WO2023193118A1 - Aptamers for porcine epidemic diarrhea virus - Google Patents

Abstract

Provided are aptamers for detecting porcine epidemic diarrhea virus (PEDv). Also provided are biosensors comprising these aptamers for detecting PEDv in a subject in need thereof, including a reagent-free, signal-on, and low background electrochemical aptamer-based sensor for rapid PEDv sensing. Also provided are methods for detecting PEDv using the aptamers and biosensors disclosed herein.

Description

APTAMERS FOR PORCINE EPIDEMIC DIARRHEA VIRUS

RELATED APPLICATION

[0001 ] This disclosure claims benefit and priority of United States Provisional Patent Application serial no. 63/328,539 filed April 7, 2022, which is incorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “3244- P68071PC00_SequenceListing.xml” (71,905 bytes), was created on April 6, 2023, is filed herewith by electronic submission and is incorporated by reference herein.

FIELD

[0003] The present disclosure relates to nucleic acid aptamers, and in particular, to aptamers capable of binding porcine epidemic diarrhea virus (PEDv) or PEDv nucleocapsid proteins and methods of making and using such aptamers.

BACKGROUND

[0004] It is widely disseminated that human, animal, and environmental health is tightly interconnected. Pathogens such as coronaviruses (CoVs) cause severe respiratory, enteric, and systemic infections in human and animal hosts,^1,2 jeopardize animal husbandry and food supply,³ and have potentially devastating environmental and global biodiversity consequences.⁴ Furthermore, viral outbreaks in livestock pose continuous threats to human health, carrying risks of human zoonotic infections or the emergence of transboundary viral strains with pandemic potential, as evidenced by the recent COVID-19 pandemic.⁵ In fact, more than 70% of all emerging infections are believed to have animal origins.⁶ Additionally, infectious animal diseases pose a clear economic threat, affecting local food production, national economies and global trade. In 2013, the introduction of porcine epidemic diarrhea virus (PEDv) - an emerging and highly transmittable CoV - devastated 10% of the US commercial swine population in 31 states within a span of 18 months, leading to economic losses of > US $400 million.²

[0005] Although biosecurity measures in the commercial swine industry is comprehensive in many countries including Canada and United States, the rapid and recurrent spread of PEDv demonstrates the vulnerability of the farming industry to emerging pathogens. The current biosecurity surveillance measures for animal diseases such as PEDv rely on polymerase chain reaction (PCR) testing, which requires analysis at centralized laboratory, specialized equipment and technical expertise, and transport and turnaround times between sample submission and diagnosis spanning 2 - 4 days.⁷ The lack of rapid and on-farm testing allows emerging animal pathogens to spread, increasing morbidity and mortality rates in animals.⁸ Immunoassays are popular alternatives to PCR for PEDv detection, which reduce sample-to-result time and assay complexity by eliminating the need for reverse transcription and target amplification. These assays rely on the use of antibodies as bio-recognition elements and are often integrated into engineered detection platforms or devices for rapid testing such as lateral flow assays (LFAs).⁹ " enzyme-linked immunosorbent assays (ELISA), ^{12 17} and electrochemical assays.¹⁸'²⁰ Although rapid, these methods, all based on antibody-based sandwich assays, either require multi-step processing such as washing, labeling, or addition of reagents (ELISA and electrochemical assays) or suffer from low clinical sensitivity and specificity partly stemming from the poor stability and cross-reactivity of the antibodies (LFAs).⁷ It is thus of utmost importance to develop rapid, simple and reagent-less on-farm tests capable of precise and reliable detection of PEDv.

[0006] DNA aptamers, a class of functional nucleic acids selected in vitro for specific target binding, offer several key advantages over antibodies for the development of rapid tests such as their small size, high chemical and thermal stability, easy and precise modification, compatibility with DNA machines and consequently reagent-less sensing, scalable production and minimal batch-to-batch variation.²¹ In addition, it has been shown that aptamers can be used to directly detect targets present in clinical samples,^22,23 making these molecular recognition elements increasingly important for developing rapid diagnostic devices. In the past few decades, many important DNA aptamers have been selected for rapid diagnosis of viral diseases including human immunodeficiency virus (HIV), influenza, porcine reproductive and respiratory syndrome virus (PRRSv), African swine fever virus (ASFv) and coronaviruses like SARS-CoV²⁴'²⁶ and MERS.²⁷ However, there are no aptamers currently available for detecting PEDv.

[0007] Existing aptamer-based electrochemical sensors typically employ target-induced structure switching.²⁸'³⁰ These assays offer a breakthrough because they enable reagent-less sensing; however, the integration of the redox label directly on the structure-switching aptamer that is immobilized on the sensing electrode often results in large background signals even before target capture due to the reversibility of the system²⁸ and/or the flexibility of the DNA aptamer.³¹'³³ Aptamers relying on the displacement of a labeled single-stranded DNA strand for signal transduction offer an alternative strategy;^28,34'³⁷ however, such assays typically operate in a signal-off configuration^{36, 37} or carry a large background current in the few reported signal-on designs.^{35, 38} The abovementioned assay designs are prone to introducing errors in heterogeneous clinical samples that generate varying background signals.

[0008] The background herein is included solely to explain the context of the disclosure. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge as of the priority date.

SUMMARY

[0009] The present disclosure describes the development of PEDv-specific aptamers and their use in a reagent-free, signal-on, and low background electrochemical aptamer-based sensor for rapid PEDv sensing, which combines the advantages of aptamers with the ultra-sensitivity of electrochemical readout. The sensor integrated these PEDv-specific aptamers into a dual-electrode electrochemical chip (DEE-Chip) aptasensor, which is designed to house the aptamer bound to a DNA barcode on one electrode and detect the released barcode on another electrode. The design enables signal-on sensing with almost no redox background. Also disclosed are mass transport enhancement strategies used to overcome slow kinetics, such as reduction of interelectrode spacing and active field mediated transport.

[0010] Accordingly, in an aspect of the present disclosure, there is provided an aptamer comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-58, or a functional fragment and/or functional variant thereof, that binds to porcine epidemic diarrhea virus (PEDv) nucleocapsid protein.

[001 1] In some embodiments, the aptamer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5 and 51-58.

[0012] In some embodiments, the aptamer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 and 51-58. [0013] In some embodiments, the aptamer comprises a nucleotide sequence of SEQ ID NO: 1 or 53.

[0014] In some embodiments, the aptamer comprises a nucleotide sequence of SEQ ID NO: 53.

[0015] In some embodiments, the aptamer consists of a nucleotide sequence of SEQ ID NO: 53.

[0016] In some embodiments, the aptamer binds to the PEDv nucleocapsid protein with at least nanomolar affinity.

[0017] According to another aspect of the disclosure, there is provided an aptamer probe, comprising the aptamer disclosed herein and a detectable label.

[0018] In some embodiments, the detectable label is a fluorescence moiety, a colorimetric moiety, an electrochemiluminescent moiety, a photoelectrochemical moiety or an electrochemical moiety.

[0019] In some embodiments, the detectable label is an electrochemical moiety.

[0020] In some embodiments, the electrochemical moiety is a redox species.

[0021] In some embodiments, the redox species comprises methylene blue or ferrocene.

[0022] In some embodiments, the aptamer probe further comprises a reporter moiety coupled to the detectable label.

[0023] In some embodiments, the reporter moiety comprises a nucleotide sequence complementary to at least a portion of the aptamer.

[0024] In some embodiments, the reporter moiety comprises a nucleotide sequence consisting of the group selected from SEQ ID NOs: 64-69.

[0025] In some embodiments, the reporter moiety comprises a nucleotide sequence of SEQ ID NO: 66 or 67.

[0026] In some embodiments, the reporter moiety is releasable from the aptamer in the presence of PEDv nucleocapsid protein. [0027] According to another aspect of the disclosure, there is provided a biosensor for detecting PEDv nucleocapsid protein comprising the aptamer disclosed herein or the aptamer probe disclosed herein functionalized on and/or in a material.

[0028] In some embodiments, the biosensor further comprises: a) a first working electrode, wherein the aptamer probe disclosed herein is functionalized on the first working electrode; b) a second working electrode configured in proximity to the first working electrode; c) a capture probe functionalized on the second working electrode; and d) a counter electrode; wherein each working electrode is configured to provide a change in signal if the PEDv nucleocapsid protein is present.

[0029] In some embodiments, the aptamer probe is functionalized on the first working electrode via chemical bonding, an intermediate linker, or physical adsorption.

[0030] In some embodiments, the capture probe is functionalized on the second working electrode via chemical bonding, an intermediate linker, or physical adsorption.

[0031 ] In some embodiments, the chemical bonding occurs via thiol and/or gold chemistry.

[0032] In some embodiments, the capture probe is for recognizing and coupling to the reporter moiety of the aptamer probe.

[0033] In some embodiments, the capture probe comprises a nucleotide sequence of SEQ ID NO: 71 or 72.

[0034] In some embodiments, the working electrode comprises a conductive material, semi-conductive material, or a combination thereof.

[0035] In some embodiments, the working electrode comprises metal, metal alloy, metal oxide, superconductor, semi-conductor, carbon-based material, conductive polymer, or combinations thereof.

[0036] In some embodiments, the working electrode comprises metal.

[0037] In some embodiments, the metal is gold. [0038] In some embodiments, the biosensor further comprises a reference electrode.

[0039] In some embodiments, the biosensor further comprises a blocking species on the first working electrode and/or second working electrode.

[0040] In some embodiments, the biosensor is on an electrochemical chip.

[0041 ] In some embodiments, the first working electrode is configured to provide a decrease in the signal and the second working electrode is configured to provide an increase in the signal in the presence of PEDv nucleocapsid protein.

[0042] In some embodiments, the change in signal comprises a change in current, potential or impedance.

[0043] In some embodiments, the change in signal is a change in current.

[0044] According to another aspect of the disclosure, there is provided a method for detecting the presence of PEDv nucleocapsid protein in a sample, comprising: a) obtaining a sample from a subject; b) combining the sample with a liquid in a sample container to form a mixture; c) exposing the mixture to the biosensor disclosed herein; whereby the reporter moiety of the aptamer probe functionalized on the first working electrode is delocalized from the first working electrode to the second working electrode upon binding of the aptamer of the aptamer probe functionalized on the first working electrode to the PEDv nucleocapsid protein; and d) measuring a change in signal at the first and/or second working electrode, wherein a change in signal is produced if the PEDv nucleocapsid protein is present in the sample.

[0045] In some embodiments, the method further comprises incubating the mixture at about 60 °C for about 25 minutes before step c).

[0046] In some embodiments, the biosensor comprises a reference electrode and the method further comprises applying a positive potential bias across the first and/or second working electrode before and/or during step d). [0047] In some embodiments, the liquid is a buffer.

[0048] In some embodiments, the mixture is exposed to the biosensor under conditions to delocalize the reporter moiety from the first working electrode to the second working electrode.

[0049] In some embodiments, the mixture is exposed to the biosensor for about 60 minutes before step d).

[0050] In some embodiments, the change in signal from the first working electrode is a decrease in signal and the change in signal from the second working electrode is an increase in signal.

[0051 ] In some embodiments, the change in signal comprises a change in current, potential or impedance.

[0052] In some embodiments, the change in signal is a change in current.

[0053] In some embodiments, the sample comprises saliva.

[0054] In some embodiments, the method detects PEDv infection in the subject.

[0055] In some embodiments, the subj ect is a porcine animal.

[0056] According to another aspect of the disclosure, there is provided a kit for detecting PEDv nucleocapsid protein, wherein the kit comprises the biosensor disclosed herein or components required for the method disclosed herein, and instructions for use of the kit.

[0057] In some embodiments, the kit further comprises at least one collection apparatus, at least one sample container, a buffer, and instructions for use.

[0058] In accordance with an aspect, there is provided a DNA aptamer that binds to a PEDv target analyte, wherein the aptamer comprises a sequence selected from the group consisting of sequences PEA1 - PEA50 (SEQ ID NOs: 1-50), PEA1-1 (SEQ ID NOs: 51), PEA1-2 (SEQ ID NOs: 52), PEA1-3 (SEQ ID NOs: 53), PEA1-4 (SEQ ID NOs: 54), PEA1-5 (SEQ ID NOs: 55), PEA1-6 (SEQ ID NOs: 56), PEA1-7 (SEQ ID NOs: 57), PEA1-8 (SEQ ID NOs: 58), or a functional fragment and/or functional variant thereof.

[0059] In some embodiments, the functional variant thereof is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or more identical to the nucleotide sequence as shown in SEQ ID NOs: 1-58.

[0060] In some embodiments, the aptamer is coupled to a DNA biobarcode moiety. In some embodiments, the DNA biobarcode moiety comprises a redox species. In some embodiments, the redox species comprises methylene blue or ferrocene.

[0061 ] In some embodiments, the DNA biobarcode moiety comprises complementary nucleic acid sequences to any of the sequences selected from the group consisting of sequences as shown in SEQ ID NOs: 1-58 or a functional fragment and/or functional variant thereof.

