DNA net nanostructure-enabled rapid and sensitive testing methods for detection of viral infections

Abstract

Provided are compositions including a DNA net nanostructure that are useful for the detection of viruses, particularly in lateral flow assay format. Also provided are lateral flow assay test strips and methods of use thereof.

Description

[0001] DNA NET NANOSTRUCTURE-ENABLED RAPID AND SENSITIVE TESTING METHODS FOR DETECTION OF VIRAL INFECTIONS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 625,683, filed January 26, 2024, which is incorporated herein by reference in its entirety.

[0004] FIELD

[0005] This disclosure relates to assays for detecting viruses, particularly lateral flow assays.

[0006] ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0007] This invention was made with government support under R01A1159454, R44DE030852, and U01AA029348 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] INCORPORATION OF ELECTRONIC SEQUENCE LISTING

[0009] The Sequence Listing is submitted as an XML file named “Sequence. xml,” created on January 24, 2025, 14,792 bytes, which is incorporated by reference herein.

[0010] BACKGROUND

[0011] The COVID-19 pandemic has brought into focus the urgent and critical need for accurate and accessible diagnostic platforms that enable individuals to test themselves and obtain rapid but still sensitive results in point-of-care (POC) and home settings (Budd et aL, Nat. Rev. Bioeng. 2023, / , 13-31; Pinheiro et al., ACS Omega 2021, 6, 29268-29290). As the world grapples with the challenges posed by the pandemic, the demand for reliable and convenient diagnostic methods has exponentially surged (Dong et al., Chem. Sci. 2023, 14, 6149-6206; Tang et al., Chin. Chem. Lett. 2023, 143, 17004-17014). Among the array of available diagnostic methods, lateral flow assays (LFAs) have emerged as the preferred and widely accepted gold standard platform for self-testing, owing to their remarkable attributes such as portability, cost-effectiveness, user-friendliness, and expeditious results. Consequently, LFAs have become the most extensively utilized diagnostic platform for COVID-19 self-testing due to their versatility in accommodating different bodily fluids and suitability for both home and POC settings (Tang et al., Chin. Chem. Lett. 2023, 143, 17004- 17014). The majority of commercially available LFAs utilize antibody or antigen-based testing methodologies. While antibody -based LFAs do not directly detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus itself, they instead assess the host immune response to SARS-CoV-2 infections, aiding in the identification of individuals with past exposure to the virus (Huang et aL, ACS Omega 2020, 5, 12550-12556). By detecting immunoglobulin antibodies produced by B cells in bodily fluids, these LFAs provide insights into the body's immune response during viral infection (Bandrowski et al., Nucleic Acids Res. 2023, 51, 358-367; Kopel et al., Bayl. Univ. Med. Cent. 2021, 34, 63-72). However, these tests exhibit low specificity during the initial week of viral exposure, with specificity improving during the subsequent second and third weeks, owing to the time required for immunoglobulin antibodies to develop following disease onset (Kopel et al., Bayl. Univ. Med. Cent. 2021, 34, 63-72; Liu et al., / ACS' Sens. 2021, 6, 593-612; Lisboa et aL, BMJ. 2020, 370, m2516; Sethuraman et al., J. Am. Med. Assoc. 2020, 323. 2249-2251). To surmount this limitation, antigen-based LFAs have been developed, targeting the nucleocapsid protein in the case of SARS-CoV-2 detection (Lee et al., Small 2023, 19, 2208035; Grant et al., Anal. Chem. 2020, 92, 11305-11309).

[0012] Antigen-based LFAs offer convenience and scalability, and the ability for viral detection in selftesting, thereby playing a crucial role in monitoring and curbing the dissemination of highly contagious mutant viral strains (Hristov et al., Anal. Chem. 2021, 93, 7825-7832). Nevertheless, they possess drawbacks when compared to nucleic acid tests such as real-time reverse transcription-PCR (RT-PCR). These limitations include reduced test specificity using N protein targeting antibodies and the inability to detect low viral loads from non-invasive samples (Yamaniha et al., J. Infect. Chemother. 2021, 27, 1112- 1114; U.S. Food and Drug Administration (FDA). “Potential for False Positive Results with Antigen Tests for Rapid Detection of SARS-CoV-2 - Letter to Clinical Laboratory Staff and Health Care Providers,” 2020; Gremmels et al., eClinical Medicine 2021 , 31, 100677). The lack of specificity can lead to both falsepositive and false-negative test results. For instance, Gremmels et al. demonstrated that the "PanbioTM COVID-19 Ag rapid test" could detect high viral loads in nasopharyngeal samples of SARS-CoV-2 infected individuals but also produced several false-negative rapid test results (Gremmels et al., eClinical Medicine 2021, 31, 100677). This becomes particularly problematic during the pre-symptomatic phase when viral loads in patient samples are exceedingly low, often resulting in false-negative antigen test outcomes (Robinson et al., J. Clin. Microbiol. 2022, 60, e00187-22; Sethuraman et al., J. Am. Med. Assoc. 2020, 323, 2249-2251).

[0013] In addition to false-negatives, rapid antigen tests have also shown instances of false-positives for SARS-CoV-2 (Mouliou et al., Expert. Rev. Respir. Med. 2021, 15, 993-1002; Patriquin et al., Microbiol. Spectr. 2023, 9, 1-15; Itoh et al., J. Infect. Chemother. 2021, 27, 1089-1091). Yamaniha et al. reported a case where a rapid antigen test demonstrated recurrent positive results for SARS-CoV-2 in an individual with acute human immunodeficiency virus (HIV) infection (Yamaniha et al., J. Infect. Chemother. 2021, 27, 1112-1114). Interestingly, the authors observed consistent negative outcomes in RT-PCR tests performed on nasopharyngeal swabs. These results may be attributed to the presence of shared epitope motifs and a certain degree of homology between the surface glycoproteins of HIV and SARS-CoV-2 (Alfie et aL, AIDS 2023, 37, 947-950).

[0014] False-positive results are concerning as they can lead to inappropriate patient care and infection control measures. Conversely, false-negative test results for asymptomatic close contacts can engender a false sense of reassurance, potentially leading to the relaxation of isolation measures and a subsequent heightened transmission rate. Thus, the test specificity of antigen-based LFAs using antibodies becomes a significant concern, as they allow the virus to go undetected during community-wide screening, thereby perpetuating the burden on the public healthcare system (Robinson et al., J. Clin. Microbiol. 2022, 60, e00187-22). This underscores the need to develop innovative solutions capable of enhancing the performance of antigen-based LFAs, augmenting specificity, and enabling the detection of low viral loads from non-invasive samples such as saliva (Frutiger et aL, Chem. Rev. 2021, 121, 8095-8160; Liu et al., ACS Nano 2021, 15, 3593-3611).

[0015] SUMMARY

[0016] Provided are lateral flow assay test strips including a lateral flow membrane including a test line, wherein the test line includes an immobilized DNA net nanostructure. The DNA net nanostructure includes DNA aptamers that bind to a target virus. In some aspects, the DNA aptamers bind a viral protein of the target virus, for example, a viral spike protein. The LFA test strip can further include a sample pad, conjugate pad, and / or wicking pad. When a conjugate pad is present, the conjugate pad includes a reporter composition. In some aspects, the reporter composition includes a DNA net nanostructure specific to the target virus linked to a reporter, for example, a gold nanoshell, gold nanoparticle, or quantum dot. Also provided are kits including a LFA test strip disclosed herein.

[0017] Further provided are methods of detecting a target virus in a sample, the methods including adding a sample to an LFA test strip disclosed herein and detecting a signal, thereby detecting the target virus in the sample. In some aspects, the methods include (i) adding the sample to a sample pad of the LFA test strip, wherein: (a) the LFA test strip includes a conjugate pad including a first DNA net nanostructure including a DNA aptamer that binds the target virus, wherein the first DNA net nanostructure is linked to a reporter, and (b) the LFA test strip includes a lateral flow membrane including a second DNA net nanostructure including a DNA aptamer that binds the target virus immobilized on a test line; and (ii) detecting a signal from the test line, thereby detecting the target virus in the sample. In other aspects, the methods include: (i) mixing the sample with a first DNA net nanostructure including a DNA aptamer that binds the target virus, wherein the first DNA net nanostructure is linked to a reporter, thereby producing a mixed sample; (ii) adding the mixed sample to a sample pad of an LFA test strip, wherein the LFA test strip includes a lateral flow membrane including a second DNA net nanostructure including a DNA aptamer that binds the target virus immobilized on a test line; and (iii) detecting a signal from the test line, thereby detecting the target virus in the sample. In some aspects, the reporter is a gold nanoshell, gold nanoparticle, or quantum dot. In some aspects, the target virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV, e.g., HIV-1 or HIV-2), porcine deltacoronavirus (PDCO), porcine epidemic diarrhea (PED), hepatitis B virus (HBV), human papillomavirus (HPV), dengue virus (DENV), or influenza A virus (IAV). In some aspects, the subject is a human or pig.

[0018] The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES

[0019] FIGS. 1A-1C. Reporter assay optimization. (FIG. 1A) Atomic force microscopy (AFM) was employed to capture high-resolution images of DNA Net structures with different dimensions, including 2 x 2, 3 x 3, and 4 x 4 DNA Nets. The AFM images reveal the intricate arrangement and organization of the DNA Net structures at a nanoscale level and scale bars indicating a length of 50 nm. (FIG. IB) Different sizes of DNA Nets were coated on gold nanoshells (AuNS), resulting in an increase in the size of AuNS- conjugated DNA Net reporter assay as the size of DNA Net increased. (FIG. 1C) The zeta potential decreases as the size of DNA Nets increases, indicating improved assay stability. Data are presented as the mean + sd, n = 5 biologically independent samples.

[0020] FIGS. 2A-2C. LFA device testing with free trimeric spike protein (TSP) as a target. (FIG. 2A) Schematic representation of the operation of the LFA device and the detection of free TSP using DNA Net. (FIG. 2B) A range of TSP concentrations from 1.7 nM - 52 nM were tested, leading to a gradual increase in the intensity of the test line on the LFA device. (FIG. 2C) The absorbance values from the test line were calculated and plotted against the corresponding TSP concentrations. The binding isotherm of TSP was analyzed using Hill fit, yielding a dissociation constant (Kn) of 7.25 ± 0.59 nM. Additionally, a linear detection range for TSP was observed, spanning from 1.7 nM - 13 nM (inset, FIG. 2C). The data are presented as the mean ± SD (standard deviation), with n = 3 biologically independent samples.

[0021] FIGS. 3A-3D. LFA device performance in different biological environments. (FIG. 3A) The LFA device was tested in a saliva environment with varying concentrations ranging from 1% to 75%. A negative symbol (-) indicates the absence of the TSP target, while a positive symbol (+) represents the presence of a 4 nM TSP target. (FIG. 3B) Similarly, the LFA device was tested in a urine environment with varying concentrations ranging from 1% to 75%, using the same TSP target indication symbols. (FIG. 3C) Furthermore, the LFA device was also tested in a serum environment with varying concentrations ranging from 1% to 75%, using the same TSP target indication symbols. (FIG. 3D) The absorbance values from the test line on the LFA device are plotted against different percentages of the target analyte (saliva / serum / urine), providing a visual representation of the device's performance in different biological environments. The data are presented as the mean + SD (standard deviation), with n = 3 biologically independent samples.

[0022] FIGS. 4A-4D. LFA device performance and selectivity. (FIG. 4A) The performance of the LFA device was evaluated in a 25% saliva environment, with a range of TSP concentrations tested from 4.3 nM to 52 nM. (FIG. 4B) The absorbance values from the test line were calculated and plotted. The binding isotherm of TSP was analyzed using Hill fit, resulting in KD value of 5.72 ± 0.28 nM. (FIG. 4C) The selectivity of the LFA device was assessed by using different trimeric spike proteins, including Influenza- HA and HIV GP-120, each at a concentration of 1 pM, along with 25 nM TSP and 1% serum. (FIG. 4D) The corresponding absorbance values from the test line were plotted against the different targets, demonstrating the selectivity of the LFA device. The data are presented as the mean ± SD (standard deviation), with n = 3 biologically independent samples. FIGS. 5A-5F. The LFA device's performance was evaluated using different strains of SARS- CoV-2 viruses, including (FIG. 5A) WA1 / 2020 / Washington, (FIG. 5B) B.1.1.7 / 2020 / UK, (FIG. 5C) B.1.351 / 2020 / South Africa, and (FIG. 5D) Omicron variant XBB.1.5 / 2022 / U. The viruses were serially diluted (107- 102 / 100 pL reaction volume) with IX PBS and 25% saliva. A buffer composed of IX PBS and 25% saliva was used as the negative control (0 virus). (FIG. 5E) To facilitate comparison, a commercially available COVID-19 antigen rapid test kit (iHealth) was utilized with the SARS-CoV-2 WA1 / 2020 / Washington strain. (FIG. 5F) The absorbance values from each test line (FIGs. 5A-5E) were plotted against the total number of viral copies in the solution, allowing for an assessment of LFA device performance. The data are presented as the mean ± SD (standard deviation), with n = 3 biologically independent samples.

[0023] FIGS. 6A-6C. An illustration of DNA Net-based LFA device design and operation. (FIG. 6A) Rhombus-shaped DNA Nets with different sizes, including “2 x 2 DNA Net (30 nm x 30 nm)”, “3 x 3 DNA Net (45 nm x 45 nm)”, and “4 x 4 DNA Net (60 nm x 60 nm)” were created. ★ shows DNA aptamer cluster positions across the DNA Net. Tri-aptamer clusters are positioned with a -6 nm intra- and a -15 nm inter- tri-aptamer spacing, mirroring the arrangement of spike trimers on viral particle. (FIG. 6B) A 3 x 3 DNA Net (10 nM) was implemented on the test line using biotin-streptavidin chemistry, while 0.15 mg / mL of SARS-CoV-2 trimcric spike protein (SARS-TSP) was placed on the control line. Streptavidin-coated gold nanoshells (SA-AuNS) were coated with a 3 x 3 DNA Net and used as a reporter assay. (FIG. 6C) Upon introducing virus particles into the LFA device, they bind in a sandwich format, utilizing the reporter assay and DNA Net present on the test line. The reporter assay also binds with the TSP on the control line through aptamer-TSP interaction. The aggregation of AuNS results in an enhancement in colorimetric signals in both the test and control lines.

[0024] FIG. 7. Exemplary demonstration of an LFA and methods of its use.

[0025] FIG. 8. Formation of the DNA Nets was characterized by 1% agarose gel electrophoresis (AGE). Yields of the 2 x 2 Net, 3 x 3 Net, and 4 x 4 Net are 96.61%, 88.01%, and 77.51%, respectively.

[0026] FIG. 9. Atomic Force Microscopy (AFM) images depicting the morphology of 2 x 2, 3 x 3 and 4 x 4 DNA Nets. The scale bars, prominently displayed in the images, indicate a length of 200 nm. These images serve to elucidate the susceptibility of the delicate DNA Net structures, based on tile-based architectures, to potential damage or displacement by the AFM probe during the scanning process.

[0027] FIG. 10. Reporter assay controls on the LFA platform. DNA Nets, aptamer and AuNS were introduced in different combinations to check test line (T) and control line (C) signal developments using (1) DNA Net (no biotin)-AuNS complex, (2) aptamer only (no biotin)-AuNS complex, (3) AuNS only (without any DNA strand), (4) DNA Net only, (5) Aptamer strand only, (6) Running Buffer only.

[0028] FIGS. 11A-11B. Time dependent signal amplification on LFA device. Signal amplification of Test line (T) and control line (C) until saturation was assessed using a buffer consisting of IX PBS and 25% saliva. (FIG. 11 A) In the absence of the virus and any spike protein, only the control line exhibited a colorimetric signal enhancement, starting at 2 minutes until reaching saturation within 10 minutes. (FIG. 1 IB) When the WA1 / 2020 / Washington strain of SARS-CoV-2 viruses (104 / reaction) was introduced into the buffer, both the test line and control line began developing colorimetric signals past 7 minutes.

[0029] FIGS. 12A-12C. Surface plasmon resonance Surface plasmon resonance (SPR) assay to determine the binding affinity of the aptamer with spike proteins of different viral strains. (FIG. 12A) SARS-CoV-2 XBB.1.5, (FIG. 12B) SARS-CoV-2 B.1.351, and (FIG. 12C) SARS-CoV-2 B.1.1.7. A range of spike protein concentrations were tested (Ci - Cs): 0.03906, 0.1563, 0.625, 2.5 and 10 LlM, respectively. Dissociation constant (KD) of the monovalent aptamer binding to trimeric spike was calculated by SPR. The reported KDvalues for XBB.1.5, B.1.351 and B.l.1.7 are 1.55xl09M, S.SlxlO8M and 2.15xl0‘7M. Experiments were repeated for 3 independent times with similar results.

[0030] FIG. 13. Detailed, stepwise serial dilution procedure for preparing virus samples with the desired virus copies per 100 pL reaction.

[0031] FIGS. 14A-14F. Exemplary LFA test strip configurations. FIGS. 14A-14E provide a top view. FIG. 14F shows a bottom view of a backing card upon which LFA test strip components can be adhered.

[0032] FIGS. 15A-15G. Surface plasmon resonance (SPR) assay. (FIG. 15 A) Schematic of the SPR assay used to determine affinity of an aptamer binding. IgG protein at a concentration of 50 pg / mL served as the negative control in Flow Cell 1 (FCl). FIGS. 15B & 15E: Flow Cell 2 (FC2) was dedicated to immobilizing 50 pg / mL of HIV-1 Group M, subtype A isolate 92RW020 spike protein (GP120MA). FIGS. 15C & 15F: Flow Cell 3 (FC3) was employed to immobilize 50 pg / mL of HIV-1 Group M, subtype B (GP120MB). FIGS. 15D & 15G: Flow Cell 4 (FC4) was utilized to immobilize 50 pg / mL of HIV-1 Group P, strain RBF168 (GP120P). Subsequently, varying concentrations of aptamers (ranging from 0.51 nM - 10000 nM) were introduced into each flow cell sequentially (FC2-FC4), for 600 s at a flow rate of 30 pL / min. FIGS. 15B-15D show interaction of HApt-1 aptamer with respective proteins present on FC1-FC4, while FIGS. 15E-15G highlight the interaction of the HApt-2 aptamer with the corresponding proteins on FC1-FC4. Each sensorgram is repeated six independent times with similar results and corrected for non-specific interaction of aptamers with negative control. The resultant sensorgrams were employed to determine binding kinetics parameters, including the association rate constant (ka), dissociation rate constant (kd), and binding equilibrium dissociation constant (KD; KD = kd / ka). These parameters were determined through global fitting of the complete association and dissociation phases using the 1 : 1 Langmuir binding model within BiaEvaluation software 4.0.1, developed by GE Healthcare Uppsala, Sweden.

