Multiplex Biosensor for Rapid Point-of-Care Diagnosis

Abstract

The present disclosure relates to carbon-based biosensors and biosensor systems. The present disclosure further relates to a method for rapidly detecting a target substance in a biological sample using the biosensors and biosensor systems described herein to characterize the antigen profile of a pathogen and / or the immune response of a subject to pathogen exposure.

Description

Technical Field

[0001] The present disclosure is directed to biosensors, systems, and methods capable of rapidly detecting a target substance in a biological sample.

Background Art

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 342,248, filed May 16, 2022, and U.S. Provisional Patent Application No. 63 / 486,144, filed Feb. 21, 2023, which are hereby incorporated by reference in their entirety.

[0003] The public health emergency due to COVID-19 has highlighted the national need for next-generation diagnostics that can be easily deployed in both conventional healthcare settings and in the field. Delays in results, inaccurate reporting, and in some cases poor access to testing hindered the reopening of the economy and contributed to the spread of COVID-19.

[0004] To achieve the goal of rapidly and effectively diagnosing emerging diseases such as COVID-19 and other viruses, rapid, cost-effective, and real-time biomarker measurement is an essential step. Currently, many biological assays rely on labeled detection molecules and optical-based detectors for diagnosis. The costs and time delays associated with these methods have a fundamental impact on patient outcomes because testing, diagnosis, and treatment typically involve multiple exchanges. There is an urgent need for innovative point-of-care biosensor devices that enable rapid and accurate disease detection.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention aims to overcome these and other drawbacks in the art.

Means for Solving the Problems

[0006] In some embodiments, a charge prevention substrate including a flat surface; at least one spatially defined active region on the flat surface of the charge prevention substrate, each active region including a carbon material, a first signal electrode and a second signal electrode operably contacting the carbon material, and at least one gate electrode; a plurality of capture molecules, different capture molecules being disposed in separate active regions and immobilized on the deposited carbon material of the active regions; and an electrical connection including a plurality of electrical contacts, each electrical contact being configured to transmit an electrical signal between the first signal electrode and the second signal electrode and the at least one gate electrode of a single active region, a biosensor is provided.

[0007] In some embodiments, a charge prevention substrate including a flat surface; at least one spatially defined active region on the flat surface of the charge prevention substrate, each active region including a carbon material, a first signal electrode, a second signal electrode, and a third signal electrode operably contacting the carbon material, and at least one gate electrode; a plurality of capture molecules, different capture molecules being disposed in separate active regions and immobilized on the deposited carbon material of the active regions; and an electrical connection including a plurality of electrical contacts, each electrical contact being configured to transmit an electrical signal between the first signal electrode and the second signal electrode and the at least one gate electrode of a single active region, a biosensor is provided.

[0008] In some embodiments, a biosensor is provided that includes: an antistatic substrate including a flat surface; at least one spatially defined active region on the flat surface of the antistatic substrate, each active region including a carbon material, a first signal electrode, a second signal electrode, a third signal electrode, and a fourth signal electrode operably contacting the carbon material, and at least a first gate electrode and a second gate electrode; a plurality of capture molecules, different capture molecules being disposed in separate active regions and immobilized on the deposited carbon material of the active regions; and an electrical connection including a plurality of electrical contacts, each electrical contact being configured to transmit an electrical signal between the first signal electrode and the second signal electrode of a single active region and at least one gate electrode.

[0009] In some embodiments, a biosensor system is provided for characterizing a subject's immune response to pathogen exposure, the biosensor system including: an electronic reader including a circuit for delivering a signal; a processing device for reading the signal; and any biosensor disclosed herein, the biosensor being operably connected to the electronic reader via the electrical connection of the biosensor and configured to receive a signal delivered by the circuit. In some embodiments, the electronic reader is configured to deliver a signal to the biosensor and obtain output impedance values before and after application of a sample to an array of active regions on the biosensor, and the processing device is configured to compare the output impedance values to determine whether a binding event has occurred in one or more of the active regions and characterize the subject's immune response to pathogen exposure.

[0010] In some embodiments, a method for characterizing an immune response of a subject to pathogen exposure includes: collecting a biological sample from the subject; providing the biosensor system described herein; delivering an electrical signal to the biosensor via a circuit of an electronic reader; determining a base resistance between a first signal electrode and a second signal electrode in each active region of the biosensor; applying the biological sample from the subject to at least one active region of the biosensor and operably contacting the biological sample with a carbon material between the first signal electrode and the second signal electrode and at least one gate electrode in the at least one active region; identifying a change in the base resistance between the first signal electrode and the second signal electrode caused by the application of the biological sample to the at least one active region; and characterizing an immune response of the subject to a pathogen or an antigen profile of the pathogen based on the change in the base resistance between the first signal electrode and the second signal electrode in the at least one active region.

[0011] In some embodiments, a method for characterizing an immune response of a subject to pathogen exposure, comprising: collecting a biological sample from the subject; providing any biosensor system disclosed herein; delivering an electrical signal to the biosensor via a circuit of an electronic reader; applying a control solution to at least one active region on the biosensor, the control solution being operably contacted with a carbon material between a first signal electrode and a third signal electrode and at least one gate electrode in the at least one active region; determining a base resistance between a second signal electrode and the third signal electrode in the at least one active region; applying a biological sample from the subject to the at least one active region, the biological sample being operably contacted with the carbon material between the second signal electrode and the third signal electrode; identifying a change in the base resistance between the second signal electrode and the third signal electrode resulting from applying the biological sample to the at least one active region; and characterizing an immune response of the subject to a pathogen, or an antigen profile of the pathogen, based on the change in the base resistance between the second signal electrode and the third signal electrode in the at least one active region.

[0012] In some embodiments, a method for characterizing an immune response of a subject to pathogen exposure, comprising: collecting a biological sample from a subject; providing any of the biosensor systems disclosed herein; delivering an electrical signal to the biosensor via a circuit of an electronic reader; applying a first control solution to at least one active region on the biosensor, the first control solution being operably contacted with a carbon material between a first signal electrode and a third signal electrode and a first gate electrode in the at least one active region; applying a second control solution to at least one active region, the second control solution being operably contacted with a carbon material between a second signal electrode and a fourth signal electrode and a second gate electrode, and the second gate electrode in the at least one active region; determining a base resistance between a third signal electrode and a fourth signal electrode in the at least one active region; applying a biological sample from the subject to the at least one active region, the biological sample being operably contacted with the carbon material between the second signal electrode and the third signal electrode; identifying a change in the base resistance between the second signal electrode and the third signal electrode resulting from applying the biological sample to the at least one active region; and characterizing an immune response of the subject to a pathogen, or an antigen profile of the pathogen, based on the change in the base resistance between the second signal electrode and the third signal electrode in the at least one active region.

[0013] In some embodiments, a method of manufacturing a biosensor is provided, the method comprising obtaining a substrate having at least two layers, wherein there is a height difference z between at least two layers; transferring graphene onto the substrate, wherein the graphene breaks along at least two layers due to the height difference z; cleaning the biosensor to remove excess graphene; and optionally adding an additional layer to the biosensor. In some embodiments, the height difference z between at least two layers is from about 50 nanometers to about 3 millimeters. In some embodiments, the substrate includes at least three layers. In some embodiments, the substrate includes at least four layers. In some embodiments, the additional layer in the method includes an insulating material, or an electrode material, or both. In some embodiments, any of the biosensors described herein can be fabricated by this contour ablation method.

[0014] The biosensors described herein utilize the excellent charge-carrying ability of graphene and provide a nearly instantaneous (~60 seconds) high-sensitivity point-of-care testing platform that can detect up to 12 antibody / antigen pairs from a single drop of saliva. This enables testing, diagnosis, and treatment to be performed simultaneously, leading to more accurate and effective treatment plans and significantly improving patient outcomes.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0016] The present disclosure is directed to biosensors, systems, and methods for rapidly detecting a target substance in a biological sample.

[0017] In some embodiments, the biosensor disclosed herein includes a substrate including an anti-static substrate and an array of spatially defined active regions on a planar surface of the substrate. Each active region on the planar surface includes a carbon material, a first signal electrode and a second signal electrode operably contacting the carbon material, and at least one gate electrode. The biosensor further includes a plurality of different detection agents disposed in separate active regions and immobilized on the deposited carbon material of the active regions; and an electrical connection portion including a plurality of electrical contacts, each electrical contact configured to transmit an electrical signal between the first signal electrode, the second signal electrode, and the at least one gate electrode of a single active region and the electrical connection portion.

[0018] In some embodiments, the biosensor disclosed herein includes a substrate including an anti-static substrate and an array of spatially defined active regions on a planar surface of the substrate. Each active region on the planar surface includes a carbon material, a first signal electrode, a second signal electrode, and a third signal electrode operably contacting the carbon material, and at least one gate electrode. The biosensor further includes a plurality of different detection agents disposed in separate active regions and immobilized on the deposited carbon material of the active regions; and an electrical connection portion including a plurality of electrical contacts, each electrical contact configured to transmit an electrical signal between the first signal electrode, the second signal electrode, the third signal electrode, and the at least one gate electrode of a single active region and the electrical connection portion.

[0019] In some embodiments, the biosensor disclosed herein includes a substrate including an anti-static substrate and an array of spatially defined active regions on a planar surface of the substrate. Each active region on the planar surface includes a carbon material, a first signal electrode, a second signal electrode, a third signal electrode, and a fourth signal electrode that are operably in contact with the carbon material, and at least a first gate electrode and a second gate electrode. The biosensor further includes a plurality of different detection agents disposed in separate active regions and immobilized on the deposited carbon material of the active regions, and an electrical connection portion including a plurality of electrical contacts, and each electrical contact is configured to transmit an electrical signal between the first signal electrode, the second signal electrode, the third signal electrode, and the fourth signal electrode, and at least the first gate electrode and the second gate electrode of a single active region and the electrical connection portion.

[0020] The basic structure of the biosensor disclosed herein is described in the pamphlet of International Patent Application Publication No. 2020 / 072966 of Hememics Biotechnologies, Inc., which is hereby incorporated by reference in its entirety as part of this specification.

[0021] The schematic diagrams of FIGS. 1 and 2 provide cross-sectional views of the active region 100 of the biosensor described herein, and the biosensor has two signal electrodes. Referring to FIGS. 1 and 2, the biosensor includes a substrate 120 having a planar surface.

[0022] In some embodiments, the biosensor includes a single-layer substrate. According to this embodiment, the single-layer substrate is a polymer material. Suitable polymer materials include, but are not limited to, poly(methyl methacrylate) (PMMA), polycarbonate (PC), epoxy resin, copolymer, polysulfone, elastomer, cyclic olefin copolymer (COC), nylon, polypropylene, polyester film, polyethylene terephthalate (PET), polyvinyl chloride, polytetrafluoroethylene, and polymeric organosilicon. In any embodiment, the polymeric substrate is modified with an antistatic agent and exhibits antistatic properties suitable for dissipating charges. The antistatic agent can be directly mixed with the polymer material to impart antistatic properties, or applied to the surface of the polymer material to impart antistatic properties to the material. Antistatic agents that can be added to the polymer to minimize static electricity are known in the art and include fatty acid esters, long-chain aliphatic amines and amides, ethoxylated amines, quaternary ammonium compounds (e.g., behentrimonium chloride or cocamidopropyl betaine), esters of phosphoric acid, polyethylene glycol esters, alkyl sulfonates, and alkyl phosphates, but are not limited thereto. The antistatic quality suitable for the substrate of the biosensor described herein is determined by the surface resistivity of the material, measured in units of ohms / square. A preferred antistatic polymer substrate material for the biosensor has a surface resistivity of 10 3 ~10 14 ohms / square. In some embodiments, the antistatic polymer substrate material of the biosensor has a surface resistivity between 10 3 ~10 5 ohms / square. In some embodiments, the antistatic polymer substrate of the biosensor has a surface resistivity between 10 7 ~10 14 ohms / square.

[0023] Referring to FIGS. 1 and 2, each active region 100 on the biosensor functions as a field effect transistor (FET) sensor unit having a liquid gate. Each active region includes a conductive carbon material 130 (e.g., graphene) deposited on a flat surface of a substrate 120 between a first signal electrode 140 and a second signal electrode 141. Suitable conductive carbon materials for the active region include, but are not limited to, graphene, carbon nanotubes, fullerenes, or combinations thereof. Alternatively, the region between the electrodes can be composed of other conductive materials known in the art. Other conductive materials include, but are not limited to, silicon, molybdenum disulfide, black phosphorus, and / or metal dichalcogenides.

[0024] In some embodiments, the carbon material is a graphene polycrystal. In some embodiments, the carbon material is a graphene single crystal. In some embodiments, the carbon material is a single layer. In some embodiments, the carbon material has multiple layers. In some embodiments, the carbon material can be laser ablated or mechanically ablated to form discrete sensor regions having a width of about 50 μm to about 500 μm and a length of about 100 μm to about 3000 μm. In some embodiments, the carbon material can be applied to the biosensor by screen printing, rotogravure printing, photolithography, mechanical ablation, or contour ablation. In some embodiments, the carbon material can be applied to the biosensor by directly screen printing on an antistatic substrate. In some embodiments, the carbon material can be applied to the biosensor by directly rotogravure printing on an antistatic substrate.

