Field of the disclosure

[0001] This disclosure relates to organic thin film transistors for use in sensing applications, the preparation of said sensors, and uses thereof.

Background of the disclosure

[0002] In recent years there has been rapid development of point-of-care (POC) devices which can be used by patients at home or medical professionals in a clinic for rapid detection or diagnosis without the need to deliver samples for pathological analysis. Electrochemical sensors and transistors such as organic thin film transistors are ideal for POC devices because of their compatibility with biological fluid and potential to interface with electronics without requiring further analytical equipment to analyse the signal. This is in contrast to absorbance, fluorescence or luminescence based assays, where a separate equipment is needed for signal analysis.

[0003] One of the obstacles in the development of electrochemical sensors, such as those in POC devices, is the rapid accumulation of proteins from biological fluids on the conductive surfaces of the sensor. This weakens the sensor signal and can deactivate the sensor. One solution has been to embed an enzyme into a porous material that the biological fluid can permeate. This approach is not suitable for all assays, for example, immunological assays.

[0004] There is a need for point-of-care diagnostic devices conducting an assay in a biological fluid contacting the surface of a sensor or transistor. Preferably, the device minimises the loss of signal to the sensor or transistor electrode.

[0005] However, deposition of proteaceous coatings on other surfaces can be more challenging. It is beneficial if the deposition of any anti-fouling coating is uniform and reproducible so that it does not variably impact electrical signal.

[0006] There is therefore a need for sensors and transistors with antifouling capabilities that can function in biological fluids with improved signal sensitivity or improved maintenance of signal sensitivity. Preferably, the sensors and transistors (i) do not impede the function of enzymes or probes such as probes, (ii) do not impede the molecular/chemical interactions resulting in the generation of charge, (iii) minimise nonspecific interactions, and/or (iv) preserve the sensitivity of the electrical sensor/transducer.

[0007] There is also a need for rapid POC diagnostic devices capable of molecular assays. Further there is a need for inexpensive and easy to manufacture diagnostic tools for POC diagnosis.

[0008] The development of organic thin film transistors (OTFTs) has grown rapidly in recent years motivated primarily by the unique physical properties of polymer devices, including their flexibility and ability to be fabricated using low-cost, solution-based techniques. Work on developing OTFTs for new and existing applications has focussed on two main areas. First, there have been systematic improvements in the materials and fabrication processes which have led to an improvement in the conventional performance parameters of organic devices making them comparable to their inorganic counterparts. Second, improvements in film morphology of the organic semiconducting layer have been made with the goal of eliminating electron and/or hole traps and enhancing free carrier transport in the polymer semiconducting materials. Progress has also been made in developing high capacitance organic dielectric layers and large improvements in OTFT performance have been reported. The inherent compatibility of organic materials with biological molecules makes OTFTs suitable for use in biosensing applications.

[0009] An OTFT device has been fabricated that is capable of detecting analyte levels across a broad range of concentrations and which is straightforward and relatively cheap to manufacture.

[0010] There is a need for OTFT devices with improved signal sensitivity and/or for sensing biologies such as proteins, antigens and antibodies. Such a device may make commercially viable biological sensors that allow estimation of blood levels of biologies and/or detection of infection such as viral infection. Such a device may be a rapid POC diagnostic device.

[0011] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Summary of the disclosure

[0012] The present inventors have successfully fabricated an organic transistor based sensor device that is capable of detecting analytes in biological fluid. The transistor is straightforward and relatively cheap to manufacture. The transistor may enable commercially viable rapid and/or POC diagnosis of various conditions including viral infections in a biological fluid such as blood or saliva.

[0013] The present disclosure provides an organic thin film transistor (OTFT) comprising an organic semiconductor and a probe or enzyme for facilitating generation of a charge carrier from an analyte, wherein either

(i) the organic semiconductor is a semiconducting antifouling layer and adapted for contact with an analyte; or

(ii) a conducting or semiconducting antifouling layer coats the surface of the OTFT and the antifouling layer is adapted for contact with the analyte.

[0014] Optionally, the probe or enzyme is at least partially embedded or attached to the surface (top) of the antifouling layer or the organic conducting or semiconducting layer, wherein, when the probe or enzyme is at least partially embedded or attached to the surface (top) of the organic conducting or semiconducting layer, the probe or enzyme is surrounded by the antifouling layer.

[0015] Optionally, the OTFT further comprises a substrate. Optionally, the substrate is on the opposite side of the OTFT to the antifouling layer. Optionally, the substrate is on the opposite side of the electrode to the conducting or semiconducting layer.

[0016] In some embodiments, the OTFT further comprises a conducting polymer gating layer between the organic conducting layer and the conducting antifouling layer. Optionally, the conducting polymer gating layer is a tetrafluoroethylene-based fluoropolymer-copolymer. The tetrafluoroethylene-based fluoropolymer-copolymer may be a copolymer of tetrafluoroethylene and perfluoro-3,6-dioxa-4-methyl-7-octene- sulfonic acid. The tetrafluoroethylene-based fluoropolymer-copolymer may be a sulfonated tetrafluoroethylene-based fluoropolymer-copolymer. Preferably the conducting polymer gating layer is nation.

[0017] In a further aspect, the present disclosure provides an OTFT comprising:

(i) a source electrode,

(ii) a drain electrode,

(iii) an organic semiconducting layer, the organic semiconducting layer connecting the source electrode to the drain electrode, wherein either:

(a) the organic semiconductor is a semiconducting antifouling layer and adapted for contact with the analyte; or

(b) a conducting or semiconducting antifouling layer coats at least a portion of the surface of the organic semiconducting layer adapted for contact with the analyte,

(iv) either the antifouling layer or the organic semiconductor is configured to be connected to an ohmic conductor for applying a gate voltage to said organic semiconducting layer and/or said organic semiconducting layer via antifouling layer, and

(v) a probe or enzyme for facilitating generation of a charge carrier from an analyte, wherein the probe or enzyme is at least partially embedded or attached to the surface (top) of the antifouling layer or the organic semiconducting layer, wherein when the probe or enzyme is at least partially embedded or attached to the surface (top) of the organic semiconducting layer the probe or enzyme is surrounded by the antifouling layer.

[0018] Optionally, the antifouling layer is configured to be connected to the ohmic conductor.

[0019] Optionally, the sensor further comprises an ohmic conductor for applying a gate voltage in contact with said organic semiconducting layer and/or said antifouling layer. Optionally, the ohmic conductor for applying a gate voltage is a gate electrode. [0020] Optionally, a voltage is applied from the ohmic conductor to the organic semiconducting layer. Optionally, a voltage is applied from the ohmic conductor to the organic semiconducting layer via the antifouling layer.

[0021] The present disclosure provides an OTFT comprising:

(i) a source electrode,

(ii) a drain electrode,

(iii) an organic semiconducting layer, the organic semiconducting layer connecting the source electrode to the drain electrode,

(iiia) a polymer gating layer, the polymer gating layer being conducting to the charge carrier and connecting the organic semiconducting layer to the antifouling layer,

(iiib) a conducting or semiconducting antifouling layer, the conducting antifouling layer coating at least a portion of the surface of the sensor adapted for contact with the analyte,

(iv) either the polymer gating layer or the antifouling layer is configured to be connected to an ohmic conductor for applying a gate voltage to said organic semiconductor via said polymer gating layer and/or said antifouling layer, and

(v) a probe for facilitating generation of a charge carrier from an analyte, wherein the probe or enzyme is at least partially embedded or attached to the surface (top) of the antifouling layer or the polymer gating layer, wherein when the probe or enzyme is at least partially embedded or attached to the surface (top) of the polymer gating layer the probe or enzyme is surrounded by the antifouling layer.

[0022] Optionally, the polymer gating layer is configured to be connected to the ohmic conductor. Alternatively, the antifouling layer is configured to be connected to the ohmic conductor. [0023] Optionally, the OTFT further comprises an ohmic conductor for applying a gate voltage in contact with the polymer gating layer and/or the antifouling layer. Optionally, the ohmic conductor for applying a gate voltage is a gate electrode.

[0024] Optionally, a voltage is applied from the ohmic conductor to the organic semiconducting layer. Optionally, a voltage is applied from the ohmic conductor to the organic semiconducting layer via the polymer gating layer and/or antifouling layer.

[0025] Optionally, the ohmic conductor is located at the top of the sensor ie opposite the source and drain electrodes. Ohmic conductors in this locations can contact the polymer gating layer or the antifouling layer.

[0026] Optionally, the ohmic conductor is in contact with the polymer gating layer and separated from the antifouling layer and the analyte ie a) at the bottom of the sensor when the antifouling layer is at the top of the sensor and offset from the source and drain electrode, or b) on the side of the OTFT when the antifouling layer is at the top and the source and drain electrodes at the bottom.

[0027] Unless otherwise stated, the following embodiments apply to a device in accordance with any one of the above aspects of the disclosure.

[0028] Optionally, the OTFT is suitable for point-of-care analysis or diagnostics (or point-of-sample collection analysis) ie it is not necessary to send the sample to a pathology centre or other diagnostic/analytical chemistry facility. Optionally, the OTFT is portable.

[0029] Optionally, the OTFT is at least a partially printed OTFT. Optionally, one or more layers of the OTFT are printed on a substrate (preferably all layers). Optionally, the organic semiconductor, antifouling layer and optionally the conducting polymer gating layer of the OTFT are printed directly or indirectly on a substrate. Optionally, the antifouling layer and optionally the conducting polymer gating layer are printed directly or indirectly onto the organic semiconductor (ie indirectly refers to printing on an intermediate layer between the organic semiconductor and the printed layer). Irrespective of the printing or non-printing of the organic semiconductor, conducting polymer gating layer, and antifouling layer, optionally, the enzyme or probe is printed onto the OTFT. Optionally, one or more layers of the OTFT and the enzyme or probe are printed on a substrate. The electrodes are optionally printed on the substrate, for example, prior to printing of the semiconductor. Optionally, one or more electrode is printed using silver ink. Optionally, the OTFT comprises one or more of a dielectric layer, and the dielectic layer is printed directly or indirectly onto a substrate or the organic semiconductor.

Methods of making the OTFT

[0030] In a further aspect, the present disclosure provides a method for preparing an OTFT in accordance with this disclosure, the method comprising: a) providing a substrate for depositing thereon components of the device; b) depositing the source electrode and the drain electrode; c) depositing the organic semiconductor; d) optionally depositing a dielectric layer; e) optionally depositing a polymer gating layer; f) depositing the antifouling layer (where the organic semiconductor is not also an antifouling layer); and g) depositing the enzyme or probe on the antifouling layer and/or optional polymer gating layer.

[0031] Preferably, the source electrode and the drain electrode are deposited on the substrate.

[0032] In at least one embodiment step b) precedes step c), step c) precedes step f), and step f) precedes step g). Optionally, a polymer gating layer is deposited and step c) precedes step e) and step e) precedes step f). Optionally, a dielectric layer is deposited and step c) precedes step d) and step d) precedes step f).

[0033] Preferably the method also includes depositing the ohmic conductor/gate electrode. Optionally, the ohmic conductor/gate electrode is in contact with said polymer gating layer to control an electric potential of said polymer gating layer. Optionally, the ohmic conductor/gate electrode is in contact with said conducting antifouling layer to control an electric potential of said conducting antifouling layer. Preferably, in this case, the ohmic conductor is deposited before the polymer gating layer and/or antifouling layer.

[0034] In other embodiments, the ohmic conductor may be connected to said polymer gating layer and/or conducting antifouling layer in use, to control an electric potential of said polymer gating layer and/or conducting antifouling layer, whereby the ohmic conductor is not integrated into sensor during manufacture of the sensor.

