Potentiometric‑ organic electrochemical transistors (POECT), methods of making and uses thereof

Abstract

An OECT configuration, called potentiometric-OECT (pOECT), which maintains the gate electrode in OCP (open circuit potential conditions is provided. The pOECT is designed to preserve the thermodynamic equilibrium necessary to define the electrochemical potential of the sensing electrode, ensuring reliability while executing the same functions of an OECT. A potentiometric OECT (pOECT) is disclosed as a four-terminal transistor which includes a source electrode (SE), a drain electrode (DE), a channel (CH), a sensing gate electrode (GsE) and a gating gate electrode (GGE). The CH electronically connects the SE and DE. This device can use dual n-type and p-type channels, simultaneously operated in the pOECT configuration, which generates a maximized signal ON response to both an increase and decrease in electrolyte pH. Multiple channels of a pOECT are operated in parallel and reliably monitor the permeability of a barrier-forming cell layer, which is otherwise not possible with conventional OECTs.

Description

[0001] POTENTIOMETRIC- ORGANIC ELECTROCHEMICAL TRANSISTORS (pOECT), METHODS OF MAKING AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to GB Application No. 2408914.6 filed on June 20, 2024, the contents of which are hereby incorporated in their entirety.

[0003] FIELD OF THE INVENTION

[0004] The invention is generally directed to polymer based electrochemical device for metabolite sensing. Specifically, the present invention is directed to polymer organic electrochemical transistors for direct metabolites sensing, which maintains the gate electrode in open circuit potential conditions.

[0005] BACKGROUND OF THE INVENTION

[0006] Potentiometry is probably the most straightforward electrochemical sensing technique1, measuring the open circuit potential (OCP) shift of a sensing electrode upon its interaction with the molecule of interest. Most potentiometric sensors rely on the classical 2-electrode setup for OCP measurements2-4, where the electrochemical potential of the sensing electrode, known as the working electrode (WE), is measured with respect to a reference electrode (RE) in an open circuit configuration. The most common and commercially available potentiometric sensors are the ion-selective electrodes (ISEs)5that usually employ ion selective membranes6,7. Potentiometric sensors can also detect the presence of other biologically relevant analytes when they are functionalized with surface-immobilized recognition elements such as enzymes8, aptamers9or antibodies10-13.

[0007] Several challenges hinder the widespread application of potentiometric sensors. One of them is the need for a stable and well-defined RE, crucial to ensure sensor reproducibility, longterm stability, minimum potential drift, fouling resistance, and biocompatibility (especially in biological applications)17. Miniaturization prevents a further obstacle; when the sensing electrode gets smaller, the electrical noise increases due to an increase in impedance. Miniaturizing a RE proves to be even more demanding than any WE due to the requirement of maintaining a protected and stable inner solution for the RE material, enclosed inside a micrometric container, alongside a micrometric ionic bridge. Moreover, materials commonly used for constructing REs, such as Ag- or Hg-based compounds, are not biocompatible, adding an extra layer of complexity, especially in biosensing applications. The organic electrochemical transistor (OECT) has emerged as an alternative bioelectronic circuit element, allowing to build RE-free and miniaturized neural interfaces18and sensors19of disease biomarkers that provide intrinsic amplification and noise reduction20-22. The OECT is a three-terminal device that uses an aqueous or solid electrolyte as the dielectric in contract with the channel. The channel is made of an organic mixed ionic and electronic conductor (OMIEC)23, connecting the Source (S) and Drain (D) electrodes, across which a voltage is applied (VDS). The current measured (IDS) reflects the conductivity of the OMIEC. The doping state of the OMIEC, hence the IDS, can be finely modulated by the voltage (VGS) applied across the electrolyte between a third electrode, the Gate (G), and the S. Therefore, the electrochemical properties of the G directly affect the magnitude of the IDS. The vast knowledge available for the functionalization methods developed for the WE of classical potentiometric sensors can be adapted for the G in an OECT. In such an OECT-based chemical sensor, the recognition event occurs on the G, analogous to the WE in a 2-electrode setup. The electrochemical potential of the G can change upon the binding of species such as ions, nucleic acids, and proteins, which change the charge distribution at the G24,25. These small variations in G electrochemical potential are then translated by the OECT into large changes in the IDS-

[0008] While OECTs bring several advantages for potentiometric sensing, they are limited by one important condition, which is also the main requirement of potentiometric sensing. Since the sensing electrode (i.e., WE) is used as the G of the OECT, both current and voltage are directly applied to the G to modulate the doping state of the OMIEC. However, in classical 2-electrode potentiometric sensing, the measurement has to be conducted in an OCP configuration with negligible current applied to the WE. The current flow / voltage biasing in OECT operation is unsuitable for potentiometric sensing because the G cannot reach the thermodynamic equilibrium43,44. In this case, the OECT input signal becomes unreliable, making the device output inaccurate. Moreover, especially for biosensing, the voltage range that can be applied to the OECT is limited because otherwise, the high current passing through the sensing interface (gate current, IGS) can damage the sensing layer45,46(which is usually made of biorecognition molecules), or trigger other parasitic faradaic reactions26. Due to a combination of these factors, the OECT in the currently utilized configuration is prone to inaccurate or inconsistent sensing readouts and cannot be reliably used for potentiometric sensing.

[0009] Thus, there is a need for OECT configurations that can overcome these limitations. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0010] BRIEF SUMMARY OF THE INVENTION

[0011] A potentiometric OECT (pOECT) is disclosed, which is a four-terminal device. The four terminals include a source electrode (hereinafter, SE), a drain electrode (hereinafter, DE), a sensing gate electrode (hereinafter, GsE) and a gating gate electrode (hereinafter, GGE). The SE and DE are placed apart, and are separated by a channel (hereinafter, CH). The CH electronically connects the SE and DE. Optionally, the pOECT includes one or more reservoirs / chambers to contain the electrolyte solution(s). In use, the device includes an electrolyte between the SE and the DE. The electrodes of the pOECT can be patterned on a supporting substrate, such as a glass substrate, a silicon substrate, or a plastic substrate, such as a polyimide substrate or a textile.

[0012] In some forms, the SE, DE, GsE and GGE are on separate planes. For example, the SE, the DE, and the CH can be patterned on the same supporting substrate, such as a glass substrate, a silicon substrate, or a plastic substrate. The GsE and GGE are positioned separately from the SE, the DE, and the CH. The GsE and GGE can be positioned vertically, relative to the SE, the DE, and the CH.

[0013] In some forms, the SE, DE, GsE and GGE are on the same plane. For example, the SE, the DE, the CH the GsE and GGE can be patterned on the same a supporting substrate, such as a glass substrate, a silicon substrate, or a plastic substrate.

[0014] In some forms, SE, DE, GsE and GGE are positioned in the same chamber.

[0015] In some forms, GsE is positioned in a separate chamber from SE, DE and GGE, and the two chambers are connected via a microfluidic channel or a floating gate. For example, the GsE is kept in a separate electrolyte with a varying pH, while the CH and GGE are in a second chamber with an electrolyte of constant composition. In this embodiment, additional electrodes are used as floating gates to connect the two chambers.

[0016] In some forms, the pOECT includes more than one CH, for example, in the form of an array, with n channels, where n is an integer (i.e., two or more pOECTs). Each SE and DE are placed apart and connected electronically by a corresponding CH. In some forms, the CH is made of an organic mixed ionic and electronic conductor (OMIEC) material. The GGE can be any type of counter electrode, such as a Pt coil (Fig.lc- middle) or an Au electrode.

[0017] In some forms, the pOECT combines p- type and n-type channels patterned on one substrate operated using the same GsE and GGE.

[0018] The pOECT allows for the use of various potentiometric sensing electrodes, including high-impedance ones, without causing damage, a capability not achievable with conventional OECTs. Moreover, since the operation is not influenced by the size of the Gs, a small sensing electrode can be used when necessary, offering advantages in terms of both cost reduction and miniaturization.

[0019] Besides potentiometric sensing, pOECT can be employed for impedimetric and faradaic sensing. pOECT allows continuous and real-time sensing on multiple devices in parallel, increasing the statistical relevancy of the experiments. This is a particularly important device design criterion for applications interfacing with living cells. Multi-marker analysis can be done from a single chip, revealing correlations between various analytes that cells consume or produce during their lifetime.

[0020] In some forms, the pOECT is coupled with a leakless RE allows to perform a reliable measurement with multiple simultaneous replicates on different locations of the cell culture. This sensor design can be used to monitor the behavior of other systems such as supported lipid bilayers70 and study kinetic interactions between biological interfaces and different drugs for long periods of time in safe conditions. For a more advanced analysis, each pOECT can be independently gated (still using a common GS and GG) to perform different techniques (impedimetric and / or faradaic) and monitor different parameters simultaneously. For example, other than the cut-off frequency, some of the pOECT channels can be functionalized with specific enzymes for the faradaic detection of metabolites.

[0021] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0023] FIG. 1A-1C. The p-OECT configuration. Comparison of the FIG. 1A) OECT, FIG. IB) gate referenced-OECT and FIG. 1C) pOECT configurations. In the top schematic, the black circle represents the electrolyte, and the dashed line represents the high-impedance connection of the potentiostat. The pink legend represents the cable labeling for the VDS application, and the purple legend represents the cable labeling for the VGS / VSG application. The 3D illustrations of the setups are in the middle panel, with G, GG, and Gs placed vertically with respect to the channel. The red spheres represent the ionic flux of the leakage current. Microscopy images of the microfabricated devices at the bottom panel depict the planar G, GG, and Gs. The channel size 100x10 pm, and G, GG, and Gs size is 500x500 pm. G and Gs represent any type of sensing interface, while GG represents any conducting material that can act as a counter electrode. FIG. ID. Schematics of the different OECT and pOECT setups used in this work. OECT-single chamber: G and OMIEC are located in the same electrolyte; pOECT-single chamber: Gs, GG and OMIEC are located in the same electrolyte; OECT-floating gate: G and OMIEC are located in two different electrolytes. The electrical and ionic communication between the sensing and amplification chambers is established through two shorted floating gates (fc,i made of Ag / AgCl glass RE). pOECT-floating gate: Gs is located in a separate electrolyte while GG and OMIEC are located in the same electrolyte. The electrical and ionic communication between the sensing and amplification chambers is established through two shorted floating gates (fc,i and G,2 made of Ag / AgCl glass RE but any other low-impedance electrode, such as Au, can be used). pOECT-microfluidic: Gs is located in a separate electrolyte while GG and OMIEC are located in the same electrolyte. The electrical and ionic communication between the sensing and amplification chambers is established through a fluidic tubing that not only guarantees the ionic bridging and the closure of the electrochemical circuit but also prevents the contamination of the OMIEC chamber.

[0024] FIG. 2A-2C. The p-OECT operation inhibits the polarization of the gate electrode. Transfer curves (left) and electrochemical potentials of the OECT / pOECT components during the corresponding voltage scan (right) when FIG. 2A) the G and Gs is a non-polarizable Ag / AgCl electrode or FIG. 2B) a polarizable Au electrode (<D = 5 mm). The devices have an n- type OMIEC in the channel. The applied gate voltage is reported as VSG for the pOECT and - VGS for the OECT. FIG. 2C) Transfer curves of the pOECT operated with either a minimally polarizable GG as a Pt coil or a highly polarizable GG (up to almost 1 V vs. Ag / AgCl) as Au electrode (<1> = 1.6 mm). FIG. 2D shows OECT / pOECT electrochemical potential measurement. Electrochemical potentials of the pOECT components during the application of the voltages of a transfer curve (VDS = +0.3 V and VSG from +0.2 to -0.5 V). FIG. 2E shows Gate electrode polarization. Left: transfer curve of an n-type OECT / pOECT when a Au <1> = 5 mm is used as G / GS and identical final IDS is reached. Right: corresponding electrochemical potentials of the OECT / pOECT components during the transfer curves. The applied gate voltage is reported as VSG for the pOECT and -VGS for the OECT. The plain lines are referred to the pOECT and the dotted lines to the OECT. The horizontal dashed arrows represent the point at which the channel of both OECT and pOECT reach the same electrochemical potential, leading to the same maximum IDS.

[0025] FIG. 3A-3F. The pOECT as a potentiometric sensor FIG. 3A) Combination of a 2- electrode potentiometric setup with the OECT to obtain a reference electrode-free IS-pOECT. FIG. 3B) Na+ sensing with a Na+ IS pOECT, where the Na+ ISE is the Gs, and a Pt coil is the GG- GS is located in a separate chamber connected to the rest of the device via a microfluidic channel. The pOECT is a p-type enhancement mode device, and its channel current varies with Na+ concentrations. FIG. 3C) The changes in the electrochemical potentials of the Na+ IS pOECT terminals during device operation when [Na+] = 0 M (left) and [Na+] = 100 mM (right). AOCP represents the electrochemical potential shift of the Gs or S due to variations in [Na+]. FIG. 3D) Na+ sensing with a Na+ ISE in a 2-electrode setup. The plot reports the electrochemical potential of the ISE at different Na+ concentrations. The inset shows the corresponding calibration curve. FIG. 3E) A schematic of the sensing-induced changes in the electrochemical potentials of the pOECT terminals (Gs, S, and D) at constant VDS and VGS. The target detected by the Gs alters its potential from EGS to EGS*. FIG. 3F) Left: Normalized IDS of the Na+-IS pOECT at various Na+ concentrations when operated in the nA regime. Right: Calibration curves of the conventional 2-electrode potentiometric setup and the Na+-IS pOECT operated in the A or the nA regime.

[0026] FIG. 4A-4F. Thermodynamic properties of the pOECT FIG. 4A) Transfer curves of an n-type OECT and pOECT operated at two different pH values with a pH-sensitive PANI electrode as the G or Gs. The applied gate voltage is reported as VSG for the pOECT and -VGS for the OECT. Inset: IDS and IGS VS. VGS of the OECT (scaled). The sensing electrode (G or Gs) is located in a separate chamber connected to the rest of the device via Ag / AgCl floating gates. FIG. 4B) Electrochemical potentials of the n-type OECT (left) and pOECT (middle) components monitored during the transfer curve recorded at pH = 6. Right: electrochemical potential of the Gs measured during the acquisition of the transfer curves at different pH. FIG. 4C) Relationship between the sensing interface (G and Gs) polarization and the electrode diameter / area in a pOECT and OECT when operated with the same n-type transistor channel. G and Gs are Au electrodes of different diameter (<D) and GG is a Au electrode of 1.6 mm diameter. FIG. 4D) n-type pOECT (left) and OECT (right) response to multiple consecutive transfer curves (50 cycles) when the G / Gs is a Au 3 mm diameter electrode and GG is Au 1.6 mm diameter. FIG. 4E) Comparison of the n-type OECT response between two consecutive sets of transfer curves (50 cycles each) when Au 3 mm diameter is used as G. The black line represents the 50thtransfer curve of the first set and the purple-pink curves represent the 50 transfer curves of the second set. FIG. 4F) Transfer curves of an n-type pOECT recorded at different pH values (4, 7, and 10). The device has a pH-sensitive glass electrode as the Gs and a Pt coil as the GG- GS is located in a separate chamber connected to the rest of the device via Ag / AgCl floating gates. The pOECT is an n-type enhancement mode device. The inset reports the calibration curve.

[0027] FIG. 5A-5F. Selecting the most efficient OMIEC for a pOECT-based sensor FIG. 5A) Top: Transfer curves of the p-type Na+-IS-pOECT at [Na+] = 0 and 100 mM (as reported in Fig. 3B). The gating voltage is referred to with respect to the electrochemical potential of Gs at different [Na+] (vs. Gs,XM = 0 M and 100 mM). The double arrow highlights the VSG distance between the two curves, corresponding to the difference in the OCP of the Gs in the two conditions (AOCP). Bottom: transfer curves at different Na+concentrations, back shifted by a AVSG = AOCP where the 0 M condition is the reference point. This back shift makes the gating voltage to be referenced with respect to the Gs at 0 M (vs. Gs, o M) FIG. 5B) Transfer curves of the n-type Na+-IS-pOECT at different Na+concentrations. The inset reports the transfer curves back-shifted by a AVSG = AOCP at 0 M Na+. FIG. 5C) Transfer curves of an n-type and p-type Na+-IS-pOECT at [Na+] = 0 and 100 mM. The target interactions with the Gs, which causes an increase in the Gs electrochemical potential, lead to a negative shift of the transfer curves. FIG. 5D) Normalized IDS of the n-type Na+-IS-pOECT recorded at different Na+concentrations. FIG. 5E) Left: Transfer curves of the n-type Cl -IS-pOECT at different Cl" concentrations Right: Calibration curves of the conventional 2-electrode potentiometric setup and the pOECT. FIG. 5F) Left: Transfer curves of a PEDOT:PSS based Na+-IS-pOECT at different Na+concentrations. Right: normalized IDS of the device.

[0028] FIG. 6A-6C. Complementary channel pOECT for exclusive turn-ON operation FIG. 6A) Top: a visual representation of the complementary channel pOECT. The Gs is kept in a separate electrolyte with a varying pH, while the channel and GG are in a second chamber with an electrolyte of constant composition (lx PBS). Two Ag / AgCl electrodes are used as floating gates to connect the two chambers. Bottom: a schematic of the setup where each channel is operated using the same Gs and GG and has its independent drain voltage (VDSI, VDS2) and gate voltage (VSGI, VSG2). The dashed lines represent the high-impedance connections of the potentiostat. The floating gate is not represented. FIG. 6B) The transfer curves of the n-type (left) and p-type (right) pOECT when the Gs is exposed to electrolytes with different pH values. The inset reports the electrochemical potential of the Gs during the measurement, demonstrating the maintained Nernstian response. FIG. 6C) Normalized response of the n-type pOECT, the p-type pOECT, and the conventional 2-electrode setup to varying pH values. FIG. 6D shows complementary pOECT performance improvement through channel size optimization. Normalized responses of the n-type and p-type pOECT with interdigitated channels and the classical 2-electrode setup when the sensing electrode is exposed to electrolytes with different pH values.