[0025] In some embodiments, the carbon material can be transferred to a substrate such that the carbon material forms a series of surfaces rather than a continuous flat surface. For example, as shown in FIGS. 16A-16F and FIGS. 17A-17F, the carbon material can be transferred to a biosensor by a contour ablation method. When the carbon material is transferred, the height difference of the biosensor breaks the carbon material, and any exposed area of the biosensor at the time of its application can be coated with the carbon material.

[0026] In some embodiments, the height difference can range from about 50 nanometers to about 3 millimeters. In some embodiments, the height difference can range from about 100 nanometers to about 3 millimeters. In some embodiments, the height difference can range from about 150 nanometers to about 3 millimeters. In some embodiments, the height difference can range from about 200 nanometers to about 3 millimeters. In some embodiments, the height difference can range from about 250 nanometers to about 3 millimeters. In some embodiments, the height difference can range from about 300 nanometers to about 3 millimeters. In some embodiments, the height difference can range from about 400 nanometers to about 3 millimeters. In some embodiments, the height difference can range from about 500 nanometers to about 3 millimeters. In some embodiments, the height difference can range from about 600 nanometers to about 3 millimeters. In some embodiments, the height difference can range from about 700 nanometers to about 3 millimeters. In some embodiments, the height difference can range from about 800 nanometers to about 3 millimeters. In some embodiments, the height difference can range from about 900 nanometers to about 3 millimeters. In some embodiments, the height difference can range from about 1 millimeter to about 3 millimeters. In some embodiments, the height difference can range from about 1.5 millimeters to about 3 millimeters. In some embodiments, the height difference can range from about 2 millimeters to about 3 millimeters. In some embodiments, the height difference can range from about 2.5 millimeters to about 3 millimeters. In some embodiments, the height difference can range from about 50 nanometers to about 2.5 millimeters. In some embodiments, the height difference can range from about 50 nanometers to about 2 millimeters. In some embodiments, the height difference can range from about 50 nanometers to about 1.5 millimeters. In some embodiments, the height difference can range from about 50 nanometers to about 1 millimeter.In some embodiments, the height difference can range from about 50 nanometers to about 900 nanometers. In some embodiments, the height difference can range from about 50 nanometers to about 800 nanometers. In some embodiments, the height difference can range from about 50 nanometers to about 700 nanometers. In some embodiments, the height difference can range from about 50 nanometers to about 600 nanometers. In some embodiments, the height difference can range from about 50 nanometers to about 500 nanometers. In some embodiments, the height difference can range from about 50 nanometers to about 400 nanometers. In some embodiments, the height difference can range from about 50 nanometers to about 300 nanometers. In some embodiments, the height difference can range from about 50 nanometers to about 250 nanometers. In some embodiments, the height difference can range from about 50 nanometers to about 200 nanometers. In some embodiments, the height difference can range from about 50 nanometers to about 150 nanometers. In some embodiments, the height difference can range from about 50 nanometers to about 100 nanometers.

[0027] In some embodiments, the contour ablation method proceeds in several steps shown in FIGS. 16A-16 and FIGS. 17A-17F. First, a substrate such as a PCB is manufactured from various layers. The substrate includes signal electrodes and / or insulating materials, and the height difference z between these layers may range from about 50 nanometers to about 3 millimeters (FIGS. 16A and 17A). In a second step, graphene grown on a donor substrate is transferred onto this PCB substrate (FIGS. 16B and 17B). At this time, the graphene is broken due to the height difference z on the substrate, showing a graphene pattern having defined graphene islands (FIGS. 16C and 17C). Excess graphene can be washed away, leaving only the graphene bound to specific regions of the biosensor (such as on the insulating material laid on the PCB substrate, etc.) (FIGS. 16D and 17D). Finally, additional insulating materials and electrodes can be printed on the top to complete the biosensor (FIGS. 16E and 17E without samples, and FIGS. 16F and 17F with samples on the biosensor). In some embodiments, the contour ablation method can be used to manufacture any of the biosensors described herein.

[0028] In some embodiments, a method of manufacturing a biosensor is provided, the method comprising obtaining a substrate having at least two layers, wherein there is a height difference z between the at least two layers, transferring graphene onto the substrate, wherein the graphene breaks along at least two layers due to the height difference z, washing the biosensor to remove excess graphene, and optionally adding an additional layer to the biosensor. In some embodiments, the height difference z between the at least two layers is from about 50 nanometers to about 3 millimeters. In some embodiments, the substrate includes at least three layers. In some embodiments, the substrate includes at least four layers. In some embodiments, the additional layer in the method includes an insulating material, an electrode material, or both.

[0029] In some embodiments, the first signal electrode 140 functions as a source electrode and the second signal electrode 141 functions as a drain electrode, or vice versa. The electrodes each include, but are not limited to, a conductive metal such as gold (Au), copper (Cu), silver (Ag), cobalt (Co), platinum (Pt), titanium (Ti), platinum (Pt), iridium (Ir), their oxides, and combinations thereof. As shown in FIGS. 1 and 2, the electrodes can be encapsulated in an insulating material 150. In some embodiments, the insulating material can be applied to the biosensor by screen printing, rotogravure printing, or photolithography. Each active region of the biosensor further includes a gate conductor (not shown), which controls the liquid gate (i.e., the electric field in the ionic fluid sample 110 applied to the graphene surface 130 during use of the sensor).

[0030] Preferably, in the presence of a preservation solution as disclosed herein, the collection of the detection agent is immobilized on the surface of the carbon material 130. Suitable detection agents, preservation agents, and methods for immobilizing the detection agent on the surface of the carbon material are described herein.

[0031] Referring to FIGS. 1 and 2, each active region 100 includes at least a first gate electrode 160. In some embodiments, the gate electrode 160 is located substantially on the upper surface of either the first signal electrode 140 or the second signal electrode 141. In some embodiments, the gate electrode 160 is mostly covered by an insulating material 150. At least a portion of the gate electrode 160 is designed to contact the ionic fluid sample 110. Without wishing to be bound by theory, in conventional FET biosensors, the faradic gate current passing through the sample remains a significant source of error and may damage the detection agent present on the graphene. In some embodiments, one or more gate electrodes on the biosensor can directly measure the ionic conductivity of the ionic fluid sample 110. This measured value can be used by the gate conductor to apply a gate voltage to the carbon material. In some embodiments, the gate electrode can apply a gate voltage. In some embodiments, the gate electrode can reduce the level of electronic noise in the system to 1 / 1, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, or 1 / 10. In some embodiments, the gate electrode 160 can apply a gate voltage.

[0032] As shown in FIG. 1, the first signal electrode 140 and the second signal electrode 141 are located on the upper surface of the carbon material 130. As shown in FIG. 2, the first signal electrode 140 and the second signal electrode 141 are located between the antistatic substrate 120 and the carbon material 130. In some embodiments, an insulating material 150 can be disposed between the first signal electrode 140 and the second signal electrode 141 to place the carbon material 130 on a uniform surface.

[0033] The schematic diagrams of FIGS. 3 and 4 provide cross-sectional views of the active region 300 on the biosensor described herein, and the biosensor has three signal electrodes. Referring to FIGS. 3 and 4, the biosensor includes a substrate 320 having a flat surface. In some embodiments, the substrate is any substrate described herein.

[0034] Referring to FIGS. 3 and 4, each active region 300 on the biosensor functions as a field effect transistor (FET) sensor unit having a liquid gate. Each active region includes a conductive carbon material 330 (e.g., graphene) deposited on the flat surface of the substrate 320. The first signal electrode 340 and the second signal electrode 341 are located at both ends of the carbon material 330, and the third signal electrode 342 is located on the upper surface of the carbon material 330 between the first signal electrode 340 and the second signal electrode 341. The carbon material 330 is any conductive carbon material disclosed herein. Alternatively, the region between the electrodes can include other conductive materials known in the art, and the other conductive materials include, but are not limited to, silicone, molybdenum disulfide, black phosphorus, and / or metal dichalcogenides.

[0035] In some embodiments, the first signal electrode 340 functions as a source electrode, and the second signal electrode 341 and / or the third signal electrode 342 function as drain electrodes. In some embodiments, the third signal electrode 342 functions as a source electrode, and the first signal electrode 340 and / or the second signal electrode 341 function as drain electrodes. In some embodiments, each of the electrodes includes any conductive material described herein. As shown in FIGS. 3 and 4, any of the electrodes can be encapsulated with an insulating material 350. Each active region of the biosensor further includes a gate conductor (not shown), and the gate conductor controls the liquid gate (i.e., the electric field in the ionic fluid sample 310 and the control solution 311 applied to the graphene surface 330 during use of the sensor).

[0036] Preferably, in the presence of a preservation solution as disclosed herein, the collection of detection agents is immobilized on the surface of the carbon material 330. Suitable detection agents, preservation agents, and methods for immobilizing the detection agents on the surface of the carbon material are described herein.

[0037] Referring to FIGS. 3 and 4, each active region 300 includes at least a first gate electrode 360. In some embodiments, the gate electrode 360 is located substantially on top of either the first signal electrode 340 or the second signal electrode 341. In some embodiments, the gate electrode 360 is mostly covered by an insulating material 350. At least a portion of the gate electrode 360 is designed to contact a reference solution 311 disposed between the first signal electrode 340 and the third signal electrode 342. Without wishing to be bound by theory, the gate electrode 360 can directly measure the ionic conductivity of the reference solution 311. Using this measurement, the biosensor system can remove, reduce, or gate the signal noise present in the system when a biological sample 310 is disposed between the second signal electrode 341 and the third signal electrode 342. In some embodiments, the gate electrode can reduce the level of electronic noise in the system to 1 / 1, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, or 1 / 10.

[0038] As shown in FIG. 3, the first signal electrode 340, the second signal electrode 341, and the third signal electrode 342 are located on the top surface of the carbon material 330. As shown in FIG. 4, the first signal electrode 340, the second signal electrode 341, and the third signal electrode 342 are located between the antistatic substrate 320 and the carbon material 330. In some embodiments, an insulating material 350 can be disposed between the first signal electrode 340, the second signal electrode 341, and the third signal electrode 342 to place the carbon material 330 on a uniform surface.

[0039] The schematic diagrams of FIGS. 5 and 6 provide cross-sectional views of the active region 500 on the biosensor described in this specification, and the biosensor has four signal electrodes. Referring to FIGS. 5 and 6, the biosensor includes a substrate 520 having a flat surface. In some embodiments, the substrate is any substrate described in this specification.

[0040] Referring to FIGS. 5 and 6, each active region 500 on the biosensor functions as a field effect transistor (FET) sensor unit having a liquid gate. Each active region includes a conductive carbon material 530 (e.g., graphene) deposited on the flat surface of the substrate 520. The first signal electrode 540 and the second signal electrode 541 are located at both ends of the carbon material 530, the third signal electrode 542 is located on the upper surface of the carbon material 530 between the first signal electrode 540 and the fourth signal electrode 543, and the fourth signal electrode 543 is located on the upper surface of the carbon material 530 between the third signal electrode 542 and the second signal electrode 541. The carbon material 530 is any conductive carbon material disclosed in this specification. Alternatively, the region between the electrodes can include other conductive materials known in the art, and the other conductive materials include, but are not limited to, silicon, molybdenum disulfide, black phosphorus, and / or metal dichalcogenides.

[0041] In some embodiments, at least one of the first signal electrode 540, the second signal electrode 541, the third signal electrode 542, and the fourth signal electrode 543 functions as a source electrode, and at least one of the remaining signal electrodes functions as a drain electrode. In some embodiments, each of the electrodes includes any conductive material described herein. As shown in FIGS. 5 and 6, any of the electrodes can be encapsulated with an insulating material 550. Each active region of the biosensor further includes a gate conductor (not shown), and the gate conductor controls the liquid gate (i.e., the electric field in the ionic fluid sample 510, the first control solution 511, and the second control solution 512 applied to the graphene surface 330 during use of the sensor).

[0042] Preferably, in the presence of a preservation solution as disclosed herein, the collection of detection agents is immobilized on the surface of the carbon material 530. Suitable detection agents, preservation agents, and methods for immobilizing detection agents on the surface of carbon materials are described herein.

[0043] Referring to FIGS. 5 and 6, each active region 500 includes at least a first gate electrode 560. In some embodiments, the gate electrode 560 is located substantially on top of either the first signal electrode 540 or the second signal electrode 541. In some embodiments, the gate electrode 560 is mostly covered by an insulating material 550. At least a portion of the gate electrode 560 is designed to contact a first reference solution 511 or a second reference solution 512 disposed between the first signal electrode 540 and the third signal electrode 542 and between the second signal electrode 541 and the fourth signal electrode 543, respectively. Without wishing to be bound by theory, the gate electrode 560 can directly measure the ionic conductivity of either reference solution 511, 512. The biosensor system can use this measurement to remove, reduce, or gate the signal noise present in the system when the biological sample 510 is disposed between the third signal electrode 542 and the fourth signal electrode 543. In some embodiments, the gate electrode can reduce the level of electronic noise in the system to 1 / 1, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, or 1 / 10.

[0044] As shown in FIG. 5, the first signal electrode 540, the second signal electrode 541, the third signal electrode 542, and the fourth signal electrode 543 are located on the upper surface of the carbon material 530. As shown in FIG. 6, the first signal electrode 540, the second signal electrode 541, the third signal electrode 542, and the fourth signal electrode 543 are located between the antistatic substrate 520 and the carbon material 530. In some embodiments, an insulating material 550 can be disposed between the first signal electrode 540, the second signal electrode 541, the third signal electrode 542, and the fourth signal electrode 543 to place the carbon material 530 on a uniform surface.

