Biofunctionalized Electronics

Abstract

The present invention relates to a device comprising a functionalized biosensor for detecting the presence of an analyte, the device comprising a field effect transistor (FET) coated with an external stimuli-responsive polymer layer, the external stimuli-responsive polymer layer configured to be modified via a localized external stimulus to expose chemical functional groups configured to bind to capture molecules. The present invention also relates to a method of making the biofunctionalized biosensor and a method of detecting the presence of an analyte using the functionalized biosensor.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 321,337, filed March 18, 2022, the disclosure of which is incorporated by reference herein in its entirety. [Background technology]

[0002] The development of new sensing paradigms underpins many exciting discoveries in life sciences. One aim of emerging sensing technologies is to enable high-throughput screening of biochemical reactions with lower cost and increased detection accuracy. Among various emerging technologies, Bergveld's pioneering work has revealed new directions for massively parallel detection of biochemical reactions using silicon field-effect transistors (FETs), a building block of modern integrated circuits. In fact, conventional bulk silicon transistors are currently used commercially for biosensing. Furthermore, the evolution of transistor technology to use nanoscale channel materials will directly benefit biochemical FET sensors (bioFETs) at a fundamental level. Specifically, improved bioFET sensor responses to external charges have been demonstrated by increasing the surface-to-volume ratio of the channel material using nanowire, nanotube, and nanoribbon structures. These bioFETs are capable of detecting a variety of biochemical reactions at concentrations below a few picomolar.

[0003] Although there has been significant progress in the development of electronic and optical sensors, one of the major problems remains the ability to functionalize the electronically active regions in a reproducible manner such that target molecules / biomolecules can be (i) selectively attached to the desired active regions, and (ii) the spatial density distribution can be controlled and is consistent among all sensors within a sensing platform. Moreover, there is currently no universal method to achieve this functionalization across different sensing platforms, devices, and materials.

[0004] In a related aspect, biosensing is usually performed in two modalities: the first is electrical, e.g., Ion Torrent DNA sensing platform; the second is optical sensing, e.g., Illumina. There is a lot of interest in this field to reduce the cost of the sensing platform and to shorten the detection and processing time. Summary of the Invention

[0005] In one aspect, the present invention relates to a device including a functionalized biosensor for detecting the presence of an analyte, the device including a field effect transistor (FET) coated with an external stimuli responsive polymer layer, the external stimuli responsive polymer layer configured to be locally modified via a local external stimulus to expose a chemical functional group configured to bind to a capture molecule within a desired sensing region. In one embodiment, the thermoresponsive polymer includes a first functional group and a second functional group, and the external stimulus removes the first functional group from the polymer such that an activated second functional group is exposed. In one embodiment, the first functional group includes at least one of tetrahydropyranyl carbamate, amine N-oxide, tetrahydropyranyl ether, triphenyl methyl ether, tetrahydropyranyl carbonate ester, S-tetrahydropyranyl carbonyl, ethyl disulfide, cyclopropenone, and tertiary butyl ester groups, and the activated second functional group includes at least one of amine, alcohol, phenol, or thiol. In one embodiment, the second functional group is an amine. In one embodiment, the polymer is produced by reversible addition fragmentation (RAFT) polymerization, atom transfer radical polymerization (ATRP), or nitroxide-mediated radical polymerization.

[0006] In one embodiment, the polymer has the formula A m -B n In one embodiment, the polymer is represented by the formula A, where m and n are independently positive integers, A is a monomer residue that includes a crosslinking functional group, B includes a monomer residue that has a protected functional group that can be deprotected by an external stimulus, and A and B are each attached to the polymer backbone (main chain). m -B n -C owherein m, n, and o are independently positive integers, A is a monomer residue comprising a crosslinking functional group, B is a monomer residue having a protected functional group that can be deprotected by an external stimulus, and C is a monomer residue comprising a solubilizing group, and wherein A, B, and C are each attached to the polymer backbone. In one embodiment, A comprises a cinnamic acid methyl ester. In one embodiment, B comprises at least one of a tetrahydropyranyl carbamate, an amine N-oxide, a tetrahydropyranyl ether, a triphenyl methyl ether, a tetrahydropyranyl carbonate ester, an S-tetrahydropyranyl carbonyl, an ethyl disulfide, a cyclopropenone, and a tertiary butyl ester group. In one embodiment, C comprises an alkyl, alkoxy, or aryl chain. In one embodiment, the polymer backbone comprises one or more polymers selected from the group consisting of poly(methacrylate), poly(acrylate), poly(ester), poly(styrene), poly(amide), poly(olefin), and combinations, copolymers, statistical copolymers, gradient copolymers, or block copolymers thereof.

[0007] In one embodiment, the external stimulus is heat and the polymer is thermoresponsive. In one embodiment, the functional groups are generated or activated upon localized heating of the thermoresponsive polymer. In one embodiment, the localized external stimulus comprises electromagnetic radiation. In one embodiment, the exposed functional groups comprise functional groups selected from the group consisting of thiols, alcohols, carboxylic acids and derivatives thereof, amines, alkynes, aldehydes, and ketones. In one embodiment, the exposed functional groups are further converted to alkyne, azide, cycloalkyne, cyclopropenone groups. In one embodiment, the exposed functional groups are configured to attach a desired capture molecule using a click chemistry reaction selected from the group consisting of thiol-ene, thiol-yne, copper catalyzed alkyne-azide cycloaddition, strain promoted alkyne-azide cycloaddition, sulfur fluoride exchange, and Diels-Alder reaction. In one embodiment, the exposed functional groups are configured to attach a desired capture molecule via 1,3-cycloaddition of diazides and diynes to form a poly(arylenetriazolylene). In one embodiment, the exposed functional groups are configured to attach a desired capture molecule via an azide and alkyne click reaction to form a polymer with 1,2,3-triazole functionality, hi one embodiment, the capture molecule is selected from the group consisting of an aptamer, an antibody, an antibody fragment, an oligonucleotide, a peptide, an enzyme, a nanobody, and a small molecule.

[0008] In one embodiment, the device further comprises a second FET that does not include a capture molecule, where the second FET is configured for differential detection. In one embodiment, the device is functionalized with a plurality of different capture molecules arranged in an array, where the device is configured for parallel detection of a plurality of analytes. In one embodiment, a local electromagnetic field is applied to the polymer layer using a scanning probe, a scanning electron beam, or a local light source. In one embodiment, the functional group or capture molecule is immobilized on the polymer or FET sensing region by a printing method. In one embodiment, a second polymer is deposited on the external stimuli responsive polymer layer, where the second polymer is configured as an anti-fouling coating to reduce non-specific binding of the capture molecule outside the sensing region. In one embodiment, the second polymer is removed by a local external stimulus to expose the external stimuli responsive polymer layer. In one embodiment, the sensing electrode is integrated on the same chip as the FET. In one embodiment, the sensing electrode is located on a different chip than the FET, where the sensing electrode is configured as an extended gate. In one embodiment, the detection circuit is integrated with the FET. In one embodiment, the detection circuitry is separate and fabricated separately from the FETs.In one embodiment, the device further comprises a detection circuitry integrated within the array of FETs.

[0009] In another aspect, the present invention relates to a method of fabricating a biofunctionalized biosensor, comprising providing a transistor comprising a semiconductor layer, coating the transistor with an external stimuli responsive polymer layer, applying a localized external stimulus to a region of the external stimuli responsive polymer layer, thereby generating activated functional groups on a region of a surface of the polymer layer, and exposing the polymer layer to a capture molecule, where the capture molecule binds to the activated functional group of the polymer layer. In one embodiment, the localized external stimulus comprises heat. In one embodiment, the heat is applied using thermal scanning probe lithography (tSPL). In one embodiment, the heat is applied using a focused light or laser. In one embodiment, the localized external stimulus comprises localized electromagnetic radiation. In one embodiment, the localized electromagnetic radiation is applied using a scanning probe, a scanning electron beam, or a localized light source.

[0010] In one embodiment, the activated functional group comprises an amine group. In one embodiment, the method further comprises functionalizing the amine to create a pattern of functional groups selected from the group consisting of amine, amide, ammonium, maleimide, aldehyde, thiol, biotin, alkyne, cycloalkyne, cyclopropenone, alkene and azide. In one embodiment, the capture molecule is selected from the group consisting of an aptamer, an antibody, an antibody fragment, an oligonucleotide, a peptide, an enzyme, a nanobody, and a small molecule. In one embodiment, the polymer is made by radical polymerization.

[0011] In one aspect, the present invention relates to a method of detecting the presence of an analyte of interest, comprising providing a device as disclosed herein, obtaining a sample, administering the sample to the device, and detecting a change in the electrical signal of the FET, thereby indicating the presence of the target analyte in the sample. In one embodiment, the target analyte is in a fluid sample. In one embodiment, the target analyte is in an air sample. In one embodiment, the target analyte is selected from the group consisting of proteins, nucleic acids, protein fragments, antigens, antibodies, surface receptors, hormones, growth factors, cells, virus particles, bacteria, secreted compounds, heavy metals, toxins, toxic molecules, explosives, pollutants, and metabolites. In one embodiment, the sample is a sample obtained from a subject. In one embodiment, the sample is an environmental sample.