[0035] In at least one embodiment, the source electrode and the drain electrode are deposited over the substrate. In at least one embodiment, the organic semiconductor layer is deposited over source electrode and the drain electrode.

[0036] In at least one embodiment, the polymer gating layer is deposited over the organic semiconductor. In some embodiments, no dielectric layer is deposited.

Alternatively, a dielectric layer may be deposited over the organic semiconductor, with said polymer of the polymer gating layer then being deposited over the organic dielectric layer. In this case, the dielectric layer may be deposited by spin coating or screenprinting.

[0037] Preferably, the enzyme is introduced by screen-printing.

[0038] Step b) may comprise depositing the source electrode and the drain electrode over the substrate such that the source electrode and the drain electrode are disposed above, and in contact with, the substrate.

[0039] Step c) may comprise depositing the organic semiconductor over the source electrode and the drain electrode such that at least part, but preferably a majority, of the semiconductor is disposed above and in between the source electrode and the drain electrode.

[0040] Preferably, in step c), the semiconductor is deposited such that it is in contact with the source electrode and the drain electrode.

[0041] In at least one embodiment, the semiconductor layer is deposited by spin coating.

[0042] In at least one embodiment the polymer gating layer is deposited by spin coating. [0043] Devices in accordance with the present disclosure may be fabricated by low- cost spin-coating and printing techniques, thereby offering the potential for affordable and disposable non-reversible devices. All of the components of the device are capable of being printed onto an (optionally removable) substrate.

[0044] The organic semiconducting layer and/or the dielectric layer (for embodiments in which such a dielectric layer is included) may be deposited in accordance with methods well known to those skilled in the art, including, but not limited to: electroplating, vapour phase deposition, spin coating, screen printing, ink-jet printing, slot-dye printing, spray coating, draw bar coating or derived coating/printing techniques thereof, painting, gravure, roller and embossing.

[0045] The organic semiconducting layer may be deposited so as to achieve a thickness between about 5 nm and about 500 nm, or between about 75 nm and about 125 nm, or about 100 nm.

[0046] Optionally, the antifouling layer is deposited as a mixture of blocking material and conducting material. Optionally, the blocking material is a protein and is denatured either: prior to mixing the protein with the conducting material; following mixing of the protein with the conducting material but prior to deposition of the antifouling layer onto the sensor; and/or after applying the antifouling layer to the sensor.

[0047] Where the probe is attached to the sensor via a connector, optionally, the connector is deposited on the sensor and the remainder of the probe attached to the connector subsequently. Alternatively, the probe with the connector is deposited on the sensor.

[0048] Optionally, the blocking material is activated prior to contact with the probe and binds to the probe directly.

[0049] In another aspect the present disclosure provides a device prepared by any of the above methods of making the sensor.

Methods of using the OTFT

[0050] In another aspect, the present disclosure provides use of the sensor/OTFT of the disclosure for sensing an analyte in a sample. Optionally, the analyte is a target for a probe. [0051] The analyte is optionally a biological analyte. The analyte may be glucose. In an embodiment the analyte/target for the probe is an antibody, antigen, protein, peptide or chemical. The chemical is optionally glucose. The antibody is optionally a coronavirus antibody. The antibody is optionally a SARS-CoV-2 antibody.

[0052] The sample may be any aqueous solution but is preferably a biological fluid, more preferably a bodily fluid, and still more preferably, blood or saliva.

[0053] In a further aspect, the present disclosure provides a method for detecting an analyte in a sample, the method comprising the following steps:

- providing an OTFT of the present disclosure;

- contacting the sample to the sensor, preferably the portion of the sensor adapted for contact with an analyte; and

- detecting the analyte based on an electrical parameter of the device.

[0054] Optionally, the method further comprises interaction between the analyte and probe or enzyme to facilitate generation of a charge carrier. Preferably, the analyte is detected by detecting the charge carrier.

[0055] Optionally, the method further comprises determining a concentration or an amount of the analyte. Preferably, the concentration or amount is determined by detecting the amount of charge carrier and/or the change in voltage

[0056] The method may comprise applying a voltage to the drain electrode. The method may comprise grounding the source electrode. The method also comprises applying a voltage to the ohmic conductor. Preferably the voltage applied to the ohmic conductor (ie the “gate voltage”) and the voltage to the drain electrode have the same polarity with respect to the source electrode.

[0057] The method may include detecting drain current through the sensor, wherein the concentration or amount of the analyte is determined based on a magnitude of the drain current.

[0058] The determination of the concentration or amount may be performed by reference to an appropriate calibration curve. [0059] Step b) may comprise contacting the sample with the polymer gating layer and/or antifouling layer.

[0060] The gate voltage and drain voltage applied may be voltages greater than that required to liberate H+ from H2O2, and lower than that required to cause electrolysis of water.

[0061] The gate voltage and drain voltage applied may be between about 0 V and -2 V, or about -1 V. Optionally, about -0.7 V and -2V or -0.7 V and -1.5 V or -0.7 and -1 V. Alternatively, about -0.55 V and -2V or -0.55 V and -1.5 V or -0.55 and -1 V or -0.6V and -2V or -0.6 V and -1.5 V or -0.6 and -1 or -0.65V and -2V or -0.65 V and -1.5 V or -0.65 and -1V.

[0062] The analyte is optionally a biological analyte. The analyte may be glucose. In an embodiment the analyte/target for the probe is an antibody, antigen, protein, peptide or chemical. The chemical is optionally glucose. The antibody is optionally a coronavirus antibody. The antibody is optionally a SARS-CoV-2 antibody.

[0063] The sample may be any aqueous solution but is preferably a biological fluid, more preferably a bodily fluid, and still more preferably, blood, urine or saliva.

[0064] In another aspect, the present invention provides a method for detecting presence of one or more analyte in a sample, the method comprising:

- contacting a sample with the one or more probe of an OTFT of this disclosure; and

- detecting binding of the analyte to the compound.

[0065] Optionally, the one or more analyte is a coronavirus analyte. Optionally, the one or more coronavirus analyte is one or more coronavirus particle, protein, peptide, nucleic acid, or antibody specific to a coronavirus antigen.

[0066] In another aspect, the present invention provides a method for detecting one or more analyte comprising: - contacting a sample comprising one or more analyte to the surface of the antifouling layer of an OTFT of this disclosure, wherein the antifouling layer is connected directly or indirectly to one or more electrode;

- allowing the analyte to bind with the probe, thereby forming a complex comprising the analyte and the probe;

- labelling the complex with a detection agent to form a detectable complex, wherein the a portion of the detection agent binds specifically with the complex and a portion of the detection agent comprises at least one reporter; and

- contacting the detectable complex with a substrate for the reporter enzyme , wherein the contact results in reaction of the substrate for the reporter with the at least one reporter to form a charge carrier.

[0067] In a further aspect, the present invention provides a method comprising:

(ia) selecting a sample in need of determination of the presence or absence of an analyte;

(i) contacting the sample to the surface of the antifouling layer of an OTFT, wherein the probe specifically binds with an analyte and the antifouling layer is connected directly or indirectly to one or more electrode;

(ii) allowing any analyte present in the sample to bind with the probe, thereby forming a complex comprising the coronavirus analyte and the probe;

(iii) labelling the analyte or any complex formed between the analyte and the probe with a detection agent to form a detectable complex, wherein a portion of the detection agent binds specifically with the complex and a portion of the detection agent comprises at least one reporter; and

(iv) contacting any detectable complex with a substrate for the reporter, wherein the contact results in reaction of the substrate for the reporter with any reporter in the detectable complex to form a charge carrier.

[0068] Optionally, the method is for detecting whether a subject has an infection

(preferably a coronavirus infection, more preferably COVID-19). Optionally, the sample is a biological sample from a subject selected as in need of determination of the presence or absence of an infection. Optionally, the method is for detecting the presence of an analyte in a non-biological sample (for example drinking water or sewerage water).

[0069] Optionally, the method further comprises (vii) determining whether the sample comprised the analyte. Optionally, the method further comprises (viii) determining whether the subject has the infection.

[0070] Optionally, the subject is selected for a SARS-CoV-2 or COVID-19 test.

[0071] Optionally, the subject is suspected of having an infection. A subject suspected of having COVID-19 infection is optionally identified as a close contact of a person with confirmed COVID-19 infection, exhibiting COVID-19 symptoms or has any other common indications of COVID-19 infections. Optionally, the sample is from a subject for the purpose of routine monitoring of a known COVID-19 infection.

[0072] In a further aspect, the present invention provides a method comprising:

(ia) selecting at least a first sample and a second sample in need of determination of the presence or absence of an analyte;

(i) contacting the first sample to the surface of the antifouling layer of an OTFT of this disclosure, wherein the probe specifically binds with an analyte and the antifouling layer is connected directly or indirectly to one or more electrode; contacting the second sample to the surface of a second conducting antifouling layer with a probe at least partially embedded or attached (top of the antifouling layer), wherein the probe specifically binds with an analyte and the antifouling layer is connected directly or indirectly to one or more electrode

(ii) allowing any analyte present in the first sample to bind with the probe of the first antifouling layer, thereby forming a complex comprising the coronavirus analyte and the probe; allowing any analyte present in the second sample to bind with the probe of the second antifouling layer, thereby forming a complex comprising the coronavirus analyte and the probe;

(iii) labelling any analyte or complex formed between analyte and probe of the first antifouling layer with a first detection agent to form a detectable complex, wherein a portion of the first detection agent binds specifically with the analyte/complex and a portion of the first detection agent comprises at least one first reporter; labelling any analyte or complex formed between analyte and probe of the second antifouling layer with a second detection agent to form a detectable complex, wherein a portion of the second detection agent binds specifically with the analyte/complex and a portion of the second detection agent comprises at least one second reporter;

(iv) contacting any first detectable complex with a substrate for the first reporter, wherein the contact results in reaction of the substrate for the first reporter with any first reporter in the detectable complex to form a charge carrier; contacting any second detectable complex with a substrate for the second reporter, wherein the contact results in reaction of the substrate for the second reporter with any second reporter in the detectable complex to form a charge carrier;

(viia) determining that the first sample includes the analyte;

(viib) determining that the second sample does not include the analyte.

[0073] Optionally, the method is for detecting whether a plurality of subjects have an infection (preferably a coronavirus infection, more preferably COVID-19). Optionally, a first subject and a second subject are identified as in need of determination of the presence or absence of an infection (preferably a coronavirus infection).

[0074] Optionally, the method further comprises (viiia) determining the first subject has the infection and the second subject does not have the infection. Options for all methods of use

[0075] Optionally, the method is conducted at the point-of-care or point-of-sample collection ie it is not necessary to send the sample to a pathology centre or other diagnostic/analytical chemistry facility.

[0076] In some embodiments, the coronavirus is a beta-coronavirus. Optionally, the coronavirus is a beta-coronavirus of lineage A. Optionally, the coronavirus is a betacoronavirus of lineage B.

[0077] Optionally, the one or more analyte is one or more coronavirus analyte.

[0078] Optionally, the reporter is a reporter enzyme.

[0079] Optionally, charge carrier precipitates on the antifouling layer. Optionally, the charge carrier adsorbs on the antifouling layer.

[0080] Optionally, the antifouling layer has a plurality of probes, for example, the probe is coated on the antifouling layer. Optionally, the probes are all of the same type. Alternatively, the sensor includes multiple probe types, for example, antibodies to more than one coronavirus antigen or multiple coronavirus antigens. Optionally, the method further comprises (v) applying a voltage resulting in a current via the conducting antifouling layer to the one or more electrode, wherein the current is impacted by the charge carrier, thereby facilitating detection of the coronavirus analyte.

