WO2025166328A1 - Dispositifs et procédés de biodétection rapide - Google Patents

Dispositifs et procédés de biodétection rapide

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Publication number
WO2025166328A1
WO2025166328A1 PCT/US2025/014271 US2025014271W WO2025166328A1 WO 2025166328 A1 WO2025166328 A1 WO 2025166328A1 US 2025014271 W US2025014271 W US 2025014271W WO 2025166328 A1 WO2025166328 A1 WO 2025166328A1
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WIPO (PCT)
Prior art keywords
polymer
biosensor
biofunctionalized
multiplexed
monomer
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PCT/US2025/014271
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English (en)
Inventor
Elisa Riedo
Davood Shahrjerdi
Alexander Wright
Moeid JAMALZADEH
Hashem NASRALLA
Rahul Deshmukh
Marcus Weck
Matthew HANNIGAN
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New York University NYU
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New York University NYU
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Publication of WO2025166328A1 publication Critical patent/WO2025166328A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors

Definitions

  • the present invention provides a method of producing a multiplexed biofunctionalized biosensor comprising: a) providing a transistor substrate comprising a semiconductive layer; b) coating the surface of the transistor substrate with an external stimulus-responsive polymer layer; c) applying a localized external stimulus to a region of the polymer surface to expose activated functional groups on the region of the polymer surface; d) contacting the polymer surface with a capture molecule, wherein the capture molecule is conjugated, directly or indirectly, to the exposed activated functional groups on the region of the polymer surface; and e) repeating steps c) and d) in different regions of the polymer surface with different capture molecules.
  • the transistor substrate of step a) is a field effect transistor (FET).
  • the semiconductive layer of step a) comprises graphene.
  • the multiplexed biofunctionalized biosensor comprises a plurality of graphene FETs (gFETs) including isolated graphene islands.
  • the external stimulus-responsive polymer of step b) comprises a first monomer A and a second monomer B; wherein monomer A comprises a cross-linking functional group; and wherein monomer B comprises a protected functional group that can be deprotected by an external stimulus to expose the reactive functional group.
  • the cross-linking functional group of monomer A is selected from the group consisting of an azide, a diaziridine, and a methacrylate. In some embodiments, the cross-linking functional group of monomer A comprises cinnamate methyl ester. [0010] In some embodiments, the reactive functional group is selected from the group consisting of a thiol, an alcohol, an amine, a carboxylic acid, an aldehyde, a ketone, and an alkyne.
  • the protected group comprises one or more selected from the group consisting of tetrahydropyranyl carbamate, amine N-oxide, tetrahydropyranyl ether, triphenylmethyl ether, tetrahydropyranyl carbonate ester, S-tetrahydropyranyl carbonyl, ethyl disulfide, cyclopropenone, and tert-butyl ester groups.
  • the backbone of the external stimulus-responsive polymer is selected from the group consisting of a poly(methacrylate), poly(acrylate), poly(ester), poly(amide), poly(styrene), poly(olefin), and combinations and co-polymers thereof.
  • the external stimulus-responsive polymer further comprises a third monomer C, wherein monomer C comprises a solubilizing group.
  • the solubilizing group of monomer C comprises an alkyl, alkoxyl, or aryl chain.
  • step b) comprises: i) coating the transistor substrate with a layer of an external stimulus-responsive polymer; and ii) coating the external stimulus-responsive polymer layer with a layer of an electrically resistive polymer.
  • the electrically resistive polymer sublimes when exposed to the same external stimulus to which the external stimulus-responsive polymer responds.
  • the external stimulus-responsive polymer is a heat-responsive polymer and wherein step c) comprises applying localized heat.
  • the heat is applied to the heat-responsive polymer through thermal scanning probe lithography (tSPL) or a focused light or laser.
  • step c) comprises applying localized electromagnetic radiation.
  • the localized electromagnetic radiation is applied by a scanning probe, electron beam, or localized light source.
  • the region exposed to the external stimulus in step c) has a surface area of between about 1 pm 2 and about 10 mm 2 . In some embodiments, the area of the region is between about 8,000 nm 2 and about 250 ⁇ m 2 .
  • the capture molecule of step d) is selected from the group consisting of aptamers, antibodies, antibody fragments, peptides, lectins, enzymes, enzyme fragments, nanobodies, and small molecules.
  • step d) comprises reacting the exposed reactive functional groups on the region of the polymer surface directly with a functional group on the capture molecule.
  • step d) comprises: i) reacting the exposed reactive functional group onto the region of the polymer surface with a functionalized linker molecule; and ii) binding the capture molecule onto the region through an interaction or reaction with the functionalized linker molecule.
  • the functionalized linker molecule comprises one or more selected from the group consisting of an amine, an aldehyde, a thiol, a Attorney Docket No.206256-0105-00WO cycloalkyne, a cyclopropenone, an alkene, an alkyne, an azide, maleimide, a maleimide derivative, biotin, a biotin derivative, streptavidin, a streptavidin derivative, avidin, and an avidin derivative.
  • the regions of the polymer surface reacted with capture molecules are arranged in a grid pattern.
  • the distance between regions of the polymer surface reacted with capture molecules is between about 100 nm and about 10 ⁇ m.
  • the method further comprises f) imaging the multiplexed biofunctionalized biosensor to determine the locations of the sensing regions.
  • step f) comprises in-situ tSPL imaging of the multiplexed biofunctionalized biosensor.
  • the present invention provides a multiplexed biofunctionalized biosensor comprising a plurality of biofunctionalized bioreceptors produced according to the method of the present invention.
  • the present invention relates to a multiplexed biofunctionalized biosensor comprising: a) a transistor substrate comprising a semiconductive layer; b) a polymer layer coating the surface of the transistor substrate; and c) a plurality of regions of the polymer surface functionalized with a plurality of capture molecules.
  • the transistor substrate is a field effect transistor (FET).
  • the semiconductive layer comprises graphene.
  • the transistor substrate comprises a plurality of graphene FETs (gFETs), wherein the gFETs comprise isolated graphene islands.
  • the polymer layer comprises a first monomer A and a second monomer B; wherein monomer A comprises a cross-linking functional group; and wherein monomer B comprises a protected functional group; and wherein capture molecules in the functionalized regions of the polymer layer are conjugated to the deprotected functional group of monomer B.
  • the cross-linking functional group of monomer A is selected from the group consisting of an azide, a diaziridine, and a methacrylate.
  • the cross-linking functional group of monomer A comprises cinnamate methyl ester.
  • the functional group is selected from the group consisting of a thiol, an alcohol, an amine, a carboxylic acid, an aldehyde, a ketone, and an alkyne.
  • the protecting group comprises one or more selected from the group consisting of tetrahydropyranyl carbamate, amine N-oxide, tetrahydropyranyl ether, triphenylmethyl ether, tetrahydropyranyl carbonate ester, S-tetrahydropyranyl carbonyl, ethyl disulfide, cyclopropenone, and tert-butyl ester groups.
  • the backbone of the polymer is selected from the group consisting of a poly(methacrylate), poly(acrylate), poly(ester), poly(amide), poly(styrene), poly(olefin), and combinations and co-polymers thereof.
  • the polymer further comprises a third monomer C, wherein monomer C comprises a solubilizing group.
  • the solubilizing group of monomer C comprises an alkyl, alkoxyl, or aryl chain.
  • the multiplexed biofunctionalized biosensor further comprises a second polymer layer comprising an electrically resistive polymer.
  • the functionalized regions of the polymer surface have a surface area between about 1 pm 2 and about 10 mm 2 . In some embodiments, the area of each functionalized region is between about 8,000 nm 2 and about 250 ⁇ m 2 .
  • the capture molecule is selected from the group consisting of aptamers, antibodies, antibody fragments, peptides, lectins, enzymes, enzyme fragments, nanobodies, and small molecules. [0031] In some embodiments, the capture molecule is directly conjugated to the functional group of the polymer layer.
  • the capture molecule is conjugated or bound to a linker molecule on a first end of the linker molecule and a second end of linker molecule is conjugated to the polymer.
  • the linker molecule is conjugated or bound to the capture molecule by an amine, an aldehyde, a thiol, a cycloalkyne, a cyclopropenone, an alkene, an alkyne, an azide, maleimide, a maleimide derivative, biotin, a biotin derivative, streptavidin, a streptavidin derivative, avidin, and an avidin derivative.
  • the regions of the polymer surface functionalized with capture molecules are arranged in a grid pattern. In some embodiments, the distance between the regions of the polymer surface functionalized with capture molecules is between about 100 nm and about 10 ⁇ m.
  • Attorney Docket No.206256-0105-00WO [0033] In some embodiments, the transistor substrate is grounded or at a fixed bias. [0034] In some embodiments, the present invention provides a method of detecting the presence of a target comprising contacting a multiplexed biofunctionalized biosensor of the present invention with a sample.
  • the sample is gaseous, airborne, or aerosolized, wherein the regions of the polymer surface functionalized with a plurality of capture molecules of the multiplexed biofunctionalized biosensor are covered with a buffer solution; and wherein contacting the multiplexed biofunctionalized biosensor with the sample comprises contacting the sample with the buffer solution.
  • Figure 1 depicts representative biochemical functionalization of FETs with sub-20 nm resolution by thermal scanning probe lithography (tSPL).
  • Figure 1A depicts a representative 3D-Render of the bioFET platform for FETs based multiplexed nanoscale biosensing.
  • Figure 1B depicts a representative schematic showing the steps of fabrication in the bioFET platform, from polymer deposition onto the FETs, to tSPL nanoscale activation of amine groups, and bioreceptor bioconjugation.
  • Figure 1C depicts a representative thermal scanning probe microscopy (SPM) topographical image of a tSPL nanoscale amine pattern fabricated in poly(phthalaldehyde) (PPA)/polymethacrylate-carbamate- cinnamate (PMCC).
  • SPM thermal scanning probe microscopy
  • PPA poly(phthalaldehyde)
  • PMCC polymethacrylate-carbamate- cinnamate
  • FIG. 1D and Figure 1E depict representative fluorescence microscopy images of biotinylated aptamers terminated with a red dye on a PPA/PMCC SiO2/Si chip. Specifically, the image shows Biotinylated Anti-SARS-CoV-2 Aptamer.
  • the sample After tSPL patterning, the sample is covered with a solution of 100 nM NHS-Biotin in DMSO and incubated for 1 hour. The sample is then functionalized with 100 nM streptavidin in 1x PBS for 30 min. After washing and drying, the sample is functionalized with the red dye- tagged aptamer.
  • Figure 1F and Figure 1G depict representative tSPL topography (f) and friction Attorney Docket No.206256-0105-00WO AFM (g) images of four PPA/PMCC coated GFETs with a channel width of 1 mm, where the areas above the GFET’s channel (graphene) sensors have been patterned by tSPL to remove PPA and activate amine groups on PMCC.
  • Figure 2 comprising Figure 2A through Figure 2C, depicts a representative comparison of non-specific binding using fluorescence intensity analysis.
  • Figure 2A depicts a representative fluorescence image comparing the fluorescent intensity of samples with squares patterned at intervals of 25 °C on a PMCC stack.
  • Figure 2B depicts a representative fluorescence image comparing the fluorescent intensity of samples with squares patterned at intervals of 25 °C on a PMMC/PPA stack.
  • Figure 2C depicts the fluorescence intensity profiles along the dashed lines in Figure 2A and Figure 2B.
  • Figure 3 comprising Figure 3A through Figure 3F, depicts representative bioFET fabrication for multiplexed nanoscale biosensing.
  • Figure 3A depicts a representative schematic of consecutive tSPL patterning and functionalization rounds to fabricate adjacent FETs, each functionalized with a different bioreceptor for multiplexed biochemical sensing.
  • Figure 3B depicts a representative fluorescence microscopy image of four different NHS-ester terminated dyes (red – squares/top left, yellow-orange – circles/top right, sky-blue – stars/bottom left, and green – triangles/bottom right) attached to amine moieties patterned by tSPL on a PPA/PMCC SiO2/Si chip.
  • Figure 3C depicts a representative fluorescence microscopy image of four representative graphene-based FETs functionalized with four different types of NHS-ester terminated dyes (green – top left, sky-blue – top right, red – bottom left, and yellow-orange – bottom right).
  • Figure 3D and Figure 3E depict representative fluorescence microscopy images of two different biotinylated aptamers with terminated red (dark grey) and green (light grey) dyes on a PPA/PMCC/SiO 2 /Si chip. Specifically, the images show biotinylated anti-SARS-CoV-2 aptamer (red, dark grey) and biotinylated anti-Hemagglutinin (HA) aptamer (green, light grey). The image in Figure 3E shows an alternating two-aptamer pattern of 500 nm circles.
  • Figure 3F depicts, representative SPM images of a bi-functionalized bioFET.
  • first in- situ tSPL topographical image of a matrix of circles produced after a first round of tSPL on a PPA/PMCC/SiO2/Si chip
  • second in-situ tSPL topographical image of the same area after conjugation of the surface with bioreceptor 1 (HA aptamer) using biotin/streptavidin as cross linker and after a second round of tSPL to pattern a second matrix of 20 nm dashes
  • third in-situ tSPL topographical image of the same area after conjugation of bioreceptor 2 (CoV-2 aptamer) Attorney Docket No.206256-0105-00WO using biotin/streptavidin as cross linker.
  • FIG. 1 The respective cross section images show the registry and robustness of multiplexed patterning and the change in depth of the patterns after functionalization due to the filling of each pattern with the NHS-biotin/streptavidin/aptamer molecules (approximately 10 -15 nm).
  • Figure 4 depicts representative topographical data at different stages of functionalization.
  • Figure 5 depicts representative spike protein– Antibody capture on the NanoBioFET platform.
  • Figure 5A depicts a representative schematic of a gFET functionalized with SARS-CoV-2 antibody using the NanoBioFET platform. The device structure, electrical measurement setup and ⁇ Debye at 1 mM HEPES buffer are indicated.
  • Figure 5B depicts representative SPR measurement at 1 mM HEPES buffer, demonstrating successful interaction between the antibody and spike protein.
  • Figure 5C depicts representative transfer characteristics of the gFET device.
  • the p-branch and n-branch are indicated.
  • the current and voltage range used in the transient measurement in panel d is indicated by the dashed box.
  • Figure 5D depicts representative transient response of the antibody-modified gFET in 1mM HEPES. Circle and diamond symbols denote, respectively, injections of buffer and spike protein of increasing concentrations. Dashed lines are a guide-to-the-eye of the measured signal.
  • Figure 5E depicts a representative sensitivity plot of the gFET, obtained from a straight line fit (dashed line) to the measured ⁇ Ids/I0 data at different spike protein concentrations.
  • FIG. 6 depicts representative stability of gFET transfer characteristics.
  • the steady-state drain current values from the transient Id(t) measurement shows the stability of the p-branch (left Attorney Docket No.206256-0105-00WO of minimum) and the distortion of the n-branch (right of minimum).
  • the solid curves are obtained from Ids-Vgs measurements, while the symbols represent the data from the transient measurement.
  • Figure 7, comprising Figure 7A and Figure 7B, depicts representative transient noise analysis.
  • Figure 7A depicts representative steady-state transient measurement.
  • Figure 7B depicts a representative noise power spectrum.
  • Figure 8 comprising Figure 8A through Figure 8D, depicts representative Spike protein– Aptamer capture on the NanoBioFET platform.
  • Figure 8A depicts a representative schematic of a gFET functionalized with SARS-CoV-2 aptamer using the NanoBioFET platform paired with a control device.
  • Figure 8B depicts representative florescence microscopy images of the sensor and control gFET devices, demonstrating successful immobilization of the fluorescently tagged aptamer exclusively on the sensor channel.
  • the dashed lines denote the metal electrodes of the gFET devices, and the rectangle denotes the graphene channel.
  • Graphene channel is denoted with a solid box.
  • FIG. 8C depicts a representative SPR measurement at 0.1X PBS of binding between the aptamer and spike protein.
