Systems and methods for rapid target molecule detection using an organic electrochemical transistor based sensing platform

Abstract

The disclosure relates to systems and methods for efficient and flexible target molecule detection using an organic electrochemical transistor (OECT). The system is particularly suited to enable a range of electrochemical sensing techniques to be utilized through a range of sensors and a multi-functional reader. Systems and methods for connecting disposable sensors to amplifiers and readers are described.

Description

SYSTEMS AND METHODS FOR RAPID TARGET MOLECULE DETECTION USING ANORGANIC ELECTROCHEMICAL TRANSISTOR BASED SENSING PLATFORMFIELD

[0001] The disclosure relates to systems and methods for efficient and flexible target molecule detection using an organic electrochemical transistor (OECT). The system is particularly suited to enable a range of electrochemical sensing techniques to be utilized through a range of sensors and a multi-functional reader.BACKGROUND

[0002] The use of organic electrochemical transistors (OECTs) to detect biological binding events is known. An OECT typically includes a sample well having a gate electrode conjugated with a biorecognition element (also referred to herein as “bait”) that can bind to a biomolecule (also referred to herein as “target”). By means of a source electrode, a drain electrode and an ion-permeable organic semi-conductor that defines a channel between the source and drain electrodes and within the sample well, binding events between the bait and target can induce changes in the capacitance of the gate electrode which can be measured and amplified to indicate that the binding event has occurred. OECTs can be functionalized with a range of biorecognition elements which individually enable the detection of specific biomolecules within a particular sample.

[0003] OECTs have been effectively used in the past to detect the presence or absence of biomolecules (such as pathogens, metabolites, hormones, proteins, nucleic acids, etc.) within a sample, where the OECT has been conjugated with a specific bait molecule to detect its target in a biological medium. When the sample well of the OECT is exposed to a solution that may contain the biomolecule target, a binding event is detected to indicate the presence of the bait-recognized target biomolecule; conversely, if the binding event is not detected, this indicates the absence of targeted biomolecule.

[0004] The process of designing an effective bait coupled gate (referred to herein as a “functionalized gate” FG) that is specific to a targeted biomolecule requires considerable time and effort to ensure the required specificity and sensitivity. Typically, different companies mayspend several years developing and validating specific sensors that may form part of the OECT and developing associated hardware specific to their biomolecules of interest.

[0005] As a result, there has been a need for improved OECT systems that can provide improved flexibility to developers of OECT systems.SUMMARY

[0006] In accordance with the disclosure, there is provided a system for detecting target molecules in a sample.

[0007] In one embodiment, the system includes a sensor having: a sample well configured with at least one functionalized gate (FG) within the sample well, each FG being configurable with a target recognition element capable of binding to a target molecule, each FG being configured to connect to a gate voltage supply and having a connector to connect to an amplifier.

[0008] In another embodiment, the system includes an amplifier having: a connector configured to connect to the sensor; a measurement gate (MG) configured to connect to the sensor via the connector; a source electrode connectable to a source voltage supply; a drain electrode connectable to the source voltage supply; and an ion-permeable organic semiconductor defining a channel between the source and drain electrodes; the channel separated by a gap to the MG and wherein the MG, channel, source electrode and drain electrode are ionically connected; and wherein target molecule binding to the target recognition element on the FG induces a change of surface potential at the FG and the MG and a change in electrical conductivity of the channel, the change in surface potential indicative of a presence of the target molecule in the sample well.

[0009] In another embodiment, the system includes a reader configured to the amplifier, the reader having a reader control system configured to provide gate and source voltage to the amplifier and sensor.

[0010] In various embodiments:• the amplifier and reader are an integrated unit having a body and the sensor is connectable to the amplifier via the connector integrated within the body.the sensor includes at least two FGs functionalized with different target recognition elements and the reader control system is configured to sequentially measure target molecule binding at each FG.• the reader control system is configured to enable at least two measurement techniques selected from potentiostatic measurement; voltametric measurement, pulsed voltammetry measurement, amperometric measurement and galvanostatic measurement.• the connector is configured with connector pins to enable at least two measurement techniques.• the reader control system is configured to enable any one or a combination of Linear Sweep Voltammetry (LSV) and Cyclic Voltammetry (CV) measurement.• the reader control system is configured to enable any one or a combination of Square Wave Voltammetry, Differential Pulse Voltammetry and Normal Pulse Voltammetry measurement.• the reader control system is configured to enable any one or a combination of Chronoamperometry, Zero Resistance Amperometry, MultiStep Amperometry, Pulsed Amperometric Detection and Multiple-Pulse Amperometric Detection measurement.• the reader control system is configured to enable Open Circuit Potentiometry measurement.• the sensor includes a control gate configured within the sample well.• the sensor is configured with at least two FGs and a multiplexer circuit configured to selectively activate FGs for measurement.• the connector is configured with FG, gate, source, drain, reference and detect pins.• the connector is configured with pins to power and control the multiplexer circuit.• the reader is configured to connect to a wide area network and to receive and send data within the wide area network.

