Method and device for detecting pathogens and molecular components thereof

Abstract

Disclosed herein are methods, systems, and devices for detecting the presence of pathogens, such as viruses (e.g., SARS-CoV-2) or molecular components thereof, in health care related samples and / or environmental samples. An exemplary system for improving the detection of a pathogen includes a biosensor device including a detection chip and at least one probe that specifically recognizes a pathogen, where the detection chip includes a graphene field effect transistor (FET) chip and the probe, the aptamer specifically binds to DNA (deoxyribonucleic acid), RNA (ribonucleic acid) or protein related to the pathogen.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent document claims priority to and the benefit of U.S. Provisional Application No. 63 / 144,460, filed on February 1, 2021, entitled “Methods and Apparatus for Detecting Viruses and Molecular Components thereof.” The entire contents of the aforementioned patent application are incorporated herein by reference as part of the disclosure of this patent document.

[0002] This invention was made with government support from the National Institutes of Health under Grant No. HL119893. The government has certain rights in this invention. Technical Field

[0003] This patent document generally relates to the detection of pathogens, and more specifically, to the use of field-effect transistors and aptamers to accurately detect viruses and their molecular components. Background Art

[0004] Pathogens such as viruses and bacteria pose a significant threat to human health. For example, coronaviruses are a large family of RNA viruses that can cause upper respiratory tract illnesses in humans, ranging in severity from mild to fatal. The novel coronavirus that has recently caused a global epidemic and health care crisis is SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 appeared in December 2019 and was declared a global pandemic by the World Health Organization (WHO) on March 11, 2020. According to recent reports, COVID-19 is highly contagious (>96 million positive cases) and causes high morbidity (>2 million deaths) worldwide.

[0005] To successfully combat a pandemic, multiple population-scale health measures need to be coordinated and implemented. One cornerstone of this multi-probe strategy is the daily, real-time delivery of reliable, rapid, and inexpensive diagnostic tests for use at home, work, and at the point of care (POC), which would be administered on simple-to-use diagnostic devices with wireless data transmission capabilities for continued large-scale pandemic surveillance. Despite significant efforts in this area, such methods and devices do not yet exist. SUMMARY OF THE INVENTION

[0006] The disclosed technology relates to methods, systems, and devices for detecting the presence of pathogens, such as viruses (e.g., SARS-CoV-2), or their molecular components in healthcare-related and / or environmental samples.

[0007] In some exemplary aspects, a biosensor device is provided to detect the presence of at least a limited count / amount of viral particles, RNA, DNA, or proteins associated with a pathogen of interest (e.g., the SARS-CoV-2 virus). However, the specificity of the biosensor can be advantageously altered to detect biological pathogens other than viruses, such as influenza, bacteria, toxins, or fungi. Thus, embodiments of the technology disclosed herein can be deployed in efforts ranging from global health to global security.

[0008] In other exemplary aspects, the biosensor device includes a detection chip, such as a graphene field effect transistor (FET) chip. In some embodiments, the detection chip includes a probe attached to the detection chip, such as an aptamer, which specifically binds to a target viral antigen, particle, RNA, DNA, or protein. In some embodiments, the aptamer is an oligonucleotide (RNA or DNA, single-stranded or double-stranded). In some embodiments, the aptamer is a peptide.

[0009] In other exemplary aspects, methods are provided for detecting whether a subject has been exposed to a particular virus, such as the SARS-CoV-2 virus, by using a biosensor device of the technology described herein to detect the presence of viral particles, RNA, DNA, or proteins associated with the virus in a sample from the subject.

[0010] In other exemplary aspects, a biosensor device for detecting one or more pathogens is provided. In one example, the biosensor device includes a detection chip, which includes (a) a substrate having a graphene surface, (b) a conductive material at the first end and the second end of the graphene surface, which form a first electrode and a second electrode, respectively, and (c) an insulating material to insulate the first electrode and the second electrode. In this example, one or more probes attached to the graphene surface specifically bind to one or more target molecules of one or more pathogens. In addition, the insulating material forms a hole to receive a biological sample so that the biological sample contacts the one or more probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figures 1A-1C and 2A-2C are schematic diagrams illustrating the design and mechanism of a portable chip-based electronic biosensor device with a wireless communication module according to the technology of the present disclosure, which facilitates the effective detection of viral RNA / DNA targets and the monitoring of large-scale viral epidemics.

[0012] Figure 3 is a schematic diagram showing the collection of biological samples and the detection of pathogens using a handheld device.

[0013] Figure 4AAn example of a graphene FET (GFET) sensor on a chip carrier and a circuit board setup for the graphene FET sensor are shown.

[0014] Figure 4B Shown for Figure 4A Example of drain-source current analysis with respect to gate voltage for the experimental setup shown in .

[0015] Figures 5A-5F Stages in an example fabrication of a graphene FET are shown.

[0016] Figure 6 Shown is a two-dimensional view of an exemplary graphene FET.

[0017] Figures 7A-7H Various stages of atomic force microscopy imaging of an exemplary graphene FET are shown.

[0018] Figure 8-11 A partial cross-sectional view of an exemplary embodiment of a detection device is shown in an assembled state.

[0019] Figure 12 Another exemplary embodiment of a detection device is shown.

[0020] Figure 13A and 13B An example of an assembled portable compact device is shown.

[0021] Figure 14-17 An example of detecting SARS-CoV-2 using aptamers specific for viral proteins with high specificity and sensitivity is shown.

[0022] Figures 18A-18C Shown are exemplary aptamer-S and aptamer-N derived GFET sensor responses due to the binding of different concentrations of the RBD region and N-protein of SARS-CoV-2.

[0023] Figures 19A-19F Shown are exemplary results based on testing the sensor specificity of aptamers-S and -N by using closely related homologous antigens of MERS-CoV, SARS-CoV, and inactive MERS-CoV viruses.

[0024] Figure 20A and 20B Example results of concentration-dependent sensor responses to different concentrations of inactive virus and 10% saliva are shown for aptamer-S derivatized GFETs and aptamer-N derivatized GFETs, respectively.

[0025] Figure 20CResults are shown for buccal swab samples corresponding to RT-PCR positive and negative tests for point-of-care (POC) antigen tests from the FDA EUA.

[0026] Figure 20D Shown are the results of blind sample testing of graphene FETs derivatized with aptamer-S.

[0027] Figure 21A and 21B An example showing the mediated transfer curve analysis of an aptamer-derived graphene FET.

[0028] Figures 22A-22C Examples of detecting SARS-CoV-2 RBD homologous proteins of mutant viruses are shown.

[0029] Figure 22D An example of detecting SARS-CoV-2 RBD homologous proteins of the original virus is shown.

[0030] Figure 23A and 23B Shown are exemplary results analyzing the specificity of aptamer-derived graphene FETs.

[0031] Figures 24A-24G Exemplary results of the current-voltage (IV) relationship of the aptamer-anylate interaction are shown.

[0032] Figures 25A-25C Exemplary GFET device responses to the Δ variant N-protein, Omicron variant N-protein, and inactivated Δ variant are shown, respectively.

[0033] Figure 26A and 26B Shown are exemplary test results of human saliva samples during the January 2022 wave of Omicron using embodiments of the disclosed technology. Detailed Description of the Invention

[0034] The present invention discloses methods, devices and systems related to the detection of proteins, nucleic acids and viruses and / or other pathogens such as bacteria, fungi or biotoxins. Specifically, a portable wireless electronic biosensor device is disclosed, which includes a biosensor chip for detecting pathogens (e.g., viruses like SARS-CoV-2) or their molecular components from biological samples or in environmental samples, such as body fluid samples from humans (e.g., saliva, sweat, tears, exhaled gas, blood, urine), human nasal, nasopharyngeal or fecal swabs; environmental samples such as air, water, airborne pathogens, and household and industrial waste. The handheld, independent, portable diagnostic device combines wireless communication technology and allows for highly specific and sensitive pathogen detection reported within minutes. The described embodiments advantageously enable rapid and accurate identification of SARS-CoV-2 and other viral antigens that may appear worldwide.