[0081] Optionally, the method further comprises (vi) measuring the current to detect the presence or determine the absence of the charge carrier.

[0082] Optionally, the sample is removed following binding of the analyte with the probe to form the complex. Optionally, the method comprises contacting a fluid comprising the detection agent to the surface of the antifouling layer with the probe. Optionally, the antifouling layer is washed before addition of the fluid comprising the detection agent.

[0083] In some embodiments, the detection agent is added to the sample. The detection agent is optionally in a fluid. This can occur following or prior to binding of the analyte to the probe. [0084] Optionally, the sample or fluid comprising the detection agent is removed following labelling the complex with the detection agent. Optionally, the method comprises contacting a fluid comprising the substrate for the reporter enzyme to the surface of the antifouling layer with the probe. Optionally, the surface of the conductive antifouling layer with the probe washed before the addition of a fluid comprising the substrate for the reporter enzyme. This washing removed unbound detection agent.

[0085] In some embodiments, the substrate for the reporter enzyme is added to the sample or the fluid comprising the detection agent. This can occur following or prior to labelling of the complex. This can occur prior to binding of the probe to the complex.

[0086] Optionally, the charge carrier is adsorbed at the surface of the conductive antifouling layer. Optionally, following adsorption of the charge carrier and before applying a voltage, the fluid including the substrate for the reporter enzyme is washed from the surface of the conductive antifouling layer with the probe. This washing removes the substrate for the reporter enzyme molecule and non-adsorbed charge carrier.

[0087] Optionally, the sample is pre-processed prior contact with the antifouling layer.

Kits

[0088] In another aspect, the present disclosure provides a kit for detection of an analyte in a sample comprising:

(i) an OTFT of this disclosure;

(ii) detection agent comprising a first portion that binds the complex of the probe and analyte and a second portion comprising at least one reporter enzyme; and

(iii) a substrate for the reporter enzyme, wherein reaction of the substrate for the reporter enzyme with the at least one reporter enzymes forms charge carrier.

[0089] Optionally, charge carrier precipitates on the antifouling layer. Optionally, the charge carrier adsorbs on the antifouling layer. Optionally, the kit further comprises a means for applying voltage to the OTFT. [0090] Further aspects of the present disclosure and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

[0091] Figure 1. Average conductance of the BSA:rGO film, in comparison with and when combined with, other components of the standard COE organic sensor.

Measurements were made when films were cast upon patterned ITO electrodes on glass substrates.

[0092] Figure 2: Output characteristic of a representative ITO/P3HT/AF coating OTFT device. Lowermost line in the figure is the curve for 0.4V increasing through to the uppermost line of -2.0V.

[0093] Figure 3: Output characteristic of a representative ITO/P3HT/Nafion OTFT. Lowermost line in the figure is the curve for 0.4V increasing through to the uppermost line of -2.0V.

[0094] Figure 4: Current-time characteristics for solution based tests on ITO/P3HT/Nafion devices. Long dashed line indicates addition of analyte, short dashed line indicates addition of TMB. The fourth inset (bottom right) shows the overlays of the first three insets.

[0095] Figure 5: Summary of results for solution based tests on ITO/P3HT/Nafion devices.

[0096] Figure 6: Current-time characteristics for solution based tests on ITO/P3HT devices. Long dashed line indicates addition of analyte, short dashed line indicates addition of TMB.

[0097] Figure 7: Results for solution based tests on ITO/P3HT devices.

[0098] Figure 8: Current-time characteristics for solution based tests on ITO/P3HT/Nafion/AF coating devices. Long dashed line indicates addition of analyte, short dashed line indicates addition of TMB. The fourth inset (bottom right) shows the overlays of the first three insets. [0099] Figure 9: Results for solution based tests on ITO/P3HT/Nafion/AF coating devices.

[0100] Figure 10: Current-time characteristics for antibody binding tests on ITO/P3HT /AF coating/antigen devices at V = 0.05 V. Dashed lines indicated time of TMB addition. The fourth inset (bottom right) shows the overlays of the first three insets.

[0101] Figure 11 : Summary of antibody binding tests on ITO/P3HT/AF coating/antigen devices at V = 0.05 V.

[0102] Figure 12: Current-time characteristics for antibody binding tests on ITO/P3HT /AF coating/antigen devices at V = 0.7 V. Dashed lines indicated time of TMB addition.

[0103] Figure 13: Summary of antibody binding tests at lt=200s/lt=65s for each analyte type. There is clear differentiation (roughly double the normalised current) between the positive COVID IgG and the two negative controls.

[0104] Figure 14: Current-time characteristics for antibody binding tests on ITO/P3HT/Nafion/AF coating/antigen devices at V = 0.7 V. Lines are from top to bottom Covid IgM + TMB, PSA Ab IgG + TMB and Covid IgG + TMB.

[0105] Figure 15: shows the structure of a device in accordance with one embodiment of the disclosure.

[0106] Figure 16: shows the structure of an alternative sensor including a dielectric layer in accordance with one embodiment of the disclosure.

[0107] Figure 17: Schematic depicting sensor device architecture (a) layer 1 , electrodes, ITO; (b) layer 2, polymer semiconductor, P3HT; (c) layer 3, polymer gating layer, Nation; (d) layer 4, antifouling layer, BSA/reduced graphene oxide; (e) layer 5, antibody probe. Each substrate pictured contains two sensors. Detailed description of the embodiments

Definitions

[0108] The following are definitions may be helpful in understanding the description of the present disclosure. These are intended as general definitions only and in no way limit the scope of the present disclosure to those terms alone.

[0109] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0110] In the context of this specification, the term "about" is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

[0111] In the context of this specification, the terms "a" and "an" refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0112] In the context of this specification, the term "bodily fluid" is understood to include any liquid which originates within a human or animal body, including fluids that are secreted or excreted. Non-limiting examples of bodily fluids include: blood, saliva, sweat, urine, breast milk, bile and peritoneal fluid.

[0113] In the context of this specification, the term "top" means farthest away from the substrate, and the term "bottom" means closest to the substrate. Where a first layer is described as "disposed above" a second layer, the first layer is disposed farther away from the substrate. Furthermore, where a first layer is described as being "disposed above" a second layer, additional intermediate layers may be present in between the first and second layers, unless it is specified that the first layer is "in direct contact with" (ie physically contacting) the second layer.

[0114] In the context of this specification, the term “sensor” refers a device that senses the presence and/or amount of something. For example, the sensor could sense the presence of a chemical such as glucose, a protein such as an antigen, or an antibody in a biological fluid.

[0115] In the context of this specification, the term “transistor” refers to a device capable of converting physical quantities into an electrical signal. Preferably, the device is a semiconductor. Preferably, the physical quantities are physical quantities of a chemical produced by an assay. The assay is optionally enzymatic. The assay is optionally a molecular diagnostic and/or involved antibody binding. The assay may be an ELISA assay.

[0116] As used herein, "to cross link" means to form one or more bonds between polymer chains so as to form a network structure such as a gel or hydrogel. The polymers are then "crosslinked" polymers. The bonding can be through hydrogen bonding, covalent bonding or electrostatic. The "cross linking agent" can be a bridging molecule or ion, or it can be a reactive species such as an acid, a base or a radical producing agent.

[0117] As used herein a "blocking material" is a compound, such as a protein or synthetic polymer, used to prevent or reduce non-specific interactions with the sensor. The blocking material when coated on the electrode, a conducting or semiconducting layer or substrate surface prevents non-specific interactions or fouling of the surface in the presence of problem compounds, such as proteins, from the sample, for example a biological fluid. The blocking material can be a protein, mixture of proteins, fragments of proteins, peptides or other compounds. Example blocking materials include proteins (eg, BSA, HSA methylated BSA, methylated HSA and Casein, nonfat dry milk, serum, gelatin), hydrophilic polymers (eg polycarbonates, PEG-based polymers, co-polymers and oligomers such as diethylene glycol dimethyl ether), hydrophobic polymers (eg polysiloxanes, PET), zwitterionic polymers (eg phospholipids), cationic surfactants (eg, DOTAP, DOPE, DOTMA), quaternary ammonium salts and polycations.

[0118] In some embodiments, the blocking material is protein, peptide, and parts or mixes thereof. Optionally the blocking material is bovine serum albumin (BSA) or human serum albumin (HSA) or peptides thereof or mixtures thereof. Optionally, the protein, peptide, parts or mixes thereof is denatured. Protein can be denatured by heating. The temperatures for denaturing and methods of denaturing are known to the skilled person. [0119] In the context of the specification, “configured to receive” means does not prevent receipt. For example, a layer “configured to receive” an ohmic conductor would not prevent contact with an ohmic conductor or receipt of voltage from that conductor as needed for function of the sensor.

[0120] In the context of this specification, the term “assay” refers to a method of detecting a biological sample suspected of having a target analyte, wherein the biological sample is contacted and incubated with the probe so that the probe binds to the analyte, if present, which can be subsequently detected in a detection step. The detection step involves the use of a labelled probe, which, when contacted with any of the bound analyte, binds to the target, and a detection means which is used to detect the label on the antibody and confirm the presence or amount of the target analyte. The assay is optionally enzymatic. The assay is optionally a molecular diagnostic, optionally involves antibody binding.

[0121] In the context of this specification, the term “coated” means that a layer of is present on a surface. For example, a layer of antifouling layer on a surface or a layer of probe on the antifouling layer. The amount of the probe used to coat the antifouling layer can vary with a number of factors such as surface area, coating density, types of probe, and binding performance.

[0122] As used herein, like reference numerals in different figures are intended to refer to the same features.

[0123] The OTFT may have a channel length, between the source and drain of electrodes, of between about 5 pm and about 50 pm, or between about 10 pm and about 30 pm, or about 20 pm, and a channel width of between about 1 mm and about 20 mm, or between about 1 mm and about 10 mm, or about 3 mm.

[0124] The OTFT may be for sensing an analyte in a sample. The analyte is optionally a biological analyte. In an embodiment the analyte is an antibody, antigen, protein, peptide or chemical. The sample may be any aqueous solution but is preferably a biological fluid, more preferably a bodily fluid, and still more preferably, saliva. The chemical is optionally glucose. Electrodes

[0125] As used herein, an “electrode” is an electrical conductor used to make contact with a nonmetallic part of a circuit (i.e., it emits or collects electrons or electron “holes”). Electrodes can comprise electrically conducting or semi-conducting material, including but not limited to metals, alloys and polymers.

[0126] Suitable electrodes are commercially available, for example pre-patterned ITO, In some embodiments, the electrodes may be fabricated by low-cost spin-coating and printing techniques.

[0127] The source and/or drain electrodes may comprise, consist, or consist essentially of an ohmic material, such as metals (eg gold or silver) or metal oxides or graphene. Preferably the source and/or drain electrodes are tin oxide. Preferably the source and/or drain electrodes are indium tin oxide (ITO), for example pre-patterned ITO. Preferably each of said source and drain electrodes consist of an ohmic material. In at least one embodiment, source electrode and drain electrode each comprise, consist, or consist essentially of ITO, for example pre-patterned ITO. In alternate preferred embodiments, the ohmic conductor is a metal based ink such as silver based ink.

Ohmic conductor for applying gate voltage

[0128] In some embodiments, the ohmic conductor comprises, consists, or consists essentially of an ohmic material, such as metals (eg gold or silver) or metal oxides or graphene. Preferably the source and/or drain electrodes are tin oxide. Preferably the source and/or drain electrodes are indium tin oxide (ITO), for example pre-patterned ITO. In alternate preferred embodiments, the ohmic conductor is a metal based ink such as silver based ink. In some embodiments the silver based ink comprises silver nanoparticles (AgNPs).