[0011] In another embodiment, a method of measuring the presence or absence of a target molecule in a fluid sample, the body fluid sample in an electrolyte, the method comprising the steps of: a) connecting a sensor to an amplifier and reader; b) introducing a baseline electrolyte to the sample well and obtaining a first baseline measurement; c) rremoving the baseline electrolyte from the sample well; d) introducing a test sample into the sensor and incubating for a time sufficient to enable target molecule binding in the sensor; e) after incubation, obtaining a sample measurement from the sensor; and, f) comparing the measurements from b) and e) relative to a threshold to determine if a target molecule is present or not or provide a quantitative description of target amount in the measuring fluid.

[0012] In various embodiments:• step b) includes applying a voltage across the source and drain electrodes in a series of steps and measuring current flow at each voltage to derive a baseline transfer measurement.• step e) includes applying a voltage across the source and drain electrodes in a series of steps and measuring current flow at each voltage to derive a sample measurement.• the method includes the step of comparing the baseline measurement and sample measurement to determine a voltage difference between the baseline and sample measurements and comparing the voltage difference to a threshold to determine if the target molecule is present or not.• the method includes the step of: wherein if the voltage difference determines the target molecule is present, the relative difference of the voltage difference to the threshold is measured to quantify the concentration of target molecule.• the method includes the step of: wherein the sensor includes at least two FGs, and binding events are measured at each FG sequentially. the method includes the step of: interpreting pin connections between the sensor and amplifier and based on the connected pins, initiating a measurement protocol based on the connected pins.• the method includes the step of: wherein at least two measurement techniques are performed, the measurement techniques selected from potentiostatic measurement; voltametric measurement, pulsed voltammetry measurement, amperometric measurement and galvanostatic measurement.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Various objects, features and advantages of the disclosure will be apparent from the following description of particular embodiments of the disclosure, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the disclosure. Similar reference numerals indicate similar components.Figure 1 is a schematic diagram of an organic electrochemical transistor (OECT) in accordance with accordance with the prior art.Figure 2 is a schematic plan view an OECT showing electrical contacts and a sample collection area in accordance with the prior art.Figure 3 is a schematic diagram of an OECT and associated block diagrams of a power and data acquisition system.Figures 4(a)-4(d) are representative (a) voltage / current transfer curves on an OECT before and after functionalization; (b) voltage / current transfer curves of SARS-CoV-2 spike protein from an OECT at varying concentrations of pathogen protein; (c) voltage / current transfer curves of MERS-CoV spike protein from an OECT at varying concentrations of pathogen; and (d) Semi-logarithmic plot of average shift in threshold voltage ( T) as a function of spike protein concentration. Functionalized OECTs showed negligible change to increasing MERS-CoV spike protein concentration and logarithmic dependence to increasing SARS-CoV-2 spike protein concentration with an average slope of -11.5 mV / dec.Figure 5 is a schematic diagram of an OECT sensor and amplifier in accordance with one embodiment of the disclosure.Figure 5A is a schematic diagram of an OECT sensor and amplifier showing a connector in accordance with one embodiment of the disclosure.Figures 5B and 5C are schematic diagrams of an OECT sensor and reader in accordance with one embodiment of the disclosure.Figure 6 is a schematic diagram of an OECT reader and amplifier circuit in accordance with one embodiment of the disclosure.Figure 7 is a flowchart showing sample testing steps using an OECT in accordance with one embodiment of the disclosure.Figures 8-8G are schematic diagrams of OECT sensors having multiple pins enabling a range of measurement techniques to be conducted utilizing one sensor / reader platform.Figure 9 are graphs showing analysis of c-reactive protein (CRP) on an anti-CRP OECT biosensor.Figure 9A are graphs showing analysis of covid-19 on an anti-covid OECT biosensor.Figure 9B are graphs showing detection of glycated albumin via an aptamer OECT and glycated hemoglobin (HbA1C via antibody) via Linear Sweep Voltammetry (LSV) analysis.Figure 9C are graphs showing non-biological bait / target detection using a non functionalized (or bare) gate sensor via LSV analysis.Figure 9D are graphs showing CRP detection using a chronoamperometry analysis technique.Figure 10 is a schematic diagram of a developer, stakeholder and user ecosystem in accordance with one aspect of the disclosure.DETAILED DESCRIPTION

[0014] With reference to the figures, systems and methods for detecting molecules including biomolecules within a sample are described.Rationale

[0015] The inventors have recognized the need for improved OECT systems to detect molecules including biomolecules that improve efficiencies of detection. In particular, the inventors have improved the ability to effectively test for chemical binding events with improved designs of OECTs and associated electronics.Introduction

[0016] Various embodiments of OECT systems will now be described with reference to the figures. For the purposes of illustration, the terms “molecule” and “biomolecule” are used interchangeably with the understanding that “molecule” is a more generic term compared to “biomolecule” and that reference to “biomolecule” is generally used for convenience. The use of the term “biomolecule” is not specifically meant to exclude non-biological chemistries (unless stated) that can be utilized as an aspect of the technology as described herein and as may be understood by those skilled in the art.

[0017] It should also be noted that components depicted in the figures are not necessarily drawn to scale. Instead, emphasis is placed on highlighting the various contributions of the components to the functionality of various aspects of the disclosure. A number of possible alternative features are introduced during the course of this description. It is to be understood that, according to the knowledge and judgment of persons skilled in the art, such alternative features may be substituted in various combinations to arrive at different embodiments of the present disclosure.