[0045] Figure 7 shows a top view of a portion of the biosensor device. This figure shows a series of five active regions 700, each of which includes a conductive carbon material 706 deposited on a substrate. The conductive carbon material 706 forms a channel between the source electrode 708 and the drain electrode 710 of the active region and is close to the gate conductor 714. Exemplary dimensions of the carbon material channel of the active region, i.e., length and width, are such that the length ranges from about 10 microns to about 3 mm and the width ranges from about 10 microns to about 1 mm. For example, the channel length may range from about 10 microns to about 3 mm, from about 25 microns to 1 mm, from about 50 microns to about 750 microns, from about 75 microns to about 250 microns. In some embodiments, the channel length is about 75 microns, about 80 microns, about 90 microns, or about 100 microns. In some embodiments, the channel length is about 90 microns. Similarly, the left and right widths of the channel may range from about 10 microns to about 1 mm, from about 25 microns to about 750 microns, from about 50 microns to about 250 microns, from about 75 microns to about 100 microns. In some embodiments, the channel width is about 75 microns, about 80 microns, about 90 microns, or about 100 microns. In some embodiments, the channel width is about 90 microns. As shown in this embodiment, the gate conductor 714 may use other numbers of active regions, but for all five active regions 700, it controls the liquid gate (i.e., the electric field in the ionic fluid sample applied to the surface of each conductive carbon material 706 on the device). Here, an active region 700 with two signal electrodes is shown, but such a configuration can be used in active regions with three, four, or more signal electrodes. In some embodiments, there are multiple liquid gates to achieve different patterns of electromagnetic fields across the biosensor.

[0046] The biosensor further includes an electrical connection portion for operatively connecting the biosensor to the electronic reading portion. The electrical connection portion includes a plurality of electrical contacts, and each contact can transmit an electrical signal between the electrode of each active region and the electrical connection portion. Referring to FIG. 7, the electrical contacts include a shared bonding pad 720 and a shared source pad 728. For example, the electrical connection portion supplies a current (i.e., an electrical signal) to each of a plurality of active regions on the biosensor from the reading portion via the shared source pad 728 and a shared source line 724 (i.e., a conductive wire), as will be described later. After passing through the source electrode 708 and the drain electrode 710 of the active region, the current is transmitted to the electrical connection portion via an independent drain line 722 and a drain bonding pad 720. As will be described later, the drain bonding pad 720 of the electrical connection portion forms components and circuits of the reading device.

[0047] The top view of the detection unit 701 of the biosensor is shown in FIG. 8. The detection unit includes all the active regions 700 of the biosensor. In this illustration, the detection unit 701 of the biosensor includes four arrays 705 of active regions 700, and each array 705 includes five active regions 700 (as shown in FIG. 7), although other numbers of active regions may be employed. The arrays of active regions are disposed on the outer periphery of the gate conductor 714. In some embodiments, the gate conductor is at least one order of magnitude larger than the dimensions of the carbon material of the active regions on the sensor. In some embodiments, the gate conductor is at least two orders of magnitude larger than the dimensions of the carbon material of the active regions. In some embodiments, the gate conductor is at least three orders of magnitude larger than the dimensions of the carbon material of the active regions. The larger the dimension of the gate conductor relative to the carbon material of the active regions, the higher the sensitivity of the sensor for detecting voltage changes. The current is supplied to each of the 20 active regions 700 via the common source pad 728 and the common source line 724 through an electrical connection. The current is supplied to the gate conductor 714 through an electrical connection passing through the conductor gate bonding pad 730. Changes in the current flowing through the active regions, for example, an increase in resistance due to the presence of a target moiety in the sample applied to the sensor and the binding of the target moiety to the immobilized biological detection agent on the graphene surface, are transmitted to the electrical connection of the sensor by the independent drain line 722 and the drain bonding pad 720.

[0048] FIG. 9 shows an electrical circuit diagram showing the aspect of FIG. 8. In this figure, the graphene active region 906 is modeled as a resistor and receives current from the common source 924. In this figure, the independent drain line and drain pad are connected to the multiplexer 936. The multiplexer 936 functions as a selector, selectively extracts the signal, and transmits the signal to the electrical connection for further transmission to the detector 938 within the reader.

[0049] As shown in FIG. 8, the detection unit of the biosensor disclosed herein includes a plurality of active regions on a flat surface of a substrate, facilitating multiplex detection of different target moieties. As will be understood by those skilled in the art, the biosensor described herein may include at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 or more active regions.

[0050] Deposit the collection of detection agents onto the carbon or other conductive carbon material of each active unit. In one embodiment, the active units of the entire biosensor device each contain a collection of different detection agents, which are pathogen proteins or their peptides, or polynucleotides such as DNA, RNA, oligonucleotides, and modified nucleotide sequences. According to this embodiment, each of the different detection agents is arranged in a separate active region and immobilized on the carbon material deposited on the active region traversing the biosensor surface. In some embodiments, the plurality of detection agents are derived from one or more infectious agents selected from viruses, bacteria, or combinations thereof. The pathogen proteins or peptides suitable for immobilization on the graphene surface generally have a length between 5 and 100 amino acid residues, between 5 and 75 amino acid residues, between 5 and 50 amino acid residues, between 10 and 50 amino acid residues, between 15 and 50 amino acid residues, between 20 and 50 amino acid residues, between 25 and 50 amino acid residues, between 30 and 50 amino acid residues, between 35 and 50 amino acid residues, between 40 and 50 amino acid residues, between 45 and 50 amino acid residues, between 45 and 75 amino acid residues, or between 45 and 100 amino acid residues. The polynucleotides suitable for immobilization on the graphene surface generally have a length of 5 to 100 nucleic acid residues, 5 to 75 nucleic acid residues, 5 to 50 nucleic acid residues, 10 to 50 nucleic acid residues, 15 to 50 nucleic acid residues, 20 to 50 nucleic acid residues, 25 to 50 nucleic acid residues, 30 to 50 nucleic acid residues, 35 to 50 nucleic acid residues, 40 to 50 nucleic acid residues, 45 to 50 nucleic acid residues, 45 to 75 nucleic acid residues, or 45 to 100 nucleic acid residues.

[0051] In some embodiments, the plurality of detection agents are derived from one or more viruses including, but not limited to, SARS-CoV-2, influenza A, influenza B, human papillomavirus, Venezuelan equine encephalitis virus, vaccinia virus, Ebola virus, Lassa fever virus, Rift Valley fever virus, and combinations thereof. Further, the pathogen proteins or peptides can be derived from paramyxovirus, paramyxovirus, adenovirus, parvovirus, enterovirus, poxvirus, rotavirus, hemorrhagic fever virus (viruses of the Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae, Togaviridae families), hepatitis virus, parechovirus, human T lymphotropic virus, Epstein-Barr virus (herpesvirus).

[0052] In one embodiment, the plurality of detection agents are derived from coronaviruses. These include both human coronaviruses (e.g., SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1) and animal coronaviruses (e.g., feline CoV [serotypes I and II], porcine epidemic diarrhea CoV (PEDV), porcine PRCV, porcine TGEV, canine CCoC, rabbit RaCoV, etc.).

[0053] In some embodiments, the plurality of detection agents are derived from heterologous viruses to enable multiplex detection of corresponding antibodies of different species in the biological sample being tested. In some embodiments, the plurality of pathogen proteins or peptides are derived from the same virus (e.g., SARS-CoV-2) to comprehensively characterize the immune (i.e., antibody) response of a subject to infection by the virus. In some embodiments, the plurality of pathogen proteins or peptides are derived from SARS-CoV-2. In some embodiments, the plurality of pathogen proteins or peptides are derived from SARS-CoV-2 and influenza A.

[0054] In some embodiments, the plurality of detection agents are derived from one or more bacteria including, but not limited to, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Bacillus anthracis, Yersinia pestis, Francisella tularensis, Burkholderia pseudomallei, Burkholderia mallei, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter species, and combinations thereof.

[0055] In one embodiment, the collection of detection agents immobilized on the deposition material of the active region includes a collection of binding molecules. Binding molecules suitable for immobilization on the active region of a biosensor include any biological substance that functions as a binding partner or pair for a detectable target substance present or potentially present in a biological sample. In some embodiments, the collection of binding molecules are antibody-based molecules. Antibody-based molecules as used herein include, but are not limited to, intact antibodies, epitope-binding fragments of intact antibodies, and antibody derivatives.

[0056] An intact antibody includes an intact immunoglobulin containing two heavy chains and two light chains, each of these chains including a variable region (i.e., V H and V L ) and a constant region (i.e., C H and C L ). Epitope-binding fragments of antibodies that exhibit epitope-binding suitable for immobilization on the active region of a biosensor (including Fab and (Fab)2 fragments) are: (i) Fab' or Fab fragments, which are monovalent fragments containing one domain each of V L , V H , C L and C H ; (ii) F(ab')2 fragments, which are divalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) essentially V H and CH An Fd fragment consisting of 1 domain; (iv) essentially V L and V H An Fv fragment consisting of domains; (v) an scFv fragment consisting essentially of a VH or VL domain and also called a domain antibody; and (vii) an epitope-binding fragment that includes, but is not limited to, isolated complementarity-determining regions (CDRs). The epitope-binding fragment can include all 1, 2, 3, 4, 5, or 6 of the CDR domains of such an antibody. Antibody derivatives suitable for immobilization to the active region of a biosensor include at least one epitope-binding domain of the antibody and typically include molecules formed using recombinant techniques. One exemplary antibody derivative includes a single-chain Fv (scFv). The scFv is formed from the two domains of the Fv fragment, the V L region and the V H region.

[0057] In some embodiments, the binding molecule of the assembly is an antibody mimic. Exemplary antibody mimics for immobilization to the active region of a biosensor are readily known in the art and include, but are not limited to, affibodies, affilins, affimers, monobodies, and DARPINs.

[0058] Other binding materials suitable for immobilization on the carbon material of the active region of the biosensor described herein include, but are not limited to, carbohydrates, lipids, nucleic acids (DNA, RNA), aptamers, recombinant proteins, proteins bound to DNA or RNA, DNA bound to carbohydrates, hybrid molecules such as molecularly imprinted polymers. Also, biological binding substances include, for example, whole cells or cell fragments of mammalian cells, prokaryotic cells, parasites, viruses, nucleated or enucleated cells.

[0059] A collection of binding molecules immobilized on the carbon surface of the active region of a biosensor binds to one or more pathogenic proteins, including pathogenic proteins derived from infectious agents such as viruses, bacteria, toxins, and combinations thereof. Detection of pathogenic proteins in a sample via binding to the binding molecules indicates the presence of pathogens in the sample. In some embodiments, the collection of binding molecules on the biosensor binds to one or more pathogenic proteins from a single infectious agent. In some embodiments, the collection of binding molecules on the biosensor binds to pathogenic proteins of heterogeneous infectious agents to enable multiplex detection of various pathogens in a single sample (e.g., multiplex detection of viruses, bacteria, and / or toxins).

[0060] In some embodiments, the collection of binding molecules binds to one or more pathogenic proteins of a virus. Exemplary viruses include, but are not limited to, SARS-CoV-2, influenza A, influenza B, human papillomavirus, Venezuelan equine encephalitis virus, vaccinia virus, Ebola virus, Lassa fever virus, Rift Valley fever virus, and combinations thereof. In some embodiments, the collection of binding molecules binds to one or more pathogenic proteins of SARS-CoV-2. In some embodiments, the collection of binding molecules binds to one or more pathogenic proteins of SARS-CoV-2 and influenza A.

[0061] In some embodiments, the collection of binding molecules binds to one or more pathogenic proteins of bacteria. Exemplary bacteria include, but are not limited to, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Bacillus anthracis, Yersinia pestis, Francisella tularensis, Burkholderia pseudomallei, Burkholderia mallei, and combinations thereof.

[0062] In some embodiments, the collection of binding molecules binds to one or more toxins to facilitate detection of the presence of toxins in a biological sample. Exemplary toxins that can be detected using appropriate antibodies include, but are not limited to, ricin toxin, botulinum toxin A / B / E, staphylococcal enterotoxin B (SEB), abrin toxin, T-2 toxin, Bacillus anthracis LF toxin, Bacillus anthracis EF toxin, Bacillus anthracis PA toxin, and combinations thereof, but are not limited thereto.

[0063] In some embodiments, the collection of detectors immobilized on the deposition material (e.g., carbon material) of the active region includes a collection of binding molecules together with a plurality of pathogenic proteins, and the heterologous binding molecules and pathogenic proteins are spatially arranged in separate active regions on the biosensor surface. Combinations of binding molecules (suitable for detecting the presence of pathogenic proteins in a sample) and pathogenic proteins or peptides (suitable for detecting the presence of antibodies in a sample) enable comprehensive characterization of biological samples. For example, when the sample is a biological sample from a subject (e.g., human mucosa, blood, plasma sample), detecting the presence of pathogenic proteins in the sample indicates the presence of an active infection, while detecting the presence of antibodies in the sample indicates a previous infection and / or provides information regarding the immune response made against that infection.

[0064] In some embodiments, the binding molecules are lyophilized before or after immobilization onto the carbon material. In some embodiments, the binding molecules are lyophilized by the use of a lyoprotectant such as Hemsol®. In some embodiments, the lyophilization process includes a freeze-drying or sublimation process.

