PORTABLE DEVICES AND METHODS FOR DETECTING AND IDENTIFYING COMPOUNDS IN A FLUID SAMPLE

TECHNICAL FIELD

[0001] This disclosure relates to the detection and identification of one or more diseases using a portable device. Individual diseases can be detected by identification of specific compounds, e.g., antibodies, antigens, RNA, etc., in a clinical specimen, such as saliva or blood. Developing a portable device that can detect one or more compounds in a sample offers a possibility to create a low-cost and efficient method for detecting and identifying a disease in real-time.

BACKGROUND OF THE INVENTION

[0002] Several assays for virus detection are currently in use, including, but not limited to immunofluorescence assays, protein microarray assays, reverse transcription loop mediated isothermal amplification assays (RT-LAMP), viral plaque assays, Hemagglutination assays, viral flow cytometry (FCM) and enzyme linked immunosorbent assays (ELISA). Despite the high sensitivity of these methods, they are not suitable for large scale screening for multiple samples because of their high cost and long analysis time. Moreover, these methods require skilled personal to perform the assays and are not suitable for point-of-care testing.

[0003] Nanobiotechnology plays a potential role in clinical applications, particularly in the development of biosensors for the detection of pathogenic microorganisms. Various immunosensors have been reported for the detection of viruses using different transducers as improved alternatives to traditional assays.

[0004] For instance, the detection of SARS associated coronavirus (SARS-CoV) in sputum in the gas phase was done by piezoelectric immunosensor. This work was based on the binding of horse polyclonal antibody of SARS-CoV to a piezoelectric crystal surface through protein A. The mass of the crystal would change as the virus was bound and the shift in the frequency was recorded. A localized surface plasmon coupled fluorescence (LSPCF) fiber-optic biosensor was also developed for the detection of SARS corona virus (SARS-CoV) nucleocapsid protein N. LSPCF was used with sandwich immunoassay technique. A label-free RNA amplification and detection method was developed for the detection of MERS-CoV by using a bio-optical sensor. The level of detection (LOD) of this assay was 10 times more sensitive than the RT-PCR method. Another new generation system was developed for the robust and facile diagnosis of MERS-CoV based on an isothermal rolling circle amplification (RCA) method. However, these methods are still time consuming and costly which limits their wide applications.

[0005] Electrochemical immunosensors have become an appealing choice due to their high sensitivity, low-cost, ease of use and possibility of miniaturization. Different electrochemical immunosensors for influenza virus were reported using differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS), linear sweep voltammetry, chronoamperometry and cyclic voltammetry

[0006] An electrochemical immunosensor based on reduced graphene oxide (RGO) that was integrated with a microfluidic chip for label-free detection of an influenza virus (e.g., H1N1) was also reported as showing good selectivity and enhanced detection limits. Human immunodeficiency virus (HIV) was detected using DPV on glassy carbon electrodes (GCE) modified with multi-walled carbon nanotubes (MWCNTs). Human papillomavirus (HPV) was also detected using glassy carbon electrodes modified with graphene/Au nanorod/polythionine via DPV and EIS.

[0007] An important application of antibody compound detection in a sample of subjects is compliance with same family virus diagnostics. Frequently, patients, due to memory loss or simple forgetfulness, fail to ingest prescribed medications in a timely fashion, or at all, which can lead to serious medical issues. Furthermore, health care professionals, who treat such patients, are not aware of the lack of compliance, which may prevent proper remedial action. Pre-existing methods require burdensome sample collection and subsequent analysis with delays in reporting of results. [0008] Accordingly, there exists a need for automated portable devices and methods for directly detecting antigen compounds in the saliva of virus infected patient, which provide analytical results in real time with concurrent reporting to remote users, such as, for example, health care professionals. Simple miniaturized devices integrated with biosensors provide significant benefits and may be commercialized as a handheld device for clinical use in a point of care setting. Such devices and methods would also be of significant value in measuring patient compliance with pharmaceutical regimens and/or determining active infection. The same sensor system could also be used for quantitative and qualitative analysis of blood or other bodily fluid samples for antibodies, e.g., for a virus.

BRIEF DESCRIPTION OF THE INVENTION

[0009] Disclosed herein are portable devices and methods for diagnosing a disease, such as viral infections, cancers, or inflammatory disorders, by detecting and identifying one or more compounds in a fluid sample from a subject. The portable devices described herein may be used for point of care testing and may provide testing results in a matter of minutes, and in some instances in under 2 minutes. The portable devices may provide identify one or more compounds with a clinical sensitivity of about 97% or greater and with a clinical specificity of about 98% or greater. The portable device described herein provides non- invasive sampling from a subject and requires no pre-processing of the sample. Further, the portable device described herein may demonstrate a limit of detection (LoD) of about 18.56 copies/mL.