[0035] Exemplary Implementations in Global Health Applications

[0036] A person infected with a pathogen such as the SARS-CoV-2 virus can expel the virus or tiny aerosolized droplets containing the virus by coughing, talking, or exhaling. A person's saliva may also contain measurable levels of the virus. A device containing a detection chip that indicates the presence of a target protein or nucleic acid associated with a specific virus, such as the SARS-CoV-2 virus, can therefore be breathed into or breathed into by the user, or droplets of saliva from the user can be deposited into the device to test the user for the presence of the SARS-CoV-2 virus. Such a device capable of signaling the presence of the SARS-CoV-2 virus would benefit from being portable and reusable for testing multiple people. However, a reusable device would require careful distinction between the reusable and disposable portions of the device, such as an interface configured to contact the user's mouth, to prevent uninfected users from inadvertently becoming infected by using a contaminated device.

[0037] In the event of a global pandemic and similar outbreaks associated with the SARS-CoV-2 virus, or even mutations thereof, there is a need to quickly, safely, and easily test people for the possible presence of the SARS-CoV-2 virus or other disease-associated viruses. It would be beneficial to be able to test people to assess the presence or absence of disease-associated viruses in their breath or saliva with minimal delay and risk of transmission between people. For example, it would be beneficial to quickly, safely, and simply test a given sample of the population or people within a location, which could include, but are not limited to, airports, libraries, movie theaters, classrooms, restaurants or bars, office environments or lobbies, hotels or lobbies / entrances, public transportation venues, hospitals / urgent care / doctors' offices, or any other interior space where people may gather and where transmission of the virus between people is possible.

[0038] Furthermore, the portability of the described embodiments will enable the biosensor device to be deployed in areas that can be identified as sources of pathogens, such as cavities in the case of SARS-CoV-2. This efficiency of deployment is supported by the wireless connectivity and capabilities of the biosensor device, which will enable periodic updates to research and medical facilities, and the sensitivity of the graphene FET, which will be able to detect very low concentrations of pathogens, enabling early detection at the source.

[0039] Exemplary Implementation in Global Security Applications

[0040] Biological threats. Biological threats can emerge without warning from nature, intentional attack, or accidental release. Infectious diseases that have swept the world, such as MERS-CoV and SARS-CoV-2 discussed above, have been controlled only after massive international collaboration with governments in affected regions and billions of dollars in expenditures. Interconnected travel and commerce, especially from the pathogen's region of origin, have quickly led to the spread of pathogens worldwide.

[0041] Governments often rely on the private sector to manufacture vaccines, pharmaceuticals, diagnostics, and medical devices needed to respond to emerging biological threats. Embodiments of the technology disclosed herein are crucial in establishing national and subnational surveillance systems capable of predicting and identifying infectious disease threats. The sensitivity of graphene FETs, capable of detecting very low concentrations, along with the portability and cost-effectiveness of the described biosensor devices, could be leveraged in deployments that could serve as large-scale early warning systems for biological threats.

[0042] Environmental Monitoring. The ability to deploy the described biosensor devices on a large scale makes them well-suited for environmental monitoring, particularly for detecting environmental pollutants such as heavy metals, small molecule agricultural toxins, and waterborne bacterial pathogens. Additional targets include aquatic toxins, pesticides, industrial byproducts, antibiotics, and pharmaceuticals. Traditional multi-step detection processes can degrade many of the environmental pollutants of interest already at low concentrations. Embodiments of the technology disclosed herein provide on-site detection and dissemination of results through built-in wireless capabilities, making the described aptamer-based biosensors particularly useful for monitoring water, soil, and air.

[0043] Advantages and benefits of the technology disclosed herein

[0044] Therefore, there is a need for a device that can detect multiple disease-related pathogens with a single test. Such a device is handheld, self-contained, and portable, and it is beneficial to produce a known test result within a few minutes of testing. Such a test would further benefit from cost savings and immediate delivery of results to non-technical personnel as well as physicians, administrators, and public health individuals. Such a device would also benefit from the ability to detect positive pathogen variants, which would allow for the diagnosis of potential future mutations of known viral genomes (e.g., SARS-CoV-2 genomes) using aptamers or nucleic acids targeting such variants.

[0045] Embodiments of the disclosed technology use graphene FET-biosensor chips, which have been used in conjunction with double-stranded probes for detecting nucleotides with single-nucleotide resolution by strand displacement (e.g., see U.S. Patent No. 10,793,898, which discloses an electrical biosensor chip based on a graphene field-effect transistor (FET)). Electrical biosensors have higher (atto-pico-molar) specificity than optical sensors, thereby reducing the need for sample amplification. In particular, FET-based sensors can sense changes in charge during biomolecular interactions and provide the highest possible sensitivity, i.e., single-electron charge sensitivity, when using every atom on the surface. Electrical biosensors also allow for faster readout, low power consumption, portability, inexpensive mass production, integrated sensor and measurement systems, and no need to label biomolecules.

[0046] However, compared to known double-stranded nucleic acid probes and chip-based devices, the technology described herein has expanded and superior capabilities, including the use of single-stranded aptamers as probes, the detection of currents generated by simple probe-sample interactions, and the ability to design aptamers specific for nucleic acid targets as well as proteins of interest (e.g., the spike protein of SARS-CoV-2). These features allow for versatility in the application of aptamers for nucleic acids, proteins, and other sensing needs, making current technology simpler and more adaptable.

[0047] Embodiments of the technology disclosed herein provide the following unique advantages, as well as other features and benefits:

[0048] (1) an aptamer, which is selected from thousands of available aptamers and is capable of binding to a specific protein;

[0049] (2) aptamers, which retain their specificity even when attached to a solid; and

[0050] (3) Aptamers that can recognize mutations of pathogens.

[0051] Publicly available aptamer databases, such as Aptagen's Apta-index, contain sequences derived from hundreds of published experiments. Entries in the database typically provide detailed, structured information about the experimental conditions under which aptamers were selected and their binding affinity quantified. A variety of analytical techniques have been employed to identify the best candidates for binding to the specific proteins considered herein, and embodiments of the disclosed technology use these aptamers to detect a variety of pathogens with high sensitivity and specificity.

[0052] Existing implementations of aptamers have always used them in solution to detect pathogens. A common limitation of aptamer-based assays has been the potential for dramatic changes in their binding properties due to their immobilization on solid substrates. However, the embodiments described herein attach aptamers to graphene surfaces, where the aptamers unexpectedly retain their specificity and can therefore be incorporated into electrical / electronic detection frameworks with high sensitivity. Existing systems using aptamers in solution do not teach or suggest immobilizing aptamers on solid graphene surfaces, as described in the technology disclosed herein.

[0053] The embodiments described herein use aptamers (or equivalently, DNA) instead of antibodies to bind to specific proteins. Thus, by using aptamers, the inherent shortcomings of antibodies in detecting mutations are avoided. As demonstrated by the results provided in this patent document, the SARS-CoV-2 virus and its Omicron and Delta variants were accurately detected using aptamer-based GFET devices that exhibited high specificity and high sensitivity.

[0054] While this disclosure can be embodied in various forms, the following description of several embodiments is with the understanding that the disclosure is to be considered exemplary of the invention and is not intended to limit the invention to the specific embodiments shown.

[0055] Section headings are used in this document to improve readability of the description and do not in any way limit the discussion or embodiments (and / or implementations) to only the corresponding section.

[0056] Example of detection chip

[0057] In some embodiments, the biosensor device of the present technology includes a detection chip. In some embodiments, the detection chip has structural and functional features similar to those disclosed in the '898 patent. For example, the detection chip can include a wafer or substrate made of silicon oxide. The substrate can be coated with a graphene surface. Electrodes are provided on the graphene layer, and a solution reservoir is created by insulating the electrodes with, for example, but not limited to, silicone rubber. In some embodiments, a probe (e.g., an aptamer) is brought into contact with the graphene layer to detect a specific target of interest.

[0058] In some embodiments, the current-voltage (IV) curve of the detection chip can be generated in the absence or presence of the target molecule. Without being bound by any particular theory, the connection of the probe (e.g., aptamer) to the graphene layer increases the resistance of the graphene layer, which results in a baseline IV curve of the detection chip in the absence of any target molecule. Hybridization of the probe to the target molecule can produce a change in affinity between the probe and the graphene layer, thereby resulting in a change in the resistance of the graphene layer, which can be detected by a shift of the IV curve compared to the baseline curve. The more probes hybridize, or if the more target molecules hybridize, the greater the change in resistance, and the greater the change in the IV curve, which provides a method for determining not only the presence of the target molecule but also the detection concentration of the target molecule.