[0029] FIG. 7A-7C. Multichannel pOECT at constant thermodynamic conditions FIG. 7A) Top: A visual representation of multichannel pOECT where a leakless RE is used as Gs and a Pt wire is used as GG- Bottom: A schematic of a multichannel pOECT setup with n channels, each operated at an individual VDS,„ but a common VSG- The dashed line represents the high- impedance connection of the potentiostat. FIG. 7B) Top: schematic representation of the cell layer grown on top of one pOECT channel before and after trypsin addition. Middle: The applied VSG pulse profile. Bottom: A representative pOECT output in the case of healthy tissue (green) and when the cells are exposed to trypsin (yellow). The measurements were conducted in parallel using multiple pOECT channels. The scale bar in the microscopy images is 100 pm. FIG. 7C) Top: The change in the cut-off frequency of four pOECT channels triggered by the trypsin addition (marked with an arrow). Bottom: The average cut-off frequency change (standard deviation displayed as shaded areas) after compensating for the delay time due to trypsin diffusion.

[0030] FIG. 8A-8F. Reference electrode current in a traditional 3-electrode setup. Schematic behavior of the electrode potential as a function of the current for FIG. 8A) an ideal non- polarizable electrode, FIG. 8B) an ideal polarizable electrode, FIG. 8C) an electrode with an intermediate behavior. The red line represents the theoretical ideal behavior, and the grey line represents the real behavior. FIG. 8D) Schematics of a 3-electrode cell with the currents involved in the circuit: the current flowing through the WE (IWE) and the current flowing through the RE (IRE). The dashed line represents the high impedance connection of the potentiostat, and the large black circle represents the electrolyte. FIG. 8E) LSV of a = 5 mm Au WE in a solution of lx PBS and 10 mM ferrocyanide, where in pink line represents the IWE and the green one is the IRE. FIG. 8F) LSV of Au WE of different areas in a solution of lx PBS and 10 mM ferrocyanide. IWE (left) and IRE (right) as a function of the applied potential (bottom) and the correlation between the peak current and the WE area (top).

[0031] FIG. 9A-9C. Currents in a pOECT. FIG. 9A) Schematics of a pOECT with only the electrodes involved in the gating system labeled. The grey arrows highlight the currents involved in the gating circuit: the current flowing through the GG WE=IGG) and the current flowing through the Gs (IRE = The dashed line represents the high impedance connection of the potentiostat, and the large black circle represents the electrolyte. FIG. 9B) Transfer curves of an n-type pOECT with either a thin or thick layer of OMEIC deposited on the channel. FIG. 9C) IWE (left) and IRE (right) recorded during the operation of the two transistors with a thin or thick layer of n-type OMIEC.

[0032] FIG. 10A-10B. The pOECT operation with a high-impedance sensing electrode. FIG. 10A) Visual representation of a pOECT in which the Gs is a high impedance electrode (i.e., a glass membrane pH-meter). FIG. 10B) Electrochemical potential of the Gs (top) and S and D (bottom) during n-type pOECT operation through a cyclic repetition of transfer curves. Right: The transfer curves.

[0033] FIG. 11A-11B. Performance comparison of a miniaturized OECT and pOECT.

[0034] FIG. 11A) Transfer curves of microfabricated pOECT and OECT with a 100x10 pm channel and a 500x500 pm G, GS and GG. The plot shows the different IDS modulation capabilities of the two configurations. FIG. 11B) Electrochemical potentials of the microfabricated pOECT and OECT components during the transfer curve.

[0035] FIG. 12A-12B. Sensing electrode polarization vs size in OECT and pOECT. FIG. 12A) Left: Transfer curves of a pOECT (blue; operated with GS of Au- 170 pm of diameter and GG of Au- 1.6 mm of diameter) and an OECT (pink; operated with G electrodes with a diameter ranging from 170 pm to 1 cm. Right: zoomed-in OECT transfer curves. FIG. 12B) Electrochemical potential changes of the OECT / pOECT components during the transfer curves, for a specific G / GS diameter (<D). The bottom panel shows the electrochemical potentials exclusively for the G / GS, reporting the total electrode polarization.

[0036] FIG. 13. Permanent alteration of the sensing electrode potential upon OECT operation. Electrochemical potential vs time of the n-type OECT components during two consecutive sets of 50 transfer curves each, separated by a resting period. No voltage is applied until the 5th second, followed by the recording of the first transfer curve of the first set (VGS -0.2 to 0.6 V); the subsequent 49 cycles are not displayed. At the end of the first set, there is a Ih resting period during which no voltage is applied. This period shows a new equilibrium potential of the G due to the IGS which leads to a permanent increase of the electrode surface potential of 97 mV. This increase represents the permanent change of the electrode properties, i.e., a new potential and loss of any analytical information. This also causes a different output current in the second set of transfer curves (after 5000 s) and a further change of the equilibrium surface potential of the sensing electrode (+132 mV).

[0037] FIG. 14. Cytotoxicity of various gate electrodes. Viability of epithelial MDCKII cells cultured in the presence of different REs as an indicator of their cytotoxicity. The first column displays bright field (BF) images. The second column displays the images of live cells expressing a green signal, cells expressing a red signal in the third column represent dead cells, and the composites in the fourth column indicate the ratio of LIVE / DEAD cells for each condition.

[0038] FIG. 15. Biocompatibility of various gate electrodes. MDCKII morphology and cell coverage as a rough indicator of RE biocompatibility. The top row displays actin-stained cells as a method to delineate their morphology, and the bottom row shows a zoomed image of the demarked area in the top image. Columns display images of actin-stained cells grown in the presence of different REs (CT = control).

[0039] FIG. 16A-16B. Effect of the electrolyte composition change on the OECT response. FIG. 16A) Electrochemical potential of a Pt wire in fresh and old cell media. FIG. 16B) Left: p- type OECT response in fresh and old cell media when a Pt wire is used as G. Right: normalized response of the OECT. In this evaluation, we used a fresh cell culture media and a conditioned cell culture media, which is a medium whose chemical composition has changed since it was left in contact with the cell culture for 3 days and contains components secreted by the cells. FIG. 17. OECT response to the use of an high-impedance gate. Left: transfer curve of a PEDOT:PSS OECT when the G is an Ag / AgCl with either a glass porous membrane or a leakless membrane for the ionic connection with the electrolyte. Right: the electrochemical potential of the two G terminals during the transfer curve.

[0040] FIG. 18. Effect of the electrolyte composition change on the pOECT response, p-type pOECT response in fresh or old cell media as electrolyte when the GS is a leakless Ag / AgCl RE.

[0041] FIG. 19A-19B. Cells coverage of the transistor channels. Phase contrast image of the pOECT channels in the presence of epithelial cells, FIG. 19A) before and FIG. 19B) after adding trypsin to the cell media.

[0042] FIG. 20A-20C. Diffusion rates compensation. FIG. 20A) Normalized response of cut-off frequency during time (blue line) and corresponding derivative (orange line). FIG. 20B) The peak position of the derivative from each pOECT channel is used to quantify the difference in diffusion time among the four pOECTs. FIG. 20C) The pOECT signals are horizontally shifted off the corresponding difference in diffusion time with respect to the pOECT 3, which is chosen as a reference.

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044] The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0045] To overcome problems associated with using OECTs for potentiometric sensing, an OECT sensing configuration called potentiometric-OECT (pOECT) was designed, which differs from the traditional 3 configuration OECT. In this configuration, the sensing electrode is maintained in OCP conditions by minimizing any current flow / voltage bias at this interface, by splitting the gate electrode GE into two different electrodes (Fig.l). The first electrode is the sensing gate (Gs), which has an electrochemical potential sensitive to the target concentrations and works as a RE for the applied gate voltage. The second electrode is the gating gate (GG), which functions as a CE and is responsible for actively applying the doping voltage.

[0046] The studies herein demonstrate the superiority of the pOECT over the classical 2- electrode system and the OECT operated in the traditional configuration in three distinct sensing scenarios with different device architectures: 1) classical potentiometric sensing, where ions were sense, namely Na+, C1-, and H+, as representative examples; 2) a combined n- and p-type pOECT for the measurement of pH fluctuations; 3) a multi-channel pOECT used to monitor barrier tissue integrity. The data show that the pOECT guarantees a higher response than the 2- electrode setup, maintaining the same accuracy despite having no RE. The pOECT showed much higher response and accuracy in sensing than the conventional OECT and has high design flexibility and low operational complexity. The setup is compatible for use with an exceptionally wider class of sensing interfaces (e.g., high-impedance electrodes) and complex configurations (e.g., parallel channels, n- and p-type channels operating simultaneously) without requiring any particular device optimization in terms of geometry or materials.

[0047] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0048] I. DEFINITIONS

[0049] As used herein, the term “channel” refers to the electrical connection established between the source and the drain electrodes by the n-type polymer.

[0050] As used herein, the term “direct electron transfer” means the process of transferring electrons directly from an enzyme or a compound to an electron acceptor such as a polymer or an electrode, or from an electron donor to a compound or an enzyme.

[0051] As used herein, the term “drain” or “drain electrode” refers to an electrode which accepts charge carriers from a channel. The terms “drain” and “drain electrode” are used interchangeably throughout the instant disclosure.

[0052] As used herein, a “electrochemical device”, “n-type electrochemical device”, “n-type polymer based electrochemical device” or “n-type polymers based accumulation mode electrochemical device” includes one or more source electrodes, one or more drain electrodes, one or more channels, a gate electrode, and an electrolyte solution containing one or more metabolites. The source electrode and the drain electrode are placed apart and connected electronically by the channel. The channel contains one or more enzymes and one or more n- type polymers. The gate electrode contains one or more n-type polymers and one or more enzymes. The gate electrode is placed separately from the source electrode, the drain electrode, and the channel to prevent electron flow between the gate electrode and the channel. The electrolyte solution is in electrical contact with the gate electrode and the channel. The electrochemical device may contain a plurality of independently addressable source and drain electrodes, a common gate electrode, and corresponding channels, wherein the corresponding channels contain different enzymes. The terms “electrochemical device”, “n-type electrochemical device”, “n-type polymer based electrochemical device” and “n-type polymers based accumulation mode electrochemical device” are used interchangeably throughout the instant disclosure. The electrochemical device disclosed herein may be used for in vitro and in vivo applications.

[0053] As used herein, the term “electrolyte solution” refers to a solution that contains ions, atoms, or molecules that have lost or gained electrons, and is electrically conductive. The electrolyte solution is in electrical communication with the bioanode and the biocathode. The electrolyte solutions contains one or more metabolites.

[0054] As used herein, the term “electron mediator” refers to a compound that can accept or donate electrons. Electron mediators can facilitate electron transfer generated from an enzymatic reaction between enzymes and metabolites to electrodes.

[0055] As used herein, the term “enzyme” refers to a protein that functions as a catalyst in a chemical reaction. Enzymes include, but are not limited to glucose oxidase, glucose dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, formate dehydrogenase, formaldehyde dehydrogenase, lactic dehydrogenase, lactose dehydrogenase, lactate oxidase, cholesterol oxidase, tyrosinase, and pyruvate dehydrogenase. Preferred enzymes include oxidase which catalyzes the oxidation of a metabolite, such as glucose oxidase (GOx). Enzymes useful for biocathodes include oxygen reductase, such as laccase and bilirubin oxidase.

[0056] As used herein, the term “gate voltage” or “VG” refers to the gate to source voltage.

[0057] As used herein, the term “metabolite” means any compound that has stored energy.

[0058] Preferred metabolites are carbon-based compound that has stored energy. Metabolites include but are not limited to nucleic acids, carbohydrates, alcohols, fatty acids and other hydrocarbons, ketones, aldehydes, amino acids, and proteins. The “metabolite” may be a biological compound within an organism. Preferred metabolites are carbohydrates, which include glucose, glucose-2, D-glucose, L-glucose, and glucose-6-phosphate.

[0059] As used herein, the term “n-type polymer” or “n-type conjugated polymer” refers to a polymer capable of accepting electrons and stabilizing electrons on its backbone.

[0060] The term “p-type polymer” refers to a polymer capable of donating electrons. As used herein, the term “planar configuration” refers to the arrangement of the components of a electrochemical device is on a common plane.

[0061] As used herein, the term “sensor” refers to a device that detects or measures an event or a change of a physical property of an analyte, and records, indicates, or responds to the event or change. The sensor can measure or sense metabolites, ions, pH, or temperature and it can measure or sense one or more analytes.

[0062] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0063] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention.

[0064] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.

[0065] These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0066] Unless otherwise indicated, the disclosure encompasses conventional techniques of molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. Unless otherwise noted, technical terms are used according to conventional usage, and in the art, such as in the references cited herein, each of which is specifically incorporated by reference herein in its entirety.

[0067] II. POTENTIOMETRIC ORGANIC ELECTROCHEMICAL DEVICE

[0068] The disclosed potentiometric OECT (pOECT) is a four-terminal transistor which includes a SE, a DE, a GsE and a, GGE. the pOECT includes a CH. The CH electronically connects the SE and DE. An important feature of the pOECT is the removal of the OMIEC gating current from the sensing electrode (GsE), which is instead provided by an auxiliary electrode (GGE).

[0069] In some forms, the SE, DE, GsE and GGE are on separate planes. For example, the SE, the DE, and the CH can be patterned on the same supporting substrate, such as a glass substrate, a silicon substrate, or a plastic substrate. The GsE and GGE are positioned separately from the SE, the DE, and the CH. The GsE and GGE can be positioned vertically, relative to the SE, the DE, and the CH (FIG. 1C, middle panel, Fig. 3A and 3B). In some forms, the SE, DE, GsE and GGE are on the same plane. For example, the SE, the DE, the CH the GsE and GGE can be patterned on the same a supporting substrate, such as a glass substrate, a silicon substrate, or a plastic substrate. (FIG. 1C, bottom panel).

[0070] Thus, the device can be fabricated to include a planar component, and the one or more SE, the one or more DE, the GsE and GGE are on the same plane. Alternatively, the one or more SE, the one or more DE are on the same plane and the GsE and GGE are in a vertical location relative to the plane of the planar component (FIG. 1C, middle panel).

[0071] In some forms, SE, DE, GsE and GGE are positioned in the same chamber (FIG. 1C). The disclosed devices can include one or more reservoirs / chambers to contain the electrolyte solution(s). FIG. ID. uses schematics of the different pOECT setups: pOECT-single chamber: Gs, GG and OMIEC are located in the same electrolyte; OECT-floating gate: G and OMIEC are located in two different electrolytes. The electrical and ionic communication between the sensing and amplification chambers is established through two shorted floating gates (fc,i and / c.2 made of Ag / AgCl glass RE). pOECT -floating gate: Gs is located in a separate electrolyte while GG and OMIEC are located in the same electrolyte. The electrical and ionic communication between the sensing and amplification chambers is established through two shorted floating gates (fc,i and fa, 2 made of Ag / AgCl glass RE but any other low-impedance electrode, such as Au, can be used). pOECT -microfluidic: Gs is located in a separate electrolyte while GG and OMIEC are located in the same electrolyte. The electrical and ionic communication between the sensing and amplification chambers is established through a fluidic tubing that not only guarantees the ionic bridging and the closure of the electrochemical circuit but also prevents the contamination of the OMIEC chamber.

[0072] In some forms, GsE is positioned in a separate chamber from SE, DE, and GGE, (FIG. 3B and 6A) and the two chambers are connected via a microfluidic channel (FIG. 3B) or a floating gate (FIG. 6A). For example, the GsE is kept in a separate electrolyte with a varying pH, while the CH and GGE are in a second chamber with an electrolyte of constant composition. In this embodiment, additional electrodes are used as floating gates to connect the two chambers.

[0073] In some forms, the pOECT includes more than one CH, for example, in the form of an array, with n channels, where n is an integer (i.e., two or more pOECTs). Each SE and DE are placed apart and connected electronically by a corresponding CH. (FIG. 6A) In some forms, the CH is made of an organic mixed ionic and electronic conductor (OMIEC) material. The GGE can be any type of counter electrode, such as a Pt coil (FIG. 1C- middle) or an Au electrode.

[0074] In some forms, the pOECT combines p- type and n-type channels patterned on one substrate operated using the same GsE and GGE. (FIG. 6A).

[0075] The pOECT may include one CH or more than one CH in the form of an array (i.e., two or more pOECTs). The SE and DE are placed apart and connected electronically by a corresponding CH. Disclosed pOECT show lowest electrochemical potential change upon ion binding to an ISE is about ±10 mV. This value translates into an LoD in the micromolar range for ion sensors, and an LoD in the attomolar range for protein sensors.

[0076] In some forms, pOECT has more than one CH in the form of an array, with n channels, where n is an integer (i.e., two or more pOECTs) for example, from 2-10, 50, 100, 1000, etc. Each SE and DE are placed apart and connected electronically by a corresponding CH. In some forms, the pOECT combines p- type and n-type channels patterned on one substrate operated using the same GsE and GGE. In some forms, the GsE is kept in a separate electrolyte with a varying pH, while the channel and GGE are in a second chamber with an electrolyte of constant composition (‘herein, complementary channel pOECT). In this embodiment, additional electrodes for example, Ag / AgCl electrodes, or two microfabricated planar Au electrodes) are used as floating gates to connect the two chambers (FIG. 6A).

[0077] The pOECT does not require any additional connections. While the conventional OECT operation involves shorting the RE2 and CE2 cables and connecting them to the same electrode (SE), in the pOECT, these cables are separated and linked to electrodes with distinct functions.

[0078] The SE, the DE, and CH can be patterned on the same supporting substrate, such as a glass substrate, a silicon substrate, or a plastic substrate, such as a polyimide substrate or a textile. In preferred embodiments, the SE, DE, and CH are patterned on a glass substrate.