[0010] Disclosed herein are portable devices for detecting and identifying one or more compounds in a fluid sample from a subject comprising one or more disposable biosensor cartridges, wherein each biosensor cartridge comprises one or more biosensors for detecting one or more compounds in the fluid sample from the subject; a cartridge housing for receiving the one or more disposable biosensor cartridges, wherein the housing comprises a communication apparatus effective to transmit data collected from the one or more biosensor cartridges to a processing apparatus; and a display to show results obtained from the processing apparatus regarding the one or more compounds in the fluid sample. [0011] Also disclosed herein are methods for detecting and identifying one or more compounds in a sample specimen of a subject comprising transmitting a fluid sample from a subject to a biosensor cartridge comprising one or more biosensors, wherein the biosensors collect data regarding one or more compounds in the fluid sample; returning the biosensor cartridge to the cartridge housing, wherein the cartridge housing comprising a communication apparatus and a display; communicating the collected data via the communication apparatus to a processing apparatus; and processing the communicated data to detect and identify the one or more compounds in the sample specimen.

[0012] In some embodiments, the processing apparatus processes the data collected by the biosensor cartridges and transmitted by the communication apparatus, thereby detecting and identifying the one or more compounds, and wherein the processing apparatus is electronically or wirelessly connected to the communication apparatus.

[0013] In some embodiments, the one or more compounds are detected in real time. In some embodiments, the one or more compounds are viral antigens and/or nucleic acids. In some embodiments, the one or more compounds are one or more SARS-CoV-2 antigens and/or nucleic acids. In one embodiment, the biosensor cartridge comprises a first biosensor for detection of a SARS-CoV-2 antigen and a second biosensor for detection of an influenza antigen. In some embodiments, the SARS-CoV-2 antigen is a SARS-CoV-2 spike glycoprotein antigen. In some embodiments, the SARS-CoV-2 antigen is a SARS-CoV-2 nucleocapsid antigen.

[0014] In some embodiments, the one or more compounds are immobilized with carbon nanostructure material or gold nanoparticles, wherein the carbon nanostructure material or gold nanoparticles comprise one or more marker compounds. In some embodiments, the one or more antibody compounds identified and detected are marker compounds. The marker compounds may be protein. In one embodiments, the marker compound is an immobilized compound with carbon nanostructure material or graphene gold nanoparticles composite. [0015] In still another aspect, an immobilization for a sensor substrate is provided. The immobilization includes a nanoparticle, one or more marking compounds embedded in the carbon-based nanomaterials, and a polymer matrix.

[0016] In still another aspect, a sensor, e.g., a biosensor, is provided. The sensor includes one or more antibodies or aptamers on a substrate form and immobilized which includes a functionalized inorganic metallic oxide nanoparticle and a polymer matrix.

[0017] In still another aspect, a sensor is provided. The sensor includes one or more electrodes, e.g., counter electrodes, working electrodes which includes multi-walled carbon nanotubes that are attached to one or more biological molecules, and reference electrodes. The sensor further includes a support on which the electrodes are disposed.

[0018] In still another aspect, the sensor includes an electrochemical three electrode system, which includes multi-walled carbon nanotubes that are attached to one or more antibody molecules which the electrodes are disposed.

[0019] In still another aspect, the sensor includes an electrochemical three electrode system. The three electrode system may include magnetic nanoparticles and antibody functionalized carbon base - gold nanoparticle hybrid electrodes which are disposed.

[0020] In still another aspect, the sensor includes an electrochemical three electrode system which includes two different shape antibody functionalized gold nanoparticles which the electrodes are disposed.

[0021] Other embodiments, systems, methods, devices, aspects, and features of the disclosure will become apparent to those skilled in the art from the following detailed description, the accompanying drawings, and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

[0022] The disclosure is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity.

[0023] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0024] FIG. 1 illustrates an example of a portable device which receives a biosensor cartridge for the analysis and detection of one or more compounds in a sample from a subject.

[0025] FIG. 2 illustrates a biosensor cartridge which receives a fluid sample from a subject.

[0026] FIG. 3 illustrates a sensor which includes an immobilized biomolecule, which may be used in the sensor module of a portable device which identifies and detects one or more antibody compounds in the sample specimen of a subject.

[0027] FIG. 4 shows DPV responses for target antibody concentrations ranging from 50 copies per ml to 680 x 10 ⁶ copies per ml which is one of the working electrodes.

[0028] FIG. 5 shows concentration peaks and calibration curves for a SARS-CoV- 2/COVID-19 nucleocapsid protein.

[0029] The following detailed description includes references to the accompanying drawings, which form part of the detailed description. The drawings depict illustrations, in accordance with example embodiments. These example embodiments, which are also referred to herein as “examples,” are described in enough detail to enable those skilled in the art to practice the present subject matter. The example embodiments may be combined, other embodiments may be utilized, or structural, logical, and electrical changes may be made, without departing from the scope of the claimed subject matter. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope is defined by the appended claims and their equivalents. DETAILED DESCRIPTION OF THE INVENTION

[0030] Illustrated embodiments herein are directed to systems, methods, and devices for detecting and identifying certain substances, such as antigen or antibody compounds in the sample specimen of a subject or person in real-time. Further, certain embodiments of the disclosure can be directed to systems, methods, and devices for diagnosis of a subject or person. Technical effects of certain embodiments of the disclosure may include providing diagnosis and treatment for identified health conditions related to the detection and identification of certain substances, such as an antigen, antibody, or DNA in the sample specimen of a subject or person in real-time.

