High-throughput electrochemical sensing device, system, and related methods

Abstract

Electrochemical biosensors are devices that convert biological signals into electrical signals for detection. Described herein are automation-compatible high-throughput electrochemical sensing devices and systems that form a high-throughput electrochemical sensing platform. In certain embodiments, the platform includes: a sensor plate assembly with a sensor board and multi-well plate, where each well contains at least one three- electrode assembly (e.g., a working electrode, a reference electrode, and a counter electrode) with a conductive antifouling coating; a control board for detecting signals from each of the multiple electrode assemblies; an optional heater and / or temperature controller for the sensor board; and an interconnect assembly to facilitate replacement of the sensor board.

Description

HIGH-THROUGHPUT ELECTROCHEMICAL SENSING DEVICE, SYSTEM, ANDRELATED METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 547,241 filed November 3, 2023, and U.S. Provisional Patent Application No. 63 / 658,229 filed June 10, 2024, the disclosures of which are incorporated by reference herein in their entireties.FIELD

[0002] The present embodiments described here relate generally to electrochemical sensing devices, and in particular to high-throughput sensing of biomolecules and biochemical reactions or interactions.BACKGROUND

[0003] Electrochemical biosensors are devices that convert biological signals into electrical signals for detection. Practical application of this technology is limited, in that the speed required to receive a useful signal for adequately quantifying reaction kinetics cannot be achieved with current high-throughput systems, and fouling remains a significant problem in most applications involving complex biofluids.SUMMARY

[0004] Described herein is an electrochemical sensing platform that, in certain embodiments, combines multi-well three-electrode sensors, a conductive antifouling coating, and a multiplexed measurement circuit for high-throughput and high-speed detection of biomolecules and biochemical reactions or interactions, thereby providing an improved electrochemical biosensor system.

[0005] The platform described herein is designed for high-throughput electrochemical sensing. The platform allows measurement of phenomena such as enzymatic activity and / or the presence and / or binding of biomolecules, and is compatible for use with existing liquid handling automation systems for significantly improved speed and consistency. For example, the platform facilitates direct analysis of electrochemically active molecules for enzymatic activities, as well as the detection of the presence and binding of biomolecules, and / or the kinetics of binding (e.g., on and / or off rate) with fast and sensitive readout.

[0006] Electrochemical measurements can detect electron transfers during enzymatic reactions in a sensitive and specific way. Most analytes (substrates, products, or even enzymes) that can be electrochemically oxidized or reduced can be measured. Moreover, the versatile coatings and measurement methods described herein can prevent or reduce fouling and record reliable signals by minimizing interventions from a complex surrounding matrix.

[0007] Thus, in one aspect, the invention is directed to an automation-compatible (e.g., high-throughput) electrochemical sensing platform (e.g., for measurement of enzymatic activity), the platform comprising: a sensor plate assembly comprising a sensor board, a multi-well plate (e.g., a bottomless multi-well plate, e.g., a 6-well, 12-well, 24-well, 48-well, 96-well plate, 384-well plate, 1536-well plate, 3456-well plate, 6144-well plate, or 9600-well plate, e.g., a plate with at least 6 well, at least 12 wells, at least 24 wells, at least 48 wells, at least 96 wells, at least 384 wells, at least 1536 wells, at least 3456 wells, at least 6144 wells, or at least 9600 wells), and an optional gasket securing the sensor board to the multi-well plate, wherein the sensor board comprises an electrode assembly (e.g., a working electrode, a reference electrode, and a counter electrode) corresponding to each well of the multi- well plate; a control board comprising hardware for detecting signals from the sensor board; and an interconnect detachably connecting the sensor board to the control board (e.g., to thereby facilitate replacement of the sensor board and / or the control board).

[0008] In some embodiments, the control board comprises: an electrochemical (EChem) front end [e.g., an EChem integrated circuit (IC), e.g., a potentiostat module, e.g., a pre-calibrated, self-contained potentiostat such as the EmStat Pico™ module manufactured by PalmSens BV of Houten, Netherlands)] to control the sensor board to obtain electrochemical measurements; a multiplexer to control temporal connection between theEChem front end and the electrode assemblies of the sensor board corresponding to the wells of the multi-well plate [e.g., wherein the multiplexer performs switching (e.g., serial switching) to automatically control which well(s) the EChem front end is connected to at a given time]; and a microcontroller to manage the EChem front end and the multiplexer (e.g., to control when the EChem front end takes measurements and to instruct the multiplexer when and where to connect corresponding signals).

[0009] In some embodiments, the electrode assembly may include multiple working electrodes and / or multiple references electrodes. For example, in some embodiments, the electrode assembly may include 1, 2, 3, 4, or more than 4 working electrodes, one or more reference electrodes, and a single counter electrode. In some embodiments, the electrode assembly may include only two types of electrodes (for example, one or more working electrodes and a single counter electrode, with no reference electrodes). It is noted that embodiments that do not include a reference electrode may not achieve the same system accuracy as embodiments that do include one or more reference electrodes.

[0010] In some embodiments, the platform further comprises a liquid handler, and the platform is programmed to perform the following steps (e.g., wherein the platform comprises a processor of a computing device and a memory having instructions stored thereon, wherein the instructions, when executed by the processor, cause the processor to perform the following steps): (i) the liquid handler dispenses substrate into required wells of the multiwell sensor plate for a reaction from which electrochemical measurements are to be obtained (e.g., to obtain enzymatic reaction kinetic data); (ii) the microcontroller is sent a signal from the liquid handler to begin a programmed measurement sequence (e.g., wherein the routine determines the next well to be measured); (iii) the microcontroller communicates with (e.g., sends a signal to) a digitally controlled analog switch (e.g., a multiplexer) to connect a working electrode, reference electrode, and counter electrode of the EChem front end to a correct corresponding well on the multi- well sensor board and disconnects all other wells; (iv) the liquid handler is sent a signal from the microcontroller such that the liquid handler dispenses a composition (e.g., a reactant, enzyme, or small molecule) to produce a reaction with the substrate and generate an electrical signal (e.g., current, voltage, and / or impedance) to be measured (e g., via amperometry, voltammetry, and / or impedance measurements); (v) the microcontroller communicates with the EChem front end and directs it to begin taking measurements for a programmed amount of time and / or datapoints, then the microcontrollerreceives the data from the EChem front end and stores the data on the microcontroller or offloads the data to an external storage space; and (vi) when the EChem front end signals it has finished a measurement, the microcontroller proceeds with at least step (iii) and (v) [e.g., and also step (iv) where reactions in wells are measured serially] for a next well in the programmed sequence and repeats this until all of the wells in the programmed sequence have been measured.

[0011] In some embodiments, obtaining the electrochemical measurements comprises performing pulsed amperometric detection (PAD) to obtain enzymatic reaction kinetic data. For example, in some embodiments, the substrate / measured species comprises uric acid, and the liquid handler dispenses uricase to produce the reaction with the substrate. In other embodiments, the product / measured species comprises hydrogen peroxide and the liquid handler dispenses lytic polysaccharide monooxygenase (LPMO) to produce the reaction with the substrate.

[0012] In other embodiments, obtaining the electrochemical measurements comprises performing differential pulse voltammetry (DPV) to obtain enzymatic reaction kinetic data. For example, DPV can be performed where the product / measured species comprises hydrogen peroxide and the liquid handler dispenses lytic polysaccharide monooxygenase (LPMO) to produce the reaction with the substrate.

[0013] In another aspect, the invention is directed to an anti-fouling coating to maintain electrochemical biosensor sensitivity and functionality when exposed to complex fluid. Accumulation of unwanted biological material (fouling) on surfaces greatly affects sensitivity and functionality. Most electrochemical sensors will suffer from signal deterioration if not functionalized with an anti-fouling coating.