  • Figure 8D depicts representative transient responses of the aptamer-modified gFET and the adjacent control gFET in 0.1X PBS.
  • the capability of the NanoBioFET platform in simultaneously monitoring the transient characteristics of the sensor and control gFETs gives confidence in fidelity of the detected signal by the sensor in response to the spike protein injections.
  • Circle and diamond symbols denote, respectively, injections of buffer and spike protein of increasing concentrations. Dashed lines are a guide-to-the-eye of the measurement signal.
  • Figure 9 depict representative Ultrasensitive and selective detection of live human SARS-CoV-2 virus.
  • Figure 9A depicts a representative schematic of a gFET functionalized with SARS-CoV-2 antibody using the NanoBioFET platform ready for detection of live SARS-CoV-2 virus.
  • Figure 9B depicts a representative transient response of the antibody-modified gFET with live virus injections in 1mM HEPES. Injections of virus medium (circle), SARS-CoV-2 virus (diamond), and H1N1 virus (star) are indicated.
  • FIG. 10 depicts a representative modelled secondary structure of the two-hairpin prediction for the SARS-CoV-2 Aptamer-6C3 (SEQ ID NO:1) using mfold software.
  • Figure 11 depicts a representative modelled secondary structure of the two-hairpin prediction for the Hemagglutinin Aptamer-RHA-0006 (SEQ ID NO:2) using mfold software.
  • Figure 12 depicts images of a representative microfluidic chamber, showing the two halves before closing the chamber for experimentation (top) and the closed chamber during electrical measurements (bottom).
  • Figure 13 depicts representative 1 H and 13 C NMR spectra for 2-((((tetrahydro-2H-pyran- 2-yl)oxy)carbonyl)amino)ethyl methacrylate.
  • Figure 14 depicts representative 1 H and 13 C NMR spectra for methyl (E)-3-(4-(3- hydroxypropoxy)phenyl)acrylate.
  • Figure 15 depicts representative 1 H and 13 C NMR spectra for (E)-3-(4-(3-methoxy-3- oxoprop-1-en-1-yl)phenoxy)propyl methacrylate.
  • Figure 16 depicts representative 1 H and 13 C NMR spectra for 3-bromopropyl methacrylate.
  • Figure 17 depicts a representative full (top) and zoomed-in (bottom) 1 H NMR spectrum of PMCC.
  • Figure 18, depicts a representative gFET device A.
  • Figure 18A depicts a representative optical image of a gFET device A before spin-coating, annotated with the source and drain electrons. The graphene channel regions are indicated by the dashed lines.
  • Figure 18B depicts a representative simple resistor model for the device shown in Figure 18A, consisting of the graphene channel resistance and the source/drain contact resistances.
  • Figure 19, comprising Figure 19A through Figure 19F, depicts representative device schematics and corresponding ionic gate characteristics of the source and drain currents for different biasing conditions of two devices A1 and A2.
  • Figure 19A depicts a representative schematic for devices 1 and 2 biased.
  • Figure 19B depicts representative ionic gate characteristics for Figure 19A.
  • Figure 19C depicts a representative schematic for devices A1 biased and device A2 grounded.
  • Figure 19D depicts representative ionic gate characteristics for Figure 19C.
  • Figure Attorney Docket No.206256-0105-00WO 19E depicts a representative schematic for device A1 grounded and device A2 biased.
  • Figure 19F depicts representative ionic gate characteristics for Figure 19E.
  • Figure 20, comprising Figure 20A and Figure 20B depicts a representative two-well device.
  • Figure 20A depicts a photograph of a representative two-well device, with drain, source, and ionic gate connections annotated.
  • Figure 20B depicts a schematic representation of a simple capacitor model of the two devices in the separate wells.
  • Figure 21, depicts representative device schematics and corresponding ionic gate characteristics of the source and drain currents for different biasing conditions of two devices B1 and B2.
  • Figure 21A depicts a representative schematic for devices B1 and B2 wherein the substrate is floating and device B1 gated.
  • Figure 21B depicts representative ionic gate characteristics for Figure 21A.
  • Figure 21C depicts a representative schematic for devices B1 and B2 wherein the substrate is grounded and device B1 gated.
  • Figure 21D depicts representative ionic gate characteristics for Figure 21B.
  • Figure 21E depicts a representative schematic for devices B1 and B2 wherein the substrate is grounded and device B2 gated.
  • Figure 21E depicts representative ionic gate characteristics for Figure 21D.
  • Figure 22 depicts representative optimization of amine exposure through fluorescence imaging.
  • Figure 22A depicts a representative fluorescence image demonstrating temperature optimization, with 5 ⁇ m squares patterned in a grid ranging from 600 °C to 1,400 °C in steps of 50 °C, with a dwell time of 120 ⁇ s, and a load of 6.5 V.
  • Figure 22B depicts representative images demonstrating UV crosslink time optimization, with 5 ⁇ m squares patterned in PMCC crosslinked for 40 minutes (left) or 60 minutes (right).
  • Figure 23 depicts representative aerosol detection of SARS-CoV-2 in a bioFET and specificity of the bioFET against other viruses.
  • the present invention relates to electrical devices for rapid multiplexed biosensing and methods of producing and using said devices.
  • the methods of producing a multiplexed biosensor comprise functionalization of a semiconductor substrate.
  • the methods of producing a multiplexed biosensor comprise coating a semiconductor substrate with a polymer, selectively activating polymer from a region of the substrate, and contacting the activated region with a capture molecule.
  • Attorney Docket No.206256-0105-00WO [0060]
  • the devices comprise a plurality of biosensors, wherein each biosensor is modified with a capture molecule.
  • the capture molecules bind a variety of targets, including, but not limited to, proteins, peptides, lipids, nucleic acids, and small molecules. In some embodiments, binding of a target to a capture molecule induces a change in charge, voltage, resistance, current, and/or capacitance of the biosensor.
  • 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.
  • an element means one element or more than one element.
  • “About” as used herein when referring to a measurable value such as an amount, a temporal 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.
  • antibody refers to an immunoglobulin molecule, which is able to specifically bind to a specific epitope on an antigen.
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules.
  • the antibodies in the present invention may exist in a variety of forms including, 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).
  • the term "analyte” refers to a substance to be detected or assayed by the method of the invention.
  • Typical analytes may include, but are not limited to proteins, peptides, nucleic acid segments, molecules, cells, microorganisms and fragments and products thereof, or any Attorney Docket No.206256-0105-00WO substance for which attachment sites, binding members or receptors (such as antibodies) can be developed.
  • aptamers refers to nucleic acids (typically DNA, RNA, or oligonucleotides) that emerge from in vitro selections or other types of aptamer selection procedures well known in the art (e.g. bead-based selection with flow cytometry or high-density aptamer arrays) when the nucleic acid is added to mixtures of molecules.
  • Ligands that bind aptamers include but are not limited to small molecules, peptides, proteins, carbohydrates, hormones, sugar, metabolic byproducts, cofactors, drugs, and toxins. 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 Kd range similar to antibody (pM to nM) and specificity similar/superior to antibody (Tuerk and Gold, 1990, Science, 249:505; Ellington and Szostak, 1990, Nature 346:818). An aptamer will typically be between 10 and 300 nucleotides in length.
  • Aptamers may comprise only deoxyribonucleotides (D-aptamers), only ribonucleotides (R-aptamers), or a combination of both (hybrid aptamers).
  • the nucleotides of an aptamer may be modified to alter binding affinity, increase their stability, or reduce aggregation.
  • Biological sample as used herein means a biological material isolated from an individual.
  • the biological sample may contain any biological material suitable for detecting a target analyte, and may comprise cellular and/or non-cellular material obtained from the individual.
  • diagnosis means detecting a disease or disorder or determining the stage or degree of a disease or disorder.
  • a diagnosis of a disease or disorder is based on the evaluation of one or more factors and/or symptoms that are indicative of the disease. That is, a diagnosis can be made based on the presence, absence or amount of a factor which is indicative of presence or absence of the disease or condition.
  • Each factor or symptom that is considered to be indicative for the diagnosis of a particular disease does not need be exclusively related to the particular disease, i.e. there may be differential diagnoses that can be inferred from a diagnostic factor or symptom.
  • there may be instances where a factor or symptom that is indicative of a particular disease is present in an individual that does not have the particular disease.
  • the diagnostic methods may be used independently, or in combination with other diagnosing and/or staging methods known in the medical art for a particular disease or disorder.
  • Attorney Docket No.206256-0105-00WO [0070]
  • the “level” of one or more target analyte means the absolute or relative amount or concentration of the analyte in the sample.
  • “Measuring” or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or amount (which can be an effective amount) of a given substance within a sample, including the derivation of qualitative or quantitative concentration levels of such substances.
  • Standard control value refers to a predetermined amount of a particular analyte that is detectable in a sample.
  • the standard control value is suitable for the use of a method of the present invention, in order for comparing the amount of a target analyte of interest that is present in a sample.
  • An established sample serving as a standard control provides an average amount of the analyte of interest in the sample type (e.g., biological sample) that is typical for an average, healthy person of reasonably matched background, e.g., gender, age, ethnicity, and medical history.
  • a standard control value may vary depending on the target analyte of interest and the nature of the sample.
  • the invention relates to devices comprising a plurality of biosensors.
  • the biosensors comprise a biofunctionalized field-effect transistor (bioFET).
  • each bioFET on a multiplexed biofunctionalized biosensor of the present invention comprises a FET comprising a source electrode, drain electrode, a gate electrode, and a semiconducting material.
  • the bioFET comprises a semiconducting material.
  • Exemplary semiconducting materials of the bioFET include, but are not limited to, silicon, silica, alumina, carbon nanotubes, III-V and II-VI semiconductors, graphene, 2D materials, MoS 2 , transition metal dichalcogenides, or other suitable materials.
  • the bioFET comprises one or more layers of semiconducting materials.
  • the bioFET comprises one, two, three, four, five, six, seven, eight, nine, or ten layers of semiconducting materials. In some embodiments, each layer comprises a different semiconducting material. In some embodiments, two or more layers may comprise the same semiconducting material. In some embodiments, the bioFET comprises a silica layer. In some embodiments, the bioFET comprises an alumina layer. In some embodiments, the bioFET comprises a graphene layer. In some embodiments, the bioFET comprises a bottom silica layer and a top alumina layer. In some embodiments, the bioFET comprises a bottom silica layer and a top graphene layer.
  • the bioFET comprises a bottom silica layer, a middle alumina layer, and a top graphene layer.
  • the bioFET comprises a polymer layer coated upon the semiconducting material or a gate.
  • the polymer is an external stimulus- responsive polymer, wherein exposure of the polymer to an external stimulus activates or functionalizes the exposed area of the polymer.
  • the polymer is sensitive to, or responsive to, an external stimulus (e.g., heat), wherein application of the external stimulus to a specific area of the polymer induces or activates the formation of a functional group (also referred to herein as a “sticky end”) in the desired specific area.
  • an external stimulus e.g., heat
  • the activated functional group of the polymer for example an amine group, is used to attach (directly or by using other functionalization intermediate steps) a probe or capture- molecule to the locally activated area of the polymer, thereby functionalizing the bioFET in the desired location (sensing area), for example the semiconducting region, the top gate, or the Attorney Docket No.206256-0105-00WO extended gate.
  • binding, or capture, of a target molecule of interest by the functionalized bioFET induces a detectable change in the electronic signals of the bioFET, thereby indicating the presence and quantity of the analyte of interest in a sample.
  • an extended gate is used, where the top gate of a FET is externally electrically connected to a gate which is coated with an external stimulus-sensitive polymer (e.g., thermally sensitive polymer) and functionalized to sense the presence of analytes.
  • an external stimulus-sensitive polymer e.g., thermally sensitive polymer
  • the “polymer” may be any polymer that can be selectively activated by a localized external stimulus. Examples of localized external stimuli include, but are not limited to, a local source of heat, electrical field, light, current, pressure, and shear force.
  • This activation can be obtained by any means of controlled delivery of the stimulus, including, but not limited to, an external probe (for example a nano-sized atomic force microscopy tip), a beam (for example a laser beam), or direct local deposition of molecules.
  • the local activation creates on the polymer surface a chemical functional group that can be used to attach a capture-molecule.
  • the polymer comprises a heat-sensitive or heat-responsive polymer. Any polymer in which localized heat or electromagnetic field induces production or activation of a functional group that can be used to attach a probe or capture-molecule, can be used in the present invention.
  • the polymer comprises a heat- sensitive or heat-responsive polymer, in which an amine group is activated upon local heating.
  • the polymer comprises a heat-sensitive or heat-responsive polymer, in which the local heat is effective to remove a first functional group from the polymer such that the surface comprises a second functional group at least at a portion of the first location; wherein the first functional group is for example but not limited to a tetrahydropyranyl carbamate, amine N-oxide, tetrahydropyranyl ether, triphenylmethyl ether, tetrahydropyranyl carbonate ester, S-tetrahydropyranyl carbonyl, or ethyl disulfide; and wherein the second functional group is a thiol, an alcohol, a carboxylic acid and its derivatives, an amine, an alkyne, an aldehyde, or a ketone.
  • Tertiary butyl ester groups can also be used as the first functional group followed by localized deprotection.
  • the tertiary butyl ester group can be converted to a carboxylic acid group with localized external stimulus.
  • the resulting carboxylic acid may be further converted to an anhydride with additional stimulus (see for example “Atomic force microscopy based thermal lithography of poly(tert-butyl acrylate) block copolymer films for bioconjugation.” Langmuir 24, 10825–10832 (2008); and “Scanning Attorney Docket No.206256-0105-00WO thermal lithography of tailored tert-butyl ester protected carboxylic acid functionalized (Meth)acrylate polymer platforms.” ACS Appl.
  • the polymer can contain groups that undergo thermal polymerization and cross- linking reactions, including Diels-Alder reactions.
  • the polymer comprises a heat-sensitive or heat-responsive polymer, in which an amine group is activated upon local heating.
  • Exemplary polymers that result in activated amines upon local heating include, but are not limited to: poly(methacrylate) copolymer: poly-((tetrahydropyran-2-yl N-(2 methacryloxyethyl) carbamate)-b-(meth-yl 4-(3- methacryloyloxypropoxy) cinnamate)) (PMCC) (Liu, X., et al., 2019, ACS Appl Mater Interfaces, 11(44):41780-41790; Liu, X., et al., 2021, Adv Funct Mater, 31(19):2008662).
  • poly(methacrylate) copolymer poly-((tetrahydropyran-2-yl N-(2 methacryloxyethyl) carbamate)-b-(meth-yl 4-(3- methacryloyloxypropoxy) cinnamate)) (PMCC) (Liu,
  • the polymer comprises a heat-sensitive or heat-responsive polymer, in which a hydroxyl or carboxylic group is activated upon local heating.
  • exemplary polymers that result in activated hydroxyl or carboxylic groups upon local heating include, but are not limited to, PMCC and copolymers of benzyl methacrylate and THP-MA, which exhibit thermal deprotection of the THP group to produce carboxylic acid around 150 °C, which is followed by conversion to anhydrides above 180 °C, a polymer where thermal deprotection of an ester group can produce a carboxylic acid group.
  • the polymer is represented by a formula (Al-Bm)n, wherein A is a monomer with a cross-linking functional group, B is a monomer with a protected functional group that can be deprotected by external stimulus such as heat or an electromagnetic field, n is an integer selected from 1-10,000, each instance of l and m is independently an integer selected from 0-100, and at least one instance of l or m is an integer selected from 1-100.
  • monomer A comprises an azide, a diaziridine, or a methacrylate.
  • monomer A comprises a cinnamate group.
  • monomer B comprises a tetrahydropyranyl carbamate, amine N- oxide, tetrahydropyranyl ether, triphenylmethyl ether, tetrahydropyranyl carbonate ester, S- tetrahydropyranyl carbonyl, or ethyl disulfide.
  • the different functional group is an amine, alcohol, phenol, or thiol.