[0012] In one aspect, the invention relates to a method for measuring a concentration of a target analyte in a volume, the method comprising providing a biosensor, exposing the biosensor to the volume, periodically measuring a sensor response, calculating a slope of the measured sensor response as the analyte binds to a capture probe on the surface, and calculating the concentration of the target analyte in the volume based on the slope of the measured sensor response. In one embodiment, the biosensor comprises a sensor surface and at least one surface capture probe. In one embodiment, the step of calculating a slope of the measured sensor response comprises calculating a slope of the measured sensor response multiple times during a binding phase and averaging the calculated slopes. In one embodiment, the biosensor comprises a pulse shaping circuit configured to increase the time resolution of the sensor. In one embodiment, the biosensor comprises a pulse shaping circuit configured to reduce noise in the sensor response during the binding phase. In one embodiment, the pulse shaping circuit comprises a (CR) n -(RC) m In one embodiment, the (CR) n -(RC) m The circuit is 1 to 1000 seconds -1 In one embodiment, the pulse shaping of a signal having a time constant of (CR) n -(RC) m The circuit is (CR) 2-(RC) 2 In one embodiment, calculating the slope of the measured sensor response comprises calibrating based on an output of the pulse shaping detection circuit. In one embodiment, the method further comprises calibrating the biosensor to quantify the association and dissociation rate constants.

[0013] In one aspect, the present invention relates to a system for measuring a concentration of a target analyte in a volume, the system comprising: a biosensor having a sensing surface and an output terminal, the output terminal configured to vary an electrical signal based on a response from the sensing surface; and a pulse shaping detection circuit configured to increase the time resolution of sensing and reduce noise in the sensor response during a binding phase. In one embodiment, the pulse shaping detection circuit comprises two first-order high pass filters connected in series having an input and an output, the input of the series being electrically connected to the output terminal. In one embodiment, the pulse shaping detection circuit comprises a second-order low pass filter having an input and an output, the input of the second-order low pass filter being connected to the output of the series. In one embodiment, the pulse shaping detection circuit comprises a gain stage having an input and an output, the input of the gain stage being connected to the output of the second-order low pass filter. In one embodiment, the pulse shaping detection circuit comprises a (CR) n -(RC) m In one embodiment, the (CR) n -(RC) m The circuit is 1 to 1000 seconds -1 In one embodiment, the pulse shaping of a signal having a time constant of (CR) n -(RC) m The circuit is (CR) 2 -(RC) 2 In one embodiment, the pulse shaping detection circuit comprises multiple amplification stages along the signal chain. In one embodiment, the electrical signal output of the pulse shaping detection circuit is a predictor of the analyte concentration. In one embodiment, the amplitude of the output of the pulse shaping detection circuit is proportional to the slope of the electrical output signal of the front-end biosensing stage.

[0014] In one embodiment, the front-end detection stage of the pulse shaping detection circuit comprises a differential amplifier pair of biosensors. In one embodiment, the output signal of the front-end detection stage feeds the signal chain of the pulse shaping detection circuit. In one embodiment, one biosensor of the differential amplifier pair has a capture probe on its surface. In one embodiment, one biosensor of the differential amplifier pair does not have a capture probe on its surface. In one embodiment, the differential amplifier pair of the biosensor cancels electrical signals due to non-specific binding. In one embodiment, the differential amplifier pair of the biosensor removes environmental noise. In one embodiment, the front-end biosensing stage is a single stage amplifier. In one embodiment, the output of the front-end biosensing stage feeds the signal chain of the pulse shaping circuit. In one embodiment, the biosensor of the front-end amplifier has a capture probe on its surface. [Brief description of the drawings]

[0015] The foregoing objects and features, as well as other objects and features, are included to provide an understanding of the present invention and will become apparent by reference to the following description and the accompanying drawings, which form a part hereof and in which like reference characters represent like elements.

[0016] [Figure 1] FIG. 1 is a diagram of a method for manufacturing a sensing chip. [Diagram 2] FIG. 2 is a diagram of a method for manufacturing a sensing chip. [Diagram 3] FIG. 3 is a diagram of a method for manufacturing a sensing chip.

[0017] [Figure 4] FIG. 4 shows an example of a biosensor.

[0018] [Diagram 5] FIG. 5 is a graph of the sensor response over time.

[0019] [Figure 6]FIG. 6 is a diagram of an example of a FET sensor (see, for example, Nature Nanotechnology, volume 7 pages 401-407 (2012), which is incorporated by reference in its entirety).

[0020] [Figure 7] FIG. 7 shows a graph of DNA+HMGB1 detection over time over a range of concentrations (see, e.g., Nature Nanotechnology, volume 7 pages 401-407 (2012), which is incorporated by reference in its entirety).

[0021] [Figure 8] FIG. 8 is an example of a pulse shaping circuit.

[0022] [Figure 9A] FIG. 9A is a schematic diagram of an example pulse shaping circuit. [Figure 9B] FIG. 9B is a schematic diagram of an example pulse shaping circuit. [Figure 9C] FIG. 9C is a schematic diagram of an example pulse shaping circuit. [Figure 9D] FIG. 9D is a schematic diagram of an example pulse shaping circuit. [Figure 9E] FIG. 9E is a schematic diagram of an example pulse shaping circuit.

[0023] [Figure 10] FIG. 10 is a graph of the simulation results.

[0024] [Figure 11A] FIG. 11A is a bulk CMOS sensing platform.

[0025] [Figure 11B] FIG. 11B is a SOI CMOS sensing platform.

[0026] [Figure 11C]FIG. 11C is a FDSOI CMOS sensing platform.

[0027] [Figure 12] FIG. 12 is an exemplary embodiment featuring airborne detection of COVID-19.

[0028] [Figure 13] Figure 13 shows an optical image of the BioFunFET extended gate device, which is coated with a thermoresponsive polymer and the graphene gate is functionalized with COVID-19 antibodies for RBD spike protein detection.

[0029] [Figure 14] FIG. 14 shows the experimental results of BioFunFET with graphene-extended gate for RBD spike protein detection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] It should be understood that the drawings and description of the present invention are simplified to show elements relevant to a clear understanding of the present invention and, for clarity, exclude many other elements found in related systems and methods. Those skilled in the art may recognize that other elements and / or steps are desirable and / or required in implementing the present invention. However, because such elements and steps are well known in the art and do not facilitate a better understanding of the present invention, a description of such elements and steps is not provided herein. The disclosure herein relates to all such variations and modifications to such elements and methods known to those skilled in the art.

[0031] definition

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0033] As used herein, each of the following terms has the meaning associated with it in this section.

[0034] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0035] As used herein, "about" when referring to a measurable value, such as an amount, duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate.

[0036] The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to a particular epitope on an antigen. An antibody may be an intact immunoglobulin derived from natural or recombinant sources, or an immunologically active portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. The antibody in the present invention may exist in various forms, such as, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1988; Houston et al., 1988; Bird et al., 1988).

[0037] The term "analyte" refers to a substance to be detected or assayed by the methods of the present invention. Exemplary analytes include, but are not limited to, proteins, peptides, nucleic acid segments, molecules, cells, microorganisms and fragments and products thereof, or any substance for which a binding site, binding member or receptor (such as an antibody) can be developed.

[0038] As used herein, "biological sample" refers to biological material isolated from an individual. A biological sample may contain any biological material suitable for detecting a target analyte and may include cells and / or non-cellular material obtained from an individual.

[0039] As used herein, the term "diagnosis" refers to detecting a disease or disorder or determining the stage or extent of a disease or disorder. Typically, a diagnosis of a disease or disorder is based on the evaluation of one or more factors and / or symptoms indicative of the disease. That is, a diagnosis can be made based on the presence, absence, or amount of factors indicative of the presence or absence of a disease or condition. Each factor or symptom considered to be indicative of a diagnosis of a particular disease need not be exclusively related to the particular disease, that is, there may be different diagnoses that can be inferred from the diagnostic factor or symptom. Similarly, factors or symptoms indicative of a particular disease may be present in individuals who do not exhibit a particular disease. The diagnostic method can be used independently or in combination with other diagnostic and / or staging methods known in the medical arts for a particular disease or disorder.

[0040] A "level" of one or more target analytes refers to the absolute or relative amount or concentration of the analyte in a sample.

[0041] "Measuring" or "measurement", or alternatively, "detecting" or "detection", means assessing the presence, absence, quantity or amount (which may be an effective amount) of a given substance in a sample, including the derivation of a qualitative or quantitative concentration level of such substance.

[0042] As used herein, a "standard control value" refers to a predetermined amount of a particular analyte detectable in a sample. A standard control value is appropriate when using the method of the present invention to compare the amount of a target analyte of interest present in a sample. An established sample that serves as a standard control provides an average amount of the analyte of interest in a sample type (e.g., a biological sample) that is typical of an average, healthy person with some matching of identity, e.g., gender, age, ethnicity, and medical history. A standard control value may vary depending on the nature of the target analyte of interest and the sample.

[0043] The terms "subject," "patient," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof suitable for the methods described herein (whether in vitro or in situ). In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0044] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6, and all whole and partial increments therebetween. This is true regardless of the breadth of the range.

[0045] Biofunctionalized field-effect transistor (biofunFET)

[0046] In one embodiment, the present invention relates to systems and devices including bio-functionalized field effect transistors (biofunFETs) and methods of using the same. The present invention improves on the current state of the bioFET by creating a CMOS-compatible biofunFET manufacturing platform that can be extended to a wide variety of biosensing applications, reduce variability, reduce noise, improve sensitivity, reproducibility and parallel detection, and increase stability and robustness.

[0047] The ability to precisely control the position or localization of molecules / biomolecules on a surface is important for several applications such as biosensing, bio-nanoreactors, and single molecule experiments. Despite recent advances, creating functional patterns with single molecule level resolution remains limited due to the lack of lithographic methods that combine high resolution, compatibility with soft polymer structures, ease of fabrication, and high throughput.