[0033] FIGS. 16A-16C. DNA-Net design, synthesis, and characterization. (FIG. 16A) A representation of the rhombus-shaped 3 x 3 DNA-Net, measuring 45 nm x 45 nm. The ★ symbols indicate the positions of DNA aptamer clusters with a total of 27 tri-aptamer clusters distributed across the DNA-Net. Tri-aptamer clusters were strategically placed with approximately 6 nm intra-tri-aptamer spacing and 15 nm inter-tri- aptamer spacing, resembling the arrangement of spike trimers on a viral particle. (FIG. 16B) Formation of 3 x 3 DNA-Net was analyzed using 1% agarose gel electrophoresis. (FIG. 16C) Atomic force microscopy (AFM) was employed in capturing high-resolution images of 3 x 3 DNA-Net structure. Scale bar in the AFM image denotes a length of 25 nm. FIGS. 17A-17F. Surface plasmon resonance (SPR) assay. (FIG. 17A) Schematic of the SPR assay used to determine affinity of the DNA-Net-aptamer complex binding. IgG protein at a concentration of 50 g / mL served as the negative control in Flow Cell 1 (FCi). FIGS. 17A and 17D: Flow Cell 2 (FC2) was dedicated to immobilizing 50 g / mL of HIV-1 Group M, subtype A (GP120MA)- FIGS. 17B & 17E: Flow Cell 3 (FC3) was employed to immobilize 50 pg / mL of HIV-1 Group M, subtype B (GP120MB). FIGS. 17C & 17F: Flow Cell 4 (FC4) was utilized to immobilize 50 pg / mL of HIV-1 Group P (GP120p). Subsequently, various concentrations of DNA-Net tethered with aptamers (0.01 nM - 150 nM) were introduced into the respective flow cells for 600 s at a flow rate of 30 pL / min. FIGS. 17A-17C show interaction of DNA-Net- HApt-1 assays with respective proteins present on FC1-FC4, while FIGS. 17D-F highlight the interaction of the DNA-Net-HApt-2 assay with the corresponding proteins on FC1-FC4. Each sensorgram is presented from six independent repetitions, yielding consistent results and corrected for the non-specific interaction of aptamers with the negative control.

[0034] FIGS. 18A-18B. Reporter assay labeling with 3 x 3 DNA-Net. (FIG. 18A) 3 x 3 DNA-Nets were coated on gold nanoshells (AuNS), resulting in an augmentation of the size of AuNS-conjugated DNA-Net reporter assays with an increase in the DNA Net size. (FIG. 18B) The zeta potential exhibited a decrease when DNA-Nets were coated on AuNS, indicating enhanced assay stability. Data are presented as the mean ± SD, with n = 5 independent samples.

[0035] FIGS. 19A-19E. DNA-Net-HApt-1 assay-based LFA device testing with GP120MB protein as a target. (FIG. 19 A) DNA-Net -based lateral flow assay (LFA) device is designed for the detection of spike proteins in free solution (FIGS. 19B, 19D in this figure) and eventually on virion outer surface (see, FIGS. 5A-5D). The ★ symbols indicate the positions of DNA aptamer clusters with a total of 27 tri-aptamer clusters distributed across the DNA-Net. Tri-aptamer clusters were strategically placed with approximately 6 nm intra-tri-aptamer spacing and 15 nm inter-tri-aptamer spacing. Streptavidin-coated gold nanoshells (SA-AuNS) were labeled with DNA-Nets and employed as a reporter assay. In the LFA setup, 10 nM DNA-Net was applied to the test line using biotin-streptavidin chemistry, while 0.15 mg / mL of GP120 trimeric spike protein was deposited on the control line. Upon introducing spike protein or virus particles into the LFA device, they bind in a sandwich format with the reporter assay and DNA-Net located on the test line. Simultaneously, the reporter assay interacts with the GP120 on the control line through aptamerprotein interactions. The aggregation of AuNS leads to an enhancement in colorimetric signals observed in both the test and control lines. (FIG. 19B) The LFA device was subjected to a range of GP120MB concentrations spanning from 1.95 nM to 250 nM, resulting in a gradual increase in the intensity of the test line (T). (FIG. 19C) Absorbance values from the test line were calculated and plotted against the respective GP120MB concentrations. The binding isotherm of GP120MB was analyzed using Hill fit. (FIG. 19D) The LFA device's performance was assessed in a 50% serum environment, with a similar range of GP120MB concentrations. (FIG. 19E) Absorbance values from the test line were calculated and plotted. The binding isotherms of DNA-Net-HApt-1 -T - GP120MB were analyzed using Hill fit. FIGS. 20A-20D. DNA-Net-HApt-1 assay based LFA device performance was tested in (FIG. 20A) Saliva, (FIG. 20B) Urine, and (FIG. 20C) Serum environments, with varying concentrations ranging from 25% to 100%. A negative symbol (-) indicates the absence of the GP120MB target, while a positive symbol (+) represents the presence of a 100 nM GP120MB target. (FIG. 20D) The absorbance values from the test line were calculated and plotted against the corresponding experimental conditions. The data are presented as the mean ± SD (standard deviation), with n = 3 biologically independent samples.

[0036] FIGS. 21A-21F. The performance of the LFA device was assessed using HIV-1 virus samples and comparison of device performance with commercially available rapid antigen tests. (FIG. 21 A) Pseudoviral (PV) and (FIGS. 21B-D) three clinical samples (clinical- 1, clinical-2, and clinical-3, respectively) were systematically diluted (from 106- 101) and (from 105- 101), respectively in 50% serum environment, while a buffer consisting of IX PBS and 50% serum served as the negative control (0 virus). (FIG. 21E) Commercial antigen rapid test (Abbott) was utilized with one clinical viral sample. Zone-1 : Control zone, Zone-2: Antigen test zone, Zone-3: Antibody test zone, C: Control line, T: Test line. (FIG. 21F) Absorbance values from the test line were plotted against the total number of viral particles per test, providing an evaluative measure of sensitivity of our LFA device and commercial rapid antigen tests. The presented data are expressed as the mean ± SD (standard deviation), with n = 3 biologically independent samples.

[0037] FIGS. 22A-22B. Specificity test for DNA-Net-HApt-1 -based LFA device. (FIG. 22A) The crossreactivity of the LFA device was evaluated using different trimeric spike proteins, including Influenza- H1N1-HA and SARS-CoV-2, each at a concentration of 1 pM, alongside 250 nM GP120MB, and 50% serum. (FIG. 22B) The resulting absorbance values from the test line were plotted against the different targets.

[0038] FIGS. 23A-23D. DNA-Net-HApt-2 assay -based LFA device testing with free GP120MB as a target. (FIG. 23A) A range of GP120MB concentrations from 0.1 uM - 1.93 pM were tested, leading to a gradual increase in the intensity of the test line (T) on the LFA device. The control line is indicated with the symbol C. (FIG. 23B) The absorbance values from the test line were calculated and plotted against the corresponding GP120MB concentrations. The binding isotherm of GP120MB was analyzed using Hill fit, yielding a dissociation constant (KD) of 0.58 ± 0.02 pM. (FIG. 23C) LFA device performance in different biological environments was checked using saliva, serum, and urine environments, each at 50% concentration. A negative symbol (-) indicates the absence of the GP120MB target, while a positive symbol (+) represents the presence of a 1.93 pM GP120MB target. (FIG. 23D) The absorbance values from the test line on the LFA device are plotted against different percentages of the target analyte (saliva / serum / urine), providing a visual representation of the device's performance in different biological environments. The data are presented as the mean ± SD (standard deviation), with n = 3 biologically independent samples.

[0039] FIGS. 24A-24D. DNA-Net-HApt-2 assay-based LFA device testing with free GP120MB as a target. (FIG. 24A) The performance of the LFA device was evaluated in a 50% serum environment, with a range of GP120MB concentrations from 0.4 pM - 1.93 pM. (FIG. 24B) The absorbance values from the test line were calculated and plotted. The binding isotherm of DNA-Net-HApt-2-GP120MB was analyzed using Hill fit, resulting in KD value of 0.63 + 0.04 pM. (FIG. 24C) The cross-reactivity of the LFA device was assessed by using different trimeric spike proteins, including Influenza-HA-HINl and SARS-CoV-2, each at a concentration of 1 pM, alongside 250 nM GP120MB, 1% BSA, and 50% serum. (FIG. 24D) The resultant absorbance values from the test line were plotted against the different targets, illustrating the selectivity of the LFA device. The data are presented as the mean ± SD (standard deviation), with n = 3 biologically independent samples.

[0040] FIG. 25. Performance assessment of the fourth-generation rapid antibody test (OraQuick®) with clinical HIV-1 samples. Commercial antibody rapid test was utilized with one clinical viral sample by serially diluting the virus (from 105to 101) in IX PBS and 50% serum, while a buffer composed of IX PBS and 50% serum served as the negative control (0 virus).

[0041] SEQUENCES

[0042] The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

[0043] In the accompanying sequence listing:

[0044] SEQ ID NO: 1 is an exemplary sequence of a DNA aptamer targeting SARS-COV-2.

[0045] CAGCACCGACCTTGTGCTTTGGGAGTGCTGGTCCAAGGGCGTTAATGGACA

[0046] SEQ ID NO: 2 is an exemplary sequence of a DNA aptamer targeting HIV-1 GATTTGTTTTCCCGATTTTGCCCAGGGTTAATT

[0047] SEQ ID NO: 3 is an exemplary sequence of a DNA aptamer targeting influenza A virus. TAGATTGGCCTTGCTATCACCCAAAACCGTTTAAAGCTGACCACGTGACGCTTCATCCGT

[0048] SEQ ID NO: 4 is an exemplary sequence of a DNA aptamer targeting influenza A virus, particularly H5N1.

[0049] GTG TGC ATG GAT AGC ACG TAA CGG TGT AGT AGA TAC GTG CGG GTA GGA AGA AAG GGA AAT AGT TGT CCT GTT G

[0050] SEQ ID NO: 5 is an exemplary sequence of a DNA aptamer targeting Porcine epidemic diarrhea virus.

[0051] AGCAGATGAC CATTCTGATT GCTTCCTAAG CGGGTTCCCT TTTAGGGGCTTGGG

[0052] SEQ ID NO: 6 is an exemplary amino acid sequence of HIV- 1 GP120MB-

[0053] KLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLVNVTENFNM WKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNI STSIRGKVQKEYAFFYKLDIIPIDNDTTSYKLTSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNK TFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTRP NNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSG GDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKA MYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTK AKRRVVQREKRAHHHHHHHHHH SEQ ID NOs: 7-15 are exemplary primer and probe sequences.

[0054] DETAILED DESCRIPTION

[0055] I. Introduction

[0056] Lateral flow assay (LFA)-based rapid antigen test is experiencing extensive global uptake as an expeditious and highly effective modality for the screening of viral infections during the COVID-19 pandemic. While these devices have played a significant role in alleviating the burden on the public healthcare system, their specificity and sensitivity fall short compared to molecular tests. New methodologies augmenting specificity and enabling the detection of low viral loads from non-invasive samples, such as saliva, are needed.

[0057] One approach lies in harnessing the potential of DNA nanotechnology and integrating aptamerbased DNA nanostructures as a replacement for antibodies on the test line of LFAs (Wandtke et al., Int. J. Mol. Sei. 2022, 23, 1412; Dalirirad et al., ACS Omega 2020, 5, 32890-32898; Wang et al., ACS Cent. Sei. 2023, 9, 72-83). Designer DNA nanostructures, products of the rapidly evolving field of DNA nanotechnology, possess unparalleled programmability and self-assembly properties (Seeman et al., Nat. Rev. Mater. 2017, 3, 17068; Dey et al., Nat. Rev. Methods Primers 2021, 1, 13; Zhang et al., Am. Chem. Soc. 2014, 136, 1 1 198-1 121 1). These engineered designer nanostructures, composed of synthetic DNA molecules, can be precisely designed to form desired shapes and patterns with highly controlled dimensions, shapes, and surface functionality (Rothemund et al., Nature 2006, 440, 297-302; Kwon et al., Nat. Chem. 2020, 12, 26-35; Ren et al., Nat. Protoc. 2022, 17, 282-326). The multivalent, pattern-matching strategy built on custom-designed star-shaped DNA nanostructures can mimic the complex spatial distribution of dengue virus epitopes with nanoscale precision and efficiently bind dengue viral particles with high avidity, present in non-invasive specimens including blood samples (Kwon et al., Nat. Chem. 2020, 72, 26-35; Ren et al., Nat. Protoc. 2022, 17, 282-326). However, the flexible and mobile nature of envelope glycoproteins in membraned viruses, including coronaviruses like SARS-CoV-2, necessitates a different approach to enable effective pattern matching and detection using DNA nanotechnology (Chauhan et al., J. Am. Chem. Soc. 2023, 145, 20214-20228).

[0058] Here, DNA nanotechnology in utilized in an LFA format, wherein target-specific antibodies are substituted with designer DNA nanostructure -based molecular probes for recognizing SARS-CoV-2 virus and human immunodeficiency virus (HIV). The design disclosed herein utilizes a net-shaped DNA nanostructure (henceforth referred to as the “DNA Net” or “DNA net nanostructure”) and strategically arranged trimeric clusters of aptamers that specifically recognize the spike proteins of SARS-CoV-2 or HIV. This approach combines pattern-matching and multivalent interactions between aptamers specific to the receptor binding domain (RBD) of SARS-CoV-2 spike proteins present on the DNA Net and trimeric spikes displayed on the viral surface. By arranging these DNA aptamers into trimeric clusters on the DNA Net, virus binding affinity was bolstered through dynamic clustering of spike proteins and precise matching of the interspacing of the trimeric clusters. To enable colorimetric or naked-eye detection, DNA Nets were employed in two distinctive ways. Firstly, they were incorporated onto the test line of LFAs, serving as capture agents for the target virus. Secondly, the DNA Nets were embellished with ultrabright gold nanoshells, endowing them with remarkable signal amplification capabilities, thereby serving as reporter assays. Upon introduction of a sample containing the target virus, a sandwich complex is formed between the DNA Nets, the target analyte, and the reporter assays, resulting in a significantly amplified colorimetric signal. This enhanced signal can be interpreted either qualitatively or quantitatively, providing valuable insights into the presence and concentration of the target analyte.

[0059] This approach is shown herein to bolster virus binding affinity and detection on LFAs. These findings indicate high LFA sensitivity, enabling the detection of viral loads ranging from 103- 108viral copies / mL. Notably, sensitivity is maintained across various viral strains, obviating the need for intricate sample preparation protocols. Without being bound by any particular theory, it is believed that the heightened sensitivity is at least in part attributable to the designer DNA net nanostructure, facilitating the detection of extremely low levels of viral loads. This not only enhances the overall reliability of self-testing but also reduces the likelihood of false-negative results, especially in cases of low viral load within patient samples.

[0060] II. Summary of Terms

[0061] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found Benjamin Lewin, Genes XII, published by Jones & Bartlett Publishers, 2018; and Krebs et al. (eds.) ISBN:9781284104493; The Encyclopedia of Cell Biology and Molecular Medicine , published by Wiley-VCH in 16 volumes, 2008; Remington’s Pharmaceutical Sciences, Adejare (Ed.), Academic Press, 23rd Edition, 2020, ISBN: 9780128223895 / 9780128200070; and other similar references. In case of conflict, the present specification, including explanations of terms, will control.

[0062] As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0063] To facilitate review of the various aspects of the disclosure, the following explanations of terms are provided:

[0064] Administration: The introduction of a composition (e.g.. a therapeutic agent or vaccine) into a subject by a chosen route. Administration can be local or systemic. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.

[0065] Analyte: A substance that can be detected, identified, and / or measured by an analytical test. For example, an analysis for a particular analyte can determines the presence, quantity or concentration, of the analyte in the sample.

[0066] Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (e.g., virus) or antigenic fragment thereof. The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen- binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1 -2, 2ndEd., Springer Press, 2010).

[0067] Aptamer: Single-stranded nucleic acid molecules (e.g., DNA or RNA) that bind to protein targets by folding into a three-dimensional conformation. In some aspects, aptamers specifically bind a protein target at a particular KD

[0068] Binding: An association between two substances or molecules, for example, the association of a DNA net nanostructure with a target (e.g., a target virus) or the association of an aptamer with a target (e.g., viral protein). The term “specifically bind” refers to preferential binding of a particular target over other substances present in the environment in which the target occurs (e.g., serum, saliva, or urine). Under designated conditions, a particular binder (e.g., an aptamer) preferentially binds to its target (e.g., an antigen present on the surface of a virus) in a sample, and does not bind in a significant amount to other substances (e.g., proteins or polysaccharides) present in the sample (off-target binding). Specific binding can be measured by methods known in the art. In some aspects, specific binding occurs when the binding equilibrium dissociation (KD) value for the binder and target is in the picomolar (pm) or nanomolar range (nm).

[0069] Class 1 Fusion Protein Virus: A membrane virus that has class 1 fusion proteins. This includes, for example and without limitation, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV, e.g., HIV-1 or HIV-2), porcine deltacoronavirus (PDCO), or porcine epidemic diarrhea (PED).

[0070] Coding Sequence: Part of a gene or cDNA which codes for the amino acid sequence of a protein, or for a functional RNA such as a tRNA or rRNA. Colorimetric Assay: An analysis where a color change or appearance of color indicates the presence or absence of an analyte. Typically, colorimetric reporters produce a visible color change based on a chemical reaction. They are distinct from fluorometric reporters, which emit a detectable light signal upon excitation at a suitable wavelength.

[0071] Complement or Complementary Sequence: A sequence of nucleotides which forms a hydrogen- bonded duplex with another sequence of nucleotides according to Watson-Crick base-pairing rules. For example, the complementary base sequence for 5'-AAGGCT-3' is 3'-TTCCGA-5'.

[0072] Control: A reference standard. In some aspects, the control is a negative control sample obtained from a healthy patient. In other aspects, the control is a positive control sample obtained from a patient diagnosed with a viral infection. In still other aspects, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients with known prognosis or outcome, or group of samples that represent baseline or normal values).

[0073] Detect: To determine if a particular agent is present or absent. Detection can be human read (a visual interpretation of the assay) or machine read. In some aspects, detection further includes quantification of the agent, if desired.

[0074] Downstream: In reference to a nucleic acid, refers to a relative position in DNA or RNA and is the region towards the 3' end of a strand.

[0075] Expression: Transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) and subsequent translation of an mRNA into a protein.

[0076] Heterologous: A protein or nucleic acid sequence originating from a different genetic source.

[0077] Homology: Refers to the extent of identity between two nucleotide or amino acid sequences.

[0078] Immobilized: Bound to a surface, such as a solid support. Suitable methods of immobilizing agents (e.g., a DNA net nanostructure) to solid supports (e.g., a nitrocellulose membrane) are known. In some aspects, a DNA net nanostructure is linked to streptavidin, which is immobilized on a nitrocellulose membrane. Exemplary methods of immobilizing peptides on a solid surface can be found, for example, in Holstein et al. “Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers” Anal Bioanal Chem. 408(5): 1335-46, 2016; see also, US20110008910A1 (Method for the immobilization of a capture molecule on a solid support).

[0079] Inhibiting or Treating a Disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease, for example a viral disease (e.g., COVID-19 or AIDS). “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. Inhibiting a disease can include preventing or reducing the risk of the disease, such as preventing or reducing the risk of viral infection. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well- being of the subject, or by other parameters that are specific to the particular disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

[0080] Isolated: An “isolated” biological component has been substantially separated or purified away from other biological components, such as other biological components in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides, nucleic acids, and viruses that have been “isolated” include those purified by standard purification methods. Isolated does not require absolute purity, and can include protein, peptide, nucleic acid, or virus molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

[0081] Linked or Attached: Forming a bond between two molecules to form one contiguous molecule. In some aspects, the two molecules are linked through Van der Waals forces, hydrogen binding, electrostatic forces, or covalent attachment. Two molecules can also be linked using streptavidin-biotin interactions or peptide linkers. Non-limiting examples of peptide linkers include glycine-serine peptide linkers. Unless context indicates otherwise, reference to “linking” a first polypeptide and a second polypeptide, or to two polypeptides “linked” together, or to a first polypeptide having a “linkage” to a second polypeptide, refers to covalent linkage by peptide bond (for example via a peptide linker) such that the first and second polypeptides form a contiguous polypeptide chain. If a peptide linker is involved, the covalent linkage of the first and second polypeptides can be to the N- and C-termini of the peptide linker. Typically, such linkage is accomplished using molecular biology techniques to genetically manipulate DNA encoding the first polypeptide linked to the second polypeptide by the peptide linker.