  • the polymer comprises poly((tetrahydropyran-2-yl N-(2- methacryloxyethyl)carbamate)-co-(methyl 4-(3-methacryloyloxypropoxy)cinnamate))
  • Attorney Docket No.206256-0105-00WO (simplified to polymethacrylate-carbamate-cinnamate copolymer; “PMCC”) as represented by the formula: wherein n is an integer between l and m is independently an integer between 0 and 100, and at least one m an between 1 and 100.
  • the polymer is represented by a formula (Al-Bm-Co)n, wherein A is a monomer with a cross-linking functional group, B is a monomer with a protected functional group that can be deprotected by external stimulus such as heat or an electromagnetic field, C is a monomer residue which contains a solubilizing group, n is an integer selected from 1-10,000, each instance of l, m, and o is independently selected from 0-100, and at least one instance of l, m, or o is selected from 1-100.
  • Exemplary solubilizing groups include, but are not limited to alkyl, alkoxyl, aryl chains, or combinations thereof, that can increase solubility of the polymeric material.
  • the bonds between monomers A, B, and C are the backbone of the polymer, for example, but not limited to, a poly(methacrylate), poly(acrylate), poly(ester), poly(styrene), poly(amide), or poly(olefin) backbone.
  • monomer A comprises an azide, a diaziridine, or a methacrylate.
  • monomer A comprises a cinnamate group.
  • monomer B comprises a tetrahydropyranyl carbamate, amine N- oxide, tetrahydropyranyl ether, triphenylmethyl ether, tetrahydropyranyl carbonate ester, S- tetrahydropyranyl carbonyl, or ethyl disulfide.
  • the different functional group is an amine, alcohol, phenol, or thiol.
  • monomer C comprises an alkyl, alkoxyl, or aryl chain, or a combination thereof. In some embodiments, monomer C comprises a short alkyl chain.
  • monomer C is a short alkoxy chain.
  • the polymer comprises poly((tetrahydropyran-2-yl N-(2- methacryloxyethyl)carbamate)-co-(n-butyl methacrylate)-co-(methyl 4-(3- methacryloyloxypropoxy)cinnamate)) (simplified to polymethacrylate-butyl-carbamate- cinnamate copolymer; “PMBCC”) as represented by the formula: wherein n is an integer between 1 and 1,000, each instance of l, m, and o is independently an integer between 0 and 100, and at least one instance of l, m, and o is an integer between 1 and 100.
  • the polymer can be represented by a formula B n , wherein B represents a monomer in a polymer backbone with a functional group that will be modified by a local external stimulus and n is a positive integer.
  • B represents a monomer in a polymer backbone with a functional group that will be modified by a local external stimulus and n is a positive integer.
  • the polymer comprises more than one polymer backbone and/or functional group.
  • 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 a substrate or platform.
  • the polymer backbone can be derived from, or can be, a monomer such as vinyl, allyl, 4-styryl, acroyl, epoxide, oxetane, cyclic-carbonate, methacroyl, acrylonitrile, or the like, which is polymerized by either a radical-, cationic-, atom transfer-, or anionic-polymerization process.
  • a monomer such as vinyl, allyl, 4-styryl, acroyl, epoxide, oxetane, cyclic-carbonate, methacroyl, acrylonitrile, or the like, which is polymerized by either a radical-, cationic-, atom transfer-, or anionic-polymerization process.
  • the polymer backbone can be derived from, or can be, an isocyanate, isothiocyanate, or epoxide, that can be copolymerized with di- Attorney Docket No.206256-0105-00WO functional amines or alcohols such as HO(CH2) ⁇ OH, H2N(CH2)XNH2, where ⁇ is a positive integer (e.g., from 1 to 25).
  • the polymer backbone can be derived from, or can be, a strained ring olefin (e.g., dicyclopentadienyl, norbornenyl, cyclobutenyl, or the like), which can be polymerized via ring opening metathesis polymerization using an appropriate metal catalyst, as would be known by those skilled in the art to which this disclosure pertains.
  • a strained ring olefin e.g., dicyclopentadienyl, norbornenyl, cyclobutenyl, or the like
  • the polymer backbone can be derived from, or can be, -(CH 2 ) n SiCl 3 , -(CH 2 ) n Si(OCH 2 CH 3 ) 3 , or -(CH 2 ) n Si(OCH 3 ) 3 , where the monomers can be reacted with water under conditions known to those skilled in the art to form either thin film or monolithic organically modified sol-gel glasses, or modified silicated surfaces, where n is a positive integer (e.g., from 1 to 25).
  • the polymer backbone can be derived from, or can be, a polymerizable group that can be photochemically dimerized or polymerized.
  • Such groups can include, but are not limited to, one or more and the following conjugated structures: 6 alkyl, C 2-6 alkenyl, alkynyl, hydroxy, thiol, C 1-6 alkoxy, halogen, haloC 1-6 alkyl, haloC 1 - 6 alkoxy, C3-8 cycloalkyl, nitrile, -NR X R Y , aryl, heteroaryl, heterocyclyl, amide, ester, sulfone, sulfonamide, sulfoxide, , -(CH2)nSiCl3, -(CH2)nSi(OCH2CH3)3, or -(CH 2 ) n Si(OCH 3 ) 3 and combinations thereof; wherein R X and R Y are independently selected from the group consisting of hydrogen, deuterium, C1-6 alkyl, haloC1-6 alkyl, C3-8 cycloalkyl, -COC1-6 alkyl, and Attorney
  • B can be chosen such that, upon exposure to local external stimuli, a protecting group is removed from the surface, leaving behind another functional group.
  • the functional group of B can be chosen form tert-butyl esters, tetrahydropyran esters, and the like.
  • the functional group of B can include tetrahydropyranyl carbamates, amine N-oxides, and the like.
  • the functional group of B can be chosen from tetrahydropyranyl ethers, triphenylmethyl ethers, tetrahydropyranyl carbonate esters, and the like.
  • the functional group of B can include S-tert-butoxy carbonyls, S- tetrahydropyranyl carbonyls, ethyl disulfides, and the like.
  • the functional group of B can be a group that undergoes thermal polymerization and cross-linking reactions, including Diels-Alder reactions between two B functional groups (e.g., furans with maleimides, and the like), ring-opening polymerization (e.g., poly(ferrocenylsilanes) and the like), ring-opening metathesis polymerization (e.g., dicyclopentadiene, and the like), reactions to form conjugated polymers (e.g., from poly(phenylene-vinylene) or other like precursors), and reactions of trifluorovinyl ethers, for example.
  • B functional groups e.g., furans with maleimides, and the like
  • ring-opening polymerization e.g., poly(ferrocenylsilanes) and the like
  • ring-opening metathesis polymerization e.g., dicyclopentadiene, and the like
  • conjugated polymers e.g.,
  • the functional group of B can be a group that volatilizes or decomposes from treatment with the local external stimuli.
  • the polymer can have more than one B monomer, with more than one functional group. These functional groups can be chosen such that each functional group of each B is modified at the same or a different temperature.
  • the polymer can have a monomer A, which can be photochemically or thermally cross-linked to control the softening temperature of the overall polymer. In some embodiments, with the use of the A group, the softening temperature can be tailored to be above or below the chemical modification temperature as desired.
  • this can be accomplished by increasing or decreasing the glass transition temperature and/or the crystallinity of the polymer.
  • the A monomer and the B monomer can be organized in blocks, ordered, or randomly oriented.
  • the A and B monomers are derived have the same functional monomer unit.
  • the A and B monomers are derived from different functional monomer units.
  • Attorney Docket No.206256-0105-00WO the functional group of the A monomer can be chosen from cinnamate esters, alkenes, chalcones, trifluorovinyl ethers, Diels-Alder reactants, or the like.
  • the polymer has an average molecular weight at least about 1 kg/mol. In some embodiments, the polymer has an average molecular weight of at least about 10 kg/mol. In some embodiments, the polymer has an average molecular weight of at least about 20 kg/mol. In some embodiments, the polymer has an average molecular weight of at least about 30 kg/mol. In some embodiments, the polymer has an average molecular weight of at least about 40 kg/mol. In some embodiments, the polymer has an average molecular weight of at least about 50 kg/mol. In some embodiments, the polymer has an average molecular weight of at least about 60 kg/mol.
  • specific polymers that can be used as the surface for reaction with local external stimuli include the following tetrahydropyran—(THP) protected carboxylic acid- functionalized poly(acrylate): [0099] In some can be thermally deprotected at about 120 °C to give a hydrophilic acid functionality. In some embodiments, the functional group will react further at about 170 °C to give a hydrophobic anhydride. This represents a surface that can undergo a so-called “read-write-overwrite process.” It is important to note that the acid to anhydride conversion is reversible by the removal and addition of water, respectively.
  • the deprotected functional groups of monomer B in the activated region of the polymer are then reacted using click-chemistry to attach a desired capture- molecule.
  • free thiol groups on the surface revealed by the local external stimulus can undergo thiol-ene click chemistry with an alkene-functionalized capture-molecule.
  • the deprotected amine groups can be converted to azides.
  • the polymer can incorporate azides as functional groups that are deprotected by external stimuli such as heat.
  • the polymer can incorporate protected alkenes or alkynes that can be deprotected via local external stimuli to react with an azide- functionalized capture-molecule.
  • thiols, alcohols, and amines can be turned into alkyne or cycloalkynes for participation in copper-catalyzed and/or strain-promoted Attorney Docket No.206256-0105-00WO alkyne azide click chemistry.
  • the click chemistry reaction is high yielding without side-reactions, byproducts, or the requirement of harsh reaction conditions.
  • a chemical reaction between the functional group and the desired capture- molecule can be initiated using 1,3-cycloaddition of diazides and diynes to form poly(arylenetriazolylene).
  • the polymer comprises two acrylate monomers (a crosslinking monomer containing an acrylate (e.g., cinnamate) and a monomer with a protected reactive functional group (e.g., an amine group) that can be deprotected under high temperatures) and is formed as a statistical mixture of these two monomers based on feed ratios using a free radical polymerization method.
  • a crosslinking monomer containing an acrylate e.g., cinnamate
  • a monomer with a protected reactive functional group e.g., an amine group
  • RAFT reversible addition fragmentation
  • Block copolymers yield materials with defined cross-linking areas and functionalization parts. Additionally, block copolymer from nanostructures on surfaces (such as gyroid and lamellar structures) allowing for predefined special distribution of functional groups. Gradient copolymers allow for the formation of gradient surface functionalization. Blocky copolymers yield smaller superstructures on surfaces with potential new functionalization schemes.
  • a living polymerization allows for complete control over polymer length, degree of polymerization and polydispersities, all variables that impact surface coverage, crosslinking and film forming properties.
  • the polymer is produced by reversible addition fragmentation (RAFT) polymerization, atom transfer radical polymerization (ATRP), or nitroxide mediated radical polymerization.
  • RAFT reversible addition fragmentation
  • ATRP atom transfer radical polymerization
  • nitroxide mediated radical polymerization is another strategy to tune polymeric surface properties.
  • a third monomer (monomer C) containing a solubilizing group such as a short alkyl chain increases solubility of the polymeric material during spin coating. This allows for the use of higher molecular weight polymers to be soluble and spin-coatable.
  • an exposed amine in an activated area of the polymer surface is further functionalized to create patterns of functional groups selected from the group of amines, amides, ammoniums, maleimides, aldehydes, thiols, biotins, and azides.
  • the shape of each activated area on the polymer surface is the same. In some embodiments, activated areas on the polymer surface vary in shape based on what capture molecule is, or will be, used to functionalize that activated area.
  • Shapes of activated areas can include, but are not limited to, circles, ovals, squares, rectangles, triangles, pentagons, hexagons, heptagons, octagons, nonagons, decagons, stars, letters, numbers, and any other shape which may reasonably be created by the technique utilized to activate the area.
  • the activated areas on the polymer surface can each independently have a surface area between about 1 pm 2 and about 10 mm 2 .
  • the surface area is about 1 pm 2 , about 5 pm 2 , about 10 pm 2 , about 15 pm 2 , about 20 pm 2 , about 25 pm 2 , about 30 pm 2 , about 35 pm 2 , about 40 pm 2 , about 45 pm 2 , about 50 pm 2 , about 60 pm 2 , about 70 pm 2 , about 80 pm 2 , about 90 pm 2 , about 100 pm 2 , about 150 pm 2 , about 200 pm 2 , about 250 pm 2 , about 300 pm 2 , about 350 pm 2 , about 400 pm 2 , about 450 pm 2 , about 500 pm 2 , about 600 pm 2 , about 700 pm 2 , about 800 pm 2 , about 900 pm 2 , about 1 nm 2 , about 2 nm 2 , about 3 nm 2 , about 4 nm 2 , about 5 nm 2 , about 6 nm 2 , about 7 nm 2 , about 8 nm 2 , about 9 nm 2 , about 10 n
  • the activated areas on the polymer surface have a depth relative to the unactivated areas on the polymer surface. In some embodiments, all activated areas on the polymer surface have the same depth. In some embodiments, the depth of the activated areas on the polymer surface varies based on what capture molecule is, or will be, used to functionalize the activated area. In some embodiments, the depth is between about 1 nm and about 100 nm. In some embodiments, the depth is between about 1 nm and about 50 nm.
  • the depth is about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 12 nm, about 14 nm, about 16 nm, about 18 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, or about 100 nm.
  • the bioFET comprises a one or more polymer layers atop a first polymer layer, which can help reduce non-specific binding of the probe/capture molecule.
  • the bioFET comprises a second polymer layer atop a first polymer layer.
  • the second polymer layer having low adhesion properties in respect to capture-molecules, is an external stimulus-responsive polymer that evaporates or sublimes upon administration of the external stimulus to which the first polymer responds.
  • the second polymer is a heat-sensitive or heat-responsive polymer where localized heating evaporates or sublimes the second polymer layer while also inducing or production or activation of the required functional group in the first polymer layer.
  • any polymer with these properties can be utilized, for example, but not limited to, is polyphthalaldehyde (PPA).
  • PPA polyphthalaldehyde
  • Another example are polymer resists composed of cyclic, low ceiling temperature poly(aldehydes).
  • Another example is molecular glasses (Howell, S. T., et al., 2020, Microsystems & Nanoengineering, 6:21).
  • the second polymer is configured as an anti-fouling coating to reduce non-specific bindings of capture molecules outside a sensing region.
  • the second polymer is removed by the localized external stimulus to expose the external stimulus-responsive polymer layer.
  • the bioFET comprises a capture-molecule attached to the functional group of the external stimulus-responsive polymer, wherein the capture-molecule specifically binds to an analyte of interest.
  • Capture-molecules for particular analytes of interest Attorney Docket No.206256-0105-00WO are known in the art, and can be selected in view of a number of considerations including analyte identity, analyte concentration, and the nature of the sample or sample conditions in which the analyte is to be detected.
  • Suitable capture-molecules include aptamers (nucleic acid or peptide), antibodies, antibody fragments, antibody mimetics (e.g., engineered affinity ligands), peptides (natural or modified peptides), proteins (e.g., recombinant proteins, host proteins), lectins, oligonucleotides, DNA, RNA (e.g., microRNAs), and organic small molecules (e.g., haptens or enzymatic co-factors, enzymes).
  • aptamers nucleic acid or peptide
  • antibodies antibody fragments
  • antibody mimetics e.g., engineered affinity ligands
  • peptides natural or modified peptides
  • proteins e.g., recombinant proteins, host proteins
  • lectins e.g., recombinant proteins, host proteins
  • lectins e.g., recombinant proteins, host proteins
  • lectins e.g
  • the polymer can be selected such that the distance between the probe or capture- molecule and the bioFET surface is such that association of an analyte of interest with the probe or capture-molecule induces a measurable change in the electronic properties of the bioFET.
  • the polymer is selected such that the distance between the capture-molecule and the surface of the FET is the range of about 1 nm to about 300 nm.
  • the distance is about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 12 nm, about 14 nm, about 16 nm, about 18 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 n
  • the bioFET 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.