[0048] In certain aspects, the biofunFET of the present invention comprises a FET comprising a source electrode, a drain electrode, a gate electrode, and a semiconductor material. In one embodiment, the biofunFET comprises a polymer coated on the FET, which can be locally activated / patterned and locally functionalized / patterned with any desired chemical molecule. For example, in certain embodiments, the polymer is sensitive or responsive to an external stimulus (e.g., heat), and a local stimulus applied to a specific region of the polymer induces or activates the formation of a functional group (also referred to herein as a "sticky end") in the desired specific region. In one embodiment, the activated functional group of the polymer, e.g., an amine group, is used to attach a probe or capture molecule (directly or using other functionalization intermediate steps) to the locally thermally activated region of the polymer, thereby functionalizing the biofunFET at the desired location (sensing region), e.g., the semiconductor region, or the top gate or extended gate. In a sensing measurement, the probe or capture molecule then specifically binds / captures the analyte of interest, such that binding of the analyte to the probe or capture molecule induces a detectable change in the electronic signal of the biofunFET, thereby indicating the presence and amount of the analyte of interest in the sample. Another possibility is the use of an extended gate, where the top gate of the FET is coated with a thermal (or other local stimuli) sensitive polymer and externally electrically connected to a gate functionalized to sense the presence of the analyte.

[0049] A "polymer" may be any polymer that can be locally activated by a localized external stimulus, such as, but not limited to, a localized heat source, electric field, light, electric current, pressure, shear force. This activation can be obtained by an external probe (e.g., a nano-sized atomic force microscope tip) or beam (e.g., a laser beam) or by direct local deposition of molecules. The local activation generates chemical functional groups on the polymer surface that can be used to bind capture molecules. The activation pattern on the polymer surface can have a lateral size ranging from a few picometers to a few millimeters.

[0050] In certain aspects, the ability to generate biofunFETs using localized heating of coated polymers reduces variability and noise, improves sensitivity, reproducibility and parallel detection, and increases stability and robustness while generating biofunFETs with a CMOS-compatible biofunFET fabrication platform that can be expanded to a wide variety of biosensing applications. For example, in one embodiment, the localized functionalization described in the present invention allows for the creation of a device with a functionalized biofunFET generated by localized heating of coated polymers as described herein and a non-functionalized FET immediately adjacent to the sensor FET that does not have any capture molecules on its sensitive area (see FIG. 13). This allows for differential detection since the device has a built-in control, and signals generated from other sources (other than the analyte), e.g., non-specific binding of other molecules to the non-functionalized control FET, can be taken into account when measuring / detecting changes in the signal generated from the biofunFET. Furthermore, the localized functionalization described herein allows for the creation of biofunFETs with various patterns of probes or capture molecules on the polymer surface. Furthermore, localized functionalization allows the ability to perform parallel detection, where different probes or capture molecules can be functionalized on the biofunFET to enable the detection of multiple analytes in a sample / microchip (see FIG. 1). Localized functionalization by localized heat can be achieved with a resolution of 8 nm, so the patterns of different capture molecules generated by localized heat can be as small as 10 nm. Importantly, since the biofunFET can be based on polymer films and localized heat spun onto any electronic device, this fabrication platform is fully CMOS compatible, low-cost, robust, and reproducible.

[0051] In one embodiment, the biofunFET comprises a semiconductor material. Exemplary semiconductor materials for the biofunFET include, but are not limited to, silicon, carbon nanotubes, III-V and II-VI semiconductors, graphene, 2D materials, MoS2, transition metal dichalcogenides, or other suitable materials.

[0052] In one embodiment, the biofunFET comprises a polymer coated on the semiconductor material or gate. In a particular embodiment, the polymer comprises a thermosensitive or thermoresponsive polymer. Any polymer in which localized heat or electromagnetic fields induce the generation or activation of functional groups that can be used to attach probes or capture molecules can be used in the present invention. For example, in one embodiment, the polymer comprises a thermosensitive or thermoresponsive polymer in which amine groups are activated upon localized heating. For example, in one embodiment, the polymer comprises a thermosensitive or thermoresponsive polymer, in which localized heat is effective to remove a first functional group from the polymer such that the surface comprises a second functional group at at least some of the first positions, where the first functional group is, for example, but not limited to, tetrahydropyranyl carbamate, amine N-oxide, tetrahydropyranyl ether, triphenylmethyl ether, tetrahydropyranyl carbonate ester, S-tetrahydropyranyl carbonyl, or ethyl disulfide, and the second functional group is a thiol, alcohol, carboxylic acid and its derivatives, amine, alkyne, aldehyde, or ketone. Tertiary butyl ester groups can also be used as the first functional group, followed by localized deprotection. In one embodiment, the tertiary butyl ester group can be converted to a carboxylic acid group by a localized external stimulus. In one embodiment, the resulting carboxylic acid can be further converted to an anhydride using an additional stimulus (see, e.g., "Atomic force microscopy based thermal lithography of poly(tert-butyl acrylate) block copolymer films for bioconjugation." Langmuir 24, 10825-10832 (2008) and "Scanning thermal lithography of tailored tert-butyl ester protected carboxylic acid functionalized (Meth)acry-late polymer platforms." ACS Appl. Mater. Interfaces 3, 3855-3865 (2011)).In other embodiments, the polymer may contain groups that undergo thermal polymerization and crosslinking reactions, such as the Diels-Alder reaction.

[0053] In some embodiments, the polymer comprises a thermosensitive or thermoresponsive polymer in which amine groups are activated upon localized heating. Exemplary polymers that yield activated amines upon localized heating include, but are not limited to, poly(methacrylate) copolymers: poly-((tetrahydropyran-2-yl N-(2 methacryloxyethyl)carbamate)-b-(methyl-4-(3-methacryloyloxypropoxy)cinnamate)) (PMCC) (see, e.g., “Sub-10 nm Resolution Patterning of Pockets for Enzyme Immobilization with Independent Density and Quasi-3D Topography Control, ACS Applied Materials and Interfaces”, 11, 2019, and “Cost and Time Effective Lithography of Reusable Millimeter Size Bone Tissue Replicas With Sub-15 nm Feature Size on A Biocompatible Polymer”, Adv. Func. Mater. 2021). Also, for example, in some embodiments, the polymer comprises a thermosensitive or thermoresponsive polymer in which hydroxyl or carboxylic acid groups are activated upon localized heating. Exemplary polymers that provide activated hydroxyl or carboxylic acid groups upon localized heating include, but are not limited to, high temperature PMCC (see, for example, "Sub-10 nm Resolution Patterning of Pockets for Enzyme Immobilization with Independent Density and Quasi-3D Topography Control, ACS Applied Materials and Interfaces", 11, 2019) and copolymers of benzyl methacrylate and THP-MA that exhibit thermal deprotection of THP groups to generate carboxylic acids at approximately 150°C, followed by thermal deprotection of ester groups to generate carboxylic acid groups, which convert to anhydrides above 180°C.

[0054] In one embodiment, the exposed amines are further functionalized to create a pattern of functional groups selected from the group consisting of amine, amide, ammonium, maleimide, aldehyde, thiol, biotin, and azide.

[0055] In some embodiments, the polymer has formula A m -B n -C o where m, n, and o are each independently a positive integer, A is a crosslinking functional group, B is a monomer residue having a protected functional group that can be deprotected by an external stimulus such as heat or an electromagnetic field, and C is a monomer residue containing a solubilizing group such as an alkyl, alkoxy, or aryl chain that can increase the solubility of the polymeric material. In one embodiment, the bond between monomer residues A, B, and C is the backbone of the polymer, which can be, for example, but not limited to, a poly(methacrylate), poly(acrylate), poly(ester), poly(styrene), poly(amide), or poly(olefin) backbone.

[0056] In one embodiment, C is a short alkyl chain. In one embodiment, C is a short alkoxy chain.

[0057] In one embodiment, B can be tetrahydropyranyl carbamate, amine N-oxide, tetrahydropyranyl ether, triphenylmethyl ether, tetrahydropyranyl carbonate ester, S-tetrahydropyranyl carbonyl, or ethyl disulfide. In one embodiment, the different functional group is an amine, alcohol, phenol, or thiol.

[0058] In some embodiments, the polymer comprises poly(p-phenylene vinylene) or poly((tetrahydropyran-2-yl N-(2-methacryloxyethyl)carbamate)-co-(methyl 4-(3-methacryloyloxypropoxy)cinnamate)) having the following formula: [ka]

[0059] In one embodiment, the polymer is a copolymer, and thus the polymer backbone comprises two or more polymeric components. In one embodiment, residues A, B and C may be on different components of the polymer backbone. In one embodiment, A comprises a cinnamic acid group. In one embodiment, B comprises a tetrahydropyran carbamate group.

[0060] According to some embodiments, the polymer has the formula B n where B represents a functional group modified by a local external stimulus, n is a positive integer, and the functional group is attached to the polymer backbone. In one embodiment, B is attached to the polymer backbone via a polymer side chain. In one embodiment, B is part of the polymer backbone. In one embodiment, the polymer comprises two or more polymer backbones and / or functional groups. In one embodiment, the polymer can be deposited on the substrate using standard film-forming techniques such as spin-coating, drop-casting, blade coating, and spray-coating onto the substrate or platform.