[0014] Thus, described herein is an electrode assembly (e.g., a working electrode, a reference electrode, and / or a counter electrode) of an automation-compatible (e.g., high- throughput) electrochemical sensing platform (e.g., for measurement of enzymatic activity), said electrode assembly corresponding to a given well of a multi-well plate (e.g., wherein the electrode assembly is planar, e.g., and circular or rectangular), wherein said electrode assembly has a conductive, anti- fouling coating. In some embodiments, the conductive, antifouling coating comprises a first component to provide anti-fouling (e.g., a hydrogel) and asecond component to provide conductivity (e.g., a conductive filler such as carbon and / or nanoparticles). For example, in some embodiments, the conductive, anti-fouling coating comprises a hydrogel matrix that allows for physical immobilization of a conductive filler, wherein the conductive filler is loaded above an electrical percolation threshold to form a conductive pathway within the hydrogel matrix (e.g., wherein the hydrogel matrix has a porosity from about 10 nm to about 20 nm).

[0015] In some embodiments, the anti-fouling coating comprises a hydrogel matrix, and wherein the hydrogen matrix comprises one or more sensing elements to facilitate detection of a desired analyte (e.g., wherein the hydrogel matrix comprises CeC nanoparticles for H2O2 detection).

[0016] In some embodiments, wherein the anti-fouling coating comprises a conductive filler [e.g., reduced graphene oxide (rGO)] in a bovine serum albumin (BSA) matrix (e.g., wherein the substrate / measured species is / comprises uric acid and is measured by differential pulsed voltammetry).

[0017] In some embodiments, the anti-fouling coating comprises a conductive hydrogel matrix [e.g., PPy / PEDOT:PSS, i.e., polypyroole (PPy)-poly(3,4- ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS)] (e.g., wherein the conductive hydrogel serves as both a matrix composite and a conductive filler).

[0018] In another aspect, described herein is a method for creating a conductive, antifouling coating of an electrode, such as an electrode assembly of the automation-compatible (e.g., high-throughput) electrochemical sensing platform described herein, as follows: a hydrogel precursor (e.g., containing hydrogel monomer, crosslinker, and conductive filler) is dropcasted directly onto the working electrode; optionally, the electrode surface is activated to allow attachment of the hydrogel; optionally, external thermal and / or UV sources are used to initiate hydrogel polymerization (e.g., if using a thermal crosslinker or UV-sensitive crosslinker and initiator); optionally, a blade (e.g., doctor’s blade) is used to ensure homogeneous precursor application, e.g., wherein the sensor board comprises a 96- well plate; a sensor chip is quenched after reaction for a period of time to ensure consistent degree of polymerization and washed to remove unreacted monomer; the sensor chip is stored inbuffer and cooled to prevent coating degradation; and optionally, an additional sensing element (e.g., antibodies, or the like) is conjugated onto the hydrogel).

[0019] In another aspect, provided herein is a method for measurement of enzymatic activity (e.g., using the automation-compatible electrochemical sensing platform described herein), the method comprising obtaining electrochemical measurements in a well of a multiwell plate, said well containing (or supporting thereon) a solution comprising a substrate (e.g., a measured species) and a composition (e.g., a reactant, enzyme, or small molecule) dispensed into the well by a liquid handler (e.g., automated liquid handler, e.g., automated liquid handler that is fully integrated and autonomously controlled), wherein said composition produces a reaction with the substrate thereby generating an electrical signal (e.g., current, voltage, and / or impedance) to be measured (e.g., via amperometry, voltammetry, and / or impedance measurements).

[0020] In some embodiments, obtaining the electrochemical measurements comprises performing pulsed amperometric detection (PAD) to produce enzymatic reaction kinetic data.

[0021] In some embodiments, the enzymatic reaction kinetic data are uricase activity measurement (e.g., wherein the substrate / measured species comprises uric acid and wherein the dispensed composition that produces the reaction with the substrate comprises uricase).

[0022] In some embodiments, the product / measured species comprises hydrogen peroxide and wherein the liquid handler dispenses lytic polysaccharide monooxygenase (LPMO) to produce the reaction with the substrate.

[0023] In some embodiments, obtaining the electrochemical measurements comprises performing differential pulse voltammetry (DPV) to produce enzymatic reaction kinetic data. For example, the substrate / measured species may comprise ascorbic acid.

[0024] In another aspect, provided herein is a system for performing high-throughput electrochemical sensing for measurement of enzymatic activity (e.g., in a complex fluid, e.g., a biological fluid), the system comprising: (i) a liquid handler programmed to dispense a composition (e.g., reactant, enzyme, or small molecule) into wells of a multi- well sensor plate to produce a reaction with a substrate and generate an electrical signal (e.g., current, voltage, and / or impedance) to be measured (e.g., via amperometry, voltammetry, and / orimpedance measurements), from which enzymatic reaction kinetic data is determined; (ii) a microcontroller that (a) receives a signal sent from the liquid handler to begin a programmed measurement sequence (e.g., wherein the routine determines the next well of the multi-well sensor plate in the sequence to be measured), (b) communicates with an electrochemical (EChem) front end [e.g., an EChem integrated circuit (IC), e.g., a potentiostat module, e.g., a pre-calibrated, self-contained potentiostat such as the EmStat Pico™ module] and directs it to begin taking measurements for a programmed amount of time and / or datapoints in a given well, (c) receives data from the EChem front end and stores the data on the microcontroller or offloads the data to an external storage space, and (d) sends a signal to a digitally controlled analog switch that may control multiple inputs or outputs (e.g., a multiplexer) to proceed to the next well in the sequence upon completion of measurements for a given well; and (iii) a digitally controlled analog switch that may control multiple inputs or outputs (e.g., a multiplexer) that receives a signal sent from the microcontroller to connect a working electrode, a reference electrode, and a counter electrode of the EChem front end to a correct corresponding well on the multi-well sensor plate and disconnects all other wells.

[0025] In some embodiments, the sensor board further includes a built-in temperature control unit. In some embodiments, the built-in temperature control unit is a built-in heating layer. In some embodiments, the built-in heating layer includes metal arranged in a serpentine pattern through the interior of the sensor board. In some embodiments, the built-in temperature control unit provides a substantially uniform temperature across the sensor board. In some embodiments, the sensor board further includes a temperature sensor. In some embodiments, the sensor board further includes a proportional-integral-derivative (PID) controller. In some embodiments, the built-in temperature control unit provides an individual temperature control to each well of the multi-well plate. In some embodiments, the sensor board further includes a plurality of temperature sensors, wherein each temperature sensor corresponds to each well of the multi- well plate.

[0026] In some embodiments, the built-in temperature control unit further includes a cooling module. In some embodiments, the cooling unit includes a Peltier module (i.e., Peltier device). In some embodiments, the sensor board includes a printed circuit board (PCB), a glass wafer, a low-conductivity silicon wafer, or an insulating substrate.

[0027] In some embodiments, the platform further includes an external temperature control unit including: a heater block including a metal block with external dimensions matching a standard well-plate, the heater block machined to have openings to accommodate electrical connections, and a plurality of grooves; a heater coil including wiring for resistive heating, disposed within the plurality of grooves; a power supply for passing current through the heater coil; and a temperature sensor disposed on a surface of the heater block.

[0028] In some embodiments, the external temperature control unit further includes a cooler. In some embodiments, the cooler includes a Peltier cooler. In some embodiments, the external temperature control unit further includes a PID controller electrically connected to the power supply, the temperature sensor, and the cooler, and wherein the PID controller is configured to maintain a setpoint temperature.

[0029] In another aspect, the present embodiments are directed to a data processing method for high-throughput electrochemical sensing using a plurality of electrochemical sensors, the method including: acquiring raw data from the plurality of electrochemical sensors; translating the raw data to a readable format by a data parser; plotting the translated data by a data visualizer; and performing automated curve fitting by a data analyzer.

[0030] In some embodiments, the uricase activity measurement includes uricase in serum. In some embodiments, the electrical connections include a plurality of metallic, spring-loaded prongs.

[0031] In some embodiments, the sensor board is a printed circuit board (PCB) that is glued to the bottomless multi- well plate with a watertight seal.