[0059] Examples of aptamer probes

[0060] In some embodiments, the detection chip of the biosensor device includes a probe specific to a target molecule associated with a pathogen of interest, for detecting the presence of a pathogen. In some embodiments, the probe includes an aptamer for specifically identifying a target molecule, such as DNA, RNA, or a protein associated with a pathogen of interest, such as a virus (e.g., SARS-CoV-2E). Aptamers are typically oligonucleotides or peptide molecules that are designed and produced to specifically bind to a target of interest, such as a protein or nucleic acid. Aptamers can be prepared from nucleic acid (RNA or DNA) or peptide. In some embodiments, aptamers include nucleic acid and are single-stranded. In other embodiments, aptamers include nucleic acid and are double-stranded.

[0061] In some embodiments, the detection chip can be configured with specific aptamers to detect the presence of specific target DNA, RNA or protein, and the aptamers can be naturally (e.g., artificially) or synthetically (e.g., using an automated high-throughput deposition system) attached to the detection chip. In some embodiments, the detection chip can include multiple aptamers, each aptamer targeting a specific target DNA, RNA or protein. In some embodiments, multiple aptamers can be arranged in an array for testing the presence of one or more target DNAs, RNAs or proteins associated with one or more pathogens of interest.

[0062] In some embodiments, an aptamer-based detection chip can detect the presence of a target molecule (e.g., a protein, nucleic acid, virus) by detecting changes in the refraction, reflection, fluorescence, absorption, and / or emission of light by the detection chip. Alternatively, an aptamer-based detection chip can utilize changes in impedance, resistance, capacitance, voltage, current, resistance, any combination thereof, or changes in the electric field to indicate the presence of a target molecule bound by an aptamer.

[0063] As shown in the working examples, the biosensor devices of the present technology can be used to quickly and accurately detect pathogens of interest, such as SARS-CoV-2 virus. However, the biosensor devices can be designed for any other pathogen of interest, or can be configured to simultaneously detect multiple viruses, such as common influenza virus, MERS-CoV, SARS-CoV, and SARS-CoV-2, by modifying aptamer probes specific for the intended target. Aptamers can also be adapted to detect potential future variants of the target pathogen, such as potential future mutations within the SARS-CoV-2 genome.

[0064] Examples of Biosensor Devices

[0065] In some aspects, a biosensor device is provided for determining the presence of proteins or viral particles in a healthcare-related environment and / or environmental sample (e.g., Figures 1A-1C and 2A-2C), collect Figure 3 The biological samples shown. Figure 3 In the embodiment shown, a biological sample containing proteins or viral particles can be collected using a nasal swab, a throat swab, or saliva. In other examples, exhaled breath or environmental samples (e.g., air, water, household and industrial waste) can be collected. Figure 3 We further show that sensor response readouts, validated against homologous protein and viral samples, can be collected on a handheld device.

[0066] In some embodiments, the biosensor device includes a housing unit having a first opening, a second opening, and a third opening. The box is adapted to be removably fixed within the housing for ease of measurement. The box includes a detection chip that is electrically connected to the surface of the box during detection of a target. The cap is removably fixed to the second opening, and the cap includes a circuit and a visual indicator. When the box is disposed within the housing and the cap is disposed above the second opening, the target sensing detection chip is configured to be in fluid communication with the first opening, and the cap is configured to be electrically connected to the surface of the box.

[0067] In some embodiments, the detection chip (which can be mounted on a Figure 4A (The panels shown above) use electron mapping or electron density mapping to distinguish energy changes between individual nucleotide pairs. This identification of energy changes determines the protein containing the nucleotide pair. Figure 4B As shown, Figure 4A The exemplary setup shown in Figure 1 results in an analysis of the drain-source current versus the gate voltage. In this example, the gate voltage is swept from +1V to -1V in 2mV steps, the drain-source voltage (Vds) is 30mV (which is optimized in 10mV increments over the 0-100mV range), the drain-source current (Ids) is in the μA range, and the Dirac voltage is analyzed at the Ids minimum.

[0068] In some embodiments, the detection chip can send this information in any of a variety of ways when one or more targets, such as viruses, are detected. In some embodiments, the detection chip can be connected to one or more colored lights, such as LEDs, via a circuit and emit illumination signals of different colors, which are preselected to represent a detection event. One or more colors, or their intensities, or the number of individual LEDs that are illuminated can also be an indication of the concentration of the detection event. In other embodiments, the detection chip can be electrically connected to a circuit including a wireless transmitter that sends data about the detection event to a computer or tablet or other portable or non-portable data storage device for analysis and / or later display.

[0069] Figures 5A-5F Graphene FETs are fabricated using sputtering on a SiO2 substrate ( Figure 5A ) are deposited on the source and drain electrodes (Au / Cr~100nm), and then a passivation layer (SiO2 or Al2O3~80nm) is deposited on the source and drain electrodes ( Figure 5B ).exist Figure 5C In the next step, graphene is wet transferred onto a patterned substrate and poly(methyl methacrylate) (PMMA) is removed by dissolving it with acetone. Figure 5DAs shown, (polymer, polymethylglutarimide) (PMGI) photoresist is applied to protect the sensor area, and the additional graphene layer is removed by O2 plasma etching. The PGMI photoresist is stripped and the graphene FET is formed in a gas atmosphere such as hydrogen / nitrogen ( Figure 5E ) is annealed, and finally polydimethylsiloxane (PDMS) or epoxy resin is applied to form wells for holding the sample liquid.

[0070] In some embodiments, the graphene FET can be configured to detect multiple different pathogens by attaching multiple probes (e.g., aptamers) to non-overlapping portions of the graphene FET. Each of the attached multiple probes is selected to bind to a different specific protein. In other embodiments, a detection chip array can be used to detect multiple different pathogens. In this example, each of the multiple probes is attached to a corresponding one of the detection chip arrays. Thus, embodiments of the technology disclosed herein provide an alternative way to detect multiple pathogens.

[0071] Figure 6 A two-dimensional schematic diagram of a graphene FET is shown, where the source, drain, and gate electrodes are fabricated using gold pads, with a passivation layer on top of the gold pads, followed by a graphene layer. In one example, the graphene FET includes a 500 μm channel between the passivation layers adjacent to the gate and drain-source electrodes.

[0072] Figures 7A-7F Shown are atomic force microscopy images of graphene FETs after different steps. Figure 7A The bare graphene surface (with a roughness of 0.7 ± 0.3 nm) is shown. Figure 7B Shown is the graphene surface after addition of 5 mM PBASE (with a roughness of 1.8 ± 0.5 nm). Figure 7G An example of a delta variant virus inactivated on polylysine-coated mica is shown, with the inset showing a single virus with a diameter of -125 nm. Figure 7H An example of a Δ variant virus inactivated on a GFET chip functionalized with an N-aptamer is shown.

[0073] Figure 7CRaman spectra of pristine graphene and PBASE-functionalized graphene FETs are shown (with the inset showing an optical microscope image of the scanned area). Graphene FETs were derivatized with PBASE (5 mM) and analyzed in variable forward (-1 to +1 V) and reverse (+1 to -1 V) gate voltage sweeps in 0.2 V steps and variable drain-source voltage (Vds) from 0 to 100 mV with 10 mV incremental steps. In addition to the expected G- and 2D-peaks on the original / untreated sample (peak intensity ratio > 1:2, indicating high-quality material), the appearance of D and D' peaks with the addition of PBASE is likely due to the incorporation of pyrene groups and the enhanced sp 3 combination.

[0074] Figure 7D and 7E The drain-source curves at 0 to -1 V are shown, where the I ds Respectively with respect to V G and V SD Measurement. Figure 7F The cyclic (backward and forward) gate voltage sweeps in the range of +1V to -1V at different V ds V under D Hysteresis analysis. Figure 7F As shown, at 20mV>V SD >50mV, in the V D There is a minimum hysteresis in .

[0075] In some embodiments, a schematic representation of a biosensor device 100 according to the present technology is shown in FIG. Figure 8 In the assembled state shown in partial cross section and in Figure 9 105. The main components are shown in exploded partial cross-section. As shown, the main components include a housing 110, which can serve as a handle and a chassis for supporting the other main components. In some embodiments, the housing 110 and the main components are arranged along the longitudinal centerline 105. In other embodiments, the components can be arranged in any geometric shape as may be desired aesthetically or functionally, such as Figure 10 and 11 As shown, and further described below.