[0065] In some embodiments, an active region containing a detection agent (i.e., a pathogenic protein or its peptide or binding molecule) is contacted with a preservation solution to maintain the integrity and stability of the detection agent immobilized thereon. In some embodiments, the preservation solution contains at least one sugar with a large MW (>40,000 Da) and at least one other sugar with a small MW (<40,000 Da). When added to the active region containing the detection agent, the detection agent is dried to a final moisture content of about 5% to about 95%. The at least one large MW sugar and the at least one small MW sugar can be present in a single preservation agent solution or can be separate solutions.

[0066] In some embodiments, the preservation solution included in the deposited material comprises at least one membrane-permeable sugar, at least one membrane-permeable sugar, at least one antimicrobial agent, at least one antioxidant, optionally salts, adenosine, and optionally albumin. In some embodiments, the preservation solution comprises at least one membrane-permeable sugar (e.g., trehalose and glucose), at least one membrane-impermeable sugar (e.g., dextran such as dextran-70), at least one antimicrobial agent (e.g., sulfanilamide), at least one antioxidant (e.g., mannitol and vitamin E), optionally adenosine, and optionally albumin. In some embodiments, the preservation solution comprises at least one membrane-permeable sugar (e.g., trehalose and glucose), at least one membrane-impermeable sugar (e.g., dextran such as dextran-70), at least one antimicrobial agent (e.g., sulfanilamide), at least one antioxidant (e.g., mannitol and vitamin E), adenosine, albumin, salts (e.g., chloride salts such as KCl, NaCl, CaCl2, and covalent chloride salts of metals or non-metals such as titanium(IV) chloride or carbon tetrachloride), buffers (e.g., K2HPO4), and chelating agents (e.g., EDTA). Suitable preservation solutions and methods of preservation are described in U.S. Patent No. 8,628,960, U.S. Patent No. 9,642,353, and U.S. Patent No. 9,943,075, which are hereby incorporated by reference in their entirety, or are available from Hememics Biotechnologies, Inc. (Hemsol™).

[0067] According to the present disclosure, each of a plurality of detection agents (i.e., pathogen proteins or peptides and / or binding molecules) is immobilized on the deposited carbon material. In some embodiments, the immobilization is via covalent interaction. In some embodiments, the binding molecule and / or protein or peptide is bound via a hydrophobic linker, which is linked to the amino or carboxy terminus of the detection agent. In some embodiments, the hydrophobic linker is a peptide linker comprising two or more linker amino acids and one or more aromatic amino acid residues. In some embodiments, the two or more linker amino acid residues are selected from glycine, alanine, serine, and combinations thereof. In some embodiments, the hydrophobic linker comprises a polycyclic aromatic hydrocarbon. Suitable polycyclic aromatic hydrocarbon linkers include, but are not limited to, pyrene.

[0068] Other methods of immobilizing the detection agent to the carbon material of the active region are known in the art and suitable for use in accordance with the biosensors described herein. These include, but are not limited to, attachment via the (EDC / NHS) chemical reaction of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), attachment via electrostatic binding, or attachment via a 1-pyrenebutanoic acid succinimidyl ester (PASE) linker (see, e.g., Pena-Bahamonde et al., "Recent Advances in Graphene-based Biosensor Technology with Applications in Life Science", J. Nanobiotechnology 16:75 (2018), which is incorporated herein by reference in its entirety).

[0069] In one aspect, the biosensor of the present disclosure further includes an electromagnet disposed under the substrate of the biosensor. The biosensor further includes means for turning the electromagnet on / off. Schematic diagrams of a biosensor including an electromagnet and a continuous process for detecting a sample antigen or antibody using the electromagnet are shown in FIGS. 13A to 13C.

[0070] As described herein, the electromagnet function of the biosensor used in combination with a sample in which an antigen or antibody of the sample is bound to magnetic beads allows the user to control the diffusion rate of the antigen / antibody of the sample to the surface of the biosensor, ensuring rapid absorption of the antigen / antibody to the active region on the surface of the biosensor. Thereby, the antigen / antibody in the sample surely approaches the active region containing the detection agent on the sensor surface, and when the target substance is present in the sample, the binding between the detection agent and the target substance (i.e., the antigen / antibody of the sample) is promoted. This reduces false negatives that may occur when relying only on diffusion. This is a particularly important function when examining samples in which the target substance may be present at very low concentrations.

[0071] Another aspect of the present disclosure is directed to a biosensor system. In one embodiment, the biosensor system is useful for characterizing a subject's immune response to pathogen exposure. In another embodiment, the biosensor system is useful for characterizing the antigen profile of a pathogen. In any of the embodiments, the biosensor system includes an electronic reader, which includes a circuit for delivering a signal and a processing device for reading the signal. The biosensor system further includes a biosensor as described herein, operably connected to the electronic reader and configured to receive a signal delivered by the circuit. The electronic reader is configured to send a signal to the biosensor and obtain output impedance values before and after application of a sample to an array of active regions on the biosensor. The processing device is configured to compare the output impedance values to determine whether a binding event has occurred in one or more of the active regions, and if the active region contains a pathogenic protein / peptide, to characterize the subject's immune response to pathogen exposure, and if the active region contains a binding molecule (e.g., an antibody or antibody-based molecule), to determine the presence of a pathogen in the sample and characterize its antigen profile. In some embodiments, the system can perform both characterization of the subject's immune response and determination of the presence and antigen profile of a pathogen in the sample if all of the active regions of the biosensor contain a binding molecule.

[0072] As described below, in some embodiments, the biosensor of the biosensor system includes an electromagnet disposed under the substrate of the biosensor.

[0073] FIG. 10 is a schematic diagram of a biosensor system including an electronic reader unit 1038 for receiving a biosensor 1002. The electronic reader unit 1038 may include a slot 1040 for receiving an electrical connection portion 1044 of the biosensor 1002. By inserting the biosensor 1002, a circuit within the electronic reader unit 1038 is completed via an electrical connection portion 1044 including a plurality of electrical contacts (i.e., a drain bonding pad 1020, a shared source bonding pad 1028, and a gate bonding pad 1034). The electronic reader unit 1038 may further include a user interface 1042 for outputting information to a user. In some embodiments, the electronic reader unit 1038 may provide signals to a monitor or other display via a user interface (e.g., Bluetooth (or other communication means)) that does not exist on the actual reader unit 1038.

[0074] In some embodiments, the biosensor system described herein further includes a communication interface coupled to the electronic reader unit for transmitting data from the electronic reader unit, and a data management computing device configured to receive data from the electronic reader unit via the communication interface. According to this embodiment, the data management computing device includes a memory coupled to a processor that executes program instructions stored in the memory to geographically map immune response data to pathogen exposure and / or pathogen antigenicity profile data (i.e., the presence or evolution of various pathogen strains) based on the data received from the electronic reader unit.

[0075] FIG. 11 is a block diagram of a circuit 1046, such as a circuit board having an arithmetic component for supplying a signal to a biosensor 1002 and receiving a feedback signal for testing a sample disposed on the biosensor 1002. In an exemplary embodiment, the circuit 1046 may include a contact 1048 that may be an electrical contact for interacting with an electrical connection 1044 that includes electrical contacts 1020, 1028, 1034 of the biosensor 1002, and completes a circuit that includes an electronic reader 1038 and the biosensor 1002.

[0076] Also, the circuit 1046 may also include an arithmetic component, which may include, but is not limited to, a microcontroller 1050, one or more I / O devices 1052, a memory or other storage component 1054, one or more sensors 1056, a signal generator 1058, and a USB or other communication hub 1060. The arithmetic component is exemplary and may be replaced with other components for implementing the disclosed embodiments for inspecting a sample via the biosensor 1002.

[0077] The microcontroller or processor 1050 may be a processing device configured to monitor and control the components of the circuit 1046 to perform setup, testing, and output processing via the electronic reader 1038. The processor 1050 may execute programmed instructions stored in the memory 1054 for any number of functions described and illustrated herein. Other types of processors (s) may be used, but the processor 1050 may include, for example, one or more central processing units (CPUs) or a general-purpose processor having one or more processing cores.

[0078] Although some or all of the programmed instructions can be stored elsewhere, the memory 1054 of the electronic reading device 1038 stores these programmed instructions for the aspects of the present technology as described and illustrated herein. Various types of memory storage devices can be used for the memory 454, such as random access memory (RAM), read-only memory (ROM), hard disks, solid state drives (SSD), flash memory, or other computer-readable media on which reading and writing are performed by magnetic, optical, or other reading and writing systems connected to the processor(s) 450.

[0079] The I / O device(s) 1052 may include, for example, a communication interface 1042 for obtaining inputs and providing outputs to and from a user. The communication interface 1042 of the electronic reading unit 1038 operably connects and communicates at least between the electronic reading unit 1038 and an external computing device, and in some embodiments, these devices are at least partially connected by one or more communication networks or public cloud networks. By way of example only, the communication network can include a local area network (LAN) or a wide area network (WAN), and the public cloud network can include a WAN (e.g., the Internet). Other types or numbers of protocols or communication networks can be used, but the communication network and / or the public cloud network can use TCP / IP over Ethernet and industry standard protocols. The communication network and / or the public cloud network in this embodiment can employ any suitable interface mechanism and network communication technology, including, for example, an Ethernet-based packet data network (PDN).

[0080] Sensor 1056 may include a voltage divider, a resistance sensor, an impedance sensor, or other devices configured to determine values related to the electrical characteristics at one or more locations on biosensor 1002. Signal generator 1058 may be configured to generate an alternating electrical signal for supply to biosensor 1002. USB port 1060 may be a connection element for receiving and providing external data to the electronic reader unit.

[0081] The biosensors and biosensor systems described herein can be utilized to analyze a number of separate biological samples to detect the presence of pathogen proteins and / or the immune response of a subject to infection by a pathogenic organism or infectious agent. Accordingly, another aspect of the disclosure is directed to a method of characterizing the immune response of a subject to pathogen exposure. The method includes collecting a biological sample from the subject. Suitable samples are any biological fluid from the subject and include, but are not limited to, whole blood, blood serum, plasma, ascites, cyst fluid, pleural fluid, peritoneal fluid, cerebrospinal fluid, tears, urine, saliva, sputum, lymph fluid, synovial fluid, amniotic fluid, follicular fluid, respiratory fluid, intestinal fluid, and urogenital fluid.

[0082] The method further includes providing a biosensor system as described herein. Suitable biosensors include those that include a plurality of pathogen proteins and / or peptides immobilized in the active region of the sensor. The method further includes delivering an electrical signal to the biosensor via the circuitry of the electronic reader and determining the base resistance between two or more signal electrodes at each active site on the biosensor. In some embodiments, the method further includes applying a control solution to at least one active site of the biosensor and operably contacting the control solution with the carbon material and the gate electrode between the two signal electrodes. In some embodiments, the signal received from the gate electrode aids in determining the base resistance between two or more signal electrodes at each active site. In some embodiments, the method further includes applying a first control solution and a second control solution to two different regions of each active site on the biosensor. In some embodiments, each of the control solutions operably contacts the carbon material, at least two signal electrodes, and at least one gate electrode.

[0083] This method further includes applying a biological sample from a subject to a biosensor and identifying a change in the base resistance between two or more signal electrodes at each active site on the biosensor resulting from the application. In some embodiments, the biological sample is operably contacted with a carbon material and at least two signal electrodes. In some embodiments, the biological sample is operably contacted with a carbon material, at least two signal electrodes, and at least one gate electrode. The change in base resistance is an indicator that an antibody from the sample is bound to an immobilized pathogen protein or peptide. Based on the identified changes in base resistance between electrodes at various active sites on the biosensor, the immune response of the subject to pathogen exposure can be characterized. Alternatively, the change in resistance value is an indicator of the presence of a pathogen in the sample, based on the binding of the pathogen protein to an immobilized binder present at the active site of the sensor. The antigenic profile of the pathogen can be characterized based on the identified changes in base resistance between electrodes at various active sites on the biosensor.

[0084] Figure 12 provides an exemplary process 1200 for detecting a target site using a biosensor 1002 and an electronic reader 1038. The biosensor 1002 is manufactured to include a collection of differential detection agents as described above.

[0085] An important feature of the biosensor device described in this specification relates to the arrangement of the detection agent across the entire detection unit of the biosensor. In some embodiments, one or more active regions on the surface of the biosensor contain a collection of positive control detection agents. The positive control detection agents include a binding agent or protein / peptide known to bind to a component of the sample (either a substance naturally present in the sample or a substance introduced into the sample to facilitate positive control detection). In addition, one or more active regions on the biosensor surface contain a collection of negative control detection agents. The negative control detection agents include a binding agent or protein / peptide that should not bind to any substance that may be present in the sample. In addition, one or more active regions on the biosensor surface do not contain detection agents immobilized on the graphene surface. The presence of the active regions containing positive control detection agents, the active regions containing negative control detection agents, and the active regions without detection agents enables accurate detection and relative quantification of the presence of true target molecules (e.g., antibodies or antigens) in the test sample by detecting the differential signal between the active regions containing control detection agents and the active regions without detection agents.

[0086] Another aspect of the present disclosure is directed to a method for characterizing the antigen profile of a pathogen. The method includes collecting a sample containing the pathogen and providing a biosensor system as disclosed herein. Suitable biosensors include those that include a collection of heterologous binding molecules immobilized on the active region of the sensor. The method further includes delivering an electrical signal to the biosensor via the circuitry of the electronic reader and determining the base resistance between two or more electrodes at each active site on the biosensor. The method further includes applying a biological sample from a subject to the biosensor and identifying a change in the base resistance between two or more electrodes at each active site on the biosensor resulting from the application. The change in base resistance is an indicator that a pathogenic protein in the sample is bound to the immobilized binding molecule. The presence of the pathogen and / or the antigenic profile of the pathogen in the sample can be characterized based on the identified changes in the base resistance between the electrodes as various active sites on the biosensor.