[0079] Potentiometric sensors can also detect the presence other biologically relevant analytes when they are functionalized with surface-immobilized recognition elements such as enzymes, aptamers, antibodies or nanobodies.

[0080] In some forms, the pOECT is integrated / functionalized with a biorecognition layer including a biorecognition element for recognition of an analyte of interest. The biorecognition element is the component that can specifically interact with its cognate target, for example, an enzyme which specifically binds a target analyte, an aptamer, antibody or fragment thereof, or nanobody which specifically binds the target analyte. Optionally, when the device contains an array of pOECTs, each pOECT may contain a SE, a DE, a corresponding CH, and GsE and GGE, such that one each of a gate electrode can be engineered with the same or different biorecognition element for detecting one or more than one analyte simultaneously. The device may be incorporated into a microfluidics configuration.

[0081] The gate electrodes GsE and GGE are placed separately from SE, DE and CH and when in use, separated by an electrolyte solution (ES). The channel is in direct contact with an ES, into which the GsE and GGE are also immersed. Optionally, the pOECT includes a reservoir to contain the electrolyte solution. The reservoir (i.e. PDMS well) is placed on top of the channel to contain the electrolyte solution such that the channel and the GsE and GGE are in contact with the electrolyte solution. The SE and DE contacts are also in contact with the electrolyte and optionally, can be insulated with an insulator such as parylene or SU-8.

[0082] When the device contains an array the device may contain multiple sets SE, DE, and a CH (i.e., (SE, DE, CH)n, where n is an integer >1) and all the sets of SE, DE, and a CH in the array use a common GsE and GGE. For example, if the pOECT array includes 2, 3, 4, 5 or 6 sets of SE, DE, and a CH where each set contains a SE, a DE, and a corresponding CH, all the sets in the array use a common GsE and GGE.

[0083] One feature of the pOECT configuration is its modularity. With the pOECT it becomes possible to couple n- and p-type OMIECs in order to create a complementary device that maximizes sensor output without any restriction on the OMIEC geometry or thermodynamic properties and operational voltages. Besides potentiometric sensing, pOECT can be employed for impedimetric and faradaic sensing, ensuring higher accuracy than the OECT. pOECT allows continuous and real-time sensing on multiple devices in parallel, increasing the statistical relevancy of the experiments. This is a particularly important device design criterion for applications interfacing with living cells. Multi-marker analysis can be done from a single chip, revealing correlations between various analytes that cells consume or produce during their lifetime. The diverse range of experiments conducted in this work underscores the pOECT setup as an accountable and higher-performing edition of the electrolyte-gated transistor operating in potentiometric mode. The pOECT configuration eliminates the errors associated with OECT sensors, propelling the transistor-based sensor technologies toward the translational value they deserve. A. Source and Drain Electrodes

[0084] The SE and DE are made from materials capable of conducting an electric current. The electrode materials can be organic or inorganic in nature, as long as it is able to conduct electrons and inject electronic charges into the channel material. The electrodes can be a polymeric electrode, a metallic electrode, a carbon-based material, a metal oxide, or a modified electrode. In some embodiments, the SE and DE are made from an electrochemically inert material such as gold, platinum, or a conductive form of carbon. In some embodiments, the SE and DE are gold electrodes. They are insulated using an insulator such as parylene. In some embodiments, SU-8 (epoxy based photoresist used to pattern electronics with photolithography (also used as an insulator)) is used.

[0085] In some embodiments, the SE and DE are made from a metallic conductor. Suitable metallic conductors include but are not limited to gold, chromium, platinum, iron, nickel, copper, silver, stainless steel, mercury, tungsten and other metals suitable for electrode construction. The metallic conductor can be a metal alloy which is made of a combination of metals disclosed above, such as gold / chromium. In addition, conductive substrates which are metallic conductors can be constructed of nanomaterials made of gold, cobalt, diamond, and other suitable metals.

[0086] In other embodiments, the SE and DE are made from carbon-based materials. Exemplary carbon-based materials are conducting polymers (in the form of films or fibers) carbon cloth, carbon paper, carbon screen printed electrodes, carbon paper, carbon black, carbon powder, carbon fiber, singe-walled carbon nanotubes, double-walled carbon nanotubes, multi-walled carbon nanotubes, carbon nanotube arrays, diamond-coated conductors, glassy carbon and mesoporous carbon. In addition, other exemplary carbon-based materials are graphene, graphite, uncompressed graphite worms, delaminated purified flake graphite, high performance graphite and carbon powders, highly ordered pyrolytic graphite, pyrolytic graphite, and polycrystalline graphite.

[0087] The electrodes can be doped semiconductors. Suitable semiconductors are prepared from silicon and germanium, which can be doped (i.e., the intentional introduction of impurities into an intrinsic semiconductor for the purpose of modulating its electrical and structural properties) with other elements. The semiconductors can be doped with phosphorus, boron, gallium, arsenic, indium or antimony, or a combination thereof.

[0088] Other electrode materials can be metal oxides, metal sulfides, main group compounds, and modified materials. Exemplary materials of this type are nanoporous titanium oxide, tin oxide coated glass, glass, cerium oxide particles, molybdenum sulfide, boron nitride nanotubes, aerogels modified with a conductive material such as gold, solgels modified with conductive material such as carbon, ruthenium carbon aerogels, and mesoporous silicas modified with a conductive material such as gold.

[0089] The SE and DE can be any shape appropriate such as cuboid, cubic, circular, and cylindrical. In some preferred embodiments, the electrodes are cuboid gold electrodes. In some embodiments, each of the source and drain electrodes has a first dimension (i.e. width), a second dimension (i.e. length), and a third dimension (i.e. thickness). For example, each of the source and drain electrodes has a width in a range from 100 pm to 1 mm (e.g. 0.8 mm), a length in a range from 100 pm to 1 mm (e.g. 0.8 mm), and a thickness of about 100 nm.

[0090] B. Channel

[0091] The channel in a pOECT is typically made of an ion-permeable organic electronic material, through which holes or electrons flow from the SE to the DE. In some forms, the channel is made of an organic mixed ionic and electronic conductor (OMIEC), connecting the Source (SE) and Drain (DE) electrodes, across which a voltage is applied (VDS). In the pOECT configuration, the IDS is modulated solely by electrochemical potential variations of Gs.

[0092] In contrast to the OECT, the pOECT prevents any polarization of the sensing interface (Gs), which is key for ensuring sensor accuracy. By removing any interference from the sensing electrode capacitance, this configuration preserves the thermodynamic behavior of the electrode, rendering the transistor solely responsive to the electrochemical potential of the Gs. It eliminates any possible current / voltage-related degradation or parasitic reactions at the sensing electrode, thereby enhancing device stability. The pOECT allows for the use of various potentiometric sensing electrodes, including high-impedance ones, without causing damage, a capability not achievable with conventional OECTs. Moreover, since the operation is not influenced by the size of the Gs, a small sensing electrode can be used when necessary, offering advantages in terms of both cost reduction and miniaturization.

[0093] Generally, the CH is configured to establish the electrochemical connection between a pair of SE and DE such that holes or electrons flow from the SE to the DE. The channel in some forms is preferably made of an organic mixed ionic and electronic conductor (OMIEC). The OMIEC type should be selected based on the direction of the electrochemical potential shift of the sensing surface upon its interactions with the target. In some forms, detecting increasing concentrations of cations is accomplished with p-type OMIECs, while detecting increasing concentrations of anions is accomplished with n-type OMIECs.

[0094] An ion-permeable organic electronic material includes the conducting polymer disclosed in, for example, Rivnay, et al., Nature Reviews, 3: 17086 (2018) and Sun, et al., J. Mater. Chem. C, 6:11778-11784 (2018). An exemplary conducting polymer for the channel is poly(3,4- ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS). The conducting PEDOT is p-type doped (oxidized), which leads to mobile holes that can hop from one chain to another, resulting in a hole current once a drain voltage is applied. These holes are compensated by the sulfonate anions of PSS. Channels made of PEDOT:PSS can work as depletion mode pOECTs. For example, in the absence of a gate voltage, a hole current flows in the CH. Once a positive gate voltage is applied, cations from the electrolyte are injected into the channel and the anions are compensated, resulting in a decrease in the number of holes in the channel. This results in a decrease in the drain current. Alternatively, the CH can be made of materials that work in accumulation mode OECT, such as a semiconductor based on a polythiophene with a sulfonate group attached to the backbone with a hexyl chain (PTHS) (Inal, et al., Adv. Mater., 26:7450-7455 (2014)), an ethylene glycol unit attached to bithiophenes (Moser, et al., Adv. Mater., 32:2002748 (2020)) or a g7-NDI-Br and NDI-T2 copolymer (P-90) (Giovannitti, et al., Chem. Mater., 30:2945-2953 (2018)).

[0095] In accumulation mode p-type pOECT, application of a negative gate voltage causes injection of anions into the channel and a corresponding accumulation of holes, leading to an increase in the drain current ID.

[0096] One can also use n-type semiconductors and build accumulation mode n-type pOECTs. In accumulation mode n-type OECT, application of a positive gate voltage causes injection of cations into the channel and a corresponding accumulation of electrons, leading to an increase in the drain current ID.

[0097] Other suitable conducting polymers for the CH include, but are not limited to, conductor based on PEDOT with a pendant sulfonate group (PEDOT-S), PEDOT doped with tosylate (PEDOT:TOS), PEDOTOH:C1O4, PEDOT-co-PEDOTOH:ClO4 (Schmode, et al., Chem. Mater., 31 (14):5286-5295 (2019)), poly(2-(3,3'-bis(2-(2-(2-methoxyethoxy) ethoxy jethoxy)- [2,2' bithiophen]-5 yl)thieno[3,2 b] thiophene), p(g2T TT), poly((ethoxy)ethyl 2-(2-(2 methoxyethoxy) ethoxy)acetate)-naphthalene 1,4, 5, 8 tetracarboxylic-diimide-co 3,3' bis(2-(2-(2 methoxyethoxyjethoxy) ethoxy)-(bithiophene)) p(gNDI g2T), P3HT (poly (3 -hexylthiophene- 2,5-diyl)), BBL (polybenzimidazo-benzoisoquioline), PTHS-TMA+-co-P3HT (poly[(6- thiophen-3-yl)hexane-l-sulfonate-co-3-(hexylthiophene)]), poly(N,N'-bis(7-glycol)-naphthalene-

[0098] 1.4.5.8-bis(dicarboximide)-co-2,2'-bithiophene-co-N,N'-bis(2-octyldodecyl)-naphthalene-

[0099] 1.4.5.8-bis(dicarboximide), naphthalene-l,4,5,8-tetracarboxylic-diimide-bithiophene (NDI-T2) based polymer with 90% glycol chain percentage (P-90), and glycolated Diketopyrrolopyrroles (DPPs) or glycolated thiophenes, bithiophenes such as p(gOT2-g6T2) (Moser, et al., Advanced Materials, 32:2002748 (2020)).

[0100] Examples of suitable polymers for the CH in a depletion mode pOECT are PEDOT polymers or copolymers thereof, such as polymers based on PEDOT with a pendant sulfonate group (PEDOT-S), PEDOT doped with tosylate (PEDOT:TOS), PEDOTOH:C1O4, PEDOT-co- PEDOTOH:C1O4, and PEDOT:PSS.

[0101] Examples of suitable polymers for the channel in an accumulation mode pOECT are poly(2-(3,3'-bis(2-(2-(2-methoxyethoxy) ethoxy)ethoxy)-[2,2' bithiophen]-5 yl)thieno[3,2 b] thiophene), p(g2T TT), poly((ethoxy)ethyl 2-(2-(2 methoxyethoxy) ethoxy)acetate)-naphthalene 1,4, 5, 8 tetracarboxylic-diimide-co 3,3' bis(2-(2-(2 methoxyethoxy)ethoxy) ethoxy)- (bithiophene)) p(gNDI g2T), P3HT (poly(3-hexylthiophene-2,5-diyl)), BBL (polybenzimidazo- benzoisoquioline), PTHS-TMA+-co-P3HT (poly[(6-thiophen-3-yl)hexane-l-sulfonate-co-3- (hexylthiophene)]), poly(N,N'-bis(7-glycol)-naphthalene-l,4,5,8-bis(dicarboximide)-co-2,2'- bithiophene-co-N,N'-bis(2-octyldodecyl)-naphthalene-l,4,5,8-bis(dicarboximide), naphthalene -

[0102] 1.4.5.8-tetracarboxylic-diimide-bithiophene (NDI-T2) based polymer with 90% glycol chain percentage (P-90), and glycolated Diketopyrrolopyrroles (DPPs) or glycolated thiophenes, bithiophenes such as p(gOT2-g6T2).

[0103] Typically, the CH has a first dimension (i.e. width) and a second dimension (i.e. length). The length is the distance between the source and drain electrodes. Width is the remaining dimension of that rectangle The length and width of the channel can be a value between about 5 pM and about 5mm, for example, between 5 pm and 1000 pm, between 5 pm and 100 pm, between 5 pm and 100 pm, or between 10 pm and 100 pm, for example, about 10 pm or about 100 pm. The width of the channel can be between 10 pm and 1000 pm, between 20 pm and 500 pm, or between 20 pm and 200 pm, for example, about 100 pm.

[0104] The first dimension and the second dimension can be the same or different. In some embodiments, the width of the channel is the same as the length of channel. For example, the channel has a width of about 100 pm and a length of about 100 pm. In some embodiments, the width of the channel is smaller than the length of the channel. In some preferred embodiments, the width of the channel is larger than the length of the channel. For example, the channel has a width of about 100 pm and a length of about 10 pm. In some embodiments, the OECT having a small channel (e.g. a width of about 100 pm and a length of about 10 pm) is more sensitive (i.e. higher NR) than the same OECT but having a larger channel (e.g. a width of about 100 pm and a length of about 100 pm) under the same measurement conditions (e.g. VG, VD, incubation time, temperature, and pressure).

[0105] In some forms, the channel remains unmodified, i.e., it is not chemically modified to anchor binding partners. The analyte recognition takes place at the functionalized gate electrode, which is not in physical contact with the channel. This configuration allows prolonged shelf life of the device compared with OECT containing channels modified with binding partners.

[0106] The channel materials can be spin casted, drop casted or inkjet or screen printed from solutions.

[0107] 1. N-Type Polymers

[0108] The polymeric material on the channel is any polymer that is capable of accepting (n- type) electrons produced in a reaction, resulting in increased charge carrier density and thus the conductivity of the polymer. The n-type polymer is a polymer capable of accepting electrons and stabilizing electrons on its backbone. Exemplary n-type polymers include OMIEC p(CeNDI-T)1(Koklu, et al.) ACS Nano 15, 8130-8141 (2021), N2300, P(NDI-T2), poly(diketopyrrolopyrrole) (DPP), poly(benzimidazobenzophenanthroline), poly(2,5-di(3,7- dimethyloctyloxyjcyanoterephthalylidene), poly(2,5-di(hexyloxy)cyanoterephthalylidene), poly(5-(3,7-dimethyloctyloxy)-2-methoxy-cyanoterephthalylidene), poly(2,5- di(octyloxy)cyanoterephthalylidene), and poly(5-(2-ethylhexyloxy)-2-methoxy- cy anoterephthalylidene) .

[0109] Optionally, an electron mediator can be used in the n-type polymer based electrochemical device. The electron mediator is a compound that can accept or donate electrons. The electron mediator can be attached onto the channel and the gate electrode. Exemplary electron mediators are pyrroloquinoline quinone (PQQ), phenazine methosulfate, dichlorophenol indophenol, short chain ubiquinones, potassium ferricyan, or equivalents of each.

[0110] 2. p-type Polymers

[0111] In some embodiments, the polymers on the channel is a single n-type polymer or a combination of several n-type polymers or a combination of n-type polymers with p-type polymers or semiconductor materials. A p-type polymer is any polymer that is capable of donating electrons to a compound, resulting in the reduction of an oxidant. In a preferred embodiment, the p-type polymer is a robust oxygen reducer, and it reduces oxygen to water. Exemplary p-type polymers include, but are not limited to, p(gaC2T2-T) (Koklu, et al., Adv. Mat. 34, 2202972 (2022); poly(3,4-ethylenedioxythiphene) (PEDOT), poly(hydrooxymethyl 3,4- ethylenedioxythiphene) (PEDOT-OH), polystyrenesulfonate (PSS), F8BT, F8T2, J51, MDMO- PPV, MEH-PPV, PBDB-T, PBDTBO-TPD, PBDT(EH)-TPD, PBDTTT-C-T, PBDTTT-CF, PBTTPD, PBTTT-C14, PCDTBT, PCPDTBT, PDTSTPD, PffBT4T-20D, PffBT4T-C9C13, PFO-DBT, Poly([2,6'-4,8-di(5-ethylhexylthienyl)benzo[l,2-b;3,3-b]dithiophene]{3-fluoro-2[(2- ethylhexyl)carbonyl] thieno [3 ,4-b] thiophenediyl } ) , Poly (3 -dodecylthiophene-2 ,5 -diyl) , Poly (3 - hexylthiophene-2, 5-diyl), Poly(3-octylthiophene-2,5-diyl), PSiF-DBT, poly(triaryl amine) (PTAA), PTB7, TQ1, and a combination thereof. Semiconductor materials may be inorganic, metal-organic, or organic, and the semiconductor may comprise small molecules, oligomers, or polymers (Kugler, US 2009 / 0040587).

[0112] C. Gate Electrodes

[0113] The GE, described herein are a combined RE2 / CE2, including into two independent electrodes, i.e., RE2 (GsE) and CE2 (GGE) (Fig. 1C). The sensing electrode remains connected to RE2. A second electrode is introduced, which can be any type of counter electrode, such as a Pt coil (Fig. IC-middle) or an Au electrode (Fig. IC-bottom), and connected to CE2. Together, RE2 and CE2 represent the “gating system”. The dual gating system (Gs and GG) can be either vertical (Fig.1 -middle), or the pOECT can be miniaturized, consolidating all the device components into a single substrate by fabricating planar Gg and Gs, thereby maintaining the overall device size in the micrometer range (Fig.1 -bottom).