[0076] refer to Figure 8 and 9In one embodiment, the housing 110 includes a first opening 120, a second opening 130, and at least one third opening 140. An insert or cartridge 150 is adapted to be removably secured within the housing 110. For example, in one embodiment, the cartridge 150 can be removably secured by a threaded connection through the second opening 130. In another embodiment, the cartridge 150 includes a shoulder 160 extending laterally from an end of the cartridge 150 such that, when the cartridge 150 is positioned within the housing 110, the shoulder 160 overhangs an edge of the second opening 130 and is compressively retained against the edge of the second opening 130 by a cap 170, which is, for example, threadedly attached to the shoulder 160 of the cartridge 150 at the second opening 130. In other embodiments, the cartridge 150 can be removably secured within the housing 110 by the mechanisms disclosed above and / or by a press fit, a magnetic connection, or any single or combination of connection mechanisms known in the art.

[0077] refer to Figure 10 and 11 In another embodiment of the device 200, the housing 210 includes a first opening 220, a second opening 230, and at least one third opening 240. In this embodiment, an insert or cartridge 250 is adapted to be removably secured within the housing 250. For example, in one embodiment, the cartridge 250 can be removably secured by a threaded connection through the second opening 230. In another embodiment, the cartridge 250 includes a shoulder 260 extending laterally from an end of the cartridge 250 such that, when the cartridge 250 is positioned within the housing 210, the shoulder 260 overhangs an edge of the second opening 230 and is compressively retained against the edge of the second opening 230 by a cap 270, which is, for example, threadedly attached to the shoulder 260 of the cartridge 250 at the second opening 230. In other embodiments, the cartridge 250 can be removably secured within the housing 210 by the mechanisms disclosed above and / or by a press fit, a magnetic connection, or any other connection mechanism known in the art, alone or in combination.

[0078] In terms of how the major components fit together, regardless of the geometry of the housing 110, 210, whether Figure 8-11 As shown in , or using other geometries known in the art for housings with insertable and removable inserts or cartridges, all embodiments of the cartridge 150 , 250 include a detection chip 300 in electrical communication with a surface 310 of the cartridge 150 , 250 .

[0079] In some embodiments, the detection chip 300 can be reused through a cleaning process, so that the cartridge 150, 250 disposed thereon can also be reused. In other embodiments, the detection chip 300 is a single-use chip, so that the cartridge 150, 250 is a disposable cartridge 150, 250. The detection chip 300 is in electrical communication with the surface 310, for example, via wires 320 or traces or an internal circuit board having wires or traces.

[0080] Still refer to Figure 8-11 In some embodiments, the cap 170, 270 is attached to the second opening 130, 230, for example, by threads, so that the circuit 330 within the cap 170, 270 is in electrical communication with the surface 310, and therefore also with the detection chip 300. In other embodiments, the cap 170, 270 is connected to the second opening 130, 230 by a press fit, a snap fit, a magnetic connection, a latch mechanism, or by any other mechanism known in the art for a removable connection.

[0081] The circuit 330 is of a type known in the art that can interface with the signals from the detection chip 300 and relay or send the independent signals to a visual indicator 340 disposed on the outside of the cap 170, 270. In one embodiment, the visual indicator 340 is one or more LEDs, but in other embodiments, the visual indicator 340 can be one or more incandescent bulbs, LED or LCD digital displays, or other types of visual indicators known in the art. As described above for the detection chip 30, the visual indicator 340 emits an illumination signal of different colors, which are pre-selected to represent a detection event. One or more colors, or their intensity, or the number of individual LEDs illuminated can also be an indication of the concentration of the detection event. In another embodiment, the visual indicator 340 is electrically connected to the circuit 330, which includes a wireless transmitter that sends data about the detection event to a computer or tablet or other portable or non-portable data storage device for analysis and / or later display. As shown in Figures 8 and 10 , when the cartridge 150 , 250 is detachably secured within the housing 110 , 210 and the cap 170 , 270 is disposed on the second opening 130 , 230 , the detection chip 300 is disposed in fluid communication with the first opening 120 , 220 .

[0082] Now refer to Figure 12In another embodiment of the device 400, the housing 410 includes a port for inserting a detection chip 420 having all the structural and functional features of the detection chip. In this embodiment, the housing 410 also includes a circuit 430 and a visual indicator 440, both of which function the same as the circuit 330 and the visual indicator 340 described above. When inserted into the housing 410, the detection chip 420 operates in the same manner as the detection chip 300 by having an electrical connection on one side that is in electrical communication with the circuit 430. In this embodiment, the detection chip 420 can be exposed to sample molecules, for example, by applying saliva to the chip 420 or by breathing or coughing onto the chip 420.

[0083] refer to Figure 8-12 In any of the embodiments, a power source 350, such as one or more batteries or batteries, is schematically shown as being disposed within the device 100, 200, or 400 and in electrical communication with the circuitry of that embodiment. For example, in the embodiment shown in FIG1 , the power source 350 is disposed within the cap 170, 270 and in electrical communication with the circuitry 330. Thus, when the cap 170, 270 is mounted on the housing 110, 210, the power source 350 is also in electrical communication with the surface 310, and thus further in electrical communication with the detection chip 300. Thus, the power source 350 can not only provide electrical power to the internal circuitry 330 within the cap 170, 270, but can also provide electrical power to the detection chip 300 when the device 100, 200 is assembled.

[0084] Still referring to Figure 1, in some embodiments, a disposable mouth 360 is removably connected to the first opening 120, 220. In some embodiments, the disposable mouth 360 is connected to the first opening 120, 220, for example, by threads, while in other embodiments, the disposable mouth 360 is connected to the first opening 120, 220 by a press fit, a snap fit, a magnetic connection, a latch mechanism, or any other mechanism known in the art for removable connection.

[0085] In some embodiments, prior to operation, a new, unused cartridge 150, 250 is removably inserted into the housing shell 110, 210, and the cap 170, 270 is secured to the housing shell 110, 210. When the device 100, 200 is so assembled, e.g., Figure 8 and 10 The detection chip 300 on the cartridge 150 , 250 is in fluid communication with the first opening 120 , 220 .

[0086] As described above, a person's breath can carry viruses or tiny aerosolized droplets containing viruses and / or molecular components of the viruses, and certain viruses can be identified by one or more target proteins that constitute the virus. The detection chip 300, 420 can indicate the presence of target molecules, proteins, or proteins associated with a specific virus, such as the SARS-CoV-2 virus. Therefore, in operation, a user who wants to test for a specific virus breathes into the interface 360 ​​(or otherwise breathes onto the detection chip 300, 420) to test whether the user has the SARS-CoV-2 virus. If the target molecule is detected, and therefore the specific virus is detected, the detection chip 300, 420 associated with the connection circuit 330, 430 sends a signal to a visual indicator 340, 440 provided on the outside of the cap 170, 270 or the housing 410. The visual indicator 340, 440 emits an illumination signal of one or more different colors preselected to represent a detection event. One or more colors, or their intensity, or the number of illuminated individual LEDs can also be an indication of the concentration of the detection event. In another embodiment, the visual indicators 340, 440 are electrically connected to the circuit 330, 430, which includes a wireless transmitter that transmits data about the detection event to a computer or tablet or other portable or non-portable data storage device for analysis and / or immediate or delayed display of the detection event. In some embodiments, the visual indicators 340, 440 can additionally flash and / or illuminate to signal a fault, low battery, or other error or problem.

[0087] Now back Figure 8-11 In the embodiment of the present invention, the third opening 140, 240 allows the user's breath to exit the housing 110, 210 without creating a pressure buildup therein. Making the mouthpiece 360 ​​disposable allows a fresh mouthpiece 360 ​​to be installed on the device 100, 200 before each use, thereby reducing the risk of contamination between people being tested.

[0088] In other embodiments, for example as Figure 8 and 10 As shown in the portion of , the cartridges 150, 250 and caps 170, 270 can be directly connected to each other by any connection method as described above or other methods known in the art, without the need for housings 110, 210. In these embodiments, the user only needs to breathe onto the detection chip 300 to be tested for the presence of the target protein, and therefore the presence of the associated virus.

[0089] In any of these embodiments, the biosensor device can be embedded in various components and products, or attached, fixed or removably attached to clothing or hats by clips, hook and loop fasteners, or other fastening mechanisms known in the art, which can accommodate the detection chip of the present invention and hook or attach it to a target surface, such as a hat or mask. In addition, the detection chip can be replaceable or disposable. In addition, the detection chip can be used to detect the presence of more than one virus by including specifically generated aptamers to detect the presence of each of a plurality of different viral nucleic acids or proteins, each nucleic acid or protein being identifiable by having a different one or more colors or lighting patterns coordinated with the detection event.