[0082] Nucleic Acid Molecule: A single- or double-stranded linear polynucleotide containing either deoxyribonucleotides or ribonucleotides that are linked by 3'-5'-phosphodiester bonds.

[0083] Operably Linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0084] Pharmaceutically Acceptable Carrier: The pharmaceutically acceptable carriers of use herein are conventional. Remington: The Science and Practice of Pharmcacy, 23rdEdition, Adejare, Academic Press, 2020, describes compositions and formulations suitable for pharmaceutical delivery of a therapeutic agent.

[0085] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular aspects, suitable for administration to a subject the carrier may be sterile, and / or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition suitable to induce the desired immune response. It may also be accompanied by medications for its use for treatment purposes. The unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage

[0086] Polypeptide: A polymer of amino acids linked by peptide bonds.

[0087] Promoter: A cis-acting DNA sequence, generally 80-120 base pairs long and located upstream of the initiation site of a gene, to which RNA polymerase may bind and initiate correct transcription. There can be associated additional transcription regulatory sequences which provide on / off regulation of transcription and / or which enhance (increase) expression of the downstream coding sequence.

[0088] Recombinant: A recombinant nucleic acid molecule is one that has a sequence that is not naturally occurring, for example, includes one or more nucleic acid substitutions, deletions or insertions, and / or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. A recombinant virus is one that includes a genome that includes a recombinant nucleic acid molecule. A recombinant protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. In several aspects, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell, or into the genome of a recombinant virus.

[0089] Sample: A composition that has or is suspected of having a target virus of interest and will be subjected to analysis. Typically, a sample for analysis is in liquid form, or can be converted into liquid form, and preferably the sample is an aqueous composition. A sample may be from any suitable source, for example, an industrial sample from a waste stream, or a biological sample such as blood, urine, or saliva. A sample may be treated, such as by extraction, dilution or filtration, or it may be a reconstituted precipitate from an industrial or biological source. In several aspects the sample is a biological sample from a patient suspected of being infected with a target virus of interest.

[0090] Sequence Identity: The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity; the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.

[0091] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. AppL Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0092] See also, BLAST tool available on National Center for Biotechnology Information’s (NCBI’s) website on the internet (blast.ncbi.nlm.nih.gov / Blast.cgi).

[0093] As used herein, reference to “at least 90% identity” (or similar language) refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.

[0094] Soluble Protein: A protein capable of dissolving in aqueous liquid at room temperature and remaining dissolved. The solubility of a protein may change depending on the concentration of the protein in the water-based liquid, the buffering condition of the liquid, the concentration of other solutes in the liquid, for example salt and protein concentrations, and the heat of the liquid. In several aspects, a soluble protein is one that dissolves to a concentration of at least 0.5 mg / ml in phosphate buffered saline (pH 7.4) at room temperature and remains dissolved for at least 48 hours.

[0095] Subject: A multi-cellular vertebrate organisms, a category that includes human and non-human mammals. In a non-limiting example, a subject is a human. In some examples, the subject is a veterinary subject, such as a cow, sheep, pig, horse, goat, turkey or chicken. In some examples, the subject is at risk or suspected of being infected with a virus.

[0096] Target Virus: A virus of interest.

[0097] Under Conditions Sufficient For: A phrase that is used to describe any environment that permits a desired activity.

[0098] Upstream: 5' end of a referenced site of a nucleic acid.

[0099] Vector: A nucleic acid molecule that is able to replicate autonomously in a host cell and can accept foreign DNA. A vector carries its own origin of replication, one or more unique recognition sites for restriction endonucleases which can be used for the insertion of foreign DNA, and usually selectable markers such as genes coding for antibiotic resistance, and often recognition sequences (e.g. promoter) for the expression of the inserted DNA. Common vectors include plasmid vectors and phage vectors.

[0100] Viral Protein: A protein made by a virus.

[0101] Virus Particle (Virion): Virions represent a virus in its extracellular phase. Virions typically include a viral genome (DNA or RNA) enclosed within a protein coat known as a capsid. Some virions also include an outer lipid envelope in which viral proteins can be embedded. The capsid and lipid envelope protect the viral genome and help determine temperature sensitivity, resistance to environmental factors, and host specificity.

[0102] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein.

[0103] One of ordinary skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this disclosure.

[0104] III. DNA Net Nanostructures

[0105] Disclosed herein are DNA net nanostructures (also referred to herein as “DNA Nets”) that bind a target virus (e.g., specifically bind). In some aspects, the DNA net nanostructures include biotin. In some aspects, the DNA net nanostructures are linked to a reporter molecule (e.g., gold nanoshell or gold nanoparticle).

[0106] The DNA net nanostructures are polynucleotide scaffolds upon which binding ligands (e.g., aptamers) are strategically attached to allow for polyvalent binding to a target virus. DNA net nanostructures specifically bind multiple targets on the surface of virus, and are compatible with targets that are mobile within and / or on the surface (e.g., have a probability of being located within an area envelope extending around a central average position), such as on a viral or cell membrane. The specific binding of the DNA net nanostructures to antigen clusters increases sensitivity and specificity of detection for a particular virus.

[0107] DNA net nanostructures that are of use in the compositions and methods disclosed herein have been previously described, for example, in US patent application 18 / 037,433, herein incorporated by reference in its entirety (see also, Chauhan et al., “Net-Shaped DNA Nanostructures Designed for Rapid / Sensitive Detection and Potential Inhibition of the SARS-CoV-2 Virus” J. Am. Chem. Soc. 145(37): 20214-20228, 2023). In brief, DNA net nanostructures are made of a network of polynucleotides creating structural units that are connected to one another via a series of arms and junctions. At least a portion of one or more strands of polynucleotides from one structural unit is complementary to at least a portion of one or more strands of polynucleotides of another structural unit, and the complementary portions of the strands of the polynucleotides hybridize to connect the units. The complementary portions of the strands of the polynucleotides form the “arms” of a predetermined length. Consequently, the arms, or at least a portion thereof may be double stranded. Intersections of three or more arms form a “junction” at a predetermined distance from one another based on the length of the arms. Aptamers are attached to the network of polynucleotides at loci on one or more of the arms forming the junctions. The loci are separated by predetermined distances such that the aptamers are positioned on the DNA net nanostructure in a two- dimensional or three-dimensional spatial pattern that matches an expected two-dimensional or three- dimensional spatial pattern of target antigens on the target virus. The structural units can be a predetermined shape, which includes, for example, a rhombus, triangle, pentagon, or hexagon. In some aspects, the structural unit is a rhombus, for example, a rhombus with a side length of 14-15 nm, for example, about 14.95 nm (see, e.g., FIG. 6A). The DNA net nanostructure includes a plurality of subunits, for example, at least 4, at least 9, at least 16, etc. In non-limiting examples, the DNA net nanostructure includes 2 to 100 subunits, for example, 2 to 80, 2 to 60, 2 to 40, 2 to 25, 2 to 20, 2 to 16, 2 to 9, 4 to 80, 4 to 60, 4 to 40, 4 to 25, 4 to 16, 4 to 9, 9 to 80, 9 to 60, 9 to 40, 9 to 25, or 9 to 16 subunits. In non-limiting examples, the DNA net nanostructure includes 2, 4, 6, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 49 subunits. In further non-limiting examples, the DNA net nanostructure includes 4, 9, or 16 subunits. In some aspects, the DNA net nanostructure includes a 2x2, 3x3, or 4x4 grid of rhombus structural units. In some aspects, the DNA net nanostructure includes a 3x3 grid of rhombus structural units (three rhombus structural units by three rhombus structural units).

[0108] The DNA net nanostructure includes one or more aptamers (e.g., a DNA aptamer) that bind a target virus (e.g., specifically binds a target virus). In some aspects, the DNA net nanostructure includes at least 8 aptamers, for example, at least 10, at least 12, at least 16, at least 20, at least 24, at least 27, at least 30, at least 36, at least 40, or at least 48. In some aspects, the DNA net nanostructure includes 8 to 60 aptamers, for example, 8 to 50, 8 to 48, 8 to 40, 8 to 36, 8 to 30, 8 to 27, 8 to 24, 8 to 20, 8 to 16, 8 to 12, 8 to 10, 10 to 50, 10 to 48, 10 to 40, 10 to 36, lO to 30, 10 to 27, 10 t 24, 10 to 20, l O to 16, 10 to 12, 12 to 50, 12 to 48, 12 to 40, 12 to 36, 12 to 30, 12 to 27, 12 to 24, 12 to 20, 12 to 16, 27 to 50, 27 to 48, 27 to 40, 27 to 36, or 27 to 30. In some aspects, the DNA net nanostructure includes 12-48 aptamers. In some aspects, the DNA net nanostructure includes 12, 27, or 48 aptamers. In some aspects, a 2x2 DNA net nanostructure includes 12 aptamers. In some aspects, a 3x3 DNA net nanostructure includes 27 aptamers. In some aspects, a 4x4 DNA net nanostructure includes 48 aptamers.

[0109] Aptamer spacing can take into account inter-aptamer spacing (according to target antigens on the target virus), as well as intra-aptamer spacing (according to target epitopes on the target antigen). In a nonlimiting example, the DNA net nanostructure is composed of rhombus shaped structural unit with a length of 14-15 nm (e.g., about 14.95 nm). In some aspects, the aptamers are arranged in an array of tri-aptamer clusters having 5-7 nm (e.g., about a 6 nm or 6.5nm) intra-aptamer spacing and about 15 nm inter-tri- aptamer spacing, which matches those of spike trimers on viral particles (e.g., SARS-CoV-2, HIV-1). The spacing of a particular target protein can be determined computationally.

[0110] In some aspects, the DNA net nanostructures specifically bind the target virus. Specific binding is preferential binding to an intended target with insignificant or tolerable off-target binding. A person of ordinary skill in the art can determine an appropriate threshold for desired specificity. In some aspects, a DNA net nanostructure disclosed herein specifically binds to a target antigen or virus when the interaction has a binding equilibrium dissociation (KD) is less than 100 nm, for example, less than 10 nm or less than 1 nm. In some aspects, a DNA net nanostructure disclosed herein specifically binds to a target antigen or virus when the interaction has a binding equilibrium dissociation (KD) between 0.1 pm to 100 nM, for example, 0.1 pm to 10 nM, 0.1 pm to 10 nM, 0.5 pm to 100 nM, 0.5 pm to 10 nM, 0.5 pm to 1 nM, 1 pm to 100 nM, 1 pm to 10 nM, or 1 pm to 1 nM. In some aspects, the KD is calculated at room temperature, for example, 22-27 °C (e.g., about 25°C).

[0111] The aptamers can be attached to the DNA net nanostructure in any suitable way, including, for example, Van der Waals forces, hydrogen binding, electrostatic forces, or covalent attachment. In some aspects, the aptamers are covalently attached. In some aspects, the aptamers are integrated into the DNA- Net by attaching complementary single-stranded DNA sequences as overhangs on each trimeric cluster.

[0112] In some aspects, a DNA net nanostructure disclosed herein includes a biotin strand (a nucleic acid molecule (e.g., DNA) that has been chemically modified by attaching one or more biotin molecules to it). In some aspects, a biotin-modified strand is incorporated into the DNA Net, preferably at a location that does not interfere with aptamer function or geometry of the DNA net nanostructure. In some aspects, the biotin modified strand is located on or near the periphery of the DNA net nanostructure. In some aspects, the DNA net nanostructure including the biotin strand is linked to a streptavidin.

[0113] In some aspects, a DNA net nanostructure disclosed herein is linked to a reporter. The reporter can be any suitable reporter that provides a detectable signal on an LFA format. In some aspects, the reporter is a colorimetric reporter. In some aspects, the reporter is a gold nanoshell, gold nanoparticle, or quantum dot. Gold nanoshells are spherical nanoparticles with a silica core and a thin gold shell. In some aspects, the gold nanoshell is between 5 nm and 400 nm. In some aspects, the gold nanoparticle is 5 nm to 350 nm, 5 nm to 300 nm, 5 nm to 250 nm, 5 nm to 200 nm, 5 nm to 150 nm, 5 nm to 100 nm, 5 nm to 50 nm, for example, 5 nm to 40 nm, 5 nm to 30 nm, 5 nm to 20 nm, 5 nm to 10 nm, etc. In a non-limiting example, the gold nanoshell is between 100 nm and 200 nm, for example, 150 nm. Gold nanoparticle size typically ranges between 5 nm to 1500 nm. In some aspects, the gold nanoparticle is between 5 nm and 800 nm, 5 nm and 700 nm, 5 nm and 600 nm, 5 nm and 500 nm, 5 nm and 400 nm, 5 nm to 350 nm, 5 nm to 300 nm, 5 nm to 250 nm, 5 nm to 200 nm, 5 nm to 150 nm, 5 nm to 100 nm, 5 nm to 50 nm, for example, 5 nm to 40 nm, 5 nm to 30 nm, 5 nm to 20 nm, 5 nm to 10 nm, 40 nm and 800 nm, 40 nm and 700 nm, 40 nm and 600 nm, 40 nm and 500 nm, 40 nm and 400 nm, 40 nm to 350 nm, 40 nm to 300 nm, 40 nm to 250 nm, 40 nm to 200 nm, 40 nm to 150 nm, 100 nm and 800 nm, 100 nm and 700 nm, 100 nm and 600 nm, 100 nm and 500 nm, 100 nm and 400 nm, 100 nm to 350 nm, 100 nm to 300 nm, 100 nm to 250 nm, 100 nm to 200 nm, or 100 nm to 150 nm. In some aspects, the gold nanoparticle is 25 nm to 200 nm. In some aspects, the gold nanoparticle is 40 nm to 150 nm. In some aspects, the gold nanoparticle is 25 nm to 200 nm. Gold nanoshells and gold nanoparticles covalently attached to streptavidin are commercially available.

[0114] In some aspects, the reporter includes quantum dots. Quantum dots (or semiconductor nanocrystals) are semiconductor particles that are a few nanometers in size with optical and electronic properties. When a quantum dot is illuminated by an appropriate wavelength (e.g., UV light) an electron in the quantum dot can be excited to a state of higher energy and emit detectable wavelengths of light. Larger quantum dots of 5- 6 nm diameter typically emit longer wavelengths, with colors such as orange, or red, while smaller quantum dots (2-3 nm) usually emit shorter wavelengths, yielding colors like blue and green. However, the specific color varies depending on the exact composition of the quantum dot. In some examples, the quantum dot is 2-6 nm in diameter, for example, 2-3 nm or 5-6 nm. Quantum dots (QDs) are commercially available, for example, Qdot™ 655, Qdot™ 525, Qdot™ 565, Qdot™ 585, Qdot™ 605, Qdot™ 655, and Qdot™ 705 (ThermoFisher, see, e.g., catalog number Q10123MP). Quantum dots covalently attached to streptavidin are commercially available. An appropriate excitation LED source is used to excite QDs and corresponding detectors are used to collect the emission spectra. Detector machines can be customized depending on which QD is required for a given application.

[0115] In some aspects, the reporter does not include an organic fluorophore. In some aspects, the reporter does not include a FAM fluorescent dye (e.g., 6-FAM).

[0116] The DNA net nanostructure can be linked to a reporter molecule in any suitable manner, including, attachment by streptavidin-biotin chemistry or covalent attachment (e.g., thiol attachment or attachment of carboxyl group modified gold nanoparticles to amine modified DNA). In some aspects, the reporter is a gold nanoshell or gold nanoparticle including streptavidin and the DNA net nanostructure includes a biotin strand, thereby facilitating linking of the DNA net nanostructure and reporter molecule by streptavidinbiotin chemistry.

[0117] The aptamers of the DNA net nanostructure bind to a target virus. In some aspects, the aptamers bind a target virus by binding a viral protein of the target virus. The viral protein can be any suitable target protein. In several aspects, the viral protein is on an outer surface of the virus, for example, embedded in or externally attached to a viral membrane or capsid. In a non-limiting example, the viral protein is viral spike protein.

[0118] In some aspects, the target virus is a class 1 fusion protein virus, for example (and without limitation): severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV, e.g., HIV-1 or HIV-2), influenza A virus (AIV), porcine deltacoronavirus (PDCO), or porcine epidemic diarrhea (PED). In other aspects, the target virus is a capsid virus, for example (and without limitation): hepatitis B virus (HBV), human papillomavirus (HPV), or dengue virus (DENV). In some aspects, the target virus is SARS-CoV-2 or HIV-1.

[0119] IV. Lateral Flow Assay Test Strips and Kits

[0120] For illustrative purposes, several non-limiting examples of LFA test strip configurations are provided in FIGS. 14A-14F. Exemplary lateral flow assay test strips 100 include a lateral flow membrane 102. The lateral flow membrane 102 includes a test line 201 comprising an immobilized DNA net nanostructure disclosed herein. The DNA net nanostructure includes one or more aptamers (e.g., a DNA aptamer) that bind a target virus (e.g., specifically binds a target virus).

[0121] In some aspects, the DNA net nanostructure includes at least 8 aptamers, for example, at least 10, at least 12, at least 16, at least 20, at least 24, at least 27, at least 30, at least 36, at least 40, or at least 48. In some aspects, the DNA net nanostructure includes 8 to 60 aptamers, for example, 8 to 50, 8 to 48, 8 to 40, 8 to 36, 8 to 30, 8 to 27, 8 to 24, 8 to 20, 8 to 16, 8 to 12, 8 to 10, 10 to 50, 10 to 48, 10 to 40, 10 to 36, 10 to 30, 10 to 27, 10 to 24, 10 to 20, 10 to 16, 10 to 12, 12 to 50, 12 to 48, 12 to 40, 12 to 36, 12 to 30, 12 to 27, 12 to 24, 12 to 20, 12 to 16, 27 to 50, 27 to 48, 27 to 40, 27 to 36, or 27 to 30. In some aspects, the DNA net nanostructure includes 12-48 aptamers. In some aspects, the DNA net nanostructure includes 12, 27, or 48 aptamers. In some aspects, a 2x2 DNA net nanostructure includes 12 aptamers, a 3x3 DNA net nanostructure includes 27 aptamers, or a 4x4 DNA net nanostructure includes 48 aptamers.

[0122] Aptamers of the DNA net nanostructure facilitate binding to the target virus. In some aspects, the aptamers bind a target virus through binding a viral protein of the target virus. The viral protein can be any suitable target protein. In several aspects, the viral protein is on an outer surface of the virus, for example, embedded in or externally attached to a viral membrane or capsid. In a non-limiting example, the viral protein is viral spike protein. In some aspects, the DNA net nanostructures preferentially bind intact viruses. In some such aspects, the test strips are particularly useful for identifying the presence of live virus / active infections. In some aspects, the DNA net nanostructure does not bind degraded viruses.

[0123] In some aspects, the target virus is a class 1 fusion protein virus, for example (and without limitation): severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV, e.g., HIV-1 or HIV-2), influenza A virus (AIV), porcine deltacoronavirus (PDCO), or porcine epidemic diarrhea (PED). In other aspects, the target virus is a capsid virus, for example (and without limitation): hepatitis B virus (HBV), human papillomavirus (HPV), or dengue virus (DENV). In some aspects, the target virus is SARS-CoV-2 or HIV-1 .