[0032] In some embodiments, the enzymatic reaction kinetic data include L-ascorbate oxidase activity measurement. In some embodiments, the enzymatic reaction kinetic data include horseradish peroxide activity measurement. In some embodiments, the substrate / measured species includes (e.g., is or includes) uric acid. In some embodiments, the substrate / measured species includes (e.g., is or includes) hydrogen peroxide.

[0033] In some embodiments, the enzymatic reaction kinetic data include biological signals from enzymatic reactions that include or are involved with glucose oxidase,peroxidase, lactate dehydrogenase, superoxide dismutase, cytochromes, and / or carbonic anhydrase.

[0034] In some embodiments, the enzymatic reaction kinetic data include biological signals from enzymatic reactions that include or are involved with a redox response.

[0035] In some embodiments, the electrochemical measurements are obtained by measuring biological signals of antibodies, DNA, enzymes, and cells and / or biological signals from binding activities of peptides and / or aptamers.

[0036] In some embodiments, the uricase activity measurement includes uricase in cell culture media. In some embodiments, cell culture media is Fetal bovine serum (FBS).

[0037] In some embodiments, FBS is mixed with buffer to make serum-based cell culture media. In some embodiments, a mouse serum-based cell culture media may be used. In some embodiments, a serum-free culture media may be used. In some embodiments, the cell culture media includes bovine serum albumin (BSA).

[0038] In another aspect, the present embodiments are directed to a sensor for measuring electrochemical activity, the sensor including: a plurality of three-electrode circuits (e.g., each three-electrode circuit including a working electrode, a counter electrode, and a reference electrode), each electrode of each three-electrode circuit including: an electrode top surface; a conductive contact pad; and a contact electrically coupling the electrode top surface to the conductive contact pad; wherein the electrode top surfaces of each electrode of the three-electrode circuits are arranged in a concentric configuration.

[0039] In some embodiments, the contacts of each of the three electrode circuits are positioned in a linear configuration. In some embodiments, the sensor further comprises a heater circuit positioned at least partially between the electrode top surfaces and the conductive contact pads, wherein the heater circuit is not electrically coupled to the electrode circuit, and wherein the heater circuit comprises a heater trace arranged in a serpentine configuration.

[0040] In another aspect, the present embodiments are directed to a high throughput system including: a multi- well plate (i.e., a 96- well plate or a well plate with a differentnumber of wells); and a sensor board including an array of sensors, wherein each sensor of the array of sensors corresponds to a well of the multi-well plate, in a one-to-one ratio.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present teachings described herein will be more fully understood from the following description of various illustrative embodiments, when read together with the accompanying drawings. It should be understood that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0042] FIG. 1 is a schematic diagram illustrating concepts in electrochemical measurement of biological reactions.

[0043] FIG. 2 is a schematic diagram illustrating direct analysis of electrochemically active molecules for enzymatic activities and representative current vs. potential and potential vs. time graphs.

[0044] FIG. 3 is a schematic diagram illustrating detection of the presence and binding of biomolecules with different examples of detection probes including DNA, peptide, antibody, and aptamer.

[0045] FIG. 4A is a graph of input potential (E) over time, according to an illustrative embodiment.

[0046] FIG. 4B is and a graph of current (I) over time associated with pulsed amperometric detection for uricase activity, according to an illustrative embodiment.

[0047] FIG. 5A is a graph of current as a function of time for uricase activity, according to an illustrative embodiment.

[0048] FIG. 5B is a graph of current as a function of time for enzymatic depletion of uric acid upon adding uricase enzyme, according to an illustrative embodiment.

[0049] FIG. 6A is a graph of a standard calibration curve for uric acid (current as a function of uric acid concentration), according to an illustrative embodiment.

[0050] FIG. 6B is a graph of current change after re-establishing equilibrium following addition of uricase enzyme (current as a function of time) at different concentrations, according to an illustrative embodiment.

[0051] FIG. 6C is a graph of initial reaction velocity as a function of uric acid concentration with Michaelis -Menten fitting, according to an illustrative embodiment.

[0052] FIG. 7 is a schematic diagram of a reference electrode, a counter electrode, and a working electrode patterned on a glass slide, according to an illustrative embodiment.

[0053] FIG. 8 is a schematic diagram of preparing a conductive antifouling coating on a working electrode on a glass slide, according to an illustrative embodiment.

[0054] FIG. 9 is a graph of current as a function of potential using reduced graphene oxide and reduced graphene oxide with poly(3,4-ethylenedioxythiophene (PEDOT) as conductive fillers in an antifouling coating, according to an illustrative embodiment.

[0055] FIG. 10A is a photograph of a 96- well sensor board, according to an illustrative embodiment.

[0056] FIG. 10B is an enlarged view of a photograph of a 96-well sensor board, according to an illustrative embodiment.

[0057] FIG. 11A is a schematic diagram of a sensor plate assembly including a bottomless 96-well plate, a gasket, and a sensor board, according to an illustrative embodiment.

[0058] FIG. 11B is a schematic diagram of a side view of a sensor board and control board connected by interconnects, according to an illustrative embodiment.

[0059] FIG. 12A is a CAD design of a 96-well sensor board, according to an illustrative embodiment.

[0060] FIG. 12B is an enlarged view of a region marked by a dashed circle in FIG. 12A, showing a working electrode, a reference electrode, and a counter electrode, according to an illustrative embodiment.

[0061] FIG. 12C is a CAD design of a 96-well sensor board with example board dimensions (in millimeters), according to an illustrative embodiment.

[0062] FIG. 13 is a schematic diagram of a single-well three-electrode setup, according to an illustrative embodiment.

[0063] FIG. 14 is a photograph of a development control board, in this case, an EmStat Pico Development control board manufactured by PalmSens B V of Houten, Netherlands.

[0064] FIG. 15 is a schematic diagram of a circuit connecting a control board and a sensor board, according to an illustrative embodiment.

[0065] FIG. 16 is a CAD diagram of circuitry in a 96- well control board, according to an illustrative embodiment.

[0066] FIG. 17 is a cross-sectional view of a sensor board, according to an illustrative embodiment.

[0067] FIG. 18A is a graph of current as a function of potential in a hydrogen peroxide I uric acid titration experiment for different hydrogen peroxide concentrations, according to an illustrative embodiment.

[0068] FIG. 18B is a graph of peak current as a function of hydrogen peroxide concentration obtained from the data in FIG. 18A, according to an illustrative embodiment.

[0069] FIG. 18C is a graph of peak current as a function of potential in a hydrogen peroxide I uric acid titration experiment for different uric acid concentrations, according to an illustrative embodiment.

[0070] FIG. 18D are two graphs of peak current as a function of uric acid concentration obtained from the data in FIG. 18C, shown on a linear scale (left) and logarithmic scale (right), according to an illustrative embodiment.

[0071] FIG. 19 is a graph of current as a function of potential for a differential pulse voltammetry (DPV) measurement with ascorbic acid alone and with ascorbic acid and uric acid together, according to an illustrative embodiment.

[0072] FIG. 20A is a graph of signal amplitude over time before signal filtering, according to an illustrative embodiment.

[0073] FIG. 20B is a graph of signal amplitude over time after signal filtering, according to an illustrative embodiment.

[0074] FIG. 21 is a flow chart diagram of a method of forming a coating, according to an illustrative embodiment.

[0075] FIG. 22 is a flow chart diagram of a method of performing automated data collection, according to an illustrative embodiment.

[0076] FIG. 23 is a flow chart diagram of a method of running a sensing test, according to an illustrative embodiment.

[0077] FIG. 24A is a schematic showing the conversion of L-ascorbic acid to L- dehydroascorbic acid enzymatically activated by L-ascorbate oxidase.

[0078] FIG. 24B is a graph of depletion of L-ascorbic acid following addition of L- ascorbate oxidase (consumption as a function of time) at different concentrations of ascorbic acid, according to an illustrative embodiment.

[0079] FIG. 24C is a graph of ascorbate oxidase activity (velocity as a function of ascorbic acid concentration) with Michaelis-Menten fitting, according to an illustrative embodiment.