[0090] The provided biosensor device has several unique features. For example, first, the biosensor device has multi-target diagnostic capabilities, including (i) detection of viral particles with a resolution of less than 7 particles / sample; (ii) detection of molecular components of the virus, including viral proteins, with a detection limit in the low nanomolar range; and (iii) detection of nucleic acids with single nucleotide resolution and femtomolar sensitivity. Second, the sensor surface is specifically treated and tuned to have a higher degree of specific charge sensitivity. Third, the electrical recording and electronic data analysis algorithms are designed to improve the S / N ratio, thereby distinguishing the smallest changes in the Dirac potential minima. This allows recording the interaction of the sample (e.g., virus, spike protein) with the probe (e.g., aptamer) at the highest resolution (lowest number) and low power consumption. These features allow miniaturization and portability of the device.

[0091] Due to its design features, the biosensor device can achieve: (i) readout within 10 minutes; (ii) samples can come from saliva, aerosols, and body fluids, such as nasal or nasopharyngeal fluid; (iii) high accuracy (~95%); (iv) sensitivity to as few as 20-30 viruses that can be detected early; (v) wireless contact tracing; (vi) portability with low power (9V battery) requirements and comparable size to a mobile phone; (vii) inexpensive mass production ($10 / test) capability; and (viii) non-technical operation requirements, i.e., easy to train lay personnel to use without any medical professional assistance.

[0092] Examples of rapid Covid-19 testing using biosensor devices

[0093] In this example, a portable diagnostic device was developed for the highly specific and sensitive detection of the coronavirus SARS-CoV-2 ( Figures 1A-1Cand 2A-2C). The device contains a high-affinity aptamer for the SARS-CoV-2 spike protein that is screened for active virus and records an electrical output to indicate a positive response. The device has built-in wireless capabilities that allow for rapid tracking and communication with interested decision-makers (e.g., physicians, administrators, policymakers). The final assembled portable and compact device, with electronics integrated with the sensor chip, is Figure 13A and 13B It includes an easily integrated genomic information workflow that uses microfluidics modules to achieve genome-scale coverage with limited pre-processing and will allow diagnosis of potential future mutations within the SARS-CoV-2 genome using aptamers specific to such variants. Patient samples can be tested using a multi-array sensor, and positive / negative results will be transmitted wirelessly.

[0094] Although originally developed for RNA / DNA detection with a sensitivity of 10XM, the device has been used to derivatize DNA aptamers onto 2D transistors to recognize their cognate partners within microfluidic devices ( Figure 2A , right). The electrical signals generated by the interaction of the specimen with its specific bait and prey are digitally recorded and transmitted by an integrated wireless system ( Figure 2C As designed, the device is capable of determining the concentration and specificity of antibodies, as well as detecting and determining the concentration of antigens.

[0095] In this example, the device focuses on specific fragments of the receptor binding domain and Spike protein of SARS-CoV-2, and uses specific DNA aptamer sequences for these antigens. The electrical output response indicates a positive response. As shown, aptamers specific for two different viral proteins were used: aptamer Np-48 (aptamer 48) specific for the N-protein and aptamer 1C (aptamer 1C) specific for the S-protein. Figure 14-17 ), an electric graphene FET (GFET) sensor can detect as few as 7 virus particles from diluted human saliva samples within 10 minutes without qPCR amplification, suggesting high specificity and sensitivity for SARS-CoV-2 detection. Specifically, Figure 16 and 17 The relationship between the sensor response to different dilutions of a saliva sample containing the SARS-CoV-2 virus, plotted on the x-axis, is shown for aptamers Np-48 and 1C, respectively. Individual data points are labeled to indicate the number of viral particles detected. The detection chip has demonstrated the ability to detect as few as seven viral particles.

[0096] Embodiments of the technology disclosed herein use aptamer-derived GFETs for label-free detection and reporting of SARS-CoV-2 and its variants (N501Y, D614G, and Y453F) antigens, which enable detection of aptamers to the SARS-CoV-2 antigen, S-protein (aptamer 1C Kd ≈ 5.8 nM) [4] and N-protein (aptamer Np-48 0.5nM) [5] , based on their affinity. Both aptamers were modified at the 3' end for GFET derivatization to functionalize the graphene surface. Furthermore, aptamers targeting the S-protein receptor binding domain (RBD) and N-protein were analyzed using homologous proteins, inactive virus, and oral samples validated by retrospective RT-PCR.

[0097] Materials: HPLC-grade 3' amino functionalized aptamers (aptamer-N) designed for N-protein [5] and Spike RBD (aptamer-S) [4] The results presented here were obtained by integration with GFETs. Molecular biology grade 1X PBS (Gibco), MgCl2, and ultrapure water (Invitrogen) were used throughout the study. Analytical grade 1-pyrenebutyric acid N-hydroxycyclobutyrimidyl ester (PBASE) and ethanolamine were used without further processing.

[0098] Aptamer derivatization on GFETs. The selected aptamers were amino-derivatized at the 3' end using a linker molecule and labeled for the spike RBD protein. [4] Aptamer-S for nucleocapsid protein and aptamer-N for nucleocapsid protein [5] . The aptamer was dissolved in 1X PBS buffer containing 0.5 mM MgCl2 and annealed by controlled heating at 94°C for 2 minutes and slowly cooling to room temperature. The annealed aptamer was stored at -20°C for further use. Derivatization was performed by adding 1 μM aptamer to the PBASE-functionalized GFET for 30 minutes. The excess aptamer was washed and the unreacted PBASE was passivated using 10 mM ethanolamine (EA) solution for 20 minutes. The excess EA was washed and the GFET measurement was performed in 1xPBS buffer.

[0099] Nucleocapsid and spike RBD domains were detected using aptamer-functionalized GFETs by scanning V in the range of ±0.5 V. G At the same time, the drain-source voltage was maintained at a fixed voltage (100 mV) for baseline correction. [4] and (nucleocapsid ~ 0.5, 1, 5, 10, 20, 50, 100 nM) [5]The concentration-dependent sensor response was analyzed under 40°C. After incubation for 10 minutes, the excess protein was washed three times with 1× PBS buffer, and the transfer function (ΔVD) of the sensor was analyzed. D Displacement calibration sensor response:

[0100]

[0101] Here, V D is the VD after adding the sample to the chip, is the VD with aptamer-derived chips and relative to (i.e., ) to calculate the percentage response. In addition, the aptamer-GFET sensor used different concentrations of recombinant RBD protein to detect SARS-CoV-2 variants of concern, such as B.1.1.7 (N501Y), Y453F, and D614G [6-8] Two different concentrations (100 fM, 100 nM) of the mutant variant were used and the responses were compared with those obtained with the RBD of SARS-CoV-2.

[0102] Inactive virus detection under simulated conditions. The relevant experiments were performed by preparing a dilution solution containing heat-inactivated SARS-CoV-2 (USA-WA1 / 2020**, 9.55×106TCID50 / ml, ZeptoMetrix). PFU / mL as Ding et al. [9] The virus dilution was calculated as described above (6.68-6.68×10 6 PFU / mL) on the GFET sensor response. 10 μl of sample was added to the chip and incubated for 10 minutes, and ds -V G Measure V in the characteristics D displacement.

[0103] Specificity Analysis. To analyze the specificity of the GFET sensor, two different concentrations of homologous proteins from MERS-CoV, SARS-CoV, and SARS-CoV-2 were analyzed. The sensor response using aptamers for the RBD and N proteins was studied at ultra-low concentrations (100 fM) and in the saturation range (100 nM).

[0104] To analyze the sensitivity and specificity of different concentrations of homologous N and RBD proteins (100 fM-100 nM) of MERS-CoV, SARS-CoV, and SARS-CoV-2, different dilutions of inactive MERS-CoV and SARS-CoV-2 viruses (670 PFU / mL-6.7×10 5 PFU / mL) for concentration-dependent analysis.