[0124] The lateral flow membrane 102 is a cellulosic membrane. In some aspects, the cellulosic membrane is nitrocellulose. The DNA Net nanostructure can be immobilized on the test line 201 by any suitable means. In some aspects, the DNA Net nanostructure is immobilized on the test line using biotin and streptavidin. For example, a DNA Net nanostructure including a biotin strand is incubated with streptavidin. Once the DNA Net nanostructures form a complex with the streptavidin protein, the complex is printed on the test line 201 using a machine dispenser.

[0125] As illustrated in FIGS. 14C-14E, the lateral flow membrane 102 can further include a control line 202. The control line 202 indicates whether the test is functioning properly by showing a visible line even if the target virus is not present in the sample. This serves as a quality control check verifying that the sample is flowing correctly through the LFA test strip and that the test components are active. In some aspects, the control line includes a viral protein recognized by the DNA Net nanostructure.

[0126] As illustrated in FIGS. 14B-14E, in some aspects, the lateral flow assay test strip 100 further includes a sample pad 101 and / or a wicking pad 103. Suitable materials for the sample pad 101 and / or wicking pad 103 have been described. The sample pad 101 receives a sample and can help facilitate controlled release of the sample onto a conjugate pad or directly to the lateral flow membrane 102, depending on the test configuration. Typically, the sample pad 101 is comprised of cellulose fiber filterse.g., filter paper) and / or woven meshes. Woven meshes (e.g. glass fiber) generally have lower bed volumes, meaning little sample is retained, however have good tensile strength resulting in easy handling. Cellulose fibers filters have higher bed volumes, lower tensile strength, and are particularly useful when a variety of buffers or blocking reagents is required. The sample pad 101 can be treated, for example, with buffers, blocking reagents, protein, detergents, or surfactants. Treating the sample pad 101 with an optimized buffer can aid in “normalizing” samples before reaching the conjugate pad and help prevent negative interactions that may occur due to differences in pH, protein composition, mucins, salt concentrations, and any other molecules that may interfere with the assay or cause non-specific interactions. Buffers can be used, for example, to normalize sample pH and salt concentration, act as blocking agent, improve flow, and / or enhance reproducibility by incorporating proteins, surfactants, salts, and / or polymers at the appropriate concentrations. Some biological sample, for example, saliva samples, may differ in viscosity. Incorporating increased salt and / or surfactant concentrations can break down mucins and proteins, thereby decreasing viscosity and improving flow.

[0127] A sample pad treatment can be performed, for example, by immersion or spraying uniformly with an automated dispenser (e.g. Isoflow, Kinematic, Biodot). After treating the sample pad, it can be cured, for example, in a forced air convection oven at 37°C for 30-60 minutes, and then allowed to dry overnight in a desiccated environment (<20% relative humidity) at 18-25°C. After overnight drying, the treated sample pad is ready to use. It may be advantageous to store treated sample pads in a dry environment (<20% relative humidity) at room temperature (18-25°C) as the pads may uptake moisture that can destabilize the reagents dried onto the surface.

[0128] The wicking pad 103 helps maintain capillary action through the LFA test strip. The wicking pad 103 is an absorbent material. Having an absorbent wicking pad 103 at the distal end of the test strip can increase the volume of sample that can be run across the lateral flow membrane 102 since it can act as a sponge for the additional volume. Typically, wicking pads 103 are made of cellulose fiber pads due to their large bed volumes. The absorption capacity of the wicking pad 103 is generally higher than the sample and running buffer volume of the assay. Suitable materials for the wicking pad are known and can be obtained commercially from companies such as Millipore, Whatman / GE, or Ahlstrom.

[0129] When present, the sample pad 101 and wicking pad 103 are on opposite ends of the lateral flow membrane 102. Due to the function of the wicking pad 103, a sample is applied on the opposite end of the LFA test strip that the wicking pad is located. In some aspects, a sample pad 101 is located on a proximal end of the lateral flow membrane 102 and the wicking pad 103 is located on a distal end of the lateral flow membrane 102. The test line 201 (and control line 202, when present) are typically located in a central portion of the lateral flow membrane 102. However, in some aspects, the test line 201 and / or control line 202 are closer to the distal end than the proximal end (e.g., located in the distal half or quadrant of the lateral flow membrane 102). Samples to be analyzed are loaded on the proximal end of the lateral flow membrane 102, or are applied to the sample pad 101 (when present). When present, the conjugate pad 104 is located between the sample pad 101 and lateral flow membrane 102.

[0130] As illustrated in FIG. 14E, in some aspects, the LFA test strip further includes a conjugate pad 104. When a conjugate pad 104 is included in an LFA test strip, it typically includes a suitable reporter composition. When the sample liquid reaches the conjugate pad 104, the reporter composition is released and mixes with the sample. If there are any target viruses in the sample that the reporter composition is designed to recognize, binding will occur. In LFA tests where a sample is mixed with a reporter composition (e.g., in a test tube or other vessel) prior to loading the LFA test strip, a conjugate pad is not needed. The conjugate pad 104 is typically a non-woven filters, for example, cellulose, glass, or plastic (e.g., polyester, polypropylene, or polyethylene) fibers woven into thin mats. In some aspects, the conjugate pad

[0131] 104 is a glass fiber pad.

[0132] In several aspects, the conjugate pad 104 includes a reporter composition, including a DNA net nanostructure disclosed herein linked to a reporter, for example, a gold nanoshell, gold nanoparticle, or quantum dot. In some aspects, the reporter composition includes a colorimetric reporter or quantum dot. In some aspects, the reporter composition does not include an organic fluorophore. In some aspects, the reporter composition does not include a FAM fluorescent dye (e.g., 6-FAM).

[0133] Individual components of a LFA test strip can be assembled on a backing card 105 for support and stability (e.g.. see, FIG. 14F). The backing card 105 is a solid material. In some examples, the backing card

[0134] 105 is a paper or plastic (e.g., polyester, polypropylene, or polyethylene). In some aspects, the backing card includes an adhesive to secure components (e.g., sample pad 101, lateral flow membrane 102, wicking pad 103, conjugate pad 104) in the proper order. When assembling components of the LFA test strip, the components can be overlapped, for example, by at least 0.5 mm, to help ensure continuous flow through the LFA test strip. In some aspects, each component is overlapped by at least 1mm. In some aspects, the components are overlapped by 1 mm to 5 mm, for example, 1 mm to 4 mm or 1 mm to 2 mm.

[0135] The LFA test strip detects the target virus at concentrations as low as 102viral copies per 100 pL sample volume. In some aspects, the LFA test strip detects the target virus in a sample at a concentration of 102-107viral copies per 100 pL sample volume, for example, 102-105viral copies per 100 pL sample volume or 102-103viral copies per 100 pL sample volume.

[0136] In some aspects, the LFA test strips disclosed herein preferentially detect intact viruses, and therefore may be particularly useful at identifying the presence of live virus / active infections. In some aspects, the LFA test strips disclosed herein do not detect degraded viruses. Without being bound by any particular theory, aptamers bind proteins in specific conformational forms. Degraded virus may have target proteins in different conformational arrangement, which could discourage aptamer binding, leading to minimal false positive signal generation.

[0137] Further disclosed are kits including a LFA test strip disclosed herein. The kits can further include, for example, reagents for running a lateral flow assay (e.g., buffers), a device for sample collection (e.g., a cotton swab, lancet, syringe, bulb dropper, etc.), a device for holding a sample (e.g., a test tube or vial), a device for transferring a sample to an LFA test strip (e.g., bulb dropper or vial with a dropper), and / or instructions for use. The kits are useful, for example, for detecting the presence of a target virus and / or diagnosing a disease caused by a target virus. V. Methods of Detecting Viruses

[0138] Further disclosed are methods of detecting a target virus in a sample, including (i) adding a sample to the sample pad of the LFA test strip disclosed herein, and (ii) detecting a signal; thereby detecting the target virus in the sample. A reporter composition (e.g., a DNA net nanostructure is linked to a reporter) can be included on a conjugate pad, or the sample can be premixed with a reporter composition (e.g., suspended in a buffer) and then loaded onto an LFA test strip. When a sample is premixed with a reporter, the LFA test strip does not need to include a conjugate pad.

[0139] In some aspects, the methods include: (i) adding a sample to a sample pad of an LFA test strip, wherein: (a) the LFA test strip includes a conjugate pad including a first DNA net nanostructure comprising a DNA aptamer that binds the target virus, wherein the first DNA net nanostructure is linked to a reporter, and (b) the LFA test strip includes a lateral flow membrane including a second DNA net nanostructure including a DNA aptamer that binds the target virus immobilized on a test line; and (ii) detecting a signal from the test line, thereby detecting the target virus in the sample.

[0140] In further aspects, the methods include: (i) mixing the sample with a first DNA net nanostructure including a DNA aptamer that binds the target virus, wherein the first DNA net nanostructure is linked to a reporter, thereby producing a mixed sample; (ii) adding the mixed sample to a sample pad of an LFA test strip, wherein the LFA test strip includes a lateral flow membrane comprising a second DNA net nanostructure comprising a DNA aptamer that binds the target virus immobilized on a test line; and (iii) detecting a signal from the test line, thereby detecting the target virus in the sample.

[0141] The first DNA net nanostructure is linked to a reporter. The reporter can be any suitable reporter. In some aspects, the reporter is a colorimetric reporter (e.g., gold nanoshell or gold nanoparticles). In some aspects, the reporter is a quantum dot. While the reporter can be linked to the DNA net nanostructure by any suitable means, an exemplary means is through an avidin-streptavidin linkage. In some such examples, gold nanoshells, gold nanoparticles, or quantum dots including streptavidin are linked to DNA net nanostructures including a biotin strand. Suitable gold nanoshells, gold nanoparticles, and quantum dots including streptavidin are commercially available. In some aspects, the reporter does not include a fluorophore. In some aspects, the reporter does not include a FAM fluorescent dye.

[0142] The LFA test strip includes a second DNA net nanostructure including a DNA aptamer that also binds the target virus, however, the second DNA net nanostructure is immobilized on a test line of the lateral flow membrane. As the sample flows through the LFA test strip, the second DNA net nanostructure binds the target virus, which in turn is bound to the first DNA net nanostructure, creating a “sandwich.” This causes an aggregation of the reporter on the test line, ultimately producing in a detectable signal on the test line.

[0143] In some aspects, the LFA test strip includes a control line. The control line indicates whether the test is functioning properly by showing a visible line even if the target virus is not present in the sample. In non-limiting examples, the methods further include detecting a signal from the control line to confirm that the sample is flowing correctly and that the test components are active. If the control line is not detected, the LFA test strip can be discarded and a new test performed.

[0144] Detecting a signal from the test line (or control line, when present) includes both human or machine detection. For example, in some aspects the signal is a color change that is visible by a human eye, which a human user can detect and interpret. In some aspects, the signal is detected by a lateral flow reader or other detection device. Lateral flow readers or other detection devices can be used, for example, to increase throughput and / or help reduce human error. Lateral flow readers or other detection devices can also be used when the signal output is not detectable by the human eye, for example, absent excitation by a suitable wavelength of light (e.g., quantum dot). Lateral flow readers are commercially available, for example, from Pacificimage Electronics® (see, e.g., RapidScan® devices), which can perform colorimetric or fluorescent signal detection. See Also, BioAssay Works® (CUBE® Lateral-Flow Reader); Biosynex Technologies® (CubePlus® lateral flow reader); and Hamamatsu (THETA® lateral flow reader). In some aspects, signal detection provides a binary (a positive or negative result). In some aspects, signal detection includes quantification of the signal, and a positive result is determined by the presence of any detectable signal, or determined by a threshold cut off value.

[0145] The LFA test strips disclosed herein preferentially detect intact viruses, and therefore are particularly useful at identifying the presence of live virus / active infections. In some aspects, the LFA test strips disclosed herein do not detect degraded viruses.

[0146] The sample is a composition suspected of having the target virus. Typically, the sample is a liquid sample, or is converted to a liquid form, for example by mixing the sample in a suitable buffer or saline solution. The sample can be from any source of interest, for example, a biological sample from a subject (e.g., blood, urine, or saliva), an industrial sample from a waste stream of interest, or a sample from a potentially contaminated surface or medical waste. Biological samples are samples originating from a body of a subject, for example, bodily fluids, cells, tissues, etc. In some aspects, the biological sample is a bodily fluid, for example, urine, saliva, sputum, mucous, sweat, lacrimal fluid, whole blood, or serum. In some aspects, the sample is a blood (whole blood or serum), urine, mucous, or saliva sample. In some aspects, the sample is a saliva or mucous sample. In several aspects the sample is a biological sample from a subject suspected at risk of, or suspected of being infected with, the target virus.

[0147] The sample can undergo one or more processing or treatment steps prior to adding the sample to the LFA test strip. Exemplary treatments include, for example, extraction, dilution, or filtration (or other separation). In some aspects, the sample is diluted by at least 25%, for example, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, etc. using a suitable buffer (e.g., PBS). In some aspects, the sample is diluted 10-99%, for example, 25% to 90%, 25% to 75%, or 25% to 50%, before adding to the LFA test strip. In some aspects, the sample is diluted 50-75% before adding to the LFA test strip. In some aspects, the sample is diluted by about 25-50% before adding to the LFA test strip. In some aspects, the sample does not undergo any processing or treatment steps prior to running the LFA test, except dilution in a suitable buffer (e.g., PBS (e.g., IX PBS), BSA (e.g., 1% BSA), MgCh (e.g., 3.5 mM MgCh - 6H2O), or polyethylene glycol sorbitan monolaurate (Tween20®) buffer (e.g, 0.05% Tween20® buffer)) when desired.

[0148] When the sample is obtained from a subject, the subject is a multi-cellular vertebrate organism at risk of, or suspected of being infected with, the target virus. In some aspects, the subject is a human or veterinary subject (e.g., farm animal or pet). In some aspects, the veterinarian subject is a domesticated animal or animal in human captivity (e.g., research animals or animals in zoos). Non-limiting examples of veterinarian subjects include, for example, dogs (Canis lupus familiaris), cats (Felis catus), pigs (Sus domesticus), cattle (Bos taunts), horses (Equus caballus), birds (e.g., chickens, ducks, turkeys), rabbits (Oryctolagus cuniculus domesticus), mice, guinea pigs, rats, etc. In some aspects the subject is a pig (Sus domesticus) or bird (e.g., chicken (Gallus gallus domesticus), duck, or turkey (Meleagris gallopavo f. domestica)). In some aspects the subject is a living subject. In some aspects, the subject is deceased (e.g. the method is performed to help determine cause of death).

[0149] In some aspects, the target virus is a class 1 fusion protein virus, for example (and without limitation): severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV, e.g., HIV-1 or HIV-2), influenza A virus (AIV), porcine deltacoronavirus (PDCO), or porcine epidemic diarrhea (PED). In other aspects, the target virus is a capsid virus, for example (and without limitation): hepatitis B virus (HBV), human papillomavirus (HPV), or dengue virus (DENV). In some aspects, the target virus includes a membrane bound spike protein. In some aspects, the target virus is SARS-CoV-2 or HIV-1.

[0150] The methods disclosed herein can further include administering a therapeutic or prophylactic agent to the subject, for example in a therapeutically effective amount. Such administrations can include one or more doses of the therapeutic or prophylactic agent. In some aspects, a therapeutic agent is administered when the target virus is detected. In some aspects, the therapeutic agent is an anti-viral therapy specific to the detected virus. In some examples, the anti-viral therapy is a monoclonal antibody therapy specific to the detected virus. In some aspects, the prophylactic agent is a vaccine or pre-exposure prophylaxis. In some aspects, the prophylactic agent is administered when the target virus is not detected, for example, a vaccine for the target virus can be administered to reduce risk of a future infection in the subject by the target virus.

[0151] In a non-limiting example, nirmatrelvir with ritonavir (Paxlovid®), remdesivir (Veklury®), and / or molnupiravir (Lagevrio®) are administered to the subject when SARS-CoV-2 is detected. In some aspects, when SARS-CoV-2 is not detected, a SARS-CoV-2 vaccine is administered to the subject.

[0152] In a non-limiting example, an anti-retroviral medication is administered to the subject when HIV is detected. In some examples, abacavir, emtricitabine, lamivudine, tenofovir disoproxil fumarate, zidovudine, efavirenz, etravirine, nevirapine, rilpivirine, enfuvirtide, atazanavir, darunavir, fosamprenavir, ritonavir, saquinavir, tipranavir, and / or maraviroc are administered to the subject when HIV is detected. In some aspects, when HIV is not detected, a pre-exposure prophylaxis (PrEP) (e.g., Truvada® (emtricitabine and tenofovir disoproxil fumarate), Descovy® (emtricitabine and tenofovir alafenamide), and / or Apretude® (cabotegravir)) is administered to the subject. In a non-limiting example, oseltamivir (Tamiflu®), zanamivir (Relenza®), peramivir (Rapivab®), amantadine, and / or rimantadine are administered to the subject when AIV is detected. In some aspects, when AIV is not detected, a flu vaccine is administered to the subject.

[0153] In a non-limiting example, when PDCO or PED is detected in an animal subject, the animal subject is treated with an anti-viral therapy, or is isolated or culled to prevent further spread of the virus.

[0154] In some aspects, the methods disclosed herein are useful for detecting or diagnosing a viral infection or disease thereof, in a subject. In some such aspects, detecting a signal from the test line indicates the viral infection or disease. In some aspects, the methods are useful for diagnosing COVID-19 in a subject when SARS-CoV-2 virus is detected. In some aspects, the methods are useful for diagnosing AIDS in a subject when HIV is detected. In some aspects, the methods are useful for diagnosing the flu or bird flu in a subject when AIV is detected. In some aspects, the methods are useful for diagnosing hepatitis B in a subject when HBV is detected. In some aspects, the methods are useful for diagnosing asymptomatic HPV infection, or HPV associated cervical cancer and / or genital warts in a subject, when HPV is detected. In some aspects, the methods are useful for diagnosing Dengue fever in a subject when DENV is detected. In some aspects, the subject is a human.

[0155] In some aspects, the methods are useful for diagnosing porcine deltacoronavirus infection in a subject when PDCO is detected. In some aspects, the methods are useful for diagnosing porcine epidemic diarrhea infection in a subject when PED virus is detected. In some aspects, the subject is a pig. In some aspects, the methods are useful for diagnosing the flu or bird flu in a subject when AIV is detected. In some aspects, the subject is a bird.

[0156] VI. Clauses

[0157] Clause 1. A lateral flow assay (LFA) test strip, comprising a lateral flow membrane comprising a test line, wherein: the test line comprises an immobilized DNA net nanostructure, and the DNA net nanostructure comprises a DNA aptamer that binds a target virus.

[0158] Clause 2. The LFA test strip of clause 1, wherein binding the target virus comprises binding a viral protein of the target virus.

[0159] Clause 3. The LFA test strip of any one of the prior clauses, wherein the viral protein is a viral spike protein of the target virus.

[0160] Clause 4. The LFA test strip of any one of the prior clauses, wherein the LFA test strip detects intact viruses. Clause 5. The LFA test strip of any one of the prior clauses, wherein the LFA test strip does not detect degraded viruses.

[0161] Clause 6. The LFA test strip of any one of the prior clauses, wherein the target virus is a class 1 fusion protein virus.

[0162] Clause 7. The LFA test strip of any one of the prior clauses, wherein the target virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV), influenza A virus (IAV), porcine deltacoronavirus (PDCO), or porcine epidemic diarrhea (PED). Clause 8. The LFA test strip of any one of the prior clauses, wherein the target virus is hepatitis B virus (HBV), human papillomavirus (HPV), or dengue virus (DENV).