[0031] Novel sensor technologies, such as nanocompositions with sensing elements, can be combined with mobile communication devices, such as smart phones, and cloud computing to create technical solutions for respiratory analysis, diagnosis, and subsequent treatment. Novel sensors used in combination with the processor of a smart phone and/or remote server, and a biomarker processing module or engine with a neural network or pattern matching algorithm, can be used to detect antibodies or virus DNA from sample specimens. Embodiments of the disclosure can have many useful and valuable applications in the biomedical industries, health care and medical care sectors.

[0032] As used herein, “target biomolecule” refers to signal and signal patterns associated with concentrations or amounts of certain substances associated with diagnosing or treating a health condition.

[0033] As used herein, “real-time” refers to an event or a sequence of acts, such as those executed by a computer processor that are perceivable by a subject, person, user, or observer at substantially the same time that the event is occurring or that the acts are being performed. By way of example, if a neural network receives an input based on sensing and identifying a target antibody, a result can be generated at substantially the same time that the target molecule was sensed and identified. The real-time processing of the input by the neural network may have a slight time delay associated with converting the sensed compound to an electrical signal for an input to the neural network; however, any such delay may typically be less than 1 minute and usually no more than a few seconds. In certain embodiments, the analysis of a sample, e.g., receipt of the sample to receipt of the processing results, takes less than 5 minutes, and in certain aspects takes less than 2 minutes.

[0034] Health conditions that can be detected by certain embodiments of the disclosure include viral infections, cancers, and inflammatory disorders. Non-limiting examples of such health conditions include adenovirus, enterovirus, human coronavirus, human metapneumovirus, rhinovirus (RV), influenza A, influenza B, parainfluenza and respiratory syncytial virus (RSV), severe acute respiratory syndrome coronavirus (SARS- CoV), middle East respiratory syndrome coronavirus (MERS-CoV), coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), adeno-associated virus, aichi virus, banna virus, bunyamwera virus, bunyavirus, cercopithecine herpesvirus, chandipura virus, dengue virus, dhori virus, dugbe virus, duvenhage virus, eastern equine encephalitis virus, ebolavirus, echovirus, encephalomyocarditis virus, GB virus C/hepatitis G virus, hantaan virus, hepatitis, human immunodeficiency virus, lassa virus, lymphocytic choriomeningitis virus, mayaro virus, measles virus, mengo encephalomyocarditis virus, mokola virus, nipah virus, rabies virus, rosavirus , rotavirus, rubella virus, salivirus, sapporo virus, Seoul virus, variola virus, yellow fever virus, zika virus, colon cancer, breast cancer, pancreatic cancer, multiple myeloma, lung cancer, melanoma, solid tumors, brain cancer, stomach cancer, esophageal cancer, prostate cancer, psoriasis, rheumatoid arthritis, crohn’s disease, ulcerative colitis, tuberculosis, etc..

[0035] In some embodiments, the one or more compounds which are detected and identified using the portable devices are detected and identified directly. In some aspects, the one or more compounds are antibodies, antigens, RNA, DNA, mRNA, and methylated DNA. In certain embodiments, the one or more compounds are nucleic acids and/or antigens. Non-limiting examples of the one or more compounds that may be detected include anti-gp41, anti-gpl20, SC2A, IgG, anti-M2, BCN antibodies, monoclonal antibodies (MAbs), anti-CD4bs, ADCVI antibody, anti-HA, anti-FLAG, SARS-CoV-2-N antibody, SARS-CoV-2 N protein, SARS-CoV-2-S antibody, SARS-CoV-2-S protein, SARS-CoV-2 E protein, SARS-CoV-2 E antibody, SARS-CoV-2 N Ab (IgG), spike glycoprotein antibody, p21 antibody, ACE2 antibody, gpl50 antibody, CD147 antigen antibody, serine 2 antibody, MERS-CoV spike (S) protein, anti-HA antibody, anti-rhinovirus antibody, anti-HCV antibodies, HCoV-229E antigen protein, prostate specific antigens, influenza A antigens, influenza B antigens, and IL6, etc.. In certain embodiments, the one or more compounds which are detected and identified using the portable device are SARS-CoV-2 antibodies. In certain embodiments, the one or more compounds which are detected and identified using the portable device are SARS-CoV-2 nucleic acids and/or antigens. For example, the one or more compounds which are detected and identified using the portable device are selected from the group consisting of SARS-CoV-2-nucleocapsid antibody, SARS-CoV-2-spike antibody, SARS-CoV-2 membrane antibody, and SARS-CoV-2 envelope antibody.

[0036] In certain embodiments, the portable devices analyze a single sample for more than one compound. For example, the portable device may analyze a sample to determine if a SARS-Cov-2 antigen, e.g., SARS-CoV-2-spike antigen, and/or an influenza A antigen is present, thereby identifying the sample as exhibiting a SARS-CoV-2 infection and/or an influenza A infection. It will be generally understood that any combination of compounds may be analyzed in a sample using the portable devices described herein.