[0062] In some embodiments, the DNA biobarcode moiety hybridizes to the aptamer. In some embodiments, the DNA biobarcode moiety is releasable and becomes delocalized in the presence of the PEDv target analyte.

[0063] In some embodiments, the aptamer changes its structure and/or conformation upon binding to the PEDv target analyte.

[0064] In some embodiments, detection of the releasable DNA biobarcode moiety results in an electrochemical signal. In some embodiments, the detectable signal comprises a fluorescent, a colorimetric, electrochemiluminescence, electrochemical or photoelectrochemical signal.

[0065] In some embodiments, the PEDv target is a protein. In some embodiments, the protein is from PEDv, or the nucleocapsid protein of PEDv.

[0066] In accordance with another aspect, there is provided a biosensor for detecting a PEDv target analyte, such as PEDv nucleocapsid protein, comprising at least two working electrodes; wherein a first working electrode comprises the aptamer disclosed herein, and a second working electrode comprises a capture probe specific to a DNA biobarcode moiety sequence. In some embodiments, the DNA aptamer disclosed herein is attached directly or indirectly to the first working electrode. In some embodiments, the DNA biobarcode moiety is configured to be delocalized in the presence of the PEDv target analyte and configured to diffuse from the first working electrode to the second working electrode. In some embodiments, the DNA biobarcode moiety is capturable on the second working electrode, wherein a change in signal is produceable at each of the first working electrode and the second working electrode. [0067] In some embodiments, a positive potential bias is applied across at least one of the working electrodes.

[0068] In accordance with another aspect, there is provided a method of detecting the presence of PEDv in a test sample, comprising: a) contacting the sample with the biosensor as disclosed herein; and b) detecting a signal, wherein detecting a signal indicates the presence of PEDv in the test sample and lack of a signal indicates that PEDv is not present.

[0069] In some embodiments, the sample comprises at least one of a tissue sample, a cell culture isolate, blood, plasma, serum, cerebrospinal fluid, lymph, tears, urine, saliva or mucus.

[0070] In accordance with another aspect, there is provided a method for detecting the presence of PEDv in a subject, comprising: a) testing a sample from the subject for the presence of PEDv by the method as disclosed herein; and b) reading the signal obtained from the biosensor disclosed herein to determine presence of PEDv.

[0071 ] In accordance with another aspect, there is provided a kit for detecting PEDv, wherein the kit comprises the biosensor as disclosed herein and instructions for use of the kit.

[0072] In some embodiments, the biosensor is used for animal testing.

[0073] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the disclosure, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.

DRAWINGS

[0074] Certain embodiments of the disclosure will now be described in greater detail with reference to the attached drawings in which:

[0075] FIGURE 1 shows aptamer selection and characterization in exemplary embodiments of the disclosure: (a) depiction of the aptamer selection process; (b) full- length aptamer PEA1 (SEQ ID NO: 1) secondary structure; (c) truncated PEA1-3 (SEQ ID NO: 53) secondary structure; (d) binding curves and resultant affinity values (Ka, nM) extracted from the aptamers binding to the nucleocapsid protein of PEDv (N- PEDv; SEQ ID NO: 73); (e) selectivity tests demonstrating PEA1-3 (SEQ ID NO: 53) binding of N-PEDv in comparison with the control proteins bovine serum albumin (BSA), thrombin and ribonuclease (RNase) - a concentration of 5 nM of each protein was used (BA: bound aptamer; UA: unbound aptamer).

[0076] FIGURE 2 shows the secondary structures and affinities (Kd) of full- length aptamer PEA1 (SEQ ID NO: 1) and truncated derivatives, PEA1-1 to PEA1-8 (SEQ ID NOs: 51-58), in exemplary embodiments of the disclosure.

[0077] FIGURE 3 shows the EMSA result of aptamer PEA1-3 (SEQ ID NO: 1) binding with N-PEDv in exemplary embodiments of the disclosure.

[0078] FIGURE 4 shows selectivity tests of PEA1-3 (SEQ ID NO: 53) binding the target N-PEDv in comparison with Spike (S) and N protein of S ARS-CoV-2 (using 10 nM of each protein) in exemplary embodiments of the disclosure.

[0079] FIGURE 5 shows a schematic illustration of the operating principles and the components of the DEE-Chip assay in exemplary embodiments of the disclosure: (a) sample-to-test workflow: liquid samples comprising N-PEDv are introduced to the DEE-Chip using a dropper; (b) signal changes: (left) prior to sample introduction, a methylene blue (MB) redox peak is exhibited by Ei while an absence of this peak is exhibited on E2; (right) following N-PEDv introduction, target induced displacement of the redox barcode on Ei and subsequent capture on E2 yields a decrease in the MB peak on Ei while a signal increase is seen on E2; (c) molecular operation: (left) aptamer probes comprised of redox barcodes are immobilized on Ei while single-stranded DNA (ssDNA) capture probes are anchored on E2; (middle) in the presence of the target protein (N-PEDv), these redox barcodes are released from Ei and diffuse towards E2; (right) following diffusion, the redox barcodes are subsequently captured on E2, yielding a decrease in the redox peak on Ei and a signal increase on E2.

[0080] FIGURE 6 shows biobarcode optimization in exemplary embodiments of the disclosure: (a) sequences of aptamer PEA1-3 (SEQ ID NO: 53) and five biobarcodes (SEQ ID NOs: 64-66, 68, 69) were created to form partial duplexes with PEA1-3 (SEQ ID NO: 53), with the complementary base pairs underlined; (b) the fraction of released barcodes from the duplexes before and after the addition of 10 nM of N-PEDv protein - 2 nM PEA1-3 (SEQ ID NO: 53) and each 2 nM radiolabeled barcode (SEQ ID NOs: 64-66, 68, 69) were annealed to establish the duplexes, and the released barcodes were measured using 8% native polyacrylamide gel electrophoresis.

[0081 ] FIGURE 7 shows SEM micrographs of the (a) Ei and (b) E2 sensing electrodes in exemplary embodiments of the disclosure.

[0082] FIGURE 8 shows DEE-Chip validation using three independent chips comprising Ei and E2 electrodes in exemplary embodiments of the disclosure: (a) (i) cyclic voltammetry (CV) scans of bare gold electrodes in sulphuric acid post acid cleaning to assess reproducibility and surface area of each Ei and E2 pair on the three chips; (ii) extracted reduction peak heights (Ah) and the associated electrochemical surface areas calculated using these peak heights for Ei and E2; (b) CV graphs of Ei and E2 electrodes, before (bare; black line) and after probe deposition (dashed line) and post surface blocking agent deposition (using PEG-6000; grey line) extracted from three independent chips - CV curves were measured in 2 M K4Fe(CN)e.

[0083] FIGURE 9 shows a dual electrode electrochemical assay for detecting PEDv nucleocapsid protein in exemplary embodiments of the disclosure: (a) raw square wave voltammetry (SWV) curves obtained from Ei and E2 following incubation with various concentrations of target protein spiked in buffer and incubated on the electrode for 45 mins; (b) calibration plots depicting the associated signal-changes in signals attained on Ei and E2 following incubation with target concentration of 10 nM (0.6 pg mL'¹), 20 nM (1.2 pg mL'¹), 100 nM (5.8 pg mL'¹), 250 nM (14.5 pg mL'¹) and 500 nM (29.0 pg mL'¹) in buffer - the linear region of the calibration curve of Ei was fitted using the equation Al Ei / 1 Ei = -0.0024 C - 0.0042 (R² = 0.9998) while E2 was fitted using the equation Al E2 / 1 E2 = 0.0131 C + 0.0502 (R² = 0.9912); (c) raw square wave voltammetry (SWV) curves obtained from Ei and E2 following incubation with various concentrations of target protein spiked in 30% porcine saliva and incubated on the electrodes for 120 mins; (d) calibration plots depicting the associated signal-changes in signal attained on Ei and E2 for target concentrations of 10 nM (0.6 pg mL'¹), 20 nM (1.2 pg mL'^lOO nM (5.8 pg mL'¹), 250 nM (14.5 pg mL'¹) and 500 nM (29.0 pg mL' ') in porcine saliva. The linear region of the calibration curve of Ei was fitted using the equation Al Ei / 1 Ei = -0.0017 C - 0.08 (R² = 0.9711) while E2 was fitted using the equation Al E2 / I E2 = 0.0419 C + 0.0262 (R² = 0.9682) - all electrochemical measurements were performed in a 25 mM phosphate buffer solution containing 25 mM NaCl (25/25 buffer) against a silver/ silver chloride (Ag/AgCl) reference electrode and a platinum (Pt) counter electrode in a potential range of 0 to - 0.6 V and a scan rate of 0.1 V sec'¹; error bars depict the standard deviation from the mean obtained using three (n=3) separate devices per sample.

[0084] FIGURE 10 shows specificity testing of the DEE-Chip in 30% porcine saliva in exemplary embodiments of the disclosure: (a) square wave voltammograms representing the signal-changes obtained from a pair of Ei and E2 electrodes - each pair was separately incubated with 250 nM N-PEDv (target protein), bovine serum albumin (BSA), thrombin and ribonuclease (RNase) spiked in 30% porcine saliva; (b) jitterplots comparing the signal responses obtained from Ei and E2 in the presence of target (N-PEDv) and control (thrombin, BSA, RNase) proteins - all electrochemical measurements were performed in a 25 mM phosphate buffer solution containing 25 mM NaCl (25/25 buffer) against a silver/ silver chloride (Ag/AgCl) electrode and a platinum (Pt) counter electrode in a potential range of 0 to - 0.6 V and a scan rate of 0.1 V sec'¹; error bars depict the standard deviation from the mean obtained using three (n=3) separate devices per sample.

[0085] FIGURE 11 shows a visual representation of the engineering changes made to the DEE-Chip for reducing assay time in exemplary embodiments of the disclosure: (a) scenario (i) demonstrates the original inter-electrode spacing ‘x=500 pm’ between Ei and E2; scenario (ii) depicts the reduction of this inter-electrode spacing to yield the new inter-electrode distance ‘y=300 pm’; scenario (iii) portrays the use of inter-electrode spacing ‘y’ in conjunction with an applied positive potential bias (+0.5 V) across E2; (b) release and capture kinetics observed in each of the three cases depicted in Figure 10(a) on Ei; (c) release and capture kinetics observed in each of the three cases depicted in (a) on E2 - dotted lines indicate signal onset extracted using tangents constructed to linearly extrapolate signal response at the first true-signal (nonnoise) data points and the data points prior (indicated by 2 reference lines per graph) - for each group, n = 3 independent devices were tested per sample, each incubated with 500 nM of N-PEDv at a sample volume of 10 pL; the data are presented as mean ± standard deviation with visual guide-lines depicting the overall trend in each scenario. [0086] FIGURE 12 shows the diagnostic workflow and associated results in exemplary embodiments of the disclosure: (a) illustration of the diagnostic workflow from sample collection to sensor operation - following sample dilution and heating, the collected porcine saliva sample is added to the DEE-Chip, incubated for about 60 mins at room temperature and scanned using a lightweight benchtop potentiostat (Palmsens); (b) jitter-plots depicting signal-changes measured on Ei for six clinically -obtained positive (black) and 6 negative (grey) porcine saliva samples; (c) jitter-plots depicting signal-changes measured from E2 as for six clinically-obtained positive (black) and 6 negative (grey) porcine saliva samples - dotted lines indicate the cut-off threshold of the assay as extracted using the ‘Analyse-it’ toolkit in excel; bars represent the mean of the signal-change extracted from square wave voltammograms on Ei and E2 for a given sample; the error bars represent the standard deviation from the mean obtained using three (n=3) separate devices per sample - all square wave voltammograms were obtained using 25 mM phosphate buffer solution containing 25 mM NaCl (25/25 buffer) as the supporting electrolyte against a silver / silver chloride (Ag/AgCl) electrode and a platinum (Pt) counter electrode in a potential range of 0 to - 0.6 V and a scan rate of 0.1 V sec^-1;E2 electrodes were biased using a bias potential of + 0.5V to reduce the detection window to 60 mins.

[0087] FIGURE 13 shows a false-negative sample study in exemplary embodiments of the disclosure: (a) jitter plots obtained from Ei incubated with 500 nM N-PEDv spiked in clinically acquired sample ‘positive 4’ (Spiked), unspiked clinically acquired sample ‘positive 4’ (Unspiked) and an unspiked clinically acquired PEDv negative sample (Negative Control); (b) jitter plots obtained from Ei incubated with 500 nM N-PEDv spiked in clinically acquired sample ‘positive 4’ (Spiked), unspiked clinically acquired sample ‘positive 4’ (Unspiked) and an unspiked clinically acquired PEDv negative sample (Negative Control) - the error bars represent the standard deviation from the mean obtained using three (n=3) separate devices per sample - all square wave voltammograms were obtained using 25 mM phosphate buffer solution containing 25 mM NaCl (25/25 buffer) as the supporting electrolyte against a silver / silver chloride (Ag/AgCl) electrode and a platinum (Pt) counter electrode in a potential range of 0 to - 0.6 V and a scan rate of 0.1 V sec'¹; E2 electrodes were biased using a bias potential of + 0.5V to reduce the detection window to 60 minutes. [0088] FIGURE 14 shows the Receiver Operator Characteristics (ROC) curve for the clinical diagnosis of PEDv as obtained from the analysis of the signal foldchange on the release electrode (grey) and the capture electrode (black) for a set of 12 anonymized, clinically sourced swine oral fluid samples in exemplary embodiments of the disclosure: TPF is true-positive fraction; FPF is false-positive fraction.