[0105] Clinical Sample Analysis. Oral samples from patients were collected in 3 ml (0.9% w / v) CDC-approved saline by trained clinicians and further tested by trained clinicians in a CLIA-certified laboratory. RT-PCR analysis was performed using the FDA-approved Promega RT-PCR test kit for SARS-CoV-2, using 10 μL aliquots of the same sample in a CLIA laboratory using both the GFET sensor and a handheld reader. Aptamer-S showed higher sensor response with inactive virus in a simulated environment and was used for all patient sample diagnostics. A total of 30 patient samples were tested and compared against RT-qPCR (Ct value ≤ 35) data. The sensor response threshold was set to 99.7% confidence interval (CI) using known negative RT-qPCR data, using ±3σ analysis to predict negative patient samples. Sensor response values ​​above the mean + 3σ were designated as positive. As per FDA guidance

[10] The positive percentage agreement (PPA) and negative percentage agreement (NPA) of the test were calculated.

[0106] The standard deviation of the response and the slope method were used to estimate the limit of detection (LoD) and limit of quantification (LoQ) of the sensor.

[10] All data presented are the mean of at least three measurements with one standard deviation (SD).

[0107] Analysis of SARS-CoV-2 and its mutant antigens. Due to the wide range of viral loads in patient samples (10 4 -10 7 copies / mL and antigen levels of 3.48 fM-58.9 nM)

[11]

[12] , analyze the concentration-dependent sensitive area and the saturation of the sensor response. Analysis of the aptamer-S and -N GFET sensor responses in the presence of different concentrations of SARS-CoV-2 RBD and N protein showed that V D The concentration-dependent index changes, such as Figures 18A-18C As shown therein, the sensor was Figure 18A ) and 100 nM N-protein ( Figure 18B saturation was achieved at 50 saturation points. Sensor responses of ≥20% were observed for all tested concentrations of antigen, and after saturation, excess antigen levels did not reduce the sensor response to below 20% response. This suggests that the GFET sensor reduces the chance of missing positive signals due to the hook effect, as in the case of flow-based antigen tests.

[13]

[14] .

[0108] Antigen tests approved by the FDA under Emergency Use Authorization (EUA) (shown in Table 1 below) do not indicate the ability to detect new variants that may evade immunity generated by available vaccines or past infection.

[15] There is also considerable concern about false negatives in recommended tests.

[16] The evolution of new mutations in SARS-CoV-2 (B.1.1.7 variant (N501Y), mink-associated mutation (Y453F), S2 domain mutation (D614G)) is a major concern.

[17] Considering the importance of these issues, the GFET sensor was deployed on different SARS-CoV-2 mutants. This test showed that aptamer-S showed more than 20% sensor response with the corresponding protein concentration of 100fM-100nM (such as Figure 18C Although, there was some variability in the observed sensor responses; however, the sensor responses were always above the threshold for positive samples (eg, >20%) (see Figure 18C Based on the concentration-dependent analysis of the homologous RBD and N proteins on various aptamer-derived GFET sensors with SARS-CoV-2 variants, embodiments of the disclosed technology can detect relevant viral antigens at fM-nM concentration levels. The enhanced sensitivity and accompanying specificity are due to the presence of aptamer-S at the spike protein amino acids T500, N437, and Q506. [4] The non-overlapping binding sites at 100 nt have novel mutations. Table 1 Comparison of Point-of-Care (POC) Antigen Test Data Collected from FDA Emergency Use Authorizations (EUAs)

[0109] Sensor specificity analysis. The specificity of the disclosed sensor embodiments with aptamers-S and -N was tested using closely related homologous antigens of MERS-CoV, SARS-CoV, and inactive MERS-CoV viruses. As shown in Figures 19A and 19B, the results clearly show that aptamers-S and -N significantly distinguish between MERS-CoV and SARS virus proteins (>20% increased sensor response). However, the graphene FET was unable to significantly distinguish between SARS-CoV and CoV-2 proteins. It was observed that the sensor response of aptamer-N (50%) was higher for all tested protein samples compared to aptamer-S (35%), and this can be attributed to the higher affinity of the aptamer (Kd, 0.5 nM, compared to Kd, 5.8 nM for aptamer-S).

[0110] To further verify the specificity of the aptamers, scrambled aptamer-S and aptamer-N (as shown in Table 2 below) were used and the graphene surface of the GFET was derivatized. Figure 19C and 19D The relative responses of the aptamers derivatized on GFETs (aptamer-S and -N) and the corresponding scrambled aptamers (aptamer-S and -N) to inactive SARS-CoV-2 virus in saliva diluted in PBS buffer are illustrated. To further analyze the specificity of the Atamer-S and N derivatized chips under simulated biological conditions, different equivalent dilutions of 10× v / v inactive SARS-CoV-2 and MERS-CoV in saliva in 1× PBS buffer were prepared. Concentration dependence analysis showed that all dilutions of the inactive MERS-CoV sample showed less than 10% sensor response (see Figure 19E and 19F One of the reasons for the high sensor response of SARS-CoV and SARS-CoV-2 may be the high affinity of aptamer-S containing threonine (T), asparagine (N) and glutamine (Q) at RBD amino acid positions 500, 437 and 506, while the low sensor response of MERS may be due to the presence of different amino acids [4]

[18] , while MERS-CoV contains a different amino acid (alanine (A)). Lysine (K) and alanine (A) at the same position of RBD. Table 2 Nucleic acid sequences screened for embodiments of the disclosed technology

[0111] These results indicate that both (aptamer-S and -N) functionalized sensors are specific for the SARS-CoV-2 protein in the virus. However, the GFET sensor with aptamer-S showed a higher sensor response in simulated biological samples compared to aptamer-N. Although we have previously observed that it showed a higher sensor response to the homologous N-protein (see Figure 19A and 19B ). Generally, surface S-proteins are more accessible than N-proteins encapsulated within lipid membranes.

[19] .

[0112] Limit of Detection (LoD) and Limit of Quantification (LoQ). To analyze the LoD and LoQ of our aptamer-based sensors, concentration-dependent sensor response analysis was performed with inactive virus under simulated biological conditions. Triplicate data were fitted using a linear fit, and LoD and LoQ were calculated according to FDA statistical data analysis guidelines. Results (e.g. Figure 20A and 20BThe figure shows LoD, which is 1.28 PFU / mL for aptamer-S (R 2 =0.98), aptamer-N was 1.45 PFU / mL (R2 = 0.99), and was inactive at room temperature in 10% v / v saliva and 1× PBS buffer. The estimated LoQs of aptamers-S and -N were 3.89 PFU / mL and 4.39 PFU / mL, respectively. Compared with the FDA-approved antigen test (see Table 1 above), this test showed higher sensitivity (lower LoD). These results demonstrate the sensitivity of aptamer-based GFET sensors for detecting SARS-CoV-2 in biological fluids and provide motivation for studies on actual patient samples.

[0113] Clinical sample collection and analysis. Based on simulated biological sample analysis, it was observed that the aptamer-S-derived GFET showed a high sensor response, while the aptamer-N response was smaller ( Figure 19D and 20B ). Therefore, the aptamer-S-derived GFET was used for patient sample analysis. To validate the GFET sensor with relevant samples, a single-blind experiment was performed with 10 each of negative and positive samples and confirmed independently by RT-PCR (e.g. Figure 20C and 20D In addition, 10 double-blind samples were tested to confirm the test. Based on these results, the RT-PCR negative samples were used to predict the sensor response with a confidence interval (CI) of 99.7% + 3σ, which yielded a V of 132.6 mV for the sample. D Displacement (<20% of sensor response). Set the threshold value to 132.6mV, above which V D Displacement is considered a positive confirmation of SARS-CoV-2 infection (see Figure 20C and 20D ).

[0114] The effectiveness of the disclosed technology is further demonstrated in the following figures: Figure 21A An example of transfer curve analysis of aptamer-S-derived GFET mediated by SARS-CoV-2 RBD binding is shown; Figure 21B An example of transfer curve analysis of aptamer-N-derived GFETs mediated by SARS-CoV-2 nucleocapsid protein binding is shown; Figures 22A-22D Examples of detecting mutations N501Y, Y453F, D614G and SARS-CoV-2 RBD homologous proteins of the original virus are shown, respectively; Figure 23A and 23B Examples of analyzing the specificity of aptamer-S and aptamer-N derived GFETs are shown separately and are shown in Tables 3-6 below. Table 3 Negative saliva sample test results (SARS-CoV-2 virus clinical test) Table 4 Positive saliva sample test results (SARS-CoV-2 virus clinical test) Table 5 Blind saliva sample test results (SARS-CoV-2 virus clinical test) Table 6. Clinical test results of SARS-CoV-2-Delta / Omicron variant

[0115] Example results of the current-voltage (IV) relationship of the aptamer-anylate interaction are shown in Figures 24A-24G middle. Figure 24A It was shown that in the absence of aptamers on the GFET, the Dirac potential shift did not change, which motivated the need for aptamers for detection. The specificity of aptamer-omicron nucleocapsid protein binding was shown in Figure 24B , where the Dirac potential is negligible when using the scrambled (or control) aptamer. A significant change in the Dirac potential is seen in Figure 24C , which shows the effect of aptamer-omicron nucleocapsid protein specific interaction. Figure 24C-24C The results in the analysis Figures 24E-24G The results shown are not available because there is currently no inactivated omicorn virus available.