[0037] One skilled in the art will recognize that various embodiments of the disclosure discuss the analysis of antibody compounds, though certain embodiments of the disclosure can also be used for the analysis of antigen compounds.

[0038] Disclosed herein are portable devices and methods for detecting and identifying one or more compounds in one or more sample specimens of a subject. Also disclosed herein are immobilization methods with different antibodies for biosensor substrates.

[0039] An example of a portable device 100 is shown in FIG. 1. The portable device may be used to detect and identify one or more compounds in a sample specimen from a subject. The portable device may be designed to receive one or more biosensor cartridges 110, e.g., disposable biosensor cartridges. In some embodiments, the portable device can receive one, two, three, four, five, six, seven, eight, nine, ten, or more biosensor cartridges at a single time. In some embodiments, the portable device includes a display 120. The display may provide the results from the analysis of a sample received within the disposable cartridge. In some aspects, the display may provide results from multiple samples received from multiple disposable cartridges. In some embodiments, the portable device includes a communication apparatus. The communication apparatus may transfer the results obtained from the sample analysis either wirelessly or electronically to another device, e.g., a mobile communication device or a computer.

[0040] The portable device may receive one or more biosensor cartridges. An exemplary biosensor cartridge 200 is shown in FIG. 2. In some embodiments, a biosensor cartridge comprises a sample inlet 210, which includes a membrane filter 220. In some embodiments, the sample enters the biosensor cartridge and flows down one or more microfluidic channels 230 to a sensor module comprising a biosensor, e.g., a sensor 240 for detecting a compound in the sample. In some embodiments, the biosensor cartridge includes one biosensor, two biosensors, three biosensors, four biosensors, five biosensors, six biosensors, seven biosensors, eight biosensors, nine biosensors, ten biosensors, or more. As shown in FIG. 2, an exemplary biosensor cartridge may have four biosensors situated in an electrode housing 250. Each biosensor may be designed to detect a specific compound, e.g., a specific protein and/or molecular target, in the sample.

[0041] A sample may be any fluid sample received from a subject, such as saliva, blood, urine, or other bodily fluid. In some embodiments, a sample is obtained from the upper or lower respiratory tract of a subject. For example, a sample may be obtained from a nasopharyngeal/oropharyngeal swab, nasal aspirate, nasal wash, saliva, sputum or tracheal aspirate, or bronchoalveolar lavage (BAL) from a subject.

[0042] It will be generally understood that a single sample received from a subject may be analyzed for multiple diseases based on the assessment of multiple compounds, e.g., specific proteins and/or molecular targets, using the multiple biosensors. For example, a single sample from a subject may be assessed to determine if the subject is infected with SARS-CoV-2, influenza A, influenza B, and/or any other disease. In addition, the single sample from the subject may be assessed to determine if it contains one or more markers for SARS-CoV-2, influenza A, and/or influenza B, such as by having a first sensor designed to identify SARS-CoV-2 spike glycoprotein, a second sensor designed to identify SARS-CoV- 2 nucleocapsid protein, a third sensor designed to identify influenza A antigens, and a fourth sensor designed to identify influence B antigens. Thereby, a medical professional processing the sample determine if the subject is experiencing an infection from SARS- CoV-2, influenza A, and/or influenza B.

[0043] In some embodiments, a subject introduces or provides, for example, a sample specimen including one or more compounds, e.g., antibody compounds, into the biosensor cartridge via a sample inlet. In some aspects, the sample specimen is breath, saliva, blood, urine, or other bodily fluid. In some aspects, the saliva of the subject enters the biosensor cartridge and passes through a filter (e.g., a 0.2 pm filter). In some aspects, the sample moves to a biosensor 240 via a microfluidic channel 230 (e.g., a 0.2 mm channel) and is deposited in a sample deposition area 260. The one or more biosensors may collect and analyze the sample for a specific compound. The biosensor cartridge may be returned to the portable device and the data obtained from individual biosensors may be transmitted via a communication apparatus (e.g., by wireless or electrical means) to the cloud. In one embodiment, the data may be stored in the cloud and may be accessed by another device, which detects and identifies the compounds in the sample of the subject and communicates the result back to the portable device where it is displayed to a user. In some embodiments, the communication apparatus directly transmits data to another device thus bypassing the cloud. In certain aspects, the analysis of the sample occurs in real time (i.e., at substantially the same time as the event is occurring, with any delay being minimal, for example less than one minute). However, in some aspects, data may be stored in the cloud before being processed by the processing unit and displayed to a user.