[0018] The working principle of the organic electrochemical transistor (OECT) is schematically illustrated in Figure 1. As shown in Figure 1 , the OECT 10 is a bio-circuit that detects small concentrations of biomolecules within a fluid sample, the OECT having a gate electrode 14c that has been functionalized with a bait molecule (e.g. an antibody) 20 specific to a biomolecule (e.g. a protein on a pathogen or a blood / urine / saliva biomarker) 22, a source electrode 14a, drain electrode 14b covered with ion-permeable channel 16. Binding of the biomolecule to the gate electrode has the effect of altering the electric field potential within the device which can be measured as a change in the drain current to determine the proportional presence or absence of biomolecule.

[0019] The OECT is formed on a substrate 12, (e.g., a silicon base) and is typically comprised of three gold electrodes including the source electrode 14a, drain electrode 14b and gate electrode 14c. An ion-permeable conducting polymer channel (for example, PEDOT:PSS; described below) 16 extends between the source and drain electrodes and connects the source 14a and drain 14b electrodes. The three electrodes are arranged in a planar arrangement on the substrate 12 as shown in a plan view in Figure 2 within a sample well area 24. Sample well walls 26 extend upwardly above the planar surface to define a sample well 26a for containing a volume of sample as shown schematically in in Figure 3. Extending beyond the sample well area 24 are electrode traces (17a-c) and corresponding contact pads (18a-c) providing an electrical interface for connection to other circuits including power and measurement circuits. The conducting traces 17a-b are passivated by an insulator layer 12a to prevent contacting with the liquid electrolyte leading to short circuit.

[0020] The dimensions of PEDOT:PSS channel (width W and length L), the width E of the source / drain traces 17a-b and the area of the gate electrode are controlled to ensure consistent voltage and current through these conductors having regard to the range of voltage and current required for functional operation of the circuit. The gold gate 14c is separated from the PEDOT:PSS channel 16 by a short distance D (typically about 300 micrometers).

[0021] The biomolecule can be a range of molecules that may have the capacity to bind to an immobilized bait molecule, for example part of a whole or fragmented pathogen, metabolites, proteins in blood, urine, saliva or swab samples, etc..

[0022] An example of a suitable ion-permeable conducting polymer is PEDOT:PSS (poly(3,4- ethylenedioxythiophene) polystyrene sulfonate). PEDOT:PSS is a polymer mixture of two ionomers. One component in this mixture is made up of sodium polystyrene sulfonate (a sulfonated polystyrene) (PSS). Some of the sulfonyl groups are deprotonated and carry a negative charge. The second component poly(3,4-ethylene-dioxythiophene) (PEDOT) is a conjugated polymer (polythiophene) and carries positive charges. The organic semiconductor, PEDOT, is positively doped by the dopant, the sulfonate groups on the PSS, and hence PEDOT: PSS shows high electrical conductivity.

[0023] In one example, as shown in Figures 1 and 3, anti-SARS-CoV-2 antibody molecules 20 are attached to the surface of the gate 14c through 3,3'-Dithiodipropionic acid di(N-hydroxy succinimide ester) (DSP) or DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)) linker molecules 21 , by a functionalization process to produce a functionalized gate (FG) 15. Thefunctionalized gate 15, source 14a and drain 14b electrodes, and channel 16 are submerged in a liquid electrolyte within the sample / well structure 26 as shown in Figure 2. The liquid electrolyte (for example, phosphate buffer saline, PBS, universal transport media, UTM, blood, serum, saliva or urine) electrically connects the channel to the functionalized gate.

[0024] When the biomolecule sample 22 is introduced to the electrolyte within the sample well, the biomolecule binds with the bait molecule 20 immobilized on the surface of the functionalized gate 15. This binding event modulates the movement of free ions 30 in the electrolyte as shown in Figure 1 and alters the surface potential at the gate. When a voltage VGis applied onto the gate electrode (i.e. , between the source 14a and gate 14c electrodes), the potential distribution VD across the gate 14c, electrolyte, and channel 16 changes, which results in a shift in the transfer characteristics of the OECT as explained below.

[0025] Current (ID) between the source and drain electrodes (i.e., current passing through the channel) is measured as a function of input gate voltage (VG) and can be plotted as a transfer curve, shown in Figures 4(a) and 4(b). In one embodiment, the gate voltage is swept from - 0.4V to 1.1V in steps of 0.05V, while a constant voltage of -0.4V is applied between the drain and source to obtain the corresponding current (ID). The voltage sweep across the OECT may be outside this range as may be determined by the dimensions and specific chemistry of the OECT. In some embodiments, particularly in the case of biomolecule detection, the voltage ranges will typically be +- 1.5V or less. In other embodiments, this range may be in the range of +- 3.0V.

[0026] Figure 4(a) shows representative transfer curves before and after functionalization whereas Figures 4(b) and 4(c) shows representative transfer curves of a functionalized electrode with bound SARS-CoV-2 and MERS-CoV spike protein respectively at different pathogen concentrations (e.g., 1 fg / ml to 1 pg / ml).