[0114] The GsE and GGE can be any shape appropriate such as cuboid, cubic, circular, and cylindrical. In some embodiments, the gate electrode is a circular electrode having a diameter un the pm range. In some forms, the diameter can be in a range from about 50 to 1000 pm, from about 100 to about 700 pm, from about 150 to about 450 pm, for example, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pm, etc.

[0115] In some forms, the diameter can be in in a range from 0.5 mm to 10 mm, from 1 mm to 10 mm, from 0.5 mm to 5 mm, or from 1 mm to 5 mm, such as 2.8 mm. In some preferred embodiments, the gate electrode is a square electrode having a length in a range from 0.1 mm to 10 mm, from 0.1 mm to 8 mm, from 0.1 mm to 5 mm, from 0.5 mm to 10 mm, or from 0.5 mm to 5 mm, such as about 0.8 mm or about 4.8 mm. In some preferred embodiments, the GsE and GGE are each a square electrode having a length of about 0.8 mm.

[0116] In some embodiments, the pOECT contains a GsE and GGE having any shape and size as described above and a channel having any shape and size as described above. In some preferred embodiments, the OECT contains square GsE and GGE having any length as described above and a rectangular channel having any size (i.e. width and length) as described above. In a particularly preferred embodiment, the pOECT contains square GsE and GGE having a length of about 0.8 mm and a rectangular channel having a width of about 100 pm and a length of about 10 pm.

[0117] Generally, the GsE and GGE can be made from a substrate coated with any conducting materials descried above for the source and the drain electrodes (e.g., Pt or Au) or a conducting polymer, such as poly(fluorine)s, polyphenylenes, polypyrenes, polyazulenes, polynaphthalenes, poly(pyrrole)s, polycarbozoles, polyindoles, polyzaepines, polyanilines, poly(thiophene)s, poly(3,4-ethylenedioxythiophene), poly(p-phenylene sulfide), poly(acetylene)s, or poly(p- phenylene vinylene). In some embodiments, the GsE and GGE contain two or more conducting materials. For example, the gate electrode contains two conducting materials, where the first conducting material can be a material disclosed above for the source and the drain electrodes and a second conducting material can be a conducting polymer. In some preferred embodiments, the GsE and GGE contain two conducting materials, where the first conducting material is a metallic conductor of a first type and the second conducting material is a metallic conductor of a second type. The two conducting materials may be coated simultaneously or subsequently on the substrate. In some forms the GsE and GGE are formed from a Kapton (polyimide) substrate sputter coated with Cr and Au.

[0118] D. Electrolyte Solution

[0119] The electrolyte solution is in electrically contact with the channel and the gate electrode, and the source electrode and the drain electrode (the last two are insulated with an insulator). The electrolyte solution is a solution that contains ions or molecules that have lost or gained electrons, and is electrically conductive and mostly aqueous. Electrolyte solutions include, but are not limited to, water, buffers such as phosphate buffered solution, phosphate buffered saline, salt water, MES buffer, Bis-Tris buffer, ADA, ACES, PIPES, MOPSO, Bis-Tris propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS. The electrolyte solution can have a pH between about 4 and about 8.5, between about 4.5 and about 8.5, between about 5 and about 8.5, between about 5.5 and about 8.5, between about 6 and about 8, or between about 6.5 and about 7.5, preferably about 7.4.

[0120] E. Electrolyte solution Reservoir / chamber

[0121] The device may include a reservoir / chamber to contain the electrolyte solution. The reservoir may be incorporated in the pOECT by any suitable methods. For example, the reservoir is glued on top of the channel. The reservoir can also be a microfluidic channel.

[0122] The reservoir is typically defined by a side wall and a bottom surface, and contains an opening configured to allow the electrolyte solution to enter the reservoir. The reservoir may have any suitable shapes, such as a cylindrical well, a cubic shape, or a cuboid shape. The bottom surface is formed from at least a portion of the channel and optionally, at least a portion of the side wall is formed from the source electrode and the drain electrode respectively. For example, the reservoir is a cylindrical well and has a bottom surface formed from at least a portion of the channel. The side wall is perpendicular to the surface of the channel and at least a portion of the side wall is in contact with the source electrode and drain electrode. The cylindrical reservoir contains an opening to allow the electrolyte solution to enter the reservoir. An exemplary pOECT employing separate chambers to contain the electrolyte solution is shown in FIG. 6A.

[0123] For example, the reservoir is a cuboid defined by a bottom surface and four side walls. The bottom surface is formed by at least a portion of the channel and each of the first and second side walls are parallel to each other and vertically placed on top of each of the source electrode and drain electrode. The third and fourth side walls are parallel to each other and perpendicular to the first and second side walls, such that the reservoir contains an opening to allow the electrolyte solution to enter the reservoir.

[0124] For example, the reservoir is a cuboid and has a bottom surface formed by the channel, a first side wall formed from a portion of the source electrode, a second side wall formed from a portion of the drain electrode, and a third side wall and a fourth side wall that are parallel to each other and perpendicular to the first side wall and second side wall, such that the reservoir contains an opening to allow the electrolyte solution to enter the reservoir. When an electrolyte solution is in the reservoir, the channel, at least a portion of the source electrode and the drain electrode, and the gate electrode are in contact with the electrolyte solution. The reservoir can be made from any suitable inert material, such as plastic, glass, or a polymeric material, such as polydimethylsiloxane (PDMS).

[0125] III. METHODS OF MAKING AND REAGENTS THEREFOR pOECT devices disclosed herein include one or more SE, one or more DE one or more CH, a GsE and GGE, and optionally, an electrolyte solution containing one or more metabolites. The SE and the DE are placed apart and connected electronically by the corresponding CH. The CH in some forms, contains one or more enzymes and one or more n-type polymers. In some embodiments, the SE and SE can be bridged by the CH, wherein the length of the channel is between 1 pm and 1000 pm. In one embodiment, the length of the channel bridging source electrode and drain electrode (i.e., the inter-electrode gap) is about 10 pm. The GsE and GGE are placed separately from the SE, the DE, and the CH. The electrolyte solution is in electrical contact with the GsE and GGE and the CH. In some forms embodiments, pOECT device has a planar configuration. Alternatively, the GsE and GGE are not provided coplanar with the SE and the DE and may be provided on a different layer of the supporting substrate or separately from the supporting substrate. In some embodiments, the pOECT contains a plurality of independently addressable SE and DE, GsE and GGE, and corresponding CHs, wherein the corresponding channels contain different enzymes for detecting multiple analytes simultaneously.

[0126] In some forms, the CH and enzymes of the CH and the gate are adjacent to one another; meaning they are physically or chemically connected by appropriate means. In a preferred embodiment, the component are physically connected by coating such as by spin-coating, dropcasting, or electropolymerization. In one embodiment, electropolymerization may be performed in a solution containing one or more monomers of the corresponding polymers. In another embodiment, polymerization may be performed on a surface modified with monomers via potential triggering or stimulus such as UV light or temperature. The components can be deposited separately, e.g. in layers, or they can be integrated into one deposition layer.

[0127] In some embodiments, the pOECT electrochemical device further includes a supporting substrate and the source, drain, and gate electrodes can be patterned on the supporting substrate. In some other embodiments, the pOECT can be incorporated into a microfluidics configuration. In one embodiment, the components are physically and chemically connected with the supporting substrate. In a preferred embodiment, the component are physically connected with the supporting substrate by coating such as by sputt-coating, spin-coating, drop-casting, or otherwise deposing the individual components on the supporting substrate.

[0128] In a preferred embodiment, gold contacts (located at the source, drain and gate) and interconnects were patterned on a glass substrate while an additional layer of Parylene C was used to insulate the gold interconnects according to established protocol (Nielsen, et al., Journal of the American Chemical Society, 138:10252-10259 (2016); Sessolo, et al., Advanced Materials, 25(15):2135-2139 (2013)). The final channel dimension is 10 pm in length and 100 pm in width. The gate electrode has an active area of 500 pm2.

[0129] In some forms, pOECT devices are fabricated using (a) gold source and drain electrodes, (b) GOx and n-type polymer coated channel, (c) GOx and n-type polymer coated gold gate electrode, and (d) phosphate buffer solution containing glucose wherein the glucose is at a physiological relevant concentration

[0130] In some forms, n-type polymer based electrochemical devices are fabricated using (a) gold source and drain electrodes, (b) LOx and n-type polymer coated channel, (c) LOx and n- type polymer coated gold gate electrode, and (d) phosphate buffer solution containing lactate wherein the glucose is at a physiological relevant concentration.

[0131] Methods for functionalizing devices for detection of biomolecular analytes such as DNA, DNA or proteins or organisms are known in the art. For example, single-strand oligonucleotide (ssODN) probes can be immobilized using a nucleophilic substitution reaction of the thiolated ssODN molecules with poly aniline (PANI); the binding of the complimentary target molecules with the immobilized probes revealed a substantial potential change (Zhou, et al., Biosensors and Bioelectronics, 24(11): 3275-3280 (2009). Peptide nucleic acid (PNA) have outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base -pairing. PNA probes immobilized on an electrode can be used for potentiometric DNA detection. Changes in the charge density as a result of hybridization at the solution / electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals (Goda, et al.,

[0132] Sensors 2013, 13(2), 2267-2278). Tang et al., (https: / / doi.org / 10.1002 / elan.200503351) disclose electrode functionalization by means of self-assembly of monoclonal diphtheria antibody (D-Ab) onto a platinum electrode based on nanoparticles mixture (containing gold nanoparticles and silica nanoparticles) and polyvinyl butyral (PVB) as matrixes). Functionalization of biosensors to include nanobodies is disclosed for example, in U.S. publication 20240280570, by modifying an OECT to include a biorecognition layer for example, the gate electrode. The biorecognition layer includes a self-assembled monolayer (SAM) of organic molecules, a linker, and a biorecognition element such as a nanobody. The biorecognition layer is designed to provide a stable complex, preferably, an immunocomplex between a biorecognition element (for example, nanobody) in the biorecognition layer, and its binding partner, which is the analyte / antigen of interest. The biorecognition layer includes two self-assembled monolayers (SAMs), the first of which is formed from organic molecules, chemically modified as disclosed therein and referred to therein as Chem-SAM, and the second of which is formed through a specific biological autocatalytic coupling strategy (therein, Bio-SAM), as disclosed therein. The Bio-SAM includes biorecognition element such as a nanobody or it can be an antibody. The biorecognition layer includes a SAM formed from an alkanedithiol (e.g. 1 ,6-hexanedithiol) and a SpyTag / SpyCatcher bioconjugation as the linker, which allows for controllable orientation of the nanobody functionalization. Methods for directly functionalizing an OECT that excludes the use of a chem-Sam are disclosed for example in U.S. Publication No. 20250027901. A cholesterol biosensor was fabricated by immobilization of cholesterol oxidase into stabilized lipid films using zinc oxide (ZnO) nanowalls as measuring electrode. Enzymes that bind to small molecule analytes of interest can be immobilized for detection of the small molecule. For example, cholesterol oxidase was incorporated into the lipid film prior polymerization on the surface of ZnO nanowalls resulting in a sensitive, selective, stable and reproducible cholesterol biosensor (Psychoyios, et al., doi.org / 10.1002 / elan.201200591); uricase, was electrostatically immobilized in conjunction with Nafion membrane on the surface of well oriented ZnO-NFs, resulting in a sensitive, selective, stable and reproducible uric acid sensor (Ali, et al., Sensors 2012, 12(3), 2787-2797); a galactose biosensor was developed by immobilizing galactose oxidase on ZnO nanorods in conjunction with glutaraldehyde as a cross-linker molecule (Khun, et al., doi.org / 10.1155 / 2012 / 696247). Aptamers specific for a target protein analyte or epitopes on the surface of a microorganism if interest can be immobilized on the disclosed POECT devices for protein detection. For example, DNA aptamers that can recognize different epitopes in thrombin were introduced in parallel or serial manners on a device the sensing surface to capture the target via multiple contacts as found in many biological systems. The spacer and linker in the aptamer probes were optimized for exerting the best performance in molecular recognition. To gain the specificity of the sensor to the target, an antifouling molecule, sulfobeaine- 3 -undecanethiol (SB), was introduced on the sensor to form a self-assembled monolayer (SAM) (Goda, et al., Biosensors and Bioelectronics, 73:174-180 (2015). DNA aptamers - tailored DNA or RNA segments acting as artificial recognition elements able to recognize conserved epitopes on the surface of the microorganism. Aptamers have been selected for human and food-borne pathogens, such as Streptococcus spp. (Hamula, et al., Anal. Chem. 83, 3640-3647 (2011); Alfavian,et al. Can. J. Microbiol. 63, 160-168. (2016)), Listeria monocytogenes (Duan, et al., Food Control 33, 239-243 (2013), Salmonella spp. (Park, et al., Appl. Biochem. Biotechnol. 174, 793-802 (2014); Lavu, et al., ACS Combin. Sci. 18, 292-301 (2016); Staphylococcus aureus (Cao, et al., Nucleic Acids Res. 37, 4621-4628 (2009), Vibrio parahaemolyticus (Duan, et al., J. Agric. Food Chem. 60, 4034-4038 (2012), Escherichia coli (Savory, et al., J. Microbiol.

[0133] Methods 104, 94—100 (2014) and Pseudomonas aeruginosa (Wang, et al., Eur. J. Clin. Microbiol. Infect. Dis. 30, 273-278 (2011).

[0134] IV. METHODS OF USING

[0135] One of the various aspects of the disclosed pOECT device is a method of detecting analytes in a sample or the state (such as pH) of a sample. Analytes that can be detected include, but are not limited to ions, nucleic acids (DNA / RNA, proteins, small molecules and organisms.

[0136] In some forms, the electrochemical device can be used as in vitro as portable devices and / or wearable electronics that measure metabolites, including, but not limited to, glucose and lactate.

[0137] The pOECT device can be used for both in vitro and in vivo applications.

[0138] In some forms, the disclosed device is used as an in vivo sensing device for measuring metabolites such as glucose and / or lactate from the blood stream. Such in vivo electrochemical sending devices may be utilized as an implanted device for continuously monitoring a single metabolite or multiple metabolites simultaneously, wherein the metabolites are derived from the organism. The device can be formulated as a wearable patch that integrates a stretchable battery and a low-power digital electrochromic display. The patch can operate as a standalone device to directly display the concentration of various electrolytes or metabolites in sweat, such as glucose and lactate, without any wired or wireless connection to external devices (Lin, et al., Nature Electronics 5:694—705 (2022)). Anastova et al ( Biosensors and Bioelectronics 93: 139- 145( 2017)) discloses a construction design which provides for continuous flow of sweat can through an array of flexible incorporated in a microfluidic channel.

[0139] In some forms, the electrochemical device can be used as in vitro as portable devices and / or wearable electronics that measure metabolites, including, but not limited to, glucose and lactate. A method of detecting metabolites using the pOECT device includes the steps of (a) applying a gate potential and (b) monitoring changes of a source-drain current (IDS) that flows through the channel that connects the SE and the DE as result of electrons transferring during uses, for example, during an enzymatic reaction between enzymes and metabolite in the electrolyte solution. The IDS changes with the change of metabolite concentration. In some forms, the IDS increases with the increase of metabolite concentration. In some embodiments, the VSG equal to or higher than a threshold voltage. In a preferred embodiment, the threshold voltage is 0.5 V. In some forms, the sensor sensitivity is tunable by varying the biasing conditions to fit the analyte range of choice in different body fluids such as blood, saliva, sweat, and tears (Pappa, et al., Science Advances, 4(6):eaat0911 (2018)).

[0140] In an n-type polymer based electrochemical device, a gate voltage (VG) is applied to dope the polymer film caused by the cations injected from the electrolyte solution, resulting in a baseline source-drain current (IBD), which is unrelated to the enzymatic reactions. The oxidation of a compound such as a metabolite with enzymes produces electrons that are directly transferred to the n-type polymers on the gate electrode and the channel. N-type polymers have conjugated backbone that have the ability to stabilize electrons. Enzymatic reactions increase the charge carrier density and thus the conductivity of the channel, leading to increased ID (signal output), thereby turning the device on. In some embodiments, a constant VG is applied to the electrochemical device for metabolite sensing. In some embodiments, VG equal to or higher than a threshold voltage, that is, the minimum VG required to result in a ID in the absence of an enzymatic reaction, i.e. oxidation reaction between enzymes and metabolites. In one particular embodiment, the threshold voltage is 0.5 V.

[0141] The present invention will be further understood by reference to the following nonlimiting examples which are incorporated herein as specific embodiments of the disclosed pOECT, including the methods of making thereof, and methods of use.

[0142] EXAMPLES

[0143] Materials and methods

[0144] Materials: Sodium chloride (NaCl), ammonium sulfate ((NEL^hSCL), Phosphate Buffered Saline (PBS, pH 7.4), ethylene glycol, sodium dodecylbenzenesulfonate, (3-glycidyloxypropyl) trimethoxysilane, chloroform, hydrochloric acid, aniline, buffer solutions pH = 4, 7 and 10, potassium hexacyanoferrate, (III), potassium hexacyanoferrate (II), phosphoric acid, boric acid, acetic acid, potassium nitrate, and propidium iodide (PI) were purchased from Sigma Aldrich. Trypsin (TrypLE™), Dulbecco's Phosphate Buffered Saline containing Ca2+and Mg2+(lx DPBS), lx DPBS (Ca2+and Mg2+free), Dulbecco's Modified Eagle's Media (DMEM), fetal bovine serum (FBS), glutaMax, penicillin-streptomycin (P / S), Bovine Albumin Serum (ABS) were purchased from Thermo Fisher. Calcein AM was purchased from Life Technologies, paraformaldehyde (PF A) from Electron Microscopy Sciences, Triton X-100, phalloidin- rhodamine and gold mounting medium from Invitrogen, Madin-Darby canine kidney cells (MDCKII) from ECACC, CLEVIOS PH1000 (PEDOT:PSS) from Heraeus. All aqueous solutions were prepared with ultrapure water (Millipore Milli-Q). The n-type OMIEC p(CeNDI- T)1and the p-type OMIEC p(gaC2T2-T)2were synthesized according to procedures reported previously. The chemical structures of the polymers are shown below. p(C6NDI-T)

[0145] The structures above are the chemical structures of the n-type OMIEC p(CeNDI-T) and the p-type OMIEC p(gaC2T2-T) used as semiconductors for the OECTs and pOECTs.