[0163] Clause 9. The LFA test strip of any one of the prior clauses, wherein the lateral flow membrane comprises a nitrocellulose membrane.

[0164] Clause 10. The LFA test strip of any one of the prior clauses, wherein the LFA test strip further comprises a sample pad and a wicking pad.

[0165] Clause 11. The LFA test strip of any one of the prior clauses, further comprising a conjugate pad.

[0166] Clause 12. The LFA test strip of any one of the prior clauses, wherein the conjugate pad comprises a reporter composition, wherein the reporter composition comprises the DNA net nanostructure linked to a gold nanoshell or gold nanoparticle.

[0167] Clause 13. The LFA test strip of any one of the prior clauses, wherein the lateral flow membrane further comprises a control line.

[0168] Clause 14. The LFA test strip of any one of the prior clauses, wherein the LFA test strip detects the target virus in a sample at a concentration of 102- 107viral copies per 100 pL sample volume.

[0169] Clause 15. The LFA test strip of any one of the prior clauses, wherein the LFA test strip detects the target virus in a sample at a concentration of 102— 105viral copies per 100 pL sample volume.

[0170] Clause 16. The LFA test strip of any one of the prior clauses, wherein the LFA test strip detects the target virus in a sample at a concentration of 102— 103viral copies per 100 pL sample volume.

[0171] Clause 17. A kit comprising the LFA test strip of any one of the prior clauses, optionally wherein the kit further comprises reagents for running a lateral flow assay, a device for sample collection, a device for holding a sample, a device for transferring a sample to an LFA test strip, and / or instructions for use.

[0172] Clause 18. A method of detecting a target virus in a sample, comprising: (i) adding a sample to the sample pad of the LFA test strip of any one of clauses 1-16, and (ii) detecting a signal, thereby detecting the target virus in the sample.

[0173] Clause 19. A method of detecting a target virus in a sample, comprising: (i) adding the sample to a sample pad of an LFA test strip, wherein: (a) the LFA test strip comprises a conjugate pad comprising a first DNA net nanostructure comprising a DNA aptamer that binds the target virus, wherein the first DNA net nanostructure is linked to a reporter, and (b) the LFA test strip comprises a lateral flow membrane comprising a second DNA net nanostructure comprising a DNA aptamer that binds the target virus immobilized on a test line; and (ii) detecting a signal from the test line, thereby detecting the target virus in the sample.

[0174] Clause 20. A method of detecting a target virus in a sample, comprising: (i) mixing the sample with a first DNA net nanostructure comprising a DNA aptamer that binds the target virus, wherein the first DNA net nanostructure is linked to a reporter, thereby producing a mixed sample; (ii) adding the mixed sample to a sample pad of an LFA test strip, wherein the LFA test strip comprises a lateral flow membrane comprising a second DNA net nanostructure comprising a DNA aptamer that binds the target virus immobilized on a test line; and (iii) detecting a signal from the test line, thereby detecting the target virus in the sample. Clause 21. The method of any one of clauses 18-20, wherein the reporter is a gold nanoshell or gold nanoparticle.

[0175] Clause 22. The method of any one of clauses 18-21, wherein the sample is a bodily fluid.

[0176] Clause 23. The method of any one of clauses 18-22, wherein the bodily fluid is saliva, urine, serum, or whole blood.

[0177] Clause 24. The method of any one of clauses 18-23, wherein the sample is obtained from a human infected with or suspected of being infected with the target virus.

[0178] Clause 25. The method of any one of clauses 18-24, wherein the sample is obtained from a pig infected with or suspected of being infected with the target virus.

[0179] Clause 26. The method of any one of clauses 18-25, wherein the target virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV), porcine deltacoronavirus (PDCO), porcine epidemic diarrhea (PED), hepatitis B virus (HBV), human papillomavirus (HPV), dengue virus (DENV), or influenza A virus (IAV).

[0180] Clause 27. A method of diagnosing a viral infection in a subject, comprising: (i) adding a sample from the subject to a sample pad of an LFA test strip, wherein: (a) the LFA test strip comprises a conjugate pad comprising a first DNA net nanostructure comprising a DNA aptamer that binds the target virus, wherein the first DNA net nanostructure is linked to a reporter, and (b) the LFA test strip comprises a lateral flow membrane comprising a second DNA net nanostructure comprising a DNA aptamer that binds the target virus immobilized on a test line; and (ii) detecting a signal from the test line, thereby diagnosing the viral infection.

[0181] Clause 28. A method of diagnosing a viral infection in a subject, comprising: (i) mixing a sample from the subject with a first DNA net nanostructure comprising a DNA aptamer that binds the target virus, wherein the first DNA net nanostructure is linked to a reporter, thereby producing a mixed sample; (ii) adding the mixed sample to a sample pad of an LFA test strip, wherein the LFA test strip comprises a lateral flow membrane comprising a second DNA net nanostructure comprising a DNA aptamer that binds the target virus immobilized on a test line; and (iii) detecting a signal from the test line, thereby diagnosing the viral infection.

[0182] Clause 29. A DNA net nanostructure that specifically binds a target virus, wherein the DNA net nanostructure is linked to a gold nanoshell or gold nanoparticle. EXAMPLES

[0183] EXAMPLE 1

[0184] DNA NET MEDIATED DETECTION OF SARS-COV-2

[0185] Materials and Methods

[0186] Reagents

[0187] DNA aptamers and other oligonucleotide strands of DNA Net were procured from Integrated DNA Technologies (Coralville, Iowa). UV-inactivated SARS-CoV-2 viruses with different strains (SARS-COV-2 Isolate hCoV-19 / USA / CA_UCSD_5574 / 2020 B.1.1.7 (UK), SARS-COV-2 Isolate hCoV-19 / South Africa / KRISP-K005325 / 2020 B.1.351 (South African), SARS-COV-2 Isolate hCoV-19 / USA / CA-SEARCH- 221363 / 2022 XBB.1.5 (Omicron Variant), SARS-COV-2 Isolate hCoV-19 / USA-WA1 / 2020 (Washington Strain)) were obtained from the University of California at San Diego through the RADx program. Each strain was diluted in cell media composed of DMEM + 1% FBS + 10 mM HEPES + 50 units mL-1 Penicillin and 50 pg mL-1 Streptomycin). Streptavidin-modified gold nanoshells, SA-AuNS (150 nm, GSIR150) were obtained from Nanocomposix (San Diego, CA). Nitrocellulose-Streptavidin (Cat# pro-338) was obtained from ProSpec Bio (US). The SARS-CoV-2 SI trimetric spike protein (9566) was sourced from Meridian Life Science. Control proteins including Influenza- A H1N1 (A / California / 04 / 2009, 11055- V08H2) and HIV-GP120 were purchased from Sino Biological Inc. Saliva samples (991-05-P-PreC-50) were obtained from Medix Biochemica USA Inc. Various reagents such as D(+)-Trehalose dihydrate (182551000), D(+)— Biotin (A14207-03), sodium chloride (S9625), magnesium chloride hexahydrate (442611), sucrose (84097), HEPES (J16924-K2, Thermo Scientific), Tween-20 (5075031), bovine serum albumin (BSA, BAH64), human serum (H5667), phosphate-buffered saline (PBS, AM9625), and gastight glass syringes (1725TLL, volume 250 pL) were obtained from MilliporeSigma (St. Louis, MO). The ALFRD Lat Flow Reagent Dispensing machine (part number: 07.711.01) was purchased from Claremont Biosolutions (CA). Paper components of the lateral flow assay, such as nitrocellulose membrane (VIVID 120 LFNC), wicking pad (CF5), and glass fiber conjugate pads (8950) were purchased from Pall Corporation, Cytiva, and Ahlstrom (Helsinki, Finland), respectively. To ensure the precise cutting of these paper components, the high-speed Rapid Test Strip Cutter (GQlOlOd) from Werfen Automation (China) was employed.

[0188] Preparation of DNA Net Complexes

[0189] DNA net complexes were prepared according to Chauhan et al., J. Am. Chem. Soc. 145(37): 20214-20228, 2023. Briefly, the preparation of each DNA complex involved a one -pot reaction in which all component strands were mixed in a final concentration of IX TAE-Mg2+K+buffer (pH 7.5). The buffer composition encompassed 40 mM Tris-acetate, 2 mM EDTA, 12.5 mM Magnesium acetate, and 10 mM potassium chloride. To construct each DNA Net complex (2 x 2, 3 x 3, and 4 x 4), the component oligonucleotides were combined in specific stoichiometric ratios, precisely aligning with the design of the respective nanostructure. For the 3 x 3 DNA Net, a molar ratio of 27: 1 was employed when attaching the aptamers to the DNA Net, considering the presence of 27 docking sites on the resulting nanostructure. Subsequently, the mixture underwent annealing by gradually reducing the temperature from 90 °C - 23 °C over a span of 14 hours, employing a state-of-the-art thermal cycler (TProfessional TRIO PCR Thermocycler). Following the annealing process, the 3 x 3 DNA Net complexes were stored at 4 °C to ensure their stability for subsequent assay.

[0190] Agarose gel electrophoresis (AGE) of DNA complexes and specific yield calculation

[0191] To validate the successful formation of the DNA Net complexes, agarose gel electrophoresis (AGE) was performed. A 1% agarose gel in IX TAE-Mg2+buffer (comprising 40 mM Tris-acetate, 2 mM EDTA, and 12.5 mM Magnesium acetate, pH 7.5) was employed for this purpose. The gels underwent electrophoresis at a constant voltage of 60 V for 2-3 hours. Subsequently, the gels were stained with SYBR Green (Thermo Fisher) and subsequently scanned using a GelDoc system (Bio-Rad). A sharp rectangular gel band in individual lanes was chosen as region of interest (ROI) for mean density calculation for different sizes of DNA Net. Background mean density was calculated in ImageJ software by selecting area outside of sharp rectangular gel band.

[0192] Atomic Force Microscopy (AFM) imaging

[0193] DNA Net samples were annealed at a concentration of 50 nM in IX TAE + 10 mM Mg2+buffer. A 5 pL drop of the buffer solution containing 2 nM DNA Net was then placed on a freshly cleaved mica surface (Ted Pella Inc.) and incubated for 5 minutes. Subsequently, IX TAE / Mg2+buffer was added, and the solution on the surface was immediately removed using a pipette. An additional 80 pL of fresh TAE / Mg2+buffer was added to the surface for AFM scanning. The imaging process was carried out using tapping mode in fluid on an Asylum AFM with an MSNL-10 probe (Bruker Nano, Inc.).

[0194] Synthesis of reporter assay

[0195] To fabricate the reporter assay, DNA Net-coated AuNS conjugates were used. The synthesis process commenced by subjecting the 150 nm SA- AuNS solution to vigorous vortexing, ensuring homogeneous dispersion. In a separate tube, the SA-AuNS solution, conjugation buffer, and DNA Net solution were combined in a precisely balanced 5:4: 1 ratio. This mixture was subsequently incubated at ambient temperature for 30 minutes, accompanied by continuous rotation to promote efficient binding between the components. To terminate the reaction and prevent further interactions, excess biotin was introduced into the mixture, effectively blocking unreacted streptavidin (SA) sites on the AuNS. The resulting concoction was further incubated at room temperature for an additional 15 minutes under continuous rotation, thereby finalizing the synthesis of the DNA Net-coated AuNS reporter assay. Dynamic Light Scattering and Zeta Potential Measurements

[0196] For the measurement of hydrodynamic diameter and zeta potential, the Malvern Zetasizer Nano ZS90 instrument (Malvern Panalytical Ltd., Malvern, UK) was employed, equipped with a backscattering detector set at an angle of 173°. This apparatus was complemented by a disposable polystyrene cuvette (DTS0012) and the ZEN1002 Universal Dip Cell for precise sample handling. To ensure optimal concentration, the AuNS stock was diluted by a factor of 106 using a 0.02 pm filtered PBS buffer. Zeta potential serves as a quantification of the electrostatic potential at the surface of the electrical double layer and the bulk medium, providing valuable information about the surface charge of the nanoparticles. To minimize the influence of Joule heating and polarization effects, a 60-second delay was implemented between each zeta potential measurement. All measurements were performed at a temperature of 25 °C, encompassing a series of five zeta potential measurements followed by five Dynamic Light Scattering size measurements, employing backscatter detection for enhanced accuracy.

[0197] Lateral flow test design and operation

[0198] The LFA design encompasses various components. Firstly, the conjugate pad plays a role in dispensing and drying the gold nanoshell-DNA Net conjugates, facilitating the controlled release onto the nitrocellulose membrane upon hydration. The nitrocellulose membrane (NCM) is strategically designed for streptavidin-biotin chemistry, enabling the immobilization of the DNA Net on the test line and a trimeric spike protein (TSP) on the control line. This configuration allows for the specific detection and confirmation of analytes. The wicking pad, on the other hand, serves as an absorption pad, ensuring a consistent capillary flow across the membrane and facilitating the seamless movement of the sample throughout the lateral flow strip. To assemble the LFA device, we prepared a “3 / 4th LFA device” by carefully aligning and affixing the NCM, wicking pad, and conjugate pad onto a backing card, ensuring a 2 mm overlap to enable uninterrupted sample flow from the sample pad to the wicking pad. This arrangement ensures the efficient operation of the lateral flow strip and prepares it for the subsequent printing of the test line and control line.

[0199] Printing the DNA Net conjugates on the test line and TSP on the control lines of the NCM

[0200] For the preparation of the test line conjugates, 3 x 3 DNA Net and nitrocellulose-streptavidin (NCSA) was mixed with a 1: 10 ratio in IX TAE-Mg-K buffer, resulting in a total volume of 200 pL. Subsequently, the test line conjugates were incubated at room temperature for 45 minutes on a rotator with intermittent shaking to ensure optimal binding. As for the control line, 200 pL of TSP at a concentration of 0.15 mg mL-1 was utilized. To ensure an even distribution of the test line and control line solutions onto the NCMs, both reagents were loaded into the glass syringes of a syringe pump connected to an ALFRD dispenser. The LFA device was securely fixed onto the dispenser table for stability. A controlled flow rate of 0.7 pL cm-1 was employed during the dispensing process. Following the printing, the NCMs were dried at 37°C for 30 minutes under a relative humidity of 17%. The amount of input material allowed for the printing of approximately 6 full-length LFA devices, each measuring 254 mm in length and 25 mm in width. Finally, the printed LFA chips were carefully stored in a laminated aluminum bag, supplemented with desiccants to maintain optimal conditions. Prior to each experiment, the sealed aluminum bag was opened, and the LFA chips / devices were precisely cut to dimensions of 60 mm in length and 4 mm in width, ensuring consistency and uniformity.

[0201] Signal detection and quantification.

[0202] Each LFA device was immersed into individual wells of 96-well plates (part number: 10062-900, VWR), containing a composition of reagents: 100 pL of running buffer encompassed IX phosphate-buffered saline, 1% BSA, potassium chloride [KC1], and magnesium chloride [MgC12], 20 pL of reporter assay, and a small volume of targets at the desired concentration. The LFA device exhibited amplified signal intensity in the test line, which was subsequently captured and interpreted using a portable reader (Lumos Leelu, Model# LUMOS-V3-03) procured from Lumos Diagnostics Inc. This reader was equipped with a built-in LED light source that emitted red illumination specifically tailored for gold nanoshells (AuNS), with an exposure time of 3 ms, facilitating the imaging of the illuminated test strip. As the AuNS absorbed a fraction of the illuminating light, it correspondingly diminished the intensity of the reflected light emanating from the test lines on the NCM. To precisely quantify the light absorbed by the test strip, we employed measurements of either absorption or reflection density. Absorbance measurements were preferred due to their superior accuracy, ensuring more reliable and consistent results across different readers and over multiple experimental sessions. An absorbance value of 1 denoted the absorption of 90% of the incident light, while an absorbance value of 0 indicated no light absorption. After adjusting for the background noise of the strip, the absorbed light was quantified and reported. The background noise of the strip was determined by calculating the average pixel values in the left and right background regions, which were centered around the peak intensity of the test line. The limit of detection (LOD) was calculated according to established methodologies, as previously reported (Armbruster et al., Clin. Biochem. Rev. 2008, 29, 49-52; Holstein et al., Anal. Chem. 2015, 87, 9795-9801).

[0203] Surface Plasmon Resonance ( SPR ) Analysis

[0204] Surface Plasmon Resonance (SPR) analysis was conducted using purified wildtype SARS-CoV-2 trimeric spike proteins from various variants, including SARS-CoV-2 XBB.1.5 (Catalog: 40589-V08H45), SARS-CoV-2 B.1.1.7 (Catalog: 40589- V08H12), and SARS-CoV-2 B.1.351 (Catalog: 40589-V08H13, Sinobiological Inc). These proteins were immobilized onto different flow cells (FC2, FC3, and FC4) of a research-grade CM5 S-Series SPR chip (GE Healthcare, Uppsala, Sweden) using a standard amine coupling protocol. In brief, carboxymethyl groups on the CM5 chip surface in Flow Cell 1 (serving as the reference) were activated. This activation involved a 420-second injection pulse at a flow rate of 5 pL / min, using a 1 : 1 mixture of N-ethyl-N-(dimethylaminopropyl) carbodiimide 5 (EDC) and N-hydroxysuccinimide (NHS) at a final concentration of 50 mM each, mixed immediately before injection. After activation, we prepared a 50 ig / mL solution of Purified Mouse IgG (from ImmunoReagents, USA) in a 10 mM sodium acetate buffer (pH 5.5) and injected it over the activated biosensor surface of Flow Cell 1. The successful immobilization of Purified Mouse IgG was confirmed by observing an increase in the baseline signal, approximately 9625 resonance units (RU). Subsequently, a 50 g / mL solution of individual stocks of SARS-CoV-2 trimeric spike protein from XBB.1.5, B.1.1.7, and B.1.351 variants was prepared in a 10 mM sodium acetate buffer (pH 5.0) and injected over the activated biosensor surfaces of Flow Cell 2, Flow Cell 3, and Flow Cell 4, respectively. The successful immobilization of the Spike Proteins was confirmed by observing an increased baseline signal of approximately 1399, 776, and 1169 RU in their respective flow channels. Any remaining unreacted carboxymethyl groups on the sensor surface were deactivated by a 600-second injection of 1 M ethanolamine in Flow Cells 1 to 4, at a flow rate of 5 pL / rnin. Flow Cell 1, with immobilized Purified Mouse IgG, served as a reference for Flow Cell 2, 3, and 4, which had immobilized spike proteins from XBB.1.5, B.1.1.7, and B.1.351, respectively. Four-fold dilutions of DNA-aptamer (0.03906, 0.1563, 0.625, 2.5, and 10 pM) were then made and injected over the sensor chip at a flow rate of 5 pL / min, using IX TAE-Mg2+K+buffer at pH 7.5 (comprising 40 mM Tris-acetate, 2 mM EDTA, 12.5 mM Magnesium acetate, and 10 mM potassium chloride) as the running buffer. After sample injection, IX TAE-Mg2+K+, pH 7.5, was flowed over the sensor surface to facilitate dissociation. Following a 5-minute dissociation period for the DNA- Aptamer, the sensor surface was fully regenerated by injecting a 10 mM Glycine buffer at pH 2.5 for 30 seconds, with a flow rate of 30 pL / min. A 5-minute stabilization interval was incorporated between each cycle of sample injection to stabilize the baseline. The SPR response (sensorgram) was monitored as a function of time at 25°C. SPR measurements were conducted using a BIAcore T200 instrument (GE Healthcare, Sweden), operated with the BIAcore T200 control software. Each sample underwent measurement in triplicate. The resulting sensorgrams were utilized to determine binding kinetics parameters, including the association rate constant (ka), dissociation rate constant (kfi, and binding equilibrium dissociation constant (KD, where KD = ka / ka), achieved by locally fitting the entire association and dissociation phases within BiaEvaluation software 4.0.1 (GE Healthcare, Sweden).