  • one or more capture-molecules are immobilized on the surface of the FET via a linking group displayed by a polymer coating the FET surface.
  • the capture-molecules are bound to the activated functional groups of the polymer via biochemical conjugation or electrostatic binding. The binding can occur directly or by using intermediate functional groups, and linkers.
  • the capture-molecule of the invention comprises an antibody, or antibody fragment.
  • the antibody capture-molecule specifically binds to a compound of interest, for example a secreted compound of interest.
  • a compound of interest for example a secreted compound of interest.
  • Such antibodies include Attorney Docket No.206256-0105-00WO polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies.
  • Such antibodies may be produced in a variety of ways, including hybridoma cultures, recombinant expression in bacteria or mammalian cell cultures, and recombinant expression in transgenic animals. The choice of manufacturing methodology depends on several factors including the antibody structure desired, the importance of carbohydrate moieties on the antibodies, ease of culturing and purification, and cost.
  • the capture-molecule of the invention comprises an isolated nucleic acid, including for example a DNA oligonucleotide and an RNA oligonucleotide.
  • the nucleic acid capture-molecule specifically binds to a compound of interest, for example a DNA molecule or an RNA molecule (e.g., mRNA, rRNA, or lncRNA).
  • the nucleic acid capture probe of the invention may be in the form of a linear oligonucleotide or may have a secondary structure (e.g., a hairpin or loop) which promotes binding and capture of the target analyte.
  • the nucleic acid comprises a nucleotide sequence that is complementary to a nucleic acid of interest.
  • nucleotide sequences of a nucleic acid capture-molecule can alternatively comprise sequence variations with respect to the original nucleotide sequences, for example, substitutions, insertions and/or deletions of one or more nucleotides, with the condition that the resulting nucleic acid functions as the original and specifically binds to the compound of interest.
  • the nucleic acid comprises a nucleic acid aptamer.
  • Nucleic acid aptamers are synthetic oligodeoxynucleotides designed according to rigorous recognition and binding affinities between nucleotides, and are obtained by screening through systematic evolution of ligands by exponential enrichments (SELEX).
  • Nucleic acid aptamers not only have features similar to antibodies, such as highly specific recognition and highly binding affinities to targets.
  • An aptamer of the invention can have one or more modified nucleosides or modified Attorney Docket No.206256-0105-00WO nucleobase linkages.
  • the aptamer may be a thioaptamer that contains one or more phosphorothioate or phosphorodithioate moieties, 2'-fluoro-ribonucleotide oligomers, NH2-substituted and OCH3-substituted ribose aptamers, and deoxyribose aptamers.
  • capture aptamer refers to an aptamer that is bound to a substrate (e.g., a functionalized polymer on a bioFET) and comprises a configuration that can locate (i.e. bind in a sample) a target analyte, thereby causing the target analyte to be attached to the substrate via the capture aptamer upon binding.
  • a substrate e.g., a functionalized polymer on a bioFET
  • Aptamers configured to bind to specific target analytes can be selected, for example, by synthesizing an initial heterogeneous population of oligonucleotides, and then selecting oligonucleotides within the population that bind tightly to a particular target analyte.
  • heat activated functionalization of the polymer-coated surface can be temporally restricted, spatially restricted or both spatially and temporally restricted such that only a portion (e.g. a single zone or pattern) of the polymer is functionalized for attaching to a capture-molecule at a given time or in a given location of the surface of the FET (e.g. the channel of the FET or the extended gate).
  • the present invention relates to a method of producing a bioFET, as described herein.
  • the method comprises a) providing a transistor substrate comprising a semiconducting material and b) coating the semiconducting material with an external stimulus-responsive polymer.
  • the transistor substrate of step a) is a field effect transistor (FET).
  • step b) comprises spin-coating the transistor substrate with an external stimulus-responsive polymer.
  • the external stimulus-responsive polymer of step b) is any polymer described elsewhere herein.
  • the external stimulus-responsive polymer comprises a first monomer A and a second monomer B, wherein the A monomer comprises a cross-linking functional group, and wherein monomer B comprises a protected functional group that can be deprotected by an external stimulus to expose activated functional groups.
  • monomer A comprises an azide, a diaziridine, or a methacrylate.
  • monomer A comprises a cinnamate group.
  • the cinnamate group is a cinnamate methyl ester.
  • monomer B comprises a tetrahydropyranyl carbamate, amine N-oxide, tetrahydropyranyl ether, triphenylmethyl ether, tetrahydropyranyl carbonate ester, S-tetrahydropyranyl carbonyl, or ethyl disulfide.
  • the activated functional group is selected from the group consisting of an amine, alcohol, phenol, thiol, carboxylic acid, aldehyde, ketone, and alkyne.
  • the polymer further comprises a monomer C, wherein monomer C comprises a solubilizing group.
  • monomer C comprises an alkyl, alkoxyl, or aryl chain, or a combination thereof.
  • monomer C comprises a short alkyl chain. In some embodiments, monomer C is a short alkoxy chain.
  • the polymer backbone of the external stimulus-responsive polymer is selected from the group consisting of poly(methacrylate), poly(acrylate), poly(ester), poly(amide), poly(styrene), poly(olefin), and combinations and co-polymers thereof.
  • the average molecular weight of the external stimulus-responsive polymer is at least about 1 kg/mol. In some embodiments, the average molecular weight of the external stimulus-responsive polymer is at least about 10 kg/mol. In some embodiments, the average molecular weight of the external stimulus-responsive polymer is at least about 20 kg/mol. In some embodiments, the average molecular weight of the external stimulus-responsive polymer is at least about 30 kg/mol.
  • step b) further comprises coating the external stimulus-responsive polymer layer with a layer of a second polymer.
  • the second polymer evaporates or sublimes when exposed to the same external stimulus to which the external stimulus-responsive polymer responds.
  • the polymer is any polymer with these properties, including, but not limited to, polyphthalaldehyde (PPA).
  • PPA polyphthalaldehyde
  • Another example are polymer resists composed of cyclic, low ceiling temperature poly(aldehydes), and molecular glasses.
  • the method further comprises c) applying to the polymer-coated FET a localized external stimulus that induces production or activation of functional groups in the polymer, to which capture-molecules can then be attached.
  • a localized external stimulus that induces production or activation of functional groups in the polymer, to which capture-molecules can then be attached.
  • the localized external stimulus is heat or electromagnetic radiation.
  • any method of locally applying heat or electromagnetic radiation may be used.
  • the method comprises using thermal scanning probe lithography (tSPL) to locally heat the polymer surface.
  • tSPL thermal scanning probe lithography
  • tSPL R. Szoszkiewicz, et al., Nano Lett.2007, 7, 1064
  • tSPL can be performed using a commercially available instrument, which uses a thermal nanoprobe to locally evaporate the thermosensitive polymer polyphthalaldehyde (PPA), leaving a void that defines the pixel size (S. T. Zimmermann, et al., ACS Appl. Mater. Interfaces 2017, 9, 41454; S. T. Howell, et al., Microsyst. Nanoeng.2020, 6, 21; X. R.
  • tSPL can write topographical and chemical features in a thermosensitive polymer resist by local heating, thereby carving and chemically activating the surface with nanoscale precision (X. Y. Liu, et al., ACS Appl. Mater. Interfaces 2019, 11, 41780; D. B. Wang, et al., Adv. Funct. Mater.2009, 19, 3696).
  • the tSPL process involves first the pixilation of a reference input image, Attorney Docket No.206256-0105-00WO and then the replication of that image by assigning at each grey level of individual pixels a particular height level.
  • step c) comprises applying the localized external stimulus to the polymer coated FET to form an activated area with a length and a width independently between about 1 pm and about 10 mm. In some embodiments, the length and the width are independently between about 1 nm and about 1 mm.
  • the length and the width are independently about 1 pm, about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 600 pm, about 700 pm, about 800 pm, about 900 pm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 pm, about 100 pm
  • the activated area on the polymer surface has an area of between about 8,000 nm 2 and about 250 ⁇ m 2 .
  • step c) comprises producing an activated area on the polymer surface with a depth relative to the unactivated area on the polymer surface. In some embodiments, the depth is between about 1 nm and about 100 nm. In some embodiments, the depth is between about 1 nm and about 50 nm.
  • the depth is about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 Attorney Docket No.206256-0105-00WO nm, about 10 nm, about 12 nm, about 14 nm, about 16 nm, about 18 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, or about 100 nm.
  • the method further comprises d) functionalizing the FET by attaching one or more capture molecules to the generated or activated functional groups generated by localized heating of the polymer.
  • the method comprises contacting the surface of the polymer with a liquid medium comprising the capture agent, where the capture agent reacts with, binds to, or otherwise associates with the functional group of the polymer.
  • capture molecules include, but are not limited to, aptamers, antibodies, antibody fragments, peptides, lectins, enzymes, enzyme fragments, nanobodies, and small molecules.
  • step d) comprises directly reacting, binding, or associating the capture molecule with a functional group of the polymer.
  • step d) comprises i) reacting the functional group of the polymer with a functionalized linker molecule and ii) reacting, binding, or associating the capture molecule with the functionalized linker molecule.
  • the functionalized linker molecule comprises a first functional group that reacts, interacts, or associates with the activated functional group of the polymer, a linker, and a second functional group that reacts, interacts, or associates with a capture molecule.
  • the first functional group is any functional group which reacts, interacts, or associates with the activated functional group of the polymer.
  • first functional groups include, but are not limited to, an amine, an aldehyde, a thiol, a cycloalkyne, a cyclopropoenone, an alkene, a diene, an alkyne, a cycloalkyne, an azide, maleimide, and a maleimide derivative.
  • suitable linkers include, but are not limited to, an alkyl chain, an alkoxyl chain, a cycloalkyl group, an aryl group, a heteroaryl group, an ester, an amide, and combinations thereof.
  • second functional groups include, but are not limited to, an alcohol, an amine, a thiol, an aldehyde, a cyclopropenone, an alkene, a diene, an alkyne, a cycloalkyne, an azide, maleimide, a maleimide derivative, biotin, a biotin derivative, streptavidin, a streptavidin derivative, avidin, an avidin derivative, and combinations thereof.
  • the method further comprises e) repeating steps c) and d) one or more times to create one or more additional functionalized areas on the bioFET.
  • all capture molecules of the bioFET are the same. In some embodiments, different functionalized areas on the bioFET have different capture molecules. In some embodiments step e) is performed 1-100,000 times. [0137] In some embodiments, steps c) and d) are performed multiple times simultaneously. In some embodiments, an array of localized external stimuli are applied to external stimulus- responsive polymer coating at different locations of the polymer coating simultaneously. In some embodiments, a capture molecule is applied to all of the activated areas of the polymer coating. In some embodiments, a capture molecule is applied to all of the activated areas of the polymer coating simultaneously.
  • a plurality of capture molecules are applied to the activated areas of the polymer coating simultaneously. In some embodiments, a capture molecule is applied to each individual activated area. In some embodiments, a capture molecule is applied to each individual activated area simultaneously. In some embodiments, a different capture molecule is applied to each individual activated area. In some embodiments, a different capture molecule is applied to each individual activated area simultaneously. [0138] In some embodiments, a linker molecule is applied to all of the activated areas of the polymer coating. In some embodiments, a linker molecule is applied to all of the activated areas of the polymer coating simultaneously. In some embodiments, the linker molecules are all the same.
  • different linker molecules are applied to different activated areas.
  • a capture molecule is applied to all the activated and linker-treated areas of the polymer coating.
  • a capture molecule is applied to all the activated and linker-treated areas of the polymer coating simultaneously.
  • a plurality of capture molecules are applied to the activated and linker-treated areas of the polymer coating.
  • a plurality of capture molecules are applied to the activated and linker- treated areas of the polymer coating simultaneously.
  • a different capture molecule is applied to each individual activated and linker-treated area.
  • a different capture molecule is applied to each individual activated and linker-treated area simultaneously.
  • the spatial density distribution of molecules on the surface of a sensing platform can be controlled and be made consistent among all sensors in the sensing platform.
  • the spatial resolution of the sensor active region is about 10 nm, Attorney Docket No.206256-0105-00WO less than 1 ⁇ m, 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.
  • the sensor performance is controlled by the thickness of the polymer, where the polymer thickness can be controlled down to a single monolayer, in increments of one, two, three, four, five, six, seven, eight, nine, or ten monolayers, for example.
  • the polymer thickness ranges from between about 1 nm and about 300 nm.
  • the limit of detection ranges from 1 fM to 1 ⁇ M.
  • Suitable types of analytes include any molecule, virus, or organism that may be captured by a capture molecule.
  • analytes include, but are not limited to, proteins, nucleic acids, protein fragments, antigens, antibodies, surface receptors, hormones, growth factors, cells, viral particles, bacteria, secreted compounds, metabolites, and the like.
  • the precise combination of compounds of interest being assayed by way of the invention is easily controllable and defined by the eventual user. For example, detection of a particular target analyte is only limited by the availability of a capture agent (e.g. antibody, aptamer, peptide, nucleic acid sequence, etc.) that specifically binds to the compound and can be functionalized to the bioFET surface.
  • a capture agent e.g. antibody, aptamer, peptide, nucleic acid sequence, etc.
  • the capture- molecule comprises an antibody or a capture aptamer
  • the target analyte comprises a region or epitope that the antibody or capture aptamer binds to.
  • the method detecting one or more analytes includes the steps of obtaining a sample containing or potentially containing a target analyte, contacting a bioFET comprising a capture probe for detection of the target analytes of the invention with at least a portion of the sample, detecting changes in the charge, voltage, resistance, current, and/or capacitance of the regions of the bioFET based on the interaction of the target analyte in the sample with the capture probes on the bioFET, and identifying the sample as containing the one or more target analytes based on the detection of a change in electric potential in one or more regions of the bioFET.
  • a sample can be contacted with the bioFET so that a target analyte present in the sample binds with the capture probe on the bioFET.
  • the Attorney Docket No.206256-0105-00WO present invention relates to methods of detecting the presence or abundance of an analyte of interest using the bioFET and sensors or devices comprising the bioFET, as described herein.
  • the present invention can be used to detect the presence or abundance of any analyte of interest.
  • any probe or capture-molecule can be functionalized using the activated functional groups of the polymer, the probe or capture-molecule can be chosen to specifically bind to the analyte of interest.
  • the parallel detection ability of the present invention allows 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, and the like, target analytes in one or more sample.
  • the spatial functionalization of the external stimulus-reactive polymer provided by the invention allows for the ability to create distinct zones for parallel capture and detection of analytes.
  • the methods comprise detecting the presence or abundance of an analyte in a sample obtained from a subject, in order to detect the presence or severity of a disease, disorder, or condition in the subject.
  • the methods can be used to diagnose the subject as having, or at being at risk for having, a disease, disorder, or condition.
  • the method is used to determine that the subject has a pathogenic infection (e.g. a viral infection or bacterial infection).
  • a pathogenic infection e.g. a viral infection or bacterial infection.
  • the invention provides methods of diagnosing a subject as having an infection, or a disease or disorder associated therewith, based on detection of one or more target molecule associated with an infectious agent (e.g., a virus, bacterium, fungus or protozoan).
  • the invention provides methods of, determining the risk of developing, or assessing progression of an infection, or a disease or disorder associated therewith, by detecting the presence of at least one target molecule in a sample.
  • the target analyte comprises a viral particle, including, but not limited to, a viral particle of 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 papilloma virus (HPV), human immunodeficiency virus-1 (HIV-1), JC virus, arborviruses, enteroviruses, West Nile virus, dengue virus, poliovirus, and varicella zoster virus.
  • a viral particle including, but not limited to, a viral particle of corona
  • the target analyte comprises a bacterial marker, including, but not limited Attorney Docket No.206256-0105-00WO to, a marker of Streptococcus pneumoniae, Neisseria meningitides, Streptococcus agalactia, or Escherichia coli.
  • the target analyte comprises a fungal or protozoan marker, including, but not limited to, a marker of Candidiasis, Aspergillosis, Cryptococcosis, and Toxoplasma gondii.