[0080] FIG. 25A is a calibration curve for uric acid (current as a function of uric acid concentration), according to an illustrative embodiment.

[0081] FIG. 25 B is a graph of reaction velocity as a function of uric acid concentration with Michaelis-Menten fitting, according to an illustrative embodiment.

[0082] FIG. 26 is a graph of uricase activity (current change as a function of time) in different fluids, according to an illustrative embodiment.

[0083] FIG. 27A is an example of a heater plate, according to an illustrative embodiment.

[0084] FIG. 27B is an example of a heater block assembly, according to an illustrative embodiment.

[0085] FIG. 28A is a graph of temperature as a function of time for a sensor board operating at set power input and without a PID controller, according to an illustrative embodiment.

[0086] FIG. 28B is a graph of temperature as a function of time for a sensor board with a PID controller to maintain a temperature setpoint, according to an illustrative embodiment.

[0087] FIG. 29 A is a CAD design of a 96-well sensor board, according to an illustrative embodiment.

[0088] FIG. 29B is a schematic diagram of a cross-section of sensor board, according to an illustrative embodiment.

[0089] FIG. 29C is a schematic diagram of another embodiment of a cross-section of sensor board, according to an illustrative embodiment.

[0090] FIGS. 30A-30I illustrate an example of an automatic procedure using a multiwell sensor plate assembly and an automated liquid handling system, according to an illustrative embodiment.

[0091] FIG. 31A shows an example of data acquired by a 96-well sensor board, according to an illustrative embodiment.

[0092] FIG. 31B shows calibration curves (current as a function of concentration) calculated from data acquired by the 96-well sensor board, according to an illustrative embodiment.

[0093] FIG. 32A is a graph of current as a function of potential obtained by multiple DPV measurements for uric acid in sodium phosphate buffer using a PCB sensor board, according to an illustrative embodiment.

[0094] FIG. 32B is a graph of current as a function of potential obtained by multiple DPV measurements for uric acid in Tris-HCl buffer using a PCB sensor board, according to an illustrative embodiment.

[0095] FIG. 33 shows an exemplary fabrication process of a sensor board on a glass substrate, according to an illustrative embodiment.

[0096] FIGS. 34A-C illustrate an example of a data acquisition and processing method, according to an illustrative embodiment.

[0097] FIGS. 35A-35L show different aspects of a sensor board, according to an illustrative embodiment.

[0098] FIGS. 36A-36J show different aspects of a control board, according to an illustrative embodiment.

[0099] FIGS. 37A-37I show different aspects of a sensor board, according to an illustrative embodiment.

[0100] FIG. 38 is a graph of eight (8) replicates of uricase activity (velocity as a function of uricase concentration) with Michaelis-Menten fitting, according to an illustrative embodiment.

[0101] FIG. 39 is a graph of ten (10) replicates of uricase activity (velocity as a function of uricase concentration) with Michaelis-Menten fitting, at different temperatures, according to an illustrative embodiment.

[0102] FIG. 40 is a graph of four (4) replicates of rasburicase activity (velocity as a function of rasburicase concentration) at different pH levels, with Michaelis-Menten fitting, according to an illustrative embodiment.

[0103] FIG. 41 is a graph of peak current as a function of time with substrate only, and after adding enzyme, according to an illustrative embodiment.

[0104] FIG. 42 is a graph of two (2) replicates of uricase activity (velocity as a function of uricase concentration) in different fluids, with Michaelis-Menten fitting, according to an illustrative embodiment.

[0105] FIG. 43 shows data change as a function of time for each one of the 96 wells, using an ultraviolet (UV) absorbance method (UV method), according to an illustrative embodiment.

[0106] FIG. 44 shows data change as a function of time for each one of the 96 wells, using an electrochemical method (EChem method), according to an illustrative embodiment.

[0107] FIG. 45 shows average of normalized statistical value in different categories, for the UV method and the EChem method, according to an illustrative embodiment.

[0108] FIG. 46 is a CAD design of an integrated heater PCB board, according to an illustrative embodiment.

[0109] FIG. 47 is a schematic of an integrated heater PCB board, according to an illustrative embodiment.

[0110] FIG. 48A is a CAD design of an individual well heater sensor board, according to an illustrative embodiment.

[0111] FIG. 48B is an enlarged view of a sensor including an individual heater trace, according to an illustrative embodiment.

[0112] FIG. 48C is a CAD design of an individual well heater sensor board, according to an illustrative embodiment.

[0113] FIG. 48D is an enlarged view of a sensor including an individual heater trace, according to an illustrative embodiment.

[0114] FIG. 49A illustrates a quadrant of an individual well heater sensor board, according to an illustrative embodiment.

[0115] FIG. 49B illustrates a sub-quadrant of an individual well heater sensor board, according to an illustrative embodiment.

[0116] FIG. 50 illustrates an example of a single well progression curve fitting method, according to an illustrative embodiment.

[0117] FIG. 51A is a CAD design of a heater sensor board, according to an illustrative embodiment.

[0118] FIG. 51B is a CAD design of a heater sensor board, according to an illustrative embodiment.

[0119] FIG. 52A is a portion of a CAD design of a heater sensor board, according to an illustrative embodiment.

[0120] FIG. 52B is a portion of a CAD design of a heater sensor board, according to an illustrative embodiment.

[0121] FIG. 52C is a portion of a CAD design of a heater sensor board, according to an illustrative embodiment.DEFINITIONS

[0122] About, Approximately: As used herein, the terms “about” and “approximately” as used in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” and “approximately” in that context. For example, in some embodiments, the terms “about” and “approximately” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0123] Amperometry: As used herein, the term, “Amperometry” refers to an electroanalytical technique that involves the application of a constant reducing or oxidizing potential to a working electrode and the subsequent measurement of the resulting steady-state current. Amperometry methods may include single-potential amperometry and pulsed amperometry (or pulsed amperometric detection, PAD).

[0124] Associated with: As used herein, the term, “associated with” refers to two events or entities when presence, level and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of and / or susceptibility to a disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non- covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0125] Conductive filler: As used herein, the term, “conductive filler” refers to materials that are added to a polymer to make it electrically conductive. In some embodiments, a conductive filler may include conductive particles, conductive filaments, conductive polymers. In some embodiments, a conductive filler may include metals, semiconductors, ceramics, organic materials, inorganic materials, and nanostructured materials. The conductive filler may form a path through the polymer material to allow electric current to flow. For conductive fillers that include discrete particles or components, the conductive path may be formed by contacts between adjacent particles. The minimum concentration of a conductive filler composed of discrete particles or components that is required for the polymer to start conducting is known as percolation threshold.

[0126] Crosslinker, crosslinking agent, crosslinking reagent: As used herein, the term, “crosslinker” refers to a molecule that contains two or more reactive ends that are capable of chemically attaching to specific functional groups on other molecules. A crosslinker is employed to create bridging or interconnecting of two chemical compounds. Insome embodiments, a crosslinker may be an agent or a molecule or a substance that is added to a polymer or a polymer blend to induce crosslinking of the polymer upon application of a stimulus (e.g., heat, ultraviolet light, etc.).

[0127] Differential pulse voltammetry (DPV): As used herein, the term “differential pulse voltammetry” refers to a technique that involves applying amplitude potential pulses on a linear ramp potential.

[0128] Doctor Blade Coating: As used herein, the term, “doctor blade coating” refers to a technique used to form films or coatings with well-defined thicknesses on surfaces. The technique includes placing a sharp blade at fixed distance from a surface to be covered. A coating solution is then placed in front of the blade and the blade is moved across parallel with the surface, creating a wet film.

[0129] Hydrogel: As used herein, the term, “hydrogel” refers to a three-dimensional network composed of hydrophobic polymers synthesized by crosslinking water-soluble polymers. Hydrogels can retain a large quantity of water within their network without disturbing their original structure which leads to their flexibility and swelling properties.