[0087] In some embodiments, the biosensor of the system includes an electromagnet disposed under the substrate of the biosensor. According to this embodiment, the method of characterizing the immune response of a subject or the antigenic profile of a pathogen described herein further includes labeling a target substance present in the collected biological sample with a magnetic moiety. In some embodiments, the target substance in the sample is an antibody present in the sample. In some embodiments, the target substance in the sample is a pathogen protein and / or peptide. In some embodiments, the target substance in the sample is a mixture of both an antibody (produced by the host subject) and a pathogen protein (derived from an infectious agent that has infected or had infected the host subject). In any case, any of the antibodies and / or proteins that are the target substances are labeled with a magnetic moiety. Mix the biological sample containing the labeled antibody and / or protein in a viscous fluid to create a viscous biological sample mixture for application to the biosensor. Once the sample is applied, turn on the electromagnet to localize the labeled antibody and / or protein of the biological sample mixture to the active region of the substrate surface and promote the binding between the labeled antibody and the cognate pathogenic protein or peptide immobilized on the active surface, the binding between the labeled protein and the cognate binding molecule immobilized on the active surface, or both. After allowing sufficient time for the binding between the immobilized detector and the magnetically labeled target substance, turn off the electromagnet to release the unbound labeled antibody and / or protein before identifying the change in the base resistance between two or more electrodes at each active site on the biosensor.

[0088] In some embodiments, labeling a target substance (antibody or protein) in a sample involves contacting the biological sample with a magnetic moiety containing azide and irradiating the contacted sample with UV light to bind the magnetic moiety to the antibody within the biological sample. In some embodiments, the magnetic moiety is magnetic beads. Suitable magnetic beads include, but are not limited to, ferrous oxide magnetic beads. Suitable magnetic beads have a diameter of 2 nm to 100 μm. For example, suitable magnetic beads have a diameter of 2 nm, 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm.

[0089] The viscous fluid containing the magnetically labeled target substance coated on the biosensor surface can include any viscous fluid. Suitable viscous fluids include, but are not limited to, fluids composed of polyethylene glycol (PEG) or glycerin. In some embodiments, the viscous fluid includes about 20% PEG, about 25% PEG, about 30% PEG, about 35% PEG, about 40% PEG, about 45% PEG, about 50% PEG, about 55% PEG, about 60% PEG, about 65% PEG, about 70% PEG, about 75% PEG, about 80% PEG, about 85% PEG, or about 90% PEG. Any PEG known in the art is suitable for use according to this aspect of the present disclosure. In some embodiments, the PEG is PEG-400.

[0090] The following are non-limiting examples of the embodiments described herein.

[0091] (Embodiment 1) An antistatic substrate including a flat surface; At least one spatially defined active region on the flat surface of the antistatic substrate, each active region including a carbon material, a first signal electrode and a second signal electrode operably contacting the carbon material, and at least one gate electrode; A plurality of detection agents, wherein different detection agents are disposed in separate active regions and immobilized on the deposited carbon material of the active region; An electrical connection portion including a plurality of electrical contacts, each electrical contact configured to transmit an electrical signal between the first signal electrode and the second signal electrode of a single active region and at least one gate electrode; A biosensor comprising the above. (Embodiment 2) The carbon material is deposited on the flat surface of the antistatic substrate, The first signal electrode and the second signal electrode are located on the opposite side of the carbon material and covered with an insulating material, and At least one gate electrode is located on the upper surface of the insulating material of either the first signal electrode or the second signal electrode. The biosensor according to Embodiment 1. (Embodiment 3) The first signal electrode and the second signal electrode are deposited on the flat surface of the antistatic substrate, and optionally, a bottom insulating material is deposited on the flat surface of the antistatic substrate between the first signal electrode and the second signal electrode, The carbon material is deposited on the upper surfaces of the first signal electrode and the second signal electrode, and the first signal electrode and the second signal electrode are located on the opposite side of the carbon material, An upper insulating material is deposited on the upper surface of the carbon material on the opposite side of the carbon material and substantially above the first signal electrode and the second signal electrode, At least one gate electrode is located on the upper surface of the upper insulating material above either the first signal electrode or the second signal electrode. The biosensor according to Embodiment 1. (Embodiment 4) The biosensor according to any one of Embodiments 1 to 3, wherein the spatially defined array of active regions includes at least two active regions. (Embodiment 5) The biosensor according to any one of Embodiments 1 to 4, wherein the carbon material is graphene, a carbon nanotube, or a combination thereof. (Embodiment 6) The biosensor according to any one of Embodiments 1 to 5, wherein at least two signal electrodes and / or gate electrodes comprise a conductive metal selected from Ti, Cu, Ag, Ir, Pt, Au, or any combination thereof, or an oxide thereof. (Embodiment 7) The biosensor according to any one of Embodiments 1 to 5, wherein at least two signal electrodes and / or gate electrodes comprise a carbon-based conductive material selected from carbon nanotubes, graphene oxide, or any combination thereof. (Embodiment 8) The biosensor according to any one of Embodiments 1 to 7, wherein each active region further comprises a preservation solution. (Embodiment 9) The biosensor according to any one of Embodiments 1 to 8, wherein each detection agent is immobilized on the deposited carbon material via a hydrophobic linker, and the hydrophobic linker is linked to a binding molecule via the amino terminus or carboxy terminus of the detection agent. (Embodiment 10) The biosensor according to Embodiment 9, wherein the hydrophobic linker is a peptide linker comprising two or more linker amino acid residues and one or more aromatic amino acid residues. (Embodiment 11) The biosensor according to Embodiment 10, wherein the two or more linker amino acid residues are selected from glycine, alanine, serine, and combinations thereof. (Embodiment 12) The biosensor according to Embodiment 11, wherein the hydrophobic linker comprises a polycyclic aromatic hydrocarbon. (Embodiment 13) The biosensor according to any one of Embodiments 1 to 12, wherein the plurality of detection agents comprise a pathogen protein or a peptide thereof, a binding molecule capable of binding to the pathogen protein or a peptide thereof, a polynucleotide, or a combination thereof. (Embodiment 14) The biosensor further includes a collection of antibody mimetics, aptamers, DNA molecules, RNA molecules, modified oligonucleotides, or combinations thereof, wherein different members of the collection bind to different pathogen proteins, different members of the collection are disposed in distinct active regions not occupied by the detection agent, and the members of the collection are immobilized on the deposited carbon material of the active region, the biosensor according to Embodiment 13. (Embodiment 15) The pathogen is one or more infectious agents selected from the group consisting of viruses, bacteria, toxins, or combinations thereof, the biosensor according to Embodiment 13 or 14. (Embodiment 16) The pathogen is one or more viruses selected from the group consisting of SARS-CoV-2, influenza A, influenza B, human papillomavirus, Venezuelan equine encephalitis virus, vaccinia virus, Ebola virus, Lassa fever virus, Rift Valley fever virus, and combinations thereof, the biosensor according to Embodiment 15. (Embodiment 17) The pathogen is one or more bacteria selected from the group consisting of Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Bacillus anthracis, Yersinia pestis, Francisella tularensis, Burkholderia pseudomallei, Burkholderia mallei, and combinations thereof, the biosensor according to Embodiment 15. (Embodiment 18) The pathogen is one or more toxins selected from the group consisting of ricin toxin, botulinum toxin A / B / E, staphylococcal enterotoxin B (SEB), abrin toxin, T-2 toxin, Bacillus anthracis LF toxin, Bacillus anthracis EF toxin, Bacillus anthracis PA toxin, and combinations thereof, the biosensor according to Embodiment 15. (Embodiment 19) Each of the plurality of pathogen peptides has a length of 5 to 50 amino acid residues, the biosensor according to any one of Embodiments 13 to 18. (Embodiment 20) The binding molecule of the collection is the biosensor according to any one of Embodiments 13 to 19, which is an antibody-based molecule. (Embodiment 21) The antibody-based molecule is the biosensor according to Embodiment 20, which is selected from an antibody, its epitope-binding domain, an antibody derivative, an antibody mimetic, or a combination thereof. (Embodiment 22) The antibody mimetic is the biosensor according to Embodiment 21, which is selected from the group consisting of an affibody, an affilin, an affimer, a monobody, and a DARPIN. (Embodiment 23) The biosensor according to any one of Embodiments 1 to 22, further comprising an electromagnet disposed under the substrate of the biosensor. (Embodiment 24) The antistatic substrate is the biosensor according to any one of Embodiments 1 to 23, which comprises a single-layer antistatic polymer material. (Embodiment 25) The antistatic substrate is the biosensor according to any one of Embodiments 1 to 24, which comprises a polymer material with an antistatic additive. (Embodiment 26) The antistatic substrate is the biosensor according to any one of Embodiments 1 to 25, which comprises an antistatic printed circuit board.

[0092] (Embodiment 27) A biosensor system for characterizing the immune response of a subject to pathogen exposure, the biosensor system comprising: an electronic reading unit including a circuit for delivering a signal and a processing device for reading the signal; a biosensor according to any one of Embodiments 1 to 26, which is operably connected to the electronic reading unit via an electrical connection part of the biosensor and is configured to receive a signal delivered by the circuit; and The electronic reading unit is configured to deliver a signal to the biosensor and obtain output impedance values before and after application of a sample to an array of active regions on the biosensor, and the processing device is configured to compare the output impedance values to determine whether a binding event has occurred in one or more of the active regions and to characterize the subject's immune response to pathogen exposure. Biosensor system. (Embodiment 28) The biosensor is the biosensor system according to Embodiment 27, including an electromagnet disposed under the substrate of the biosensor. (Embodiment 29) A communication interface coupled to the electronic reading unit for transmitting data from the electronic reading unit; A data management arithmetic unit configured to receive data from the electronic reading unit via the communication interface, the data management arithmetic unit including a memory coupled to a processor configured to execute program instructions stored in the memory to geographically map immune response data to pathogen exposure based on the data received from the electronic reading unit. The biosensor system according to Embodiment 27 or 28, further including the above.

[0093] (Embodiment 30) Collecting a biological sample from a subject; Providing the biosensor system according to any one of Embodiments 27 to 29; Delivering an electrical signal to the biosensor via the circuit of the electronic reading unit; Determining a base resistance between a first signal electrode and a second signal electrode in each active region of the biosensor; Applying a biological sample from the subject to at least one active region of the biosensor and operably contacting the biological sample with a carbon material between the first signal electrode and the second signal electrode and at least one gate electrode in the at least one active region; Identifying a change in base resistance between a first signal electrode and a second signal electrode resulting from application of a biological sample to at least one active region; Characterizing an immune response of a subject to a pathogen, or an antigen profile of the pathogen, based on a change in base resistance between a first signal electrode and a second signal electrode in at least one active region; A method of characterizing an immune response of a subject to pathogen exposure, comprising: (Embodiment 31) The biosensor of the system includes an electromagnet disposed under a substrate of the biosensor, and the method comprises: After the collecting step, labeling an antibody present in the collected biological sample with a magnetic moiety; Mixing the biological sample containing the labeled antibody in a viscous fluid to create a viscous biological sample mixture for the applying step; During the applying step, turning on the electromagnet to localize the labeled antibody of the biological sample mixture to the active region of the substrate surface and promoting binding between the labeled antibody and a homologous detector immobilized on the active surface; Turning off the electromagnet before the identifying step to release unbound labeled antibody and / or protein; The method according to Embodiment 30, further comprising: (Embodiment 32) The labeling step comprises: Contacting the biological sample with a magnetic moiety containing azide; Irradiating the contacted sample with UV light to bind the magnetic moiety to an antibody in the biological sample; The method according to Embodiment 31, further comprising: (Embodiment 33) The method according to Embodiment 32, wherein the magnetic moiety is magnetic beads. (Embodiment 34) The method according to Embodiment 33, wherein the magnetic beads are ferrous oxide magnetic beads. (Embodiment 35) The method according to Embodiment 33, wherein the magnetic beads have a diameter of 2 nm to 100 μm. (Embodiment 36) The method according to Embodiment 31, wherein the viscous fluid comprises polyethylene glycol (PEG) or glycerin. (Embodiment 37) The method according to Embodiment 36, wherein the PEG is PEG-400. (Embodiment 38) The method according to Embodiment 36 or 37, wherein the viscous fluid comprises about 20% to about 90% PEG.