[0027] That is, the binding of the virus particles 22 to the antibodies 20, results in a shift of the transfer curve proportional to the concentration of the virus detected. This shift is quantitatively represented by the shift in a threshold voltage ( VT). The threshold voltage (VT) is a quantity extracted from the transfer curve where VT is the x axis intercept of a line tangent to the linear slope of the transfer curve.

[0028] AVT is the difference of VT before and after the virus particles are introduced to the electrolyte solution. VT as a function of virus concentration is shown in Figure 4(d). The slope of this represents the sensor sensitivity to the SARS-CoV-2 virus.

[0029] In various embodiments, the arrangement and geometry of the source, drain, channel and gate can be further altered to improve / optimize sensitivity of the OECT sensor. For example, various dimensions including the width, length and the thickness of the channel between the source and drain and the surface area of the gate electrode may be varied to optimize the electrical current level and device sensitivity. Other design features such as arranging the source and drain electrodes in an interdigitated electrode fashion can also improve performance.

[0030] In a preferred embodiment, the composition of the gate electrode is gold, which is important for the functionalization process for the selected DSP or (DTSSP ligand, a preferred choice to immobilize bait molecules to the gold surface). Other chemistries involving 3- Mercaptopropionic acid (3-MPA), 11- mercaptoundecanoic acid (11-MIIA), 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC) or LIV crosslinking for aptamers can also be utilized. The composition of the source and drain electrodes are conductive metals, preferably metals such as gold or platinum.

[0031] The substrate material (e.g., silicon oxide) may be substituted for other insulating substrates such as glass, ceramic or other plastic materials.

[0032] PEDOT:PSS may be substituted with other ion-permeable conducting polymers.Sensor and Amplifier Design

[0033] As described above, an OECT provides an effective means of amplifying surface potential changes as a result of binding events. However, as can be appreciated, each binding event that utilizes an OECT is a single-use event. That is, sample is applied to a functionalized sample well, the test is performed and the OECT is discarded. As a result, there has been a need for testing systems that reduce the disposable components of the system.

[0034] As shown in Figures 5-5C, a biomolecule sensing system (BSS) 50 is described which generally includes various components separable from one another enabling multi-use of reader and amplifier components and disposability of sensor components.

[0035] As shown in Figure 5, the BSS includes a sensor component 52, an amplifier component 51 and a reader 56. As compared to Figure 1 , the amplifier 51 is configured within the reader separated from the sensor 52, enabling sensors to be selectively connected to the amplifier. Thus, in use, a disposable sensor may be connected to the amplifier / reader, a binding event (i.e., a “test”) monitored, and upon completion of the binding event, the sensor may be disconnected from the amplifier / reader and discarded. A new test may then be completed by connecting a new sensor to the amplifier / reader and repeating.

[0036] In one embodiment, with reference to Figures 5A and 5B, the sensor 52 includes a functionalized gate (FG) and a control gate (CG) within a test well 52a. The FG is functionalized with a bait molecule Y and the CG is connectable to a gate voltage (VG) supply. The sensor may be connected to a reader through a connector 54 showing separate sides 54a, 54b in Figure 5A. The CG is an unfunctionalized electrode that may be separate from the FG but may be electrically connected to the FG in the presence of an ionic solution, i.e., by a calibration / test sample that is placed within the test well 52a that allows the exchange of ions between the CG and FG. Accordingly, in use, when an ionic solution is placed within the test well 52a and gate voltage applied, charge is transferred to the FG. In this particular embodiment, the combination of FG and CG forms a capacitor / resistor / capacitor (CRC) circuit.

[0037] The reader 56 is configured to receive the sensor through at least 2 connector halves 54a, 54b as explained in greater detail below. For the example shown in Figures 5 and 5A, the FG is connected to the gate voltage source that energizes the FG and to the amplifier via a low resistance connector 54, 54a, 54b.

[0038] The amplifier 51 includes a measurement gate (MG) connected to connector 54a, which in turn is connectable to the FG via connector 54b.

[0039] Accordingly, when the sensor 52 and amplifier 51 are connected, the FG and MG are connected such that a binding event at the FG has an effect at the MG. That is, after a sample is added to the sample well and the gate voltage applied, the gate voltage induces charge on the FG and MG (when connected). If a binding event occurs, there is a capacitance change at both the FG and MG.

[0040] Adjacent to the MG, the amplifier further includes an ion-permeable conducting polymer channel 51c (for example, PEDOT:PSS; as described above) between source 51a and drain 51 b electrodes. The MG and ion-permeable channel 51c are overlain with a conductive medium(e.g., an ionic thin film) 51 d, enabling the movement of charge between the MG and polymer channel 51c. As such, the conductive mediums of the amplifier and sensor are separated from one another.

[0041] The amplifier 51 is configured within the reader 56 as a permanent or semi-permanent component of the reader and as a component of the measurement / amplification circuit of the reader as shown in Figure 6.

[0042] In one embodiment, as shown in Figure 5C, a sensor 52 having a sample well 52a and connector 54 is inserted into slot 53 of reader 56.Test Procedure

[0043] In operation, the system may be utilized to conduct a chemical detection test as shown in Figure 7.