  • the method of the invention is a method of diagnosing a disease or disorder associated with the presence or absence of a target analyte.
  • 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 a bioFET comprising a capture probe for detection of the target analyte with at least a portion of the sample, detecting a change in electric potential based on the interaction of the target analyte in the sample with the capture probe on the bioFET, identifying the sample as containing the target analyte based on the detection of a change in electric potential, and diagnosing the subject as having the disease or disorder associated with the presence of the target analyte.
  • 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 a bioFET comprising a capture probe for detection of the target analyte with at least a portion of the sample, detecting a lack of change in electric potential based on the absence of an interaction of target analyte with the capture probe on the bioFET, identifying the sample as lacking the target analyte based on the detection of a lack of change in electric potential, and diagnosing the subject as having the disease or disorder associated with the absence of the target analyte.
  • the method of the invention is a method of treating a disease or disorder associated with the presence or absence of a target analyte.
  • 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 a bioFET comprising a capture probe for detection of the target analyte with at least a portion of the sample, detecting a change in electric potential based on the interaction of the target analyte in the sample with the capture probe on the bioFET, identifying the sample as containing the target analyte based on the detection of a change in electric potential, identifying the subject as having the disease or disorder associated with the presence of the target analyte, and administering a therapeutic agent for the treatment of the disease or disorder associated with the presence of the target analyte.
  • the method includes the steps of obtaining a sample from a Attorney Docket No.206256-0105-00WO subject having or at risk of having a disease or disorder associated with the absence of a target analyte, contacting a bioFET comprising a capture probe for detection of the target analyte with at least a portion of the sample, detecting a lack of change in electric potential based on the absence of an interaction of target analyte with the capture probe on the bioFET, identifying the sample as lacking the target analyte based on the detection of a lack of change in electric potential, identifying the subject as having the disease or disorder associated with the absence of the target analyte, and administering a therapeutic agent for the treatment of the disease or disorder associated with the absence of the target analyte.
  • FIG. 12 An exemplary embodiment featuring airborne detection of COVID-19 is shown in Figure 12.
  • the air sample suspected to contain a SARS-CoV-2 viral particle is mixed with liquid and applied to a bioFET sensor of the invention functionalized with an aptamer specific for binding to a SARS-CoV-2 antigen.
  • the invention provides methods of diagnosing a subject as having a SARS-CoV-2 infection or a disease or disorder associated therewith such as COVID- 19.
  • the invention provides methods of, determining the risk of developing, or assessing progression of a SARS-CoV-2 infection or a disease or disorder associated therewith (e.g., COVID-19) though detection of a SARS-CoV-2 antigen or nucleic acid molecule in a sample.
  • the method comprises detecting the presence or abundance of the analyte in an environmental sample, such as a water sample, sewage sample, air sample, or the like, to determine the presence of the analyte within the environment.
  • the method comprises detecting a change in electrical properties of the bioFET, wherein a change indicates the presence of the analyte of interest.
  • the presently described sensors reduce noise by having one functionalized bioFET and one non-functionalized FET, thereby allowing for a more accurate determination and quantification of analyte presence in a sample.
  • the electrical signals of the bioFET are analyzed using the rapid sensing methodology described below in further detail.
  • the bioFET and methods of use are not specifically limited to any analysis method.
  • the invention provides systems comprising the bioFET of the invention and a computing device.
  • the computing device may include a desktop computer, laptop computer, tablet, smartphone or other device and includes a software platform for control of the system components, display of raw data, and analysis of acquired data.
  • the computing devices may include at least one processor, standard input and output devices, as well as all hardware and software typically found on computing devices for storing data and running programs, and for sending and receiving data over a network.
  • the system of the invention comprises hardware and software which detect and quantify detection signals from the bioFET. The signals may be quantified using any suitable analysis software package, or using custom made analysis algorithms.
  • the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for the purpose of clarity, many other elements found in related systems and methods. Those of ordinary skill in the art may recognize that other elements and/or steps are desirable and/or required in implementing the present invention.
  • Embodiment 1 is a method of producing a multiplexed biofunctionalized biosensor comprising: a) providing a transistor substrate comprising a semiconductive layer; b) coating the surface of the transistor substrate with an external stimulus- responsive polymer layer; c) applying a localized external stimulus to a region of the polymer surface to expose activated functional groups on the region of the polymer surface; Attorney Docket No.206256-0105-00WO d) contacting the polymer surface with a capture molecule, wherein the capture molecule is conjugated, directly or indirectly, to the exposed activated functional groups on the region of the polymer surface; and e) repeating steps c) and d) in different regions of the polymer surface with different capture molecules.
  • Embodiment 2 is the method of embodiment 1, wherein the transistor substrate of step a) is a field effect transistor (FET).
  • Embodiment 3 is the method of embodiment 1 or 2, wherein the semiconductive layer of step a) comprises graphene.
  • Embodiment 4 is the method of any one of embodiments 1-3, wherein the multiplexed biofunctionalized biosensor comprises a plurality of graphene FETs (gFETs) including isolated graphene islands.
  • gFETs graphene FETs
  • Embodiment 5 is the method of any one of embodiments 1-4, wherein the external stimulus-responsive polymer of step b) comprises a first monomer A and a second monomer B; wherein monomer A comprises a cross-linking functional group; and wherein monomer B comprises a protected functional group that can be deprotected by an external stimulus to expose the reactive functional group.
  • Embodiment 6 is the method of embodiment 5, wherein the cross-linking functional group of monomer A is selected from the group consisting of an azide, a diaziridine, and a methacrylate.
  • Embodiment 7 is the method of embodiment 5, wherein the cross-linking functional group of monomer A comprises cinnamate methyl ester.
  • Embodiment 8 is the method of any one of embodiments 5-7, wherein the reactive functional group is selected from the group consisting of a thiol, an alcohol, an amine, a carboxylic acid, an aldehyde, a ketone, and an alkyne.
  • Embodiment 9 is the method of any one of embodiments 5-8, wherein the protected group comprises one or more selected from the group consisting of tetrahydropyranyl carbamate, amine N-oxide, tetrahydropyranyl ether, triphenylmethyl ether, tetrahydropyranyl carbonate ester, S-tetrahydropyranyl carbonyl, ethyl disulfide, cyclopropenone, and tert-butyl ester groups.
  • Embodiment 10 is the method of any one of embodiments 5-9, wherein the backbone of the external stimulus-responsive polymer is selected from the group consisting of a poly(methacrylate), poly(acrylate), poly(ester), poly(amide), poly(styrene), poly(olefin), and combinations and co-polymers thereof.
  • Embodiment 11 is the method of any one of embodiments 5-10, wherein the external stimulus-responsive polymer further comprises a third monomer C, wherein monomer C comprises a solubilizing group.
  • Embodiment 12 is the method of embodiment 11, wherein the solubilizing group of monomer C comprises an alkyl, alkoxyl, or aryl chain.
  • Embodiment 13 is the method of any one of embodiments 1-12, wherein step b) comprises: i) coating the transistor substrate with a layer of an external stimulus-responsive polymer; and ii) coating the external stimulus-responsive polymer layer with a layer of an electrically resistive polymer.
  • Embodiment 14 is the method of embodiment 13, wherein the electrically resistive polymer sublimes when exposed to the same external stimulus to which the external stimulus- responsive polymer responds.
  • Embodiment 15 is the method of any one of embodiments 1-14, wherein the external stimulus-responsive polymer is a heat-responsive polymer and wherein step c) comprises applying localized heat.
  • Embodiment 16 is the method of embodiment 15, wherein the heat is applied to the heat- responsive polymer through thermal scanning probe lithography (tSPL) or a focused light or laser.
  • Embodiment 17 is the method of any one of embodiments 1-16, wherein step c) comprises applying localized electromagnetic radiation.
  • Embodiment 18 is the method of embodiment 17, wherein the localized electromagnetic radiation is applied by a scanning probe, electron beam, or localized light source.
  • Embodiment 19 is the method of any one of embodiments 1-18, wherein the region exposed to the external stimulus in step c) has a surface area of between about 1 pm 2 and about 10 mm 2 .
  • Embodiment 20 is the method of embodiments 1-19, wherein the area of the region is between about 8,000 nm 2 and about 250 ⁇ m 2 .
  • Embodiment 21 is the method of any one of embodiments 1-20, wherein the capture molecule of step d) is selected from the group consisting of aptamers, antibodies, antibody fragments, peptides, lectins, enzymes, enzyme fragments, nanobodies, and small molecules.
  • Embodiment 22 is the method of any one of embodiments 1-21, wherein step d) comprises reacting the exposed reactive functional groups on the region of the polymer surface directly with a functional group on the capture molecule.
  • Embodiment 23 is the method of any one of embodiments 1-22, wherein step d) comprises: Attorney Docket No.206256-0105-00WO i) reacting the exposed reactive functional group onto the region of the polymer surface with a functionalized linker molecule; and ii) binding the capture molecule onto the region through an interaction or reaction with the functionalized linker molecule.
  • Embodiment 24 is the method of embodiment 23, wherein the functionalized linker molecule comprises one or more selected from the group consisting of an amine, an aldehyde, a thiol, a cycloalkyne, a cyclopropenone, an alkene, an alkyne, an azide, maleimide, a maleimide derivative, biotin, a biotin derivative, streptavidin, a streptavidin derivative, avidin, and an avidin derivative.
  • Embodiment 25 is the method of any one of embodiments 1-24, wherein the regions of the polymer surface reacted with capture molecules are arranged in a grid pattern.
  • Embodiment 26 is the method of embodiment 25, wherein the distance between regions of the polymer surface reacted with capture molecules is between about 100 nm and about 10 ⁇ m.
  • Embodiment 27 is the method of any one of embodiments 1-26, wherein the method further comprises f) imaging the multiplexed biofunctionalized biosensor to determine the locations of the sensing regions.
  • Embodiment 28 is the method of claim 27, wherein step f) comprises in-situ tSPL imaging of the multiplexed biofunctionalized biosensor.
  • Embodiment 29 is a multiplexed biofunctionalized biosensor comprising a plurality of biofunctionalized bioreceptors produced according to the method of any one of claims 1-28.
  • Embodiment 30 is a multiplexed biofunctionalized biosensor comprising: a) a transistor substrate comprising a semiconductive layer; b) a polymer layer coating the surface of the transistor substrate; and Attorney Docket No.206256-0105-00WO c) a plurality of regions of the polymer surface functionalized with a plurality of capture molecules.
  • Embodiment 31 is the multiplexed biofunctionalized biosensor of embodiment 30, wherein the transistor substrate is a field effect transistor (FET).
  • FET field effect transistor
  • Embodiment 32 is the multiplexed biofunctionalized biosensor of embodiment 30 or 31, wherein the semiconductive layer comprises graphene.
  • Embodiment 33 is the multiplexed biofunctionalized biosensor of any one of embodiments 30-32, wherein the transistor substrate comprises a plurality of graphene FETs (gFETs), wherein the gFETs comprise isolated graphene islands.
  • Embodiment 34 is the multiplexed biofunctionalized biosensor of any one of embodiments 30-33, wherein the polymer layer comprises a first monomer A and a second monomer B; wherein monomer A comprises a cross-linking functional group; and wherein monomer B comprises a protected functional group; and wherein capture molecules in the functionalized regions of the polymer layer are conjugated to the deprotected functional group of monomer B.
  • Embodiment 35 is the multiplexed biofunctionalized biosensor of embodiment 34, wherein the cross-linking functional group of monomer A is selected from the group consisting of an azide, a diaziridine, and a methacrylate.
  • Embodiment 36 is the multiplexed biofunctionalized biosensor of embodiment 34, wherein the cross-linking functional group of monomer A comprises cinnamate methyl ester.
  • Embodiment 37 is the multiplexed biofunctionalized biosensor of any one of embodiments 34-36, wherein the functional group is selected from the group consisting of a thiol, an alcohol, an amine, a carboxylic acid, an aldehyde, a ketone, and an alkyne.
  • Embodiment 38 is the multiplexed biofunctionalized biosensor of any one of embodiments 34-37, wherein the protecting group comprises one or more selected from the group consisting of tetrahydropyranyl carbamate, amine N-oxide, tetrahydropyranyl ether, triphenylmethyl ether, tetrahydropyranyl carbonate ester, S-tetrahydropyranyl carbonyl, ethyl disulfide, cyclopropenone, and tert-butyl ester groups.
  • the protecting group comprises one or more selected from the group consisting of tetrahydropyranyl carbamate, amine N-oxide, tetrahydropyranyl ether, triphenylmethyl ether, tetrahydropyranyl carbonate ester, S-tetrahydropyranyl carbonyl, ethyl disulfide, cyclopropenone, and tert-butyl ester groups.
  • Embodiment 39 is the multiplexed biofunctionalized biosensor of any one of embodiments 30-38, wherein the backbone of the polymer is selected from the group consisting of a poly(methacrylate), poly(acrylate), poly(ester), poly(amide), poly(styrene), poly(olefin), and combinations and co-polymers thereof.
  • Embodiment 40 is the multiplexed biofunctionalized biosensor of any one of embodiments 34-39, wherein the polymer further comprises a third monomer C, wherein monomer C comprises a solubilizing group.
  • Embodiment 41 is the multiplexed biofunctionalized biosensor of embodiment 40, wherein the solubilizing group of monomer C comprises an alkyl, alkoxyl, or aryl chain.
  • Embodiment 42 is the multiplexed biofunctionalized biosensor of any one of embodiments 30-41, wherein the multiplexed biofunctionalized biosensor further comprises a second polymer layer comprising an electrically resistive polymer.
  • Embodiment 43 is the multiplexed biofunctionalized biosensor of any one of embodiments 30-42, wherein the functionalized regions of the polymer surface have a surface area of between about 1 pm 2 and about 10 mm 2 or width and length each independently of between about 100 nm and about 50 ⁇ m.
  • Embodiment 44 is the multiplexed biofunctionalized biosensor of any one of embodiments 30-43, wherein the area of each functionalized region is between about 8,000 nm 2 and about 250 ⁇ m 2 .
  • Attorney Docket No.206256-0105-00WO [0201]
  • Embodiment 45 is the multiplexed biofunctionalized biosensor of any one of embodiments 30-44, wherein the capture molecule is selected from the group consisting of aptamers, antibodies, antibody fragments, peptides, lectins, enzymes, enzyme fragments, nanobodies, and small molecules.
  • Embodiment 46 is the multiplexed biofunctionalized biosensor of any one of embodiments 30-45, wherein the capture molecule is directly conjugated to the functional group of the polymer layer.
  • Embodiment 47 is the multiplexed biofunctionalized biosensor of any one of embodiments 30-45, wherein the capture molecule is conjugated or bound to a linker molecule on a first end of the linker molecule and a second end of linker molecule is conjugated to the polymer.
  • Embodiment 48 is the multiplexed biofunctionalized biosensor of embodiment 47, wherein the linker molecule is conjugated or bound to the capture molecule by an amine, an aldehyde, a thiol, a cycloalkyne, a cyclopropenone, an alkene, an alkyne, an azide, maleimide, a maleimide derivative, biotin, a biotin derivative, streptavidin, a streptavidin derivative, avidin, and an avidin derivative.
  • Embodiment 49 is the multiplexed biofunctionalized biosensor of any one of embodiments 30-48, wherein the regions of the polymer surface functionalized with capture molecules are arranged in a grid pattern.
  • Embodiment 50 is the multiplexed biofunctionalized biosensor of any one of embodiments 30-49, wherein the distance between the regions of the polymer surface functionalized with capture molecules is between about 100 nm and about 10 ⁇ m.
  • Embodiment 51 is the multiplexed biofunctionalized biosensor of any one of embodiments 30-50, wherein the transistor substrate is grounded or at a fixed bias.
  • Attorney Docket No.206256-0105-00WO is a method of detecting the presence of a target comprising contacting a multiplexed biofunctionalized biosensor of any one of embodiments 30-52 with a sample.