[0094] (Embodiment 39) An antistatic substrate including a flat surface; At least one spatially defined active region on the flat surface of the antistatic substrate, each active region including a carbon material, a first signal electrode, a second signal electrode, and a third signal electrode that are operably in contact with the carbon material, and at least one gate electrode; A plurality of detection agents, wherein different detection agents are disposed in separate active regions and are immobilized on the deposited carbon material of the active regions; An electrical connection portion including a plurality of electrical contacts, each electrical contact being configured to transmit an electrical signal between the first signal electrode and the second signal electrode of a single active region and at least one gate electrode; A biosensor including the above. (Embodiment 40) The carbon material is deposited on the flat surface of the antistatic substrate. The first signal electrode and the second signal electrode are located at both ends of the carbon material and are covered by an insulating material. The third signal electrode is located on the upper surface of the carbon material between the first signal electrode and the second signal electrode. At least one gate electrode is located on the upper surface of the insulating material of the first signal electrode. The biosensor according to Embodiment 39. (Embodiment 41) The first signal electrode, the second signal electrode, and the third signal electrode are deposited on the flat surface of the antistatic substrate, and optionally, a bottom insulating material is deposited on the flat surface of the antistatic substrate between the first signal electrode, the second signal electrode, and the third signal electrode. The carbon material is deposited on the upper surfaces of the first signal electrode, the second signal electrode, and the third signal electrode. The first signal electrode and the second signal electrode are located at both ends of the carbon material, and the third signal electrode is located between the first signal electrode and the second signal electrode. The upper insulating material is deposited on the upper surface of the carbon material substantially above the first signal electrode, the second signal electrode, and the third signal electrode, and At least one gate electrode is located on the upper surface of the upper insulating material above the first signal electrode. The biosensor according to Embodiment 39. (Embodiment 42) The spatially defined array of active regions includes at least two active regions, and the biosensor according to any one of Embodiments 39 to 41. (Embodiment 43) The biosensor according to any one of Embodiments 39 to 42, wherein the carbon material is graphene, a carbon nanotube, or a combination thereof. (Embodiment 44) The biosensor according to any one of Embodiments 39 to 43, wherein at least two signal electrodes and / or gate electrodes include a conductive metal selected from Ti, Cu, Ag, Ir, Pt, Au, or any combination thereof, or an oxide thereof. (Embodiment 45) The biosensor according to any one of Embodiments 39 to 43, wherein at least two signal electrodes and / or gate electrodes include a carbon-based conductive material selected from carbon nanotubes, graphene oxide, or any combination thereof. (Embodiment 46) The biosensor according to any one of Embodiments 39 to 45, wherein each active region further includes a preservation solution. (Embodiment 47) Each detection agent is immobilized on the deposited carbon material via a hydrophobic linker, and the hydrophobic linker is linked to a binding molecule via the amino terminus or carboxy terminus of the detection agent. The biosensor according to any one of Embodiments 39 to 46. (Embodiment 48) The hydrophobic linker is a peptide linker containing two or more linker amino acid residues and one or more aromatic amino acid residues. The biosensor according to Embodiment 47. (Embodiment 49) The two or more linker amino acid residues are selected from glycine, alanine, serine, and combinations thereof. The biosensor according to Embodiment 48. (Embodiment 50) The hydrophobic linker contains a polycyclic aromatic hydrocarbon. The biosensor according to Embodiment 49. (Embodiment 51) The plurality of detection agents include a pathogen protein or a peptide thereof, a binding molecule capable of binding to the pathogen protein or a peptide thereof, a polynucleotide, or a combination thereof. The biosensor according to any one of Embodiments 39 to 50. (Embodiment 52) The biosensor further includes a collection of antibody mimics, aptamers, DNA molecules, RNA molecules, modified oligonucleotides, or combinations thereof. Heterogeneous members of the collection bind to heterogeneous pathogen proteins. Heterogeneous members of the collection are arranged in distinct active regions not occupied by the detection agent. The members of the collection are immobilized on the deposited carbon material of the active region. The biosensor according to Embodiment 51. (Embodiment 53) The pathogen is one or more infectious agents selected from viruses, bacteria, toxins, or combinations thereof. The biosensor according to Embodiment 51 or 52. (Embodiment 54) The biosensor according to embodiment 53, wherein the pathogen is one or more viruses selected from SARS-CoV-2, influenza A, influenza B, human papillomavirus, Venezuelan equine encephalitis virus, vaccinia virus, Ebola virus, Lassa fever virus, Rift Valley fever virus, and combinations thereof. (Embodiment 55) The biosensor according to embodiment 53, wherein the pathogen is one or more bacteria selected from the group consisting of Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Bacillus anthracis, Yersinia pestis, Francisella tularensis, Burkholderia pseudomallei, Burkholderia mallei, and combinations thereof. (Embodiment 56) The biosensor according to embodiment 53, wherein the pathogen is one or more toxins selected from ricin toxin, botulinum toxin A / B / E, staphylococcal enterotoxin B (SEB), abrin toxin, T-2 toxin, Bacillus anthracis LF toxin, Bacillus anthracis EF toxin, Bacillus anthracis PA toxin, and combinations thereof. (Embodiment 57) The biosensor according to any one of embodiments 51 to 56, wherein each of the plurality of pathogen peptides has a length of 5 to 50 amino acid residues. (Embodiment 58) The biosensor according to any one of embodiments 51 to 57, wherein the binding molecule of the collection is an antibody-based molecule. (Embodiment 59) The biosensor according to embodiment 58, wherein the antibody-based molecule is selected from an antibody, its epitope-binding domain, an antibody derivative, an antibody mimic, or a combination thereof. (Embodiment 60) The biosensor according to embodiment 59, wherein the antibody mimic is selected from the group consisting of an affibody, an affilin, an affimer, a monobody, and a DARPIN. (Embodiment 61) The biosensor according to any one of embodiments 39 to 60, further comprising an electromagnet disposed under the substrate of the biosensor. (Embodiment 62) The antistatic substrate is the biosensor according to any one of Embodiments 39 to 61, including a single-layer antistatic polymer material. (Embodiment 63) The antistatic substrate is the biosensor according to any one of Embodiments 39 to 62, including a polymer material with an antistatic additive. (Embodiment 64) The antistatic substrate is the biosensor according to any one of Embodiments 39 to 63, including an antistatic printed circuit board.

[0095] (Embodiment 65) A biosensor system for characterizing a subject's immune response to pathogen exposure, the biosensor system comprising: an electronic reading unit including a circuit for delivering a signal and a processing device for reading the signal; a biosensor according to any one of Embodiments 39 to 64, operably connected to the electronic reading unit via an electrical connection part of the biosensor and configured to receive a signal delivered by the circuit; comprising The electronic reading unit is configured to deliver a signal to the biosensor and obtain output impedance values before and after application of a sample to an array of active regions on the biosensor, and the processing device is configured to compare the output impedance values to determine whether a binding event has occurred in one or more active regions and characterize the subject's immune response to pathogen exposure. The biosensor system. (Embodiment 66) The biosensor is the biosensor system according to Embodiment 65, including an electromagnet disposed under the substrate of the biosensor. (Embodiment 67) a communication interface connected to the electronic reading unit for transmitting data from the electronic reading unit; A data management arithmetic unit configured to receive data from an electronic reading unit via a communication interface, the data management arithmetic unit including a memory coupled to a processor configured to execute program instructions stored in the memory to geographically map immune response data against pathogen exposure based on the data received from the electronic reading unit, and the data management arithmetic unit The biosensor system according to embodiment 65 or 66, further comprising.

[0096] (Embodiment 68) Collecting a biological sample from a subject; Providing the biosensor system according to any one of embodiments 65 to 67; Delivering an electrical signal to the biosensor via a circuit of the electronic reading unit; Applying a control solution to at least one active region, the control solution being operably contacted with a carbon material between a first signal electrode and a third signal electrode in at least one active region; Determining a base resistance between a second signal electrode and a third signal electrode in at least one active region; Applying a biological sample from a subject to at least one active region, the biological sample being operably contacted with a carbon material between a second signal electrode and a third signal electrode; Identifying a change in base resistance between a second signal electrode and a third signal electrode resulting from applying a biological sample to at least one active region; Characterizing an immune response of the subject against a pathogen or an antigen profile of the pathogen based on a change in base resistance between a second signal electrode and a third signal electrode in at least one active region; and A method for characterizing an immune response of a subject against pathogen exposure, comprising. (Embodiment 69) The biosensor of the system includes an electromagnet disposed under the substrate of the biosensor, and the method includes: After the collection step, a step of labeling antibodies present in the collected biological sample with a magnetic moiety; A step of mixing the biological sample containing the labeled antibody in a viscous fluid to create a viscous biological sample mixture for the application step; During the application step, turning on an electromagnet to localize the labeled antibody of the biological sample mixture to the active region of the substrate surface and promoting the binding between the labeled antibody and the homologous detector immobilized on the active surface; Before the identification step, turning off the electromagnet to release unbound labeled antibody and / or protein; The method according to embodiment 68, further comprising. (Embodiment 70) The labeling step comprises: Contacting the biological sample with a magnetic moiety containing azide; Irradiating the contacted sample with UV light to bind the magnetic moiety to the antibody in the biological sample; The method according to embodiment 69, comprising. (Embodiment 71) The method according to embodiment 70, wherein the magnetic moiety is magnetic beads. (Embodiment 72) The method according to embodiment 71, wherein the magnetic beads are ferrous oxide magnetic beads. (Embodiment 73) The method according to embodiment 71, wherein the magnetic beads have a diameter of 2 nm to 100 μm. (Embodiment 74) The method according to embodiment 69, wherein the viscous fluid comprises polyethylene glycol (PEG) or glycerin. (Embodiment 75) The method according to embodiment 74, wherein the PEG is PEG-400. (Embodiment 76) The method according to embodiment 74 or 75, wherein the viscous fluid comprises about 20% to about 90% PEG.

[0097] (Embodiment 77) An antistatic substrate including a flat surface; At least one spatially defined active region on the flat surface of the antistatic substrate, each active region comprising a carbon material, a first signal electrode, a second signal electrode, a third signal electrode and a fourth signal electrode operably contacting the carbon material, and at least a first gate electrode and a second gate electrode; A plurality of detection agents, wherein different detection agents are arranged in separate active regions and immobilized on the deposited carbon material of the active regions; An electrical connection part including a plurality of electrical contacts, each electrical contact being configured to transmit an electrical signal between the first signal electrode, the second signal electrode, the third signal electrode and the fourth signal electrode of a single active region and at least the first gate electrode and the second gate electrode, and the electrical connection part; A biosensor comprising. (Embodiment 78) The carbon material is deposited on the flat surface of the antistatic substrate, The first signal electrode and the second signal electrode are located at both ends of the carbon material and covered by an insulating material The third signal electrode is located on the upper surface of the carbon material between the first signal electrode and the fourth signal electrode and surrounded by an insulating material, The fourth signal electrode is located on the upper surface of the carbon material between the second signal electrode and the second signal electrode and surrounded by an insulating material, At least the first gate electrode is located on the upper surface of the insulating material of the first signal electrode, and At least the second gate electrode is located on the upper surface of the insulating material of the second signal electrode The biosensor according to Embodiment 77. (Embodiment 79) The first signal electrode, the second signal electrode, the third signal electrode and the fourth signal electrode are deposited on the flat surface of the antistatic substrate, and optionally, together with a bottom insulating material deposited on the flat surface of the antistatic substrate, between the first signal electrode, the second signal electrode, the third signal electrode and the fourth signal electrode, The carbon material is deposited on the upper surfaces of the first signal electrode, the second signal electrode, the third signal electrode, and the fourth signal electrode. The first signal electrode and the second signal electrode are located at both ends of the carbon material. The third signal electrode is located between the first signal electrode and the fourth signal electrode. The fourth signal electrode is located between the second signal electrode and the third signal electrode. The upper insulating material is deposited on the upper surface of the carbon material substantially above the first signal electrode, the second signal electrode, the third signal electrode, and the fourth signal electrode. At least the first gate electrode is located substantially above the upper insulating material of the first signal electrode, and At least the second gate electrode is located substantially above the upper insulating material of the second signal electrode. The biosensor according to Embodiment 77. (Embodiment 80) The spatially defined array of active regions includes at least two active regions, and the biosensor according to any one of Embodiments 77 to 79. (Embodiment 81) The carbon material is graphene, a carbon nanotube, or a combination thereof, and the biosensor according to any one of Embodiments 77 to 80. (Embodiment 82) At least two signal electrodes and / or gate electrodes include a conductive metal selected from Ti, Cu, Ag, Ir, Pt, Au, or any combination thereof, or an oxide thereof, and the biosensor according to any one of Embodiments 77 to 81. (Embodiment 83) At least two signal electrodes and / or gate electrodes include a carbon-based conductive material selected from carbon nanotubes, graphene oxide, or any combination thereof, and the biosensor according to any one of Embodiments 77 to 81. (Embodiment 84) Each active region further includes a preservation solution, and the biosensor according to any one of Embodiments 77 to 83. (Embodiment 85) Each detector is immobilized on the deposited carbon material via a hydrophobic linker, and the hydrophobic linker is linked to a binding molecule via the amino or carboxy terminus of the detector, the biosensor according to any one of embodiments 77 to 84. (Embodiment 86) The hydrophobic linker is a peptide linker containing two or more linker amino acid residues and one or more aromatic amino acid residues, the biosensor according to embodiment 85. (Embodiment 87) The two or more linker amino acid residues are selected from glycine, alanine, serine, and combinations thereof, the biosensor according to embodiment 86. (Embodiment 88) The hydrophobic linker contains a polycyclic aromatic hydrocarbon, the biosensor according to embodiment 87. (Embodiment 89) The plurality of detectors includes a pathogen protein or a peptide thereof, a binding molecule capable of binding to the pathogen protein or a peptide thereof, a polynucleotide, or a combination thereof, the biosensor according to any one of embodiments 77 to 88. (Embodiment 90) The biosensor further includes a collection of antibody mimics, aptamers, DNA molecules, RNA molecules, modified oligonucleotides, or combinations thereof, wherein different members of the collection bind to different pathogen proteins, different members of the collection are located in separate active regions not occupied by the detector, and the members of the collection are immobilized on the deposited carbon material of the active region, the biosensor according to embodiment 89. (Embodiment 91) The pathogen is one or more infectious agents selected from viruses, bacteria, toxins, or combinations thereof, the biosensor according to embodiment 89 or 90. (Embodiment 92) The biosensor according to embodiment 91, wherein the pathogen is one or more viruses selected from SARS-CoV-2, influenza A, influenza B, human papillomavirus, Venezuelan equine encephalitis virus, vaccinia virus, Ebola virus, Lassa fever virus, Rift Valley fever virus, and combinations thereof. (Embodiment 93) The biosensor according to embodiment 91, wherein the pathogen is one or more bacteria selected from the group consisting of Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Bacillus anthracis, Yersinia pestis, Francisella tularensis, Burkholderia pseudomallei, Burkholderia mallei, and combinations thereof. (Embodiment 94) The biosensor according to embodiment 91, wherein the pathogen is one or more toxins selected from ricin toxin, botulinum toxin A / B / E, staphylococcal enterotoxin B (SEB), abrin toxin, T-2 toxin, Bacillus anthracis LF toxin, Bacillus anthracis EF toxin, Bacillus anthracis PA toxin, and combinations thereof. (Embodiment 95) The biosensor according to any one of embodiments 90 to 94, wherein each of the plurality of pathogen peptides has a length of 5 to 50 amino acid residues. (Embodiment 96) The biosensor according to any one of embodiments 90 to 95, wherein the binding molecule of the collection is an antibody-based molecule. (Embodiment 97) The biosensor according to embodiment 96, wherein the antibody-based molecule is selected from an antibody, its epitope-binding domain, an antibody derivative, an antibody mimetic, or a combination thereof. (Embodiment 98) The biosensor according to embodiment 97, wherein the antibody mimetic is selected from the group consisting of an affibody, an affilin, an affimer, a monobody, and a DARPIN. (Embodiment 99) The biosensor according to any one of embodiments 77 to 98, further comprising an electromagnet disposed under the substrate of the biosensor. (Embodiment 100) The antistatic substrate is a biosensor according to any one of Embodiments 77 to 99, including a single-layer antistatic polymer material. (Embodiment 101) The antistatic substrate is a biosensor according to any one of Embodiments 77 to 100, including a polymer material with an antistatic additive. (Embodiment 102) The antistatic substrate is a biosensor according to any one of Embodiments 77 to 101, including an antistatic printed circuit board.