[0130] High-Throughput: As used herein, the term, “high-throughput” refers to a method for scientific experimentation in which the use of automated equipment such as liquid handling robots, sensitive detectors, and data processing / control software allows a researcher to conduct a large number of tests within a certain period of time. In some embodiments, high-throughput refers to the ability to rapidly conduct many experiments in parallel or in series.

[0131] Liquid Handling: As used herein, the term, “liquid handling” refers to transferring liquid from one location to another in a laboratory, via a diverse range of large- scale robotic platforms to handheld single channel pipettes. Use of robotic liquid handling minimizes contamination, reduces inconsistencies in sample yield and prepares uniform quantities. In some embodiments, liquid handling may refer to automated liquid-handling systems or robots.

[0132] Pulsed Amperometric Detection (PAD): As used herein, the term, “pulsed amperometric detection (PAD)” refers to a technique that is used to detect certain classes of compounds that tend to foul the surface of an electrode. In PAD, a rapid sequence of potential pulses is applied. Part of the ‘pulse sequence’ is used to sample the signal and therest is used for oxidative and reductive cleaning. During this cleaning, the working electrode is regenerated to ensure stable and reproducible detection conditions.

[0133] Voltammetry, Cyclic Voltammetry: As used herein, the term, “Voltammetry” refers to a collection of techniques in which the relationship between current and voltage is observed during electrochemical processes. In voltammetry, a range of potentials are scanned where the generated current is directly proportional to the concentration of electroactive species present in the sample. As used herein, the term, “cyclic voltammetry” is based on a rapid voltage scan technique in which the direction of voltage scan is reversed. When a potential is applied at the working electrode in both forward and reverse directions the resulting current can be recorded as a single cycle or multiple cycles.

[0134] Microcontroller: As used herein, the term, “microcontroller” refers to a compact integrated circuit designed to govern a specific operation in an embedded system. A typical microcontroller may include a processor, a memory and input / output peripherals on a single chip.

[0135] Multiplexer and Demultiplexer: As used herein, the term “multiplexer” refers to a combinational logic circuit that selects between several input signals and forwards the selected input to a single common output line by the application of a control signal. In some embodiments, a multiplexer may be an analog multiplexer or a digitally controlled analog switch. Analog multiplexers / switches can act as either multiplexers or demultiplexers as they are not directional like digital multiplexers I demultiplexers are. In some embodiments, analog switches may be referred to as demultiplexers or multiplexers depending on which direction the analogic signal is moving, but the hardware remains the same. In general, the circuit may be a digitally controlled analog switch that may control multiple inputs or outputs.

[0136] Multiwell Plate: As used herein, the term, “multiwell plate” or “multi- well plate” (used interchangeably) refers to a flat plate with multiple wells that are used as small test tubes. Multiwell plates are standard tools in research and clinical diagnostic testing laboratories and have standard sizes. Common multiwell plates may have 6, 12, 24, 48, 96, 384, 1536, 6144, or 9600 wells arranged in an array. In certain embodiments, a “multi-well” has at least 6 wells, at least 12 wells, at least 24 wells, at least 48 wells, at least 96 wells, atleast 384 wells, at least 1536 wells, at least 3456 wells, at least 6144 wells, or at least 9600 wells.

[0137] SBS Microplate Standards: As used herein, the term, “SBS Microplate Standards” refers to dimensional standards for a microplate which was first introduced by the members of the Society for Biomolecular Screening (SBS). This standard defines the well center positional requirements of a microplate and outlines the conditions required for making necessary measurements.

[0138] Three-Electrode System: As used herein, the term, “three-electrode system” refers to a configuration of a potentiostat, which is an electronic instrument that controls the voltage between two electrodes. The three-electrode system consists of a working electrode, a counter electrode, and a reference electrode. The working electrode is where the chemistry of interest occurs. The reference electrode acts as a reference in measuring and controlling the working electrode potential, without passing any current. The counter electrode passes all the current needed to balance the current observed at the working electrode.ABBREVIATIONS

[0139] BSA = bovine serum albumin

[0140] CAD = computer-aided design

[0141] CeO2 = Cerium(IV) oxide

[0142] EDC / NHS = (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide) / (N- hydroxysuccinimide)

[0143] ENIG = electroless nickel immersion gold

[0144] EPIG = electroless palladium immersion gold

[0145] FBS = Fetal Bovine Serum

[0146] GelMA = gelatin methacrylate

[0147] GO = Graphene Oxide

[0148] H2O2 = Hydrogen peroxide

[0149] IC = integrated circuit

[0150] LPMO = lytic polysaccharide monooxygenase

[0151] pAA = polyacrylic acid

[0152] PAD = Pulsed Amperometric Detection

[0153] PANI = polyaniline

[0154] PBS = phosphate-buffered saline

[0155] PCB = printed circuit board

[0156] PEDOT = poly (3, 4-ethylenedioxy thiophene)

[0157] PEDOT:PSS = poly(3,4-ethylenedioxythiophene) polystyrene sulfonate

[0158] PID = proportional-integral-derivative controller

[0159] PNIPAM = poly(N-isopropylacrylamide)

[0160] PPy = polypyrrole

[0161] PVD = Physical Vapor Deposition

[0162] rGO = reduced graphene oxide

[0163] SBS = Society of Biomolecular Screening

[0164] Tris-HCl = Tris Hydrochloride

[0165] UV = UltravioletDETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0166] It is contemplated that methods, systems, compositions, and processes described herein encompass variations and adaptations developed using information from theembodiments described herein. Adaptation and / or modification of the methods, systems, compositions, and processes described herein may be performed, as contemplated by this description.

[0167] Throughout the description, where methods, systems, compositions, and / or processes are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are embodiments of the systems and / or compositions that consist essentially of, or consist of, the recited components, and that there are embodiments of the processes and methods that consist essentially of, or consist of, the recited steps.

[0168] It should be understood that the order of steps or order for performing certain action(s) is immaterial so long as the method and / or process remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0169] The mention herein of any publication, for example, in the Background section, is not an admission that the publication serves as prior art with respect to any of the claims presented herein. The Background section is presented for purposes of clarity and is not meant as a description of prior art with respect to any claim.

[0170] Documents are incorporated herein by reference as noted. Where there is any discrepancy in the meaning of a particular term, the meaning provided in the Definition section above is controlling.

[0171] Headers are provided for the convenience of the reader; the presence and / or placement of a header is not intended to limit the scope of the subject matter described herein.

[0172] Provided herein are improved technologies for electrochemical measurements of biological, biochemical, and chemical reactions.I. Overview of High-Throughput Electrochemical Detection Platform

[0173] Many reactions that occur in biological, biochemical, or chemical systems involve electron transfer but are commonly measured by optical or other means. However, detection of signals such as changes in color and emission of light is slower than the electron transfer step, and can be affected by other components in a complex fluid environment. By combining a multiplexed electrochemical detection platform with an antifouling coating,high-throughput and high-speed measurement capabilities, a broad range of reactions can be measured. Described herein is a sensor board with an array of three-electrode wells that is compatible with automated liquid handling systems, a conductive antifouling coating on the array of electrodes to prevent fouling of the electrodes for multiple measurements, and a control board to rapidly process signals from the array of electrodes.

[0174] FIG. 1 is a schematic diagram illustrating key concepts in electrochemical measurement of biological reactions. Electrochemical biosensors are devices that can directly convert biological signals into electrical signals for fast, real-time readout. As shown in FIG. 1, electrochemical biosensors may include biological sensing elements 10, transducer element electrodes 20 with a readable output 22, and a signal processor 24. The biological sensing elements 10 may be housed on (or otherwise operatively coupled to) the transducer element electrodes 20. The biological sensing elements 10 may include DNA 12, antibodies, cells 16, and / or enzymes 18.