[0089] FIGURE 15 shows binding assays using the DEE-Chips featuring a 300 pm inter-electrode distance and a 0.5V bias applied to E2 in exemplary embodiments of the disclosure: (a) calibration plot, demonstrating the associated signal-change in the signal obtained for E2 after incubation in 10, 20, 100, 250, and 500 nMN-PEDv in 30% swine saliva; (b) specificity test.

[0090] FIGURE 16 shows the effect of saliva dilution on the DEE-Chip assay in exemplary embodiments of the disclosure: N-PEDv protein (500 nM) was spiked into both undiluted and 30% diluted saliva samples, and the measurements were conducted following a one-hour incubation period.

DETAILED DESCRIPTION

I, Definitions

[0091 ] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0092] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps.

[0093] Terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies. In addition, all ranges given herein include the end of the ranges and also any intermediate range points, whether explicitly stated or not.

[0094] As used in this disclosure, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise.

[0095] In embodiments comprising an “additional” or “second” component, the second component as used herein is chemically different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0096] The term “and/or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present.

[0097] The abbreviation, “e.g.” is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” The word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0098] The term “subject" as used herein includes all members of the animal kingdom including mammals such as a mouse, a rat, a dog, a pig, and a human.

[0099] The term "sample" or "test sample" as used herein may refer to any material in which the presence or amount of a target analyte is unknown and can be determined in an assay. The sample may be from any source, for example, any biological (e.g. human or animal samples, including clinical samples), environmental (e.g. water, soil or air) or natural (e.g. plants) source, or from any manufactured or synthetic source (e.g. food or drinks). The sample may be comprised or is suspected of comprising one or more analytes. The sample may be a "biological sample" comprising cellular and non-cellular material, including, but not limited to, tissue samples, urine, blood, serum, saliva, other bodily fluids and/or secretions.

[00100] The term “target”, “analyte” or “target analyte” as used herein may refer to any agent, including, but not limited to, a small inorganic molecule, small organic molecule, metal ion, biomolecule, toxin, biopolymer (such as a nucleic acid, carbohydrate, lipid, peptide, protein), cell, tissue, and virus, for which one would like to sense or detect. In an embodiment, the analyte is either isolated from a natural source or is synthetic. The analyte may be a single compound or a class of compounds, such as a class of compounds that share structural or functional features. The term analyte also includes combinations (e.g. mixtures) of compounds or agents such as, but not limited, to combinatorial libraries and samples from an organism or a natural environment.

[00101 ] The term “nucleic acid” as used herein refers to a biopolymer comprising monomers of nucleotides, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and other polynucleotides of modified nucleotides and/or nucleotide derivatives, and can be either double stranded (ds) or single stranded (ss). In some embodiments, modified nucleotides contain one or more modified bases (e.g. unusual bases such as inosine, and functional modifications to the bases such as amino), modified backbones (e.g. peptide nucleic acid, PNA) and/or other chemically, enzymatically, or metabolically modified forms.

[00102] The term "aptamer" as used herein may refer to a short, chemically synthesized nucleic acid molecule or oligonucleotide sequence which can be generated by in vitro selection to fold into specific three-dimensional (3D) structures that bind to a specific analyte with dissociation constants, for example, in the pico- to nano-molar range. Aptamers may be single-stranded DNA, and may include RNA, modified nucleotides and/or nucleotide derivatives. Aptamers may also be naturally occurring RNA aptamers termed “riboswitches”. Functional aptamer sequences can also be rationally designed, truncated, conjugated or otherwise modified from original parent (or full length) sequences. A functional fragment of an aptamer is the portion of an aptamer that retains aptameric function, for example, function in binding to molecules such as protein, lipid, carbohydrate, and nucleic acid. A functional variant of an aptamer refers to an aptamer that has been modified, with nucleotide derivatives or otherwise, elongated or truncated, and still retains aptameric function.

[00103] The term “hybridizes”, “hybridized” or “hybridization” as used herein refers to the sequence specific non-covalent binding interaction with a complementary, or partially complementary, nucleic acid sequence. When, for example, the 5'-end region of an aptamer hybridizes to the 3'-end region, it can form a duplex DNA element.

[00104] The term “biosensor” as used herein refers to a device that incorporates a biological entity as a molecular recognition element and is capable of producing a measurable signal upon binding of a target analyte to the molecular recognition element. The biosensor can also be part of a larger device.

[00105] The term “working electrode” as used herein refers to an electrode in an electrochemical system on which the reaction of interest is occurring. The working electrode can be used in conjunction with a counter electrode in a two-electrode system, and further a reference electrode in a three-electrode system. The counter electrode, also called the auxiliary electrode, is an electrode used in, for example, a three-electrode electrochemical cell for voltammetric analysis or other reactions in which an electric current is expected to flow. The counter electrode can also be part of a two-electrode system. The counter electrode is distinct from the reference electrode, which establishes the electrical potential against which other potentials can be measured, and the working electrode, at which the cell reaction takes place. As such, a reference electrode has a stable and well-known electrode potential. Depending on whether the reaction on the electrode is a reduction or an oxidation, the working electrode is called cathodic or anodic, respectively. Working electrodes can, for example, comprise materials ranging from inert metals such as gold, silver or platinum, to inert carbon such as glassy carbon, boron doped diamond or pyrolytic carbon, and mercury drop and fdm electrodes.

[00106] The term “functionalizing” or “functionalized on” as used herein refers to various common approaches for functionalizing a material, which can be classified as mechanical, physical, chemical and biological. Any suitable form of coupling may be utilized (e.g. coating, binding, etc.). The functionalized material, for example, an aptamer or a blocking species, is also immobilized. [00107] It will be understood that any component defined herein as being included may be explicitly excluded by way of proviso or negative limitation, such as any specific compounds or method steps, whether implicitly or explicitly defined herein.

II. Aptamers. Biosensors. Kits and Methods of the Disclosure

[00108] Disclosed herein is an aptamer comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-58, or a functional fragment and/or functional variant thereof, that binds to porcine epidemic diarrhea virus (PEDv) nucleocapsid protein. In some embodiments, the aptamer comprises a sequence for the aptamers listed in Table 2 or Table 3. In some embodiments, the aptamer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5 and 51- 58. In some embodiments, the aptamer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 and 51-58. In some embodiments, the aptamer comprises a nucleotide sequence of SEQ ID NO: 1 or 53. In some embodiments, the aptamer consists of a nucleotide sequence of SEQ ID NO: 1 or 53. In some embodiments, the aptamer comprises a nucleotide sequence of SEQ ID NO: 53. In some embodiments, the aptamer consists of a nucleotide sequence of SEQ ID NO: 53.

[00109] In some embodiments, the aptamer binds to the PEDv nucleocapsid protein with at least nanomolar affinity. In some embodiments, the aptamer binds to the PEDv nucleocapsid protein with at least at least 2 nM, at least 3 nM, at least 4 nM at least 5 nM, at least lOnM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM or at least 100 nM affinity. In some embodiments, the aptamer binds to the PEDv nucleocapsid protein with affinity ranging from about 1 nM to about 100 nM, about 1 nM to about 90 nM, about 1 nM to about 80 nM, about 1 nM to about 70 nM, about 1 nM to about 60 nM, about 1 nM to about 50 nM, about 1 nM to about 40 nM, about 1 nM to about 30nM, about 1 nM to about 20 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, about 1 nM to about 3 nM, about 2 nM to about 100 nM, about 2 nM to about 90 nM, about 2 nM to about 80 nM, about 2 nM to about 70 nM, about 2 nM to about 60 nM, about 2 nM to about 50 nM, about 2 nM to about 40 nM, about 2 nM to about 30nM, about 2 nM to about 20 nM, about 2 nM to about 10 nM about 2 nM to about 5 nM, about 2 nM to about 4 nM, or about 2 nM to about 3nM. [00110] Also provided herein is an aptamer probe comprising the aptamer disclosed herein and a detectable label. In some embodiments, the detectable label is a fluorescence moiety, a colorimetric moiety, an electrochemiluminescent moiety, a photoelectrochemical moiety or an electrochemical moiety.

[0011 1] In some embodiments, the detectable label is an electrochemical moiety. In some embodiments, the electrochemical moiety comprises a redox species. In some embodiments, the electrochemical moiety is a redox species. Examples of redox species include, but are not limited to, ruthenium hexamine chloride, methylene blue, methylene blue succinymide, methylene blue maleimide, Atto MB2 maleimide (Sigma Aldrich) and other methylene blue derivatives, ferrocene and Fe²⁺ and/or Fe³⁺ ions. In some embodiments, the redox species comprises methylene blue or ferrocene. In some embodiments, the redox species comprises methylene blue.

[00112] In some embodiments, the aptamer probe further comprises a reporter moiety coupled to the detectable label. In some embodiments, the reporter moiety comprises a biomolecule. In some embodiments, the reporter moiety comprises a nucleotide sequence complementary to at least a portion of the aptamer. In some embodiments, the reporter moiety is capable of hybridizing to at least a portion of the aptamer. In some embodiments, the reporter moiety comprises a nucleotide sequence selected from SEQ ID NOs: 64-69. In some embodiments, the reporter moiety comprises a nucleotide sequence of SEQ ID NO: 66 or 67. In some embodiments, the reporter moiety consists of a nucleotide sequence of SEQ ID NO: 66 or 67. In some embodiments, the reporter moiety is releasable from the aptamer in the presence of PEDv nucleocapsid protein. In some embodiments, the reporter moiety is capable of dehybridizing from the aptamer in the presence of PEDv nucleocapsid protein.

[00113] Provided herein is also a biosensor for detecting PEDv nucleocapsid protein comprising the aptamer disclosed herein or the aptamer probe disclosed herein functionalized on and/or in a material. In some embodiments, the biosensor is a lateral flow device. In some embodiments, the biosensor is an electrochemiluminescent, photoelectrochemical or electrochemical sensor.

[00114] In some embodiments, the biosensor further comprises a) a first working electrode, wherein the aptamer probe further comprising a reporter moiety disclosed herein is functionalized on the first working electrode; b) a second working electrode positioned in proximity to the first working electrode; c) a capture probe functionalized on the second working electrode; and d) a counter electrode; wherein each working electrode is configured to provide a change in signal if the PEDv nucleocapsid protein is present.

[00115] In some embodiments, the capture probe is for recognizing and coupling to the reporter moiety of the aptamer probe. In some embodiments, the capture probe comprises a nucleotide sequence of SEQ ID NO: 71 or 72. In some embodiments, the capture probe consists of a nucleotide sequence of SEQ ID NO: 71 or 72.

[00116] In some embodiments, the biosensor further comprises a blocking species on the first working electrode and/or second working electrode. In some embodiments, the blocking species comprises proteins, nucleic acids, sugars and/or synthetic polymers. In some embodiments, the blocking species comprises bovine serum albumin (BSA), fish sperm DNA, mercapto hexanol (MCH), polyethylene glycol (PEG), or polyadenylic acid (poly A). In some embodiments, the blocking species comprises polyethylene glycol (PEG). In some embodiments, the blocking species comprises PEG 6000.

[00117] Functionalizing the electrode with the aptamer probe, capture probe and/or blocking species can be done by any suitable technique, for example, as described in Putzbach, W., & Ronkainen, N. J. (2013), incorporated herein by reference. Immobilization techniques in the fabrication of nanomaterial-based electrochemical biosensors: A review. In Sensors (Switzerland) (Vol. 13, Issue 4, pp. 4811-4840) MDPI AG, incorporated herein by reference.

[00118] In some embodiments, the aptamer probe is functionalized on the electrode via chemical bonding, an intermediate linker, or physical adsorption. In some embodiments, the capture probe is functionalized on the electrode via chemical bonding, an intermediate linker, or physical adsorption. In some embodiments, the blocking species is functionalized on the electrode via chemical bonding, an intermediate linker, or physical adsorption. In some embodiments, the aptamer probe, capture probe and/or blocking species is directly linked to the working electrode. In some embodiments, the linker is a bifunctional linker comprising complementary reactive functional groups. Examples of reactive functional groups include, but are not limited to, thiol, amine, epoxy, carboxylic acid, azide, and alkyne. In some embodiments, the linker comprises a thiol. In some embodiments, the aptamer probe capture, probe and/or blocking species is functionalized on the electrode via chemical bonding. In some embodiments, the chemical bonding occurs via thiol and/or gold chemistry. In some embodiments, the aptamer probe, capture probe and/or blocking species is indirectly linked to the working electrode. In some embodiments, the linker is a biotin, streptavidin, cystamine, or glutaraldehyde. In some embodiments, the aptamer probe, capture probe and/or blocking species is linked to the working electrode via biotin-streptavidin interactions.