  • Embodiment 53 is the method of embodiment 52, wherein the sample is gaseous, airborne, or aerosolized, wherein the regions of the polymer surface functionalized with a plurality of capture molecules of the multiplexed biofunctionalized biosensor are covered with a buffer solution; and wherein contacting the multiplexed biofunctionalized biosensor with the sample comprises contacting the sample with the buffer solution.
  • the invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
  • Example 1 Transistor Platform for Rapid and Parallel Detection of Multiple Pathogens by Nanoscale-Localized Multiplexed Biological Activation
  • FETs Field-effect transistors
  • biosensors serve as a suitable platform, compatible with modern semiconductor manufacturing, for label-free rapid sensing by converting interactions between target analytes and surfaces into real-time electrical Attorney Docket No.206256-0105-00WO signals (Bergveld, P., 1972, IEEE Transactions on Biomedical Engineering, 1972, 19(5):342- 351; Bergveld, P., 2003, Sensors and Actuators B: Chemical, 88(1):1-20; Wu, T., et al., 2017, ACS Nano, 11(7)7142-7147; Rothberg, J.M., et al., 2011, Nature, 475(7356):348-352).
  • the proposed approach so far to achieve multiplexing is to create physical macroscopic barriers between different FETs using for example polydimethylsiloxane (PDMS) barriers and chambers measuring several millimeters in size (Table 1) (Nakatsuka, N., et al., 2018, Science, 362(6412):319-324; Kumar, N., et al., 2023, Attorney Docket No.206256-0105-00WO ACS Nano, 17(18):18629-18640; Sun, J. and Liu, Y., 2018, Micromachines, 9(4):142).
  • PDMS polydimethylsiloxane
  • Nanoscale thermal biofunctionalization the NanoBioFET platform [0217]
  • desired bioreceptors from antibodies to aptamers
  • desired channel material e.g., graphene, oxides or silicon
  • thermal scanning probe lithography tSPL
  • tSPL thermal scanning probe lithography
  • tSPL uses a hot nanotip to expose amine groups with nanoscale resolution on a thermally sensitive biocompatible polymer resist, which is spin coated on a fully-fabricated array of FETs (see Figure 1B)
  • Figure 1B Finnisetti, E., et al., 2016, Nanotechnology, 27(31):315302; Liu, X., et al., 2021, Advanced Functional Materials, 31(19):2008662; Liu, X.Y., et al., 2019, ACS Applied Materials & Interfaces, 11(44):41780-41790; Wang, D.B., et al., 2009, Advanced Functional Materials, 19(23):3696-3702; Zanut, A., et al., 2023, Adv Healthc Mater, 12(10):e2201503).
  • NanoBioFET This spatially selective activation process enables subsequent modification of individual or a group of FETs Attorney Docket No.206256-0105-00WO with a desired bioreceptor, resulting in an array of FET-based biosensors configured for simultaneously detecting various target analytes (NanoBioFET; see Figure 1A).
  • NanoBioFET See Figure 1A.
  • the implementation of the NanoBioFET platform begins by fabricating a FET array.
  • a monolayer graphene was adopted for realizing the FET-based sensors , due to its potential for ultrahigh sensitivity (Kim, J.E., et al., 2017, Applied Physics Letters, 110(20); Yeh, C.-H., et al., 2016, Biosensors and Bioelectronics, 77:1008-1015).
  • the fully fabricated graphene FETs (gFETs) are then coated by spin coating two thermally sensitive polymer resist films .
  • the stack comprises a first film of about 70-nm-thick polymethacrylate- carbamate-cinnamate copolymer (PMCC), which provides amine groups on demand in designated FET regions upon local heating by tSPL (Liu, X., et al., 2021, Advanced Functional Materials, 31(19):2008662; Liu, X.Y., et al., 2019, ACS Applied Materials & Interfaces, 11(44):41780-41790; Wang, D.B., et al., 2009, Advanced Functional Materials, 19(23):3696- 3702).
  • PMCC polymethacrylate- carbamate-cinnamate copolymer
  • PMCC can be locally patterned through local heat-induced deprotection of amine groups from tetrahydropyranyl carbamates in the carbamate block of PMCC (Albisetti, E., et al., 2016, Nanotechnology, 27(31):315302; Liu, X., et al., 2021, Advanced Functional Materials, 31(19):2008662; Liu, X.Y., et al., 2019, ACS Applied Materials & Interfaces, 11(44):41780- 41790; Wang, D.B., et al., 2009, Advanced Functional Materials, 19(23):3696-3702).
  • the second film is poly(phthalaldehyde) (PPA) which serves as a top layer resist (10-20 nm thick) to reduce non-specific binding outside the FET channel region (Albisetti, E., et al., 2022, Nature Reviews Methods Primers, 2(1):32; Zheng, X., et al., 2019, Nature Electronics, 2(1):17-25).
  • PPA poly(phthalaldehyde)
  • Figure 2A and Figure 2B show the fluorescence images for PMCC and PMCC/PPA stacks with 5 ⁇ m squares patterned at intervals of 25 °C. Both the samples are functionalized with NHS Biotin followed by streptavidin tagged with Dylight 633 dye. By observing the fluorescence intensity profiles along the dashed lines, we observe that the average floor of the intensity for the PMCC only sample is ⁇ 185 counts per sec, compared to 165 counts per sec for the PMCC/PPA stack, showing 11% reduction in fluorescence from non-specific binding. [0220] After forming the bilayer polymer resist stack, the process proceeds with the localized functionalization of individual FETs with desired bioreceptors.
  • the bilayer Attorney Docket No.206256-0105-00WO polymer resist stack is modified atop the channel region of each target gFET with amine groups.
  • This process utilizes a hot nano-tip in a commercial tSPL system , which removes PPA completely above the channel region, while simultaneously applying heat to the PMCC polymer surface above the amine deprotection temperature ( ⁇ 120 °C), exposing amine groups in the desired area (Figure 1B).
  • a hot nano-tip in a commercial tSPL system , which removes PPA completely above the channel region, while simultaneously applying heat to the PMCC polymer surface above the amine deprotection temperature ( ⁇ 120 °C), exposing amine groups in the desired area (Figure 1B).
  • amine deprotection temperature ⁇ 120 °C
  • Figure 1B For differential sensing, only the FET used as a sensor is patterned, while leaving un-patterned FET for use as a control. Ensuring signal fidelity is a key challenge for commercialization of F
  • the amine groups in the channel region can subsequentially be functionalized with ad-hoc bioreceptors such as antibodies or aptamers .
  • ad-hoc bioreceptors such as antibodies or aptamers .
  • the sensor/control FETs are ready for biodetection of a specific target, such as SARS-CoV-2 virus.
  • the target is immobilized by the bioreceptor near the polymer surface, giving rise to a change in the electronic signal of FET-based sensors.
  • Figure 1C demonstrates the resolution of the NanoBioFET fabrication method.
  • Figure 1C shows an in-situ tSPL topographical image of an amine pattern produced by tSPL in a PPA/PMCC bilayer polymer resist stack deposited on a SiO 2 /Si wafer (without graphene).
  • the pattern consists of a 10 ⁇ 10 matrix of 15 nm-diameter amine circles with 40 nm pitch.
  • This image shows a resolution compatible with the 5 nm-node silicon FET technology and below.
  • this image is taken in-situ during the tSPL patterning process, since thermal probes can also allow local nanoscale imaging (Methods). This feature enables localization of target regions on a chip with nanoscale precision without requiring sophisticated and costly pattern alignment procedures.
  • Figure 1D and Figure 1E present two fluorescence optical microscopy images of 100 squares (with 5 ⁇ m and 500 nm sides, respectively) of biotinylated anti-SARS-CoV-2 aptamers, which have been fluorescently tagged with a red dye (see Methods for details on the aptamers used here).
  • aptamer patterns have been produced first by exposing amine groups by tSPL on the surface of the PPA/PMCC polymer resist, and then by conjugating the amine groups to NHS-biotin, followed by streptavidin, and biotinylated aptamers (the details of the chemical functionalization steps are reported in the Methods).
  • the PPA/PMCC double polymer stack is spin-coated on a silicon Attorney Docket No.206256-0105-00WO oxide/silicon wafer.
  • multi-polymer stacks are spin-coated onto a silicon oxide/silicon wafer comprising PPA, PMCC, and one or more additional polymers.
  • Figure 1F and Figure 1G show how the NanoBioFET fabrication process is implemented on a gFET array chip.
  • an array of four gFETs was fabricated with a channel length of 1 ⁇ m, where each sensor FET is adjacent to a control FET.
  • the PPA and PMCC resists are then spin-coated on the gFETs, and tSPL is used to remove PPA and pattern amine groups in the channel region of the two gFET sensors, leaving the FET controls unpatterned.
  • Figure 1F and Figure 1G show, respectively, an in-situ tSPL topographical image, and an ex-situ friction atomic force microscopy (AFM) image of the resulting gFET sensors and controls after tSPL patterning.
  • the graphene region underneath the polymer is indicated with a dashed red rectangle.
  • the tSPL topography image shows that the patterns above the channel region of the gFET sensors are at a depth of approximately 10 nm, corresponding to the thickness of the PPA film, which is sublimated during tSPL patterning.
  • Figure 3 demonstrates the capability of the NanoBioFET fabrication process to pattern each FET with an independent bioreceptor to permit parallel sensing on the same chip.
  • Figure 3A shows a schematic representing the steps required for patterning the different FETs with distinct bioreceptors. Initially, an array of FETs was fabricated and a PPA/PMCC polymer stack spin- coated on them.
  • NanoBioFET fabrication as depicted in Figure 1B, is Attorney Docket No.206256-0105-00WO performed on FET-1 to attach bioreceptor-1 (red square), followed by a second round to attach bioreceptor-2 (green square) to FET-2, and so on until all FETs are functionalized up to the desired n-number of bioreceptors.
  • Each round of NanoBioFET fabrication includes: first, in-situ tSPL topographical imaging of the surface to locate where the pattern needs to be made (e.g., the desired FET); second, tSPL local patterning of an individual FET channel; third, a series of biochemical functionalization steps, and selective attachment of the desired bioreceptor to the required FET as described in the Methods part.
  • Figure 3B presents a fluorescence optical microscopy image of biochemical patterns generated by four sequential rounds of NanoBioFET fabrication as depicted in Figure 3A.
  • Figure 3B shows the fluorescence of four types of NHS-esters terminated dyes (red squares – top left, yellow-orange circles – top right, green stars – bottom left, and sky-blue triangles – bottom right) attached directly to the amine moieties exposed during the tSPL process on the surface of the PPA/PMCC polymer resist deposited on a silicon oxide/Si wafer.
  • Figure 3C shows the implementation of the multiplexed chemical patterning in an array of gFETs.
  • FIG. 1 For brevity, four representative gFETs channel regions have been functionalized with four different types of NHS-esters terminated dyes (red – bottom left, yellow-orange – bottom right, green – top left, and sky-blue – bottom right) attached directly to the amine moieties exposed by tSPL on the surface of a PPA/PMCC polymer resist spin-coated on a gFET chip.
  • Figured 3D and Figure 3E demonstrate the capability of the NanoBioFET fabrication method to produce independent patterns of different types of bioreceptors sensitive to different types of target molecules, e.g., different viruses.
  • a fluorescence image of patterns of two types of fluorescently labelled aptamers is shown, influenza A anti-hemagglutinin (HA) aptamer tagged with a green fluorophore, and anti-SARS-CoV-2 aptamer (tagged with a red fluorophore).
  • the patterns are created by exposing amine groups by tSPL, and subsequently by attaching NHS-ester biotin to the amine patterns. Biotin patterns are then exposed to streptavidin, and finally to the biotinylated HA aptamer (green, light grey).
  • a second identical round of NanoBioFET fabrication is performed to attach the biotinylated CoV-2 aptamer (red, dark grey) to the desired area on the surface.
  • Figure 3E and Figure 3F demonstrate the high spatial resolution with which patterns/bits of different bioreceptors can be fabricated on the channel of parallel FETs with this approach.
  • Figure 3D and Figure 3E produces patterns of two Attorney Docket No.206256-0105-00WO types of aptamers (HA and CoV-2 aptamers).
  • the fluorescence image in Figure 3E shows alternating green-aptamer (light grey) and red-aptamer (dark grey) patterns with a “bit” dimension of 500 nm.
  • two- aptamer circle/dash patterns were fabricated with a 20 nm width and a minimum 200 nm pitch, i.e., minimum distance between circle (HA aptamer) and dash (CoV-2 aptamer) pattern centers.
  • the patterns are then imaged in-situ by tSPL imaging ( Figure 3F). Three measurements were performed on the PPA/PMCC surface. First, the pattern type-circle is imaged after a first round of tSPL. Second, pattern type-circle is functionalized with NHS-biotin/streptavidin/HA aptamer and the pattern region imaged after a second round of tSPL to produce pattern type-dash.
  • the polymer surface is imaged after functionalization of pattern type-dash with NHS- biotin/streptavidin/CoV-2 aptamer.
  • the cross-sections show the high registry and robustness of the fabrication process and the change in depth of the patterns after functionalization due to the filling of each pattern with the NHS-biotin/streptavidin/aptamer molecules (approximately 10-15 nm).
  • the two types of patterns have been produced with different shapes to add clarity to the image and show that tSPL can also produce patterns at different depths and shape. See also Figure 4.
  • Electronic sensing using the NanoBioFET platform [0228] Having established the versatility and nanoscale spatial precision of the NanoBioFET fabrication strategy, its feasibility in electronic detection of target analytes was examined.
  • gFETs are promising biosensor candidates due to their potential for high sensitivity and ease of fabrication (Scotto, J., et al., 2022, ACS Applied Electronic Materials, 4(8):3988-3996; Béraud, A., et al., 2021, Analyst, 146(2):403-428; Hwang, M.
  • FIG. 5A shows a schematic illustration of an antibody-modified gFET.
  • the sensor is solution-gated, where a fixed bias gate voltage (Vgs) is applied to the Attorney Docket No.206256-0105-00WO solution (300 mV in our experiments) using an Ag/AgCl reference electrode.
  • Vgs fixed bias gate voltage
  • the amplitude of I ds changes upon conjugation of target analytes (e.g., spike protein or virus) with bioreceptors, due to a change in electronic charge on the surface of the biosensor.
  • the recorded Ids signal can be used to quantify the concentration of target analytes through creation of a sensitivity calibration curve, as we explain later.
  • a key experimental challenge in FET-based biosensing is the electronic screening of charges with increasing distance from the surface, characterized by the Debye length ⁇ Debye .
  • the concentration of spike proteins is limited to a low nanomolar range, which is the typical sensitivity of this type of measurements (Zhang, Y., et al., Attorney Docket No.206256-0105-00WO 2023, Trends in Biotechnology, 41(4):528-544; Guo, Y., et al., 2021, Nature Communications, 12(1):2623).
  • the electronic sensing measurements were performed with the NanoBioFET platform.
  • the bioreceptor-modified gFETs are placed in a microfluidic chamber.
  • An initial screening of a gFET sensor quality was performed by measuring its transfer characteristics (Ids vs. Vgs) using a buffer solution gate.
  • FIG 5C the typical transfer characteristics of an antibody-modified gFET are shown, obtained by sweeping the solution gate voltage, V gs , and recording I ds at a fixed V ds of 50 mV.
  • V gs modulates the gFET charge carrier concentration and carrier type, from holes in the p-branch, to electrons in the n-branch, passing through the charge neutrality point (at V CNP ).
  • V CNP charge neutrality point
  • the data are centered around V CNP .
  • Figure 5E shows a sensitivity plot of the antibody-modified gFET, plotting the sensor response (the amplitude of the exponential decay fitting function) against its corresponding spike concentration. Since the sensor response must be null when the target analyte concentration is zero, a linear fit to the data must go through the origin and hence a meaningful fit could be made, obtaining a sensitivity of 0.59 ⁇ 0.04 % aM. Analysis of the electronic noise in these measurements reveals a 36 nArms input-referred noise (0.3% of I0), corresponding to an estimated limit of detection of 1.5 aM.