[0205] Virus copies number / reaction quantification

[0206] For virus copy number / reaction quantification, RNA was extracted from 140pl inactivated viral stock using the QIAamp Viral RNA Mini kit (QIAGEN) following manufacturer’s instructions and eluted in 140pl nuclease-free H2O. RNA copies of nucleocapsid (N) and ORFla were quantified by digital droplet PCR: A 1:10,000 dilution of eluted RNA was made, and lOpl of RNA dilution was run in triplicate and amplified in a 20pL reaction (One-step RT-ddPCR Advanced Kit, Bio-Rad) with 900nM each primer and 250nM probe_FAM / ZEN / IBFQ targeting 2019-nCoV_N3 and ORFla with RNAse P as an internal control (Table 1). Droplets were generated (QX200 droplet generator, Bio-Rad), placed in ddPCR™ 96-Well Plates (Bio-Rad). Plates were heat-sealed and transferred to an Applied Biosystems Veriti 96 Well Thermal Cycler. PCR reaction conditions were 25°C for 3 min, at 45°C for 60 min, at 95 °C for 10 min. and then cycled 45 times at 95°C for 30 sec and at 55°C (N3 & RNAse P) or 60 °C (COVID19 ORFla) for 60 sec. Droplets were read on a QX200 droplet reader (Bio-Rad). Copies per pl viral stock were calculated in Microsoft Excel as follows: Copies per ml viral stock = copies per 20pl reaction / lOpl template per reaction x 10,000 (dilution factor of eluted RNA) x 1000 (convert to ml).

[0207] Table 1: Primers and probes used for RT digital droplet PCR.

[0208] Optimization of DNA Net on Gold Nanoshells

[0209] Rapid antigen-based LFAs are commonly employed for self-testing purposes, aiming to detect viral antigens or antibodies for confirming infection. However, the limited sensitivity and selectivity of these assay often lead to the generation of false-negative or false-positive results, both of which have significant implications. False-negative outcomes create a false sense of security, increasing the risk of unknowingly transmitting the virus, while false-positive cases require appropriate patient care. To tackle these challenges and enhance the specificity of LFAs for SARS-CoV-2 virus detection which can be readily expanded to other viral detections, a distinctive approach can be used that harnesses the power of designer DNA nanostructure (DDN) based viral capture probes. Despite the emergence of DNA nanotechnology nearly four decades ago, accompanied by the pioneering development of the first home pregnancy test kit ("Clearblue One Step”) based on LFA technology during a similar timeframe, the convergence of these two scientific disciplines has been scarce (Armburster et al., Clin. Biochem. Rev. 2008, 29, 49-52; Holstein et al., Anal. Chem. 2015, 87, 9795-9801). The untapped potential of incorporating DNA nanostructures into LFAs represents a vast reservoir for scientific innovation and progress in this realm. An application of DNA nanotechnology is presented herein that significantly enhances the sensitivity of LFAs, thereby revolutionizing the field of early viral detection.

[0210] To achieve this goal, DDN design principles were applied to create intricate net-shaped DNA nanostructures (called “DNA Net”), composed of rhombus units with a ~ 15 nm long edge. The DNA Net was designed to arrange RBD-targeting aptamers into an intricate array of tri-aptamer clusters with a ~6 nm intra- and a -15 nm inter-tri-aptamer spacing, matching those of the spike timers on viral particles. The resulting DNA Net architecture offers excellent flexibility and broader coverage, enabling the high affinity binding of multiple trimeric spike proteins on viral surfaces for sensitive and selective detection of SARS- CoV-2 virus via multivalent, pattern-matching interactions (Chauhan et al., J. Am. Chem. Soc. 2023, 145, 20214-20228). DNA Nets with various sizes, including "2 x 2" (30 nm x 30 nm), "3 x 3" (45 nm x 45 nm), and "4 x 4" (60 nm x 60 nm) Nets, were constructed and characterized herein to determine the best Net size we should use on the LFA devices (FIG. 6A) (Chauhan et al., . J. Am. Chem. Soc. 2023, 145, 20214-20228). To confirm the successful formation of DNA Nets, atomic force microscopy (AFM) imaging and agarose gel electrophoresis was used (FIGs. 1 A, 8, 9). The yields of forming the monomeric 2 x 2 Net, 3 x 3 Net, and 4 x 4 Net are estimated as 96.61%, 88.01%, and 77.51%, respectively, as shown in Table 2. Due to the delicate nature of the thin tile-shaped DNA Net structures, they can be susceptible to deformation or damage during the process for AFM characterization (May et al., Am J Obstet Gynecol 1991, 165, 2000-2002). However, despite this inherent fragility, all the desired DNA Net structures were effectively formed with a high degree of yield and purity as shown by our gel electrophoresis assay (Chauhan et al., J. Am. Chem. Soc. 2023, 145, 20214-20228), enabling their direct utilization in these assays without the need for further purification.

[0211] Table 2: Yield calculation for DNA Net using agarose gel electrophoresis image data

[0212] This approach aimed to effectively incorporate DNA Nets into LFAs, serving two primary objectives: first, employing them as reporter assay, and second, integrating them onto the test line. In pursuit of the former objective, DNA Nets of various sizes were evaluated on AuNS to assess the overall stability of the DNA Net-AuNS conjugates. In this work, the use of commercially available 150 nm gold nanoshells were explored as an alternative to 40 nm gold particles commonly used in LFA. Gold nanoshells are composed of a 20 nm thick gold shell deposited around 110 nm silica spheres, exhibiting unique optical properties that yield highly contrasting signals. The presence of the silica core reduces particle density, enabling easy resuspension in water and smooth flow through the LFA without precipitation (Seeman et al., Nano Lett. 2020, 20, 1477-1478). To synthesize the reporter assay, DNA Nets were attached with 150 nm streptavidin-coated gold nanoshells (SA-AuNS) via a biotin strand. Zeta potential and hydrodynamic diameter measurements were performed to evaluate the characteristics of the resulting conjugates. The size of the AuNS increased from approximately 159.94 ± 2.96 nm to 178.04 ± 3.16 nm upon the introduction of the "2 x 2" DNA Net, followed by further growth to 264.84 ± 5.55 nm and 270.66 ± 6.18 nm with the "3 x 3" and "4 x 4" DNA Nets, respectively (FIG. IB). Of note, the DLS measurements for AuNS coated with 2 x 2 and 4 x 4 Nets did not simply agree with the theoretical sizes of the corresponding Nets. The geometric orientation (2 x 2 Net), and steric hindrance (4 x 4 Net) may influence these size measurements. Controls for the reporter assay are shown in FIG. 10.

[0213] These findings substantiated the effective coverage of the AuNS surface area by the DNA Nets, as anticipated based on their theoretical dimensions. Such coverage is important for stabilizing individual AuNS, which initially exhibit very low zeta potential (-18.16 ± 2.67 mV). Consequently, the overall zeta potential of the AuNS gradually decreased with the increasing sizes of the DNA Nets, indicating the stability of the AuNS-DNA Net complex (FIG. 1C). More specifically, the zeta potential of the AuNS decreased from -18.16 ± 2.67 mV (without DNA Net) to -27.56 ± 1.67 mV in the presence of the "2 x 2" DNA Net, further dropping to -35.6 ± 2.95 mV with the "3 x 3" DNA Net, and ultimately attaining a value of -40.0 ± 1.36 mV when the "4 x 4" DNA Net was employed. Notably, no significant alterations in zeta potential were observed between the ”3 x 3" and "4 x 4" DNA Nets. Consequently, for the subsequent experiments in this study, we opted to utilize the more cost-effective "3 x 3" DNA Net to develop both the reporter assay (DNA Net - AuNS) and integrating DNA Nets onto the test line of the LFA.

[0214] LFA Device Testing Using SARS-CoV-2 Trimeric Spike Protein as a Target

[0215] In previous investigations, it has been demonstrated that the binding affinity of DNA Nets to trimeric spike proteins (TSP) immobilized on an SPR chip is within the femtomolar to nanomolar range (Chauhan et al., J. Am. Chem. Soc. 2023, 145, 20214-20228). Encouraged by these findings, a comprehensive exploration was undertaken to ascertain whether DNA Nets, when immobilized on the test line of a nitrocellulose membrane (NCM), would exhibit comparable binding behavior towards free TSP in solution. Test line conjugates, consisting of a 3 x 3 DNA Net, as well as control line conjugates of TSP were strategically affixed onto the NCM, utilizing working concentrations of 10 nM and 0.15 mg mL1, respectively (as expounded upon in the Materials and Methods section as well as in the FIG. 2A).

[0216] Subsequently, a spectrum of TSP concentrations ranging from 1.7 nM - 52 nM was assessed as input samples in the test strips. Remarkably, distinct gray lines with high contrast forming on both the test line and control line were observed as TSP was added at increasing concentrations, ultimately reaching the saturation state (FIG. 2B) (May et al., Am J Obstet Gynecol 1991, 165, 2000-2002; Kuzuya et al., Nat. Commun. 2011, 2, 449). Representative images depicting the colorimetric changes in the test line (T) and control lines (C) signals in the presence of TSP are presented in FIG. 2C. These findings revealed that the LFA exhibited a wide linear range of detection spanning from 1.7 nM - 13 nM with a dissociation constant (KD) of 7.25 ± 0.59 nM indicating its ability to accurately detect free TSP in solution within the tested concentration range, thereby establishing its potential for virus particle detection. Notably, this method does not necessitate the use of blocking reagents to passivate or pretreat the NCM and conjugate pad, as reported in previous reports (Liu et al., ACS Nano 2021, 15, 3593-3611; Dalirirad et al., ACS Omega 2020, 5, 32890- 32898; Gonzalez-Moa et al., ACS Infect. Dis. 2018, 4, 912-917; Srinivasan et al., Biotechnol. 2021, 3, 288- 299; Bishop et al., Lab on a Chip 2019, 2486-2499). This approach not only enhances the overall assay sensitivity but also reduces the preparation time required for the assay.

[0217] LFA Performance in Saliva, Serum, and Urine Environment

[0218] An ideal LFA device must possess the capacity to navigate intricate biological environments encountered during patient sample testing. To assess the compatibility of this LFA device with such environments, a 4 nM TSP targets spiked with various levels (ranging from 1% - 75%) of serum, saliva, and urine samples was introduced. Remarkably, when utilizing saliva and urine samples at concentrations ranging from 1% - 25%, successful binding was observed between the TSP target and the 3 x 3 DNA Net anchored on the test line, as evidenced by the discernible colorimetric signals (FIGs. 3A-3B).

[0219] However, upon increasing the concentrations of saliva and urine samples beyond 25%, a discernible gray color on the test line in the absence of TSP targets was observed, indicating the presence of falsepositive signals. An analogous pattern of false-positive signals was observed when the concentration of human serum samples exceeded 10% (FIG. 3C). It was hypothesized that the presence of excess proteins in concentrated serum / saliva / urine may interfere with the test line, contributing to the enhanced color bands observed in the absence of TSP. Furthermore, when the concentrations were further elevated to 75% in both saliva and urine samples, the signals on the control line were absent in the presence of the target protein. This phenomenon may be due to the heightened sample viscosity, impeding the fluid flow of the reporter assay. Based on these discernments, the optimal analyte quantity (serum / saliva / urine) that can be employed on the LFA device was established, effectively minimizing the occurrence of false -positive signals (FIG. 3D). These findings provide invaluable insights for the design of assays that proficiently navigate complex biological environments encountered during the testing of patient samples.

[0220] Comparing the Target Binding Isotherm in the LFA Device

[0221] FIG. 3A demonstrates the effective binding of the DNA Net, located on the test line of the LFA, with the target TSP. To compare the binding isotherms in biological (25% saliva) and aqueous (0% saliva) environments, TSP was introduced into the LFA device under both environments (FIGs. 4A-4B). The KD value of 5.72 ± 0.28 nM was found in the biological environment, which closely matched the value obtained in the aqueous environment (KD of 7.25 ± 0.59 nM, FIG. 2C). The minimal disparity in K values may be ascribed to the presence of diluted salivary proteins within the samples, exerting an influence on the binding of TSP with the DNA Net. These findings unequivocally substantiate the excellent binding capability of the LFA device to its intended target, in this case, the spike proteins of SARS-CoV-2, even within biologically relevant conditions.

[0222] Target Selectivity of the LFA Device

[0223] Specificity stands as a pivotal determinant in effectively mitigating false-positive occurrences resulting from cross-reactivity. The LFA device developed here is meticulously tailored for the precise detection of the TSP target. It is imperative to consider the potential for cross-reactivity with other trimeric- shaped proteins. Previous studies have reported instances of cross-reactivity using viral antigen-targeting antibody -based LFA with influenza hemagglutinin (HA) trimers and HIV-1 GP120 trimers (Yamaniha et al., J. Infect. Chemother. 2021, 27, 1112-1114; Hsieh et al., Diagnostics 2017, 7, 29; Grant et al., PLoS One 2021, 16, e0256352; Copeland et al., J. Cell Biol. 1986, 103, 1179-1191). Cross-reactivity can occur when the target molecule shares structural similarities or exhibits some degree of homology with other molecules present in the sample. To comprehensively evaluate the cross-reactivity and selectivity of this LFA built on DNA Net probe, tests using influenza HA trimers (H1N1) and HIV-1 GP-120 trimers proteins at a much higher concentration of 1 mg mL1were conducted. The results were satisfactory, as the LFA device exclusively manifested a positive colorimetric signal in the presence of TSP while displaying minimal test line signals in response to other proteins and serum samples (FIGs. 4C-4D). These findings provided evidence ruling out protein cross-reactivity within our LFA, thereby establishing its great specificity. This is an important feature considering specificity concerns associated with commercially available lateral flow assay kits (Murray et al., Nat. Rev. Immunol. 2023, 23, 304-316; Liu et al., Nature 2008, 455, 109-113; Berger et al., PLoS One 2021, 16, e0248921; Kohmer et al., J. Med. Virol. 2020, 92, 2243-2247).

[0224] For sensitivity assessments, the LFA device performance was evaluated in a 25 nM TSP with a 25% saliva environment that comprises 102 samples. The device exhibits a sensitivity of 96.08%, with 98 true positives (TP) and 4 false negatives (FN). Regarding specificity, the LFA device was examined under the same environmental conditions, excluding TSP from the solution. The device demonstrates a specificity of 91.18%, with 93 true negatives (TN) and 9 false positives (FP), as outlined in Table 3.

[0225] Table 3: Possible outcomes of a binary classification test for sensitivity and specificity measurement

[0226] Device Performance with SARS-CoV-2 Viruses with Different Strains

[0227] The principle aim of this study was to develop a rapid, reliable, and sensitive LFA device capable of detecting minute viral loads of SARS-CoV-2 viruses while adeptly managing intricate biological assay. A comprehensive assessment of SARS-CoV-2 viruses was conducted, encompassing four strains of substantial concern, namely WA1 / 2020 / Washington, B.1.1.7 / 2020 / UK, B.1.351 / 2020 / South Africa, and Omicron variant XBB.1.5 / 2022 / USA, all rendered non-infectious through UV inactivation but with the binding function of viral surface spikes maintained (Osterman et al., Med. Microbiol. Immunol. 2021, 210, 65-72). The selection of SARS-CoV-2 variants in this study was driven by two principal considerations: (1) the alpha and beta variants had previously been classified as highly contagious variants, and (2) the XBB.1.5 Omicron variant had maintained its status as a circulating variant of interest according to the World Health Organization as of August 17, 2023 (Corman et al., Lancet Microbe. 2021, 2, 311-319). Furthermore, the quick accessibility of these variants was facilitated through the RADx program. The DNA aptamer employed in our investigation was initially developed against the RBD of the WA1 / 2020 / Washington SARS-CoV-2 strain (Song et al., Anal. Chem. 2020, 92, 9895-9900). Subsequently, surface plasmon Resonance (SPR) assays were used to assess the binding affinity between this aptamer and the spike proteins of different SARS-CoV-2 variants. These findings show the KD values of 1.55xl0'9M for XBB.1.5, 8.81x108M for B.1.351, and 2.15x107M for B.1.1.7 SARS-CoV-2 variants (FIGs. 12A-12C). These results demonstrate the versatility of the aptamer as a ligand binding different spike variants, which can be integrated into a 3 x 3 DNA Net for the effective detection of various variants of interest. As these viral strains were pre-diluted in cell media, a 10-fold serial dilutions of SARS-CoV-2 viruses was done by adding 10 pL of the virus stock into 90 nL of a reaction buffer containing IX phosphate-buffered saline (PBS) and 25% saliva (FIG. 13). A normal buffer, consisting of IX PBS with 25% saliva served as the negative control. A time dependent signal amplication is shown in FIGs. 11 A-l IB. Subsequently, the diluted viral strains, in triplicate, were subjected to a 20 pL reporter assay solution in 96-well plates, exposing each well to our LFA device (Figs. 6B-6C).

[0228] A gradual increase in the intensity of the test line signals as the viral load in the samples increased was observed (FIGs. 5A-5D). This LFA device exhibited a broad detection range, spanning from 102- 107viral copies per 100 pL reaction volume (or 103- 108viral copies / mL), thus satisfying the acceptable analytical sensitivity delineated within the World Health Organization’s target product profile (Tan et al., Diagnostics 2021, 77, 1190). To determine the LOD, a previously reported statistical method was used employing a four-parameter logistic curve fit (World Health Organization. Tracking SARS-CoV-2 variants. 2023). By using this method, the theoretical LOD values were calculated to be 42,830 viral copies; 26,720 viral copies; 5,590 viral copies; and 2,280 viral copies / mL for the B.1.1.7, B.1.351, XBB1.5, and WAI viral strains, respectively (FIG. 5F). To further benchmark this LFA device, a commercially available gold nanoparticle-based rapid antigen test for SARS-CoV-2 was accessed (iHealth COVID-19 Antigen Rapid Test), which has a reported LOD of 350 TCID50 / swab. This product was selected because it has received authorization from the FDA under an Emergency Use Authorization (EUA) and was widely adopted in the market, being available for purchase on various e-commerce platforms. To facilitate a comparison between this kit and our LFA, the SARS-CoV-2 virus derived from the WA1 / 2020 / Washington strain in the antigen rapid test was used. A serial dilution of the viral stock was generated by combining 10 pL of the virus stock with 90 pL of the manufacturer-supplied extraction solution. Relative to this developed LFA device, the commercial antigen rapid test demonstrated a suboptimal linear detection range, ranging from 10s-107total viral copies per 100 pL reaction volume (or 106- 108viral copies / mL). Notably, the test line signals were markedly weak for the viral stock containing 103-105viral copies / mL (FIGs. 5E-5F). These results highlight the role of DNA nanostructures in improving the sensitivity of LFAs to detect extremely low levels of viral loads. Importantly, this level of LFA sensitivity was achieved without using extensive sample preparation steps such as sample lysis, purification, and nucleic acid amplification steps (Liu et al., ACS Appl. Nano Mater. 2021, 4, 13826-13837; Parolo et al., Nat. Protoc. 2020, 75, 3788-3816). In the specificity test involving H1N1 (FIG. 4C), the test line signals exhibited comparable intensities to those observed with a low concentration of SARS-CoV-2 (FIG. 5). This observation raises the concern of potential false-positive cases attributable to other viral infections. To address this issue, several experimental strategies can be employed to minimize the occurrence of false-positive results. For instance, pretreating nitrocellulose membranes (NCM) with various blocking reagents, such as polymers, surfactants, and proteins (e.g., gelatin, casein, and bovine serum albumin), has shown promise in preventing the occurrence of false positive results (Dalirirad et al., ACS Omega 2020, 5, 32890-32898). However, it's worth noting that NCM pre-treatment may impact assay sensitivity. In commercial settings, addressing these challenges can involve pretreating the conjugate pad with these blocking reagents before the application and drying of reporter assays.