Offset ohmic conductors/gate electrodes

[0129] The ohmic conductor or gate electrode is optionally laterally offset from the source and drain electrodes, as is part of the polymer layer. When the gate voltage is applied via the ohmic conductor / gate electrode to the polymer gating layer, a substantial electric field results in a vertical plane (ie in a plane perpendicular to the top surface of the semiconductor layer).

Organic semiconducting layer

[0130] In at least one embodiment, the organic semiconducting layer consists of one organic semiconductor.

[0131] The organic semiconducting layer may be disposed above and in between the source electrode and the drain electrode. In an embodiment, the organic semiconducting layer is connected to and between a source electrode and a drain electrode. The organic semiconducting layer may be disposed above and in between the source electrode and the drain electrode, and in direct contact with, the source electrode and the drain electrode.

[0132] The organic semiconducting layer is preferably in contact with, the source electrode and the drain electrode. At least part of the organic semiconducting layer is disposed above the source electrode and the drain electrode. Preferably a majority of the organic semiconducting layer is disposed above the source electrode and the drain electrode.

[0133] The organic semiconducting layer is configured to enable flow of electrical current between the source electrode and the drain electrode as a result of the generation of these charge carriers.

[0134] The organic semiconducting layer includes one or more organic compounds. Any organic compound having semiconducting properties is suitable for use. However, it is preferred that the one or more organic compounds are selected from the group consisting of: polyacetylenes, porphyrins, phthalocyanins, fullerenes, polyparaphenylenes, polyphenylenevinylenes, polyfluorenes, polythiophenes, polypyrroles, polypyridines, polycarbazoles, polypyridinevinylenes, polyarylvinylenes, poly (p-phenylmethylvinylenes), including derivatives and co-polymers thereof, and further including combinations thereof. More preferably, the one or more organic compounds are selected from the group consisting of: poly(9,9-dioctylfluorene-2,7-diyl- co-bis-N,N-(4-butylphenyl)-bis-N,N-phenyl-1,4-phenylenediami ne), poly(9,9- dioctylfluorene-2,7-diyl-co-benzothiadiazole), poly(3-hexylthiophene), (6,6)-phenyl-Cei- butyric acid methyl ester, poly(2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene), and combinations thereof.

[0135] Most preferably, the semiconducting layer includes, consists of, or consists essentially of poly(3-hexyl-thiophene) (P3HT).

[0136] Optionally, the semiconducting layer does not include poly(4-vinylphenol) (PVP).

[0137] The organic semiconducting layer may have a thickness between about 5 nm and about 500 nm, or between about 75 nm and about 125 nm, or about 100 nm. In at least one embodiment, the organic semiconductor layer has a thickness of less than about 390 nm. In at least one embodiment, the organic semiconductor layer has a thickness of between about 36 nm and about 9 nm. In at least one embodiment, the organic semiconductor layer has a thickness of between about 22 nm and about 9 nm. In at least one embodiment, the organic semiconductor layer has a thickness between about 22 nm and about 390 nm. In at least one embodiment, the organic semiconductor layer has a thickness between about 74 nm and about 108 nm (such as between 75 nm and 100 nm).

[0138] In embodiments with a polymer gating layer, preferably, said thickness spans at least between the polymer gating layer and inner ends of the respective source and drain electrodes, the inner ends being at opposite ends of a channel between the source and drain electrodes. However, preferably said thickness is a minimum thickness between the polymer gating layer and all of the source electrode and drain electrode.

Polymer gating layer

[0139] In aspects of the disclosure not requiring a polymer gating layer there is optionally a polymer gating layer. In one embodiment, the organic semiconductor layer is in contact with said polymer gating layer. In at least one embodiment, at least part of the polymer gating layer is disposed above the semiconductor layer. Optionally, at least part of the ohmic conductor may be beneath another part of the polymer gating layer. [0140] In at least one embodiment, a layer of the probe is formed on a surface of the antifouling layer. The antifouling layer is optionally directly above the polymer gating layer.

[0141] In at least one embodiment, neither said polymer gating layer does not include poly(4-vinylphenol) (PVP).

[0142] Preferably said polymer gating layer forms a proton-conductive membrane. Preferably the polymer gating layer has a conductivity to protons that is greater than a conductivity to protons that is possessed by said organic semiconductor layer.

[0143] In at least one embodiment, at least part of the polymer gating layer is disposed above the semiconductor layer. Optionally, at least part of the ohmic conductor may be beneath another part of the polymer gating layer.

[0144] Preferably the polymer gating layer is not covered by the organic semiconductor. In at least one embodiment this is achieved by having the polymer gating layer as a top-most layer of the device, ie furthest from the substrate (other than the antifouling layer and probe). In another embodiment this is achieved by having the polymer gating layer beneath the organic semiconductor, but extending laterally beyond the organic semiconductor so that a portion of the polymer gating layer is not covered by the semiconductor.

[0145] Optionally, the polymer gating layer is 10 nm to 750 nm, 100 to 600 nm or about 400 nm thickness between the organic semiconductor or dielectric layer and the antifouling layer.

[0146] The polymer gating layer may comprise, consist, or consist essentially of a sulfonated tetrafluoroethylene-based fluoropolymer-copolymer, for example a copolymer comprising a tetrafluoroethylene backbone and perfluoroalkyl ether groups terminated with sulfonate groups.

[0147] The sulfonated tetrafluoroethylene-based fluoropolymer-copolymer may be a copolymer of tetrafluoroethylene and perfluoro-3,6-dioxa-4-methyl-7-octene-sulfonic acid. It is preferred that the sulfonated tetrafluoroethylene-based fluoropolymer- copolymer is a tetrafluoroethylene-perfluoro-3,6-dioxa-4-methyl-7-octenesul fonic acid copolymer which is commonly referred to as Nation. [0148] In an embodiment, the tetrafluoroethylene-based fluoropolymer-copolymer has the following structure:

[0149] In an embodiment, the tetrafluoroethylene-based fluoropolymer-copolymer has the following structure:

Organic dielectric layer

[0150] In some embodiments, the sensor or OTFT of the disclosure further comprises a dielectric layer intermediate the polymer gating layer and the organic semiconductor layer.

[0151] In an embodiment, the ohmic conductor is in contact with the dielectric layer and configured to apply a gate voltage to the dielectric layer.

[0152] The dielectric layer may comprise, consist of, or consist essentially of an organic dielectric material. Preferably, the organic dielectric material has a conductivity to protons that is greater than the conductivity of said semiconductor layer. Preferably the dielectric layer intermediate said polymer gating layer and said semiconductor layer is a homogenous layer. Preferably the organic dielectric material is a hygroscopic insulator, such as for example polyvinyl phenols. More specifically, the dielectric layer may comprise, consist of, or consist essentially of, poly(4-vinylphenol).

[0153] Alternative dielectric materials that may be used in the devices will be readily apparent to those skilled in the art. Non-limiting examples include polyimide and poly(methyl methacrylate) (PMMA). In alternative embodiments the dielectric layer may comprise a doped dielectric material, for example lithium perchlorate doped poly(4- vinylpyridine).

[0154] The dielectric layer intermediate said polymer gating layer and said semiconductor layer may be in contact with the organic semiconductor. Additionally or alternatively the dielectric layer may be in contact with said polymer gating layer.

[0155] The gate electrode/ohmic conductor may be disposed above the dielectric layer. The gate electrode may be disposed above, and in direct contact with, the dielectric layer or disposed above the dielectric layer, and in indirect contact with the dielectric layer via the polymer gating layer and/or the antifouling layer.

[0156] The gate electrode/ohmic conductor may be offset with respect to the dielectric layer.

[0157] The dielectric layer may be disposed above the semiconducting layer. The dielectric layer may be disposed above, and in direct contact with, the semiconducting layer. The dielectric layer may be disposed below the polymer gating layer. The dielectric layer may be disposed below, and in direct contact with, the polymer gating layer. The dielectric layer may comprise, consist of, or consist essentially of, poly(4- vinylphenol). The dielectric layer may have a thickness between about 50 nm and 750 nm, or between about 300 nm and about 500 nm, or about 400 nm.

Antifouling layer

[0158] The antifouling layer prevents, reduces or minimises fouling of one or more of the enzyme assay, binding of the probe, binding of the detection molecule, generation of charge carrier and conduction of the charge carrier by binding of undesirable protein to the surface of the sensor. [0159] The antifouling layer optionally includes semiconducting particulates or polymers and a blocking material. This is particularly desirable when the antifouling layer is the organic semiconductor or part thereof or the antifouling layer is in contact with the organic semiconductor. One or more of the organic semiconductor materials describes elsewhere in this disclosure are suitable for use as a polymeric semiconducting material with a blocking material in the antifouling layer.

[0160] Preferably, the antifouling layer is conductive to the charge carrier. Such antifouling layers include a blocking material and conducting material. Optionally, the conducting material is a particulate and/or polymer. In some embodiments, the conducting material includes a metal (eg aluminium, copper, silver, gold, or platinum), a metalloid, a conducting polymer, conducting nanoparticles (eg metal nanoparticles), a conducting carbon based material (graphite, reduced graphene oxide, or carbon nanotubes), conductive polymers or any combination of these.

[0161] The conducting material can be in any form compatible with the blocking material (ie that does not degrade the blocking material). Optionally, the conducting material is a particulate material. Suitable particulate materials include carbon nanotubes or graphene oxide. Optionally, the conducting material is a polymeric material. Optionally, the conducting material is dielectric. Optionally, the conducting material is a conductor or semi-conductor. Optionally, the conducting material comprises an allotrope of carbon atoms arranged in a hexagonal lattice. Preferably, the conducting material is functionalised graphene oxide. In some embodiments, the particular material is reduced graphene oxide.

[0162] Suitable conducting polymers include the conducting polymers and polymeric gating layer materials described in this disclosure.

[0163] In some embodiments, the conducting material includes one or more conducting polymers. In some embodiments, the polymer is one or more of a eg fluorinated polymers eg poly methyl methacrylate (PMMA), Polyhydroxyethylmethacrylate (Poly H EMA), polyacetylenes, porphyrins, phthalocyanins, fullerenes, polyparaphenylenes, polyphenylenevinylenes, polyfluorenes, polythiophenes, polypyrroles, polypyridines, polycarbazoles, polypyridinevinylenes, polyarylvinylenes, poly(p-phenylmethylvinylenes), including derivatives and co-polymers thereof, and further including mixtures thereof and copolymers and blends of these. [0164] In at least one embodiment of the disclosure the polymer one or more of: poly(9,9-dioctylfluorene-2,7-diyl-co-bis-N,N-(4-butylphenyl) -bis-N,N-phenyl-1,4- phenylenediamine), poly(9,9-dioctylfluorene-2,7-diyl-co-benzothiadiazole), poly(3- hexylthiophene), (6,6)-phenyl-C61 -butyric acid methyl ester, poly(2-methoxy-5-(2'-ethyl- hexyloxy)-1 ,4-phenylene vinylene). Also contemplated are mixtures of one or more of the above noted organic compounds.

[0165] In at least one embodiment, the conducting polymer is poly(3-hexyl- thiophene), ie P3HT. Alternatively, the conducting polymer is nation.