[00119] In some embodiments, the working electrode comprises a conductive material, semi-conductive material, or a combination thereof. In some embodiments, the working electrode comprises metal, metal alloy, metal oxide, superconductor, semiconductor, carbon-based material, conductive polymer, or combinations thereof. Examples include, but are not limited to, gold, platinum, palladium, carbon-based materials such as glassy carbon, graphite, graphene, or carbon nanotubes, nickel oxide, bismuth oxide, indium tin oxide, and titanium dioxide. In some embodiments, the working electrode comprises metal. Typically, the metals are selected from gold, other noble metals, or combinations thereof. In some embodiments, the metal is gold. In some embodiments, the working electrode comprises gold.

[00120] In some embodiments, the first working electrode is configured to provide a decrease in the signal and the second working electrode is configured to provide an increase in the signal in the presence of PEDv nucleocapsid protein.

[00121] In some embodiments, the change in signal comprises a change in current, potential or impedance. In some embodiments, the change in signal is a change in current. In some embodiments, the change in current is measured using square wave voltammetry (SWV).

[00122] The term “proximity” as used herein refers to a distance sufficient to provide the reporter moiety capability to transport from the aptamer probe to the capture probe. In some embodiments, the second working electrode is positioned in about 50 pm to about 800 pm, about 50 pm to about 700 pm, about 50 pm to about 600 pm, about 50 pm to about 500 pm, about 100 pm to about 800 pm, about 100 pm to about 700 pm, about 100 pm to about 600 pm, about 100 pm to about 500 pm, about 200 pm to about 800 pm, about 200 pm to about 700 pm, about 200 pm to about 600 pm, about 200 pm to about 500 pm, about 300 pm to about 800 pm, about 300 pm to about 700 pm, about 300 pm to about 600 pm, or about 300 pm to about 500 pm proximity to the first working electrode. In some embodiments, the second working electrode is positioned in about 300 pm proximity to the first working electrode. In some embodiments, the second working electrode is positioned in about 500 pm proximity to the first working electrode.

[00123] In some embodiments, the biosensor further comprises a reference electrode. In some embodiments, the counter electrode is also a reference electrode. In some embodiments, the reference electrode is a platinum (Pt) reference electrode.

[00124] In some embodiments, the biosensor is configured to apply a positive potential bias across the first and/or second working electrode. In some embodiments, the biosensor is configured to apply a positive potential bias across the second working electrode. In some embodiments, the biosensor is configured to apply a positive potential bias on the second working electrode. In some embodiments, the positive potential bias is about +0.5 V.

[00125] In some embodiments, the biosensor is on an electrochemical chip. In some embodiments, the biosensor comprises an electrochemical chip. In some embodiments, the biosensor comprises a multi-electrode electrochemical chip.

[00126] Any suitable electrodes and multiple electrode designs may be used in the biosensors disclosed herein, in particular, in the differential signaling approach. Two or more working electrodes may be used. The electrodes may be any suitable shape, geometry or pattern such as, and without being limited thereto, squares, rectangles, parallelograms, rhombuses, stars, spiral, serpentine, circles, triangles, jigsaw-shaped, etc. In addition, the electrodes may be interdigitated, intermeshed, interleaved or intertwined geometries or patterns such as fingers, dendritic or sawtooth electrode patterns, or indeed any of a number of possibilities that will now become apparent to those skilled in the art, including irregular patterns. Further examples include concentric spiral and serpentine line electrodes, brush electrodes, and intertwined star electrodes and multiple intertwined electrodes such as, and without being limited thereto, multi-star electrodes. Therefore, the biosensor design is also compatible with multiple intertwined electrodes integrated on the same chip for multiplexed analyte detection. In some embodiments, the biosensor provided herein comprises nanostructures with high-aspect ratio on the electrode surface. In some embodiments, the biosensor comprises nanostructures with high-aspect ratio on the first and/or second working electrode.

[00127] In some embodiments, the electrodes are nanostructures. In other embodiments, the electrodes have high-aspect ratios. The high-aspect ratio may be any suitable ratio. Examples of the high-aspect ratio include a vertical-to-lateral ratio ranging from about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10. about 8 to about 10, about 2 to about 9, about 3 to about 9, about 4 to about 9, about 5 to about 9, about 6 to about 9, about 7 to about 9, about 8 to about 9, about 2 to about 8, about 3 to about 8, about 4 to about 8, about 5 to about 8, about 6 to about 8, about 7 to about 8, about 2 to about 7, about 3 to about 7, about 4 to about 7, about 5 to about 7, or about 6 to about 7. In embodiments, the electrodes have multiple edges and/or points. Such edges and/or points may contribute to high-aspect ratio electrodes (e.g. nanostructured electrodes). The edges and/or points may be sharp. Typical surface areas are about 7 to about 14 times higher in electrodes with such edges and/or points. In certain embodiments, the electrodes disclosed herein have surface areas of about 1.0 cm² to about 5.0 cm², about 1.5 cm² to about 5.0 cm², about 1.5 cm² to about 4.5 cm², about 1.5 cm² to about 4.0 cm², about 1.5 cm² to about 3.9 cm², about 1.7 cm² to about 3.8 cm², or about 1.7 cm² to about 3.7 cm², about 1.7 cm² to about 3.6 cm², about 1.7 cm² to about 3.5 cm², about 1.7 cm² to about 3.4 cm², about 1.7 cm² to about 3.3 cm², or about 1.7 cm² to about 3.2 cm². In some embodiments, the nanostructuring of the electrodes enhanced their limit-of- detection in detecting electroactive oligonucleotides by about 1 to about 3 orders of magnitude compared to their planar counterparts.

[00128] The electrodes may be constructed of any suitable conductive and/or semi- conductive material(s) comprising a metal, metal alloy, metal oxides, superconductor, semi-conductor, carbon-based materials, conductive polymer, or combinations thereof. The metal may be selected from aluminum (Al), antimony (Sb), bismuth (Bi), boron (B), cadmium (Cd), carbon (C), cerium (Ce), chromium (Cr), cobalt (Co), copper (Cu), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), germanium (Ge), gold (Au), graphite (C), halhium (Elf), holmium (Ho), indium (In), iridium (Ir), iron (Fe), lanthanum (La), lutetium (Lu), magnesium (Mg), manganese (Mn), molybdenum (Mo), neodymium (Nd), nickel (Ni), niobium (Nb), palladium (Pd), platinum (Pt), praseodymium (Pr), rhenium (Re), ruthenium (Ru), samarium (Sm), selenium (Se), scandium (Sc), silver (Ag), silicon (Si), tantalum (Ta), terbium (Tb), thulium (Tm), tin (Sn), titanium (Ti), tungsten (W), vanadium (V), ytterbium (Yb), yttrium (Y), zirconium (Zr) and/or zinc (Zn). Typically, the metals are selected from gold, other noble metals, or combinations thereof. The metal alloy may be selected from the group consisting of aluminum copper (AICu), aluminum chromium (AlCr), aluminum magnesium (AlMg), aluminum silicon (AlSi), aluminum silver (AlAg), cerium gadolinium (CeGd), cerium samarium (CeSm), chromium silicon (CrSi), cobalt chromium (CoCr), cobalt iron (CoFe), cobalt iron boron (CoFeB), copper cobalt (CuCo), copper gallium (CuGa), copper indium (Culn), copper nickel (CuNi), copper zirconium (CuZr), hafnium iron (HfFe), iron boron (FeB), iron carbon (FeC), iron manganese (FeMn), iridium manganese (IrMn), iridium rhenium (IrRe), indium tin (InSn), molybdenum silicon (MoSi), nickel aluminum (NiAl), nickel chromium (NiCr), nickel chromium silicon (NiCrSi), nickel iron (NiFe), nickel niobium titanium (NiNbTi), nickel titanium (NiTi), nickel vanadium (NiV), samarium cobalt (SmCo), silver copper (AgCu), silver tin (AgSn), tantalum aluminum (TaAl), terbium dysprosium iron (TbDyFe), terbium iron alloy (TbFe), titanium aluminum (TiAl), titanium nickel (TiNi), titanium chromium (TiCr), tungsten rhenium (WRe), tungsten titanium (WTi), zirconium aluminum (ZrAl), zirconium iron (ZrFe), zirconium nickel (ZrNi), zirconium niobium (ZrNb), zirconium titanium (ZrTi), zirconium yttrium (ZrY), zinc aluminum (ZnAl) and/or zinc magnesium (ZnMg).

[00129] The electrodes, and more specifically, the nanostructured electrodes may be made from any suitable method, for example, a seed layer for the nanostructured electrodes may be made by sputter-coating, evaporation, chemical vapor deposition, or a pulsed laser method, ink jet printing. The nanostructures may be made using direct solution-based deposition using covalent bonding, electroless or electroplating, electrospinning, template-based synthesis, electrophoretic deposition, etching, printing, self-assembly of nanoparticles, etc. In an example, the method comprises applying a patterned mask to a substrate, depositing a conductive material on the mask forming a conductive layer, exposing the conductive layer to pre-conductive material, and using a potentiostatic technique to convert the pre-conductive material to the conductive material, forming the nanostructured electrodes.

[00130] In some embodiments, the biosensor is an electrochemical biosensor as described in PCT/CA2020/050375, incorporated herein by reference.

[00131] Also provided herein is a device comprising the biosensor disclosed herein. In some embodiments, the device is a hand-held device. In some embodiments, the device is a hand-held electrical reader. In some embodiments, the biosensor or electrochemical chip disclosed herein are inserted into the reader.

[00132] In some embodiments, the biosensor or device disclosed herein is for use in animal screening and/or diagnostics, environmental monitoring, health monitoring, and/or pharmaceutical development. In some embodiments, the biosensor or device disclosed herein is for use in animal health screening, diagnostics, and/or monitoring. In some embodiments, the biosensor or device disclosed herein is for use in detecting PEDv infection. In some embodiments, the biosensor or device disclosed herein is for use without the need for sample pre-treatment, target labeling, and/or amplification.

[00133] Also provided herein is a method for detecting the presence of PEDv nucleocapsid protein in a sample from a subject, comprising: a) obtaining a sample from the subject; b) combining the sample with a liquid in a sample container to form a mixture; c) exposing the mixture to the biosensor disclosed herein; whereby the reporter moiety of the aptamer probe functionalized on the first working electrode is delocalized from the first working electrode to the second working electrode upon binding of the aptamer of the aptamer probe functionalized on the first working electrode to the PEDv nucleocapsid protein; and d) measuring a change in signal at the first and/or second working electrode, wherein a change in signal is produced if the PEDv nucleocapsid protein is present in the sample. [00134] In some embodiments, the method further comprises lysing viral cells in the mixture to release PEDv nucleocapsid protein, if present in the sample. In some embodiments, the method further comprises incubating the mixture at about 55 °C to about 95 °C, about 55 °C to about 90 °C, about 55 °C to about 85 °C, about 55 °C to about 80 °C, 55 °C to about 75 °C, about 55 °C to about 70 °C, about 55 °C to about 65 °C, about 55 °C to about 60 °C, 60 °C to about 95 °C, about 60 °C to about 90 °C, about 60 °C to about 85 °C, about 60 °C to about 80 °C, 60 °C to about 75 °C, about 60 °C to about 70 °C, or about 60 °C to about 65 °C for about 5 minutes to about 45 minutes, about 5 minutes to about 40 minutes, about 5 minutes to about 35 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 25 minutes, for about 10 minutes to about 45 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 35 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 25 minutes, about 15 minutes to about 45 minutes, about 15 minutes to about 40 minutes, about 15 minutes to about 35 minutes, about 15 minutes to about 30 minutes, about 15 minutes to about 25 minutes, about 25 minutes to about 45 minutes, about 25 minutes to about 40 minutes, about 25 minutes to about 35 minutes, or about 25 minutes to about 30 minutes, before step c). In some embodiments, the method further comprises incubating the mixture at about 60 °C for about 25 minutes before step c).

[00135] In some embodiments, the sample is diluted to about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, or about 70% in the liquid. In some embodiments, the sample is diluted to about 30% in the liquid. In some embodiments, the liquid is a buffer. In some embodiments, the buffer is Tris.

[00136] In some embodiments, exposing the mixture to the biosensor comprises contacting the mixture with the first working electrode and second working electrode. In some embodiments, exposing the mixture to the biosensor comprises contacting the mixture with the first working electrode. In some embodiments, the mixture is exposed to the biosensor under conditions to delocalize the reporter moiety from the first working electrode to the second working electrode. In some embodiments, the mixture is exposed to the biosensor in a buffer. In some embodiments, the mixture is exposed to the biosensor in a phosphate buffer solution containing NaCl. [00137] In some embodiments, the mixture is exposed to the biosensor from about 10 minutes to about 120 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 45 minutes, 15 minutes to about 120 minutes, about 15 minutes to about 90 minutes, about 15 minutes to about 60 minutes, about 15 minutes to about 45 minutes, about 30 minutes to about 120 minutes, about 30 minutes to about 90 minutes, about 30 minutes to about 60 minutes, about 30 minutes to about 45 minutes, about 45 minutes to about 120 minutes, about 45 minutes to about 90 minutes, or about 45 minutes to about 60 minutes before step d). In some embodiments, the mixture is exposed to the biosensor from about 30 minutes to about 120 minutes before step d). In some embodiments, the mixture is exposed to the biosensor for about 45 minutes before step d). In some embodiments, the mixture is exposed to the biosensor for about 60 minutes before step d).