[0061] In some embodiments, the polymer backbone can be derived from or can be monomers such as vinyl, allyl, 4-styryl, acroyl, epoxide, oxetane, cyclic carbonate, methacryloyl, acrylonitrile, etc., polymerized by either radical, cationic, atom transfer, or anionic polymerization processes. In some embodiments, the polymer backbone can be HO(CH2) λ OH, H2N(CH2) λThe polymer backbone can be or be derived from an isocyanate, isothiocyanate, or epoxide that can be copolymerized with a difunctional amine or alcohol, such as NH2, where λ is a positive integer (e.g., 1 to 25). In some embodiments, the polymer backbone can be or be derived from a strained ring olefin (e.g., dicyclopentadienyl, norbornenyl, cyclobutenyl, etc.) that can be polymerized by ring-opening metathesis polymerization using an appropriate metal catalyst, as known to those of skill in the art to which this disclosure pertains. In some embodiments, the polymer backbone can be or be a -(CH2) n SiCl3, -(CH2) n Si(OCH2CH3)3 or -(CH2) n The polymer backbone may be derived from or may be Si(OCH3)3, where the monomer may react with water under conditions known to those skilled in the art to form either thin films or monolithic organically modified sol-gel glasses, or modified silylated surfaces, where n is a positive integer (e.g., 1-25). In some embodiments, the polymer backbone may be derived from or may be a polymerizable group that may be photochemically dimerized or polymerized. It may include one or more of such groups, which include, but are not limited to, the following conjugated structures: [ka]

[0062] In some embodiments, the functional group B can be selected such that upon exposure to a local external stimulus, the protecting group is removed from the surface, leaving another functional group. For example, to obtain a carboxylic acid, B can be selected from tert-butyl ester, tetrahydropyran ester, etc. To obtain an amine, B can include tetrahydropyranyl carbamate, amine N-oxide, etc. If an alcohol or phenol is desired, B can be selected from tetrahydropyranyl ether, triphenylmethyl ether, tetrahydropyranyl carbonate ester, etc. If a thiol is desired exposure to a local external stimulus, B can include S-tert-butoxycarbonyl, S-tetrahydropyranyl carbonyl, ethyl disulfide, etc.

[0063] In other cases, B may be a group that undergoes thermal polymerization and crosslinking reactions, such as, for example, a Diels-Alder reaction between two B groups (e.g., furan and maleimide), ring-opening polymerization (e.g., poly(ferrocenylsilane)), ring-opening metathesis polymerization (e.g., dicyclopentadiene), reactions to form conjugated polymers (e.g., from poly(phenylenevinylene) or other similar precursors), and reactions of trifluorovinyl ethers. In some embodiments, B may be a group that volatilizes or decomposes upon treatment with a local external stimulus.

[0064] As noted above, the polymer may have two or more functional groups B. These functional groups may be selected such that each B is modified at the same or different temperatures.

[0065] In one embodiment, the polymer can have a group A, which can be photochemically or thermally crosslinked to control the softening temperature of the entire polymer. In one embodiment, such a polymer can have the formula A m -B nIn one embodiment, using the A group, the softening temperature can be adjusted to be above or below the chemical modification temperature as desired. In one embodiment, this can be accomplished by increasing or decreasing the glass transition temperature and / or crystallinity of the polymer. In one embodiment, the A and B groups can be attached to the polymer backbone via side chains and organized into blocks, which can be aligned or randomly oriented. In one embodiment, the A and B groups are derived from the same functional monomer unit. In one embodiment, the A and B groups are derived from different functional monomer units. In some embodiments, the A group can be selected from cinnamates, alkenes, chalcones, trifluorovinyl ethers, Diels-Alder reactants, and the like.

[0066] By way of example, certain polymers that can be used as surfaces for reaction with localized external stimuli include the following tetrahydropyran-(THP)-protected carboxylic acid-functionalized poly(acrylates): [ka]

[0067] In one embodiment, this type of polymer, which is hydrophobic, can be thermally deprotected at about 120 degrees Celsius (°C) to give hydrophilic acid functionality. In one embodiment, the functional groups are further reacted at about 170°C to give hydrophobic anhydrides. This represents a surface that can undergo the so-called "read-write-overwrite process." It is important to note that the conversion from acid to anhydride is reversible by the removal and addition of water, respectively.

[0068] In one embodiment, another polymer that can be used for treatment with a local external stimulus is the following poly(amide): [ka]

[0069] In one embodiment, the starting surface composition (i.e., poly(amide)) is hydrophilic, but it can be modified to a hydrophobic poly(imide) at about 300° C. In one embodiment, this reaction is reversible by the addition of acid.

[0070] For example, in one embodiment, the polymer includes functional groups that can be deprotected by a local external stimulus, such as heat. In one embodiment, the deprotected functional groups in the activated regions of the polymer are then reacted using click chemistry to attach the desired capture molecule. In one embodiment, free thiol groups on the surface revealed by the local external stimulus can undergo thiol-ene click chemistry with an alkene-functionalized capture molecule. In one embodiment, the deprotected amine groups can be converted to azides. In one embodiment, the polymer can incorporate azides as functional groups that are deprotected by an external stimulus, such as heat. In one embodiment, the polymer can incorporate protected alkenes or alkynes that can be deprotected via a local external stimulus to react with an azide-functionalized capture molecule. In one embodiment, thiols, alcohols, and amines can be converted to alkynes or cycloalkynes to participate in copper-catalyzed and / or strain-promoted alkyne-azide click chemistry. In one embodiment, the click chemistry reaction is high yielding without side reactions, by-products, or harsh reaction conditions. In one embodiment, 1,3-cycloaddition of diazides and diynes can be used to initiate a chemical reaction between the functional groups and a desired capture molecule to form a poly(arylenetriazolylene). In one embodiment, the exposed functional groups are configured to attach a desired capture molecule via a click reaction of azides and alkynes to form a polymer with 1,2,3-triazole functionality.

[0071] For example, in one embodiment, the polymer contains two acrylate monomers (a crosslinking monomer containing cinnamic acid and a monomer with a protected amine group that can be deprotected under high temperature) and is formed as a statistical mixture of these two monomers based on the feed ratio using a free radical polymerization method. The formation of other polymer architectures such as block copolymers, gradient copolymers, brush copolymers and block copolymers requires living polymerization methods. To polymerize the two monomers living and access the above polymer structures, the two monomers can be pseudo-living polymerized using reversible addition-fragmentation (RAFT) polymerization. The livingness of the RAFT polymerization of the two monomers allows the formation of different polymer structures. Block copolymers result in materials with a predetermined crosslinking area and functionalized moieties. Furthermore, block copolymers with nanostructures on the surface (such as gyroid and lamellar structures) allow for a predetermined and special distribution of functional groups. Gradient copolymers allow for the formation of gradient surface functionalization. Block copolymers result in smaller superstructures on the surface with potential new functionalization schemes. Finally, living polymerization allows complete control of all variables that affect polymer length, degree of polymerization and polydispersity, as well as surface coverage, crosslinking and film-forming properties.

[0072] In one embodiment, the polymer is produced by reversible addition-fragmentation (RAFT) polymerization, atom transfer radical polymerization (ATRP), or nitroxide-mediated radical polymerization.

[0073] In some embodiments, another way to tailor polymer surface properties is to expand the monomer. A third monomer containing a solubilizing group, such as a short alkyl chain, increases the solubility of the polymeric material during spin coating. This allows the use of higher molecular weight polymers that are soluble and spin coatable. Finally, a third monomer under RAFT polymerization conditions provides further access to new polymer film formation morphologies and thus new polymer film properties.

[0074] In one embodiment, the surface may be formed from a self-assembled monolayer or multilayer of molecules. The molecules may have the basic structure X k -RB n where X represents an anchor group for attachment of the molecule to a substrate or platform, R represents a cross-linking group, C represents a functional group that is modified by a local external stimulus, and k and n are independently positive integers. In one embodiment, these molecules are represented by thiol-terminated X k -RB n Reaction of silane-terminated X with gold surfaces k -RB n The material can be processed by standard self-assembled monolayer or multilayer formation techniques, including reaction of the material with a glass surface.

[0075] In some embodiments, the anchor group X can be selected from phosphonic acids, phosphinic acids, sulfonic acids, carboxylic acids, carbamates, dithiocarbamates, thiols, selenols, phosphines, amines, amides, carbohydroximic acids, sulfonohydroxamic acids, phosphohydroxamic acids, monochlorosilanes, dichlorosilanes, trichlorosilanes, mono(alkoxy)silanes, di(alkoxy)silanes, tri(alkoxy)silanes, and the like, or a conjugate base of any of the foregoing, and the bridging group R can be selected from linear or branched C-C 50 G can be any of the functional group types described above for the polymer surface, and can be an aliphatic or cycloaliphatic, fluoroalkyl, oligo(ethylene glycol), aryl, amine, etc. group.

[0076] In one embodiment, the biofunFET includes a second polymer layer on top of the first polymer layer that can help reduce non-specific binding of the probe / capture molecules. For example, in one embodiment, the second polymer layer, which has low adhesion properties for the capture molecules, is a thermosensitive or thermoresponsive polymer, where localized heating evaporates the second polymer layer while simultaneously inducing or generating or activating the required functional groups in the first polymer layer. Any polymer with these properties can be utilized, such as, but not limited to, polyphthalaldehyde (PPA). Another example is a polymer resist composed of a cyclic low ceiling temperature poly(aldehyde). Another example is molecular glass. (See, for example, Microsystems & Nanoengineering (2020) 6:21, which is incorporated herein by reference in its entirety).

[0077] In one embodiment, the second polymer is configured as an anti-fouling coating to reduce non-specific binding of capture molecules outside the sensing region. In one embodiment, the second polymer is removed by a localized external stimulus to expose the external stimulus responsive polymer layer.