  • gFET transient analyte buffers and injection procedure Measurement buffers were chosen according to an appropriate Debye length for the specific measurement.
  • Antibody experiments were performed in a 1 mM HEPES buffer containing 600 ⁇ M NaCl.
  • Aptamer experiments were performed in 0.1X PBS buffer with 1 mM MgCl 2 .
  • Viral protein solutions were prepared in 1X PBS and diluted in the measurement buffer to the required concentration, resulting in a dilute but finite amount of PBS in the protein solutions.
  • gFET transient experiments commenced with an injection of 180 ⁇ L of the measurement buffer into the microfluidic chamber using a micropipette. Dilutions of the spike protein or viral samples were prepared in a matching buffer beforehand. The injections of viral suspensions were methodically administered, starting with 20 ⁇ L of the analyte to achieve the final virus concentration. [0236] To study the stability of the gFETs, a gFET sample with no polymer coating was loaded into the microfluidic chamber with 0.01X PBS buffer.
  • V g 760 mV
  • the Attorney Docket No.206256-0105-00WO system was allowed to stabilize (i.e., the current reaches a constant minimum) and then the gate voltage was decremented in 50 mV steps every 30 seconds to 585 mV (sample rate 1 Hz).
  • the Ids values measured 15 seconds after change in V gs were then graphed against the corresponding gate voltage over the original characteristic curve, shown in Figure 6.
  • the sensing mechanism of an aptamer- modified FET-based sensor is attributed to the fact that the analyte-induced conformational changes of the aptamer alter the surface charge within ⁇ Debye , generating a detectable electronic Attorney Docket No.206256-0105-00WO signal.
  • Aptamer-modified gFETs were implemented following an identical tSPL fabrication and biofunctionalization procedure as in the experiments in Figure 1 and Figure 3, and a biotinylated anti-SARS-CoV-2 aptamer utilized as bioreceptor.
  • FIG. 8C presents SPR experiments confirming the binding between the anti-SARS- CoV-2 aptamer and spike protein in 0.1X PBS, corresponding to a ⁇ Debye of approximately 2 nm.
  • the subsequent electronic sensing experiments in a microfluidic chamber demonstrate the ability of aptamer-modified gFETs in generating detectable electronic signals in response to spike protein injections in the same buffer concentrations (see Figure 8D).
  • the simultaneous monitoring of an adjacent control device in this experiment provides confidence about the fidelity of the electronic signals generated by the gFET biosensor.
  • FIG. 9A The measurement setup and procedure are nearly identical to that for the antibody–spike protein measurements earlier, with a 1 mM HEPES buffer ( Figure 9A).
  • the temporal changes in Ids were monitored during the course of the sensing experiment.
  • the experimental sensing procedure involves injection of the blank virus medium at the beginning of the experiments, followed by a few alternating injections of SARS-CoV-2 virus and the human H1N1 influenza virus.
  • Figure 9B shows the transient response of the antibody-modified gFET with live virus injections in 1mM HEPES. Injections of virus medium (circle), SARS-CoV-2 virus (diamond), and H1N1 virus (star) are marked in Figure 9B.
  • the data demonstrate the sensitive and selective detection by the SARS-CoV-2 antibody-modified gFET.
  • the SARS-CoV-2 virus (diamond) and H1N1 virus (star) in Figure 9 show that the SARS-CoV-2 virus repeatedly produces an exponential-like response in the sensor, whereas the H1N1 virus (a negative control) or buffer produce no response.
  • the first four virus injections are at a concentration of 20 TCID 50 /ml and the last at 200 TCID50/ml, estimated to be about 10 infectious virus particles per ml, Attorney Docket No.206256-0105-00WO demonstrating ultra-sensitive detection of the live virus.
  • the insensitivity of the sensor to H1N1 injections highlights the selectivity of the platform.
  • This methodology can be used to chemically functionalize individual FETs with different bioreceptors at sub-20 nm resolution and 200 nm pitch, a distance comparable to the pitch of modern FETs array in CMOS chip. Functionalization of target regions with sub-micron pitch is a crucial ingredient to achieve massively parallel FET detection of multiple target pathogens. The ability to pattern individual FET also allows for in-situ differential sensing, a key feature to ensure signal fidelity. [0245] The versatility of NanoBioFET is demonstrated by modifying gFET chips with antibody and aptamer bioreceptors and subsequently employing them in electronic detection of spike protein and human SARS-Cov-2 live virus.
  • the polymer film covering the FET allows nano- functionalization, and it functions as a coupling capacitor between the surface-anchored bioreceptors and the buried FET sensors, allowing the electronic detection of interactions between target analytes and specific bioreceptors.
  • the electronic sensing experiments reveal ultrasensitive and selective sensing performance of the NanoBioFET platform. Robust detection was achieved for 5 aM of SARS-CoV-2 spike proteins and 10 human SARS-CoV-2 infectious live virus particles/mL, as well as selectivity against human influenza A (H1N1) live virus. [0246]
  • a key asset of this functionalization strategy is its generalizability to various FET materials from silicon to graphene.
  • the polymer stack is spin coated atop the fully fabricated chips, a process scalable to commercial silicon and other types of substrates with up to 300-mm diameter. Therefore, the NanoBioFET platform is compatible with commercial semiconductor manufacturing for producing CMOS chips that can contain many FETs (thousands to millions), where each FET or a group of FETs can be modified for detecting a Attorney Docket No.206256-0105-00WO specific target pathogen.
  • the tSPL process could be parallelized with the use of hot probes arrays, while the scanner head could work in parallel with picoliter piezotype printing.
  • tSPL is a flexible and sustainable nanofabrication method that does not require vacuum or high energy or alignment marks.
  • the multiplexed parallel sensing enabled by the NanoBioFET platform will offer unprecedent opportunities in the fields of in-home diagnostics, wearables, AI for health data, and e-health.
  • the methods used are described herein.
  • FET devices fabrication [0247] The FETs are fabricated using a four-stage process. The first stage involves the preparation of the substrate and graphene material. The substrate is prepared by covering SiO 2 - coated silicon substrates with a 10-nm aluminum oxide (Al2O3) film grown by atomic layer deposition at 270 °C, followed by the densification of Al 2 O 3 at 500 °C for 1 hour in an oxygen ambient.
  • Al2O3 aluminum oxide
  • fabricating of gFET array is complete through the formation of source and drain metal electrodes using a combination of EBL patterning, e-beam evaporation of 5 nm Cr/ 20 nm Ti/ 25 nm Au metal stack, and lift-off in acetone, followed by covering the metal electrodes with an EBL-patterned SU8 isolation film.
  • Stable connections are made from the contact pads to external test circuit using spring loaded pogo pins.
  • the contact pads are designed to lie outside the microfluidic chamber during electrical measurements and allow space for the pogo pins.
  • Single layer graphene on copper foil from ACS Material is used for the graphene transfer.
  • 2% PMMA in anisole was spin coated on a piece of the copper foil at 5000 RPM for 60 seconds, followed by soft-bake on a hot-plate at 110 °C for 2 minutes.
  • the copper is then etched in copper etchant (Sigma Aldrich) for 30 minutes, leaving a graphene/PMMA stack, and washed 4 times by successive transfers to fresh DI water baths using a glass microslide. This was followed by a Attorney Docket No.206256-0105-00WO final transfer onto the prepared substrate and dried for >12 hours.
  • the PMMA layer is removed by UV exposure at 254 nm for 30 mins followed by chemical etching in acetone.
  • the samples are dried using nitrogen and annealed in a tube furnace at 500 °C (ramp rate ⁇ 5 °C/min) in Ar/H 2 (100 scc/min flow rate) for 5 hours to remove PMMA residue.
  • the graphene monolayers are spin-coated with 495 PMMA A11 (Kayaku), and islands regions are defined in the resist using electron beam lithography, developed in IPA:DI Water (3:1 ratio).20 nm gold is deposited in the patterned areas using electron beam evaporation to form a metal mask.
  • the PMMA is removed in acetone and exposed graphene is removed by plasma cleaning in oxygen atmosphere for 10 minutes.
  • Two-polymer resist stack [0251] The two-polymer resist stack is spin coated on the desired chip in a two-step process. First, a PMCC film (about 70 nm thick) is spin-coated on the chip.
  • the PMCC film is obtained by using a 15 mg/ml solution of PMCC dissolved in cyclohexanone (Wang, D. B., et al., 2009, Attorney Docket No.206256-0105-00WO Advanced Functional Materials, 19(23):3696-3702; Liu, X. Y., et al., 2021, Advanced Functional Materials, 31(19):2170129).
  • the chips are spun at 1000 RPM for 5 seconds and 1500 RPM for 15 seconds to disperse the PMCC solution, then 4000 RPM for 30 seconds to evenly apply a thin film to the substrate.
  • the chips are then treated with 302 nm UV light to crosslink the polymer and increase adhesion to the substrate.
  • a PPA film (10-20 nm thick) is spin coated on PMCC.
  • PMCC coated chips are spin-coated with a solution of 0.5% PPA in anisole to cover the PMCC surface.
  • the spin-coating of PPA is performed at 6000 RPM for 30 seconds.
  • Thermal Scanning Probe Lithography and Microscopy [0252] Patterning of the PMCC/PPA polymeric stack is performed using a commercial tSPL system (NanoFrazor, Heidelberg Instruments, Germany), which utilizes a heated silicon probe (Albisetti, E., et al., 2022, Nature Reviews Methods Primers, 2(1):32). For patterning, the probe on the head of the thermal cantilever is heated up by a resistive micron-heater.
  • the probe works as a separate thermal reading sensor for in-situ topography thermal imaging when the micro- heater is turned off. This is a key feature that allows imaging of the FETs underneath the polymer resist, permitting the local functionalization without the need of markers.
  • the thermal reading sensor probes the topography of the patterned structure right after each patterning line when retracing back in contact mode, which leads to the simultaneous patterning and imaging capability of the NanoFrazor system as well as a closed feedback loop correction (Liu, X. Y., et al., 2019, ACS Applied Materials & Interfaces, 11(44):41780-41790; Wang, D.
  • the probe temperature is automatically calibrated through the system software according to the current-voltage characteristics of the Si tip (During, U., et al., 2005, Journal of Applied Physics, 98(4):044906).
  • the tSPL patterning parameters (such as write temperature, dwell time, and load) are adjusted to achieve the amine deprotection on the PMCC surface and PPA removal, while controlling the depth of the lithographic indentation.
  • the NanoFrazor enables fully automated calibration routines and Python scripting, allowing for the rapid calibration of reading and writing parameters through the software. This includes the importing of various geometries, for layout writing, and patterning variables that control writing characteristics. The information provided will encompass several key parameters, Attorney Docket No.206256-0105-00WO but it is not exhaustive and may include additional relevant aspects; geometry import, pixel size, pixel time, heat pulse time, force pulse time, write force load, write temperature, depth feedback mode, forward height and height offset. [0254] Geometry import and pixel size will determine the patterning shape and the dimensions of the target geometry.
  • Pixel time, heat pulse time, and force pulse time while writing is the time interval between adjacent pixels, writer heating time for each pixel, and interval of force voltage applied between the cantilever and substrate for each pixel, respectively.
  • the write temperature is the set temperature of the heater, not the temperature of the tip in contact with the polymer surface; this variable is translated to a voltage according to a measurement of the current-voltage (IV) characteristic for the cantilever during the calibration.
  • the software uses an extrapolation of this measurement to adjust the applied voltage during patterning to maintain the desired set temperature.
  • Depth feedback mode is used to optimize the writing force application during patterning according to different correlations.
  • the forward height is the height of the tip from the surface when not writing; electrostatic forces are used to bring the tip in contact with the surface.
  • the ability of the NanoFrazor to simultaneous read and write allows for the patterning of multiplexed samples (as shown in Figure 1 and Figure 3).
  • Figure 3B an entire image file is prepared in Illustrator, considering the desired pixel size, pattern sizes and pitch. Then, the said file will be exported to .bmp or .png files in grayscale four times; each time, the desired shape grayscale value will be set higher than the rest. The first image file is then imported into the software so that target geometry may be viewed.
  • the area is then scanned with a large Attorney Docket No.206256-0105-00WO read field so that the previously patterned squares appear in the world map (a tool that overlays the optical camera with existing scanned topography for reference).
  • the shapes are maneuvered by manipulating both the layout position and angle as to perfectly overlay the previously patterned squares, as per the scanned topography) with the total layout.
  • the undesired fields i.e., the squares
  • the next shape i.e., the circles
  • Nanoscale local biochemical functionalization is used as the bio-conjugation strategy to attach ad-hoc bioreceptors in designated regions of chips.
  • the chip is covered with a solution of 100 nM NHS-Biotin in dimethyl sulfoxide (DMSO) and incubated for 1 hour.
  • DMSO dimethyl sulfoxide
  • the NHS ester groups react with the surface NH2 groups, conjugating the biotin to the surface.
  • the chip is then washed with a 1X phosphate buffered saline solution (PBS) and DI-H 2 O to remove any non-reacted material, and dried using compressed N 2 gas.
  • PBS 1X phosphate buffered saline solution
  • the chip is then functionalized with 100 nM streptavidin in 1X PBS for 30 min. Following this step, the sample is functionalized with the desired biotinylated bioreceptor: 100 nM anti-SARS-CoV-2 antibody in 1X PBS, 100 nM anti-SARS-CoV-2 aptamer in DI water or 100 nM Influenza A anti-Hemagglutinin aptamer in DI water.
  • All solvents are acquired from Sigma-Aldritch, unless stated otherwise.
  • the (+)-Biotin N-hydroxysuccinimide (NHS) ester, DyLightTM-conjugated esters, streptavidin are acquired from ThermoFisher Scientific.
  • Biotinylated Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody (S1N-VM226), as well as SARS-CoV-2 spike proteins for detection are purchased from ACRO Biosystems. Aptamers are synthesized per order from Integrated DNA Technologies (IDT) using the following sequences from previous studies: Anti-SARS-CoV-2 Aptamer-6C35′- CGCAGCAC CCAAGAAC AAGGACTG CTTAGGAT TGCGATAG GTTCGG-3′ (SEQ ID NO:1; Figure 10) (Sun, M., et al., 2021, Angewandte Chemie-International Edition, 60(18):10266-10272).
  • IDT Integrated DNA Technologies
  • HA Anti-Hemagglutinin
  • Aptamer-RHA-0006 Integrated DNA Technologies, 5’-GGGTTTGG GTTGGGTT GGGTTTTT GGGTTTGG GTTGGGTT GGGAAAAA-3’ (SEQ ID NO:2, Figure 11) (Shiratori, I., et al., 2014, Biochemical and Attorney Docket No.206256-0105-00WO Biophysical Research Communications, 443(1):37-41).
  • Aptamers are modified with biotin on the 5’ end and tagged with TYE TM 665 (Aptamer-6C3) or 6-Carboxyfluorescein (Aptamer-RHA- 0006) fluorescent dye on the 3’ end, formulated by Integrated DNA Technologies, Inc.
  • the square patterns are formed initially using tSPL and then quenched with conjugated 100 nM DyLightTM 633 NHS Ester in DMSO (red fluorophore). Subsequently, each patterned shape undergoes a thorough wash in 1X PBS and DI-H2O and drying with compressed N2 gas to halt the amine-reaction. Utilizing in-situ tSPL imaging ensures precise and rapid patterning of designated geometries across multiple instances. This enables simultaneous imaging, facilitating the creation of aligned patterns with high-resolution registry (approximately 1 nm precision) after each functionalization step.
  • DyLight NHS Ester derivatives 50 nM DyLightTM 550 for circles (yellow-orange fluorophore), 500 nM DyLightTM 405 for stars (blue fluorophore), and 200 nM DyLightTM 488 for triangles (green fluorophore).
  • Each round follows the same process of tSPL patterning, functionalization, washing, and drying, resulting in distinct shapes corresponding to their designated fluorophores.
