWB), EnOcean, and a Wi-Fi communication module.

29. The vertical flow assay device of any of claims 1-28, wherein the communication module and the power module are integrated into a single component.

30. The vertical flow assay device of claim 29, wherein the single component facilitates wireless power delivery and wireless communication.

31. The vertical flow assay device of claim 29 or 30, wherein the single component is a near-field magnetic induction (NFMI) module operably connected to the assay device to support wireless power delivery and wireless communication.

32. The vertical flow assay device of any of claims 1-31, further comprising a delay membrane or fluidic arrangement to hold samples in the electrode chamber for detection.

33. The vertical flow assay device of any of claims 1-32, further comprising a blood filter.

34. The vertical flow assay device of any of claims 1-33, further comprising a reagent zone.

35. A system for analyte detection comprising: an assay device of any of claims 1-34; andAttny Docket No. STDU243535.601Client Ref No. 24-301 a mobile user device configured to receive and send communications from the communication module.

36. The system of claim 35, wherein the mobile user device comprises a smart phone, tablet computer, smart wearable device, portable laptop computer, voice command device, or other mobile computing device.

37. The system of claim 36, wherein the mobile user device sends and / or receives communications to the assay device using an application.

38. The system of any of claims 35-37, wherein the system comprises two or more assay devices.

39. The system of any of claims 35-38, further comprising a sample.

40. The system of claim 39, wherein the sample is a biological sample, an environmental sample, an industrial sample, or a research sample.

41. A method for detecting one or more target analyte(s) in a sample, comprising loading the sample in the sample application region of an assay device of any of claims 1-34 or a system of any of claims 35-40; and measuring changes in current or potential of working electrode.

42. The method of claim 41, further comprising determining the concentration of the analyte in the sample based on the changes in current or potential of working electrode.

43. The method of claim 42, wherein the sample is a biological sample, an environmental sample, an industrial sample, or a research sample.

44. The method of any of claims 41-43, wherein each of the one or more target analytes is individually selected from the group consisting of: a small molecule, an amino acid, a peptide, a protein, a nucleic acid, a lipid, and a carbohydrate.

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