[0044] With the sensor configured to a power and data acquisition system / reader as shown in Figures 3, 5B, 5C and 6, clinical samples can be tested according to the following general protocol shown in Figure 7. In the following protocol, the sensor 52 is disposable and cannot be re-used. Thus, for each clinical test a separate sensor is utilized.

[0045] A sensor is connected 71 to the amplifier / reader system, and a patient sample 72 (e.g., a swabbed sample from a patient) is immersed in buffer 72a to suspend the patient sample.

[0046] A volume of blank buffer 73 (i.e., without patient sample) is loaded into the sensor (about 80-100 pl) and a first transfer curve measurement is taken 74. This is a reference or blank measurement.

[0047] Thereafter, blank buffer is removed 74a and the suspended patient sample 72a is loaded into the sample cell 75 (about 80-100 pl), allowed to incubate for about 5 minutes 75a and a second transfer curve measurement is taken 76.

[0048] The transfer curves are obtained by measuring the drain current (ID) when applying a constant voltage between the source and drain (VD) contact and stepping the voltage between the gate and source (VG). In one embodiment for the purpose of general description, the voltage is ranged from 0 to 1.121 V with a step size of approximately 49 mV. It is understood that theparticular design of the BSS and the bait / molecule chemistries involved will require adjustments to this particular procedure and range.

[0049] The threshold voltage is extracted using both the reference and sample transfer curve. The linear extrapolation method is used to extract the threshold voltage by finding the maximum slope (e.g., using every 4 points on the curve) for the transfer curve and adding VD / 2 to the extrapolated x-intercept (ID = 0).

[0050] The change in threshold voltage (AVT) 77 is calculated as VT of the reference transfer curve minus the VTof the sample transfer curve. A cut-off threshold 78 AVTof -10 mV may be used for a clinical sample for determining positive or negative results. If the change is larger than -10mV, then the sample may be considered as returning a positive result 79 and if less than -10mV, then the sample may be considered as returning a negative result 80. A larger T may indicate a higher concentration of the biomolecule in the sample.

[0051] A new test may then be conducted with a new sensor. As such, the amplifier component of the reader is available for subsequent readings.

[0052] In one embodiment, the amplifier 51 is also exchangeable / removable from the reader. That is, over time and multiple uses, the amplifier may degrade and / or lose precision whereas reader electronics may not degrade at the same rate such that replacement of the amplifier makes economic sense.Multiplex Sensor and Reader

[0053] In various embodiments, the sensor and reader are configured to enable different binding events to be conducted with a single sensor. In these examples, the system is designed such that a single sample may be tested for multiple biomolecules, referred to herein as a multiplex assay.

[0054] In these embodiments, as shown in Figures 8-8G, multiple functionalized gates (FGs) are positioned on the sensor within a common sample well 52a, where each FG has been functionalized with different baits. In one example, as shown in Figure 8, there are 7 different FGs (A-G) with one control gate CG within a sample well 52a. The sensor 52 may be connected to a reader via connector 54 as described above. Typically, one FG will remain unfunctionalized and be used as a baseline gate.

[0055] In this generalized example, the sensor is connected to the reader and prepared as described above. A test sample is placed in the sample well 52a. In this case, with 7 different FGs A-G, the sensor and reader are controlled to sequentially measure binding events at each FG. For example, after the test sample has been placed in the sample well, FG A is energized, and the drain / source measured. Once an FG A reading is obtained, the system turns off FG A and turns ON FG B, obtains a reading, turns OFF FG B and progresses in a similar manner to FG C and FG G.

[0056] Thus, the system can obtain information about multiple binding events from a single sample.Reading Techniques

[0057] Each FG within a single sensor may be functionalized by various techniques including micro-dosing and / or micro pipetting techniques.

[0058] The ability to integrate multiple FGs to a reader can take advantage of a range of electrochemical inspection techniques including but not limited to:• Potentiostatic and Galvanostatic methods.• Voltammetric techniques i. Linear Sweep Voltammetry (LSV) ii. Cyclic Voltammetry (CV)• Pulsed techniques i. Square Wave Voltammetry ii. Differential Pulse Voltammetry iii. Normal Pulse Voltammetry• Amperometric techniques i. Chronoamperometryii. Zero Resistance Amperometry iii. Chronocoulometry iv. MultiStep Amperometry v. Pulsed Amperometric Detection vi. Multiple-Pulse Amperometric Detection• Galvanostatic techniques i. Open Circuit Potentiometry

[0059] Depending on the specific technique being used, the reader can be configured with a number of active electrodes to support various sensors.

[0060] Examples of systems having different electrodes are shown in Figures 8-8G. Figure 8 shows a sensor having multiple FGs configured to a multiplexer 56a, a microcontroller 56b and a connector 54. The connector is configured with a number of pins 54d-m that enable sensors utilizing different measurement techniques to share a common connection scheme.

[0061] In the generic embodiment as shown in Figure 8, the reader connector 54 is configured to include a floating gate 54d, gate 54e, source 54f, drain 54g, reference 54h and detect pins 54i enabling the connection of different sensors to the same reader.