[0166] The antifouling layer is optionally a porous nanocomposite material comprised of a protein matrix interspersed with conducting material. Incorporation of the conducting material into the porous matrix improves the charge carrier transfer to the underlying transistor while still reducing the off-target adsorption at the surface. Alternatively, the antifouling layer is a matrix of conducting material interspersed with blocking material, for example, polymeric conducting material and protein blocking material.

[0167] In some embodiments, the antifouling layer comprises a conducting material and a blocking material.

[0168] Optionally, the blocking material is a protein. The blocking material is optionally denatured or partially denatured. Optionally, the protein is a globular protein. Additionally and/or alternatively the protein is a non-glycosylated protein. Optionally the protein may be a serum albumin protein, such as BSA (bovine serum albumin) or HSA (human serum albumin). Preferably, the blocking material comprises bovine serum albumin. The small pore size of the BSA matrix size-excludes proteins found in blood and plasma.

[0169] Optionally, the mixture further comprises a cross-linking agent. For example, the blocking material includes a cross linking agent attached to or as part of the blocking material structure.

[0170] Optionally the blocking material/protein can be cross-linked with a compatible cross linker or cross linking agent. Common cross linkers include functional groups such as maleimide,, sulfhydryl reactive groups, succinimidyl esters, sulfosuccinidimyl esters, or disulfides. Suitable cross linkers are commercially available from for example bioloqy/crosslinkers. Preferably, the cross linking agent is glutaraldehyde.

[0171] Optionally, the antifouling layer is a 3D matrix consisting of bovine serum albumin (BSA) crosslinked with glutaraldehyde and supported by a network of conducting material such as polymer or nanomaterials, such as carbon nanotubes.

[0172] Optionally, the antifouling layer comprises from 1:10 to 10:1 w/w conducting material to blocking material. Optionally, 1:10 to 5:1 w/w, 1:5 to 5:1 w/w, 1 :5 to 1 :1 w/w or 1 :2 to 1 :3 w/w conducting material to blocking material. In some embodiments the w/w ratio of conducting material to blocking material is 10:1, or 9:1 or 8:1 or 7:1 or 6:1 or 5:1 or 4:1 or 3:1 or 2:1 or 1 :1 or 1:2 or 1 :3 or 1:4 or 1 :5 or 1:6 or 1 :7 or 1 :8 or 1:9 or 1:10 w/w. In a particularly preferred embodiment, the antifouling layer comprises 1:2.5 w/w (ie 2:5 w/w) conducting material to blocking IQ3Corp Ltd.

[0173] In some embodiments, the antifouling coating comprises conducting material and a denatured protein blocking material. For example, to form a denatured protein I conducting material mixture that is coated on the OTFT. The denatured protein is optionally functionalised with a probe, such as a probe such as an antibody or antigen. The probed is detected with a detection agent (eg antibody conjugated to streptavidin- polyHRP). The TMB is oxidized, and precipitates onto the OTFT where it can be detected electronically.

[0174] Optionally, the conducting material is in continuous contact from the top to the bottom of the antifouling layer to facilitate conduct of a charge through the antifouling layer.

[0175] The surface may be further functionalised, eg with antibodies.

[0176] Without wishing to be bound by theory, the minimum amount of blocking material required for a functioning anti-fouling layer is expected to depend on the type of conducting material (ie particulate v polymer) and the morphology of the layer and the distribution of the blocking material). The antifouling layer requires blocking material to block the deposition of off-target species to the surface of the antifouling layer. It is not essential for the blocking material to be throughout the antifouling layer so long as it is attached to the surface. [0177] In some embodiments, the antifouling layer is a homogeneous matrix of conducting material and blocking material. This is preferred for embodiments where the antifouling layer is affixed to the layer below by bonding with the blocking material, for example, adsorption of the blocking material to the layer of the OTFT below the antifouling layer.

[0178] Nanocomposites were prepared by mixing, and sonicating, and centrifugation where homogenization was required, before drop casting on the surface.

[0179] Advantageously, the antifouling layer may be deposited by ink-jet printing, potentially improving manufacturing costs.

[0180] Antifouling layers similar to those of the present invention have previously been applied to metal substrates. It was not known, prior to this disclosure, whether conducting or semiconducting layers could be fixed to organic conducting or semiconducting materials and maintain both their conducting or semiconducting features and the antifouling effect.

Enzyme

[0181] For embodiments involving an enzyme probe, the enzyme is selected to facilitate generation of charge carriers when an analyte contacts the device. The charge carriers are typically electrons, anions, or cations (e.g. hydrogen ions/protons). The generation of the charge carriers may be further facilitated by the presence of an electric field. As will be described, these generated charge carriers can then contribute to electric current through the device. It will be recognised that a range of enzymes could be used for any one particular analyte. Further given the diversity of enzymes available, the device, by following the disclosure herein can be adapted or developed for detection of a range of analytes.

[0182] A particularly preferred class of enzyme is an oxidoreductase. An oxidoreductase for use in the device may act on any one of the following donor groups: the CH-OH group of donors (alcohol oxidoreductases), the aldehyde or oxo group of donors, the CH-CH group of donors (CH-CH oxidoreductases), the CH-NH2 group of donors (amino acid oxidoreductases, monoamine oxidase), CH-NH group of donors, NADH or NADPH, other nitrogenous compounds as donors, a sulfur group of donors, a heme group of donors, diphenols and related substances as donors, peroxide as an acceptor (peroxidases), hydrogen as donors, single donors with incorporation of molecular oxygen (oxygenases), paired donors with incorporation of molecular oxygen, superoxide radicals as acceptors, CH or CH2 groups, iron-sulfur proteins as donors, reduced flavodoxin as a donor, phosphorus or arsenic in donors, X-H and Y-H to form an X-Y bond, or oxidoreductases that oxidize metal ions.

[0183] The enzyme may be glucose oxidase.

[0184] In some embodiments, the enzyme is printed, eg ink-jet printed, on the antifouling layer and/or polymer gating layer. Printing methods include gravure, flexographic, screen-printing and doctor blade. Alternatively, non-printing methods known to those skilled in the art may used, such as drop casting, vapour deposition and sputtering.

[0185] An exemplary device of the prior art using an enzyme is disclosed in International patent application PCT/AU2013/000207, filed 5 March 2013. That patent application provides an example of organic transistor with a nation polymer gating layer and a hygroscopic dielectric layer. A device can also operate effectively without a hygroscopic dielectric layer between the polymer gating layer and the organic semiconductor layer as disclosed in International patent application PCT/AU2016/050555, filed 28 June 2016.

Probe

[0186] For embodiments involving a probe, the probe is selected to bind the analyte.

[0187] As used herein, a "probe” is a natural or synthetic receptor (eg, a molecular receptor) that binds to a target molecule such as an analyte. In some embodiments, the probe is an "antibody probe." The probe is optionally attached to the sensor (for example to the antifouling layer or other part of the OTFT) via a connector such as a connector protein.

[0188] In some embodiments, the binding is a specific binding such that it is selective to that target above non-targets.

[0189] For example, a probe may be, but is not limited to an antibody, an antigen, an antibody mimetic, a peptide, protein or nucleic acid (eg, an RNA or DNA aptamer) or enzyme. The probe may be a sugar, oligosaccharide, polysaccharide, lipopolysaccharides. The probe may be a receptor, for example a hormone receptor, cytokine receptor, or a synthetic receptor. Additionally or alternatively the probe may be a small molecule such as a pharmacological active substance, alkaloid, steroids, vitamins, or amino acids.

[0190] In the context of this specification, the term “antibody” includes a protein comprising an antigen binding domain and capable of specifically binding to an antigen. Preferably, the antigen binding domain is contained within a Fv. An antibody can be a polyclonal antibody, monoclonal antibody, humanized or chimeric antibody. Antibody fragments include single chain Fv antibody fragments, Fab fragments, and F(ab)2 fragments.

[0191] In some embodiments, the probe is printed, eg ink-jet printed, on the antifouling layer and/or polymer gating layer. Printing methods include gravure, flexographic and doctor blade. Alternatively, non-printing methods known to those skilled in the art may used, such as drop casting, vapour deposition and sputtering

[0192] Optionally, the antifouling layer has a plurality of probes, for example, the probe is coated on the antifouling layer. Optionally, the probes are all of the same type. Alternatively, the sensor includes multiple probe types, for example, antibodies to more than one coronavirus antigen or multiple coronavirus antigens.

[0193] The probe is selective to a specific analyte or class of analyte.

Detection agent and generation of charge carrier

[0194] If the analyte is not a charge carrier or able to generate a charge carrier the analyte bound probe can be further bound to a detection agent that can generate a charge carrier. Optionally, the detection agent includes a first portion that binds to the analyte or analyte and probe and a second portion for generation of a charge carrier. Optionally, the second portion is an enzyme. Preferably, the enzyme is a redox active enzyme, for example, a peroxidase such as horseradish peroxidase (HRP) that will generate a charge carrier in the presence of a the substrate for the reporter enzyme such as 3,3'-diaminobenzidine (DMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS); o-orthophenylenediamine (OPD); amplexred; 3,3'-diaminobenzidine (DAB); 4-chloro-1-naphthol (4CN); AEG; 3,3',5,5'-tetramethylbenzidine (TMB); homovanilllic acid; lumininol; nitro blue tetrazolium (NBT); hydroquinone; benzoquinone; or mixtures of these.

[0195] The probe/target/detection agent and substrate for the reporter enzyme facilitate generation of charge carriers when the target contacts the sensor in the presence of detection agent and substrate for the reporter enzyme and a minimum electric potential (ie gate voltage or potential) is applied via the ohmic conductor, the voltage being selected relative to at least one of the drain and source electrodes. As for embodiments of the sensor with an enzyme, the charge carrier impacts the current in the device allowing detection of the target.

[0196] In some embodiments the probe facilitates generation of a charge carrier indirectly. For example, following binding of the analyte to the probe, the analyte can be detected by binding with a detection agent such as an antibody, protein or other molecule that generates a charge carrier by catalyzing, directly or indirectly, a redox reaction close to the surface of the OTFT. Optionally, the detection agent, antibody, protein or other molecule in the presence of a substrate for the reporter enzyme causes deposition of a charge carrier on the sensor surface (eg, on the antifouling layer and/or polymer gating layer of the sensor). The charge carrier alters the charge applied by the ohmic conductor/gate electrode. The difference in the charge (eg voltage and/or current) is detected by the sensor. For example, the detection agent can be conjugated with a redox catalyst and, in the presence of the substrate for the reporter enzyme, substrate for the reporter enzyme is oxidized or reduced and precipitated onto the OTFT surface, thereby generating a charge carrier.

[0197] Embodiments include known immunoassays or modifications of these to be detectable by an OTFT.

[0198] In some embodiments, the detection agent is used at a concentration between about 1 and about 500 mg/mL. In embodiments, the detection agent is used at a concentration between about 10 and 100 mg/mL, such as between about 20 and 100 mg/mL, or between about 20 and about 100 p/mL. In some embodiments, the detection agent is (eg, detection antibody) is used at a concentration between about 0.1 and 100 mg/mL, such as between about 0.5 and 50 mg/mL, between about 1 and 30 mg/mL, between about 10 and 30 mg/mL, or between about 15 and 25 mg/mL. [0199] In embodiments, the substrate for the reported enzyme includes rabbit antihuman IgG-HRP, HPR:AB, or a similar molecule for signal augmentation. In embodiments, the HRP concentration is between about 0.1 and about 100 pg/mL, such as between about 1 and 50 p/mL, or between about 5 and 20 p/mL. The ranges of concentrations of substrate for the reported enzyme and detection agent can be used in any combination, such as about 20 mg/mL of substrate for the reported enzyme in combination with 10 pg/mL of detection agent. The ranges of concentrations of substrate for the reported enzyme, detection agent and HRP may also be used in any combination, such as 10 pg/mL of probe, 20 mg/mL of detection agent and 10 pg/mL HRP.