[0044] In some embodiments, the portable device collects data about the presence and identity of compounds of a disease, e.g., a virus, in a sample specimen of a subject via the biosensor cartridge. In some aspects, a sensor module converts collected data to a signal (e.g., provides significantly high electrical conductivity, thermal conductivity, optical conductivity, etc.) Data may be transmitted via a communication apparatus to a processing apparatus which analyzes the data to provide information about the presence and identity of compounds in the sample specimen of the subject. The processing apparatus may receive signals providing information about the presence and identity of one or more compounds, e.g., antibodies, in a sample specimen of a subject and, in some aspects, may transmit the presence and identity of the one or more compounds to a display on the portable device. [0045] In some embodiments, an inlet, such as a sample well, allows a sample specimen of a subject to enter the biosensor cartridge. In one embodiment, a biosensor cartridge comprises one or more sensor modules. In some aspects, the sensor module includes an array of sensors, e.g., biosensors, which may be independently capable of detecting the presence of one or more compounds in the sample specimen of a subject. In some aspects, the array of biosensors is attached to a base, which may be molded plastic or ceramic. In some embodiments, the biosensors are selected to detect the presence of one or more compounds in the sample specimen of the subject. In certain embodiments, sensor modules are replaceable, which allows the biosensor cartridge to be rapidly adapted to recognize the presence and identity of many different types of compounds. In some embodiments, the biosensor cartridge comprises one or more screen printed electrodes including working electrodes, counter electrodes and reference electrodes. In some aspects, the electrodes are connected to a main circuit having a printed circuit board (PCB).

[0046] In some embodiments, the biosensors are present in the biosensor cartridge as a biosensor array. In one embodiment, a biosensor array is a microfluidic system including amplification and detection reagents for electrochemical detection. In some embodiments, a device is configured to be insertable into an electrochemical reader for electronic readings. In some aspects, the detection of signals, such as electrical current, voltage, and other electric signals known in the art, is contemplated. In some aspects, a biosensor array includes a first cellulosic layer which includes one or more amplification agents in a sample deposition zone. In some embodiments, cellulosic layer (e.g., paper)-based test strips are used thereby reducing the sample volume and therefore cost of reagents. In some embodiments, the cellulosic layer is a patterned layer, e.g., a patterned paper layer. For example, the sample deposition zone is a hydrophilic porous area in the cellulosic layer defined by a fluid-impermeable material which permeates through the thickness of layer and surrounds the sample deposition zone. In some embodiments, the biosensor array further includes a second cellulosic layer, with an electrode assembly printed or attached onto layer. In one embodiment, the electrode assembly contains a one, two, three, four, five, six, seven, eight, nine, or ten electrodes. In some aspects, the electrode assembly includes one or more counter electrodes, one or more reference electrodes, and one or more working electrodes. In one embodiment, electrode assembly contains a three electrodes system that includes a counter electrode, reference electrode and five working electrodes. In other embodiments, the electrode assembly contains three, four, or more electrodes, e.g., positive, negative and reference electrodes. The electrodes in the assembly, e.g., screen-printed electrodes (SPE), connect electrically to a set of contact pads from the test zone. Optionally, a spacer layer is disposed between the first cellulosic layer and the second cellulosic layer. In certain embodiments, the spacer layer is non-porous, e.g., is plastic or glass. In certain embodiments, the spacer can be made from double-sided tape to join the first cellulosic layer and the second cellulosic layer. In some aspects, the spacer layer includes an opening to allow fluidic contact between portions of the first cellulosic layer and the second cellulosic layer, e.g., the sample deposition zone and the test zone. After a fluidic sample is deposited in the sample deposition zone, an optional cover layer may be placed on top of the first cellulosic layer to prevent fluid evaporation. In some aspects, sample deposition zone is in fluidic communication with a test zone on the second cellulosic layer. In certain embodiments, one or more amplification agents are embedded in the test zone and/or the sample deposition zone. In one embodiment, one or more amplification agents interact with the genetic material in the fluidic sample to provide copies of the genetic material using one or more methods described herein. In certain embodiments, one or more binding agents are embedded in the test zone and/or the sample deposition zone and are selected for binding the amplified genetic material to result in a change of the concentration of a signaling chemical. In some aspects, the test zone is in fluidic communication with the electrode assembly. In some embodiments, a hydrophilic channel connects test zone to the electrode assembly fluidically. In other embodiments, at least part of the electrode assembly is located in, printed in, or overlaps with the test zone. In some embodiments, when the signaling chemical's concentration is changed or altered as a result of the binding between the binding agents and the amplified genetic material, such a change may be detected by the electrochemical reader or biosensor to generate an electronic readout. Other variations in the arrangement of the cellulosic layers, sample deposition zone and detection zone are contemplated and will be apparent to one of skill in the art.

[0047] In one embodiment, a simple electrochemical sensor identifies and detects one or more compounds in a sample specimen. In some embodiments, the compound is an antibody, protein, DNA, or RNA in the blood of a subject. In certain embodiments, the compound is an antigen and/or nucleic acid, e.g., a viral antigen and/or viral nucleic acid. In some aspects, a sample encounters an electrochemical sensor which may include a counter electrode, a plurality of working electrodes, and a reference electrode. The counter electrode is, for example, made of carbon paint, while the reference electrode is, for example, made of silver paint. In other aspects, counter and reference electrodes may be made of boron doped diamond, silver (Ag) or platinum (Pt). Electrodes which may be used in the sensors described herein include, but are not limited to, multiwalled carbon nanotubes (MWCNT)/Ag nanohybrids/ Au, Ag nanoparti cles/DNA/glassy carbon electrodes (GCE), nano-CuO/Ni/Pt, PtPdFe3O4 nanoparticles/GCE, Co3O4 nanoparticles/GCE, Cu2O/Ni/Au, AgMnO2-MWCNTs/GCE, immobilized with antibody with carbon nanostructure or graphene gold nanocomposites, etc. In some aspects, application of voltage to working electrodes, which includes carbon paint and multi-wall carbon nanotubes, leads to recognition of the presence and identity of one or more antibody compounds in the sample specimen of the subject.