[0062] In one embodiment, the reader connector may also be configured with microcontroller pins 54j-54m to power a sensor-based microcontroller 56b configured to control the sensor. Such features may include controlling the multiplexer to switch specific channels on and off as described above.

[0063] As shown in Figure 8A, a simple sensor having a single FG and CG are configured to the floating gate 54d and gate 54e pins only.

[0064] Figure 8B shows an example of 2 FGs (FG A and FG B) using a different measurement technique with active gate 54e, source 54f, drain 54g and reference 54h pins. In potentiostatic mode, a potentiostat / galvanostat (PGSTAT) will accurately control the potential of the Counter Electrode (CE) against the Working Electrode (WE) so that the potential difference between the working electrode (WE) and the Reference Electrode (RE) is defined.

[0065] Figure 8C shows an example of a sensor having active gate 54e, source 54f and drain 54g pins. In this case, the output voltage on the gate is held and the source / drain amperage is read.

[0066] Figure 8D shows an example of a sensor having active gate 54e, source 54f and reference 54h pins and 2 functionalized gates.

[0067] Figure 8E shows an example of a sensor having a referenced floating gate.

[0068] Figure 8F shows an example of a sensor having an integrated redox OECT. This example includes a reference electrode configured to leverage REDOX reporters being sensed instead of the electric double layer capacitance change.

[0069] Figure 8G shows an example of a sensor having a reference electrode configured to leverage REDOX reporters being sensed instead of the electric double layer capacitance change. This examples also shows the sampling areas separated from the OECT amplifier.ExperimentalOECT Fabrication

[0070] OECTs were fabricated using Optomec Aerosol Jet 5X 3D printer with commercially available materials. Gold nanoparticle (Au NP) ink (LIT Dots, Inc.) was deposited onto a 25.4 pm thick Kapton HN film to form source, drain, and gate electrodes and all the metal interconnects and annealed at 280°C for 1 hour. The printed Au traces have an average thickness of 584 nm and an average resistivity of 8.32x1 O'6Q-cm. The channel of OECT was printed using poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) mixture composed of 94% Heraeus Clevios™ PH 1000, 5% ethylene glycol (EG), 0.1 % dodecylbenzenesulfonic acid solution (DBSA) (70 wt. % in isopropanol), and 1wt% (3- Glycidyloxypropyl) trimethoxysilane (GOPS) (> 98 %) and annealed at 130°C for 20 minutes. Finally, a 1 .05 pm thick layer of UV-curable polydimethylsiloxane (PDMS) (Shin-Etsu Chemical Co., Ltd.) was printed to passivate the metal traces, acting as an insulating layer to prevent shorting from the liquid electrolyte. The PDMS insulating layer was cured with LIV on-the-fly while printing and then annealed at 130°C for 30 minutes.CRP Detection

[0071] Detection of C-reactive protein (CRP) in whole blood was undertaken and compared to detection in serum using LSV for CPR detection. Figure 9 shows controls (left panels) and test results for sensors configured with anti-CRP antibody (right panels). Both current and voltage changes are shown. P values for buffer vs CRP in whole blood or serum for both parameters are < 0.0001. P value between CRP in whole blood vs CRP in serum for both parameters are < 0.0001 indicating that there is a difference in results. This result illustrates the detection of CRP in whole blood with p value > 0.5 for CRP detection in whole blood vs serum.

[0072] Figure 9A shows implementation of an OECT pathogen biosensor configured with anti- covid 19 spike protein antibodies. The figure shows that COVID-19 positive patient samples produced a spread of 50-100 mV shifts whereas non-COVID patient samples produced mV shifts in the 10 - 50 mV range. Examples of output transfer curves are shown on the right and an illustration of the rightward voltage shifts.

[0073] Figure 9B detection of purified glycated human serum albumin (HSA, top left panel) and glycated hemoglobin (Gly Hb or HA1c, bottom left panel) vs buffer or non-Gly Hb (Hb) to an OECT anti-Gly Hb based bait to demonstrate specificity of our Gly Hb bait. Each curve is a separate run of the same sample. Right panel, both current and voltage changes are shown for glycated albumin detection and data not shown for glycated HbA1c.

[0074] Figure 9C shows detection of non-biological targets using LSV. The results show a dynamic range of detection of non-biological targets such as streptavidin / biotin and for seawater detection. Similar to CRP described above, transfer curve data is converted to quantitative values to be used to determine target molecule concentration in the biological medium.

[0075] Figure 9D shows detection of CRP using chronoamperometry. In this example, the sensor was connected to the reader and allowed to equilibrate with baseline buffer. Thereafter, sample was added directly to the well and allowed to equilibrate for 5 minutes. The electric potential of the working electrode was stepped and the resulting current at the reference electrode was monitored as a function of time. The appropriate controls are shown for CRP detection in Figure 9D (1). N values are between 2-4 and p values < 0.05 between CRPdetection and non-CRP detection (e.g., BSA). Results are obtained in < 10 min and all samples in 1 / 20 diluted serum.Biorecognition Elements

[0076] As shown above, sensors can be designed for 1 or more gates that can be configured to allow for wider range of multiplex assays.