[0116] Figure 24D Shown are IV plots of the dose response of the aptamers to various concentrations of inactivated Δ variant virus. Figure 24D The results in were used to detect the Δ variant in clinical samples, e.g. Figures 24E-24G As discussed in this document, the disclosed embodiments can detect as few as 5-10 viruses.

[0117] Figures 24E-24G Exemplary results including representative IV curves for clinical testing in December 2021 are shown. As shown therein, in the absence of the aptamer (sensor), for individuals who tested positive for RT-PCR ( Figure 24E ) and RT-PCR negative individuals ( Figure 24F ), there is little or no change in the Dirac potential. However, for RT-PCR positive individuals (asymptomatic individuals), there is a significant shift in the Dirac potential when the aptamer is present ( Figure 24G ).

[0118] Figures 25A-25C Example GFET device responses to Δ variant N-protein, Omicron variant N-protein, and inactivated Δ variant virus are shown, respectively. Results shown in Figure 1. Serial dilution method was used to obtain Figure 25A and 25C The results shown in

[15] were used to determine the dose response of the GFET system for detecting the Δ variant. The GFET sensor was functionalized using an N-aptamer. The nucleocapsid protein (N-protein) of the Δ variant SARS-CoV-2 (B.1.617.2) (2-4×10 9 copies / ml). Various dilutions were tested serially on the PIVOT device using a single detection chip. Figure 25B The results shown in , and Figure 25C Each bar in the graph shows the shift from baseline readings in saline to administration of 10 μM viral omicron N-protein. Control experiments included those without any aptamer ( Figure 25C aptamer) or a detection chip functionalized with a control aptamer (e.g., a random sequence; Figure 25C "Scrambled N aptamer" bar).

[0119] Figure 26A and 26B Figures 2 and 3 show exemplary detection results of human saliva samples using N-aptamers and S-aptamers using embodiments of the disclosed technology during Nabo Omicron on January 10, 2022. Figure 26A As shown, a total of 15 samples were analyzed using the N-aptamer GFET device, of which 12 samples resulted in a positive score (indicating Omicron infection), yielding an estimated 80% infection rate in US. Figure 26B As shown, a total of 17 samples were analyzed using the S-aptamer GFET device, of which 10 samples resulted in a positive score (indicating Omicron infection), resulting in an estimated infection rate of 59% in the US. In combination, 32 samples were analyzed using either the N- or S-aptamer GFET device, of which 22 samples resulted in a positive score, resulting in an estimated infection rate of 69% in the US population.

[0120] Compared to commercially available diagnostic devices, the biosensor device of this technology is cheaper (~$10 / test), faster (~10 minutes), and represents a portable point-of-care (POC) system with general utility for the current COVID-19 epidemic. The device also has the potential to be adapted for rapid and accurate detection of other coronaviruses (e.g., MERS-CoV, SARS-CoV), as well as mutants and / or variants of viruses with minimal modification. It requires non-technical operation and allows for rapid delivery of results to physicians, administrators, and public health personnel, thereby contributing to efforts to combat current and future pandemics.

[0121] Implementation and implementation of the disclosed technology

[0122] The disclosed technology includes a biosensor device for detecting one or more pathogens. In one example, the biosensor device includes a detection chip, which includes (a) a substrate having a graphene surface, (b) a conductive material at a first end and a second end of the graphene surface, which form a first electrode and a second electrode, respectively, and (c) an insulating material to insulate the first electrode and the second electrode. In this example, one or more probes attached to the graphene surface specifically bind to one or more target molecules of one or more pathogens. In addition, the insulating material forms a hole to receive a biological sample so that the biological sample is in contact with the one or more probes. For example, in Figure 1B , 2B, 3 and 6 show biosensor devices.

[0123] In some embodiments, at least one of the one or more probes is an aptamer.

[0124] In some embodiments, the aptamer comprises a nucleic acid or a peptide. In some instances, the nucleic acid aptamer is selected from Table 2. In other instances, the nucleic acid is double-stranded. In other instances, the nucleic acid is single-stranded.

[0125] In some embodiments, the one or more pathogens are one or more variants of coronavirus. In some instances, the one or more variants of coronavirus include SARS-CoV, SARS-CoV-2, and MERS-CoV.

[0126] In some embodiments, the target molecule is a nucleic acid or a protein.

[0127] In some embodiments, the target molecule includes the S protein of SARS-CoV-2, the N protein of SARS-CoV-2, a variant thereof, or a subunit thereof.

[0128] In some embodiments, the one or more probes comprise multiple probes attached to different portions of the graphene surface. In some instances, a single detection chip, e.g., Figure 1B 、2B , 3 or 6, can be configured to detect multiple pathogens using multiple probes attached to the graphene surface of the GFET of the detection chip. This advantageously enables the described embodiments to be used in environmental monitoring and global security applications.

[0129] In some embodiments, a first probe in the plurality of probes is attached to a first portion of the graphene surface, and a second probe in the plurality of probes is attached to a second portion of the graphene surface that does not overlap with the first portion. In some instances, the plurality of probes specifically bind to different target molecules of the same pathogen. In other instances, the plurality of probes specifically bind to different target molecules of different pathogens.

[0130] In some embodiments, the biosensor device further comprises a plurality of detection chips, the plurality of detection chips comprising a detection chip, and each of the plurality of probes is attached to a corresponding detection chip in the plurality of detection chips. In this embodiment, the described detection chip can be implemented as an array, which enables its deployment in environmental monitoring and global security applications, and also allows for simultaneous processing of multiple detection chips.

[0131] In some embodiments, a handheld device, such as Figure 13A and 13B As shown, the handheld device is configured to receive a biosensor device. In some examples, the handheld device is configured to perform detection of one or more pathogens based on one or more probes that specifically bind to one or more target molecules of the one or more pathogens. In some examples, the handheld device includes a wireless transceiver configured to transmit the detection results. The wireless transceiver can support at least one of a Bluetooth protocol, a Wi-Fi protocol, or a cellular protocol.

[0132] In some embodiments, the handheld device includes a power source, one or more visual indicators coupled to the power source, and a display coupled to the power source, the indicators configured to indicate the start and completion of detection of one or more pathogens, the display presenting the detection results for each of the one or more pathogens. In some examples, the one or more visual indicators include LEDs, the display includes an LCD, and the power source includes one or more batteries.

[0133] Embodiments of the disclosed technology also provide a method for detecting the presence of one or more pathogens in a biological sample obtained from a subject. The method comprises contacting the biological sample with a biosensor device according to any one of the embodiments or implementations described above.

[0134] In some embodiments, the biological sample comprises saliva, exhaled breath, nasal swab, or nasopharyngeal swab from the subject.

[0135] In some embodiments, the presence of fewer than 10 pathogen particles is detected in a biological sample.

[0136] Embodiments of the disclosed technology support, among other things, a solution to the technical problem of accurately detecting one or more pathogens using a reliable, inexpensive, and portable device.

[0137] 1. A biosensor device for detecting one or more pathogens, the biosensor device comprising a detection chip, the detection chip comprising a substrate having a graphene surface, a conductive material forming a first electrode and a second electrode at a first end and a second end of the graphene surface, respectively, and an insulating material, the insulating material insulating the first electrode and the second electrode, wherein one or more probes are attached to the graphene surface, wherein the one or more probes specifically bind to one or more target molecules of the one or more pathogens, and wherein the insulating material forms a hole to receive a biological sample so that the biological sample contacts the one or more probes.

[0138] 2. The biosensor of embodiment 1, wherein at least one of the one or more probes is an aptamer.

[0139] 3. The biosensor according to claim 2, wherein the aptamer comprises a nucleic acid or a peptide.

[0140] 4. The biosensor of claim 3, wherein the nucleic acid is double-stranded.