[00138] In some embodiments, the change in signal is produced from about 30 minutes to about 120, about 30 minutes to about 90 minutes, about 30 minutes to about 60 minutes, about 30 minutes to about 45 minutes, about 45 minutes to about 120 minutes, about 45 minutes to about 90 minutes, or about 45 minutes to about 60 minutes after step c). In some embodiments, the change in signal is produced about 45 minutes after step c). In some embodiments, the change in signal is produced about 60 after step c).

[00139] In some embodiments, the biosensor of the method comprises a reference electrode and the method further comprises applying a positive potential bias across the first and/or second working electrode before and/or during step d). In some embodiments, the positive potential bias applied across the first and/or second working electrode counter electrode is applied against the reference electrode. In some embodiments, the reference electrode is a platinum (Pt) reference electrode. In some embodiments, the positive potential bias is applied before step d) when the redox species comprises methylene blue. In some embodiments, the positive potential bias is applied during step d) when the redox species comprises ferrocene. In some embodiments, the positive potential bias is applied across the second working electrode. In some embodiments, the positive potential bias is about +0.05 V to about +1.0 V, about +0.05 V to about +0.9, about +0.05 V to about +0.8 V, about +0.05 V to about +0.7 V, about +0.05 V to about +0.6 V, +0.05 V to about +0.5 V, about +0.1 V to about +1.0 V, about +0.1 V to about +0.9, about +0.1 V to about +0.8 V, about +0.1 V to about +0.7 V, about +0.1 V to about +0.6 V, +0.1 V to about +0.5 V, +0.2 V to about +1.0 V, about +0.2 V to about +0.9, about +0.2 V to about +0.8 V, about +0.2 V to about +0.7 V, about +0.2 V to about +0.6 V, +0.2 V to about +0.5 V, +0.3 V to about +1.0 V, about +0.3 V to about +0.9, about +0.3 V to about +0.8 V, about +0.3 V to about +0.7 V, about +0.3 V to about +0.6 V, +0.3 V to about +0.5 V, +0.4 V to about +1.0 V, about +0.4 V to about +0.9, about +0.4 V to about +0.8 V, about +0.4 V to about +0.7 V, about +0.4 V to about +0.6 V, +0.4 V to about +0.5 V, +0.5 V to about +1.0 V, about +0.5 V to about +0.9, about +0.5 V to about +0.8 V, about +0.5 V to about +0.7 V, or about +0.5 V to about +0.6 V. In some embodiments, the positive potential bias is about +0.5 V. In some embodiments, the positive potential bias provides active field mediated transport between the first working electrode and the second working electrode. In some embodiments, the positive potential bias provides active field mediated transport for delocalizing the reporter moiety of the aptamer probe from the first working electrode to the second working electrode.

[00140] In some embodiments, the change in signal from the first working electrode is a decrease in signal and the change in signal from the second working electrode is an increase in signal. In some embodiments, wherein the change in signal comprises a change in current, potential or impedance. In some embodiments, the change in signal is a change in current. In some embodiments, the change in current is measured using square wave voltammetry (SWV).

[00141] In some embodiments, the sample comprises saliva, sputum, oropharyngeal and/or nasopharyngeal secretions. In some embodiments, the sample comprises saliva. In some embodiments, the sample is a saliva sample.

[00142] In some embodiments, the method detects PEDv infection in the subj ect.

[00143] In some embodiments, the subject is a porcine animal.

[00144] Also provided is kit for detecting PEDv nucleocapsid protein, wherein the kit comprises the biosensor disclosed herein, device disclosed herein or components required for the methods disclosed herein, and instructions for use of the kit.

[00145] In some embodiments, the kit further comprises at least one collection apparatus, at least one sample container, a buffer, and instructions for use. [00146] Also provided is the use of the aptamer, aptamer probe, biosensor, device or kit disclosed herein for detecting PEDv in a sample.

EXAMPLES

[00147] The following non-limiting examples are illustrative of the present disclosure:

[00148] Methods

[00149] Materials and Reagents. All oligonucleotides were purchased from Biosearch and Integrated DNA Technologies (IDT), and purified by standard 10% denaturing (8 M urea) polyacrylamide gel electrophoresis (dPAGE) before use. The sequences are listed in Tables 1 and 2. The nucleocapsid protein of PEDv (N-PEDv, molecular weight: 60 kDa) was expressed from E. coli cells and purified using standard methods. Bovine serum albumin (BSA) and human-a-thrombin were purchased from Sigma-Aldrich (Oakville, Canada). SARS-CoV-2 nucleocapsid protein (catalog number: 40588-V08B) and spike protein (catalog number: 40591-V08B1) were purchased from Sino Biological Inc. RNase H2 was expressed from E. coli and purified in the Li’s lab at McMaster University.⁷⁰ Taq DNA polymerase was purchased from GenScript. y-[³²P]- ATP was acquired from PerkinElmer. Phosphate buffer solution (1.0 M, pH 7.4), sodium chloride (NaCl, >99.0%), magnesium chloride (MgCh, >99.0%), 4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid (HEPES), sodium phosphate dibasic (Na2HPO4), potassium phosphate monobasic (KH2PO4), potassium chloride (KC1), Tween-20, polyethylene glycol 6000 (PEG 6000), 6-mercapto-l -hexanol (MCH, 99%), tris(2- carboxyethyl)phosphine hydrochloride (TCEP), potassium hexacyanoferrate(II) trihydrate ([Fe(CN)e]⁴ . >99.95%), gold(III) chloride solution (HAuCL, 99.99%) were purchased from Sigma-Aldrich. HisPur Ni-NTA magnetic beads (catalog number: 88831), T4 DNA ligase, T4 polynucleotide kinase (PNK), adenosine triphosphate (ATP) and deoxyribonucleoside 5 '-triphosphates (dNTPs) was from Thermo Scientific. Sulfuric acid (H2SO4, 98%) and 2-propanol (99.5%) were purchased from Caledon Laboratories. Hydrochloric acid (HC1; 37% w/w) was purchased from LabChem. Swine saliva samples were obtained from South West Ontario Veterinary Services, Stratford, Ontario. Milli-Q water was used for all experiments.

[00150] Expression and Purification of N-PEDv. The nucleocapsid protein of PEDv (N-PEDv; SEQ ID NO: 73; NCBI Accession Number ALF39593) was produced as described by Deejai et al. 2017, herein incorporated by reference.⁷¹ The optimized sequence coding N-PEDv (SEQ ID NO: 72) was purchased from GENEWIZ (Suzhou, China) based on the genome of strain CH/HNQX-3/14 and cloned in pET28-a (+) vector for expression in E coli. The E. coli BL21-DE3 clones harboring the pET28-a (+) vector was cultured in Luria-Bertani (LB) broth medium containing 50 pg/mL kanamycin with shaking at 210 rpm at 37 °C. Isopropyl-P-d-thiogalactoside (IPTG, 1 mL, 0.5 M) was added to the culture (1 L) when the optical density of cells at 600 nm (OD600) reached 0.5 to induce expression of N-PEDv for 24 hours at 20 °C. The harvest cells from 1 L of culture were suspended in 20 mL of PBS and lysed by sonication. After centrifugation at 40,000 g for 40 mins, the supernatant fraction of recombinant N-PEDv was purified using His-trap-affinity column (catalog number: 45-000-323, Fisher Scientific) run on Cytiva AKTA Start chromatography system (36-104-1137, Fisher Scientific). Purity of >90% N-PEDV was eluted in PBS with 400 mM imidazole. The imidazole was removed by dialysis in PBS with 50 mM Arginine. Purified N-PEDv protein aliquots were stored at - 80 °C for aptamer selection and tests.

[00151 ] Conjugation of N-PEDv protein on magnetic beads. HisPur Ni-NTA magnetic beads (16 pL, 5% w/v, 12.5 mg/mL) were first washed with PBST buffer (0.5 mL, 1.8 mM KH₂PO₄, 10 mM Na₂HPO₄. 2.7 mM KC1, 137 mM NaCl, 0.01% v/v Tween- 20). Magnetic bead pellets were then resuspended in 5* PBST buffer (40 pL). Imidazole (4 pL, 1 M), N-PEDv protein with His-tag (100 pL, 0.5 mg/mL) and water (60 pL) were mixed with the magnetic beads and incubated at 4 °C for 12 h. The protein-conjugated magnetic beads (20 pL) were then washed twice with PBST and resuspended in PBST buffer with 200 mM imidazole. The free and bound N-PEDv were analyzed by SDS PAGE and the bound N-PEDv on magnetic beads was determined to be 0.16 mg/mL. The protein-conjugated magnetic beads were stored at 4 °C for selection.

[00152] Selection of DNA aptamers for N-PEDv. DNA aptamers for N-PEDv protein were selected using magnetic bead-based SELEX method as described in the previous study.³⁹ Briefly, the DNA library was diluted in selection buffer (1 x SB; 50 mM Tris-HCl, pH 7.26, 150 mM NaCl, 1 mM MgCh, 0.01% Tween-20, 10 mM Imidazole) and heated at 90 °C for 1 min, followed by annealing at room temperature for 10 mins. Then, the N-PEDv protein-conjugated magnetic beads were washed twice with l SB and mixed with the DNA library at 23 °C for 30 mins. After washing three times with 1 x SB (1 mL), the magnetic beads were resuspended in elution buffer (50 mM Tris-HCl, pH 7.26, 150 mM NaCl, 1 mM MgCE, 0.01% Tween-20, 500 mM Imidazole) to elute the bound DNA on magnetic beads. The eluted DNA in the supernatant was collected by magnetic separation, followed by the addition of the reverse primer RP1 (10 pL, 10 pM), the forward primer FP1 (10 pL, 10 pM), Taq DNA polymerase (2 pL, 5 U/pL), Taq buffer (200 pL, 50 mM KC1, 10 mM Tris-HCl, 1.5 mM MgCh, 1% v/v Triton X-100, pH 9.0) and dNTPs (20 pL, 2 mM) for PCR1. PCR1 was carried out using the following temperature profile: preheating at 94 °C for 30 s; thermo cycles of 94 °C for 30 s, 50°C for 30 s, and 72 °C for 30 s; annealing at 72 °C for 5 mins. Next, the PCR1 product was used as the template for PCR2. The PCR2 mixture was prepared by mixing the PCR1 product (50 pL), FP1 (25 pL, 10 pM), RP2 (25 pL, 10 pM), 10x Taq buffer (50 uL), Taq DNA polymerase (5 pL, 5 U/pL), dNTPs (10 pL, 10 mM), and water (335 uL). The amplification reaction used the same temperature profile as PCR1. After amplification, the PCR2 product was pelleted by ethanol precipitation, purified by dPAGE, quantified by UV-Vis absorbance at 260 nm and utilized for the next round of selection. A total of 10 rounds of selection were carried out. The SELEX pressure was gradually increased by decreasing the concentrations of the DNA library and N-PEDv target (from 1 - 10 round: DNA library (nM): 2000, 200, 200, 200, 200, 50, 10, 5, 2.5, 2.5 nM; N-PEDv (nM): 100, 100, 50, 25, 10, 5, 5, 1.6, 1, 0.5 nM). Selected DNA libraries were amplified by PCR using primers with sequencing tags and analyzed with the MiSeq (Illumina) sequencing platform using previously published protocols.⁷²

[00153] Radiolabeling of DNA Aptamers. DNA aptamers were labeled with y- [³²P] ATP at the 5'-end using PNK reactions according to the manufacturer's protocol. Briefly, 2 pL of 1 pM DNA aptamers were mixed with 2 pL of y-[³²P] ATP, 1 pL of 10 x PNK reaction buffer A, 10 U (U: unit) of PNK and 4 pL water. The mixture was incubated at 37 °C for 20 mins, and then purified by 10% dPAGE.

[00154] Electrophoresis mobility shift assays (EMSA). y-[³²P] labelled DNA aptamers (1 nM) were dissolved in IxSB and heated at 90 °C for 5 mins, and then cooled at room temperature for 20 mins. N-PEDv protein were dissolved and diluted in the same buffer. 5 pL of the above aptamer solution was mixed with 15 pL of N-PEDv solution with different concentrations. The mixture was incubated at room temperature for 1 h, followed by adding 6 x glycerol loading buffer (4 pL) and analyzed by native PAGE (10% w/v, 100 V, 20 mins, room temperature). The PAGE gel was then analyzed using a Typhoon 9200 imager. Aptamer binding with other proteins were tested using the same method.