[0077] Various biorecognition elements may be configured to an FG. For example, a biorecognition element may be an element capable of specifically binding to an antigen of a pathogen such as an antibody, aptamer, chemical, nucleic acid probe, protein bait that, once bound, is capable of detecting a change in surface potential that is proportional to the concentration of a target molecule (e.g. pathogens, toxins, metabolites, nucleic acids, protein biomarkers in blood / serum / saliva / urine / swab medium etc.).Power and Data Acquisition Circuits

[0078] Returning to Figure 3, schematics of the OECT based SARS-CoV-2 biosensor are illustrated with a representative circuit block diagram for power, data acquisition and data processing. The power / data acquisition / data processing system 31 generally includes a power supply 30a, voltage regulator for G, voltage regulator for D, a transimpedance amplifier and filter 30d, analogue-to-digital converter 30e, display / user input 30f, microcontroller 30g and datalogging system 30h.

[0079] Figure 6 is a schematic diagram showing a reader having a multi-functional sensor connection system 54 and integrated OECT amplifier 51 enabling one or more of the above measurement techniques to be conducted through the pin connector system. That is, as described above, the reader is configured with a connector system enabling sensors having different configurations to be connected to the reader. The reader, upon recognizing active pins of a particular sensor, will initiate a measurement protocol based on the active pins and / or other information received from the sensor.

[0080] This connection allows for hardware extensibility using the data and power pins and allowing for precise potentiostatic and galvanostatic tactics using various combinations of drain, source, gate, floating gate, and reference pins.

[0081] Importantly, this circuit / system can be configured to enable the ability to use potentiostatic tactics on next generation OECT based sensors supporting features such as a reference electrode as part of the sensor design and / or any existing potentiostatic technique that may be leveraged on three electrode sensors that do not leverage OECTs in their designs.

[0082] This circuit / system may also be configured to enable holding the source / drain bias constant while allowing the source (counter electrode) to change in potential in relation to the gate (working electrode). This unique behavior allows for OECTs to operate in their usual manner while leveraging the ability of the working electrode to reference electrode relationship to be closely controlled.

[0083] Further, in various embodiments, the circuit / system may be configured to include a sine wave generator and phase comparator that enable electrochemical impedance spectroscopy to be used during interrogation.Wide Area Deployment

[0084] Figure 10 illustrates a BSS deployed across a wide area involving multiple parties. The BSS may be configured to enable a range of parties to utilize the BSS including sensor developers 100, reader developers 101 , and various stakeholders 102 such as jurisdictional healthcare providers, hospitals, clinics, funding agencies, non-governmental organizations, patients, and others in order to obtain and communicate information between the various parties.

[0085] For example, as the reader can support a range of sensors, individual developers can concentrate development efforts on specific sensor chemistries for a particular assay. For example, one developer may wish to develop a sensor utilizing a particular Hb A1C test whereas another is interested in commercializing a sensor for rapid SARS-CoV2 testing. Each developer may imagine different markets for their sensors that may include individual patients, caregivers, clinics, hospitals and / or jurisdictional health care providers. Depending on the commercialization objectives of a developer, the developer may develop sensors appropriate for different users utilizing different sampling / assaying procedures. For example, some assays may utilize saliva / swabbed samples and be more appropriate for any user, whereas others may utilize blood that is better suited to be managed by trained medical personnel.

[0086] In either case, a developer may utilize the reader platform as a foundation to develop a particular sensor which may be marketed / sold to a variety of end-users.

[0087] Depending on specific functionality, the use of devices and sensors may be monitored / reported to stakeholders as a means of collecting and utilizing relevant data.

[0088] For example, in the case of a Covid or flu outbreak, testing data including positive test results, negative test results or invalid test results may be reported to one or more jurisdictional bodies to whom such data may be relevant as well as back to the developer and / or the reader developer.

[0089] Although aspects of the disclosure have been described and illustrated with respect to preferred embodiments and preferred uses thereof, it is not to be so limited since modifications and changes can be made therein which are within the full, intended scope of the disclosure as understood by those skilled in the art.References1. Hartley DM, Perencevich EN. Public health interventions for COVID-19: emerging evidence and implications for an evolving public health crisis. Jama. 2020;323(19): 1908-9.2. Nouvellet P, Bhatia S, Cori A, Ainslie KE, Baguelin M, Bhatt S, et al. Reduction in mobility and COVID-19 transmission. Nature communications. 2021 ; 12(1 ):1-9.3. Ji T, Liu Z, Wang G, GuoX, Akbar Khan S, Lai C, et al. Detection of COVID-19: A review of the current literature and future perspectives. Biosens Bioelectron. 2020; 166: 112455.4. Wang W, Xu Y, Gao R, Lu R, Han K, Wu G, et al. Detection of SARS-CoV-2 in Different Types of Clinical Specimens. JAMA. 2020;323(18):1843-4.5. Tang YW, Schmitz JE, Persing DH, Stratton CW. Laboratory Diagnosis of COVID-19: Current Issues and Challenges. J Clin Microbiol. 2020;58(6).6. Ravi N, Cortade DL, Ng E, Wang SX. Diagnostics for SARS-CoV-2 detection: A comprehensive review of the FDA-EUA COVID-19 testing landscape. Biosens Bioelectron. 2020; 165: 112454.7. Doris SE, Pierre A, Street RA. Dynamic and Tunable Threshold Voltage in Organic Electrochemical Transistors. Adv Mater. 2018;30(15):e1706757.