[0098] (Embodiment 103) A biosensor system for characterizing a subject's immune response to pathogen exposure, the biosensor system comprising: an electronic reading unit including a circuit for delivering a signal and a processing device for reading the signal; a biosensor according to any one of Embodiments 77 to 102, operably connected to the electronic reading unit via an electrical connection part of the biosensor and configured to receive a signal delivered by the circuit; comprising The electronic reading unit is configured to deliver a signal to the biosensor and obtain output impedance values before and after application of a sample to an array of active regions on the biosensor, and the processing device is configured to compare the output impedance values to determine whether a binding event has occurred in one or more of the active regions and characterize the subject's immune response to pathogen exposure. A biosensor system. (Embodiment 104) The biosensor is a biosensor system according to Embodiment 103, including an electromagnet disposed under the substrate of the biosensor. (Embodiment 105) A communication interface connected to the electronic reading unit for transmitting data from the electronic reading unit; A data management arithmetic unit configured to receive data from an electronic reading unit via a communication interface, the data management arithmetic unit including a memory coupled to a processor configured to execute program instructions stored in the memory to geographically map immune response data against pathogen exposure based on the data received from the electronic reading unit, and the data management arithmetic unit The biosensor system according to Embodiment 103 or 104, further including

[0099] (Embodiment 106) Collecting a biological sample from a subject; Providing the biosensor system according to any one of Embodiments 103 to 105; Delivering an electrical signal to the biosensor via a circuit of the electronic reading unit; Applying a first control solution to at least one active region, the first control solution being operably contacted with a carbon material between a first signal electrode and a third signal electrode in the at least one active region; Applying a second control solution to at least one active region, the second control solution being operably contacted with a carbon material between a second signal electrode and a fourth signal electrode in the at least one active region; Determining a base resistance between a third signal electrode and a fourth signal electrode in at least one active region; Applying a biological sample from a subject to at least one active region, the biological sample being operably contacted with a carbon material between a second signal electrode and a third signal electrode; Identifying a change in base resistance between a second signal electrode and a third signal electrode resulting from applying the biological sample to at least one active region; Characterizing an immune response of the subject against a pathogen or an antigen profile of the pathogen based on a change in base resistance between a second signal electrode and a third signal electrode in at least one active region, and A method for characterizing an immune response of a subject against pathogen exposure, including (Embodiment 107) The biosensor of the system includes an electromagnet disposed under the substrate of the biosensor, and the method includes: after the collection step, labeling the antibodies present in the collected biological sample with a magnetic moiety; mixing the biological sample containing the labeled antibodies in a viscous fluid to create a viscous biological sample mixture for the application step; during the application step, turning on the electromagnet to localize the labeled antibodies of the biological sample mixture to the active region of the substrate surface and promoting the binding between the labeled antibodies and the homologous detector immobilized on the active surface; before the identification step, turning off the electromagnet to release unbound labeled antibodies and / or proteins; The method according to Embodiment 106, further comprising. (Embodiment 108) The labeling step includes: contacting the biological sample with a magnetic moiety containing azide; irradiating the contacted sample with UV light to bind the magnetic moiety to the antibodies in the biological sample; The method according to Embodiment 107, further comprising. (Embodiment 109) The method according to Embodiment 108, wherein the magnetic moiety is magnetic beads. (Embodiment 110) The method according to Embodiment 109, wherein the magnetic beads are ferrous oxide magnetic beads. (Embodiment 111) The method according to Embodiment 109, wherein the magnetic beads have a diameter of 2 nm to 100 μm. (Embodiment 112) The method according to Embodiment 107, wherein the viscous fluid contains polyethylene glycol (PEG) or glycerin. (Embodiment 113) The method according to Embodiment 112, wherein the PEG is PEG-400. (Embodiment 114) A viscous fluid, a method according to Embodiment 112 or 113, comprising about 20% to about 90% PEG.

[0100] (Embodiment 115) A method for manufacturing a biosensor according to any one of Embodiments 1-26, 39-64, or 77-102, the method comprising depositing a graphene material on the surface of the biosensor using a contour ablation method.

[0101] (Embodiment 116) Obtaining a substrate having at least two layers, wherein there is a height difference z between at least two layers; Transferring graphene onto the substrate, wherein the graphene breaks along at least two layers due to the height difference z; Washing the biosensor to remove excess graphene; Optionally, adding an additional layer to the biosensor, A method for manufacturing a biosensor, comprising. (Embodiment 117) The height difference z between at least two layers is from about 50 nanometers to about 3 millimeters, the method according to Embodiment 116. (Embodiment 118) The substrate comprises at least three layers, the method according to Embodiment 116 or 117. (Embodiment 119) The substrate comprises at least four layers, the method according to Embodiment 116 or 117. (Embodiment 120) The additional layer comprises an insulating material, or an electrode material, or both, the method according to any one of Embodiments 116-119.

Examples

[0102] The following examples are intended to illustrate embodiments of the present disclosure and are in no way intended to limit its scope.

[0103] (Example 1: Comparison of Biological Attachment and Target Incubation on Graphene-Silicon Sensor Chips and Graphene-Polyethylene Terephthalate (PET) Sensor Chips) To utilize these graphene surfaces as a diagnostic platform, first, the antibody or protein of interest is attached. This process (referred to herein as "attachment") is initiated by immobilizing pyrene molecules on the graphene surface. Pyrene and graphene form strong interactions through π-π stacking of sp 2 carbons within their respective ring structures. Subsequently, as described by Goldsmith et al., "Digital Biosensing by Foundry-Fabricated Graphene Sensors", Sci. Rep. 9:434 (2019), each circuit undergoes a 7-step attachment process. This process involves activating pyrene and then covalently bonding the protein or antibody of interest to pyrene. The surface is then blocked with PEG-amine and ethanolamine respectively to stop the reaction. After attachment, the chip is washed prior to target incubation. In this part of the experiment, the chip is calibrated (an important step for data normalization) by adding PBS buffer, and then the antibody or protein of interest is added.

[0104] (Graphene-Silicon Chip (Comparison Measuring Instrument)) The graphene-silicon chip (graphene chip) is an independent graphene circuit purchased from Graphenea (Cambridge, Massachusetts). Each chip contains 12 circuits that can be used for multiplexing.

[0105] Attachment: The process of attaching BSA or Spike 1 protein to the graphene chip followed the seven-step process described above. A graph of the circuit resistance through the seven-step attachment process is shown in Figure 14. The small jumps in the data occur when buffer is added to maintain surface saturation and can be considered negligible when observing the entire protein attachment process. Unfortunately, this method cannot quantify the degree of surface attachment of proteins and antibodies. However, multiplexing is possible with these chips, and multiple positive and negative controls can be attached in a single operation. Furthermore, the graphene chips show a consistently low standard deviation between circuits in the same experiment (run), giving confidence in the overall reliability of these circuits.

[0106] Target Incubation on Graphene Chip: Generally, graphene chips are overall low-noise and have an especially excellent signal / noise ratio during target incubation experiments. The addition of BSA antibody (1 mg / mL) to the circuit was used to test specific binding to the attached BSA. The addition of BSA antibody (1 mg / mL) to the circuit was used to test non-specific binding to the attached Spike 1 protein. The blank control standard is an unlabeled circuit to which BSA antibody (1 mg / mL) was added. The results summarized in Table 1 below show the specific binding, non-specific binding, and blank control standard experiments performed on one chip by multiplexing. This data is normalized using the addition of PBS at the -300 s time point to remove the influence of drift and accurately compare the data. Selective binding was observed from this test.

[0107]

Table 1

[0108] (General Graphene PET - Graphene Sheet) A general graphene PET-graphene sheet (PET-graphene) was purchased as a 4.5-inch × 3.8-inch PET plastic sheet coated with a single layer of graphene. These sheets were cut into rectangles of 0.7 inches × 0.3 inches, and silver paint was spotted to establish the electrical connection of the entire circuit.

[0109] Adhesion: In experiments using a simplified three-step adhesion process (omitting the blocking and reaction termination steps), the presence of the adhered antibody (BSA or Pseudomonas) or protein was confirmed using AFM and SEM on a silicon wafer. This three-step process was used for PET-graphene, and the presence of protein was confirmed using Coomassie blue staining. From the results of this adhesion process, the binding proteins used in the following target incubation experiments were obtained.

[0110] Target Incubation on PET-Graphene Chips: Target incubation in PET-graphene initially showed promising results (see the results in Table 2 below). A dose-response relationship existed in the specific binding samples. Specific binding (2-fold) to the adhered BSA antibody was tested by adding BSA protein (14 μg / mL) to the circuit. Specific binding (1-fold) to the adhered BSA antibody was tested by adding BSA protein (7 μg / mL) to the circuit. For non-specific binding to the adhered Pseudomonas antibody, BSA protein (7 μg / mL) was added to the circuit. The blank control standard was adding BSA antibody (1 mg / mL) to the circuit with nothing adhered (i.e., unlabeled circuit).

[0111] [Table 2]

[0112] (Example 2: Electromagnetic Substrate Enhancement Target) Summary: As described herein, the biosensors of the present disclosure can include an electromagnet located beneath the biosensor surface. This electromagnet includes an on / off switch, enabling the user to control the diffusion of magnetically labeled sample components to the biosensor surface. Generally, the method of use involves binding antibodies and / or proteins in a biological sample to magnetic moieties, such as magnetic beads, using UV or chemical activation as known in the art. The labeled sample is mixed with a high-density fluid, the magnetic bead-protein complex is suspended in solution, and the mixture is added to the biosensor. FIG. 13A is a schematic diagram showing the magnetically labeled target components of a sample applied to the biosensor when the electromagnet disposed beneath the sensor surface is off.

[0113] When the electromagnet is turned on, as shown in FIG. 13B, the magnetically labeled proteins and antibodies in the sample approach the surface and the active region containing the detector. Thereby, the target substance in the sample binds to its cognate binding partner, which can be any of an antibody, an antibody-based molecule, or a protein / peptide immobilized on the sensor surface.

[0114] After a period of time has elapsed to allow the binding interaction to occur, the electromagnet is turned off, and the unbound magnetically labeled target substances are released into the solution, as shown in FIG. 13C below. The target substances specifically bound to the immobilized binding partners on the surface remain bound to the surface, and the change in current between circuits due to the binding of the target substances specifically bound to the immobilized binding partners on the surface is measured.

[0115] Experimental analysis: A blank circuit (die number 56 of GG2) was rehydrated with 2.5 μL of 80% PEG-400 and 0.01X PBS. PEG-400 gives viscosity to the solution, limits the evaporation of water (and thus signal drift), and enables the investigation of binding kinetics (by retarding the diffusion of particles). Readings were obtained from two circuits. Two sets of ground magnets were placed under the wafer, and the die to be analyzed was placed at the center of the magnets.