[0048] Gold nanoparticles (AuNPs) are the most stable metal nanoparticles, due to their unique optical, electrical, and catalytic activity, as well as high biocompatibility properties and enhanced electron transfer rate. Therefore, they have shown wide applications in various electrochemical biosensors. Gold nanoparticles can be prepared by the chemical or electrochemical reduction of gold salt. Electrode deposition of AuNPs on the surface of carbon electrodes is appealing due to its direct, fast and easier preparation method.

[0049] Other materials which may be used in working electrodes include but are not limited to immobilized antibody gold nanoparticles, graphene, carbon nanotubes, reduced graphene, graphene oxide, carbon nanofibers, quantum dots, fullerene, carbon polymer nanocomposites, glassy carbon, carbon fiber nanocomposite, carbon black, etc. In an exemplary biosensor the working electrodes are arrayed on a glass epoxy chip with an intervening insulating layer. Other supports for disposing the electrodes are generally known to those of skill in the art.

[0050] An exemplary sensor for use in a biosensor module as described herein is illustrated in FIG. 3. Sensor 500 includes one or more biological molecules 506 immobilized on support 508. Biological molecules or receptors 506 include, for example, enzymes, cells, protein receptors, antibodies, nucleic acids, etc. Exemplified in FIG. 5 are constituents 502 which are present in a specimen sample but do not bind to immobilized biological molecules 506 on support 508. Also shown in FIG. 5 is compound 504 which specifically binds to biological molecule 506. In some embodiments, upon binding of compound 504 to immobilized biological molecule 506, a signal is generated which is communicated from transducer 510 to communication apparatus (not shown) and then to processing apparatus 514, via signal amplifier 512, where the data is processed to confirm the presence and identity of the compound. In some aspects, signal amplifier 512 reduces instability and noise and may be purchased from commercial sources. Optionally, the signal may be directly communicated to the processing apparatus without the intermediacy of a signal amplifier.

[0051] In some embodiments, a sensor, e.g., a biosensor, based on biological materials can be constructed by covalently attaching a biological molecule to the carbon nanostructure component of the working electrodes. In some embodiments, the biological molecule is an enzyme. In other embodiments, the biological molecule is an oxidase. In still other embodiments, the biological molecule is antibodies, spike protein or DNA. In one embodiment, the sensitivity of the antibody system, e.g., a glycoprotein p41 antibody system, is very high with the ability to detect some phenols at a sensitivity as low as 50 copies per ml. For example, glycoprotein p41 can be attached to an electrode by incubation with a binding agent for one to four hours, or in certain aspects for two hours, in buffer solution. The immobilized glycoprotein p41 antibody will be selectively interacting with glycoprotein p41 of HIV 1 in sample specimen solution as demonstrated by differential pulse voltammetry.

[0052] In some embodiments, a base material of a multilayer biosensor includes a graphene-polymer nanocomposite. The graphene-polymer composite may be coated on to the biosensor to make it suitable for absorbing the fluid component of a sample. In some embodiments, electrochemical immunosensing is based on the principle of measuring the changes in electrical properties of a conductive material due to the adsorption of an analyte on the surface functionalized with antibodies. [0053] In one embodiment, electrochemical impedance spectroscopy and/or cyclic voltammetry are utilized to detect one or more compounds on the sensor surface. In one embodiment, one or more electrochemical techniques are used to detect the one or more compounds, such as differential pulse voltammertry (DPV) or square wave voltammetry (SWV). A low-cost, easy-to-use, simple and re-configurable miniaturized electronics sensor module is described herein to quantify the biosensor before and after treating a fluid (e.g., saliva) sample with immobilized surface antibodies.

[0054] In some embodiments, DC voltage of 5V is applied directly to the microcontroller embedded in the sensor module which then transfers signals to the communication device and display device. Electrical signals may monitor on Mat lab in real-time fashion. Readings record in terms of electrical resistance. In some aspects, electrical resistance can be displayed into current and voltage gain if required by reconfiguring the program for the microcontroller.

[0055] An exemplary battery is a lithium ion battery, which are conventional and available from many commercial sources (e.g., Panasonic DMW-BCM14 battery). Many batteries are known in the art and may be used in the portable devices described herein.

[0056] In some embodiments, the results from the sample analysis may be transmitted to another device, such as a conventional general-purpose computer which includes a display device and a communication interface which allows reception and transmittal of information from other devices and systems via any communication interface. Any general-purpose computer known in the art which has sufficient processing power to analyze data provided by the sensor module may be used in conjunction with the portable devices described herein.

[0057] In some embodiments, data from sensors in the biosensor cartridge is analyzed using pattern and recognition systems such as, for example, artificial neural networks, which include, for example, multi-layer perception, generalized regression neural network, fuzzy inference systems, etc., and statistical methods such as principal component analysis, partial least squares, multiples linear regression, etc. Artificial neural networks are data processing architectures that use interconnected nodes (i.e., neurons) to map complex input patterns with a complex output pattern. Importantly, neural networks can learn from using various input output training sets.