Charge carrier

[0200] The charge carrier may be any one or more of the following charge carrier types: anions, cations or electrons. However, in at least one embodiment the charge carriers are cations, and more preferably hydrogen ions (eg protons).

[0201] Preferably, the organic semiconductor is doped by interaction with said charge carriers, preferably protons, to increase an electrical conductivity between the drain electrode and the source electrode.

Substrate layer

[0202] In one or more embodiments, the OTFT further comprises a substrate layer. Preferably, at least the source electrode and drain electrode are disposed on the substrate. In at least one form, the source electrode, drain electrode and organic semiconductor are each in contact with the substrate. The substrate may be glass, or any other suitable substrate known to those skilled in the art, for example metal, paper or a low-cost plastic, such as polyethylene terephthalate (PET).

[0203] The source electrode and the drain electrode may be disposed above the substrate. The source electrode and the drain electrode may be in direct contact with the semiconductor layer. Analyte

[0204] The one or more probe and one or more analyte in each embodiment are compatible ie each probe specifically binds an analyte.

[0205] The analyte can be an ion, molecule, oligomer, polymer, protein, peptide, antigen, antibody, nucleic acid, toxin, biological threat agent such as spore, viral, cellular and protein toxin, carbohydrate (eg, monosaccharide, disaccharide, oligosaccharide, polyol, and polysaccharide), lipid, fatty acid, or combinations of these. The analyte is optionally an antigen or antibody indicative of infection or resistance to infection. The analyte is optionally a clinical chemistry analyte.

[0206] In some embodiments the analyte can be redox active and the probe/enzyme directly responsible for generation of the charge carrier that is detected by an electrode. For example, the binding of the analyte to the probe facilitates generation of the charge carrier near the antifouling layer surface, the conducting antifouling layer conducts the charge carrier and this impacts the applied voltage and/or current resulting in detection of the analyte by the sensor. In some embodiments, the electrode is a gate electrode of an OTFT.

[0207] In some embodiments, the analyte is immunological or serological, for example an antigen or antibody. In preferred embodiments the analyte is an antigen or antibody a coronavirus, Hepatitis A, Hepatitis B, Hepatitis C, or HIV.

[0208] Preferably the analyte indicates a coronavirus infection or immunity, more preferably COVID-19 infection or immunity.

[0209] In some embodiments, the analyte is a coronavirus antigen. Preferably, the coronavirus antigen is a SARS-CoV-2 antigen. Examples of SARS-CoV-2 antigens include, but are not limited to, nucleocapsid, glycoprotein spike including individually the S1 and S2 subunits and the RBD domain, membrane glycoprotein, small envelope protein, accessory proteins, non-structural proteins and any combinations thereof.

[0210] In some embodiments, the analyte is a coronavirus antibody. Optionally, a SARS-CoV-2 antibody. Optionally, the SARS-CoV-2 antibody is an immunoglobulin M (IgM), immunoglobulin A (IgA) or immunoglobulin G (IgG) antibody. Examples of IgG subtypes are lgG1, lgG2, lgG3 and lgG4. Optionally, the analyte is anti-SARS-CoV-2 nucleocapsid IgG.

[0211] In some embodiments multiple probes may be used to detect multiple analytes, for example, multiple antigens for the same infection or an antigen and antibody for the same infection.

[0212] In some embodiments the probes are selected to cover one or more analytes for one or more diseases of the TORCH Screen ie for detecting one or more of Toxoplasmosis, HIV, hepatitis A, B or C, varicella, parvovris, rubella, cytomegalovirus, herpes simplex and syphilis.

[0213] In some embodiments the analyte is a hormone, for example a gynaecological hormone such as luteinizing hormone (LH), progesterone, estradiol or follicle-stimulating hormone. In preferred embodiments the probe detects LH. In some embodiments the probe is a LH specific antibody. In some embodiments the probe is an LH monoclonal antibody. Additionally or alternatively the hormone may be a pregnancy hormone such as human chorionic gonatropin (hCG).

[0214] In some embodiments the analyte is a clinical chemistry analyte such as an ion, salt, mineral, metabolite, therapeutic drug, toxicology marker, drug of abuse, transport protein, enzyme, specific protein, lipoprotein or marker, for example diabetes or myocardial infarction markers. In some embodiments the analyte is a metabolite selected from the group of glucose, cholesterol, urea, lactic acid, bilirubin, creatinine, triglycerides. In preferred embodiments the probe is selected to detect glucose or cholesterol.

[0215] In some embodiments, the analyte is a tumour marker. Tumour markers can be used in guiding treatment decisions, monitoring treatment, predicting the change of recovery and to predict or monitor for tumour recurrence.

[0216] Once the analyte is selected, a probe is selected to bind the analyte. Many combinations of analyte and probe are known in the art. Coronaviruses

[0217] Coronaviruses, belong to the Coronaviridae family in the Nidovirales order, are minute in size (65-125 nm in diameter) and contain a single-stranded RNA as a nucleic material, ranging from 26 to 32kbs in length. The subgroups of coronaviruses family are alpha (a), beta (P), gamma (y) and delta (5) coronavirus. The beta-coronaviruses are of the greatest clinical importance concerning humans. Beta-coronavirus of lineage A include OC43 and HKLI1 (which can cause the common cold). Beta-coronaviruses of Lineage B include the severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2 (which causes the disease COVID-19). Middle East respiratory syndrome coronavirus (MERS-CoV) is a beta-coronavirus from lineage C. These viruses cause acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) which leads to pulmonary failure and death.

Voltage

[0218] The gate voltage VG and drain voltage VD provide a sufficiently strong electric field to liberate H+ from H2O2, but not strong enough to cause electrolysis of water, as electrolysis of water may lead to a decrease in the signal-to-noise ratio of the sensor (ie below -1.23 V). Typically, the gate voltage and drain voltage applied are between about 0 V and -2 V, e.g. about -1 V. Where a simple sensor is used, low voltages can be used including 250 to 500 mV to 300-400 mV. The voltage range can then be -0.25 to -2V or - 0.25 to -0.1 V.

[0219] When the sensor is an OTFT with an organic semiconductor with a conducting layer (eg nation), the voltage applied (eg by an ohmic conductor/gate electrode) is at least -0.55 V, -0.6 V, -0.65 V or -0.7V (ie -0.55 V to -2 V). -0.6 to -0.8V and -0.65 to - 0.75 V are preferred ranges.

[0220] The voltage applied by the ohmic conductor/gate electrode is optionally at least 0.7 V.

Printing

[0221] Printing methods include gravure, flexographic, screen-printing and doctor blade. Alternatively, non-printing methods known to those skilled in the art may used, such as drop casting, vapour deposition and sputtering. Sample

[0222] In accordance with various embodiments described herein, a sample, including any fluid or specimen (processed or unprocessed) that is intended to be evaluated for the presence of an analyte can be subjected to methods, compositions, kits and systems described herein. The sample or fluid can be liquid, supercritical fluid, solutions, suspensions, gases, gels, slurries, and combinations thereof. The sample or fluid can be aqueous or non-aqueous.

[0223] In some embodiments, the sample can be an aqueous fluid. An aqueous fluid includes biological fluids as described below. Optionally, if the sample is water-based but not fluid, an aqueous solution can be added to produce a fluid sample.

[0224] In some embodiments, the sample can include a biological fluid obtained from a subject. Exemplary biological fluids obtained from a subject can include, but are not limited to, blood (including whole blood, plasma, cord blood and serum), lactation products (e.g., milk), amniotic fluids, sputum, saliva, urine, semen, cerebrospinal fluid, bronchial aspirate, perspiration, mucus, liquefied stool sample, synovial fluid, lymphatic fluid, tears, tracheal aspirate, and any mixtures thereof. In some embodiments, a biological fluid can include a homogenate of a tissue specimen (e.g., biopsy) from a subject. In one embodiment, a test sample can comprises a suspension obtained from homogenization of a solid sample or a fragment thereof obtained from a subject.

[0225] In some embodiments, the sample can include a fluid or specimen obtained from an environmental source. For example, the fluid or specimen obtained from the environmental source can be obtained or derived from food products or industrial food products, food produce, poultry, meat, fish, beverages, dairy products, water (including wastewater), surfaces, ponds, rivers, reservoirs, swimming pools, soils, food processing and/or packaging plants, agricultural places, hydrocultures (including hydroponic food farms), pharmaceutical manufacturing plants, animal colony facilities, and any combinations thereof.

[0226] In some embodiments, the sample can be a non-biological fluid. As used herein, the term “non-biological fluid” refers to any fluid that is not a biological fluid as the term is defined herein. Exemplary non-biological fluids include, but are not limited to, water, salt water, brine, drinking water, industrial water, brown water, sewerage, and mixtures thereof. Preferred non-biological fluids are drinking or industrial water or sewerage.

Determining concentrations

[0227] The impact of a specific concentration of analyte on the current in the semiconductor in a specific sensor can be calibrated and the sensor used to identify the concentration of analyte in a sample.

Exemplary devices

[0228] An exemplary organic thin film transistor based sensor 100 in accordance with one embodiment of the disclosure is illustrated in Figure 15, which provides a conceptual representation of the structure of the sensor 100. Sensor 100 includes a drain electrode 102 and source electrode 104 disposed on the surface of a substrate 106. The sensor includes a three-layered structure that includes: an antifouling layer 108, a polymer gating layer 120, and an organic semiconducting layer 122. The organic semiconducting layer 122 covers a portion of the drain and source electrodes, with the organic semiconducting layer 108 in contact with, and extending between the drain electrode 102 and the source electrode 104. The polymer gating layer 120 is disposed on a surface of the organic semiconducting layer 122. An ohmic conductor 132 is in contact with polymer gating layer 120 to enable a gate voltage to be applied to the polymer gating layer 120. An antifouling layer 108 is disposed on the surface of the polymer gating layer 120. The surface of the antifouling layer 108 is exposed to receive a fluid sample. An enzyme or probe is on the surface of the antifouling layer 108. The antifouling layer 100 is not located between gate and drain electrodes. The antifouling layer 100 is optionally configured to enable an ohmic conductor to connect to the antifouling layer. In alternative embodiments the ohmic conductor 132 is in contact with the antifouling layer to enable a gate voltage to be applied.

[0229] The polymer gating layer 120 is disposed between the antifouling layer 108 and the organic semiconducting layer 122. The function of polymer gating layer 120 is to facilitate transport of charge carriers generated above the antifouling layer 108 from an analyte on application of a gate voltage and transported via the conducting antifouling layer. A range of different polymers may be used to form the polymer gating layer 120 depending on the nature of the analyte, enzyme, and/or charge carrier. In a preferred form, the charge carriers are protons (such as hydrogen ions) and the polymer gating layer 120 and antifouling layer 108 are proton conducting. Preferably the polymer layer has a conductivity to protons that is greater than a conductivity to protons of the organic semiconductor layer 122. The conductivity may be due to permeability of the polymer gating layer 120 to charge carriers, such as where conduction occurs via migration of the charge carriers; alternatively, conduction may occur via another mechanism, such as the Grotthuss mechanism. Where a charge carrier is a proton, or is an electron, the polymer gating layer may be proton conducting or electron conducting.

[0230] The organic semiconducting layer 122 is configured to enable flow of electrical current between the source electrode and the drain electrode as a result of the generation of these charge carriers.

[0231] The use of the OTFT 100 will now be described below in relation to a preferred embodiment in which the OTFT 100 is for detecting the presence of glucose in a saliva sample.