[0078] In one embodiment, the biofunFET comprises a probe or capture molecule attached to a functional group of a thermosensitive or thermoresponsive polymer. The probe or capture molecule specifically binds to an analyte of interest. Any type of probe or capture molecule known in the art that can be attached to the biofunFET via a functional group can be used in the present invention, including but not limited to antibodies, aptamers, and peptides.

[0079] The polymer can be selected such that the distance between the probe or capture molecule and the biofunFET surface is such that association of the analyte of interest with the probe or capture molecule induces a measurable change in the electronic properties of the biofunFET. In some cases, the polymer is selected such that the distance between the capture molecule and the surface of the FET is in the range of 1-300 nm.

[0080] In some embodiments, the biofunFET comprises one or more capture molecules immobilized on the surface of the FET via a linking group or by direct adsorption to a polymer coating the FET surface. In some embodiments, the one or more capture molecules are immobilized on the surface of the FET via a linking group presented by a polymer coating the FET surface.

[0081] In some embodiments, the biofunFET comprises a surface coated with a polymer in which a localized stimulus (such as a localized heat source) generates a free chemical group or "sticky end" such as a free functional amine, hydroxy group, carboxylic acid group, alcohol, phenol, or thiol or azide. In some embodiments, the "sticky end" serves as a binding site for attaching one or more capture molecules for detecting an analyte of interest. In some embodiments, the biofunFET comprises a surface coated with a thermosensitive polymer that includes groups that undergo a thermoresponsive change (e.g., thermally induced cleavage) such that after thermal activation, a free chemical group or "sticky end" such as a free functional amine is available. In some embodiments, the "sticky end" serves as a binding site for attaching one or more capture molecules for detecting an analyte of interest.

[0082] In some embodiments, the biofunFET comprises a surface coated with a thermosensitive polymer that contains groups that undergo a thermoresponsive change (e.g., thermally induced cleavage) after thermal activation to become free chemical groups or "sticky ends" such as free functional amines, hydroxyl groups, carboxylic acid groups, alcohols, phenols, or thiols or azides. In some embodiments, the "sticky ends" serve as binding sites for attaching one or more capture molecules for detecting an analyte of interest.

[0083] The biofunFET sensor of the present invention further comprises a capture molecule for an analyte of interest immobilized in proximity to the FET surface, such that association of the analyte of interest with the capture molecule induces a measurable change in the electrical properties of the FET.

[0084] Desired capture molecules for a particular analyte are known in the art and can be selected taking into account a number of considerations, including the identity of the analyte, the concentration of the analyte, and the nature or condition of the sample in which the analyte is to be detected. Suitable capture molecules include aptamers (nucleic acids or peptides), antibodies, antibody fragments, antibody mimetics (e.g., engineered affinity ligands), peptides (natural or modified peptides), proteins (e.g., recombinant proteins, host proteins), oligonucleotides, DNA, RNA (e.g., microRNA), and small organic molecules (e.g., haptens or enzyme cofactors, enzymes).

[0085] In some embodiments, the capture molecules are attached to activated functional groups of the polymer via biochemical conjugation or electrostatic binding, which can occur directly or by using intermediate functional groups and linkers.

[0086] In one embodiment, the capture molecule of the present invention comprises an antibody or an antibody fragment. In a particular embodiment, the antibody capture molecule specifically binds to a compound of interest, for example, a secreted compound of interest. Such antibodies include polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies.

[0087] Such antibodies can be produced in a variety of ways, including hybridoma culture, recombinant expression in bacterial or mammalian cell culture, and recombinant expression in transgenic animals. The choice of production method depends on several factors, including the desired antibody structure, the importance of carbohydrate moieties to the antibody, ease of culture and purification, and cost. Many different antibody structures can be produced using standard expression techniques, including full-length antibodies, antibody fragments such as Fab and Fv fragments, and chimeric antibodies containing components from different species. Small size antibody fragments such as Fab and Fv fragments with no effector function and limited pharmacokinetic activity can be produced in bacterial expression systems. Single chain Fv fragments exhibit low immunogenicity.

[0088] In one embodiment, the capture molecule of the present invention comprises isolated nucleic acids, such as DNA oligonucleotides and RNA oligonucleotides. In certain embodiments, the nucleic acid capture molecule specifically binds to a compound of interest, such as a DNA molecule or an RNA molecule (e.g., mRNA, rRNA, or lncRNA). The nucleic acid capture probe of the present invention may be in the form of a linear oligonucleotide or may have a secondary structure (e.g., hairpin or loop) that facilitates binding and capture of the target analyte.

[0089] For example, in one embodiment, the nucleic acid comprises a nucleotide sequence complementary to the nucleic acid of interest. Alternatively, the nucleotide sequence of the nucleic acid capture molecule may comprise sequence mutations, such as substitutions, insertions and / or deletions of one or more nucleotides, relative to the original nucleotide sequence, provided that the resulting nucleic acid functions as the original compound and specifically binds to the compound of interest.

[0090] In one embodiment, the nucleic acid comprises a nucleic acid aptamer. A nucleic acid aptamer is a synthetic oligodeoxynucleotide designed according to strict recognition and binding affinity between nucleotides, obtained by screening through systematic evolution of ligands by exponential enrichment (SELEX). A nucleic acid aptamer not only has similar characteristics to an antibody, such as highly specific recognition and high binding affinity to a target. The aptamer of the present invention can have one or more modified nucleoside or modified nucleobase linkages. For example, in some embodiments, the aptamer can be a thioaptamer containing one or more phosphorothioate or phosphorodithioate moieties, 2'-fluoro-ribonucleotide oligomers, NH2- and OCH3-substituted ribose aptamers, and deoxyribose aptamers. In some embodiments, the aptamer can be an LNA aptamer.

[0091] As used herein, the term "capture aptamer" refers to an aptamer that includes a configuration that can bind to a substrate (e.g., a functionalized polymer on a biofunFET) and position a target analyte (i.e., bind in a sample), thereby allowing the target analyte to bind to the substrate via the capture aptamer upon binding.

[0092] The term "aptamer" refers to a nucleic acid (typically DNA, RNA or oligonucleotide) that emerges when added to a mixture of molecules by in vitro selection or other types of aptamer selection procedures known in the art (e.g., bead-based selection by flow cytometry or high-density aptamer arrays). Ligands that bind to aptamers include, but are not limited to, small molecules, peptides, proteins, carbohydrates, hormones, sugars, metabolic by-products, cofactors, drugs and toxins. The aptamers of the present invention are specific for a particular target analyte of interest. Aptamers can have diagnostic, target validation and therapeutic applications. The specificity of the binding is defined in terms of the dissociation constant Kd of the aptamer for its ligand. Aptamers can have high affinity with a Kd range (pM to nM) similar to antibodies and specificity similar to / better than antibodies (Tuerk and Gold, 1990, Science, 249:505; Ellington and Szostak, 1990, Nature 346:818). Aptamers are typically 10-300 nucleotides in length.

[0093] Aptamers configured to bind to a particular target analyte can be selected, for example, by synthesizing an initial heterogeneous population of oligonucleotides and then selecting oligonucleotides within the population that bind strongly to a particular target analyte. Once an aptamer that binds to a particular target molecule has been identified, it can be replicated using a variety of techniques known in the biological and other arts, for example, by cloning and polymerase chain reaction (PCR) amplification followed by transcription.

[0094] In some embodiments, the thermally activated functionalization of the polymer-coated surface can be temporally restricted, spatially restricted, or spatially and temporally restricted such that at a given time or at a given location on the surface of the FET (e.g., the channel or extended gate of the FET), only a portion of the polymer (e.g., a single zone or pattern) is functionalized to bind capture molecules. Such embodiments enable the creation of multi-probe biofunFET sensors in which different capture molecules are functionalized at different regions across the surface of the sensor, or multiplexed sensors in which different samples can be applied to different regions of the sensor.

[0095] Devices including probes and multiwell plates incorporating the biofunFET sensors of the present invention are also provided.

[0096] In one embodiment, the invention relates to a method of fabricating a biofunFET as described herein. In one embodiment, the method includes coating a semiconductor material of the FET with a heat-sensitive or thermally responsive polymer. For example, in one embodiment, the method includes spin-coating a semiconductor material of the FET with a heat-sensitive or thermally responsive polymer. Other examples include, but are not limited to, physical vapor deposition, chemical vapor deposition, sputtering, or other suitable methods.

[0097] As described herein, localized heat applied to a polymer induces the creation or activation of functional groups to which probe or capture molecules can then be attached. Any localized heat source can be used to apply to the polymer surface.

[0098] For example, in one embodiment, the method includes heating or activating the polymer surface with an electromagnetic field, e.g., a laser, an electron beam, an electric field, or other suitable method.

[0099] In one embodiment, the method includes locally heating a polymer surface using thermal scanning probe lithography (tSPL). tSPL (R. Szoszkiewicz, et al., Nano Lett. 2007, 7, 1064) has been shown to be capable of patterning complex quasi-3D topographies on polymer surfaces with lateral resolutions of 15 nm or less and depth resolutions of 2 nm or less (D. Pires, et al., Science 2010, 328, 732; R. Garcia, et al., Nat. Nanotechnol. 2014, 9, 577; XY Liu, et al., ACS Appl. Mater. Interfaces 2019, 11, 41780). tSPL can be performed using commercially available equipment that uses a heat-sensitive nanoprobe to locally evaporate the heat-sensitive polymer polyphthalaldehyde (PPA), leaving behind a void that defines the pixel size (ST Zimmermann, et al., ACS Appl. Mater. Interfaces 2017, 9, 41454; ST Howell, et al., Microsyst. Nanoeng. 2020, 6, 21; XR Zheng, et al., Nat. Electron. 2019, 2, 17). In one embodiment, a scanning probe, a scanning electron beam, or a localized light source is used to irradiate the polymer layer with localized electromagnetic radiation.