[0175] FIG. 2 is a schematic diagram illustrating direct analysis of electrochemically active molecules for enzymatic activities and representative current vs. potential and potential vs. time graphs. For example, enzymes 18, electrons 28, and / or other products 30 from various substrates 26 may be sensed by a recognition element based on 1) measured current as a function 38 of voltage (potential) and / or 2) measured voltage (potential) as a function 40 of time. The recognition element(s) 34 may be communicatively and / or electrically coupled with one or more electrodes 36, as described herein. Electrochemical sensing of electron transfer enables accurate measurement of enzymatic activity. For example, electrochemical measurement can detect electron transfers during enzymatic reactions in a sensitive and specific way. Any analyte (for example, substrate 26, product 30 or even enzyme 18) that can be oxidized or reduced can be measured electrochemically.

[0176] FIG. 3 is a schematic diagram illustrating detection of the presence and binding of biomolecules with different examples of detection probes including DNA 66, peptides 68, antibodies 42, and aptamers 72. As illustrated in FIG. 3, the detection methodologies may include multistep sandwich sensing 50, binding-induced folding sensing 60, and proximity binding-based affinity sensing 64. Multistep sandwich sensing 50 may include the use of a first antibody 42 (for example, including a tag 74) interacting with a second antibody 44 that is coupled to the working electrode 48, in combination with one or more DNA strands 66 interacting with the working electrode 48, thereby producing adetectable electrical response 52 (for example, a detectable increase or spike in current as a function of potential). Binding-induced folding sensing 60 and proximity binding -based affinity sensing 64 may both also using working electrodes 48 (for example, negatively- charged working electrodes 48) according to aspects of the present embodiments.Example: Pulsed Amperometric Detection of Enzymatic Depletion of Uric Acid by Uricase

[0177] Pulsed Amperometric Detection (PAD) is a technique that may be used to sense electrochemically active analytes. Amperometry measures the current flow between electrodes during an oxidation or reduction. During mixing, once diffusion of the analyte reaches equilibrium (i.e., in the diffusion limited regime), the current can be quantified byI = nFADC, where I is the current, n is the number of electrons transferred, F is the Faraday constant, A is the area of electrode, D is the diffusion constant of the analyte, and C is the concentration. This indicates for a known analyte (for example, uric acid), amperometry may be performed at the oxidation potential of the analyte, such that the following relation can be derived: =nFAD is constant for the same analyte in the same experiment. Theconcentration change can be calculated by measuring the current change over time (for example, as shown in FIG. 4B), according to the following: dC _ 1 di dt nFAD dt‘The constant nFAD can be acquired by linearly fitting I=nFADC at different substrate concentrations (for example, as shown in FIG. 6A).

[0178] FIGS. 4A and 4B include a graph of input potential (E) over time 101 (FIG. 4A), and a graph of current (I) over time 101 (FIG. 4B) associated with pulsed amperometric detection for uricase activity, according to an illustrative embodiment. PAD may be used to measure the current at a pulsed oxidation potential of uric acid (for example) for a brief duration. When not measuring, the potential is set to clean the electrode (i.e., during pretreatment 102) to prevent possible fouling on the surface. Uric acid may be added to the electrode at varying concentrations. Once the current has stabilized, the diffusion hasreached equilibrium (i.e., pre-equilibrium 108 shown in FIG. 4B), and a current baseline 107 may be obtained. In some embodiments, the current may be considered to be stable when the max-min change in current is less than about 5% (or less than about 4-6%, 3-7%, or 2-8%). After the baseline is stable, diluted enzyme may be added 110 to the electrode. Once the current has stabilized, the diffusion has reestablished equilibrium (i.e., re-equilibrium 112). The decrease in current 109 (for example dl / df), which is related to the decrease in uric acid concentration, may then be recorded.

[0179] Referring still to FIGS. 4A and 4B, the illustrated amperometry was performed at the oxidation potential of the substrate (uric acid) using pulsed amperometric detection method (PAD). The experiments may be run in two parts. The first part may include a standard calibration, where different concentrations of uric acid are added separately, and the slope 109 from current and concentration plot (FIG. 4B) is used to convert current decay to substrate consumption. The second part may include running the enzymatic kinetics assays. The same volume of different concentration of substrate may be added. Once the baseline 107 is stabilized (max-min current change is smaller than 5%, for example), the same amount of enzyme is added 110 to the substrate. The current decrease may then be recorded. The initial linear decrease slope 109 is used to calculate substrate consumption rate, which reflects enzymatic velocity at different substrate concentrations.

[0180] FIG. 5A is a graph of current as a function of time for uricase activity using 300uM uric acid as substrate, according to an illustrative embodiment. Enzyme (uricase) is added after pre-equilibrium 114. Once re-equilibrium 116 is reached, the initial linear decrease slope 109 can be measured during the enzymatic reaction 118.

[0181] FIG. 5B is a graph of current as a function of time for complete enzymatic depletion of uric acid upon adding uricase enzyme, according to an illustrative embodiment. As illustrated in FIG. 5B, enzyme is added 122, and the decrease in current of 300uM uric acid with uricase 124 is plotted with Tris-HCl buffer 126 on the same platform, according to aspects of the present embodiments. The steadiness (i.e., constant current signal) of the Tris- HCl buffer 126 demonstrates that the platform is not negatively affecting uricase activity (for example, not introducing sources of variation to the measured current signal response and / or enzymatic reaction).

[0182] FIG. 6A is a graph of a standard calibration curve for uric acid (current as a function of uric acid concentration), according to an illustrative embodiment. As illustrated in FIG. 6A, the standard calibration curve is linear through the full range of uric concentrations tested.

[0183] FIG. 6B is a graph of current change after re-establishing equilibrium following addition of uricase enzyme (current as a function of time) at different concentrations, according to an illustrative embodiment. The current change for concentrations from 0 pM to 300 pM are illustrated in FIG. 6B. At 0 pM, there is no current change, and at concentrations below 75 pM, (for example, at 25 pM and 50 pM, the current change is very small. At higher concentrations (for example, at 150 pM, 200 pM, and 300 pM) the current change is much larger. As illustrated in FIG. 6B, each of the current changes following re-equilibrium may include in initial steep drop 127 in current following by a more linear drop 129 in current. The data shown in FIG. 6B were taken with 0.1 second pulse durations, recorded every second, and following a 60 second equilibrium period.

[0184] FIG. 6C is a graph of initial reaction velocity as a function of uric acid concentration with Michaelis -Menten fitting, according to an illustrative embodiment. The plots illustrated in FIGS. 6A-6C are based on measurements with uricase from Arthrobacter globiformis bacteria. The same methodology can be used in connection with measurements of substrates, products, enzymes, etc. The measurements were run with uricase with a uric acid substate that was electrochemically measured to evaluate enzymatic activities. When the methodology is used with other enzymes, such as LPMO (lytic polysaccharide monooxygenase), the resulting enzymatic product is hydrogen peroxide, which can be measured; in some cases the enzyme itself can be measured. The methodologies of the present embodiments can be used with smaller time intervals (for example, from about 1 ps, 5 ps, and / or 10 ps to several seconds, for example from about 10 ps to about 1 second), which can be used in connection with the progress curve described herein (FIG. 6C) to measure all six rate constants. The methodologies of the present embodiments can also be used to compare enzymatic activity at different conditions, including pH, temperature, interaction with ions, specificity, viability etc.Example: Depletion of Ascorbic Acid by Ascorbate Oxidase

[0185] FIG. 24A is a schematic showing the conversion of L-ascorbic acid to L- dehydroascorbic acid enzymatically activated by L-ascorbate oxidase. L-ascorbate oxidase is an enzyme that catalyzes the chemical reaction between L-ascorbate acid and O2, which results in dehydroascorbate and H2O.

[0186] FIG. 24B is a graph of depletion of L-ascorbic acid following addition of L- ascorbate oxidase (consumption as a function of time) at different concentrations of ascorbic acid, according to an illustrative embodiment. The depletion of ascorbic acid at concentrations from 0 pM to 4000 pM are illustrated. At 0 pM, there is no depletion, and the depletion increases with increasing concentrations (250 pM, 500 pM, 1000 pM, 2000 pM and 4000 pM). The measurements were performed using a 0.2M sodium phosphate buffer with 10 mM EDTA, at a pH of 7.4 and a temperature of 21.3 °C.