[0232] In use, a gate voltage VG and a drain voltage VD are applied to the OTFT100. The gate voltage applied by via ohmic conductor or gate electrode. The drain voltage applied via the drain electrode. The voltages being with respect to the source electrode 104. A liquid sample comprising glucose, for example a bodily fluid such as saliva, is contacted with the antifouling layer 108 upon which glucose oxidase enzyme (GOX) has been deposited/attached. The glucose is degraded via an enzymatic reaction with GOX thereby producing H2O2. The gate voltage VG and drain voltage D provide a sufficiently strong electric field to liberate H ⁺ from H2O2, but not strong enough to cause electrolysis of water, as electrolysis of water may lead to a decrease in the signal-to-noise ratio of the sensor (ie below -1.23 V). Typically, the gate voltage and drain voltage applied are between about 0 V and -2 V, e.g. about -1 V.

[0233] When the is an OTFT with an organic semiconductor with a conducting layer (eg nation), the voltage applied (eg by an ohmic conductor/gate electrode) is at least 0.55 V, 0.6 V, 0.65 V or 0.7V (ie -0.55 V to -2 V). -0.6 to -0.8V and -0.65 to -0.75 V are preferred ranges.

[0234] The H ⁺ ions are conducted though the conductive antifouling layer 108 though the polymer gating layer 120 (e.g. Nation) to the organic semiconducting layer. In embodiments without a polymer gating layer the H+ ions are conducted through the antifouling layer directly to the organic semiconducting layer. This results in doping of the semiconductor (from the Nation and optionally also from the AF layer), and consequentially, current between the drain and the source electrodes. Without wishing to be bound by theory, the inventors are of the view that the gate potential controls the doping and de-doping of the semiconducting compound(s) via ion migration from the site of ion generation to the active channel in the organic semiconductor. Thus, the increase in H ⁺ ions results in an increase in drain current, such that a relationship is established between the amount of glucose present in the sample and the magnitude of the drain current. The drain current is then measured which provides an indication of the presence and optionally concentration of glucose within the saliva sample.

[0235] The skilled person will be aware that a similar outcome can be achieved using different enzyme, analyte and charge carrier options.

[0236] In an alternative form of the disclosure, as illustrated in Figure 16, the OTFT 200 is similar to OTFT 100 of Figure 15, but further comprises a dielectric layer 234 between and in contact with polymer gating layer 220 and organic semiconductor layer 222.

[0237] The dielectric layer 234 comprises, consists of, or consists essentially of an organic dielectric material. Preferably, the organic dielectric material has a conductivity to protons that is greater than the conductivity of organic semiconductor layer 222. Preferably the organic dielectric material is a hygroscopic insulator, such as for example polyvinyl phenols. More specifically, the dielectric layer may comprise, consist of, or consist essentially of, poly(4-vinylphenol).

[0238] The device operates at least for an organic semiconductor layer thickness of less than about 390 nm. Further, there is advantageous device behaviour when the organic semiconducting layer has a thickness in the range of about 75 nm and about 100 nm, between the polymer gating layer and the source and drain electrodes. The advantage is that in this range the device has a calibration curve that has a one-to-one correspondence between a calibration parameter and glucose concentration for concentrations between 0.1mM and 100mM. Further their results have shown the calibration curve as being is essentially linear over that range. [0239] Further, there is also advantageous behaviour when the organic semiconducting layer instead has a thickness in the range of about 36 nm or less, between the polymer gating layer and the source and drain electrodes. The advantage in this case is a faster response time for the device.

[0240] It will be understood that the disclosure disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the disclosure.

Examples

Example 1 - Preparation of the AF coated substrates

Materials and Reagents

[0241] Tetraethylene pentamine functionalised-reduced graphene oxide (rGO), glutaraldehyde (GA), N-hydroxysuccinimide (NHS), 2-(N-morpholino)ethanesulfonic acid hydrate (MES), 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric substrate, bovine serum albumin (BSA), ethanolamine, Tween 20, 0.01 M phosphate buffer saline (PBS, pH 7.4) were purchased from Sigma-Aldrich. 1-ethyl-3-(3- dimethylamonipropyl)carbodiimide (EDC), blocker blotto blocking buffer were purchased from Thermo Scientific. Recombinant SARS-CoV-2 Nucleocapsid Protein (His-tag) (230-30164) (SARS-CoV-2 N protein) was purchased from Raybiotech. SARS-CoV-2 Spike S1-His Recombinant Protein (HPLC-verified) (40591-V08H) (SARS-CoV-2 S1 protein) was purchased from Sino Biological. SARS-CoV-2 Spike S1 antibody (hlgM2001), human chimeric (A02046) (SARS-CoV-2 IgM) and SARS-CoV-2 Spike S1 antibody (HC2001), human chimeric (A02038) (SARS-CoV-2 IgG) were purchased from GenScript. Rabbit anti-human Prostate Specific Antigen antibody (IgG) (ab53774) (PSA IgG) and rabbit anti-human IgG H&L (HRP) (ab6759) (anti-human IgG-HRP or HRP:AB) were purchased from Abeam.

Preparation of substrates

[0242] Patterned indium tin oxide (ITO) on glass substrates were cleaned using liquid Pyroneg solution, deionised water, acetone and isopropanol. The ITO pattern consists of two sets of three electrodes: two of which are separated by a 20 pm long and 3 mm wide channel and the third offset by 2 mm orthogonal to the channel.

Preparation of Thin Films

[0243] Where required, P3HT (synthesised in accordance with the method of Pappenfus et al. Macromol. Chem. Phys., 2018, 219: 1800272; section 2.2.2 GRIM Synthesis of Poly(3-hexylthiophene), 20 mg/mL or 10 mg/mL in chloroform) was spin coated on to ITO-on-glass substrates at 2000 rpm and 1596 rpm/s for 60 seconds to produce a thin film approximately 150 nm in thickness. Nation (N-(3-acetylphenyl)-4-(2- phenylethyl)thieno[3,2-b]pyrrole-5-carboxamide) solution (Sigma Aldrich, part number 274704) was used as received and spin coated on to P3HT films as appropriate at 550 rpm and 399 rpm/s for 120 seconds to produce a thin film approximately 400 nm in thickness.

Preparation of rGO/BSA nanocomposite

[0244] A BSA stock solution was first prepared at a concentration of 5 mg/mL in 10mM PBS solution (pH 7.1 - 7.5). A 2 mg of pentamine functionalised rGO was then dispersed in 1 mL of BSA solution (5 mg/mL) by sonication at 1s on/off cycles for 30 min. For protein denaturation, the rGO/BSA dispersions were heated at 105.5°C for 5 min. Subsequently, the mixture was centrifuged at 4800 x g for 15 min and the supernatants were obtained to prepare anti-fouling film.

Deposition of the Anti-fouling (AF) Coating

[0245] The substrate onto which the AF coating will be deposited (here, either ITO on glass, ITO/P3HT or ITO/P3HT/Nafion) is appropriately prepared as described above. Thereafter, the previously prepared rGO/BSA supernatants were mixed with GA in the ratio of 1 :69. A 200-pL aliquot of rGO/BSA/GA mixture was deposited on the substrate immediately and incubated overnight at room temperature in the dark to fabricate the AF film. Subsequently, the film is rinsed with 0.01 M PBS (500 rpm, 5 min) and spin- dried at 4800 rpm for 1 min. Antigen-functionalisation of the AF Coating

[0246] The AF coating was prepared as described in the section above. The carboxyl groups of BSA were activated by incubation in 200 pL of a freshly EDC/NHS mixture solution (77 mg/mL of EDC and 23 mg/mL of NHS in 0.05 M MES buffer, pH 6.2) for 30 min at room temperature. The specific coating protein were covalently attached onto activated surface by drop-casting 5 pL of the capture N/S1 protein (0.43 mg/mL) and incubated overnight at 4 °C. Subsequently, the capture N/S1 proteins were rinsed with 0.01 M PBS at 500 rpm for 5 min, spin-dried at 4800 rpm for 1 min, and incubated with 20 pL of 1 M ethanolamine solution for 30 min to deactivate the remaining NHS esters. Washing and spin-drying (4800 rpm, 1 min) were performed with PBST (0.1% Tween 20 in PBS), followed by a 2 h blocking step with 20 pL of 5% Blotto at room temperature. Thereafter, the blotto was washed with PBST and spin-dried at 4800 rpm for 1 min.

Example 2 - Detection of SARS-CoV-2 IgG through antigen binding with functionalised AF coated devices

[0247] Antigen-functionalised devices were incubated with 10 pL of the target antibody (SARS-CoV-2 IgG, 10 pg/mL in 0.01 M PBS) or negative controls (SARS-CoV- 2 IgM or PSA IgG, 10 pg/mL in 0.01 M PBS) for 30 min at room temperature.

Subsequently, the devices were washed with PBST and spin-dried (4800 rpm, 1 min) to remove nonspecific binding, and the devices were then incubated with 10 pL of a rabbit anti-human IgG-HRP (10 pg/mL) prepared in 0.01 M PBS buffer for 30 min. After one final washing/spin-drying (4800 rpm, 1 min) with PBST, the devices were ready for testing.

[0248] Using a semiconductor parameter analyzer coupled with a probe station, a drain voltage of up to 0.7 V was applied and the resulting current measured for a period of up to 600 s. Once the background current was stabilised, 10 pL of TMB solution was added at 60 s. The device response after the TMB addition was monitored relative to the current at t = 65 s.

Solution-based detection of HRP:AB

[0249] ITO/P3HT, ITO/P3HT/Nafion or ITO/P3HT/Nafion/AF coating devices were prepared as described above. To test, a voltage of 0.05 V was applied for 300 s across the two electrodes separated by the 20 pm channel and the current recorded. At t = 30 s, 5 pL of the analyte solution (either HRP:AB, a non-HRP conjugated antibody or PBS) was deposited over the channel area, and at t = 60 s, 10 pL of TMB solution was added. The device response after the TMB addition was monitored relative to the current at t = 55 s.

[0250] The results demonstrated that current could be detected via an electrode in an organic semiconducting sensor with a nation doping layer and an antifouling coating.

[0251] Example 3 - Characterisation of a coated transistor

[0252] The AF coated ITO/glass, ITO/P3HT or ITO/P3HT/Nafion and AF coated ITO/P3HT/Nafion of example 1 was characterised using two-terminal conductance measurements, optical microscopy and Raman mapping.

Electrical characterisation

[0253] The electrical characterisation results are shown in Figure 1. The AF film alone has very low electrical conductivity (<10 ^-9 S). When the AF film was deposited on a P3HT substrate, electrical conductivity was increased compared to the native P3HT substrate (10 ⁷ -10 ⁶ S and approx. 10 ^-8 S respectively). The P3HT/Nafion substrate demonstrated the highest electrical conductivity of all substrates (10 ⁶ -10 ⁵ S), with slight dedoping observed when the AF film was deposited (approx. 10 ^-6 S). The slight reduction in conduction with addition of the AF coating is a minimal cost for the antifouling effect demonstrating efficacy of this technology. (

Optical Microscopy

[0254] Optical characterisation of the AF coating on ITO/glass shows a dendritic distribution of small aggregates in the film (sub 1 pm). When the film is deposited on P3HT, the dendritic nature is maintained, but a secondary distribution of aggregates is observed. This is believed to be following the topography of the P3HT film. When the AF coating is deposited on Nation, a significant morphology change occurs. Larger aggregates are now observed (> 1 pm) which are randomly distributed throughout the AF matrix.