[0100] tSPL writes topographical and chemical features into a thermosensitive polymer resist by localized heating, which can engrave and chemically activate surfaces with nanoscale precision (XY Liu, et al., ACS Appl. Mater. Interfaces 2019, 11, 41780; DB Wang, et al., Adv. Funct. Mater. 2009, 19, 3696). The tSPL process involves first pixelating a reference input image and then replicating that image by assigning a specific height level to each gray level of each individual pixel. Additional information regarding tSPL and other lithography techniques can be found in U.S. Patent Application No. 17 / 592,169, filed February 3, 2022, and U.S. Patent No. 8,468,611, issued June 18, 2013, which are incorporated herein by reference in their entireties.

[0101] In one embodiment, the method includes functionalizing the FET by attaching one or more agents or capture molecules to generated or activated functional groups generated by localized heating of the polymer. In one embodiment, the method includes contacting the surface of the polymer with a liquid medium containing a probe or capture agent, where the probe or capture agent binds to or is otherwise associated with the functional groups of the polymer.

[0102] FIG. 9 shows an exemplary method of producing a biofunFET of the present disclosure, where a polymer can be spin-coated onto a transistor, then a local heat source is used to change the surface chemistry and create a "sticky" amine nanopattern on the transistor. Aptamers then attach only to the patterned amines. In some embodiments, multimeric patterns can be used with different types of aptamers for sensing multiple analytes. In this way, a control FET is created on the same chip. Another view of the fabrication process is shown in FIG. 2 and FIG. 3. The steps in FIG. 1 correspond to steps A) and B) in FIG. 2.

[0103] Disclosed herein is a new sensing platform that includes the integration of the above-mentioned tSPL patternable polymers with electronic and optoelectronic devices, and in some embodiments, can utilize such sensing methods. The integration includes placing a polymer over a sensor, e.g., electronic, magnetic, and / or optical sensor, followed by localized nanopatterning with localized heat, tSPL, nanopatterning means, or other heat source (e.g., laser) to functionalize the target area. Novel features of this platform include, but are not limited to, the selective attachment of molecules and biomolecules to desired active areas of the sensor after sensor fabrication, e.g., via patterning of voids or thinner areas of polymer or other covering material, e.g., masking polymer, placed over the sensor active area.

[0104] In some embodiments, the spatial density distribution of molecules on the surface of the sensing platform can be controlled and consistent among all sensors within the sensing platform. In some embodiments, functionalization patterns of any shape can be created with a spatial resolution of the order of 10 nm, less than 1 μm, less than 500 nm, less than 200 nm, less than 100 nm, less than 50 nm, less than 20 nm, less than 10 nm, less than 8 nm, less than 5 nm, or less than 3 nm within the sensor active area.

[0105] Sensor performance (detection limit) can be controlled in some embodiments by the thickness of the polymer, which can be controlled down to a single monolayer, for example in increments of 1, 2, 3, 5, or 10 monolayers. The thickness of the polymer can range from 1 nm to 300 nm. In some embodiments, multiple biomolecules can be bound to the same electroactive region and sensed. In some embodiments, the limit of detection can range from femtomolar to nanomolar.

[0106] The platform of the present disclosure is a universal method to achieve this functionalization across different sensing platforms, devices, and materials.

[0107] In some embodiments, disclosed herein is a method of fabricating a patterned or customized sensing surface, comprising providing a sensor having an active sensing area, disposing or depositing a masking material having a deposited thickness or thickness range, e.g., a polymer, on the active sensing area, determining a desired sensing surface profile, and lithographically patterning a set of voids or thin regions, the thin regions having a thickness less than the deposited thickness or thickness range in the masking material such that the voids or thin regions form exposed or partially exposed areas, respectively, of the sensing surface configured to act as the desired sensing surface profile. The size of the active sensing area can range from a few hundred nanometers. 2 ~several thousand mm 2 The range may be:

[0108] The devices and methods of the present invention can be used to determine the presence and / or amount of any target analyte. Suitable types of analytes include proteins, nucleic acids, protein fragments, antigens, antibodies, surface receptors, hormones, growth factors, cells, virus particles, bacteria, secreted compounds, metabolites, and the like. The exact combination of compounds of interest that are assayed by the present invention is easily controllable and defined by the ultimate user. For example, detection of a particular target analyte is limited only by the availability of a capture agent (e.g., antibody, aptamer, peptide, nucleic acid sequence, etc.) that can specifically bind to the compound and functionalize to the biofunFET surface. In one embodiment, the capture molecule comprises an antibody or capture aptamer, and the target analyte comprises a region or epitope to which the antibody or capture aptamer binds.

[0109] In one embodiment, the method of the invention includes obtaining a sample containing or suspected of containing a target analyte, contacting at least a portion of the sample with a biofunFET including a capture probe for detecting a target analyte of the invention, detecting a change in electrical potential based on an interaction of the target analyte in the sample with the capture probe on the biofunFET, and identifying the sample as containing the target analyte based on the detection of the change in electrical potential.

[0110] In one aspect, a sample can be contacted with a biofunFET such that target analytes present in the sample bind to the capture probes on the biofunFET. In one aspect, the invention relates to a method for detecting the presence or abundance of an analyte of interest using a biofunFET and a sensor or device comprising a biofunFET as described herein. The invention can be used to detect the presence or abundance of any analyte of interest. Any probe or capture molecule can be functionalized using an activated functional group of the polymer, so that the probe or capture molecule can be selected to specifically bind to the analyte of interest.

[0111] The parallel detection capabilities of the present invention allow for the detection of 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 40 or more, 50 or more, 100 or more, etc. target analytes in one or more samples. For example, as described elsewhere herein, the spatial functionalization of thermally activatable polymers provided by the present invention allows for the ability to create distinct zones for the parallel capture and detection of analytes.

[0112] In certain embodiments, the method comprises detecting the presence or abundance of an analyte in a sample obtained from a subject to detect the presence or severity of a disease, disorder, or condition in the subject. For example, in certain embodiments, the method can be used to diagnose a subject having or at risk of having a disease, disorder, or condition. In one embodiment, the method is used to determine whether a subject has a pathogenic infection (e.g., a viral or bacterial infection).

[0113] In one embodiment, the invention provides a method for diagnosing a subject as having an infectious disease, or a disease or disorder associated therewith, based on detection of one or more target molecules associated with an infectious agent (e.g., a virus, a bacteria, a fungus, or a protozoa). In one embodiment, the invention provides a method for determining the risk of developing, or assessing the progression of, an infectious disease, or a disease or disorder associated therewith, by detecting the presence of at least one target molecule in a sample.

[0114] In some embodiments, the target analytes include virus particles, such as, but not limited to, coronavirus, influenza virus, Zika virus, Ebola virus, Japanese encephalitis virus, mumps virus, measles virus, rabies virus, varicella zoster, Epstein-Barr virus (HHV-4), cytomegalovirus, herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2), herpes papillomavirus (HPV), human immunodeficiency virus 1 (HIV-1), JC virus, arbovirus, enterovirus, West Nile virus, dengue virus, poliovirus, and varicella zoster virus. In some embodiments, the target analytes include bacterial markers, such as, but not limited to, markers for Streptococcus pneumoniae, Neisseria meningitidis, Streptococcus agalactiae, or Escherichia coli. In some embodiments, target analytes include fungal or protozoal markers, such as, but not limited to, markers for candidiasis, aspergillosis, cryptococcosis, and toxoplasmosis.

[0115] In one embodiment, the method of the present invention is a method for diagnosing a disease or disorder associated with the presence or absence of a target analyte. In one embodiment, the method includes the steps of obtaining a sample from a subject having or at risk of having a disease or disorder associated with the presence of a target analyte, contacting at least a portion of the sample with a biofunFET comprising a capture probe for detecting the target analyte, detecting a change in electrical potential based on the interaction of the target analyte in the sample with the capture probe on the biofunFET, identifying the sample as containing the target analyte based on the detection of the change in electrical potential, and diagnosing the subject as having a disease or disorder associated with the presence of the target analyte. In one embodiment, the method includes the steps of obtaining a sample from a subject having or at risk of having a disease or disorder associated with the absence of a target analyte, contacting at least a portion of the sample with a biofunFET comprising a capture probe for detecting the target analyte, detecting the absence of a change in electrical potential based on the absence of the interaction of the target analyte with the capture probe on the biofunFET, identifying the sample as being absent of the target analyte based on the detection of the absence of the change in electrical potential, and diagnosing the subject as having a disease or disorder associated with the absence of the target analyte.

[0116] In one embodiment, the method of the present invention is a method for treating a disease or disorder associated with the presence or absence of a target analyte. In one embodiment, the method includes the steps of obtaining a sample from a subject having or at risk of having a disease or disorder associated with the presence of the target analyte, contacting at least a portion of the sample with a biofunFET that includes a capture probe for detecting the target analyte, detecting a change in electrical potential based on the interaction of the target analyte in the sample with the capture probe on the biofunFET, identifying the sample as containing the target analyte based on the detection of the change in electrical potential, identifying the subject as having a disease or disorder associated with the presence of the target analyte, and administering a therapeutic agent to treat the disease or disorder associated with the presence of the target analyte. In one embodiment, the method includes obtaining a sample from a subject having or at risk of having a disease or disorder associated with the absence of a target analyte, contacting at least a portion of the sample with a biofunFET including a capture probe for detecting the target analyte, detecting an absence of change in electrical potential based on an absence of interaction between the target analyte and the capture probe on the biofunFET, identifying the sample as lacking the target analyte based on the detection of an absence of change in electrical potential, identifying the subject as having a disease or disorder associated with the absence of the target analyte, and administering a therapeutic agent to treat the disease or disorder associated with the absence of the target analyte.