[00155] DEE-Chip Fabrication. The DEE-Chip was fabricated on polystyrene sheets (Graphix Shrink Film, Graphix). The polystyrene was cleaned with ethanol and deionized water, after which a vinyl mask (FDC 4304, FDC Graphic Films) was applied onto the sheet. The vinyl mask was then patterned (as defined by Adobe Illustrator) using a Robo Pro CE5000-40-CRP cutter (Graphtec America). A 100 nm gold film was sputtered onto the masked substrate using direct current sputtering (MagSput, Torr International) and the vinyl mask was removed. Subsequently, gold nanostructures were electrodeposited onto the two working electrodes by applying a static potential of -0.6 V for 600 s in a solution of 10 mM gold chloride (HAuCU) and 5 mM HC1 using a potentiostat (PalmSens4, PalmSens BV, Netherlands) with Ag/AgCl as the reference and platinum wire as the counter electrode.

[00156] DEE-Chip Biofunctionalization. All of the electrochemical measurements were performed using a potentiostat (Palmsens). The DEE-Chips were rinsed in isopropanol and ddFEO. This was followed by electrochemical cleaning using cyclic voltammetry in 0.1 M H2SO4 (0-1.5 V, 100 mV s '. 40 cycles). After cleaning of the DEE Chips, the sensing electrodes (Ei and E2) were functionalized with their respective biorecognition elements. To functionalize Ei, 0.5 pM thiol-terminated e- aptamer sequence was reduced with 150 pM TCEP for 2 h in the dark at room temperature and then deposited onto the sensing electrode. Simultaneously, TCEP reduced single-stranded DNA (ssDNA) capture probes (3 pL, 1.5 pM) specific to the biobarcode sequence of the e-aptamer were drop functionalized onto E2. The DEE-Chips containing their respective solutions were then enclosed in foil-backed petri dishes at room temperature and stored in a dark environment for 20 h.

[00157] Electrochemical Characterization. Electrochemical characterization of the DEE-Chips was carried out using square wave voltammetry (SWV) over a voltage range of 0 V to -0.45 V (all of the voltages are reported as anodic negative) before and after the e-aptamer deposition in 25 mM PBS and 25 mM NaCl buffer (25:25 buffer). The immobilization of a thiol-terminated ssDNA probe on the capture channel was characterized using a cyclic voltammetry scan from 0 V to 0.5 V at a scan rate of 50 mV s ¹ in 2 mM potassium hexacyanoferrate(II) solution. [00158] Detection in Buffer and Saliva. Following biofunctionalization, a 100 mM MCH solution was used to backfill the surface for 10 mins in the dark at room temperature prior to buffer (50 mM Tris (pH 7.4), NaCl 150 mM, MgCh 1 mM, Tween- 20 0.01%) based analysis. Buffer spiked N-PEDv samples (8 pL) of varying concentrations were then deposited onto the DEE-Chips such that both Ei and E2 were covered at the same time. The sample containing DEE-Chips were subsequently incubated for a period of 45 mins.

[00159] For saliva spiked analysis, a ImM thiolated PEG 6000 solution was used to backfill the sensing surfaces for lOO mins in the dark at 4°C. Healthy swine saliva samples were then thawed, redispersed and diluted to 30% (v/v) in buffer. N-PEDv proteins of various concentrations were subsequently spiked into these saliva samples (10 pL) and incubated on the DEE-Chips at room temperature for 120 mins.

[00160] Following incubation, redox currents on each electrode were probed using SWV over a voltage range of 0 V to -0.45 V (all voltages are reported as anodic negative) and a scan rate of 0.1 V sec'¹ in 25 mM PBS and 25 mM NaCl buffer (25/25 buffer).

[00161 ] Specificity Testing. Individual sets of DEE-Chips (n > 3) were incubated with 250 nM N-PEDv (target protein), bovine serum albumin (BSA), thrombin and ribonuclease (RNase) spiked in 30% porcine saliva as per the protocol for detection in saliva (described above). Square wave voltammograms for each of the sensing electrodes were subsequently acquired in a 25 mM phosphate buffer solution containing 25 mM NaCl (25/25 buffer) against a silver/silver chloride (Ag/AgCl) electrode and a platinum (Pt) counter electrode in a potential range of 0 to - 0.45 V and a scan rate of 0. 1 V sec'¹.

[00162] Kinetic Characterization. Three different electrode configurations were designed to manipulate mass transport times. Towards this end, DEE-Chips pertaining to the first two configurations were obtained by vinyl masking and sputtering gold star electrodes with interelectrode distances of 500 pm and 300 pm, respectively. A third configuration was then generated by applying a positive potential bias (+0.5 V against a Pt reference electrode) across E2 for a set (n = 3) of DEE-Chips possessing an interelectrode spacing of 300 pm. Each set of electrode configurations was then cleaned, characterized and incubated with 500 nM of N-PEDv (10 pL) spiked in 30% porcine saliva samples for incubation times spanning 0 - 120 mins. Square wave voltammograms for each of the sensing electrodes were subsequently acquired at each predetermined incubation interval. All electrochemical measurements were performed in 25 mM phosphate buffer solution containing 25 mM NaCl (25/25 buffer) against a silver/silver chloride (Ag/AgCl) electrode and a platinum (Pt) counter electrode in a potential range of 0 to - 0.6 V and a scan rate of 0.1 V sec'¹.

[00163] Clinical Evaluation. A set of 12 porcine saliva samples was obtained through Southwest Vets following a PEDv outbreak. Of these, six were established as PEDv-positive and six were determined to be PEDv -negative by RT-PCR (Table 1). Each of these 12 samples was then anonymized and tested in a double-blind study. Following sample dilution to a 30% saliva concentration in buffer (50 mM Tris (pH 7.4), NaCl 150 mM, MgC12 1 mM, Tween-20 0.01%), whole saliva samples were heat lysed (off-chip) at 60°C for 25 mins to release N-PEDv proteins from within the viral cells, if present. A sample volume of 10 pL was then added to each DEE-Chip, with a positive potential bias (+0.5 V) applied across E2 using a potentiostat (Palmsens). The DEE-Chips were incubated for 60 mins at room temperature and scanned using a handheld potentiostat. All electrochemical measurements were performed in 25 mM phosphate buffer solution containing 25 mM NaCl (25/25 buffer) against a silver/silver chloride (Ag/AgCl) electrode and a platinum (Pt) counter electrode in a potential range of 0 to - 0.45 V and a scan rate of 0.1 V sec'¹. Changes in signal were then reported against the potential window according to the following equation:

Signal Change El (Al El /I El) = (I El after - I El before) I El before

Signal Change E2 (Al E2/ 1 E2) = (I E2 after - I E2 before) 1 1’>2 before

[00164] Clinical sensitivity and specificity were calculated as:

Sensitivity = True positive (True positive + False negative)

Specificity = True negative (True negative + False positive)

[00165] Every data point corresponds to the mean of three (n = 3) individual data points measured for the same conditions on three separate devices, with the error bar indicating the standard deviation.

[00166] Limit-of-detection Calculation. In order to determine the limit-of- detection (LOD) of the assay, the linear portions of the signal change for Ei and E₂ were individually plotted against the concentrations of N-PEDv (buffer spiked and saliva spiked). For the buffer spiked data, the linear portion of the resultant Ei curve was fitted using the regression line equation Signal Change Ei = -0.0024 C - 0.0042 (R² = 0.9998) while E₂ was fited using the regression line equation Signal Change E₂ = 0.0131 C + 0.0502 (R² = 0.9912); here, ‘C’ represents the concentration of N-PEDv.

[00167] Similarly, for the saliva spiked samples, the linear region of the calibration curve of Ei was fited using the regression line equation Signal Change Ei = -0.0017 C - 0.08 (R² = 0.9711) while E₂ was fitted using the regression line equation Signal Change E₂ = 0.0419 C + 0.0262 (R² = 0.9682); where ‘C’ represents the concentration of N-PEDv. For the reduced inter-electrode spacing with an applied bias of 0.5 V the regression line equation Signal Change E₂ = 0.0391 C + 3.2984 (R² = 0.9296).

[00168] The limit-of-blank (LOB) for the graphs was subsequently calculated as:

LOB EI ⁼ PB - 3 * OB

LOB _B2 = p_B + 3 * o_B

[00169] Here, ‘pB’ is the mean and ‘oB’ is the standard deviation of the background signal, i.e. the signal obtained upon incubation of the DEE-Chips with unspiked buffer and saliva samples. The LOD was then calculated by determining the concentration (C) where the ‘Signal Change Ef and ‘Signal Change E₂’ value of the regression line becomes equal to the value of LOB _Bi and LOB _B2, respectively.

[00170] Receiver Operator Characteristics Curve. The Receiver Operating Characteristics (ROC) curve analysis was performed using the program Analyse-it for Microsoft Excel (Analyse-it, Leeds UK) on the Signal Change El (Al El / 1 El) and Signal Change E2 (Al E2 / 1 E2) data obtained from the clinical evaluation to determine the decision threshold required to maximize the sensitivity and specificity of the assay.

[00171 ] Results and Discussion

[00172] Aptamer selection and characterization. In order to develop the DEE- Chip aptasensor for detecting PEDv, DNA aptamers specific to the nucleocapsid protein of PEDv (N-PEDv) were selected using previously described magnetic bead-based SELEX technique (Figure la) ^26,39 N-PEDv was chosen as the target analyte due its high degree of expression and ability to conserve large genetic regions during PEDv infection, which minimizes the extent of mutation relative to the spike (S) protein sites.^40,41 [00173] Following ten rounds of selection with a library containing 6*10¹⁴ DNA molecules comprising a random 40-nucleotide sequence capped by fixed-sequence primers on both the 5'-end and a 3 ' -end (see Table 2 for the sequences of the primers used), high-throughput sequencing was conducted using the 10^th selection pool, consistent with a previously described protocol.⁴² The top 50 aptamer sequences based on their abundance in the final pool are listed in Table 3 (named according to their rank). The top five ranked aptamers, PEA1-PEA5 (SEQ IDNOs: 1-5) were selected for binding studies using a gel -based electrophoresis mobility shift assay (EMSA). The extracted KA values (binding affinity) of these aptamers ranged from 2.3 to 26.3 nM (Table 3), values that are comparable with most reported aptamers for pathogen detection and thus validate the quality of the selected sequences for subsequent use in diagnostic assays.⁴³ As PEA1 (SEQ ID NO: 1) yielded the highest affinity KA = 2.3 nM) compared to the other four sequences, it was chosen for further investigation.

[00174] It is widely disseminated that shorter aptamers are more stable, cheaper to produce and easier to immobilize onto electrodes.⁴⁴ Considering these factors, PEA1 was truncated (containing 79 nucleotides; Figure lb) to yield shorter variants. In total, 8 variants, named PEA1-1 to PEA1-8 (SEQ ID NOs: 51-58) were designed and tested for N-PEDv binding using EMSA (Figure 2). A close analysis of the data revealed that PEA1-3 (SEQ ID NO: 53; shortened to 54 nucleotides), shown in Figure 1c, possessed an affinity (KA = 2.8 nM, Figure Id and Figure 3) similar to that the full length PEA1 (SEQ ID NO: 1), while a significant loss in affinity was observed for all other derivatives (Figure 2). This result shows that the LI and L2 loops in PEA1 (SEQ ID NO: 1) play important roles in recognizing the protein target (Figure lb).

[00175] In addition to affinity, the specificity of PEA1-3 (SEQ ID NO: 53) was tested by evaluating its binding to the following four proteins: bovine serum albumin (BSA), human a-thrombin (Tb), RNase H2 of Clostridium difficile (RNase H2) and the target N-PEDv protein (Figure le). BSA is commonly used as a control protein to test the aptamer specificity. Human a-thrombin, on the other hand, was chosen due to the existence of a high-affinity DNA aptamer that specifically recognizes this human protein⁴⁵ while RNase H2 was chosen to represent a nucleic acid binding protein. There was no observance of any significant binding of PEA1-3 (SEQ ID NO: 53) to the control proteins. Further testing of aptamer-specific interactions of PEA1-3 (SEQ ID NO: 53) with the spike (S) and nucleocapsid (N) proteins of SARS-Cov-2 (another virus from the coronavirus family) did not reveal any significant binding (Figure 4), further validating the specificity of the aptamer. Owing to its ability to specifically identify N-PEDv, PEA1- 3 (SEQ ID NO: 53) was chosen for constructing the reagent-less electrochemical assay for detecting PEDv.