[0229] Given that false positive and negative results are the major limitation of existing LFAs, the integration of machine learning, probabilistic algorithms, and automated image collection providing a measure of uncertainty can help reduce user confusion stemming from false positives or negative results, fostering wider LFA adoption (Wang et al., Front. Bioeng. Biotechnol. 2023, 11, 1020430). The existing infrastructure developed during COVID pandemic could be leveraged to automate image collection of digital LFT data within these pipelines, facilitating the continuous refinement of image classification models. For instance, due to visual similarities in qualitative LFT, existing algorithms can be updated with a smaller dataset once a substantial dataset is accumulated for a specific test.

[0230] Conclusions

[0231] The LFA device developed herein has three main components, DNA Net-based virus probes, AuNSs, and paper strips. The described technology addresses the limitations inherent in rapid antigen-based COVID- 19 testing by introducing a DNA nanostructure -based assay that significantly improves sensitivity and allows for testing non-invasive sampling, such as saliva samples. This LFA device exhibits a sensitivity of 96.08% and a specificity of 91.18% when subjected to testing with the SARS-CoV-2 TSP. Notably, the device demonstrates a broad detection range spanning from 103-108viral copies / mL and the capability to detect as few as 2,280 viral copies / mL of the SARS-CoV-2 WAI strain. In contrast, the commercial kit, when subjected to the same strains and reaction volume, failed to produce any detectable signals on the test line below 105viral copies / mL in comparative analysis. These findings demonstrate the overall reliability of self-testing and reduce the occurrence of false-negative results in case of low viral load in patient samples.

[0232] EXAMPLE 2

[0233] DNA NET MEDIATED DETECTION OF HIV

[0234] Overview

[0235] Human immunodeficiency virus (HIV) continues to stand as a prominent and pressing concern, in 2022 it was estimated that 39.0 million individuals were living with HIV. Current molecular diagnostics tools are fundamentally limited by their reliance on the amplification and detection of specific viral genetic materials even if there are no intact / infectious viruses. For example, in case of current nucleic acid amplification tests, HIV RNA becomes detectable in plasma approximately 10 days post-infection (Gulinaizhaer, et al., Analyst, 148:1189, 2023). Expression of HIV-1 p24 antigen does not become detectable until 4 to 10 days after the initial detection of HIV-1 RNA, as discerned by Lateral Flow Assay (LFA)-based fourth-generation rapid diagnostic tests (RDTs) such as Determine™ HIV- 1 / 2 Ag / Ab Combo (Abbott) kits (Fox, et al., Sex Transm Infect. 87: 178, 2011). This precedes the detection of HIV antibodies during the acute infection phase, known as the “window period,” which may extend for several weeks to months. Furthermore, as antibodies evolve, their interaction with the p24 antigen leads to the formation of immune complexes, introducing interference with p24 assay detection. Accurate detection of the virus during this early stage holds particular significance for prevention efforts, given that the rate of HIV transmission is markedly elevated during the acute phase, accounting for up to 50% of new HIV infections (Brenner et al., Journal of Infectious Diseases 195:951, 2007). It is equally important to address the second limitation associated with commercially RDTs in sensitive detection of diverse HIV variants, further compromising their overall sensitivity (Aghokeng et al., PLoS One, 4(11): e7702, 2009). To address such gaps in current HIV diagnostics, there is a unmet need for highly sensitive and selective detection assays capable of identifying diverse strains of HIV-1 and overcoming the limitations associated with the window period.

[0236] The HIV-1 virion includes spike trimeric envelope glycoproteins (GP120), exclusively functioning as virally encoded antigens on the surface and facilitating viral entry into host cells through interactions with CD4 receptors. However, considering the limited number of trimeric GP120 spikes on the viral surface, averaging only fourteen, achieving multivalent antigen-binding poses a considerable challenge. Notably, HIV-1 employs a unique antibody-evasion mechanism, strategically preventing immunoglobulin G (IgG) antibodies from accomplishing bivalent binding to viral spikes through both antigen-binding fragments (Fabs). This evasion tactic exploits the sparse distribution of envelope spikes, positioned at least 15 nm apart based on the spike dimensions, exceeding the reach of IgG's two Fab arms.

[0237] To overcome these challenges, two HIV spike-targeting DNA aptamers (Zarandi, et al., SLAS Discovery, 25, 1087, 2020; Liu et al., Biosens Bioelectron, 228, 115197, 2023; Rizvi, et al., Anal Chem, DOI 10.1021 / acs.analchem.2c03467, 2022) were strategically organized into a geometrical arrangement of triaptamer clusters on a net-shaped DNA nanostructure (henceforth referred as DNA-Net) to mirror the spatial pattern of trimeric GP120 proteins on the outer surface of the virion (Klein, PLoS Pathog, 6, 1, 2010). Like DNA scaffold-guided clustering of cell surface mobile proteins, clustering of GP120 proteins guided by the DNA-Net scaffold can achieve global tri-aptamer-GP120 spatial pattern matching by correcting deviations of inter-GP120 distance through the multiple sites of attachment, resulting in enhanced virus binding affinity. The DNA-Net nanostructure was designed using the multi-layer designer DNA nanostructure (DDN) design principle (Kwon et al., Nat Chem, 12, 26, 2020), which offers needed structural bendability for the comprehensive coverage of spike proteins on a viral surface. The resulting DNA-Net-based viral probes are shown herein to enable direct sensing of intact virions within their native environment, thereby enhancing assay sensitivity and reducing false-negative instances during early infection, and further bind spike proteins from various HIV-1 viral strains.

[0238] The DNA-Net-based viral probes were successfully translated to a LFA rapid diagnostic platform, resulting in the creation of a versatile, direct viral recognition platform capable of delivering rapid diagnostic results within 10 mins. The assay is user-friendly, requiring no complex chemistry, elaborate reagent synthesis, or laborious sample preprocessing. Additionally, it offers heightened sensitivity comparable to FDA-approved molecular tests and the convenience of home-testing with results visible to the naked eye for direct detection of HIV infection. The LFA device demonstrated detection of viral loads as low as 328 in clinical samples. In comparison, commercial tests failed to detect viral loads below 10’ under similar experimental conditions. These findings advance HIVST, and provide methodologies for sensitive detection of viral infections.

[0239] Materials and Methods

[0240] DNA-Net complex synthesis.

[0241] The DNA complex preparation involved a one-pot reaction, where all constituent strands were mixed in a final concentration of IX TAE-Mg2+K+buffer (pH 7.5). This buffer solution (IX Net buffer) consisted of 40 mM Tris-acetate, 2 mM EDTA, 12.5 mM Magnesium acetate, and 10 mM potassium chloride. For the construction of the 3 x 3 DNA-Net complex, the individual oligonucleotides were combined in specific stoichiometric ratios, precisely matching the design of the intended nanostructure. To attach the aptamers to the DNA-Net, a molar ratio of 27 : 1 was employed, accounting for the 27 available docking sites on the resultant nanostructure. Subsequently, the mixture was annealed, with the temperature gradually decreasing from 90 °C to 23 °C over a period of 14 hours, using a thermal cycler (TProfessional TRIO Thermocycler). Following the annealing process, the 3 x 3 DNA-Net complexes were stored at 4 °C for use in subsequent assays.

[0242] Agarose gel electrophoresis of DNA-Net.

[0243] Gel electrophoresis was performed using a 1% agarose gel prepared in IX TAE-Mg2+buffer (comprising 40 mM Tris-acetate, 2 mM EDTA, and 12.5 mM Magnesium acetate, pH 7.5). The electrophoresis process was carried out at a constant voltage of 70 V for a period lasting 2-3 hours. Following electrophoresis, the gel was stained with SYBR Green (Thermo Fisher) and then scanned using a GelDoc system (Bio-Rad). To determine the output yield, a clearly defined rectangular gel band in each lane was designated as the region of interest (ROI), and the mean density for the DNA-Net was computed. Additionally, in ImageJ software, the background mean density was calculated by selecting an area external to the distinct rectangular gel band. Atomic Force Microscopy (AFM) imaging.

[0244] DNA-Net samples were annealed at 50 nM concentration in IX Net buffer. A 10 pL aliquot of the buffer solution, containing 2 nM DNA-Net, was then gently placed on a freshly cleaved mica surface (Ted Pella Inc.) and allowed to incubate for 5 minutes. Subsequently, IX TAE / Mg2+ buffer was introduced, and the solution on the surface was promptly aspirated using a pipette. An additional 100 pL of fresh TAE / Mg2+buffer was applied to the mica surface to prepare it for Atomic Force Microscopy (AFM) scanning. The imaging process was conducted in tapping mode within a fluid environment using an Asylum AFM equipped with an MSNL-10 probe (Bruker Nano, Inc.).

[0245] Reporter assay synthesis.

[0246] For the synthesis of the reporter assay, we employed commercially available streptavidin-modified gold nanoshells (AuNS). The synthesis process commenced with a brief vortexing of the AuNS solution to ensure the uniformity of the solution. Subsequently, we combined the AuNS solution, conjugation buffer, and DNA-Net solution in a ratio of 5:4:1, followed by a 30-minute incubation with continuous rotation. After this incubation period, we introduced an excess of biotin molecules into the solution to halt the reaction and block any remaining open streptavidin sites on the AuNS. The solution was further incubated for an additional 15 minutes under continuous rotation before applying and drying it onto the conjugate pad at 37°C for 20 minutes.

[0247] Dynamic Light Scattering and Zeta Potential measurements.

[0248] To determine both the hydrodynamic diameter and zeta potential, we employed the Malvern Zetasizer Nano ZS90 instrument (Malvern Panalytical Ltd., Malvern, UK). This device features a backscattering detector set at an angle of 173°, and for precise sample handling, we complemented the setup with the ZEN1002 Universal Dip Cell. In preparation, we diluted the AuNS stock by a factor of 106using a 0.02 pm filtered PBS buffer to optimize the measurement signals. For zeta potential assessments, we introduced a 60-second delay between each measurement to mitigate the potential influence of Joule heating and polarization effects. The procedure encompassed five zeta potential measurements, immediately followed by five Dynamic Light Scattering size measurements, with the added advantage of backscatter detection, ensuring enhanced precision. These measurements were consistently carried out at a temperature of 25 °C.

[0249] Lateral flow assay device operation.

[0250] The LFA has several components: (i) a conjugate (or conjugation) pad, which is responsible for dispensing and drying the gold nanoshell-DNA-Net conjugates, enabling controlled release onto the nitrocellulose membrane upon hydration; (ii) a nitrocellulose membrane (NCM), which serves to immobilize the 3 x 3 DNA-Net on the test line and the GP120MB protein on the control line; and (iii) The absorption pad which allows for consistent capillary flow across the membrane, facilitating the seamless movement of the sample throughout the lateral flow strip.

[0251] LFA devices were assembled by aligning and affixing the NCM, wicking pad, conjugate pad and sample pad onto a backing card. Each LFA strip was 60 mm in length and 4.5 mm in width, with a 2 mm overlap between each component to ensure continuous sample flow from the sample pad to the wicking pad.

[0252] Printing the conjugates on the test and control lines of the nitrocellulose membrane.

[0253] In the preparation of the test line conjugates, a mixture of 3 x 3 DNA-Net, which carries a biotin strand, and nitrocellulose-streptavidin (NC-SA) was created in a 1:10 ratio within IX DNA-Net buffer, resulting in a total volume of 400 pL. This mixture underwent incubation at room temperature for 45 minutes on a rotator with intermittent agitation to ensure optimal binding. The resultant assay was loaded into a glass syringe. For the control line, a separate glass syringe was filled with 400 pL of GP120MB at a concentration of 0.25 mg mL1. Both glass syringes were then connected to a syringe pump, which was subsequently interfaced with the ALFRD dispenser machine. A controlled flow rate of 0.7 pL cm'1was employed in the syringe pump to initiate the dispensing process. Once the assays were printed onto the nitrocellulose membrane (NCM), they were allowed to dry at 37°C for a duration of 30 minutes under a relative humidity of 17%. Subsequently, these prepared devices were stored in a laminated aluminum bag, supplemented with desiccants to maintain optimal conditions. Prior to the commencement of each experiment, the sealed aluminum bag was opened, and the lateral flow assay (LFA) devices were cut to dimensions of 60 mm x 4.5 mm (length x width) to ensure uniformity across all experiments.

[0254] Colorimetric signal acquisition and quantification.

[0255] The detection process using the lateral flow assay (LFA) device was initiated by immersing each device into individual wells of a 96-well plate. Each well within the 96-well plate was pre-loaded with a composition consisting of 100 pL of running buffer (comprising IX PBS, 1% BSA, KC1, and MgCL), 20 pL of the reporter assay, and a specific volume of the target molecules at the designated testing concentration. The LFA device demonstrated heightened signal intensities in both the test and control lines within a time frame of 10-15 minutes. Subsequently, these signals were captured and quantified using a portable reader (Lumos Leelu, Model# LUMOS-V3-03, Lumos Diagnostics Inc). The Lumos reader utilized an integrated LED light source, emitting red illumination onto the LFA strip with an exposure time of 3 milliseconds. This red illumination was tailored specifically for the gold nanoshells (AuNS) present on both the control and test lines. AuNS effectively absorbed a portion of the incident light, resulting in the reduction of reflected light intensity. The corresponding absorption measurements, following background corrections, were quantified. The determination of the limit of detection (LOD) and limit of quantification (LOQ) values was carried out following well-established methodologies. Handling of clinical HIV-1 virus samples.

[0256] Three HIV-1 clinical samples were received and processed according to the following protocol: 5 mL of 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) was prepared, and 200 pL of the viremic plasma sample was added to this solution. The mixture was incubated at room temperature for 2 hours, after which it was filtered through a 0.22 pm syringe filter. The filtered solution was then aliquoted and stored at -80 C.

[0257] Virus copy quantification.

[0258] In the pursuit of precise RNA isolation and subsequent reverse transcription-quantitative PCR (RT- qPCR), a meticulous and systematic approach was implemented to ensure the reliability and accuracy of the results. The protocol initiates with the thawing of virus samples and standards at 4°C, coupled with sterilization of all surfaces, including gloves, and pre-chilling of the centrifuge and rotor. The subsequent steps involve the transfer of samples to RNase-free microfuge tubes, sequential addition of Trizol-LS solution, ultrapure water, and chloroform, accompanied by mixing and incubation stages. The centrifugation steps, performed at 12,000g for 15 minutes at 4°C, effectively segregate the aqueous phase for further processing. Following isopropanol precipitation and a subsequent centrifugation cycle, the RNA pellet undergoes drying, reconstitution, and incubation before being chilled on ice. The protocol transitions to the preparation of a 2X reverse transcription master mix on ice, according to the manufacturer's instructions. The master mix is then dispensed into a 96-well plate or PCR reaction tubes, followed by the addition of 10 pL of each sample to their respective wells or tubes. Post-mixing and optional centrifugation, the samples are subjected to a controlled incubation in a thermocycler at specified temperatures (25°C for 10 mins, 37°C for 120 mins, 85°C for 5 mins, and a subsequent hold at 4°C).

[0259] Subsequently, a Taqman probe master mix is prepared following the manufacturer's protocol. Employing a qPCR thermocycler, 7.5 pL of the prepared mix, in 4x replicates for each sample, is pipetted into the bottom of a barcoded 384-well PCR plate, with a exclusion of outer wells to mitigate the risk of sample evaporation. An additional 2.5 pL of the sample is introduced to the base of each well, gently mixed by swirling, and the plate is subjected to pulsation on a centrifuge if deemed necessary. The final phase entails executing the protocol in the thermocycler, with the anticipated observation of amplification between 30 and 35 cycles for the 150,000 copy standards. Subsequent data analysis is conducted using a delta-delta CT method against the established quantification standard.

[0260] Surface plasmon resonance study with aptamer and GP120 proteins.

[0261] Experimental assays utilizing the surface plasmon resonance (SPR) technique were conducted to characterize the aptamer-GP120 interactions (FIG. 15A). This allowed evaluation of binding affinity for both aptamers (HApt-1 and HApt-2) across various subtypes and groups of HIV proteins. HIV-1 group M class proteins were first examined, as this HIV-1 class has attained pandemic prevalence, constituting over 99% of the 40 million-plus global HIV infections (Wymant, Science, 375(6580), 540-545, 2022). Specifically, the binding affinity of both aptamers were evaluated against two subtypes of HIV-1 group M, namely subtype-A (GP120MA) and subtype-B (GP120MB) spike proteins. Subsequent analysis was extended to encompass different HIV-1 Group P (GP120p) proteins.

[0262] Based on acquired SPR sensorgram data, HApt-1 aptamer displays a nanomolar binding affinity for all three HIV-1 proteins, showcasing dissociation constant (KD) values of 11.09 nM, 42.03 nM, and 80.67 nM for GP120MA, GP120MB and GP120p respectively, as shown in FIGS. 15B-D. In contrast, the binding behavior of the H Apt-2 aptamer to HIV-1 proteins proved to be more intricate, manifesting a limited response at higher aptamer concentrations (FIGS. 15E-G). Overall, the binding affinity of HApt-2 across all tested proteins was poor, with indeterminate KD values. The SPR results, in conjunction with our simulation outcomes, collectively suggest that HApt-2 aptamer demonstrates more robust binding not only with different subtypes of HIV-1 group M but also with distinct HIV-1 group P.

[0263] DNA-Net Design, Synthesis, and Characterization.

[0264] Building upon previous research, a "three-layer" DDN design principle was implemented to construct rhombus-shaped units of a 3 x 3 DNA-Net structure, approximately measuring 45 nm x 45 nm (Chauhan et al., J Am Chem Soc 145, 20214, 2023; Umrao et al., Anal Chem 96(8): 3291-3299, 2023). Within this structure, spike -targeting aptamers were arranged into an array of tri-aptamer clusters with a ~6 nm intra-trimer spacing and ~15 nm inter-trimer spacing, as shown in FIG. 16A. The intra-trimer cluster spacing was determined based on the trimeric GP120 structure and tentative aptamer-GP120 interactions. The 15 nm inter-tri aptamer spacing on the DNA-Net represents the smallest allowable spacing between two adjacent GP120 on the viral surface and thus, maximizing simultaneous inter-spike interaction with the triaptamers on the DNA-Net (Chauhan et al., J Am Chem Soc 145, 20214, 2023; Galimidi, Cell, 160, 433, 2015). The resultant pattern-matching guided multivalent interactions provide an optimal framework for rapid virus binding with high avidity as employed in the build of LFA device in this study.

[0265] The successful assembly of 3 x 3 DNA-Net was first verified using 1% agarose gel electrophoresis (AGE, FIG. 16B). Subsequently, the structural formation of the DNA-Net was confirmed using atomic force microscopy (AFM) imaging (FIG. 16C). A quantitative assessment of DNA-Net yield was performed using the "Gel Analysis" tool within ImageJ software, resulting in an approximate yield of 88% of DNA-Net monomer. The high purity of the desired DNA-Net structures obviated the necessity for additional purification steps. While the AGE analysis revealed the presence of DNA-Net multimers in trace amounts, this minor occurrence would not exert a significant influence on the overall sensing capabilities of the DNA- Nets.