[0117] An exemplary embodiment featuring airborne detection of COVID-19 is shown in Figure 12. In such an embodiment, an air sample suspected of containing SARS-CoV-2 viral particles is mixed with a liquid and applied to a biofunFET sensor of the invention functionalized with an aptamer specific for binding to the SARS-CoV-2 antigen.

[0118] Thus, in one embodiment, the invention provides a method of diagnosing a subject as having a SARS-CoV-2 infection or a disease or disorder associated therewith, such as COVID-19. In one embodiment, the invention provides a method of determining the risk of developing or assessing the progression of a SARS-CoV-2 infection or a disease or disorder associated therewith (e.g., COVID-19) via detection of a SARS-CoV-2 antigen or nucleic acid molecule in a sample.

[0119] Figure 13 shows an optical image of a BioFunFET extended gate device, where the device is coated with a thermoresponsive polymer and the graphene gate is functionalized with COVID-19 antibodies for RBD spike protein detection. Figure 14 shows the experimental results of the BioFunFET with graphene extended gate for RBD spike protein detection, which shows the V T Demonstrate a shift in

[0120]

[0121] In certain embodiments, the methods include detecting the presence or abundance of an analyte in an environmental sample, such as a water sample, a sewage sample, an air sample, etc., to determine the presence of the analyte in the environment.

[0122] In certain embodiments, the method includes detecting a change in the electrical properties of the biofunFET, the change being indicative of the presence of the analyte of interest. As described herein, in certain embodiments, the sensor described herein reduces noise by having one functionalized biofunFET and one non-functionalized FET, thereby allowing for a more accurate determination and quantification of the presence of the analyte in the sample. In a particular embodiment, the electrical signal of the biofunFET is analyzed using a rapid sensing method, which is described in more detail below. However, the biofunFET and the method of use are not limited to any particular analysis method.

[0123] The disclosed device and method include three important advantages. The first is biofunctionalization by local heating of ad-hoc polymers spin-coated on the FET. This provides robust chemical functionalization of biofunFETs, allows compatibility with CMOS (complementary metal oxide semiconductor), FDSOI (fully depleted silicon-on-insulator), and DG FDSOI (double-gate fully depleted silicon-on-insulator) (which is advantageous for the microelectronics industry), and allows localized functionalization for differential sensing, resulting in a significant increase in signal-to-noise ratio and reproducibility. In some embodiments, parallel sensing capabilities can be realized by sequentially activating the polymers, for example, for sensing multiple analytes or viruses on the same biochip. Local heating may be available in CMOS, which allows for localized and on-demand functionalization. DG FDSOI has two independent gates for device-level "capacitive" amplification. In some embodiments, only a portion of the polymer is functionalized, and the non-functionalized portion is utilized as a control.

[0124] The second advantage is fast detection using a transient response as disclosed herein, as opposed to steady-state measurement methods, where the response time (time to steady state) depends on the concentration of the sample being measured. The lower the concentration, the longer it takes to reach steady state. The third advantage is that aptamers are inexpensive, allowing easier functionalization and adaptation of biofunFETs to new viruses and bacteria, and have high reaction rates for fast detection.

[0125] High-speed biosensing

[0126] In one aspect, a CMOS biochip and method for high speed sensing applications are disclosed herein. An exemplary biosensor is shown in FIG. 4. The biosensor includes a sensor surface 103 functionalized with one or more capture probes 102 for detecting at least one target analyte. The target analyte 101 is shown either bound or unbound to the surface probe 102. The target analyte 101 and the surface probe 102 result in a chemically specific detection. The combination of the target analyte 101 and the surface probe 102 results in a signal being generated from the sensor 103. Exemplary target analytes that may be detected include those described elsewhere herein.

[0127] Referring to FIG. 5, a graph of the sensor response 202 over time during a typical detection is shown. In some embodiments, the sensor response is measured in volts. Sensing moves from initialization to a region designated (I), which occurs when the analyte 101 begins to bind to the surface probe 102. The first stage is defined by a transient to a steady state in a second region designated (II). In some embodiments, the kinetics can be determined using a two-compartment model defined in Equation 1 below:

number

[0128] Region II is defined as the steady-state region where conventional detection is performed. Once the measured signal is sufficiently constant, the sensor value is recorded. Thus, only one measurement can be taken to determine the concentration of the analyte.

[0129] Finally, region III is the dissociation region, which corresponds, for example, to sensor washing, in which analyte particles are removed from the sensor surface and the sensor is ready for the next measurement.

[0130] A key finding of this disclosure is that rise time (shown by dashed box 201) increases with decreasing analyte concentration (and conversely decreases with increasing analyte concentration). Thus, rise time measurements can be used to calculate concentration more quickly. By tuning appropriate circuit parameters, as described in later embodiments, detection times can go from a few seconds to sub-second. In some embodiments, rise time measurements can be a less accurate estimate, so multiple measurements in parallel from multiple sensors, or sequential measurements from a single sensor, can be used to improve accuracy. These two approaches by themselves have the disadvantage of loss of spatial and temporal resolution, respectively.

[0131] Examples of using FET sensors are shown in Figures 6 and 7 and further described in Nature Nanotechnology, Volume 7, pages 401-407 (2012), which is incorporated herein by reference in its entirety. An exemplary FET sensor is shown in Figure 6. Figure 7 shows a graph of DNA+HMGB1 detection over time in a range of concentrations, where curve 401 corresponds to 500 nM, curve 402 corresponds to 400 nM, curve 403 corresponds to 300 nM, curve 404 corresponds to 200 nM, curve 405 corresponds to 150 nM, curve 406 corresponds to 100 nM, curve 407 corresponds to 50 nM, curve 408 corresponds to 30 nM, and curve 409 corresponds to 3 nM. As shown, the time resolution at 3 nM is less than 0.33 Hz.

[0132] According to the two-compartment model, when the analyte / surface probe reaction is not mass transport limited (i.e., the mass transfer coefficient is large), the transient is exponential and depends on the association rate constant (k1) and the analyte concentration [A]. One aspect of the present disclosure is to use fundamentals of analyte / surface probe reaction kinetics to predict the analyte concentration from the slope of the transient curve. This principle is applicable, in some embodiments, to all sensing modalities and sensor types.

[0133] The disclosed methods are advantageous because they improve time resolution, and in some embodiments, the disclosed measurement schemes can be applied multiple times during the same "A+B" reaction transient (i.e., t1-t0 timescale), allowing for averaging to reduce error and improve reliability.

[0134] An example for a FET sensor is given below: The output V(t) of the FET sensor is given by Equation 2 below.

number

number

number

number

number

[0135] The derivative of V(t) can then be used to determine the concentration based on the slope of the transient (rise time portion, region I) of the sensor response curve.

[0136] In some embodiments, a pulse shaping circuit can be used to condition the sensor output, for example as shown in FIG. 8. In some embodiments, the pulse shaping circuit is designed to be insensitive to noise in the sensor response, especially in region I from t0 to t1, and / or in the initial sensor response region 201. In some embodiments, (CR) n -(RC) m Pulse shaping circuits of this type are used. These circuits are used in particle detectors for high energy physics (having time scales of μs). Such circuits have time scales of 1 to 1000 seconds. -1 The 10-bit 12 ...

[0137] In some embodiments, a front-end sensing stage of the detection circuit comprises a differential amplifier pair of the biosensor. In some embodiments, the output signal of the front-end detection stage feeds a signal chain of a pulse shaping circuit. In some embodiments, one biosensor of the differential amplifier pair has a capture probe on its surface. In some embodiments, one biosensor of the differential amplifier pair does not have a capture probe on its surface. In some embodiments, the differential amplifier pair of the biosensor cancels electrical signals due to non-specific binding. In some embodiments, the differential amplifier pair of the biosensor removes environmental noise. In some embodiments, the pulse shaping detection circuit includes multiple amplification stages along the signal chain. In some embodiments, the front-end biosensing stage is a single stage amplifier. In some embodiments, the output of the front-end biosensing stage feeds the signal chain of the pulse shaping circuit. In some embodiments, the biosensor of the front-end amplifier has a capture probe on its surface. In some embodiments, the electrical signal output of the pulse shaping detection circuit is a predictor of the analyte concentration. In some embodiments, the amplitude of the output of the pulse shaping detection circuit is proportional to the slope of the electrical output signal of the front-end biosensing stage.

[0138] (CR) 2 -(RC) 2 Assuming the waveform shaping structure, an analytical model for predicting the peak value of the voltage output of the waveform shaping circuit is given by the following Equation 7 and Equation 8. Equation 7 is (CR) 2 -(RC) 1 Equation 8 is for the waveform shaping circuit, (CR) 2 -(RC) 2 This is an equation for the waveform shaping circuit.

number

number

[0139] An exemplary wave shaping circuit for use with the system of the present disclosure is shown in Figure 9A with detailed diagrams as follows: a current generator is shown in Figure 9B, two first order high pass filters are shown in Figure 9C, a second order low pass filter is shown in Figure 9D, and a gain stage is shown in Figure 9E. In some embodiments, all GΩ resistors (shown as white ovals) are replaced with active or pseudo resistors. In some embodiments, the gain stage is realized by a programmable-gain amplifier (PGA).

[0140] Simulation results using the waveform shaping circuit of FIG. 9A are shown in FIG. 10, where τ L =0.1s, τ H =0.3s.