[0146] Device fabrication: The single channel OECTs / pOECTs (W-L = 100- 10 pm) with various OMIEC patterning protocols were fabricated according to a procedure reported previously2. Large channel OECTs / pOECT capable to reach mA currents were obtained by using commercially available interdigitated electrodes. The PANI film was electropolymerized on a Au electrode (<D = 5 mm) using a procedure reported in literature3. Na+ and Cl- ISE were purchased from NT Sensors, Pt coil / wire, Au electrodes and Ag / AgCl glass RE from als-japan, AgCl pellet from Warner Instruments, leakless Ag / AgCl from eDAQ, glass pH meter from Metrohm, interdigitated electrodes (ED-IDE3-Au) from MicruX Technologies. For interfacing the pOECT channels with epithelial cells, pOECT devices were firstly sterilized using 70% ethanol for 30 min, rinsed with water and dry inside biosafety cabinet. To improve the adhesion of cells, pOECT devices were plasma activated using plasma cleaner 20W for 20 sec. MDCKII cells were then seeded on the device and incubated at 37 °C in a humidified atmosphere with 5% CO2 until a barrier tissue was formed, which appeared typically after 2-3 days.

[0147] Device operation, characterization, and sensing measurements: All electrochemical measurements were performed with a BioLogic VSP-300 multichannel potentiostat. The transfer curves of the OECT / pOECT were recorded by connecting the devices according to the schematics shown in FIG. ID. Specifically, two channels of the multichannel potentiostat were used, the first one for the application of VDS and the second one for the application of VGS or VSG. The instrument can be operated in either floating or grounded mode. The same device operation can be obtained also by combining a source measure unit for the application of VDS and a single channel potentiostat for the application of VGS or VSG.

[0148] If otherwise stated, the applied VDS for all measurements was +100 mV for the n-type and -100 mV for the p-type devices, while the gate voltage was cyclically scanned multiple times (100 mV / s), according to the ranges reported on each plot, until the IDS stabilized. A glass well with a PDMS base was placed on top of the OECT / pOECT channel and filled with the electrolyte (100 mM (NH4)2SO4 or lx PBS), and G / GG (or the Gs when the single chamber configuration was used) was also placed in the same solution.

[0149] The OECT and pOECT characterization (no sensing) was done with the OECT-single chamber and pOECT-single chamber setup with lx PBS electrolyte. For sensing measurements, the sensing electrode (G or Gs) was kept in a separate compartment from the OMIEC (and GG when pOECT used). Since variations of ion concentration / composition in the electrolyte can significantly alter the behavior of the OMIEC4, the separation of the channel from the sensing interface allows to maintain a constant electrolyte composition in the OMIEC compartment. This allows to eliminate any interference coming from OMIEC and measure solely the effect of the change of electrochemical potential of the Gs on output characteristics. The measurements related to pOECT Na+and CP sensing were performed with the pOECT -microfluidic setup, where the two compartments were connected through a microfluidic tubing to establish the ionic connection. 100 mM (NH4)2SO4 was used as the electrolyte in both chambers. Consecutive aliquots of NaCl from a standard solution were added to the sensing chamber to increase the concentration of the target ion. The 2-electrode OCP measurement for Na+sensing was performed by measuring the open circuit voltage between the Na+-ISE (WE) and the Ag / AgCl (RE) when the two electrodes were placed in the same electrolyte (100 mM (NIE SCE) and consecutive aliquots of NaCl from a standard solution were added to the electrolyte. (NH 2SO4 was selected as the electrolyte instead of the more common lx PBS since it contains only minimal traces of Na+and CP, which are otherwise present in PBS.

[0150] The measurements related to pH sensing were performed with the OECT -floating gate and pOECT -floating gate setup, where the two compartments were connected through two Ag / AgCl RE to establish the ionic connection, lx PBS was used as the electrolyte in the OMIEC chamber, and different buffer solutions were added to the sensing chamber. For sensing measurements where the pH variations were of 3 units (4, 7, and 10), commercially available buffer solutions were used. For sensing measurements where the pH variation was of 1 unit (6 and 7), a universal buffer (Britton-Robinson Buffer: 0.01 M H3PO4, 0.01 M H3BO3, 0.01 M CH3COOH in 0.1 M KNO3 adjusted to the desired pH with NaOH(aq)) was used. The floating gate and the microfluidic setup are identical in terms of device performance and reported to show the system's versatility.

[0151] The measurement related to the barrier tissue monitoring was performed with the pOECT-single chamber setup, with a Pt wire as GG and a leakless Ag / AgCl RE as Gs. The pOECTs with cells were taken out of the incubator and washed 3 times with DPBS after removing the cell media. 100 pL of lx DPBS (Ca2+and Mg2+free) was added as the electrolyte. A constant VDS = -0.1 V was applied while the VSG was continuously pulsed (3 s) between 0 and -0.3 V for the whole experiment, with no interruption. After about 100 s of initial pulsing, to establish a baseline, 100 pL of trypsin solution was added to the initial volume of 100 pL DPBS. The pulse recording was performed for 1 h; however, after 15 min, the cells completely detached.

[0152] The electrochemical potential measurements of the OECT / pOECT components were performed by measuring the open circuit voltage of each electrode connected to the WE of the potentiostat with respect to a common Ag / AgCl RE if the components were in the same electrolyte. The measurement of n components of the OECT / pOECT were performed in parallel during the device operation by utilizing n synchronized potentiostat channels.

[0153] The cyclic voltammetry (CV) measurements were performed according to the schematics shown in FIG. 8D with an Ag / AgCl RE and Pt coil CE, by cyclically applying the voltage range reported in each plot, repeated until IWE stabilization. The scan rate was 100 mV / s. The electrolyte was lx PBS with 5 mM potassium hexacyanoferrate (III) and 5 mM potassium hexacyanoferrate (II).

[0154] The current flowing through the RE connection was measured by using the zero resistance amperometry (ZRA) technique and an ultra-low current module (BioLogic) where one side of the RE cable was connected to the RE / CE and the other side was connected to the WE of the potentiostat, and a constant 0 V was applied.

[0155] To quantify the device response in the ion sensing measurements and minimize the device-to-device variations, the normalized response of the pOECT was reported as NR (IDS). This parameter is also used as a metric to compare the performance of the pOECT in different conditions. It is calculated by normalizing the IDS change after target exposure to the sensing unit with respect to the blank solution: blank Equation (2) where IDS M± is the pOECT IDS current after interaction of the Gs with the target M± at a specific bulk concentration, and IDS, blank isthe pOECT IDS current before interaction of the Gs with the target. The NR values reported in the calibration curves correspond to the voltages where the device showed the highest response (highest NR).

[0156] An analogous normalization was performed for the 2-electrode OCP measurement, reported as NR (V):

[0157] NR(V) = EWE M± / EWEiblankEquation (3)

[0158] Since it is not possible to directly compare the signal (IDS VS EWE) or the sensitivity (AIDS / C VS A EWE / C) of the pOECT and the classical 2-electrode setup, due to different working regimes (current regime vs voltage regime), we compared their performance in terms of NR.

[0159] All the devices performance comparison were conducted in analogous condition. In particular, for pOECT vs 2-electrode setup with WE size = Gs size and for OECT vs pOECT with G size = Gs size and OECT channel size = pOECT channel size.

[0160] To quantify the device response in the cell barrier tissue monitoring and minimize the device-to-device variations, the cut-off frequency change was normalized (NR(fC0)) to a range from 0 to 1, where 0 corresponds to the lowest cut-off frequency (fco.min) and 1 to the highest (fco.max)-

[0161] NR(fco)=(fco—fco.min) / (fco.max—fco.min) Equation (4) Cell culture: Madin-Darby Canine Kidney cells (MDCK II) were routinely maintained in DMEM media at 37 °C in a humidified atmosphere of 5% CO2. The media was supplemented with 5% fetal bovine serum (FBS), 2 mM Glutamax and 1% PenStrep 100X (10000 U mL-1Penicillin, 10 000 pg mL-1Streptomycin).

[0162] Assessment of cytotoxicity in different reference electrodes: For a general evaluation of the cytotoxicity on various reference electrodes (pellet, platinum, leakless, and glass), two characteristics of cells were evaluated after their culture in contact with the electrodes for 72 hours: cell viability using a LIVE / DEAD assay and cell morphology with actin staining. The different electrodes were carefully placed in a 48-well plate, avoiding contact with the bottom of the well, where MDCKII cells (6000 cells / cm2, P.34) were seeded.

[0163] Cell viability: After 72 hours of culture, cells were incubated with 3 pM, Calcein AM and 3.5 pM PI in PBS containing Ca2+and Mg2+, for 10 min at 37°C and fluorescent images were captured using a 10X objective in an inverted Leica DMi8 microscope. Calcein AM is converted to a fluorescent molecule, green, when it interacts with viable cells, while PI reacts with the nuclei content of dead cells, generating a red signal.

[0164] Cell morphology: After 72 hours of culture, cells were fixed with 4% (w / v) paraformaldehyde for 15 minutes and stained with rhodamine-phalloidin for 60 min. Phalloidin binds and stabilizes F-actin in the cell cytoskeleton, and rhodamine works as a marker and allows fluorescent visualization of F-actin with a red signal. This method delineates the cells and allows the visualization of differences in morphology between cells in contact with different electrodes. Cells were imaged using a 20X objective in an inverted confocal laser scanning microscope with Airyscan (Zeiss LSM 880).

[0165] Results and Discussion

[0166] The pOECT configuration

[0167] The top panel in Fig. 1A shows the electrical connections of a conventional OECT, from the point of view of an electrochemical cell. To apply VDS, the DE is connected to the WEi, while the SE is connected to a combined RE1 / CE1. The electrochemical potential of the DE is defined with reference to SE (due to the REi connection), and the current flowing between WEi and CEi is the IDS- Simultaneously, VGS is applied between the GE (used as the sensing electrode in OECTs), which is connected to the WE2, and the SE, connected to the RE2 / CE2. The potential of the GE is defined with respect to SE due to the RE2 connection, and for this reason, the gating system can be considered as source-referenced. The current between GE and SE is IGS, also called leakage current.

[0168] The roles of SE and GE for the VGS application can be swapped, resulting in the SE being the WE2 and the sensing interface (GE) being a combined RE2 / CE2, as shown in Fig. IB. This configuration is named herein as gate referenced-OECT . The gate voltage should now be called VSG, and the gate current is ISG - to maintain an accurate nomenclature. The gate referenced-OECT will work exactly like the regular OECT, with the same IDS and IGS values, but with only the sign of the gating voltage reversed. However, having re-wired the S-G connection, the system gains an extra degree of freedom. The GE, which is now a combined RE2 / CE2, can be decomposed into two independent electrodes, i.e., RE2 and CE2 (Fig. 1C). The sensing electrode remains connected to RE2. A new electrode is introduced, which can be any type of counter electrode, such as a Pt coil (Fig. IC-middle) or an Au electrode (Fig. IC-bottom), and connected to CE2. Together, RE2 and CE2 represent the new “gating system”. This configuration is referred to as the ‘fotentiometric-OECT” (pOECT). The pOECT does not require any additional connections. While the conventional OECT operation involves shorting the RE2 and CE2 cables and connecting them to the same electrode (SE), in the pOECT, these cables are separated and linked to electrodes with distinct functions.

[0169] Both RE2 and CE2 gate the channel but serve different roles. RE2 (sensing electrode) serves as the reference point for the electrochemical potential of the SE. CE2, on the other hand, actively provides the voltage and current to bring the SE to the required potential difference with respect to RE2, thereby doping / dedoping the channel. Since this electrode provides the necessary current to gate the channel, this design eliminates any electrochemical stress from the sensing electrode interface. Considering their distinct functions, RE2 is designated as the sensing gate (Gs), and CE2 as the gating gate (GG). In the pOECT configuration, the IDS is modulated solely by electrochemical potential variations of Gs.

[0170] In Fig. 1A-C (middle and bottom), shows schematics and microscope images of the same channel gated in these three configurations. Like in the OECT, the dual gating system (Gs and GG) can be either vertical (Fig.l-middle), or the pOECT can be miniaturized, consolidating all the device components into a single substrate by fabricating planar GG and Gs, thereby maintaining the overall device size in the micrometer range (Fig.1 -bottom). The additional electrode introduced has, therefore, no impact on device geometry but offers significant advantages in device performance and sensor accuracy, as described below. Fig. 2A-left shows the transfer curves of an n-type enhancement mode device wired in the conventional way (dotted lines, “OECT”), and reconfigured as described in Fig. 1c (solid lines, “pOECT”). First, to characterize the behavior of the pOECT setup as a transistor, non- polarizable Ag / AgCl electrodes were used as G (for OECT) and Gs, and a Pt coil as the Gs. On the right-hand side of Fig.2A shows the evolution of electrochemical potentials of the OECT and pOECT terminals during the acquisition of transfer characteristics. Since the applied voltages are the same for the OECT and the pOECT (except for the sign of the gate voltage; VSG = -VGS), the electrochemical potential profiles overlap when a non-polarizable electrode is used as G / Gs. Consequently, the corresponding transfer curves are identical for both configurations, demonstrating no loss or improvement of transistor performance in the pOECT configuration compared to the classical one.

[0171] The advantage of the pOECT becomes evident when using a polarizable electrode as the G of the OECTs. Fig. 2B shows the transfer curves of the n-type OECT / pOECT gated with a polarizable vertical Au electrode, with a diameter of 5 mm, used as G or Gs, along with the electrochemical potentials of device components. Polarizable electrodes, such as Au, are commonly used as the functionalized bio-sensing surfaces of OECTs, with the size of the G / Gs chosen to be similar to several biosensing applications reported in the literature.25,30In the case of the OECT (dotted lines), there is a clear voltage drop on the G during the VGS scan, polarizing the G to positive potentials. This, in turn, drags the S and D also toward positive potentials, preventing the OMIEC from reaching a high doping state. Conversely, for the pOECT (solid lines), no voltage drop occurs on the Gs across the entire biasing range. This allows the S and D to be linearly brought toward doping potentials without any distortion. As a result, the transfer curves of OECT and pOECT look dramatically different, with the pOECT channel achieving much larger current modulation than that of the OECT. Despite the use of a polarizable electrode as the Gs, the pOECT behaves as if the channel is gated with a non-polarizable electrode.

[0172] When using a polarizable gate electrode, obtaining the same IDS modulation with the OECT in a comparable biasing range necessitates applying a much higher VGS (X2.5) up to 1.26 V. This marks the first advantage of the pOECT : it enables the safe use of polarizable gate electrodes to attain stable and full modulation of the channel. Secondly, in the pOECT, negligible current (pA or less) flows through the Gs due to the high impedance of the RE2 connection (100 TQ). This design thus secures a nondestructive biasing method for the sensing interface, which often contains the biorecognition units that may be damaged due to the leakage current (IGS). Moreover, high voltages applied for better modulation in the OECTs push the sensing electrode to electrochemical potentials, which can be detrimental to the biofunctionalized gate electrode (0.9 V vs. Ag / AgCl, Fig. 2E). Although the Gs nonpolarizability is not an absolute condition, in most practical sensing applications, this issue does not arise for the pOECT.

[0173] In the pOECT, any possible polarization of the Gs is bypassed by redirecting the polarizing current to the GG. Importantly, the choice of the GG in the pOECT design does not affect device characteristics. Fig. 2C-right shows the electrochemical potentials of two pOECTs operated with different GG electrodes. One GG is a large and minimally polarizable electrode, such as a Pt coil (solid lines), and the other is an extremely polarizable electrode, such as a small Au ( = 1.6 mm, dotted lines). Both devices effectively prevent polarization on the Gs, resulting in identical electrochemical potential profiles of S, D, and Gs, as well as transfer curves (Fig. 2C-left). This result underscores the versatility of the pOECT, as any material can be employed as the GG without imposing limitations based on its polarizability / size or specific electrochemical potential. The absence of specific requirements for the newly introduced electrode in our configuration streamlines device fabrication and design. The results depicted in Fig. 2A-2C are derived from devices equipped with vertical gating electrodes. A similar enhancement in device performance can also be achieved with fully microfabricated devices, as demonstrated in Fig. IC-bottom, and Fig. 11A-11B.

[0174] The operation of the pOECT as a sensor

[0175] Since the pOECT renders any sensing electrode non-polarizable, hence, eliminating interferences arising from its capacitance, it serves as an ideal platform for pure potentiometric sensing. To demonstrate how pOECT operates as a potentiometric sensor and benchmark its capabilities, ion sensing was used, that is, the most representative type of potentiometric sensing. Ions were selected as the target instead of other biomarkers, such as proteins or nucleic acids, because ion sensing can be easily described by the thermodynamic Nernst equation. This a comparison of the pOECT and OECT experimental behavior with the theoretically expected one (Nernstian behavior). Surface potential changes resulting from protein or DNA binding range from 10 to 150 mV for LoD and saturation concentrations, respectively25 ,53“55. These values are in the same range as those reported here for the ISEs. Despite the different origins of the surface potential change (ion capture or DNA / protein binding), the response of the pOECT remains the same since the device detects these changes as a simple equivalent gating voltage variation. Therefore, the conclusions drawn regarding the pOECT’ s operation in ion sensing and its advantages extend to the detection of any other target through a potentiometric mechanism.