[0187] FIG. 24C is a graph of ascorbate oxidase activity (velocity as a function of ascorbic acid concentration) with Michaelis-Menten fitting, according to an illustrative embodiment. The plots illustrated in FIGS. 24A-24C are based on measurements with ascorbate oxidase from Cucurbita sp.Example: Horseradish Peroxidase activity using hydrogen peroxide as substrate

[0188] This example demonstrates the capability of the electrochemical platform to measure Horseradish Peroxidase activity. FIG. 41 is a graph of peak current as a function of time after adding substrate (27 mM H2O2) only 600, and enzyme 602, according to an illustrative embodiment. The slight decrease in the signal, after adding substrate 600, is due to the natural decomposition of hydrogen peroxide. The significant decrease in the signal, after adding Horseradish Peroxidase 602, is due to enzymatic activities.Electrochemical Measurement Accuracy

[0189] To evaluate the accuracy of the electrochemical measurements, uricase activity was measured using electrochemical and UV absorbance methods, and the results were compared with literature. FIG. 25A is a calibration curve for uric acid (current as a function of uric acid concentration), according to an illustrative embodiment. FIG. 25B is a graph of reaction velocity as a function of uric acid concentration with Michaelis-Mentenfitting, according to an illustrative embodiment. The Michaelis -Menten fitting method may determine catalytic constant (kcat) and Michaelis constant (Km) of uric acid, which may be used to define specificity constant (fcCfl / / Km) of uric acid.

[0190] The UV absorbance method may include a standard approach to measure uric acid (the substrate) concentration at its absorbance wavelength. To evaluate uricase activity, the decrease in absorbance at 292 nm wavelength resulting from the conversion of uric acid to allantoin is measured. Table 1 shows the measurements results obtained from literature (A.C. Nyborg et al., “A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties”, PLoS One 11(11): ee0167935 (2016)), UV absorbance, and the presently disposed electrochemical method. The values obtained by the electrochemical method show similar accuracy to the values obtained by the UV absorbance method and the reported values from Nyborg et al. (i.e., with kcat / Kmvalue very close to the value from Nyborg et al., and within a factor of 2 of the value from UV absorbance measurements).Table 1:

[0191] The consistency of substrate measurements across a 96-well plate by the electrochemical platform, was evaluated using commercial enzyme uricase from E.coli. The activity of uricase was measured at room temperature and pH of 7.4, and the measurement was replicated 8 times. FIG. 38 is a graph of eight (8) replicates of uricase activity (velocity as a function of uricase concentration) with Michaelis-Menten fitting, according to an illustrative embodiment. The consistent results across the 96-well plate demonstrate the electrochemical platform may provide highly reproducible enzymatic activity measurements.

[0192] The consistency of enzymatic activity measurements across a 96-well plate was compared for the EChem and the UV methods, using 300pM uric acid as substrate. The uric acid signal was measured continuously over time by each method. FIG. 43 shows the data change as a function of time for each one of the 96 wells, using the UV method,according to an illustrative embodiment. FIG. 44 shows the data change as a function of time for each one of the 96 wells, using the EChem method, according to an illustrative embodiment. FIG. 45 shows average of normalized statistical value in different categories, for the UV method 608 and the EChem method 610, according to an illustrative embodiment. The categories include Root Mean Square of the Derivative (RMSD), Standard Deviation of Data Variations (STDEV), and Variance of the Derivative (VOD). The normalized RMSD and STDEV of the UV method 608 and the EChem method 610 are comparable, while VOD of the EChem method 610 is significantly lower than the UV method 608. The EChem method 610 shows a more consistent and predictable behavior, whereas the UV method 608 exhibits significant variability, rendering it more erratic and less stable. Accordingly, the EChem method is more reliable for applications where predictability and / or stability of changes are crucial.

[0193] The enzymatic activity measurements across a 96- well plate, at different temperatures, by the electrochemical platform, was evaluated using commercial recombinant enzyme uricase from E.coli. The activity of uricase was measured at a pH of 7.4, and measurement at each temperature was replicated 10 times. FIG. 39 is a graph of ten (10) replicates of uricase activity (velocity as a function of uricase concentration) with Michaelis- Menten fitting, at different temperatures, according to an illustrative embodiment. The velocity for uricase at a temperature of 37° (series 590) and at room temperature (series 592) are plotted. The measured activities are consistent at each temperature, and the increase in activity at higher temperature (37° C, series 590) is consistent with literature reporting higher activities of at 37° C compared to room temperature. These results demonstrate the capability of the electrochemical platform of the present disclosure to measure activities with and without a heater.

[0194] The enzymatic activity measurements across a 96- well plate, at different pH levels, by the electrochemical platform, was evaluated using commercial enzyme rasburicase. The activity of rasburicase was measured at different pH levels, and measurement at each pH level was replicated 4 times. FIG. 40 is a graph of four (4) replicates of rasburicase activity (velocity as a function of uricase concentration) at different pH levels, with Michaelis- Menten fitting, according to an illustrative embodiment. The velocity at pH level of 6 for rasburicase in PBS (series 594), pH level of 7.4 for rasburicase in PBS (series 596), and pH level of 8.5 for rasburicase in borate buffer (series 598) are plotted. The measured activities are consistent at each pH level, and the increase in activity at a higher pH level (8.5, series598) is consistent with literature reporting higher rasburicase activity at higher pH levels. These results demonstrate the capability of the electrochemical platform of the present disclosure to measure activities across a range of pH levels.

[0195] FIG. 26 is a graph of uricase activity (current change as a function of time) in different fluids, according to an illustrative embodiment. The current change for uricase in buffer 394, uricase in serum 396, and serum only 392 are plotted. The serum is a mouse serum and includes 1 / 5 of the volume of uricase and serum mixture. The uricase activity was measured at room temperature. The successful measurement of uricase activity in serum demonstrates the compatibility of the electrochemical assay with complex fluids (e.g., serum), whereas traditional methods are not compatible with complex fluids (e.g., UV absorbance methods may be complicated by the presence of additional molecules in complex fluid that may absorb at similar wavelengths at uric acid, thus making it difficult to distinguish the absorbance of uric acid from the background signal).

[0196] The enzymatic activity measurements across a 96- well plate, in different complex fluids, by the electrochemical platform, was evaluated using commercial enzyme uricase from E.coli. The activity of uricase was measured at a temperature of 37° C and pH level of 7.4, and measurement in each fluid was replicated 2 times. FIG. 42 is a graph of two (2) replicates of uricase activity (velocity as a function of uricase concentration) in different fluids, with Michaelis-Menten fitting, according to an illustrative embodiment. The velocity for uricase in cell culture media (series 604), and uricase in buffer (series 606) are plotted. In this example, the cell culture media is Fetal Bovine Serum (FBS). The measured activities in FBS 604 and in buffer 606 are comparable, demonstrating the capability of the electrochemical platform of the present disclosure to measure activities in a wide range of fluid conditions.Planar Three-Electrode System

[0197] FIG. 7 is a schematic diagram of a sensor 178 comprising a reference electrode 142, a counter electrode 144, and a working electrode 146 patterned on a glass slide 148, according to aspects of the present embodiments. In some embodiments, the working electrode 146 may include a substantially circular geometry and may be concentrically disposed about a sensing center point 141. In some embodiments, the counter electrode 144may be substantially ring-shaped, and may be disposed concentrically around each of the working electrode 146 and the sensing center point 141. In some embodiments, the reference electrode 142 may be substantially ring-shaped, and may be disposed concentrically around each of the counter electrode 144, the working electrode 146, and the sensing center point 141. In some embodiments, the sensor 178 may include a first gap 143 separating an outer circumference of the working electrode 146 from an inner circumference of the counter electrode 144. In some embodiments, the sensor 178 may include a second gap 145 separating an outer circumference of the counter electrode 144 from an inner circumference of the reference electrode 142. In some embodiments, each of the first and second gaps 143, 145 are substantially constant along their respective circumferential lengths. In some embodiments, the shapes of the planar electrodes may be other geometries or shapes (i.e., any non-circular shape). In some embodiments, the shapes of the planar electrodes can be, e.g., rectangular, triangular, square, oval, etc.