Claims

CLAIMS1. A system for detecting target molecules in a sample comprising: a sensor having: a sample well configured with at least one functionalized gate (FG) within the sample well, each FG being configurable with a target recognition element capable of binding to a target molecule, each FG being configured to connect to a gate voltage supply and having a connector to connect to an amplifier.

2. The system as in claim 1 further comprising an amplifier having: a connector configured to connect to the sensor; a measurement gate (MG) configured to connect to the sensor via the connector; a source electrode connectable to a source voltage supply; a drain electrode connectable to the source voltage supply; and an ion-permeable organic semi-conductor defining a channel between the source and drain electrodes; the channel separated by a gap to the MG and wherein the MG, channel, source electrode and drain electrode are ionically connected; and wherein target molecule binding to the target recognition element on the FG induces a change of surface potential at the FG and the MG and a change in electrical conductivity of the channel, the change in surface potential indicative of a presence of the target molecule in the sample well.

3. The system as in claim 2 further comprising a reader configured to the amplifier, the reader having a reader control system configured to provide gate and source voltage to the amplifier and sensor.

4. The system as in claim 3 wherein the amplifier and reader are an integrated unit having a body and the sensor is connectable to the amplifier via the connector integrated within the body.

5. The system as in any one of claims 1-4 wherein the sensor includes at least two FGs functionalized with different target recognition elements and the reader control system is configured to sequentially measure target molecule binding at each FG.

6. The system as in any one of claims 3-5 wherein the reader control system is configured to enable at least two measurement techniques selected from potentiostatic measurement; voltametric measurement, pulsed voltammetry measurement, amperometric measurement and galvanostatic measurement.

7. The system as in claim 6 wherein the connector is configured with connector pins to enable at least two measurement techniques.

8. The system as in claim 6 wherein the reader control system is configured to enable any one or a combination of Linear Sweep Voltammetry (LSV) and Cyclic Voltammetry (CV) measurement.

9. The system as in claim 6 wherein the reader control system is configured to enable any one or a combination of Square Wave Voltammetry, Differential Pulse Voltammetry and Normal Pulse Voltammetry measurement.

10. The system as in claim 6 wherein the reader control system is configured to enable any one or a combination of Chronoamperometry, Zero Resistance Amperometry, MultiStep Amperometry, Pulsed Amperometric Detection and Multiple-Pulse Amperometric Detection measurement.11 . The system as in claim 6 wherein the reader control system is configured to enable Open Circuit Potentiometry measurement.

12. The system as in any one of claims 1-11 wherein the sensor includes a control gate configured within the sample well.

13. The system as in any one of claims 1-12 wherein the sensor is configured with at least two FGs and a multiplexer circuit configured to selectively activate FGs for measurement.

14. The system as in any one of claims 1-13 wherein the connector is configured with FG, gate, source, drain, reference and detect pins.

15. The system as in claim 13 wherein the connector is configured with pins to power and control the multiplexer circuit.

16. The system as in any one of claims 3-15 wherein the reader is configured to connect to a wide area network and to receive and send data within the wide area network.

17. A method of measuring the presence or absence of a target molecule in a fluid sample, the body fluid sample in an electrolyte, the method comprising the steps of: a) Connecting a sensor to an amplifier and reader as in claim 3; b) Introducing a baseline electrolyte to the sample well and obtaining a first baseline measurement; c) Removing the baseline electrolyte from the sample well; d) introducing a test sample into the sensor and incubating for a time sufficient to enable target molecule binding in the sensor; e) after incubation, obtaining a sample measurement from the sensor; and, f) comparing the measurements from b) and e) relative to a threshold to determine if a target molecule is present or not.

18. The method as in claim 17 wherein step b) includes applying a voltage across the source and drain electrodes in a series of steps and measuring current flow at each voltage to derive a baseline transfer measurement.

19. The method as in claim 17 or 18 wherein step e) includes applying a voltage across the source and drain electrodes in a series of steps and measuring current flow at each voltage to derive a sample measurement.

20. The method as in claim 19 further comprising the step of comparing the baseline measurement and sample measurement to determine a voltage difference between the baseline and sample measurements and comparing the voltage difference to a threshold to determine if the target molecule is present or not.

21. The method as in claim 20 further comprising the step of: wherein if the voltage difference determines the target molecule is present, measuring the relative difference of the voltage difference to the threshold to quantify the concentration of target molecule.

22. The method as in any one of claims 17-21 wherein the sensor includes at least two FGs, further comprising the step of: measuring binding events at each FG sequentially.

23. The method as in any one of claims 17-22 further comprising the step of interpreting pin connections between the sensor and amplifier and based on the connected pins, initiating a measurement protocol based on the connected pins.

24. The method as in claim 23 wherein at least two measurement techniques are performed, the measurement techniques selected from potentiostatic measurement; voltametric measurement, pulsed voltammetry measurement, amperometric measurement and galvanostatic measurement.

25. The method as in claim 17 wherein step f) includes providing a quantitative description of target amount in the measuring fluid.

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