[00176] Engineering the dual-electrode electrochemical chip (DEE-Chip). In order to integrate PEA 1-3 (SEQ ID NO: 53) into a reagentless electrochemical biosensor, this aptamer sequence was annealed with a methylene-blue (redox molecule) tagged biobarcode (SEQ ID NO: 67), to create an electroactive aptamer probe (e-aptamer). The e- aptamer was then integrated into a dual-electrode electrochemical chip (DEE-Chip) with two sensing electrodes, Ei and E2 (Figure 5). In this assay, Ei was used for housing the e- aptamers on the electrochemical chip at the time of fabrication to enable reagent-less operation and for validating the chip and assay quality; whereas, E2 was designed for generating the electrochemical signal needed for sample analysis. For constructing the DEE-Chip, the e-aptamers were immobilized onto Ei and E2 was modified with singlestranded DNA (ssDNA) capture probes (SEQ ID NO: 70) specific to the biobarcode sequence of the e-aptamer (Figure 5). Thiolation of the e-aptamers (SEQ ID NO: 63) and capture probes (SEQ ID NO: 71) allowed their covalent attachment onto the electrodes. In the presence of the target protein, the redox barcode (SEQ ID NO: 67) was designed to be released from Ei, diffuse on the DEE-Chip, and then be recaptured on E2 for signal generation (Figure 5c). The two related yet separate events on Ei and E2 are expected to yield a signal attenuation on Ei and signal increase on E2upon target capture (Figure 5b). To facilitate the barcode release from Ei, five barcode sequences (SEQ ID NO: 64-66, 68, 69) were designed with several mismatched base pairs to form partial duplexes with PEA1-3 (SEQ ID NO: 53) and the release of each barcode was assessed using native polyacrylamide gel electrophoresis with radiolabeled barcodes and PEA1-3 (SEQ ID NO: 53) in the presences of N-PEDv protein (Figure 6). Biobarcode-3 (SEQ ID NO: 66), which showed the highest release (91.2 %), was chosen as the bio-barcode for setting up the electrochemical tests (Figure 6).

[00177] Electrochemical chips comprised of star-shaped Ei and E2 gold electrodes patterned onto polystyrene substrates were fabricated for small volume (10 pL) bioanalysis. Electrodeposition of gold was then employed to derive the three-dimensional nanostructured architecture required for sensitive detection at both electrodes^{46, 47} (Figure 7). Diffusion-limited growth stemming from the presence of sharp edges along the star electrodes served as the main driving force in generating this dense nanostructured topography.^{48, 49} To ensure successful reproducibility and function of the devised platform, (1) extensive electrochemical cleaning of the electrodes was conducted prior to e-aptamer and probe bio-functionalization, (2) reproducible electroactive surface area generation was conducted on both Ei and E2 using cyclic voltammetry in sulfuric acid (Figure 8a), and (3) sufficient e-aptamer and probe bio-functionalization was verified for Ei and E2 using cyclic-voltammetry measurements in 2 mM K4Fe(CN)e (Figure 8b). Electrodes that significantly deviated from the expected electroactive surface area and those that did not present the expected redox signature and peak values were not used for further experiments to reduce chip and manufacturing related variabilities.

[00178] The storage stability of DNA functionalized chips was evaluated in a previous study.⁵⁰ Vacuum-packed chips stored at 4 °C demonstrated a 20 % decrease in signal after 30 days, which is in agreement with reported literature on storage stability of similarly functionalized gold electrodes.⁵¹ The high signal-to-background ratio of the assay is expected to enable the assays to be usable despite the signal loss; however, the use of preservation chemistries⁵² might be required in the future to overcome aging effects.

[00179] Electrochemical assessment against viral protein load. Given its importance to the farming industry and the lack of rapid tests currently available, PEDv was chosen as the clinical target. However, this assay design can be easily adapted to other viral and non-viral targets by changing the e-aptamer to one specific to the newly chosen analyte.⁵³'⁵⁸

[00180] Aiming to assess the performance of the DEE-Chip in detecting clinically relevant amounts of viral protein, the platform was challenged with known concentrations of N-PEDv spiked in buffer. Measured current changes at both Ei and E2 (using square wave voltammetry, SWV), were used to probe the DEE-Chip response to viral protein loads of 10 nM (0.6 pg mL'¹) to 500 nM (29 pg mL'¹) following an incubation period of 45 mins and a small sample volume of 10 pL (Figure 9a). The redox currents on each electrode were generated through the electrochemical reduction of methylene blue that was tagged on the DNA barcode (SEQ ID NO: 67).

[00181 ] The ensuing signal-changes were quantified by measuring the redox current before (baseline; 1 Ei before, I E2 before) and after (signal; 1 Ei after, I E2 after) viral protein loading of the DEE-Chip, with the resultant signal-changes ( Signal Change Ei (AIE/ IEi) = (I Ei after - 1 Ei before) 1 Ei before and Signal Change E2 (Al E2 / IE2) = (I E2 after - 1 E2 before) I E2 before) then extracted to yield the sensor response calibration curve (Figure 9b). Prior to target introduction, e-aptamers immobilized on Ei bring methylene blue moieties close to the gold surface. As this electrode is stepped through a series of potential pulses, reduction of methylene blue at the electrode gives rise to a characteristic voltametric peak at - 0.3 V (baseline; Figure 5b (left, inset)).⁵⁹ Concurrently, an absence of this redox signature is observed on E2 due to the lack of methylene blue (baseline; Figure 5b (right, inset)). In this manner, a device comprising a signal-off (Ei) electrode and a signal-on electrode (E2) is designed on a single chip, in which both electrodes are operated under identical experimental conditions.

[00182] As anticipated, opposing trends in signal-changes were observed on Ei and E2 that monotonically decreased (Figure 9b, left) and increased (Figure 9b, right) with target concentration, respectively. Signal saturation was exhibited at both electrodes at a target concentration of 500 nM (29 pg mL'¹), likely due to the saturation of available capture sites on Ei and E2. A regression fit to the linear region of the calibration curve yielded a limit-of-detection (LOD) of 14 nM (0.82 pg mL'¹; 8.2 ng for a sample volume of 10 pL/electrode) for the signal-off electrode, whereas aLOD of 10 nM (0.60 pg mL'¹; 6.0 ng) was exhibited by the signal -on electrode in buffer (Figure 9b).

[00183] To evaluate the effectiveness of the DEE-Chip in working in relevant matrices, the viral protein targets (10 pL sample volume) were spiked in 30 % swine saliva . However, in order to compensate for the increased non-specific adsorption in this biological matrix, thiol terminated polyethylene glycol (PEG) was used as the antifouling agent. Furthermore, the incubation period was increased from 45 mins to 120 mins to account for the increased diffusion time required by the bio-barcode within the more viscous saliva matrix.⁶⁰ Trends mirroring those demonstrated in the buffer-spiked study were seen in SWV curves obtained using saliva-spiked samples (Figure 9c). The signal-change extracted from the voltammetry curves revealed that while both the buffer and saliva study were able to distinguish between samples with and without PEDv on Ei, starting at a concentration of 10 nM (0.6 pg mL'¹), the signal-change was considerably higher in the saliva study (Figure 9d). This is largely attributed to the increased incubation time used in the saliva-spiked study. Even though signal saturation on E2 was observed in buffer starting at a concentration of 250 nM (Figure 9b), this was not observed when saliva samples were used (Figure 9d). This is atributed to the increased viscosity of saliva and slower diffusion of barcode from Ei to E2 as well as non-specific binding between released barcodes and saliva components, all of which could reduce the amount of barcode available at E2. The associated calibration curve, pertaining to the signalchanges at Ei and E2, revealed a LOD of 15.3 nM (0.89 pg mL'¹; 8.9 ng) for Ei and a LOD of 8.0 nM (0.46 pg mL'^ . ng) for E2 in saliva (Figure 9d). Despite minute differences in LOD between buffer spiked and saliva spiked samples, both samples yielded LODs that fall well within the clinically relevant range reported. More specifically, considering the 10 uL analysis volume, the method was capable of detecting as low as 4.6 ng of the protein in saliva, which is in line with the reported values of 440 ng on day 3 or 66.3 ng on day 7 in rectal swabs.⁶¹ This is further bolstered by the finding that a significantly higher magnitude of shedding is observed in oral fluids as compared to rectal swabs during the 14 days-post-infection with PEDv.⁶² The large signal-changes achieved on E2 and the resultant LOD were facilitated by the unique two-electrode design that suppressed background contributions on E2.⁶³ Rapid antigen tests often require sample dilution to both reduce non-specific binding and control sample viscosity.⁶⁴ It was examined whether this was necessary for the newly developed assay (Figure 16). The data demonstrated that despite the observed signal change on Ei in the presence of N- PEDv in undiluted saliva, negligible signals are generated on E2. However, the signal on E2 is recovered when saliva is diluted to 30 %. The lack of signal on E2 in undiluted saliva is likely related to the reduced mass transport from Ei to E2 due to the high viscosity of undiluted saliva, non-specific binding between saliva components and the biobarcode, and the non-specific binding of saliva components with the capture probe on E2, all of which could have affected the signal generation at E2.

[00184] Specificity of the DEE-Chip assay. To further explore the integrity of the devised platform, the engineered DEE-Chip assay was investigated to determine if it could specifically recognize N-PEDv targets. The sensor was challenged by introducing a panel of non-target proteins including thrombin, bovine serum albumin (BSA), and ribonuclease (RNase) spiked in 30% healthy porcine saliva. The DEE-Chip displayed high specificity, accurately distinguishing the N-PEDv samples from the nonspecific panel on both Ei and E2 (Figure 10a- 10c). Moreover, a significantly high signal-change of 10.80 was observed on E2 when it was incubated with the N-PED sample compared to relatively insignificant signal-changes of 0.0, 0.09 and 0.41 for thrombin, BSA and RNase, respectively, demonstrating the specificity of the assay. The use of two separate, yet related biorecognition events - target binding on Ei and biobarcode binding and signal transduction on E2 - reduces the effect of nonspecific binding occurring on Ei on the signal generated on E2, contributing to the high specificity of this assay.

[00185] Kinetics analysis and optimization. While the engineered DEE-Chip embodies the analytical sensitivity and specificity required for N-PEDv detection in saliva, a detection period of 120 mins is undesirable for on-farm testing. To overcome this limitation, strategies such as reduction of inter-electrode distance and electric field- mediated biobarcode transport were explored to determine whether they could be leveraged to accelerate mass transport and reduce the required incubation times.⁶⁵ Towards this end, the kinetics of the sensor response were examined by measuring the signal-changes on the two electrodes as a function of incubation time (Figure 11) for three different electrode configurations (Figure I la). The first configuration represents the original sensor design, wherein an original inter-electrode spacing ‘x’ of 500 pm between Ei and E2 (Figure 11 a(i)). This was used to probe and identify the limitations inherent to the original design (as used in Figure 9). The second configuration features a reduced inter-electrode spacing ‘y’ of 300 pm between the two electrodes (Figure l la(ii)), to directly investigate whether decreasing the inter-electrode distance is sufficient to overcome the challenge of transporting the barcode through the viscous salivary medium to E2 in under 120 mins. The third configuration explored the use of a positive bias potential, applied on E2, in conjunction with an interelectrode spacing of 300 pm (Figure 1 la(iii)) to drive the negatively charged DNA barcode via electro-migration from Ei to E₂.

[00186] Plots representing the release and capture kinetics observed in each of these three scenarios revealed that on Ei, a linear decrease in signal-change is observed between 15 and 60 mins, with the slope of the curve decreasing after 60 mins (Figure 11b) indicating redox-tagged biobarcodes were increasingly released between 15 and 60 mins as target proteins bind to the surface immobilized e-aptamers. A plateau in signalchange was subsequently attained at 120 mins as accessible e-aptamer sites on Ei are exhausted. In scenario ‘i’ (x = 500 pm), capture of the released barcodes begins at 43 mins, rising exponentially until a capture of >50% is attained by 120 mins (Figure 11 c(i)). Using a diffusion coefficient (D) of ~1.18* 10 ¹⁰ m² s ', as deduced from a fluorescein- tagged 33-nucleotide oligomer navigating an aqueous solution,⁶⁶ it wass calculated to take 17.7 mins (r = x²/2D') for the barcode to diffuse through an inter-electrode distance (x) of 500 gm. Given that the signal onset (43 mins) takes longer than the diffusion time (estimated ~18 mins in aqueous media), it can be logically derived that the viscosity of the salivary matrix lowers the reaction kinetics. Reducing the inter-electrode distance to 300 gm to overcome this inherent diffusion time barrier causes the theoretical diffusion time to reduce to 6.4 mins, which correlates to a practical observed signal onset at 33 mins (Figure l lc(ii)). Inspired by the electrophoretic motion of DNA fragments through gels, a positive potential bias was applied across E2 to further aid in diffusion of the barcode through saliva. Time-resolved electrochemical measurements revealed that while signal onset mirrored that of detection without the application of a bias-potential (scenario ii), the signal-change increased at a much faster rate between 30 and 60 mins, reaching 75% of its final value at 60 minutes compared to much lower values for the first (20%) and second (45%) configurations (Figure l lc(iii)). The improved signal detection achieved for sensors employing both the reduction of inter-electrode distance and the application of a bias potential supported the reduction of detection time from 120 mins (scenario i; the original design) to 60 mins (using an optimized design) for facilitating rapid on-farm analysis. The specificity and limit-of-detection of DEE-Chips were measured with a reduced inter-electrode distance and under an applied bias (scenario (iii)). The redesigned chips resulted in an improvement in the limit-of-detection measured on E2 (6 nM in saliva and in 60 minutes). Additionally, the chip redesign did not have any effect on the assay specificity (Figure 15).