  • Repeating rounds with alternative incubation produce patterns with different baits. Extended incubation time is used to ensure total coverage of the exposed amine sites to avoid “fluorescence bleeding” with subsequent ester additions.
  • Aptamers are prepared in nuclease-free distilled water to a dilution of 100 nM, heated at 95 °C for 5 min, and then allowed to cool to room temperature before application to ensure correct conformation.
  • Aptamer-6C3 and Aptamer-RHA-0006 are incubated on the surface for 1 hour. After incubation of the bioreceptors, the sample is thoroughly washed in 1X PBS and DI water. In Figure 3D through Figure 3F this process is repeated twice for the two aptamers. Electrical measurements [0261]
  • the gFET chip is placed inside a custom-made microfluidic chamber with a ⁇ 350 ⁇ l volume, ( Figure 12) and the gFETs are then covered with a buffer solution.
  • An Ag/AgCl reference electrode is utilized as the gate.
  • the FET electrical response is continuously monitored by means of a custom-made Printed Circuit Board.
  • This circuit convers the drain-source current into a voltage using a transimpedance amplifier, and subsequently, the output voltage is digitized with a data acquisition instrument (NI USB6353X series, National Instruments).
  • a custom LabVIEW control interface is employed to operate these instruments.
  • the transfer curve of the device is measured multiple times for reproducibility.
  • the drain-source voltage (V ds ) is set to 50 mV throughout the experiments.200 ⁇ l of buffer is placed in the microfluidic chamber at the start of the measurement.20 ⁇ l of the analyte is injected at a concentration accounting for dilution in the chamber.
  • Viral stocks were prepared ahead of the experiments conducted in the ViroStatics facility in Alghero, Italy, within a Biosafety Level 3 Laboratory.
  • a VERO E6 cell line (Cercopithecus aethiops, kidney) was purchased from American Type Culture Collection (ATCC) and was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine Attorney Docket No.206256-0105-00WO serum (FBS) (Biowest), 1% antibiotic solution penicillin/streptomycin (Biowest), 1% L- glutamine (Biowest), i.e., complete medium, at 37 °C with 5% CO2.
  • TCID 50 Human 2019-nCoV strain 2019- nCoV/Italy-INMI1, isolated in Italy (ex-China) from a sample collected on January 29, 2020, was provided by the Istituto Lazzaro Spallanzani, Rome, Italy (Archive E, “Human 2019- nCoV strain 2019- nCoV/Italy-INMI1, clade V,” 2020).
  • the virus was propagated in Vero E6 cells as described above to obtain high titer virus (>10 6 TCID 50 /mL) and was stored in DMEM 2% FBS at -80 °C until use.
  • Tissue culture infectious dose (TCID 50 ) is defined as the dilution of a virus required to infect 50% of a given cell culture.
  • H1N1 H1N1
  • A/PR/8/34 a high titer (>10 6 TCID 50 /mL) viral suspension of H1N1 (A/PR/8/34) was produced from infected cultured of MDCK (Madin-Darby canine kidney).
  • DMEM 2% FBS was used to store the virus at -80 °C.
  • Live virus suspensions for the gFETs experiments are prepared using a buffer containing 1 mM HEPES and 0.6 mM NaCl.
  • SPR experiments [0264] Putative interactions between probes and target analytes are confirmed using a 2-Channel Surface Plasmon Resonance System (Reichert® 2SPR, Reichert Inc, NY).
  • SPR system is equilibrated with the target buffer system and all the subsequent probes, analyte samples, and blank samples are produced with the same buffer.
  • Biotin-conjugated aptamers IDT Corporation, IA
  • antibodies Acro Biosystems, DE
  • probes 100 ⁇ L injection, 100 nM
  • streptavidin-coated gold chips Reichert Inc. Only the left channel is functionalized with the probe while the right channel is used as a reference.
  • Analyte interactions is assessed by flowing through (25 ⁇ L/min) solutions of target analyte samples SAR Cov-19 spike protein (Acro Biosystems) and live Covid particle (patient nasal swabs) with increasing concentrations through both channels.
  • Figure 1D and Figure 1F were taken using a Zeiss LSM 880 Airyscan Fast Live Cell confocal microscopy unit, Plan Apo 40x/1.2 Type W Oil objective and submersion, and HeNe 633 nm laser line.
  • Figure 3B through Figure 3D and Figure 3F fluorescence imaging was done using a Nikon Ti2-E Motorized Microscope equipped with a 7-line solid state light source, Attorney Docket No.206256-0105-00WO CFI60 Plan Apochromat Lambda 10 ⁇ and 40 ⁇ lenses and ORCA-Fusion Gen-III sCMOS camera.
  • Example 2 Polymethacrylate-carbamate-cinnamate copolymer was synthesized as needed. Synthesis of 2-((((tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)ethyl methacrylate isocyanatoethyl)methacrylate (3.00 g, 2.72 mL, 19.3 mmol, 1.00 equiv.) as a colorless oil, and 2H-tetrahydropyran-2-ol (2.17 g, 1.97 mL, 21.3 mmol, 1.10 equiv.), pyridine (0.05 g, 0.05 mL, 0.51 mmol, 0.03 equiv.), 2,6-Di-tert-butyl-4-methylphenol (10 mg, 45 ⁇ mol, 0.002 equiv.).
  • reaction flask was sealed with a rubber septum, placed under an atmosphere of argon, and allowed to stir, neat, at rt. After 20 h, the reaction mixture was viscous and pale yellow; it was purified directly via column chromatography (stationary phase: silica, eluent: 20% ethyl acetate in hexanes). Pure fractions were identified via TLC (using a KMnO 4 stain), combined, and condensed in vacuo to give a white solid (2.374 g, 47% yield) – NMR spectra are consistent with previously reported spectra (Underwood, W.
  • Route 1 The benefit of Route 1 is that the intermediate is stable indefinitely – but the purification of the intermediate and coumarin monomer from Route 1 can sometimes be challenging, requiring multiple long columns.
  • Route 2 The benefit of the Route 2 is that the intermediate and resulting coumarin monomer are easily purified via a short column – however, the 3-bromopropyl methacrylate intermediate is not stable and will autopolymerize at ambient conditions in open air if left more than an hour without an inhibitor (e.g., BHT).
  • an inhibitor e.g., BHT
  • the solution Upon addition of the coumarate, the solution turned bright Attorney Docket No.206256-0105-00WO yellow.
  • the reaction was allowed to stir as a yellow suspension at rt for 48 h. Afterwards, the reaction was poured into a separatory funnel with 250 mL water and 100 mL dichloromethane. The organic layer was collected, and the aqueous layer was extracted with 100 mL DCM twice more. The combined organic layers were then washed 5 times with water, 100 mL each time, to remove remaining DMF. The organic layer was dried over magnesium sulfate, filtered, and condensed in vacuo to give a pale yellow solid.
  • reaction mixture was quenched by pouring into 100 mL EtOAc and 100 mL water, shaking, and collecting the organic layer.
  • the organic layer was dried over magnesium sulfate, filtered, and condensed in vacuo to give a white solid – this solid was redissolved in 100 mL DCM with 2 g silica, and solvent was removed in vacuo to deposit onto silica for purification via column chromatography (stationary phase: silica, eluent: 10% ethyl acetate in hexanes).
  • Fractions were identified via TLC with KMnO 4 staining, and pure fractions were combined and condensed in vacuo to give a pale-yellow oil (0.754 g, 51% yield).
  • reaction was capped and allowed to stir at rt for 18 h – after which, the reaction mixture was poured into diethyl ether (140 mL) and water (200 mL) in a separatory funnel, shaken, and the organic layer was collected and washed with water (3 ⁇ 100 mL) and then dried over magnesium sulfate, filtered, and condensed in vacuo to give a white solid.
  • This solid was purified via column chromatography (stationary phase: silica, eluent: 20% ethyl acetate in hexanes) – pure fractions were identified via TLC, combined, and condensed to a white solid (4.293 g, 60%).
  • the polymerization is a free-radical polymerization – given this, the polymerization is not a chain-growth polymerization, so it is difficult to control molecular weight (Grubbs, R. B. & Grubbs, R. H., 2017, Macromolecules, 50:6979-6997).
  • the polymerization must be set up in an air free environment (such as a glovebox) using unstabilized THF which was degassed using a freeze- pump-thaw technique (Borys, A. M., 2023, Organometallics, 42:182-196).
  • the reaction mixture was capped and allowed to stir in a 70 °C aluminum heating block in the glovebox overnight (16 h). After stirring, the reaction appeared to be pale yellow and very viscous.
  • the reaction mixture was taken out of the glovebox, quenched by exposing to ambient air, then the reaction mixture was dissolved in 3 mL dichloromethane and slowly added (dropwise) to stirring hexanes (100 mL, 0o C) giving a white solid.
  • the white solid was collected via vacuum filtration, and dried under high vacuum for 5 h, yielding a fine white powder (1.015 g, 97% yield).
  • the powder was dissolved in THF for GPC analysis, and CDCl 3 for NMR analysis.
  • NMR Spectroscopy Attorney Docket No.206256-0105-00WO [0282] All NMR spectra were recorded on a Bruker AVANCE III HD 400 MHz NMR spectrometer with a 5mm X-nuclei-optimized CryoProbe probe (operating at 400 MHz for 1 H NMR spectra or 101 MHz for 13 C NMR spectra). All spectra were taken at room temperature under ambient atmosphere. All 1 H NMR spectra were 13 C decoupled.
  • Glovebox [0283] The glovebox in which specified procedures were carried out was an MBraun LABmaster 130 with an N 2 atmosphere (O 2 levels ⁇ 100 ppm, H 2 O levels ⁇ 0.1 ppm). Freeze-pump-thaw degassing technique: [0284] To prepare tetrahydrofuran for polymerization, non-stabilized tetrahydrofuran purchased from Millipore-sigma was dried over molecular sieves (4 ⁇ ) for 3 days, then distilled into a Schlenk tube using Schlenk technique (making sure to not fill the tube more than halfway) and degassed via a freeze-pump-thaw procedure (Williams, D. B. G.
  • This solution was filtered through 0.45 ⁇ m PTFE membrane syringe filters.50 ⁇ L of the filtered solution was injected into a Shimadzu GPC system consisting of a SIL-20A autosampler, LC-20AT liquid chromatograph, CTO-10ASvp column oven, CBM-20A communications bus module, SPD-10AVP UV-Vis detector, RID-20A refractive index detector, and Shodex GPC KF-804 (7 ⁇ m, 8.0 ⁇ 300 mm) columns.
  • Molecular weight data presented corresponds to data collected on the UV detector module with absorbance at 254 nm.
  • Example 3 Improved Polymer Design
  • PMCC polymethacrylate-carbamate-cinnamate copolymer
  • external stimulus-responsive polymers need to include a cross-linking moiety and a protected functional group that can be removed upon exposure to the external stimulus.
  • a copolymer comprising three different monomers was prepared.
  • the reaction mixture was capped and allowed to stir in a 70 oC aluminum heating block in the glovebox overnight (16 hours). After stirring, the reaction was pale yellow and very viscous.
  • the reaction mixture was taken out of the glovebox, quenched by exposure to ambient air, and dissolved in 3 mL dichloromethane. Dropwise addition of the reaction mixture to stirring hexanes (100 mL, 0 oC) yielded a white solid precipitate. The white solid was collected via vacuum filtration, and dried under high vacuum for 5 hours, yielding a fine white powder (0.621 g, 85% yield).
  • Example 4 Mitigation of Electronic Crosstalk Interference Attorney Docket No.206256-0105-00WO [0289]
  • 2D two-dimensional
  • signal fidelity from individual sensors may be compromised due to the existing non- idealities of 2D electronic devices.
  • graphene field effect transistors gFETs
  • Two potential sources of crosstalk interference between sensors are identified in such a platform: resistive interference through shared gFET channels, and capacitive coupling through the shared device substrate. Having illustrated these sources of interference, solutions to mitigate them for ensuring fidelity of the biosensor signal are proposed.
  • Device fabrication Device fabrication.
  • ⁇ and n0 can be independently calculated as a function of the impurity concentration nimp: ⁇ ⁇ 33 ⁇ /h ⁇ and ⁇ ⁇ 0.2 ⁇ , where ⁇ and h are the electronic charge and Planck’s constant respectively (Adam, S., et al., 2007, Proceedings of the National Academy of Sciences USA, 104(47):18392-18397). Accordingly, reasonable agreement is found between ⁇ ⁇ 7.7 ⁇ 10 ⁇ ⁇ calculated from ⁇ and ⁇ ⁇ ⁇ ⁇ ⁇ 6 ⁇ 10 ⁇ from ⁇ , giving confidence in the extracted parameters.
  • the Schottky barrier can be reduced through a proper choice of metal, use of low-residue nano-patterning methods, or local treatment of graphene in S/D regions (Jia, Y., et al., 2016, Nano-Micro Letters, 8(4):336-346; Liu, X., et al., 2021, APL Materials, 9(1):011107; Leong, W. S., et al., 2014, ACS Nano, 8(1):994-1001; English, C.
  • a V ds 50 mV was applied to gFET A1 while setting the V ds of device A2 to 0 V (configuration 2), illustrated by Figure 19B.
  • current through gFET A1 is expected to be unchanged from the previous test of Figure 19A and Figure 19D, and the current through gFET A2 to be zero.
  • the measured currents deviated from the expected behavior, shown in Figure 19E.
  • FETs made of 2D materials, such as graphene are commonly fabricated on SiO2-coated silicon substrates.
  • the silicon substrate primarily serves as a mechanical support during fabrication and operation of the gFET array.
  • the silicon substrate is Attorney Docket No.206256-0105-00WO not required to perform an electrical function as the biasing of the gate electrode of gFETs is achieved through the reference electrode inserted inside the buffer solution.
  • silicon is conductive even at low concentrations, it forms capacitances with the gFETs and the solution gate through the SiO2 dielectric, as shown in Figure 20B.
  • the gate bias applied to the B1 device in the left well couples to the buffer solution in the opposite fluid well through the C bt capacitances.
  • This plate capacitor is formed between the ionic gate and the silicon substrate.
  • the Cbt capacitance in both wells are connected in a series configuration. Therefore, the solution in the right well follows the gate bias applied to the solution in the left well.
  • the B1 device is biased by applying an external V gs to the ionic solution of the left well, while no voltage is applied to the solution in the opposite well (see Figure 21B).
  • the results Attorney Docket No.206256-0105-00WO of this experiment are shown in Figure 21E, indicating that the external Vgs modulates only the current of the B1 device and not the B2 gFET.
  • Applying the external Vgs to the B2 gFET while electrically floating the B1 device produces consistent results, as shown in Figure 21F. While these experiments confirm the capacitive interference through the share substrate, they also provide a solution for mitigating this source of cross-talk between gFETs on a chip.
  • the results presented here highlight two common sources of crosstalk interference that can occur in gFET sensors: resistive interference and capacitive coupling.
  • the resistive interference arises from the non-ideal contacts in an array design where the neighboring devices share a source electrode on a continuous graphene active region. This issue can be mitigated by fabricating isolated graphene islands and connecting shared sources away from the graphene.
  • capacitive coupling arises from the shared Si substrate when it is electrically floating.
  • this form of cross-talk can be effectively addressed by applying a fixed bias (e.g., ground) to the substrate.

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Abstract

La présente invention concerne un procédé de production d'un biocapteur biofonctionnalisé électrique, le biocapteur et des procédés d'utilisation du biocapteur pour détecter simultanément une pluralité de molécules biologiques cibles. Sont également proposés des procédés de réduction des interférences dans le biocapteur pour améliorer la sensibilité et la spécificité des biocapteurs.
PCT/US2025/014271 2024-02-01 2025-02-03 Dispositifs et procédés de biodétection rapide Pending WO2025166328A1 (fr)

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US8468611B2 (en) 2009-05-29 2013-06-18 Georgia Tech Research Corporation Thermochemical nanolithography components, systems, and methods
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