[0176] Fig. 3A schematically represents how the 2-electrode setup and the OECT are merged into a single configuration in the pOECT. By using a Na+ISE as the Gs, a Na+Ion-Selective pOECT (Na+-IS-pOECT) is obtained. To demonstrate the modality of the platform, a p-type OMIEC operating in enhancement mode was used. Fig. 3B shows the transfer curves of the Na+-IS-pOECT when the Gs is exposed to different concentrations of Na+. An increase in the Na+concentration in the sensing chamber increased the IDS. Fig. 3C depicts the electrochemical potential changes that the device terminals undergo during Na+detection. A higher cation concentration shifts the electrochemical potential of the Gs to higher positive potentials (AOCP), which consequently drags the entire channel (S and D) to a new potential in the same direction and of the same amount as AOCP. At this new potential, the p-type OMIEC channel is in a more oxidized / doped state, resulting in the higher IDS observed in Fig. 3B.

[0177] The origin of the AOCP is reported in Fig. 3D, showing the real-time Na+-induced change in the ISE potential monitored with respect to a RE (Ag / AgCl). The electrochemical potential of the ISE (EWE) follows the Nernst equation (Equation 1), which translates increasing concentrations of Na+in the electrolyte to a log-linear increase in electrochemical potential. where k is a specific constant of the electrochemical cell that depends on the setup and represents the sum of potential differences at all of the other interfaces other than the ISE, R is the gas constant (8.316 J mol1K1), T is the temperature in K, F is the Faraday constant (96485 C mol1), +n is the charge of the ion detected by the ISE, aM±n and [M-n] are the activity and the concentration of the target ion in solution, respectively. The sensitivity of the ISE for a monovalent ion is theoretically calculated as 59 mV / dec in standard conditions but can be less depending on the quality, storage conditions, and history of the ISE. When the ISE is used as the Gs of the pOECT, the electrochemical potential change reported in Fig. 3D becomes the input signal for the device (EWE= ECs) and is implicitly reflected in an equivalent electrochemical potential shift (AOCP) of the whole channel, where it will be amplified as a current output. Fig.3 E describes the working mechanism of the pOECT. Fig. 3F-left shows the normalized response (NR) of the Na+-IS-pOECT calculated as the change in IDS at different VSG in the nA current regime. Fig. 3F-right shows the corresponding calibration curves of the pOECT working in the nA or A current regime, and the conventional 2-electrode potentiometric setup. NR values of the 2-electrode setup are calculated from the change in the OCP of the Gs, and the maximum NR obtained is 1.4 for the highest Na+concentration. The relatively low NR values can be explained by the Nernst equation (constant sensitivity at 59 mV / dec) and the device operation, which is limited by the voltage regime. On the other hand, the NR of the IS-pOECT is much higher because it is obtained in the current regime, which can span across several orders of magnitude and can also be tuned by selecting the VSG that provides the highest signal shift from the baseline current (0 M), as shown in Fig. 3F-left. The NR of the pOECT working in the pA regime reaches 8.5 for the highest Na+concentration. When accurate electronics are available to operate the OECT in the nA regime, where the device shows higher transconductance efficiency, the NR for the highest Na+concentration can reach up to 35.

[0178] Performance comparison of pOECT versus the conventional OECT

[0179] The Nernst equation (Equation 1) describes the electrochemical potential that the sensing electrode reaches in OCP conditions, i.e., without applied current or voltage. These conditions represent an equilibrium state for the electrode, describing the system’s thermodynamics rather than its kinetics. The monitoring of this thermodynamic state is the analytical signal in a classical OCP measurement using a 2-electrode setup (Fig. 3D) and is also what is aimed to be used as input signal in an OECT and pOECT (Fig. 3E). For this reason, the accuracy of the setups compared in this work (2-electrode, OECT and pOECT) can be considered as the agreement between the measured electrochemical potential of the sensing interface and the theoretical expected Nernstian behavior.

[0180] To compare the performance of the OECT and pOECT, a 5 mm diameter pH-sensitive electrode was created, that is, an electrodeposited film of polyaniline (PANI), which was used as the sensing interface (G and Gs, respectively). PANI was selected as the sensing electrode because using the commercially available electrodes (pH-meter or the Na+-ISE) as the G of the OECT deteriorates them, as discussed above. Fig. 4A shows the OECT and pOECT transfer curves recorded when the PANI G and PANI Gs were exposed to buffer solutions with a difference of 1 pH unit (6 to 7). While the pOECT shows a significant current modulation due to the pH variation, the OECT has no response. The two insets of Fig.4A show the IDS and IGS of the OECT on a zoomed scale, almost identical in terms of order of magnitude, demonstrating no OMIEC modulation and no signal amplification. The sensing interface of the OECT (i.e., the G) is immediately polarized due to the application of VGS with 63% of the VGS dropping on G (Fig. 4B-left). This drop causes the loss of the initial Nernstian potential, which represented the analytical input signal, making the device output inaccurate. On the other hand, with the pOECT, the Gs potential remains completely unperturbed (Fig.4B-middle). Since the thermodynamics of the electrode used as Gs remains unaltered, the behavior of the pOECT is ruled by the Nernst equation. Fig.4B-right shows the electrochemical potential changes of the PANI-Gs pH-pOECT at different pH values. The pH-sensitive interface maintains its expected Nernstian response, exhibiting a -59 mV / pH slope, which is then amplified by the pOECT as an output current.

[0181] These experiments demonstrate that the pOECT preserves the Nernstian behavior of the sensing electrode at the same level as a 2-electrode setup, leading to a transistor that provides the maximum accuracy.

[0182] To construct an OECT with comparable accuracy as the pOECT, the G area could be increased to reduce the polarization. However, the geometry required to attain the same pOECT performance exceeds any reasonable size for a small and portable device. Fig. 4C shows how the Gs diameter can be reduced down to 170 pm while still keeping the electrode polarization not larger than 1 mV. On the other hand, with an OECT, it is necessary to use a G of 4 cm diameter to minimize the electrode polarization at the same level as the pOECT, requiring a sensing electrode area 55-thousand times larger. Thus, the pOECT allows for the use of smaller sensing interfaces, a significant advantage over the OECT in terms of device miniaturization, while increasing device performance and ensuring accuracy.

[0183] The pOECT demonstrates significant advantages over the OECT also in terms of stability. Since the Gs is not polarized, the pOECT output response remains constant upon consecutive biasing cycles. In contrast, the OECT shows deviations with a continuous drift of the transfer curve (Fig. 4D). Figure 4E shows the OECT output during a second set of biasing cycles. The second set of transfer curves differs from the first one due to the leakage current (IGS), which permanently alters the electrochemical potential of the gate (+97 mV, Fig. 13). Fig. 4F, is a plot the OECT and pOECT IDS at constant operating voltages during the entire duration of these biasing experiments, evidencing the unstable and non-reproducible OECT response compared to the remarkable stability of the pOECT channel current. In addition to these performance improvements, the pOECT demonstrates exceptional modularity, enabling the integration of virtually any type of potentiometric sensing interface as Gs, even materials with extremely high impedance. This capability surpasses that of traditional OECTs, which cannot accommodate high-impedance materials as the G. One extreme example of such a potentiometric sensor is the commercially available pH-meter glass electrode. The glass membrane of pH-meter glass electrodes, responsible for the sensing mechanism, has a resistance typically between 50-500 MQ56, which would drop the entire VGS on the glass membrane itself if it was used as the G of the OECT. Moreover, since the pH-meter is not designed to work with current or voltage applied, the highly sensitive and delicate glass membrane would be damaged by the flow of IGS, and the thermodynamic equilibrium of the H+ions bound on the membrane would be disrupted. Fig. 4F illustrates the large current change of the pH-pOECT to a pH increase from 4 to 10, with an NR reaching a maximum of 200 (Fig. 4F, inset) along with an output signal spanning three orders of magnitude (nA to pA).

[0184] These ion and pH sensing experiments prove that the pOECT combines the accuracy of the 2-electrode setup with the amplification capability of the OECT, all without a conventional RE. Accuracy is the most important trait of all sensors, which is guaranteed for potentiometric OECT sensors through this configuration. Of note, the lowest electrochemical potential change upon ion binding to an ISE is about ±10 mV. This is also the lowest value that has been recorded upon protein or nucleic acid binding to the gate of an OECT.25,53-55This value translates into an LoD in the micromolar range for ion sensors, and the physical reason that limits the LoD of ISEs has been discussed by A. J. Bard et al57, and an LoD in the attomolar range for protein sensors. An effective strategy to further improve the LoD for OECT-based potentiometric sensors is to optimize the chemistry of the sensing interface for amplifying the input signal, i.e., surface potential change25. Moreover, the Nernstian or Super-Nernstian sensitivity (59 mV / dec or > 59 mV / dec, respectively, for a monovalent ion) is an intrinsic property of the ion-sensitive material and cannot be improved by either the OECT or the pOECT, as often improperly claimed in studies reporting OECT-based ion sensors. In fact, as shown for the pOECT, the electrochemical potential modulation of the Gs does not exceed the expected Nernstian response (Fig. 4B-right). However, only with the pOECT, it is possible to retain any Nernstian or Super-Nernstian sensitivity of the sensing material, which is converted into an amplified current output signal. Given that in most OECT-based sensing applications, polarizable electrodes are used as the G, there are inaccuracies in these measurements arising from G polarization. Importantly, without a GG, the changes observed in the transfer characteristics, due to a parasitic polarization, can be falsely attributed to a biorecognition event.

[0185] Choosing the OMIECfor the pOECT sensor

[0186] When developing a pOECT-based sensor, selecting the right OMIEC (n-type or p-type) and device operation (enhancement or depletion mode) is important to achieve the highest signal amplification. The OMIEC type should be selected based on the direction of the electrochemical potential shift of the sensing surface upon its interactions with the target. For example, a positive electrochemical potential shift of the ISE-based Gs due to Na+binding (AOCP, Fig. 3C) is translated as a shift of the transfer curve towards the negative VSG direction, without any distortion (Fig. 3B). The magnitude of this transfer curve shift corresponds precisely to the AOCP of the Gs (AOCP = AVSG), as shown in Fig. 5A-top. In fact, when the transfer curve recorded at 0 M Na+is used as a reference point and horizontally shift all the other curves of the sensing experiment by a VSG value corresponding to their own AOCP with respect to the 0 M condition (AOCPX= OCP^Na+^x— OCPNa+o M), all the curves collapse onto a single plot (Fig. 5A-bottom). If the same Na+-selective Gs is used, this time with an n-type channel, the transfer curve still shifts towards negative VSG (Fig. 5B). The electrochemical potential distribution of the pOECT terminals is consistent with those reported in Fig. 3C. An increase in Na+concentrations leads to increased channel potentials at which the n-type material becomes more de-doped at the given VSG, reducing the IDS- The pure electrochemical potential contribution to the sensing signal is shown in the inset of Fig. 5B. Fig. 5C summarizes and compares the sensing-induced changes in the transfer curves of the two types of devices (n- and p-type) operated with the same Na+- selective Gs. Regardless of the OMIEC type employed in the pOECT, the transfer curve shifts towards negative VSG (positive VSG) for positive AOCP (negative AOCP) of the Gs. For a p-type channel, positive AOCP causes a current increase, leading to an NR that can easily overcome 1 , thus providing high amplification. For an n-type channel, instead, this type of sensing causes a current decrease, leading to low amplification, i.e., the NR values are limited between 0 and 1 (Fig. 5d). For the detection of the same target (Na+), with the same pOECT configuration, and the same pOECT working mode (enhancement), the maximum NR is 35 for the p-type (Fig. 3F), while it cannot exceed 1 for the n-type channel (Fig. 5D).

[0187] If this “low performing” n-type OMIEC is used for the detection of anions, instead of cations, the device performs as effectively as the p-type Na+-IS-pOECT (Fig. 5E-left). An increase in Cl" concentration turns ON the pOECT, and thus, the NR values are no longer constrained between 0 and 1 and are similar to those achieved with the p-type Na+sensors for the same range of concentrations and at the same nA working regime (Fig. 5E-right). The similarity in NR values follows the Nernst equation; the slope of the electrochemical potential shift of the Gs is the same but opposite in direction, since Na+and Cl" have the same charge but opposite signs.

[0188] This comparison of two devices with similar transconductance efficiencies shows that detecting increasing concentrations of cations is better accomplished with p-type OMIECs, while detecting increasing concentrations of anions is more effective with n-type OMIECs, since, in both cases, we have a progressive turn-ON behavior of the OMIEC, main requirement for maximizing the NR. This conclusion extends beyond ion detection and can be applied to any biosensing event where a target-recognition unit binding induces a potentiometric change. It is then important to know in advance the polarity of the electrochemical potential shift of the sensing electrode in order to couple it with an OMIEC that can be doped as a result of the potential shift.

[0189] A popular channel material for OECTs is poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), which is used in p-type depletion-mode devices25,58. The main disadvantage of PEDOT:PSS based OECT sensors is that a depletion-mode device can only be turned OFF, and hence, the maximum NR that can be achieved is 1. Fig. 5F shows how this limit can be overcome in the pOECT configuration where the Na+-ISE based Gs increases the electrochemical potential of the channel with an increase in Na+concentration, leading to a doped film. In this case, the maximum NR for the highest cation concentration is 4, which is not as high as the enhancement-mode p-type pOECT but is still above the limit. The NR is lower because the highest sensitive region of this transistor is close to the OFF state of PEDOT:PSS. The intrinsic doping provided by the PSS" matrix does not allow complete de-doping without reaching an electrochemically unstable region that damages the polymer.

[0190] Complementary channel pOECT sensor for exclusive turn- ON operation

[0191] When monitoring parameters such as pH or other ions, work is usually performed in an environment with a constant concentration of the target (set point), equivalent to, for example, the physiological concentration in the healthy state. When using sensors that can perform continuous monitoring, the interest lies in tracking positive or negative fluctuations of the target. Since positive or negative concentration changes can cause an increase or decrease of the electrochemical potential of the Gs, the pOECT channel will either turn ON or OFF, depending on the OMIEC type employed, p or n. As demonstrated above, the pOECT will be more sensitive to fluctuations that turn ON the OMIEC and less sensitive to fluctuations that turn it OFF. To achieve the highest sensitivity for fluctuations in both directions, the design can combine an n-type and a p-type channel, both gated by the same Gs.

[0192] The conventional device type that combines n-type and p-type channels is the complementary amplifier59-61. Despite the good sensor performance of OECT-based complementary amplifiers, this configuration requires direct gating using the sensing electrode, which, as discussed earlier, has adverse effects on the sensor’s thermodynamic behavior. Moreover, given that the two OMIECs are in series and share the same drain voltage (VDD), the relative geometry and thickness of the OECT channels must be optimized so that the current output of both channels is similar in absolute value. This usually means a small channel for the p-type and an interdigitated and thick channel for the n-type device. Another constraint is that since the two materials are gated using the same voltage, their threshold voltage should be matched to avoid overlapping ON states of the OMIECs. Furthermore, the channels should share common gate voltages for the OFF state, conditions that are challenging to achieve using the current library of OMIECs62.

[0193] The pOECT configuration can overcome these challenges and monitor all the variations of a target concentration by combining p-type and n-type channels patterned on one substrate operated using the same Gs and GG (Fig. 6A). In contrast to a complementary amplifier, in the complementary pOECT, all applied voltages (VDS and VSG for n-type and p-type) are independent. This allows the system to obtain similar IDS values in the ON states if needed (in our case, VDSI = 100 mV and VDS2 = 10 mV) without optimizing the device geometry. In addition, two independent gating voltages can be applied (VSGI and VSG2), removing any threshold voltage matching requirement, allowing to work efficiently in an OFF state for both channels at a specific electrochemical potential of the Gs (set point). In Fig. 6A, depicts the complementary pOECT with a pH-sensitive PANI electrode as the Gs. Ag / AgCl electrodes were used as floating gates that establish communication between the sensing chamber and the channels’ chamber. The two Ag / AgCl electrodes play the role of a floating gate and not of conventional reference electrodes. The integration of the floating gates here is equivalent to that of a salt bridge or a microfluidic channel. The same floating gate configuration can also be achieved by using two microfabricated planar Au electrodes instead of Ag / AgCl41. Fig. 6B shows that it is possible to keep both channels in an OFF state when the pH is at the set point (7 in this example) by selecting the appropriate VSGI and VSG2- When the pH increases, the electrochemical potential of the Gs decreases (Fig. 6B-inset), and the n-type channel turns ON, while the p-type channel, which was already OFF, remains in an OFF state. When the pH decreases, the positive electrochemical shift of the Gs drags the pOECT channels to positive potentials, and only the p-type device turns ON. Working in conditions where both channels are OFF when the system is at the set point allows the system to maximize the response to pH shifts in both directions, as shown in Fig. 6C. The complementary pOECT can reach an average NR of 44 for a pH shift of 3 units in both directions, while the classical 2-electrode setup can only reach 1.6. Fig. 6B-inset evidences the retention of the Nernstian behavior of the PANI pH-sensitive electrode (Gs) in this complementary setup (59 mV / pH). The sensor performance of each pOECT can be further improved by changing the device geometry (FIG. 6D).

[0194] Multichannel pOECT for cell monitoring at constant OMIEC thermodynamic conditions The devices demonstrated above used the pOECT configuration to amplify the electrochemical potential changes that an external electrode (Gs) undergoes during sensing. While the pOECT is not designed for faradaic or impedimetric sensing at the gate contact due to the constant OCP condition of this electrode, it is possible to use this configuration to perform such sensing directly at the channel / electrolyte interface. Reported here is an example of OECT- based impedimetric sensing, where the pOECT provides a fundamental advantage over the traditional OECT. OECTs can measure the transepithelial / transendothelial electrical resistance of barrier-forming cells with high sensitivity, which is a widely accepted electrical parameter to assess tissue health, generating a label-free platform for screening drug toxicity63-66. In this application, the cells are grown on top of the channel; hence, they partially block the ion flow toward the OMIEC, which is necessary for its doping / de-doping. The permeability of the tissue over time is thus indirectly assessed through IDS, by monitoring how easily the G can inject ions inside the OMIEC, passing across the cell layer. However, for accurate measurements of the tissue permeability, the channel must be biased at the same electrochemical potential, which means that it is at constant thermodynamic conditions and under a constant driving force for ion injection during the entire experiment. This requirement can be achieved only if the electrochemical potential of the G remains constant across all the measurements.