[00187] Clinical Detection. Given the literature evidence supporting the presence of sufficient viral loads in the oral fluid samples for up to 35 days post-infection as measured by reverse transcription PCR (RT-PCR), viral detection in saliva from clinical samples was expected to be feasible using this assay.² Next, the capability of the DEE- Chip was assessed for detecting PEDV in clinical swine saliva samples. 12 anonymized, clinically-sourced swine oral fluid samples were analyzed (Figure 12), six of which were established as PEDv-positive and 6 of which were determined to be PEDv-negative by RT-PCR (Table 1). Following lysis and dilution, the porcine saliva samples were analyzed using DEE-Chips for 60 mins (Figure 12a).

[00188] PEDv-positive samples exhibited a larger signal change (0.44-0.75) compared to PEDv-negative samples (0.05-0.30) on Ei (Figure 12b). This was paralleled by the much larger signal differences observed between positive (3.0-20.8) and negative samples (<0.90) on E2 (Figure 12c). The DEE-Chip was unable to identify the presence of PEDv in positive sample 4. In addition, small variations in the magnitude of signalchange were observed with the different samples, which is likely due to the differences in the composition of the individual saliva samples and their respective viral loads. Despite these variations, the electrochemical assay retains its performance over a range of statistically significant cycle threshold (Ct) values derived from PCR (i.e., Ct of 28.9 - 28.6 for positive samples 1 - 3 and Ctof33.2 - 33.1 for positive samples 5 - 6). The falsenegative sample 4 had the largest Ct value (33.9) amongst the samples and, as such, the lowest viral load. There are two concepts for the rationale behind the false-negative result generated using sample 4: (1) an insufficient viral load and (2) the presence of salivary inhibitory factors that prevent bio-recognition by the aptamer on EL These two concepts were tested by re-analyzing the false-negative sample and manually spiking it with PEDv at a concentration of 500 nM (Figure 13). The signal-changes elicited were then compared against an unspiked false-negative analogue and a PEDv negative sample. Of these, only the PEDv spiked samples exhibited a significant signal-change (0.53) on Ei and a large gain in signal-change (17.83) on E2. The ability of the DEE-Chip to detect the spiked sample rules out salivary inhibitory factors, pointing to the insufficient viral load as the reason for the negative response (consistent with the high Ct value of sample 4).

[00189] Receiver Operator Characteristics (ROC) curves constructed from the data from the DEE-Chip (Figure 12b, 12c), were subsequently used to assess the clinical performance characteristics of this assay (Figure 14). ^{24, 67} An overall accuracy, or area under the curve (AUC), of 0.975 (CI, 0.936 -1.014), sensitivity of 83%, and specificity of 100% was obtained at a decision threshold of -0.34 (signal-change) on EL A corresponding AUC of 0.873 (CI, 0.735 -1.012), sensitivity of 83%, and specificity of 100%, at a threshold of 1.16 (signal-change) was obtained using E2 data. A calibration curve (Figure 15a) was utilized to convert electrochemical metrics into analytical metrics (Table 4). The signal ranges for the positive samples on E2 ranged from 12-613 nM (0.68-27 pg mL ¹). Inspection of the clinical performance metrics (sensitivity, specificity and AUC) revealed that the DEE-Chip performance fell well within the standard threshold of a reliable diagnostic test (AUC > 0.8, ⁶⁸ sensitivity > 80%⁶⁹ and specificity > 90%⁶⁹), thereby indicating the potential of this assay for future clinical use.

[00190] Discussion [00191 ] Considering recurrent surges in PEDv outbreaks coupled with increasing reports of more highly communicable and infective variants of concern, the need for a simple, on-farm, saliva-based PEDv test is more critical than ever. In response, a rapid, simple, and reagent-free assay for on-farm detection of PEDv was developed. More specifically, a dual-electrode electrochemical (DEE) chip featuring functional e-aptamers to bridge the current diagnostic gap — the reliance on centralized laboratories — for PEDv testing. Towards this end, SELEX was first employed to select a novel high- affinity DNA aptamer (K = 2.3 nM) that discriminately targeted the highly conserved, mutation-resistant N-protein of the PEDv virus. The DEE-Chip used a unique approach for integrating these PEDv aptamer for reagent-less sensing; it included one electrode for housing the aptamer annealed with a redox DNA barcode and another electrode for capturing the DNA barcode and transducing an electrochemical signal. Using this approach, the DEE-Chip yielded a clinically relevant LODs of 14 nM (0.82 pg mL'¹; on Ei) and 10 nM (0.60 pg mL'¹ ; on E2) in 10 pL of buffer-spiked N-PEDv samples; while clinically relevant LODs of 15 nM (0.89 pg mL'¹ ; Ei) and 8 nM (0.46 pg mL'¹ ; E2) were obtained using saliva-spiked N-PEDv samples.

[00192] To reduce the assay time of the DEE-Chip from (initially 120 mins) for rapid on-farm testing, a focus was placed on engineering the inter-electrode spacing of the chip and the use of a bias potential to enhance the mass transport of the DNA barcode from one electrode to another. Using the rationally-engineered DEE-Chip, 12 clinically- derived swine saliva samples were analyzed. The DEE-Chip successfully analyzed 92% (and 100% of all samples for Ct < 33.17) of the clinical samples (sample volume of 12 pL) with a clinical sensitivity of 83%, specificity of 100%, and LOD of 6 nM (0.37 pg mL'¹; E2) in 60 mins without target amplification, target enrichment, target labelling, or the addition of readout reagents. The DEE-Chip is versatile and can be integrated with other e-aptamers for detecting various bioanalytes, including other bacterial and viruses, for fulfilling the unmet need of rapid and on-farm animal disease surveillance.

[00193] While the present disclosure has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole. [00194] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present disclosure is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

TABLES

Table 1. RT-PCR testing results of the clinically acquired swine oral fluid samples.

Table 2. DNA oligonucleotides used in this disclosure. Sequences are written 5’-3’. N40: 40-nucleotide random region; L: non-amplifiable linker. [5ThioMC6-D] : Thiol modifier; [MB]: methylene blue. Complementary bases between PEA1-3 and biobarcode are underlined.

Table 3. DNA sequences in pool 10 ranked by their percentage.

Table 4. Concentration of PEDv positive clinical samples.

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Claims

CLAIMS:

1. An aptamer comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-58, or a functional fragment and/or functional variant thereof, that binds to porcine epidemic diarrhea virus (PEDv) nucleocapsid protein.

2. The aptamer of claim 1, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5 and 51-58.

3. The aptamer of claim 1 or 2, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 and 51-58.

4. The aptamer of any one of claims 1 to 3, comprising a nucleotide sequence of SEQ ID NO: 1 or 53.

5. The aptamer of any one of claims 1 to 4, comprising a nucleotide sequence of SEQ ID NO: 53.

6. The aptamer of any one of claims 1 to 5, consisting of a nucleotide sequence of SEQ ID NO: 53.

7. The aptamer of any one of claims 1 to 6, wherein the aptamer binds to the PEDv nucleocapsid protein with at least nanomolar affinity.

8. An aptamer probe, comprising the aptamer of any one of claims 1 to 7 and a detectable label.

9. The aptamer probe of claim 9, wherein the detectable label is a fluorescence moiety, a colorimetric moiety, an electrochemiluminescent moiety, a photoelectrochemical moiety or an electrochemical moiety.

10. The aptamer probe of claim 9, wherein the detectable label is an electrochemical moiety.

11. The aptamer probe of claim 10 or 11, wherein the electrochemical moiety is a redox species.

12. The aptamer probe of claim 11 , wherein the redox species comprises methylene blue or ferrocene.

13. The aptamer probe of any one of claims 8 to 12, further comprising a reporter moiety coupled to the detectable label.

14. The aptamer probe of claim 13, wherein the reporter moiety comprises a nucleotide sequence complementary to at least a portion of the aptamer.

15. The aptamer probe of claim 13 or 14, wherein the reporter moiety comprises a nucleotide sequence consisting of the group selected from SEQ ID NOs: 64-69.

16. The aptamer probe of any one of claims 12 to 15, wherein the reporter moiety comprises a nucleotide sequence of SEQ ID NO: 66 or 67.

17. The aptamer probe of any one of claims 13 to 16, wherein the reporter moiety is releasable from the aptamer in the presence of PEDv nucleocapsid protein.

18. A biosensor for detecting PEDv nucleocapsid protein comprising the aptamer of any one of claims 1 to 7 or the aptamer probe of any one of claims 8 to 17 functionalized on and/or in a material.

19. The biosensor of claim 18, further comprising: a) a first working electrode, wherein the aptamer probe of any one of claims 13 to 17 is functionalized on the first working electrode; b) a second working electrode positioned in proximity to the first working electrode; c) a capture probe functionalized on the second working electrode; and d) a counter electrode; wherein each working electrode is configured to provide a change in signal if the PEDv nucleocapsid protein is present.

20. The biosensor of claim 19, wherein the aptamer probe is functionalized on the first working electrode via chemical bonding, an intermediate linker, or physical adsorption.

21. The biosensor of claim 19 or 20, wherein the capture probe is functionalized on the second working electrode via chemical bonding, an intermediate linker, or physical adsorption.

22. The biosensor of claim 20 or 21, wherein the chemical bonding occurs via thiol and/or gold chemistry.

23. The biosensor of any one of claims 19 to 22, wherein the capture probe is for recognizing and coupling to the reporter moiety of the aptamer probe.

24. The biosensor of any one of claims 19 to 23, wherein the capture probe comprises a nucleotide sequence of SEQ ID NO: 71 or 72.

25. The biosensor of any one of claims 19 to 24, wherein the working electrode comprises a conductive material, semi-conductive material, or a combination thereof.

26. The biosensor of any one of claims 19 to 25, wherein the working electrode comprises metal, metal alloy, metal oxide, superconductor, semi-conductor, carbonbased material, conductive polymer, or combinations thereof.

27. The biosensor of any one of claims 19 to 26, wherein the working electrode comprises metal.

28. The biosensor of claim 27, wherein the metal is gold.

29. The biosensor of any one of claims 19 to 28, further comprising a reference electrode.

30. The biosensor of any one of claims 19 to 29, further comprising a blocking species on the first working electrode and/or second working electrode.

31. The biosensor of any one of claims 19 to 30, wherein the biosensor is on an electrochemical chip.

32. The biosensor of any one of claims 19 to 31 , wherein the first working electrode is configured to provide a decrease in the signal and the second working electrode is configured to provide an increase in the signal in the presence of PEDv nucleocapsid protein.

33. The biosensor of any one of claims 19 to 32, wherein the change in signal comprises a change in current, potential or impedance.

34. The biosensor of any one of claims 19 to 33, wherein the change in signal is a change in current.

35. A method for detecting the presence of PEDv nucleocapsid protein in a sample, comprising: a) obtaining a sample from a subject; b) combining the sample with a liquid in a sample container to form a mixture; c) exposing the mixture to the biosensor of any one of claims 19 to 34; whereby the reporter moiety of the aptamer probe functionalized on the first working electrode is delocalized from the first working electrode to the second working electrode upon binding of the aptamer of the aptamer probe functionalized on the first working electrode to the PEDv nucleocapsid protein; and d) measuring a change in signal at the first and/or second working electrode, wherein a change in signal is produced if the PEDv nucleocapsid protein is present in the sample.

36. The method of claim 35, further comprising incubating the mixture at about 60 °C for about 25 minutes before step c).

37. The method of claim 35 or 36, wherein the biosensor comprises a reference electrode and the method further comprises applying a positive potential bias across the first and/or second working electrode before and/or during step d).

38. The method of any one of claims 35 to 37, wherein the liquid is a buffer.

39. The method of any one of claims 35 to 38, wherein the mixture is exposed to the biosensor under conditions to delocalize the reporter moiety from the first working electrode to the second working electrode.

40. The method of any one of claims 35 to 39, wherein the mixture is exposed to the biosensor for about 60 minutes before step d).

41. The method of any one of claims 35 to 40, wherein the change in signal from the first working electrode is a decrease in signal and the change in signal from the second working electrode is an increase in signal.

42. The method of any one of claims 35 to 41, wherein the change in signal comprises a change in current, potential or impedance.

43. The method of any one of claims 35 to 42, wherein the change in signal is a change in current.

44. The method of any one of claims 35 to 43, wherein the sample comprises saliva.

45. The method of any one of claims 35 to 44, wherein the method detects PEDv infection in the subject.

46. The method of any one of claims 35 to 45, wherein the subject is a porcine animal.

47. A kit for detecting PEDv nucleocapsid protein, wherein the kit comprises the biosensor of any one of claims 18 to 34 or components required for the method of any one of claims 35 to 46, and instructions for use of the kit.

48. The kit of claim 47, further comprising at least one collection apparatus, at least one sample container, a buffer, and instructions for use.