[0116] After the start of the experiment, a sharp decrease in the signal was observed at the 0 - 200 second mark, which is thought to be due to signal stabilization (Figure 15, leftmost box). The baseline signal was recorded at the 200 - 600 second mark, and the average gradient was 0.038. At the 594 second mark, 2.5 μL of 2 nm ferrous oxide magnetic beads bound to BSA in 0.01X PBS was added to give final concentrations of 40% PEG - 400, approximately 0.12 mg / mL BSA, and 0.01X PBS. Due to this addition, the signal increased at the 600 - 800 second mark, and its gradient was 1.09 (see the middle box in Figure 15). This transition stabilized at the 800 second mark and baselined with a ΔV of approximately 200 mV up to the 1200 second mark (gradient = 0.0057). At the 1188 second mark, a second addition of 2.5 μL of 2 nm ferrous oxide magnetic beads bound to BSA in 0.01X PBS was made to give final concentrations of 27% PEG - 400, approximately 0.12 mg / mL BSA, and 0.01X PBS. Due to this addition, as shown in Figure 15 (see the rightmost box), a voltage increase of approximately 200 mV was seen over 200 seconds (gradient = 0.948). Subsequently, the sample baselined over the remaining 400 seconds (gradient = 0.154).

[0117] (Example 3: Biosensor with added graphene by the contour ablation method) Graphene, a two-dimensional material composed of carbon atoms, has attracted attention for its potential application in biosensors due to its unique electrical properties. However, despite its promising properties, the low reproducibility of graphene-based biosensors has hindered their widespread use. This problem is caused by the difficulty of obtaining a clean graphene layer due to residues of photoresists (such as PMMA) that affect the performance of graphene, etching processes that affect the physical and chemical properties of graphene, and variations in the coating of bioreceptors on graphene. All of these problems affect the high reliability and accurate sensor performance. Due to these challenges, it has been difficult for graphene-based biosensors to fully realize their potential as commercial products, and there have been limitations to their widespread adoption. Therefore, a unique contour ablation method has been developed for synthesizing and manufacturing graphene-based biosensors. Graphene (or other thin materials) is transferred onto a pre-patterned substrate. In this transfer process, graphene is broken along pre-patterned lines and adheres to specific parts of the substrate but not to others. This represents a major breakthrough in the commercialization of graphene-based biosensors, meaning that several steps in sensor manufacturing can be omitted, and it has the additional effect of assisting in maintaining sensitivity by reducing graphene contamination.

[0118] Figures 16A - 16F and Figures 17A - 17F show two modified examples of the contour ablation method. First, a substrate such as a PCB is manufactured using various layers. The substrate includes signal electrodes and / or insulating materials, and the height difference z between these layers ranges from 50 nanometers to 3 millimeters (Figures 16A and 17A). In the second step, graphene grown on a donor substrate is transferred onto this PCB substrate (Figures 16B and 17B). At this time, the graphene is broken due to the height difference z on the substrate, and a graphene pattern with defined graphene islands is shown (Figures 16C and 17C). Excess graphene can be washed away, leaving only the graphene bound to specific regions of the biosensor, such as on the insulating material placed on the PCB substrate (Figures 16D and 17D). Finally, additional insulating materials and electrodes can be printed thereon to complete the biosensor (Figures 16E and 17E without samples, and Figures 16F and 17F with samples on the biosensor).

[0119] To investigate whether the biosensor fabricated by the contour ablation method functions, a PCB substrate chip with 24 separate biosensors was fabricated using the contour ablation method. The bioreceptor molecules described in this specification were attached onto graphene and freeze - dried using Hemsol (trademark). An application example of such a sensor was the detection of lysine. The biosensor with patterned graphene was coated with appropriate insulating ink and conductive ink, and then functionalized with an aptamer for lysine toxin. Lysine toxoid was spiked into mud mixed with various concentrations of PBS buffer. Nonspecific control standards spiked in the mud were streptococcal enterotoxin B (SEB) and bovine serum albumin (BSA). The response of the biosensor is shown in Figure 17. The lowest detection concentration was 0.28 pM (0.01 ng / mL), and the responses to off - target SEB and BSA samples were very low.

[0120] The second use was the detection of porcine reproductive and respiratory syndrome virus (PRRSV). cDNA complementary to the viral RNA was functionalized on the biosensor, and the biosensor was fabricated as described above, except that it was lyophilized. The viral RNA was spiked in a buffer containing lysis buffer and a known negative porcine saliva sample. Functionalized cDNA and mismatched influenza virus RNA were used as negative control standards. The response of the biosensor is shown in Figure 18. There is a dramatic difference between the complementary RNA and the mismatched RNA, and the complementary RNA has a minimum detectable concentration of 0.1 pM.

[0121] Finally, the functionality of the graphene-based biosensor was tested by measuring the distribution of the Dirac points. The biosensor was fabricated as described above. The results showed that the distribution of the Dirac points follows a Gaussian distribution with a very narrow spread of about 35 mV and a median of 80 mV. This indicates that most of the Dirac points are concentrated around the median of 80 mV. Furthermore, the standard deviation of the Dirac points is in the range of 50 mV to 120 mV. In contrast, the standard published process for manufacturing graphene-based biosensors shows a much wider distribution of Dirac points, from 250 to 1500 mV.

[0122] Although the preferred embodiments have been described and explained in detail herein, it will be apparent to those skilled in the relevant technical fields that various changes, additions, substitutions, etc. can be made without departing from the true spirit of the present invention, and thus these are considered to be within the scope of the present invention as defined in the claims described below.

Claims

1. An antistatic substrate (120) including a flat surface; An active region (100) on a flat surface of an antistatic substrate, each active region comprising a carbon material (130), a first signal electrode (140) and a second signal electrode (141) operably in contact with the carbon material, and at least one gate electrode (160); Multiple detection agents, wherein different detection agents are arranged in separate active regions and immobilized on a deposited carbon material in the active regions; An electrical connection part comprising multiple electrical contacts, wherein each electrical contact (720, 728) is configured to transmit an electrical signal between the electrical connection part and a first signal electrode and a second signal electrode of a single active region and at least one gate electrode, A biosensor containing [unspecified].

2. The carbon material is deposited on the flat surface of the antistatic substrate. The first and second signal electrodes are located on the opposite side of the carbon material and are covered with an insulating material, and At least one terminal electrode is located on the upper surface of the insulating material of either the first signal electrode or the second signal electrode. The biosensor according to claim 1.

3. The first signal electrode and the second signal electrode are deposited on the flat surface of the antistatic substrate, and optionally, a bottom insulating material is deposited on the flat surface of the antistatic substrate between the first signal electrode and the second signal electrode. The carbon material is deposited on the upper surface of the first signal electrode and the second signal electrode, and the first signal electrode and the second signal electrode are located on the opposite side of the carbon material. The upper insulating material is deposited on the upper surface of the carbon material, opposite to the carbon material and approximately above the first and second signal electrodes. At least one gate electrode is located on the upper surface of the upper insulating material above either the first signal electrode or the second signal electrode. The biosensor according to claim 1.

4. Optionally: A spatially defined array of active regions includes at least two active regions; Carbon materials include graphene, carbon nanotubes, or combinations thereof; At least two signal electrodes and / or gate electrodes comprise a conductive metal selected from Ti, Cu, Ag, Ir, Pt, Au, or any combination thereof, or an oxide thereof; At least two signal electrodes and / or gate electrodes comprise a carbon-based conductive material selected from carbon nanotubes, graphene oxide, or any combination thereof; and / or Each active region contains the preservation solution. The biosensor according to claim 1.

5. Each detection agent is immobilized on the deposited carbon material via a hydrophobic linker, the hydrophobic linker being linked to a binding molecule via the amino or carboxyl terminus of the detection agent, and Optionally: A hydrophobic linker is a peptide linker comprising two or more linker amino acid residues and one or more aromatic amino acid residues; Two or more linker amino acid residues are selected from glycine, alanine, serine, and combinations thereof; and / or Hydrophobic linkers contain polycyclic aromatic hydrocarbons. The biosensor according to claim 1.

6. Multiple detection agents include pathogen proteins or their peptides, binding molecules capable of binding to pathogen proteins or their peptides, polynucleotides, or combinations thereof, and Optionally: The biosensor further comprises a collection of antibody mimetic bodies, aptamers, DNA molecules, RNA molecules, modified oligonucleotides, or combinations thereof, wherein heterogeneous members of the collection bind to heterogeneous pathogen proteins, the heterogeneous members of the collection are located in separate active regions not occupied by the detection agent, and the members of the collection are immobilized on a deposited carbon material of the active region; A pathogen is one or more infectious agents selected from viruses, bacteria, toxins, or combinations thereof; The pathogen is one or more viruses selected from SARS-CoV-2, influenza A, influenza B, human papillomavirus, Venezuelan encephalitis virus, cowpox virus, Ebola virus, Lassa fever virus, Rift Valley fever virus, and combinations thereof; The pathogen is one or more bacteria selected from the group consisting of Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachoma, Treponema pallidum, Bacillus anthrax, Plague bacillus, Tularemia tularensis, Glandersinus meridianus, Glandersinus rhinoplasia, and combinations thereof; The pathogen is one or more toxins selected from lysine toxin, botulinum toxin A / B / E, Staphylococcus enterotoxin B (SEB), abrin toxin, T-2 toxin, Bacillus anthracis LF toxin, Bacillus anthracis EF toxin, Bacillus anthracis PA toxin, and combinations thereof; Each of the multiple pathogen peptides has a length of 5 to 50 amino acid residues; The binding molecules in the collection are antibody-based molecules; Antibody-based molecules are selected from antibodies, their epitope-binding domains, antibody derivatives, antibody mimetic compounds, or combinations thereof; and / or The antibody mimetic is selected from the group consisting of affibody, affilin, affimer, monobody, and DARPIN. The biosensor according to claim 1.

7. The biosensor further includes an electromagnet placed beneath the substrate, and Optionally: The antistatic substrate comprises a single layer of antistatic polymer material; The antistatic substrate comprises a polymer material with an antistatic additive; and / or The antistatic substrate includes an antistatic printed circuit board. The biosensor according to claim 1.

8. A biosensor system for characterizing a subject's immune response to pathogen exposure, wherein the biosensor system is An electronic reading unit (1038) including a circuit (1046) for transmitting a signal and a processing unit (1050) for reading a signal; A biosensor (1002) according to any one of claims 1 to 7, wherein the biosensor is operably connected to an electronic reading unit via an electrical connection part of the biosensor and is configured to receive a signal delivered by a circuit, Includes, The electronic reading unit is configured to deliver a signal to a biosensor and obtain output impedance values ​​before and after application of a sample to an array of active regions on the biosensor. The processing unit is configured to compare the output impedance values ​​to determine whether a binding event occurred in one or more active regions and to characterize the subject's immune response to pathogen exposure. Biosensor system.

9. A communication interface connected to the electronic reader unit, which transmits data from the electronic reader unit; A data management computing device configured to receive data from an electronic reader via a communication interface, the data management computing device includes a memory connected to a processor configured to execute program instructions stored in memory to geographically map immune response data to pathogen exposure based on the data received from the electronic reader, and The biosensor system according to claim 8, further comprising:

10. The process of collecting biological samples from the subject; A step of providing the biosensor system according to claim 8; The process involves delivering an electrical signal to a biosensor via the circuit of the electronic reading unit; A step of determining the base resistance between the first signal electrode and the second signal electrode in each active region of the biosensor; The process involves applying a biological sample from a subject to at least one active region of a biosensor, and bringing the biological sample into operable contact with the carbon material between the first signal electrode and the second signal electrode, and with at least one gate electrode, in at least one active region; A step of identifying the change in base resistance between a first signal electrode and a second signal electrode, which occurs upon application of a biological sample to at least one active region; A step of characterizing the subject's immune response to a pathogen, or the antigen profile of a pathogen, based on a change in base resistance between a first signal electrode and a second signal electrode in at least one active region, A method for characterizing the immune response of a subject to pathogen exposure, including [specific method / method].

11. The system's biosensor includes an electromagnet placed beneath the biosensor's substrate, and the method is: Following the collection step, the process involves labeling the antibodies present in the collected biological sample with a magnetic portion; A step of mixing a biological sample containing labeled antibodies in a viscous fluid to create a viscous biological sample mixture for the application step; During the application process, the electromagnet is turned on to localize the labeled antibody of the biological sample mixture to the active region on the substrate surface and to promote binding between the labeled antibody and the congeneral detection agent immobilized on the active surface; Prior to the identification step, the electromagnet is turned off to release the unbound labeled antibody and / or protein. The method according to claim 10, further comprising:

12. The aforementioned labeling step is, The process involves bringing a biological sample into contact with a magnetic portion containing azide; The process involves irradiating the sample with UV light to bind the magnetic portion to the antibody in the biological sample, including, and Optionally: The magnetic part is a magnetic bead; The magnetic beads are iron oxide magnetic beads; Magnetic beads have a diameter of 2 nm to 100 μm; The viscous fluid contains polyethylene glycol (PEG) or glycerin; and / or Viscous fluids contain approximately 20% to 90% PEG. The method according to claim 11.

13. A method for manufacturing a biosensor according to claim 1, comprising the step of depositing a graphene material on the surface of the biosensor using a contour ablation method.

14. A step of obtaining a substrate having at least two layers, wherein there is a height difference z between the at least two layers; A step of transferring graphene onto a substrate, wherein the graphene is fractured along at least two layers due to a height difference z; The process involves cleaning the biosensor to remove excess graphene; The process of optionally adding an additional layer to the biosensor, A method for manufacturing a biosensor, including [a specific component].

15. The height difference z between at least two layers is approximately 50 nanometers to approximately 3 millimeters, and Optionally: The substrate includes at least three layers; The substrate includes at least four layers; and / or The additional layer includes an insulating material, or an electrode material, or both. The method according to claim 14.

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