[0058] An exemplary artificial neural network can process data received from the biosensor cartridge. In general, the neural network can use three different layers of neurons. The first layer is input layer, which receives data from sensor module, the second layer is hidden layer while the third layer is output layer, which provides the result of the analysis. In some aspects, each neuron in hidden layer is connected to each neuron in input layer and each neuron in output layer. In the exemplified neural network, hidden layer processes data received from input layer and provides the result to output layer. Although only one hidden layer is described herein, any number of hidden layers may be used, with the number of neurons limited only by processing power and memory of the general-purpose computer or smart phone. In certain aspects, the inputs to the input neurons are inputs from the sensors in the sensor module. If, for example, seven sensors are in the sensor module, then the input layer will have seven neurons. In general, the number of output neurons corresponds to the number of compounds that the sensor module is trained to detect and identify. The number of hidden neurons may vary considerably. In some embodiments, the number of hidden neurons is between about 4 to about 10.

[0059] In some embodiments, the nanoparticle is chitosan and a polymer, polyvinyl alcohol nanoparticles or polyvinylpyrolidine nanoparticles, which may be made by methods well known in the art. In other embodiments, the polymer used with chitosan is tripolyphosphate, HPMC, HPC, PVP, ethyl cellulose, PEG, cellulose acetate phthalate and derivatives thereof, bioadhesive coatings such as, for example, poly(butadiene-maleic anhydride-co-L-DOPA) (PBMAD), etc.

[0060] The example mobile communication device shown in FIG. 3, can include one or more processors; a memory device including the biomarker processing module or engine, a diagnostic module, and an operating system (O/S); one or more activity sensors; a network and input/output (I/O) interface; and an output display. The example remote server computers can include one or more processors; a memory device including a biomarker processing module or engine, a diagnostic module, an operating system (O/S), and a database management system (DMBS); a network and input/output (I/O) interface; and an output display.

[0061] Those of skill in the art will appreciate that combinations of nanocomposite immobilized antibodies with antibody marker compounds at varying concentrations can be used to create a vast library of sequential viral detection. For example, glycoprotein p41, which includes the immobilized carbon nanostructure material, can be detected at different concentrations ranging from 50 copies per ml to 5 x 10 ⁶ copies per ml by differential pulse voltammetry and can be readily distinguished as illustrated in FIG. 4. FIG. 4 depicts an example user interface output by a diagnostic module, according to one example embodiment of the disclosure. In some embodiments, target biomolecules are also useful for diagnosis, monitoring virus diseases progression, and predicting disease recurrence. A virus target biomolecule refers to a substance or process that is indicative of the presence of same virus or not in the body.

[0062] In some embodiments, the devices and methods described herein are used to diagnose a subject as suffering from or having suffered from a viral infection. In certain embodiments, the devices and methods described herein are used to diagnose or identify a patient as having or had a coronavirus. For example, a subject may be diagnosed as suffering from or recovering from a SARS-CoV-2 infection. In one embodiment, a sample specimen from the subject is positive for SARS-CoV-2 antibodies. In certain embodiments, the sample specimen from the subject comprises one or more antibody compounds selected from the group consisting of SARS-CoV-2-nucleocapsid antibody, SARS-CoV-2-spike antibody, SARS-CoV-2 membrane antibody, and SARS-CoV-2 envelope antibody.

[0063] In some embodiments, the biosensor cartridge comprises a first biosensor or biosensor array that includes a customized molecular probe using a specific and customized aptamer sequence for very low level and highly accurate detection of COVID-19 (e.g., via SARS-CoV-2 antigens and/or nucleic acids) and further comprises a second biosensor or biosensor array that includes a customized probe for the detection of protein targets.

[0064] In some aspects, aptamers have unique attributes that are highly attractive for the development of field deployable or ‘point-of-care’ (POC) tests for the detection of a disease, such as those described herein. Aptamers are DNA- or RNA-based ligands capable of binding practically any molecular target. Aptamers are uniquely suited to address the challenges associated with viral antigen detection. They are usually identified by an in vitro method of selection referred to as Systematic Evolution of Ligands by Exponential enrichment or “SELEX.” Aptamers are additionally heat stable and can be lyophilized on sensor electrodes and exhibit an extended shelf life. In one embodiment, multiple aptamers or similar nucleic acid probes, such as oligonucleotide probes, are used as a multiplexing sensor where different targets can be targeted by each biosensor or biosensor array of the biosensor cartridge or electrode system simultaneously. Oligonucleotide probes are singlestranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions.