[0195] The straightforward solution to bias the OECT channel always at the same and reproducible electrochemical potential is to use a non-polarizable electrode such as the common Ag / AgCl pellet as the G. However, the release of Ag+from the pellet into the cell media causes a toxic environment for the cells67(Figs. 14 and 15). A Pt electrode, which has a low polarizability, could be a nontoxic alternative (data not shown). However, since this electrode is directly exposed to the cell media, and cell media composition changes throughout the lifetime of the tissue, it cannot guarantee a constant electrochemical potential (Figs. 16A and 16B). Isolating the G from the cell media and placing it in its own electrolyte with a porous glass frit could be a solution; hence, a regular Ag / AgCl RE or a Pt wire in a separate compartment could be used. However, the glass frit can be easily contaminated with species in the complex cell media,68leading to a variation of the junction potential and a non-constant G biasing. Moreover, the Ag+ions of the Ag / AgCl RE can still leak in the cell media through the frit - even though at a lower rate - being toxic to the cells14,68’69(Figs. 14 and 15). A leakless Ag / AgCl RE seems to be the only and best option as the G that features all the necessary requirements (constant electrochemical potential and no toxic compound leakage, Figs. 14 and 15). However, because of the high impedance of the solid electrolyte that prevents leakage or internal contamination, leakless Ag / AgCl RE cannot be used directly as a G in a regular OECT.

[0196] Experiments were conducted to identify the optimal GS electrode for cell monitoring with pOECT.

[0197] In experiments involving cells, increasing the number of devices is crucial. Probing the behavior of the cells on a single micrometric spot might lead to over- or under-estimation of the system behavior due to the low statistical relevancy of sampling a surface containing a few tens of cells, compared to the several thousand present in the whole cell culture. Instead of handling multiple individual devices, it is more convenient to work with a single chip containing multiple sensors (n) and operate these devices in parallel. Unfortunately, with a conventional OECT, increasing the number of devices progressively increases the IGS, which leads to strong G polarization and low reproducibility.

[0198] The pOECT allows for the integration of high-impedance gate electrodes, including the leakless RE (Fig. 18) as well as the fabrication of multiple micronscale channels patterned on a small area (6 mm2) (Fig. 19A). FIG. 20A shows normalized response of cut-off frequency during time (blue line) and corresponding derivative (orange line). FIG. 20B shows the peak position of the derivative from each pOECT channel is used to quantify the difference in diffusion time among the four pOECTs. FIG. 20C shows that pOECT signals are horizontally shifted off the corresponding difference in diffusion time with respect to the pOECT 3, which is chosen as a reference.

[0199] Fig.7A shows the device design with multiple channels gated by the same GG (CEa), and their doping potential is defined with respect to a common Gs (REa, leakless Ag / AgCl). The number of transistor channels is virtually unlimited, as long as the GG is capacitive enough not to be polarized at too high or low potentials (due to the IG). Considering the usually small size of the pOECT channels, a regular CE, for example, at Pt wire, easily serves the purpose.

[0200] Figs. 7B and 19A show the healthy layer of epithelial cells covering the pOECT channels (PEDOT:PSS). When a VSG pulse is applied, the equilibrium IDS is reached slowly as the cells present a tight barrier against the injection of ions towards the channel. When trypsin is added to the media, the proteins responsible for forming the barrier tissue and those responsible for the adhesion to the substrate are attacked (Fig. 19B). This leads to the progressive detachment of the cells and, hence, easier movement of ions that penetrate the channel in a much shorter time. The response time of the pOECT channels can be modeled with an exponential equation (JDS= IDs min + ^DS ’exP (—(t—IO) / TOFF) from which a time constant (TOFF) can be extracted and the cut-off frequency of the device can be calculated (fco= 1 / 27TTOFF). In Fig.7C, top, shows the fcochange for four cell-interfacing pOECT channels belonging to the same culture, as trypsin is added to the media. All channels show a sigmoidal current change with an initial latency, a fast frequency increase due to progressive cell detachment, and a final saturation when the cells are completely detached. The variability between the speeds of each channel at the beginning of the growth phase is due to the different diffusion rates of trypsin toward the cells, which is related to their differentiation. After compensating for this variable delay by horizontally shifting the curves, the average fcochange is obtained as shown in Fig.7C-bottom.

[0201] The multiple pOECT setup coupled with a leakless RE allows to perform a reliable measurement with multiple simultaneous replicates on different locations of the cell culture, providing data of statistical relevance. This sensor design can be used to monitor the behavior of other systems such as supported lipid bilayers70and study kinetic interactions between biological interfaces and different drugs for long periods of time in safe conditions. For a more advanced analysis, each pOECT can be independently gated (still using a common Gs and GG) to perform different techniques (impedimetric and / or faradaic) and monitor different parameters simultaneously. For example, other than the cut-off frequency, some of the pOECT channels can be functionalized with specific enzymes for the faradaic detection of metabolites71-73, whose quantification through OECT / pOECT may require a different bias than the one used for the cutoff frequency.

[0202] Supplementary Discussion 1: OECT / pOECT electrochemical potential measurement The electrochemical potential of the OECT / pOECT terminals is measured during its operation by using n independent potentiostat channels, where each OECT / pOECT component (S, D, G, GS, GG) is the WEnsharing the same RE. As an example, here in FIG. 2Dshows the electrochemical potentials of an n-type enhancement-mode pOECT device components with Ag / AgCl as GS and a Pt coil as GG (see pOECT transfer curve in Fig. 2A-left). The transfer curve is recorded at a constant VDS = + 0.3 V, which can be seen in the graph since the D (red line) is constantly 300 mV above the S (black line). At the same time, the VSG is linearly scanned from +0.2 V to - 0.5 V, which can be seen in the graph as the distance from the S and the GS (blue line). For a full picture, 2D also reports the electrochemical potential of the GG (yellow line). During the transfer curve, the GG is slightly positively polarized when the channel is pushed to the most doping potentials.

[0203] Supplementary Discussion 2: Gate electrode polarization

[0204] Fig. 2E-left reports the transfer curves of an n-type OECT and pOECT when the G or GS is an Au electrode of 5 mm diameter. For the OECT, it is necessary to apply a much higher gating voltage to reach the same maximum IDS of the pOECT. This is due to the high G polarization caused by an increase of OMIEC capacitance / doping when pushed to negative electrochemical potentials. Fig. 2E-right shows how it is necessary to push the VGS up to +1.26 V to reach the same level of OMIEC doping as with the pOECT, which then results in the same maximum IDS (Fig. 2E-left). This progressive and non-linear polarization of the G leads also to a significant distortion of the transfer curve shape.

[0205] Supplementary Discussion 3: Currents involved in the pOECT gating system and effect on the sensing electrode polarization

[0206] Fig. 8A shows the behavior of an ideal non-polarizable electrode, which is an electrode able to keep a constant and stable potential regardless of the current flowing through it. This is a feature typical of a RE such as Ag / AgCl. Fig. 8B shows an ideal polarizable electrode whose potential can be easily modulated even through minuscule current variations. Fig. 8C represents an intermediate behavior, which is a characteristic of most of the electrodes, including those employed in biosensing applications. In the case of sensing based on variation of the electrochemical potential of the sensing electrode, it is desirable work in conditions that resemble Fig. 8As o as not to lose the analytical information represented by the electrode potential. To render an electrode presented in Fig. 8C more like the one in 8A, one can either (i) increase the size / capacitance of the electrode or (ii) decrease the current flowing through the electrode. Increasing the size of the sensing electrode is not a practical solution for several reasons. It is not cost-efficient and does not allow miniaturization. In the case of OECTs, where the capacitance of the OMIEC is significantly higher than the materials employed in electrolytegated organic field effect transistors (EGOFETs), the electrode size required to achieve nonpolarizability is too big for practical applications. For this reason, reducing the current passing through the sensing electrode represents a better alternative, which maintains the analytical information of the electrode potential while using an electrode of reasonable size (mm or pm range).

[0207] The main purpose of the pOECT is the removal of the OMIEC gating current from the sensing electrode (Gs), which is instead provided by an auxiliary electrode (GG). Theoretically, through the RE cable at which the Gs is connected, no current should pass so that the potential of the RE is not disturbed and any polarization is prevented. For this reason, the impedance of the RE line is usually in the range of 1 to 100 TQ in most of the potentiostats. However, since this impedance is high but not infinite, a small current flows through the RE. Moreover, a minimal current is required to pass through the connection, otherwise it would not be possible to measure the potential of the WE with respect to the RE, and apply the required VSG, because the RE would result as completely disconnected from the circuit. As a demonstration of this feature of the potentiostat circuit, the schematic of a classical 3-electrode setup operated with a potentiostat in FIG. 8D. The presence of the electrical currents involved in the circuit is highlighted with grey arrows. One of these currents is the one flowing between the WE and the CE (IWE), which is normally reported in a cyclic voltammetry (CV) or linear scan voltammetry (LSV) plot. The other one is the current flowing through the RE (IRE). Fig. 8E shows these two characteristic currents in the case of the oxidation scan of an Au <l> = 5 mm WE in a lx PBS solution of 10 mM ferrocyanide. Due to the high impedance of the circuitry associated with the RE, the IRE is 9 orders of magnitude lower than IWE- However, the shape of the IRE follows the same trend as the IWE, demonstrating that any increase in the IWE will also be reflected in the IRE but on a much lower order of magnitude. The magnitude of the IRE is proportional to the total current flowing through the circuit, which can be estimated from the IWE. This total current depends on the properties of the electrochemical cell, such as WE size and concentration of redox active components. In order to demonstrate the proportionality of the IRE with the IWE, we report an analogous measurement performed with WEs of different sizes in Fig. 8F. A WE of larger size (larger area) provides a higher IWE (Fig. 8F-left, bottom) whose intensity follows the proportionality with the electrode size (Fig. 8F-left, top). The same current trend is also reflected on the IRE (Fig. 8F-right, bottom) with an analogous proportionality (Fig. 8F-right, top) but with an intensity 9 orders of magnitude lower with respect to the IWE; the relationship for this specific case between the IRE and IWE is about 2 PA(IRE) / 1 mA(IwE), R2= 0.998. This experiment demonstrates that despite the negligible IRE, if the size of the WE increases or the electrochemical cell is miniaturized, the non-polarizability of the RE is sacrificed. As such, the RE could become polarized, reducing the reliability of the analytical method, a condition, however, which rarely occurs.

[0208] For the pOECT, the current flowing through the GG ( / GG), which is the current that effectively dopes and dedopes the OMIEC, can be considered as the IWE, and the current flowing through the Gs ( / Gs)asthe IRE, due to the analogous cable connections to the 3-electrode setup, as shown in Fig.9A. It is important not to confuse the IGS(also called / G) of a classical OECT setup and the IGsof the pOECT setup, where ideally IG= IGs+ IGcsince RE and CE connections are shorted on the same electrode. To increase the current flowing through the gating system, analogous to what is shown in Fig. 8F, thickness of the OMIEC film in the channel can be increased . FIG. 9B shows transfer curves for two pOECTs with either a thick or thin layer of OMIEC channel. The thick pOECT shows a slightly lower IDS (for a constant scan rate) due to its slower behavior. If the amount of OMIEC that has to be doped at the applied voltage (VSG) increases (Thin Thick), also the doping current (IGG) has to increase, as shown in FIG. 9C-left. Similar to what we saw in the LSV curve (Fig. 9E), the IGis higher, but remaining always 6 or 7 orders of magnitude lower than the IGc(FIG.9C-right). This experiment demonstrates that, even if the pOECT guarantees no-polarization for the sensing electrode, this is not true in absolute terms. In fact, if the size of the Gs is extremely small (nm or tens of pm), the Gs material is poorly conductive, or the amount of OMIEC in the channel is high, the Gs could be polarized even with few tens of pA of current. However, for the most common biosensing applications, where the channels have pm size, and the sensing electrode sizes are in the mm range and are made of noble metals, metal oxides, OMIECs, or carbon-based materials, the pOECT setup allows prevention of any polarization of the sensing electrode. Moreover, if Gs is required to be in pm dimensions, such as for brain activity monitoring5, the electrodes can be functionalized with an OMIEC layer like PEDOT:PSS6to increase their capacitance and reduce the polarizability.

[0209] The Gs can be polarized by the IRE / / GS5apOECT in which the Gs is a classical pH-meter glass electrode was constructed (FIG. 10A). The experiment reported here is the same as the one presented in Fig. 4G at pH = 10. One of the cases that could lead to a Gs polarization even with the small pA current passing through the RE (IRE) is the poor conductivity / high resistance of the Gs. In the case of a classical pH-meter, the pH sensitive component is a glass membrane whose resistance is typically 50-500 MO. This would normally lead to an extremely high gating voltage drop across the glass membrane if used as a direct G in a classical OECT setup (Vdrop= ^membrane X IG= 50 ■ 106X 100 ■ 10-6= 5 kV). However, since in a pOECT the current flowing through the Gs is several orders of magnitude lower, the voltage drop reaches an acceptable value. Considering the glass membrane resistance as 50 MQ and an IRE of 20 pA, the Vdrop is equal to 1 mV (Vdrop= Rmembrane X IG= 50 ■ 106X 20 ■ 10-12= 1 mV). This value is in agreement with what is measured by monitoring the electrochemical potential of the pOECT components (FIG. 10B -left) where the potential of the Gs is oscillated / polarized around an average value with an amplitude of ± 1 mV (FIG. lOB-left, top). Despite the small voltage drop, this polarization is not harmful. The Vdrop measured for the Gs is one order of magnitude lower than the usually applied amplitude modulation in electrochemical impedance spectroscopy (EIS), which is considered a non-destructive technique (if the modulation is applied vs. OCP hence, analogous conditions as in y Fig. 10B for the Gs). We can further verify that this small polarization has no effect on the electrochemical state of our electrode if we compare the electrochemical potential of the Gs before the device operation (-180.2 ± 3 mV vs. Ag / AgCl) and during the device operation (-180.5 ± 6 mV vs. Ag / AgCl). Moreover, the drift of the Gs potential during the pOECT operation is about -2 pV / s, which is widely inside the usual drift range of a resting RE7.

[0210] Studies compared the electrochemical performance of microfabricated OECT and pOECT of similar size. The devices have identical channel (100x10 pm) and sensing interface (500x500 pm, G and Gs) dimensions. The only difference for the pOECT is the additional electrode (GG) of the same size as the sensing interface. The overall substrate size and active area are exactly the same in both devices. Similar to the devices with vertical gating electrodes, the pOECT allows a much larger IDS modulation (data not shown). In the OECT, the G polarizes during the VGS application, even if the electrode has a surface area 250 times higher than the channel (data not shown). This polarization is instead efficiently prevented by the pOECT (data not shown), where Gs remains unperturbed at its stable equilibrium electrochemical potential, and the auxiliary electrode (GG) plays the role of doping the OMIEC, being consequently polarized. In addition to its stable and high performance, the pOECT is as miniaturized as the conventional OECT, as exemplified here.

[0211] Supplementary Discussion 4: pOECT miniaturization capabilities and comparison with OECT dimensions

[0212] As demonstrated in Fig. 11A-11B and Fig. 4A-4B, an OECT with a comparable electrode size as a pOECT (OMIEC and sensing interface area) has an output signal with severe interference from gate polarization. The polarization renders the OECT in this geometry either a poor sensor (due to low modulation) or a sensor with inaccurate output (as the polarization affects the channel current). To achieve the same accuracy as the pOECT, we need to operate the OECT with a very large G. However, the necessary size to make this electrode non-polarizable exceeds the acceptable dimensions for a portable device. A partial demonstration of this is reported in Fig. 2C-right and Fig. 2D. Despite the large capacitance of the GG granted by a 23 cm long Pt wire, the electrode is still polarized (+65 mV) by the leakage current necessary to dope the channel. Hence, if this electrode is used as G in an OECT, it will still be polarized.

[0213] With organic semiconductors employed for EGOFETs, a millimeter gate size is sufficient to prevent any polarization and provide reliable performance, as demonstrated in previous works4-6. With OMIECs, the doping mechanism involves the entire bulk of the transistor channel, requiring a larger number of ionic charges and, in turn, a larger IGS. The result is that for OMIEC-based transistors, even the large area gates7of EGOFETs biosensors are not large enough to prevent the polarization of the G.

[0214] For example, Fig. 12A shows the transfer curves of a pOECT in which the GS is a very small Au electrode (170 pm in diameter) and an OECT in which the G is a Au electrode, the size of which is progressively increased from a diameter of 170 pm to 1 cm. The pOECT shows a full IDS modulation up to 1.5 mA, with a minimal GS polarization of 1 mV as reported in Fig. 12B- i. Note that, as already demonstrated in Fig. 2C, the size optimization of the GG is not crucial for determining the pOECT performance. Hence, in this experiment, we used an Au electrode of 1.6 mm diameter as GG. When the same small GS is used as G in an OECT (Fig. 12A, right), the IDS modulation is absent because the applied VGS drops almost entirely on the G itself (684 mV polarization), as demonstrated in Fig. 12B-ii. If we want to bring the IDS modulation of the OECT to the same level as the pOECT, we need to increase the size of the G. By progressively increasing the G diameter from 170 pm up to 1 cm, we observe a gradual increase in IDS modulation, Fig. 12A-right. The increase in G size reduces the G polarization gradually, as shown in Fig. 12B-ii-iv. However, even with a G diameter of 1 cm, which corresponds to an area 4200 times larger than the GS of the pOECT, the G is still polarized (97 mV), Fig. 12B-iv, leading to a lower IDS modulation compared to that of a pOECT operated with a much smaller sensing interface.