[0266] Surface Plasmon Resonance Study with Aptamer Coupled DNA-Net and GP120 Proteins.

[0267] SPR assays were conducted aiming to assess the impact of DNA-Net coupled with aptamers on the binding affinities for GP120 proteins, in comparison with free aptamers. The results show a significant enhancement in the binding strength of HApt-1 aptamer trimers when incorporated into the DNA-Net structure, herein referred to as DNA-Net-HApt-1. Specifically, compared to the monomeric HApt-1 aptamer (FIGS. 15B-15D), the DNA-Net-HApt-1 assay demonstrated a notable 2 x 102-fold improvement for GP120MA, displaying a KD value of 50.81 pM (FIG. 17A); a 6 x 104-fold enhancement for GP120MB with a KD value of 0.71 pM (FIG. 17B), and a 1 x 104-fold improvement for GP120p with a KD value of 7.48 pM (FIG. 17C).

[0268] The DNA-Net platform has also shown its power and capability to enhance the binding affinity of HApt-2 aptamer trimers, denoted as DNA-Net-HApt-2. The SPR assays exhibited KD values of 1.03 nM, 0.10 nM, and 1.31 nM binding between DNA-Net-HApt-2 with GP120MA, GP120MB, and GP120p proteins, respectively (FIG. 17D-17F). Without being bound to a specific theory, the organization of HApt-2 aptamer (a weak binder) into trimeric clusters on the DNA-Net may contribute to the collective efforts of aptamers, potentially stabilizing and enhancing the binding affinity with the GP120 trimer. This may explain the observed nanomolar binding affinity of DNA-Net-HApt-2 compared to free HApt-2 aptamers (FIGS. 15E-15G). Collectively, these findings provide demonstrate the efficacy of the 3 x 3 DNA-Net design in proficiently capturing GP120 proteins, and for detecting HIV.

[0269] Reporter Assays Formulation.

[0270] A reporter or detector assay was developed for an HIVST device. Here, streptavidin-coated gold nanoshells (SA-AuNS) were utilized. A methodology wherein the DNA-Nets (DNA-Net-HApt-1 or DNA- Net-HApt-2) were affixed to SA-AuNS through a biotin strand was employed, leading to the development of AuNS-based reporter assays. A thorough characterization was carried out, encompassing assessments of zeta potential and hydrodynamic diameter, to evaluate the characteristics of the resulting conjugate. Notably, the introduction of the 3 x 3 DNA-Nets led to a discernible increase in the size of the AuNS, from an initial dimension of approximately 161.74 ± 2.50 nm to 261.44 ± 1.91 nm (FIG. 18A). These findings corroborated the anticipated effective coverage of the AuNS surface area by the DNA-Nets, in line with their theoretical dimensions. Such coverage may be influenced by the geometric orientation of DNA-Net on the AuNS surface. This coverage is afactor in stabilizing individual AuNS, as these nanoshells initially exhibit a notably low zeta potential of -15.22 + 0.44 mV. The overall zeta potential of the AuNS experiences a further decrease to -35.4 ± 1.13 mV upon the introduction of the 3 x 3 DNA-Nets, indicating the stability achieved within the AuNS when coated with DNA-Net complex (FIG.18B).

[0271] DNA-Net Assay-Based LFA Device Testing using GP120MB Protein as a Target.

[0272] Sensitivity of the LFA device investigated by immobilizing 3 x 3 DNA-Net- HApt- 1 assay on the test line (DNA-Net-HApt-1 -T) and GP120MB protein on the control line (FIG. 19A for the convenience of illustration). Subsequently, a range of GP120MB concentrations was introduced, spanning from 1.95 nM - 250 nM in a running buffer. In the presence of the GP120MB target, a sandwich complex is formed using AuNSDNA-Net-HApt-i, and DNA-Net-HApt-1 -T on the test line (" / ) and another complex is formed between AuNS DNA-Net-HApt-i and GP120MB on the control line (C). This resulted in an elevated gray color signal amplification by the reporter assay, producing high-contrast gray colors on both the T and C lines in direct response to the increasing GP120MB concentrations (FIG. 19B). The heightened signal intensities offer a foundation for both qualitative and semi-quantitative interpretations, providing valuable insights into the presence and concentration of the target in the tested sample. These findings demonstrated that the LFA device exhibited a broad linear range of target protein detection extending from 1.95 nM - 62.5 nM with KD value of 62.97 ± 9.46 nM (FIG. 19C).

[0273] Impact of Different Biological Matrices on LFA Performance.

[0274] Considering that RDT for HIV screening are typically performed on blood, oral, and sometimes urine fluids, adaptability of the LFA device for clinical use was evaluated by subjecting it to various levels of serum, saliva, and urine. The 100 nM GP120MB target, supplemented with varying proportions of saliva, urine, and serum samples ranging from 25% to 100% (FIGS. 20A-20D) was introduced. The results demonstrated that the device could robustly detect the protein target in the presence of 25% - 75% saliva or urine, and 25% - 50% in serum fluids. However, elevating the body fluid concentration beyond 50% resulted in false positive signal generation in case of serum and false negative results in case of saliva and urine. Without being bound by any particular theory, this may be attributed to the presence of higher concentration of mixed proteins, which might interfere with the sandwich complex formation process of DNA-Net-HApt-l-T with the reporter assay and the protein target. Nonetheless, these findings provide exemplary body fluid compositions without compromising device sensitivity.

[0275] Subsequently, GP120MB, at concentrations ranging from 1.95 nM - 250 nM, was introduced into a biological milieu containing 50% serum in a running buffer (FIG. 19D). This setup facilitated a comparative analysis of the binding isotherm in contrast to aqueous environments lacking serum (0% serum, FIG. 19B). The LFA exhibited a broad linear detection range, from 1.95 nM - 62.5 nM, with a KD of 79.62 ± 11.80 nM (FIG. 19E). The K values closely mirrored those obtained in the aqueous environment (FIG. 19C). The marginal difference in KD values can be attributed to the presence of diluted mixed proteins within the serum containing samples, influencing the binding of GP120MB with the DNA-Net-HApt-l-T. These findings underscore the robustness of the LFA device in detecting the spike protein of the HIV-1 virus under biologically relevant conditions. Additionally, sensitivity and specificity tests of the device were performed within a 50% serum environment. The LFA device exhibited a sensitivity of 99%, identifying 100 true positives (TP) and registering only 1 false negative (FN) when exposed to 100 nM GP120MB protein. In terms of specificity assessments, the LFA device was examined under identical environmental conditions, omitting GP120MB from the solution. The device demonstrated a specificity of 98.02%, correctly identifying 99 true negatives (TN) and recording 2 false positives (FP) cases.

[0276] To address potential false-positive occurrences arising from cross-reactivity with other trimeric- shaped proteins such as influenza H1N1 hemagglutinin (HA) trimers and SARS-CoV-2 trimeric spike proteins, we conducted specificity testing (FIGS. 22A-22B). The LFA device exclusively exhibited a positive colorimetric signal only in the presence of GP120MB protein, with minimal test line signals in response to other control samples. These findings provided evidence to rule out the potential for other protein cross-reactivity with the disclosed LFA device.

[0277] The DNA-Net-HApt-2 assay-based LFA device was also subjected to testing using the GP120MB protein. A consistent experimental approach was employed similar to the one employed in the DNA-Net- HApt-1 assay-based LFA device. However, in this instance the 3 x 3 DNA-Net-HApt-2 molecules were immobilized on the test line (DNA-Net-HApt-2-T) and an AUNSHAPI-2 reporter assay was utilized. The experimental conditions involved the introduction of increasing concentrations of GP120MB ranging from 0.1 pM - 1.93 pM in a running buffer. DNA-Net-HApt-2 exhibited weak binding with the GP120MB protein, manifesting a KD value of 0.58 + 0.02 pM (FIG. 23A-23B) highlighting the limited binding performance of the HApt-2 aptamer with the target protein. The DNA-Net-HApt-2 assay -based LFA device demonstrated a lack of non-specific binding events when tested in diverse biological environments, including saliva, serum, and urine samples (FIG. 23C and 23D). The device exhibited consistent functionality even in a 50% serum environment, as evidenced by comparable KD values of 0.63 + 0.04 pM (FIGS. 24A-24B), relative to the aqueous environment (0% serum, FIG. 24B). Furthermore, the DNA-Net-HApt-2 - based LFA device exhibited a good specificity towards the GP120MB protein, as illustrated by its performance compared to similar trimeric spike proteins (FIGS. 24C and 24D).

[0278] Device Performance with HIV-1 Viruses and Comparison with Commercial RDTs.

[0279] Provided is a rapid, reliable, and user-friendly HIVST device capable of detecting low HIV-1 viral loads during the acute infection stage. The device was tested with different sets of HIV-1 viruses, including HIV-1 pseudoviruses (PV) and three HIV-1 clinical samples. Serial dilution of viruses were performed using IX PBS in 50% serum as dilution buffer. 100 pL of each diluted viral sample was applied to the sample pad of the LFA device. A gradual intensification in the test line signal was observed as DNA-Net- HApt-l-T assay formed a sandwich complex with the whole virus and reporter assay, correlating with increased viral loads in the samples (FIGS. 21A-21D). The device demonstrated a broad detection range spanning from 103- 106viral copies for PV, and 103- IO3copies for clinical HIV-1 viral samples. To benchmark the device, performance was compared to commercially available fourth-generation Abbott Determine™ H IV- 1 / 2 Ag / Ab Combo RDT, which is widely accessible over the counter and on e-commerce platforms. For a comparative study, clinical HIV-1 sample diluted in 50% serum was applied directly to the sample pad of the commercial RDT and the results were read within 20-30 mins. As compared to the disclosed LFA device, the commercial RDT failed to show detectable test line signals below 103viral copies per test (FIG. 21E). To determine the sensitivity of the disclosed device, a previously established statistical method utilizing a four-parameter logistic curve fit for calculating the limit of detection (LOD) was used. The device could detect 446 (PV), 328 (clinical-1), 341 (clinical-2), and 345 (clinical-3) viral copies using PV and clinical samples, respectively (FIG. 21F). Additionally, another antibody-based RDT, OraQuick, was evaluated following the same testing conditions with clinical HIV-1 sample (FIG. 25). Similar to the Abbott RDT, OraQuick RDTs failed to produce detectable signals below 103viral copies. The enhanced sensitivity of the disclosed device demonstrates it can be used to detect active viral infections during the acute phase, where viral load in plasma samples varies from 102-104copies per mL (McMichael et al, Nat Rev Immunol, 10, 11, 2010). This enhanced sensitivity significantly improves the likelihood of individuals receiving prompt results at an early stage, thereby reducing the risk of disengagement from follow-up care. Furthermore, such sensitivity is important for prescribing intrapartum prophylactic therapy and antiretroviral drugs to prevent mother-to-child transmission, making a valuable contribution to HIV prevention programs.

[0280] In the context of the evolving HIV epidemic, developing innovative tools, particularly HIVST products, is crucial. These advancements are key to managing HIV infection during the acute stage, alleviating the burden on public healthcare systems, and improving overall health outcomes. The aptamer - GP120 binding was characterized using SPR, which quantified the nanomolar binding affinity of the HApt-1 aptamer across diverse groups (M / P) and protein subtypes (A / B). To further enhance the aptamer binding affinity, they were incorporated into a DNA-Net platform, which provides flexibility and extensive coverage for spike proteins on the viral surface. The aptamer-integrated DNA-Net exhibited up to a 104-fold improvement in detecting GP120 proteins compared to free aptamers. Subsequently, the DNA-Net-Aptamer construct was adapted for use on the LFA platform, demonstrating its suitability for point-of-care and home settings. The DNA-Net-HApt-1 -based LFA device demonstrated a sensitivity of 99% and specificity of 98.02% when subjected to GP1 0MB proteins in biologically relevant conditions. To assess the practical application of the disclosed LFA device, it was tested with PV and three patient samples in a 50% serum environment. The device successfully detected as few as 328 viral copies in clinical samples, surpassing the sensitivity of commercial RDTs, which were unable to detect viral loads below 105copies. Without being bound by any particular theory, the enhanced sensitivity may be attributed to aptamer integration into the DNA-Net platform, which improves the detection of extremely low viral loads while minimizing false negative occurrences.

[0281] EXAMPLE 3

[0282] DNA NET-BASED LFA TESTS FOR RAPID AND SENSITIVE DETECTION OF VIRAL INFECTIONS

[0283] While data herein discloses detection of SARS-COV-2 and HIV-1, virus specificity is mediated by the DNA aptamer. Thus, DNA net-based LFA assays can readily be adapted to detect other viruses of interest by changing the DNA aptamer. Specific aptamers are integrated into the DNA-Net by attaching complementary single-stranded DNA sequences as overhangs on each trimeric cluster, which are strategically distributed throughout the DNA-Net.

[0284] Aptamers are short, single-stranded DNA or RNA molecules that fold into specific three- dimensional structures, allowing them to bind target molecules with high affinity and specificity. A typical aptamer selection process begins with synthesizing a large, diverse library of nucleic acid sequences, typically containing 1013- 1015unique sequences, each with a degenerate sequence in the central region flanked by fixed primer-binding regions for amplification. The primary method for aptamer selection is the Systematic Evolution of Ligands by Exponential Enrichment (SELEX), a process involving repeated cycles of binding, partitioning, and amplification. In the binding step, the library is exposed to the target molecule under specific conditions, retaining sequences that bind to the target. These bound sequences are then separated from non-binders using techniques such as affinity chromatography, magnetic beads, or microfluidics and subsequently amplified through polymerase chain reaction (PCR) or RT-PCR plus in vitro transcription for RNA aptamers. The iterative selection process, typically repeated for 8-15 cycles, enriches the library for high-affinity binders with high specificity. After SELEX, enriched sequences are cloned and sequenced, with dominant aptamer candidates identified using NGS. These candidates are characterized for binding affinity, specificity, and secondary structure using techniques like Biolayer Interferometry or Surface Plasmon Resonance (SPR).

[0285] Many DNA aptamers that specifically bind a virus, particularly a surface vial protein, have been described. Non- limiting examples of suitable aptamer sequences are provided below:

[0286] It will be apparent that the precise details of the methods or compositions described herein may be varied or modified without departing from the spirit of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

We claim:

1. A lateral flow assay (LFA) test strip, comprising a lateral flow membrane comprising a test line, wherein: the test line comprises an immobilized DNA net nanostructure, and the DNA net nanostructure comprises a DNA aptamer that binds a target virus.

2. The LFA test strip of claim 1, wherein binding the target virus comprises binding a viral protein of the target virus.

3. The LFA test strip of claim 2, wherein the viral protein is a viral spike protein of the target virus.

4. The LFA test strip of claim 1, wherein the LFA test strip detects intact viruses.

5. The LFA test strip of claim 1, wherein the LFA test strip does not detect degraded viruses.

6. The LFA test strip of claim 1 , wherein the target virus is a class I fusion protein virus.

7. The LFA test strip of claim 6, wherein the target virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV), influenza A virus (IAV), porcine deltacoronavirus (PDCO), or porcine epidemic diarrhea (PED).

8. The LFA test strip of claim 1, wherein the target virus is hepatitis B virus (HBV), human papillomavirus (HPV), or dengue virus (DENV).

9. The LFA test strip of claim 1, wherein the lateral flow membrane comprises a nitrocellulose membrane.

10. The LFA test strip of claim 1, wherein the LFA test strip further comprises a sample pad and a wicking pad.

11. The LFA test strip of claim 10, further comprising a conjugate pad.

12. The LFA test strip of claim 11, wherein the conjugate pad comprises a reporter composition, wherein the reporter composition comprises the DNA net nanostructure linked to a gold nanoshell or gold nanoparticle.

13. The LFA test strip of claim 1, wherein the lateral flow membrane further comprises a control line.

14. The LFA test strip of claim 1, wherein the LFA test strip detects the target virus in a sample at a concentration of 102-107viral copies per 100 pL sample volume.

15. The LFA test strip of claim 1, wherein the LFA test strip detects the target virus in a sample at a concentration of 102-105viral copies per 100 pL sample volume.

16. The LFA test strip of claim 1, wherein the LFA test strip detects the target virus in a sample at a concentration of 102-l 03viral copies per 100 pL sample volume.

17. A kit comprising the LFA test strip of any one of claims 1-16.

18. A method of detecting a target virus in a sample, comprising:(i) adding a sample to the sample pad of the LFA test strip of any one of claims 1-16, and(ii) detecting a signal, thereby detecting the target virus in the sample.

19. A method of detecting a target virus in a sample, comprising:(i) adding the sample to a sample pad of an LFA test strip, wherein:(a) the LFA test strip comprises a conjugate pad comprising a first DNA net nanostructure comprising a DNA aptamer that binds the target virus, wherein the first DNA net nanostructure is linked to a reporter, and(b) the LFA test strip comprises a lateral flow membrane comprising a second DNA net nanostructure comprising a DNA aptamer that binds the target virus immobilized on a test line; and(ii) detecting a signal from the test line, thereby detecting the target virus in the sample.

20. A method of detecting a target virus in a sample, comprising:(i) mixing the sample with a first DNA net nanostructure comprising a DNA aptamer that binds the target virus, wherein the first DNA net nanostructure is linked to a reporter, thereby producing a mixed sample;(ii) adding the mixed sample to a sample pad of an LFA test strip, wherein the LFA test strip comprises a lateral flow membrane comprising a second DNA net nanostructure comprising a DNA aptamer that binds the target virus immobilized on a test line; and(iii) detecting a signal from the test line, thereby detecting the target virus in the sample.

21. The method of any one of claims 18-20, wherein the reporter is a gold nanoshell or gold nanoparticle.

22. The method of any one of claims 18-21, wherein the sample is a bodily fluid.

23. The method of any one of claims 18-22, wherein the bodily fluid is saliva, urine, serum, or whole blood.

24. The method of any one of claims 18-23, wherein the sample is obtained from a human infected with or suspected of being infected with the target virus.

25. The method of any one of claims 18-24, wherein the sample is obtained from a pig infected with or suspected of being infected with the target virus.

26. The method of any one of claims 18-25, wherein the target virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV), porcine deltacoronavirus (PDCO), porcine epidemic diarrhea (PED), hepatitis B virus (HBV), human papillomavirus (HPV), dengue virus (DENV), or influenza A virus (IAV).

27. A method of diagnosing a viral infection in a subject, comprising:(i) adding a sample from the subject to a sample pad of an LFA test strip, wherein:(a) the LFA test strip comprises a conjugate pad comprising a first DNA net nanostructure comprising a DNA aptamer that binds the target virus, wherein the first DNA net nanostructure is linked to a reporter, and(b) the LFA test strip comprises a lateral flow membrane comprising a second DNA net nanostructure comprising a DNA aptamer that binds the target virus immobilized on a test line; and(ii) detecting a signal from the test line, thereby diagnosing the viral infection.

28. A method of diagnosing a viral infection in a subject, comprising:(i) mixing a sample from the subject with a first DNA net nanostructure comprising a DNA aptamer that binds the target virus, wherein the first DNA net nanostructure is linked to a reporter, thereby producing a mixed sample;(ii) adding the mixed sample to a sample pad of an LFA test strip, wherein the LFA test strip comprises a lateral flow membrane comprising a second DNA net nanostructure comprising a DNA aptamer that binds the target virus immobilized on a test line; and(iii) detecting a signal from the test line, thereby diagnosing the viral infection.

29. A DNA net nanostructure that specifically binds a target virus, wherein the DNA net nanostructure is linked to a gold nanoshell or gold nanoparticle.

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