[0065] In some embodiments, the biosensor cartridge comprises biosensors or biosensor arrays that are tailored or designed for the detection of ultralow (e.g., about 1 fg/ml) to ultrahigh (e.g., about 1 mg/ml) detection of viral load. In some aspects, the sensitivity of the portable device for detecting a disease in a subject is 18.56 copies viral RNA/ml to 1856 copies viral RNA/ml. In some embodiments, the sensitivity of the portable device for detecting a disease in a subject is about 100 copies viral RNA/mL. In some aspects the portable device provides results in viral load as outlined in the table below. [0066] In some embodiments, the devices and methods described herein are used to calculate and quantify the viral load within a sample of a subject. For example, a fluid sample may be received from a subject and inserted into the biosensor cartridge through the sample inlet. The fluid sample will flow through the microfluidic channel and drop onto a sensing area of one or more biosensors (e.g., a sensing area of a customized gold electrode attached with specific antibodies for SARS-CoV-2 proteins). Based on the interaction between these antibodies and target proteins, electrochemical signals will be captured by electronic circuits embedded within the portable device. The captured data will be collectively processed to predict accurate results for the quantitative confirmation of viral load for COVID-19.

[0067] In one embodiment, the devices and methods described herein are used to quantify the viral load in a sample from a subject. After quantification of the viral load within a subject, a therapeutic regimen may be designed. For example, a subject suffering from a viral infection (e.g., COVID19, HIV, etc.) may have a therapeutic regimen, including specific medications, adjusted as the viral load decreases.

[0068] In one embodiment, the devices and methods described herein are used to monitor the administration and response of a therapeutic regimen to a subject. For example, a subject receiving treatment for a disease may be monitored by having samples taken consecutively over a period of time, e.g., daily, weekly, or monthly, to monitor the viral load within the sample to determine if the subject is improving from the disease. In addition, a subject receiving treatment for a disease may be monitored by having samples taken over a period of time, e.g., daily, weekly, or monthly, to confirm the subject is maintaining a specific therapeutic regimen, e.g., samples may be taken and screened for compounds signifying specific medication is being taken by the subject. For example, a subject may be monitored to confirm the subject is maintaining a therapeutic regimen of dabigatran, rivaroxaban, apixaban, or Levicetam.

[0069] Many modifications and other embodiments of the example descriptions set forth herein to which these descriptions pertain will come to mind having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Thus, it will be appreciated that the disclosure may be embodied in many forms and should not be limited to the example embodiments described above. Therefore, it is to be understood that the disclosure is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0070] EXEMPLIFICATION OF PORTABLE DEVICE

[0071] Electrodes

[0072] Surface modification and functionalization: Clean gold electrode was immersed overnight under ambient condition to generate targeted functional groups. The gold electrode was gently washed with absolute ethanol to remove unbound thiol groups and dried.

[0073] Chemical cross- linking: For coupling of the antibodies on gold electrode surface, the carboxyl group on modified electrode was activated by adding an optimized concentration of cross linkers and incubated for 2h. Subsequently, the gold electrode was washed with membrane filtered de-ionized water to remove unbound cross-linkers.

[0074] Antibody immobilization- Optimized antibody dilution was added carefully over the activated surface of gold electrode followed by overnight incubation at 4 °C. The coupled gold electrode was washed gently to remove unbound antibody.

[0075] Blocking of the antibody: Unbound antibodies from the gold electrode surface were blocked by using an optimized concentration of a non-specific protein for 1 h and washed subsequently with membrane filtered de-ionized water.

[0076] Now the gold electrodes are ready for assembly in the biosensor cartridge. The biosensor cartridges are utilized with the portable device to test saliva samples from subjects. Based on the interaction between COVID-19 protein containing saliva and specific antibodies, in the presence of customized nanoprobes, a signal will be generated. The signals will be collected by the electronic set-up and collectively processed through Al techniques to predict the viral load quantity.

[0077] Device hardware

[0078] Connector interface: It involves the generation of the ramped square wave signal with a defined and fixed frequency and amplitude step, with an incremental increase in step size. This incremental signal is provided to the electrode system over a fixed interval of time.

[0079] Equivalent current for each step voltage is then obtained using a voltagecurrent divider. This current is then measured using a current sense circuit. This current sense circuit is then fed to an analog-to-digital convertor. This signal is then further processing using a microcontroller system.

[0080] Pre-processing stage: Each of the 4-connector has a dedicated 3-Pin connector interface to which the electrode is connected. Connector ensures the electrode has a proper snug fit and there is no artifact.

[0081] Processing: Processing of the signal is then performed using ESP32 microcontroller. Data from analog-to-digital convertor is then further sampled and mapped with the metadata and is ready for further processing.

[0082] Internal storage: Data samples are stored in an internal storage system which consists of a non-volatile storage card. This is for data back-up, in events of connection failure while streaming the data using wired or wireless connection.

[0083] Real-time clock: Hardware consist of an on-board clock which provides data and timestamp. This data forms the metadata for events when the data is to be stored in a storage card, so data can be co-related.

[0084] Unique identifier: Each biosensor cartridge with 4-electrodes that consist of a unique ID, which is stored in an RFID TAG. This information is stored in an encrypted format and can be interpreted only by the RFID READER in the device. [0085] Power Supply: Device is powered using a 3.7v Lithium-ion battery. Dedicated 3.3v, 5v supply is derived using switching power supplies which meet all RoHS grade components. Dedicated battery management system is provided which ensures that battery status is monitored and relevant indications are provided. This ensures the battery health is maintained.