[0215] If the measured G polarization is correlated with the G diameter or area, the trend reported in Fig. 4C is obtained. An exponential decrease of the G polarization with size was observed, but the G polarization was comparable to the GS polarization only when the G reaches a diameter of 4 cm. This demonstrates that an OECT with an equivalent accuracy as the pOECT (minimal sensing interface polarization) requires the G to be 55-thousand times larger. Even when the total gating area of the pOECT (i.e., the size of the GG plus that of the GS) is considered, the pOECT gating system area is 625 times smaller than that of the OECT (AG / (AGS+AGG)).

[0216] In conclusion an OECT with comparable performance as a pOECT requires a gate electrode of unpractical size. Hence, the pOECT, despite the integration of a second electrode, remains smaller than an OECT designed for potentiometric sensing applications.

[0217] Supplementary Discussion 5: Nernstian and Super-Nernstian response of pOECT and OECT

[0218] An OECT / pOECT cannot improve the response of the sensing interface from Nernstian or sub- Nernstian to super-Nernstian but just convert the voltage response to an amplified current response or another form of OECT-related voltage. The Nernstian sensitivity is defined, for example, as ±59 mV / dec for an ISE for monovalent ions or a pH sensor. What is frequently mistakenly considered correct is that any output voltage of an OECT (or more complex configurations), such as VDS, gm peak position or more, can be attributed to the Nernstian voltage. The Nernst equation describes the electrochemical potential of the sensing interface in an OCP condition, which is in the absence of current and with the sensing interface at its thermodynamic equilibrium. Most of the works claiming OECTs with Nernstian or Super- Nernstian sensitivities do not report any electrochemical potential measurement of the sensing interface during the OECT operation that can allow to verify if this claim is correct. For this reason, only the results obtained by Gualandi et al., who performed a systematic analysis and reported all the necessary data for different examples of OECTs driven by potentiometric signals are discussed here. In their first OECT-based pH sensor8, they employ poly(3,4- e thy lenedioxy thiophene: bromothymol blue (PEDOT:BTB) as G and monitor as output signal the position of the OECT gm peak as a function of pH. The resulting shift of the gm peak is -93 mV / pH, which is claimed to be a Super-Nernstian response. This claim is theoretically incorrect since the reported voltage is not an electrochemical potential of PEDOT:BTB during its operation in the OECT. In subsequent work,9the authors show how PEDOT:BTB in a 2- electrode OCP measurement exhibits a sensitivity of -43 mV / pH, below the super-Nernstian response. In this work, they also monitor the electrochemical potential of this material when used in the OECT channel during the pH sensing, revealing a sub-Nernstian sensitivity of 5 mV / pH, which, however, still allows to have an appreciable calibration curve thanks to the amplification provided by the OECT. The loss of 38 mV / pH when the pH sensitive material is under the application of a voltage / current that disturbs the thermodynamic equilibrium established by the Nernst equation is analogous to what is shown in Fig. 4B. This conclusion does not mean that a Super-Nernstian OECT is not possible to achieve. Gualandi et al. developed another OECT- based pH sensor using PEDOT:PSS / iridium oxide (IrOx) nanoparticles10. This material already showed a Super- Nernstian response of -81 mV / pH when employed in a classical 2-electrodes setup for OCP measurement. The electrochemical potential of the material, when used in an OECT, showed a sensitivity of -64 mV / pH. Only in this case the OECT also have a Super- Nernstian response because the intrinsic material behavior is partially retained in the OECT configuration. The partial loss of sensitivity of 17 mV / pH is probably again due to the voltage applied to the OECT components that do not allow the material to settle at the thermodynamic equilibrium.

[0219] Supplementary Discussion 6: details on the thermodynamic advantages of pOECT over OECT

[0220] To the best of our knowledge, the first example of OECT sensor designed to respond to variations of the electrochemical potential of the G in a Nernstian manner (hence thermodynamic response), is reported by Gualandi et al.11Their OECTs represent the closest analogous to classical potentiometric sensing in a 2-electrode setup, and for this reason, their work will be taken as a reference point. The authors showed how an Ag / AgCl gate electrode can modulate the IDS of an OECT depending on the concentration of Cl" in the electrolyte in contact with the AgCl wire, following the Nernst equation. The same group developed other OECT-based potentiometric sensors, for the detection of other ions12and pH8-10, with different OECT architectures, such as dual terminal and Wrighton-configuration. In these works, the authors list or indirectly show a series of limitations that the pOECT disclosed herein can overcome. For example, the use of redox active species as a sensing element11narrows down the range of possible voltages that can be applied to the OECT due to the parasitic faradaic reactions of the sensing material, leading to its oxidation or reduction. This problem is avoided in the pOECT because the sensing interface is maintained in an OCP configuration.

[0221] The authors also introduced the concept of electrochemical gating13, where the conductivity of a channel, electrically shorted with the gate12, or inside which the gating material is dispersed11, can be modulated through spontaneous electrochemical reactions without a gate voltage. This configuration simplifies the OECT design because it removes the application of the gate voltage12or completely removes the gate electrode11but, at the same time, is equivalent to operating the OECT at VGS = 0 V. This operating condition may not represent the voltage at which the OECT guarantees the highest sensor response, as shown for example in Fig. 3F. Moreover, while the sensitivity of an ISE depends only on the charge of the detected ion (according to the Nernst equation), in an electrochemically gated OECT, the behavior is different. Since the electrochemical potential of the S is equal to the electrochemical potential of the G (ES = EG) at any time, the channel is biased around different electrochemical potentials (doping states), depending on the intercept value of the Nernst equation that describes the specific potentiometric interface, which then corresponds to different sensitivity regimes for the OECT. This leads to anomalous behavior compared to the Nernstian one, where despite the same charge of the ion, different ionic species (such as CT, Br", I ) show different sensitivity due to different k values. With the pOECT configuration, it is still possible to apply any desired VGS, not only 0 V. The voltage is provided by an independent CE (GG) and the channel can always be at the voltages where the pOECT shows the highest response, regardless of the k that describes the thermodynamic equilibrium of the GS. This allows us to operate the device at an electrochemical potential close to the onset potential of the OMIEC12at the maximum transconductance efficiency14.

[0222] The authors also point out that the electrochemical gating can work only if the gate or the gating material has a much higher capacitance than the channel, to bring the Fermi level of the channel to the same level of the G, without affecting the Fermi level of the G itself. This requirement can be reached only with a large size or a non-polarizable G. Also, in this case, with the pOECT, the modulation of the Fermi level of the channel is provided by an independent electrode (GG), whose characteristics do not affect the pOECT performance, removing the requirement of non-polarizability of the G. They also show that a voltage applied to the potentiometric electrode hampers the spontaneous redox processes required to reach the thermodynamic equilibrium and stable potential9.

[0223] Since intrinsically-undoped OMIECs have naturally low OFF currents, increasing the size of the transistor channel increases the maximum ON current, leading to a higher NR for the same electrochemical potential shift of the Gs. Fig. 6D shows the NR of a complementary pOECT where the channel size increased from W-L=l 00- 10 pm to 980 mm- 5pm, leading to channels capable of reaching mA current ranges. In this case, the pH shifts tested are only one unit (smaller than the ones reported in Fig. 6B), leading to a p-type NR of 51 and an n-type NR of 594. With a larger channel, the performance of the n-type device is significantly higher since it corresponds to one order of magnitude more for an input signal three times smaller (3 orders of magnitude smaller if we consider the ion concentration and not the logarithm) than the experiment reported in Fig. 6B. The increase in performance of the p-type device is still remarkable because it retains a similar NR value but for a pH shift three times smaller. However, the n-type device performed better because, regardless of the channel size, the material can keep its OFF current in the range of units of nA or even hundreds of pA. Instead, the p-type OMIEC has a higher OFF current when the channel is larger (tens of nA vs. tens of pA), due to the spontaneous oxygen doping19.

[0224] In Fig. 16 A, the electrochemical potential of a Pt wire in fresh and conditioned cell media is reported, showing a progressive negative shift as the media ages. The potential of the Pt changes with a shift of 33 mV after 3 days of incubation. The consequence of this shift in the OECT results is reported in Fig. 16B-left, where a Pt wire is used to modulate a p-type enhancement mode OECT when the electrolyte is fresh or old, in the absence of cells. The measurement in the conditioned media has a lower current. If these measurements were made in the presence of the cells, this low current would be mistakenly attributed to a successful formation of the insulating cell layer. However, in reality, it is only related to a reduction of the potential of the G. As reported in Fig. 16B -right, this artifact led to a modulation of the IDS up to 100% for the most sensitive VGS values. These conclusions are valid for any other G material, regardless of its safety for the cells (such as PEDOT:PSS, Ag, Ti, Cr, and Au16). When the polarizable electrodes are exposed to cell media, their electrochemical potential may vary with the composition of the media itself.

[0225] The leakless RE cannot be used as a direct G due to the high resistance of the solid electrolyte membrane (10 kQ), which causes a high voltage drop on the gate itself, hampering the channel modulation. In Fig. 17-left, a transfer curve of a PEDOT:PSS-based OECT, when gated with a porous glass membrane RE and with a leakless RE is reported. The leakless RE provides a much smaller modulation of the IDS, and the reason for this is reported in Fig. 17-right. While the electrochemical potential of the glass RE remains constant during the OECT operation, the leakless RE shows a high polarization, up to 41 mV, that affects the transmission of the gate voltage to the OECT channel.

[0226] In Fig. 18, a pOECT is gated by a leakless RE as GS, a Pt wire is used as GG and is operated in two different electrolytes, a fresh cell media and the cell media that the cells produced after 3 days of culture. In these conditions, the response of the pOECT is identical, regardless of the freshness of the media, also showing that the OMIEC behavior is not significantly affected by the different composition of the electrolyte. With this setup, if the measurement is taken in the presence of cells on top of the channel, any variation of the pOECT response is solely related to the permeability of the cell layer and completely free from any interference from changes in the gate electrode’s properties.

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[0245] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0246] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWe claim:

1. A potentiometric organic electrochemical transistor (pOECT) device for metabolite sensing comprising:(a) one or more source electrodes (SE);(b) one or more drain electrodes (DE);(c) one or more channels (CH) which comprise (a) one or more p-type and / or n-type polymers and optionally, (b) one or more enzymes;(d) one or more sensing gate electrodes (GsE) which comprises (a) one or more n-type polymers and (b) one or more enzymes; and(e) one or more gating gate electrodes (GGE); wherein each SE and DE is electrically connected by a CH and optionally, the IDS is modulated solely by electrochemical potential variations of Gs.

2. The pOECT device of claim 1 further comprising a supporting substrate, wherein the one or more source SE, the one or more DE and / or the one or more GsE and GGE are patterned on the supporting substrate.

3. The pOECT device of claim 1 or 2, wherein the one or more GsE and GGE are patterned on the supporting substrate.

4. The pOECT device of any one of claims 1-3, wherein: (a) the device is planar and the one or more SE, the one or more DE, the GsE and GGE are on the same plane or (b) the device comprises a planar component, wherein the one or more SE, the one or more DE are on the same plane and the GsE and GGE are in a vertical location relative to the plane of the planar component.

5. The pOECT device of any one of claims 2-4, where the supporting substrate is selected from the group consisting of glass, polyethylene terephthalate, polyethylene naphthalene dicarboxylate, polyethylene, polypropylene, polycarbonate, paper, coated paper, resin-coated paper, paper laminates, paperboard, and corrugated board.

6. The pOECT device of any one of claims 1-5, wherein the CH is made from:(a) an n-type polymer selected from the group consisting of p(CeNDI-T), N2300, P(NDI- T2), poly(diketopyrrolopyrrole) (DPP), poly(benzimidazobenzophenanthroline), poly(2,5-di(3,7- dimethyloctyloxy)cyanoterephthalylidene), poly(2,5-di(hexyloxy)cyanoterephthalylidene), poly(5-(3,7-dimethyloctyloxy)-2-methoxy-cyanoterephthalylidene), poly(2,5-di(octyloxy)cyanoterephthalylidene), and poly(5-(2-ethylhexyloxy)-2-methoxy- cy anoterephthalylidene) ;(b) a p-type polymer selected from the group consisting of p(gaC2T2-T), poly(3,4- ethylenedioxythiphene) (PEDOT), poly(hydrooxymethyl 3,4-ethylenedioxythiphene) (PEDOT- OH), polystyrenesulfonate (PSS), F8BT, F8T2, J51, MDMO-PPV, MEH-PPV, PBDB-T, PBDTBO-TPD, PBDT(EH)-TPD, PBDTTT-C-T, PBDTTT-CF, PBTTPD, PBTTT-C14, PCDTBT, PCPDTBT, PDTSTPD, PffBT4T-20D, PffBT4T-C9C13, PFO-DBT, Poly([2,6'-4,8- di(5 -ethylhexylthienyl)benzo [ 1 ,2-b ; 3 ,3 -b ] dithiophene] { 3 -fluoro-2 [(2- ethylhexyl)carbonyl] thieno [3 ,4-b] thiophenediyl } ) , Poly (3 -dodecylthiophene-2 ,5 -diyl) , Poly (3 - hexylthiophene-2, 5-diyl), Poly(3-octylthiophene-2,5-diyl), PSiF-DBT, poly(triaryl amine) (PTAA), PTB7, TQ1; or(c) a combination n-type and p-type polymers.

7. The pOECT device of claim 6, wherein the n-type polymer is p(CeNDI-T), and the p-type polymer is p(g3C2T2-T).

8. The pOECT device of any one of claims 1-7, comprising more than one CH in the form of an array, with n channels, where n is an integer (i.e., two or more pOECTs), from 2 to 10, 50, or 100.

9. The pOECT device of any one of claims 1-8 wherein:(a) the GsE and GGE each has a dimension between about 100 pm2and 250000 pm2, optionally wherein the GsE and GGE each have a dimension of about 500 x500 pm;(b) the length of the CH is between 1 pm and 1000 pm;(c) the width of the CH is between 1 pm and 1000 pm; optionally, wherein the channel size is about 100 x 10 pm;(d) the Gs diameter if from about 150 to about 450 pm;(e) an electrodeposited film of polyaniline (PANI) is uses at the sensing interface Gs and / or(f) the thickness of the CH is between 50 nm and 1 pm.

10. The pOECT device of any one of claims 1-9, wherein the dimension of the electrochemical device is between 1000 and 1000000 pm2.

11. The pOECT device of any one of claims 1-8, wherein: (a) the channel is arranged such that flow of electrons between the source electrode and the drain electrode is controllable by a voltage applied to the gate electrode; and / or (b) wherein during use, the GsE is kept in a separateelectrolyte chamber with a varying pH, while the CH and GGE are in a second chamber with an electrolyte of constant composition; optionally, wherein the pOECT device comprises additionally electrodes used floating gates to connect the two chambers.

12. The pOECT device of any one of claims 1-11, having a configuration as shown in FIG. ID.

13. The pOECT device of any one of claims 1-12, wherein the pOECT has improved stability when compared to a standard OECT, as measured by multiple repetition of the electrical characterization, until a stable response is obtained, when operated with the same electrode as G and GS (Au 3 mm diameter).

14. The pOECT device of any one of claims 1-13, wherein the device is configured as an implantable device or as a wearable.

15. The pOECT device of any one of claims 1-14, wherein the device is configured as a sensor for monitoring a metabolite.

16. The pOECT of any one of claims 1-15, wherein the pOECT combines p- type and n-type CH patterned on one substrate operated using the same GsE and GGE.

17. The pOECT device of any one of claims 1-16, wherein the device is configured as an array of sensors for monitoring multiple metabolites.

18. A method of detection using the pOECT device of any one of claims 1-17, comprising (a) applying a gate potential, and (b) monitoring changes of a source-drain current (IDS) that flows through the CH that connects the SE and the DE.

19. The method of claim 18, wherein the pOECT device detects pH or one or more analytes selected from the group consisting of ions, small molecules, nucleic acids and microorganisms.

20. The method of claims 18 or 19, wherein the IDS increases with increasing concentration of metabolites.

21. The method any one of claim 18-20, two independent gating voltages can be applied (VSGI and VSG2), removing any threshold voltage matching requirement.

22. The method of any one of claims 18-21, wherein the a Pt coil is the GG and Gs is located in a separate chamber connected to the rest of the device via a microfluidic channel..

23. The method of claim 22, wherein, detecting increasing concentrations of cations is accomplished with a p-type organic mixed ionic and electronic conductor (OMIEC) and while detecting increasing concentrations of anions is accomplished with an n-type OMIECs.

24. The pOECT of any one of claims 1-17, further comprising one or reservoirs to contain the electrolyte solution(s).

25. The pOECT of any one of claims 1-17 wherein the electrodes of the pOECT are patterned on a supporting substrate, such as a glass substrate, a silicon substrate, or a plastic substrate, such as a polyimide substrate or a textile.

26. The pOECT of any one of claims 1-17, wherein the SE, DE, are on an separate plane from GsE and GGE.

27. The pOECT of any one of claims 1-17, wherein the SE, DE, GsE and GGE are on the same plane.

28. The pOECT of claim 24, wherein the SE, DE, GsE and GGE are positioned in the same chamber.

28. The pOECT of claim 24, wherein GsE is positioned in a separate chamber from SE, DE and GGE, and the two chambers are connected via a microfluidic channel or a floating gate.

29. The pOECT of any one of claims 1-17 comprising at least one p- type and one n-type channel patterned on one substrate operated using the same GsE and GGE.

30. The pOECT of any one of claims 1-17 or 25-29, further comprising a biorecognition element.

31. The pOECT of claim 30, wherein the biorecognition element is selected from the group consisting of an enzyme, an aptamer, a nucleic acid, an antibody or a fragment thereof, or a nanobody.

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