[0141] In some embodiments, the extreme values ​​P L and P H The peak values ​​are determined using an analytical model that utilizes: In some embodiments, the determined peak values ​​are correlated to concentration after being calibrated, and can therefore be used to determine concentration. In some embodiments, according to Equation 8, the peak amplitude is inversely proportional to τ, and τ (according to Equation 5) is proportional to k1, k2, k3, k4, k5, k6, k7, k8, k9, k10, k11, k12, k13, k14, k15, k16, k17, k18, k19, k20, k21, k22, k23, k24, k25, k26, k27, k28, k31, k32, k33, k34, k35, k35, k36, k37, k38, k39, k40, k41, k42, k43, k44, k45, k46, k47, k48, k49, k50, k51, k52, k53, k54, k55, k56, k57, k58, k59, k60, k61, k62, k63, k64, k65, k65, k75, k86, k97, k10, k11, k12, k13, k14, k15, k15, k25, k16, k17, k26, k18, k27, k31, k32, k33, k44, k45, k51, k52, k63, k64, k75, k86, k97, k10, k11, k12, k13, k14, k15, k25, k26, k27, k32, k33, k34, k35, k45, k46, k47, k5 -1 , and [A].

[0142] Three exemplary structures with a biofunFET monolithically integrated with a CMOS readout circuit are shown in Figures 11A, 11B, and 11C: Figure 11A shows a bulk CMOS sensing platform, Figure 11B shows a SOI CMOS sensing platform, and Figure 11C shows a FDSOI CMOS sensing platform.

[0143] In Figures 11A, 11B and 11C, the letter "S" refers to source, the letter "D" refers to drain, the letter "TG" refers to top gate, the letter "BG" refers to bottom gate, the letter "G" refers to gate and the letter "DG" refers to double gate. "Sensing pixel" refers to biofunFET + readout circuit. "SOI" refers to silicon-on-insulator. "Target" refers to target molecules, viruses, etc. "BOX" refers to buried oxide. Vg is the gate bias voltage. REF is the reference electrode.

[0144] In various embodiments of Figures 11A, 11B, and 11C, the biofunFET can have a single gate for SOI and bulk CMOS technologies. The biofunFET can operate in single-gate or double-gate configurations in FDSOI technology. In these configurations, the biofunFET is monolithically fabricated in silicon. The biofunFET may be heterogeneously integrated on top of a fully fabricated CMOS chip in some embodiments.

[0145] In some configurations, the biofunFET can be made of different functional semiconductors. Furthermore, heterogeneously integrated biofunFETs can operate in single-gate or double-gate configurations. In some disclosed configurations, the polymer disposed on the sensing surface can be locally functionalized using a CMOS integrated heater or an external heat source (e.g., thermal scanning chip, laser, etc.).

[0146] system

[0147] In one embodiment, the present invention provides a system including the biofunFET, CMOS chip, or combination thereof of the present invention. In some embodiments, the system includes a computing device. The computing device may include a desktop computer, laptop computer, tablet, smartphone, or other device, and includes a software platform for controlling system components, displaying raw data, and analyzing acquired data. The computing device may include at least one processor, standard input and output devices, and all hardware and software typically found on a computing device for storing data, executing programs, and transmitting and receiving data over a network.

[0148] In certain embodiments, the system of the present invention includes hardware and software for detecting and quantifying the detection signal from the biofunFET or CMOS chip. The signal can be quantified using any suitable analysis software package or using a custom analysis algorithm.

[0149] The disclosures of each of the patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and modifications of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

[0150] References The following publications are incorporated herein by reference in their entireties:

[0151] X.Liu,et al.,Sub-10nm Resolution Patterning of Pockets for Enzyme Immobilization with Independent Density and Quasi-3D Topography Control,ACS Applied Materials and Interfaces,11,2019

[0152] X.Liu,et al.,Cost and Time Effective Lithography of Reusable Millimeter Size Bone Tissue Replicas With Sub-15nm Feature Size on A Biocompatible Polymer,Adv.Func.Mater.2021

[0153] D.Wang,et al.,Thermochemical Nanolithography of Multifunctional Nanotemplates for Assembling Nano-Objects,Adv.Func.Mater.,2009

[0154] T.Wu,et al.,Experimental Study of the Detection Limit in Dual-Gate Biosensors Using Ultrathin Silicon Transistors,ACS Nano,2017

[0155] E.Cuniberto,et al.,Nano-engineering the material structure of preferentially oriented nano-graphitic carbon for making high-performance electrochemical micro-sensors,Scientific Reports,2020

[0156] X.Zheng,et al.,Patterning metal contacts on monolayer MoS2 with vanishing Schottky barriers using thermal nanolithography,Nature Electronics,2019

Claims

1. A device comprising a functionalized biosensor for detecting the presence of an analyte, It includes a field-effect transistor (FET) coated with an external stimulus-responsive polymer layer, The external stimulus-responsive polymer layer is configured to be locally modified via a local external stimulus to expose chemically functional groups configured to bind to a capture molecule within a desired sensing region of the device. device.

2. The thermoresponsive polymer comprises a first functional group and a second functional group, The external stimulus removes the first functional group from the polymer so that the activated second functional group is exposed. The device according to claim 1.

3. The first functional group comprises at least one of tetrahydropyranylcarbamate, amine N-oxide, tetrahydropyranyl ether, triphenyl methyl ether, tetrahydropyranyl carbonate ester, S-tetrahydropyranyl carbonyl, ethyl disulfide, cyclopropenone, and tertiary butyl ester group. The activated second functional group comprises at least one of an amine, an alcohol, a phenol, or a thiol. The device according to claim 2.

4. The second functional group is an amine. The device according to claim 2.

5. The aforementioned polymer is, Formula A ｍ -B ｎ It is represented as, In the formula, m and n are independent positive integers. A is a monomer residue containing a crosslinking functional group, B contains a monomer residue having a protected functional group that can be deprotected by external stimuli. A and B are bonded to the polymer backbone, The device according to claim 1.

6. The aforementioned polymer is, Formula A ｍ -B ｎ -C ｏ It is represented as, In the formula, m, n, and o are independent positive integers. A is a monomer residue containing a crosslinking functional group, B contains a monomer residue having a protected functional group that can be deprotected by external stimuli. C is a monomer residue containing a solubilizing group, preferably C includes an alkyl chain, an alkoxy chain, or an aryl chain. In the formula, A, B, and C are each bonded to the polymer backbone. The device according to claim 1.

7. A contains methyl cinnamate, The device according to claim 5 or claim 6.

8. B comprises at least one of the following: tetrahydropyranyl carbamate, amine N-oxide, tetrahydropyranyl ether, triphenyl methyl ether, tetrahydropyranyl carbonate ester, S-tetrahydropyranyl carbonyl, ethyl disulfide, cyclopropenone, and tertiary butyl ester group. The device according to claim 5 or claim 6.

9. The polymer skeleton comprises one or more polymers selected from the group consisting of poly(methacrylate), poly(acrylate), poly(ester), poly(styrene), poly(amide), poly(olefin), and combinations thereof, copolymers, statistical copolymers, gradient copolymers, or block copolymers. The device according to claim 5 or claim 6.

10. The exposed functional groups are further converted into alkyne, azide, cycloalkyne, and cyclopropenone groups. The device according to claim 1.

11. a) The exposed functional group is configured to bind a desired capture molecule using a click chemistry reaction selected from the group consisting of thiol-ene, thiol-yine, copper-catalyzed alkyne-azide cycloaddition, strain-accelerated alkyne-azide cycloaddition, sulfur fluoride exchange, and Diels-Alder reaction, or b) The exposed functional group is configured to bind the desired capture molecule via a click reaction between the azide and the alkyne to form a polymer having a 1,2,3-triazole functional group. The device according to claim 1.

12. The capture molecule is selected from the group consisting of aptamers, antibodies, antibody fragments, oligonucleotides, peptides, enzymes, nanobodies, and small molecules. The device according to claim 1.

13. Functionalized with multiple different capture molecules arranged in an array, for parallel detection of multiple analytes. It consists of, The device according to claim 1.

14. A second polymer is deposited on the external stimulus-responsive polymer layer. The second polymer is configured as an antifouling coating to reduce nonspecific binding of captured molecules outside the sensing region. The device according to claim 1.

15. a) Sensing electrodes are integrated on the same chip as the FET, or b) The sensing electrode is located on a different chip from the FET, and the sensing electrode is configured as an extended gate. The device according to claim 1.

16. A method for manufacturing a biofunctionalized biosensor, To prepare a transistor that includes a semiconductor layer, The transistor is coated with an external stimulus-responsive polymer layer. Applying a local external stimulus to the region of the external stimulus-responsive polymer layer thereby generating activated functional groups on the surface region of the polymer layer, and Exposing the polymer layer to a capturing molecule, wherein the capturing molecule binds to the activated functional group of the polymer layer, method.

17. The aforementioned local external stimulus includes local heat, or The aforementioned local external stimulus includes local electromagnetic radiation. The method according to claim 16.

18. The aforementioned activating functional group includes an amine group. The method according to claim 16.

19. The capture molecule is selected from the group consisting of aptamers, antibodies, antibody fragments, oligonucleotides, peptides, enzymes, nanobodies, and small molecules. The method according to claim 16.

20. A method for detecting the presence of a target analyte, To provide the device according to claim 1, To obtain a sample, Administering the sample to the device, and This includes detecting a change in the electrical signal of the FET, thereby indicating the presence of a target analyte in the sample, preferably the target analyte is in a fluid sample, and more preferably the target analyte is in an air sample. method.

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