[0255] Surprisingly, the AF layer functioned to conduct current and prevent fouling despite these morphology differences. OTFT Characterisation

[0256] In order to assess the viability of using the AF coating as a dielectric material in the fabrication of an organic thin film transistor (OTFT) device for use in sensors (to allow for potential increase in device sensitivity) the output characteristic of an ITO/P3HT/AF coating device was recorded (Figure 2). This output characteristic was compared with that of a reference device using Nation as a dielectric layer (Figure 3). For the ITO/P3HT/AF coating devices, the AF coating was extended to the third (gate) electrode on the ITO-on-glass substrate. The current modulation ratio for the reference device for a gate voltage (VGS) range was 2.25 at drain voltage (VDS) of -1.5 V. For the AF coating dielectric device, the corresponding current modulation ratio was 1.67.

[0257] The characterisation shows that the AF film is a complex matrix within which functionalised rGO is restricted to aggregated domains which tend to follow the morphology of any underlying P3HT. A drastic morphology change is observed when the antifouling layer is deposited onto Nation, with a larger-scale, random distribution of aggregated material now being observed in the film. Conductance measurements of AF, P3HT, P3HT/Nafion, P3HT/AF and P3HT/Nafion/AF film stacks show that the BSA-rGO film has lower conductance than native P3HT, that the addition of Nation to P3HT results in highly doped P3HT: Nation interface and a corresponding increase in conductance above that of P3HT and that the AF film also dopes the P3HT:AF interface, increasing the conductance of the bilayer, but to a lesser extent than Nation (Figure 1).

Example 4 - Design of an antigen integrated AF coated OTFT architectures and characterisation of antibody response

[0258] On a P3HT/Nafion bilayer, the initial conductivity of the channel material was measured to be relatively high and the P3HT/Nafion interface is doped. Addition of plain PBS or antibody solutions in PBS (at t = 30 s) results in a lowered current due to dedoping of the P3HT/Nafion interface due to removal of protons from the interface. The subsequent addition of the TMB solution (at t = 60 s) resulted in a further reduction in current. This reduction is attributed to a reaction between doped P3HT and the neutral TMB. This reaction results in oxidation of the TMB to TMB2+ and dedoping of the P3HT was observed in all cases. In the absence of HRP, this dedoping continues during the testing period until approximately t = 200 s at which point the current stabilises (Figure 4 (a) and (b)). When the HRP-conjugated antibody is present in solution a competitive reaction occurs. The TMB is partially oxidised by the HRP (again to form TMB2+ which aggregates on the film surface as a blue precipitate). Consequently, less TMB is available in solution to dedope the P3HT and the current remains higher (Figure 4 (c)), indicating a positive response for the presence of HRP as shown in the composite plot of representative data in Figure 4 (d).

[0259] The ratio of lt=200s/lt=55s was chosen as a calibration parameter to compare the response of devices to the applied analyte. The choice of 55 s for the calibration reference point was made in order to provide a reference where the hydration level of devices has had time to equilibrate (accounting for any difference due to storage or testing conditions) but prior to the addition of TMB. Figure 5 shows the average value of this calibration parameter for devices with either PBS, COVID antibody or a HRP- conjugated antibody (HRP:AB) and a TMB developer solution used in all cases as the secondary addition. The graph clearly shows that the addition of the HRP:AB results in a four-fold higher normalised current value indicating a positive response (i.e. less dedoping of the P3HT/Nafion interface) in comparison to the other two analytes. Due to the similarity in the responses of the devices to PBS and COVID antibody additions, in subsequent experiments PBS was judged to be a suitable negative control.

[0260] In order to further probe the mechanism of device operation, a film of P3HT without the addition of Nation was used in the device. The initial conductivity of the undoped P3HT is much lower than the P3HT/Nafion devices, and hence the current for the same applied voltage (50 mV) is also lower. Upon addition of PBS solutions (t = 30 s), an initial increase in current as the buffer partially dopes the low conductivity P3HT was observed. Upon addition of the TMB (at t = 60 s), a current pulse was observed which is attributed to ionic solution-based conductivity and then a current drop as TMB dedopes the P3HT and forms TMB2+ on the film surface. When HRP is present in solution the competing oxidation of the TMB was again observed. The amount of neutral TMB in solution is lowered (having already formed TMB2+) and therefore the P3HT is less dedoped. Indeed, for the native P3HT films used here with a low initial doping level, it is likely that TMB2+ on the surface of the film may even further dope the P3HT, as evidenced by the slowly increasing current observed for the 1 pg/mL HRP:AB devices (Figure 6(a)). [0261] Due to the large difference between the responses to 1 pg/mL HRP:AB solution and the negative PBS control, the response to a lower concentration of HRP:AB (chosen to be 0.1 pg/mL) was also recorded (Figure 6 (b)). Encouragingly, there is a distinct difference between the response to 0.1 pg/mL HRP:AB solution and the negative control (Figure 6 (c)) indicating that the devices are currently operating well above the limit of detection. A composite plot of representative data for each analyte is presented in Figure 6 (d).

[0262] Figure 7 shows the average value of lt=2oos/lt=55s for each analyte type. The graph clearly shows that the addition of 1 pg/mL HRP:AB results in a greater than 20 times increase in normalised current in comparison to the negative control and the 0.1 pg/mL HRP:AB response lying between the other two analytes with clear separation from the negative control.

[0263] Having elucidated the mechanism for current modulation upon HRP/TMB addition to P3HT-based sensors, the architecture of the P3HT/Nafion devices was changed to include a capping layer of the antifouling material described elsewhere (as a step towards the covalent bonding of the antigen as a recognition element described below). Note that the antifouling layer used does not include functionalisation by a COVID antigen, and is simply composed of BSA and functionalised-rGO crosslinked with glutaraldehyde.

[0264] The addition of the antifouling layer to the P3HT/Nafion bilayer results in a small relative dedoping of the P3HT/Nafion interface (resulting in an initial current level which falls between those observed for pure P3HT and P3HT/Nafion as seen above in Figure 1). Upon addition of the PBS buffer solutions an initial drop in the current was observed (due to dedoping of the interface) similar to that observed for the P3HT/Nafion bilayer devices. However, in this case the antifouling layer addition dedopes the P3HT interface sufficiently and now the initial ionic current pulse is observed upon the addition of the TMB. Note that the ionic (capacitive charging) current observed here is also observed in the native P3HT devices, but not in the P3HT/Nafion devices where the interface is highly doped and the associated higher current levels mask this response.

[0265] Again, after the initial current increase, dedoping of the P3HT by neutral TMB results in a gradual decrease in current and the formation of blue TMB2+. When HRP is present the competing oxidation of TMB reduces this effect, resulting in the observed higher current and a positive response for HRP (Figure 8). However, the inventors postulate that the addition of the antifouling layer obscures this positive response at lower (0.1 mg/mL) HRP concentrations in contrast to the response observed in the native P3HT devices presented above. This result may be a consequence of changes to the antifouling layer morphology since, as discussed above, the morphology of the antifouling film is significantly different when cast on Nation as opposed to P3HT or just glass.

[0266] Figure 9 shows the average value of I t=200s/l t=55s for each analyte type. The graph clearly shows that the addition of 1 pg/mL HRP:AB results in approximately three fold increase in normalised current in comparison to the negative control and the 0.1 pg/mL HRP:AB response.

[0267] Despite the apparent hindering of device response by the inclusion of the antifouling (AF) layer on P3HT/Nafion devices, the AF coating importantly facilitates covalent binding of the COVID antigen recognition element at the solution-device interface, in addition to reducing non-specific binding of interfering organic species.

[0268] ITO/P3HT, ITO/P3HT/Nafion and ITO/P3HT/AF devices all show reproducible and characteristic current changes when the HRP:AB and TMB are added to the device as solutions. These current changes are clearly distinguishable from negative control experiments and provide an excellent basis for sensor operation. The proposed mechanism for the detection is discussed. The addition of the antifouling layer appears to reduce the current change, resulting in little differentiation of current between low (0.1 pM HRP) analyte concentrations and negative response controls.

Example 5 - Fabrication of sensors for antibody detection

[0269] Devices suitable for binding of antibodies were fabricated and tested.

[0270] First the functional antifouling coating was applied to P3HT films. Then subsequently Nation was included as an intermediary layer. Initially, the device voltage was chosen to be 0.05 V.

[0271] Similar to the results observed in the solution-based tests above, addition of TMB to the ITO/P3HT/antifouling coating/antigen device, results in an initial current spike which is attributed to ionic (or capacitive) current in solution. During these tests, HRP should only be present for the COVID IgG analyte test as the HRP-conjugated secondary antibody interacts selectively with IgG antibodies, and due to the selective nature of the antigen recognition element, only COVID IgG should be present upon addition of the HRP-conjugated secondary antibody. Unlike the solution-based tests, there is rapid stabilisation of the current, however no difference is observed between the different analytes in the output of the devices (Figure 10).

[0272] Figure 11 summarises the results of the above experiment using a similar analysis method as previously described for the solution-based tests. However, for these binding tests, since the antibody analyte is already present on the device and there is only a single solution addition during testing (that of TMB at t = 60 s), it was not possible to normalise to 55 s as it is expected that there is some variation in the hydration level of devices. Consequently, a normalisation time of t= 65 s was chosen for these experiments; this choice was made to avoid complication around the initial current peak after analyte addition due to the buffer (i.e. between t = 60 s and ~ 62 s) early enough to minimise changes due to the analyte itself. This graph clearly shows that there is no differentiation between the target analyte (COVID IgG) and the negative controls (PSA IgG and COVID IgM).

[0273] In considering how to improve the response of the devices, it was considered that the voltage seen at the device-solution interface may be critical to sensor operation. Consequently, the device voltage was initially increased ten-fold (to 0.5 V) in an attempt to compensate for the voltage drop across the less conductive AF coating. However, preliminary results when conducting the same experiment at a device voltage of 0.5 V resulted in a lack of discrimination between analytes similar to the 0.05 V data. Subsequently, the device voltage was increased further to 0.7 V.

[0274] When 0.7 V was applied to the ITO/P3HT/AF coating/antigen device, the initial ionic current pulse is observed upon the addition of TMB, as observed previously. Subsequently, a reduction in current is observed for all three tests as TMB dedopes the P3HT film and forms TMB2+ on the film surface. However, unlike the 0.05 V and 0.5 V cases, at 0.7 V, when HRP is present in solution, competing oxidation of the TMB was not observed. Consequently, there is a lower concentration of TMB in solution, and therefore the P3HT is less dedoped and a higher current is observed, indicating a positive response for the HRP. Detection of COVID IgG due to competitive oxidation of TMB by HRP is observed for the ITO/P3HT/AF coating/antigen device.

[0275] Figure 13 shows the average value of lt=200s/lt=65s for each analyte type. There is clear differentiation (roughly double the normalised current) between the positive COVID IgG and the two negative controls. This result clearly demonstrates the suitability of this approach for antibody sensing.

[0276] When 0.7 V is applied to the ITO/P3HT/Nafion/AF coating/antigen device, an initial ionic current pulse is observed upon the addition of TMB, as observed for the solution tests above.

[0277] ITO/P3HT/AF coating/antigen and ITO/P3HT/Nafion/AF coating/antigen devices were tested by the sequential addition of COVID IgG antibodies (or negative control COVID IgM of PSA IgG antibodies), washing, addition of HRP:AB, washing and addition of TMB. At an operating voltage of 0.7 V, a clear and reproducible difference in device current is observed between the positive and negative controls for the ITO/P3HT/AF coating/antigen (a positive COVID IgG antibody and negative control).