[0141] 5. The biosensor of claim 3, wherein the nucleic acid is single-stranded.

[0142] 6. The biosensor of any one of claims 1 to 5, wherein the one or more pathogens are one or more variants of a coronavirus.

[0143] 7. A biosensor as in Scheme 6, wherein the one or more variants of coronavirus include SARS-CoV, SARS-CoV-2 and MERS-CoV.

[0144] 8. The biosensor according to any one of embodiments 1 to 7, wherein the target molecule is a nucleic acid or a protein.

[0145] 9. A biosensor as described in any one of Schemes 1 to 8, wherein the target molecule includes the S protein of SARS-CoV-2, the N protein of SARS-CoV-2, a variant thereof or a subunit thereof.

[0146] 10. The biosensor of any one of claims 1 to 9, wherein the one or more probes comprise a plurality of probes attached to different portions of the graphene surface.

[0147] 11. The biosensor of claim 10 , wherein a first probe of the plurality of probes is attached to a first portion of the graphene surface, and wherein a second probe of the plurality of probes is attached to a second portion of the graphene surface that does not overlap with the first portion.

[0148] 12. The biosensor of scheme 10 or 11, wherein the plurality of probes specifically bind to different target molecules of the same pathogen.

[0149] 13. The biosensor of scheme 10 or 11, wherein the plurality of probes specifically bind to different target molecules of different pathogens.

[0150] 14. The biosensor as described in Option 10 comprises a plurality of detection chips, wherein the plurality of detection chips include the detection chip, wherein each of the plurality of probes is attached to a corresponding detection chip among the plurality of detection chips.

[0151] 15. A biosensor as described in any of options 1 to 14, wherein the handheld device is configured to receive the biosensor device, and wherein the handheld device is configured to perform detection of the one or more pathogens based on the one or more probes that specifically bind to the one or more target molecules of the one or more pathogens.

[0152] 16. The biosensor of claim 15, wherein the handheld device comprises a wireless transceiver configured to transmit a result of the detection.

[0153] 17. The biosensor of claim 16, wherein the wireless transceiver supports at least one of a Bluetooth protocol, a Wi-Fi protocol, or a cellular protocol.

[0154] 18. A biosensor as described in Option 15, wherein the handheld device includes a power source, one or more visual indicators coupled to the power source, and a display, wherein the one or more visual indicators are configured to indicate the start and completion of detection of the one or more pathogens, and the display is coupled to the power source to present the detection results of each of the one or more pathogens.

[0155] 19. The biosensor of claim 18, wherein the one or more visual indicators comprise light emitting diodes (LEDs), the display comprises a liquid crystal display (LCD), and the power source comprises one or more batteries.

[0156] 20. A method of detecting the presence of one or more pathogens in a biological sample obtained from a subject, the method comprising contacting the biological sample with the biosensor device of any one of claims 1 to 19.

[0157] 21. The method of claim 20, wherein the biological sample is selected from the subject's saliva, exhaled breath, nasal swab or nasopharyngeal swab.

[0158] 22. The method of claim 20 or 21, wherein the presence of fewer than 10 pathogen particles in the biological sample is detected.

[0159] 23. A method for environmental monitoring, comprising collecting a sample selected from a soil sample, an aerosol sample, an air sample or a water sample, contacting the sample with the biosensor device of any one of schemes 1 to 19, and detecting the presence of the one or more pathogens in the at least one sample.

[0160] 24. The method of claim 23, wherein the one or more pathogens comprise one or more of heavy metals, small molecule agricultural toxins, waterborne bacterial pathogens, aquatic toxins, pesticides, industrial byproducts, antibiotics, or drugs.

[0161] References

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[0181] in conclusion

[0182] The above detailed description of the embodiments of the technology is not intended to be exhaustive or to limit the technology to the precise form disclosed above. Although specific embodiments and examples of the technology are described above for illustrative purposes, as those skilled in the relevant art will recognize, various equivalent modifications are possible within the scope of the technology. For example, although the steps are presented in a given order, alternative embodiments may perform the steps in a different order. The various embodiments described herein may also be combined to provide other embodiments.

[0183] As will be appreciated from the foregoing, specific embodiments of the present technology have been described herein for illustrative purposes, but well-known components and functions have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments of the technology. Where the context permits, singular or plural terms may also include plural or singular terms, respectively. In addition, although the advantages associated with some embodiments of the present application technology have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit such advantages to fall within the scope of the present technology. Therefore, the present disclosure and associated technology may encompass other embodiments not explicitly shown or described herein.

Claims

1. A biosensor device for detecting one or more pathogens, comprising: Detection chip, including: a substrate having a graphene surface; A conductive material forming a first electrode and a second electrode at a first end and a second end of the graphene surface, respectively; and an insulating material that insulates the first electrode from the second electrode, wherein one or more probes are attached to the graphene surface, wherein the one or more probes specifically bind to one or more target molecules of the one or more pathogens, and The insulating material forms a hole to receive a biological sample so that the biological sample contacts the one or more probes. 2 . The biosensor of claim 1 , wherein at least one of the one or more probes is an aptamer. The biosensor according to claim 2 , wherein the aptamer comprises a nucleic acid or a peptide. The biosensor according to claim 3 , wherein the nucleic acid is double-stranded. The biosensor according to claim 3 , wherein the nucleic acid is single-stranded.

6. The biosensor according to any one of claims 1 to 5, wherein the one or more pathogens are one or more variants of a coronavirus.

7. The biosensor of claim 6, wherein the one or more variants of coronavirus include SARS-CoV, SARS-CoV-2, and MERS-CoV.

8. The biosensor according to any one of claims 1 to 7, wherein the target molecule is a nucleic acid or a protein.

9. The biosensor according to any one of claims 1 to 8, wherein the target molecule comprises the S protein of SARS-CoV-2, the N protein of SARS-CoV-2, a variant thereof, or a subunit thereof.

10. The biosensor according to any one of claims 1 to 9, wherein the one or more probes comprise a plurality of probes attached to different portions of the graphene surface.

11. The biosensor of claim 10, wherein a first probe of the plurality of probes is attached to a first portion of the graphene surface, and wherein a second probe of the plurality of probes is attached to a second portion of the graphene surface that does not overlap with the first portion.

12. The biosensor according to claim 10 or 11, wherein the plurality of probes specifically bind to different target molecules of the same pathogen.

13. The biosensor according to claim 10 or 11, wherein the plurality of probes specifically bind to different target molecules of different pathogens.

14. The biosensor according to claim 10, comprising: A plurality of detection chips including the detection chip, Each of the plurality of probes is attached to a corresponding detection chip among the plurality of detection chips.

15. The biosensor according to any one of claims 1 to 14, wherein a handheld device is configured to receive the biosensor device, and wherein the handheld device is configured to detect the one or more pathogens based on the one or more probes that specifically bind to the one or more target molecules of the one or more pathogens.

16. The biosensor of claim 15, wherein the handheld device comprises a wireless transceiver configured to transmit the detection result.

17. The biosensor of claim 16, wherein the wireless transceiver supports at least one of a Bluetooth protocol, a Wi-Fi protocol, or a cellular protocol.

18. The biosensor of claim 15, wherein the handheld device comprises: power supply; one or more visual indicators coupled to the power source and configured to indicate initiation and completion of detection of the one or more pathogens; and A display is coupled to the power source to present a result of the detection of each of the one or more pathogens.

19. The biosensor of claim 18, wherein the one or more visual indicators comprise light emitting diodes (LEDs), the display comprises a liquid crystal display (LCD), and the power source comprises one or more batteries.

20. A method of detecting the presence of one or more pathogens in a biological sample obtained from a subject, the method comprising contacting the biological sample with the biosensor device of any one of claims 1 to 19.

21. The method of claim 20, wherein the biological sample is selected from the group consisting of saliva, exhaled breath, nasal swab, or nasopharyngeal swab of the subject.

22. The method of claim 20 or 21, wherein the presence of fewer than 10 pathogen particles in the biological sample is detected.

23. Environmental monitoring methods, including: collecting a sample selected from a soil sample, an aerosol sample, an air sample, or a water sample; contacting the sample with the biosensor device of any one of claims 1 to 19; and The presence of the one or more pathogens is detected in at least one sample.

24. The method of claim 23, wherein the one or more pathogens comprise one or more of heavy metals, small molecule agricultural toxins, waterborne bacterial pathogens, aquatic toxins, pesticides, industrial byproducts, antibiotics, or drugs.

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