[0198] Referring still to FIG. 7, the sensor 178 may include a first lead 147 coupled (for example, electrically coupled) to the working electrode 147, a second lead 149 coupled (for example, electrically coupled) to the counter electrode 144, and a third lead 151 coupled (for example, electrically coupled) to the reference electrode 152. In some embodiments, each of the first, second, and third leads 147, 149, 151 are aligned such that they substantially parallel to each other. In some embodiments, the first lead 147 is disposed between, and spaced apart from, the second and third leads 149, 151. In some embodiments, each of the counter electrode 144 and reference electrode 142 extends substantially around the full outer circumference of the working electrode 146. For example, in some embodiments, each of the counter electrode 144 and reference electrode 142 extends from about 270° to about 360°, or from about 290° to about 350°, or from about 300° to about 340°, or from about 310° to about 330° (and / or other subranges therebetween) about the outer circumference of the working electrode 146. In some embodiments, the circumferential angular range through which each of the counter electrode 144 and reference electrode 142 does not extend allows space for each of the first, second, and third leads 147, 149, 151 to extend externally from the sensor 178 (or stated otherwise, toward an exterior of the sensor 178) such that they may be electrically coupled to other external components, as described herein, while still being coupled to the respective working electrode 146, counter electrode 144, and reference electrode 142.

[0199] According to aspects of the present embodiments, sensors 178 (for example, bio-sensors, for example, three-electrode sensors) may include other arrangements, configurations, and / or geometries of the three electrodes. For example, FIG. 10B illustrates an enlarged view of the 96-well sensor board 184 shown in FIG. 10A. Similarly, FIG. 12B illustrates an enlarged view of the 96-well sensor board CAD design 192 shown in FIG. 12A. In some embodiments, each of the sensors 178 shown in FIGS. 10B and 12B may include both a counter electrode 196 that extends the full 360° around the working electrode 194 as well as a reference electrode 198 that extends the full 360° around the counter electrode 196. In addition, in some embodiments, each of the sensors 178 shown in FIGS. 10B and 12B may include both a first gap 143 separating the working electrode 194 from the counter electrode 196 as well as a second gap 145 separating the counter electrode 196 from the reference electrode 198. In some embodiments, the shapes of the electrodes may be other geometries or shapes (e.g., circular, non-circular, rectangular, square, triangular, or other shapes). The shapes of electrodes may vary and will perform the necessary electrochemical functions, as long as they fit within the area contained within a well in a multi- well plate (i.e., within the area at the bottom of the volume of an opening in a gasket).

[0200] FIG. 13 is a schematic diagram of a single-well three-electrode setup, according to aspects of the present embodiments. The sensor 178 shown in FIG. 13 includes a three-electrode setup with a similar topology to that of the sensor 178 shown in FIG. 7, though with a different geometry. For example, in some embodiments, the working electrode 200 may be coupled (i.e., electrically coupled) to a first contact 205 (for electrically coupling to external components) via a first lead 199. The counter electrode 202 may be electrically coupled to a second contact 207 via a second lead 201. The reference electrode 204 may be electrically coupled to a third contact 209 via a third lead 203. In some embodiments, the second contact 207 may be disposed between the first and third contacts 205, 209, while the second lead 201 may be disposed between the first and third leads 199, 203. In some embodiments the second lead 201 may include a first bend 211 (for example, a 90-degree bend) and a second bend 213 (for example, a 90-degree bend). In some embodiments, the first, second, and third leads 199, 201, 203 may each include zero bends. In some embodiments, the first, second, and third leads 199, 201, 203 may each include one or more bends (for example, 1 bend, 2 bends, 3 bends, 4 bends, 5 bends, or more bends), each bend of a different angle (for example, a 30 degree bend, a 45 degree bend, a 90 degree bend, a 135 degree bend, or other angles).

[0201] Referring still to FIG. 13, the sensor 178 may include a first gap 143 separating the working electrode 200 and the counter electrode 202 as well as a second gap 145 separating the counter electrode 202 and the reference electrode 204. In some embodiments, the working electrode 146 may include a substantially circular geometry and may be concentrically disposed about a sensing center point 141. While each of the counter electrode 202 and the reference electrode 204 may be disposed and centered around the working electrode 200 and sensing center point 141, in some embodiments, the counter electrode 202 may include a partially square shaped with one or more rounded vertices. Accordingly, in some embodiments, the first gap 143 may be substantially ring-shaped while the second gap 145 may be at least partially square-shaped. In some embodiments, each of the counter electrode 202 and reference electrode 204 extends from about 270° to about 360°, or from about 290° to about 350°, or from about 300° to about 340°, or from about 310° to about 330° (and / or other subranges therebetween) about the outer circumference of the working electrode 200 and sensing center point 141. In some embodiments, each of the first, second, and third leads 199, 201, 203 comprises a thickness that is substantially the same as (for example, within about 5% or with about 10%) of a width of the first and / or second gaps 143, 145.

[0202] Referring to FIGS. 7, 10B, 12B, and 13, the three-electrode configurations described herein may also be referred to as an electrochemical cell since these sensor arrangements (and / or devices or biosensors that include them) are used for performing the electrochemical measurements. As described herein, the electrochemical front end (the EM Stat Pico module or equivalent such as the Analog Devices AD5940) sends the signal to the counter electrode 202 on the electrochemical cell or sensor 178 based on feedback from the reference electrode 204. The electrochemical front end (i.e., the EM Stat Pico module or equivalent) sends a signal (e.g., voltage) to the counter electrode 202 on the electrochemical cell. This signal (e.g., voltage) is adjusted by the EChem front end based on the feedback (e.g., voltage) measured from the reference electrode 204 on the electrochemical cell / sensor 178. The electrochemical front end measures the current from the working electrode 200 on the electrochemical cell I sensor 178. According to aspects of the present embodiments, and depending on the design and / or layout of various control, sensor, and / or circuit boards, the configurations of FIGS. 7, 10B, 12B, and / or 13 (all of which include planar layouts) may be used. For example, in some embodiments, the leads I contacts may need to be on, or connect to, one side of the board or the other (and as such, the topology of the layout of FIG. 13 (oralternatively, the layout of the other figures) may be chosen based on a specific configuration).IL Conductive Antifouling Coating

[0203] Accumulation of unwanted biological material (fouling) on surfaces greatly affects sensitivity and functionality of sensors and other equipment. Most electrochemical sensors may have the tendency to suffer from signal deterioration if they are not functionalized with an anti-fouling coating or other surface treatment. Anti-fouling surface medications (e.g., coatings) therefore may be required for electrochemical biosensors in order to prevent accumulation of foulants on the sensor.

[0204] FIG. 8 is a schematic diagram of preparing a conductive antifouling coating on a working electrode on a glass slide, according to aspects of the present embodiments. As shown in FIG. 8, a hydrogel precursor layer 152 may be added 150 to the working electrode 154 (which is disposed on a glass slide 156). In some embodiments, the electrode surface may be activated to allow attachment of the hydrogel (e.g., using piranha etch to hydroxylate the surface). In some embodiments, a hydrogel precursor may be deposited by drop casting, spin coating, printing (e.g., screen printing, inkjet printing, or other printing methods), doctor blading, or stamping. Hydrogel crosslinking 158 is performed to convert the hydrogel precursor layer 152 into a crosslinked hydrogel 160. In some embodiments, hydrogel crosslinking may include external thermal or ultraviolet (UV) sources to initiate hydrogel polymerization in cases where a thermally activated or UV-sensitive crosslinker and initiator, respectively, are used. In some embodiments, the hydrogel precursor 152 may include hydrogel monomer plus a crosslinker 166 disposed within a conductive filler 168, which yields a hydrogel matrix 172, as described herein. In some embodiments, a razor blade 170 may be used to ensure consistent thickness of the conductive antifouling coating and to remove any excess precursor. In some embodiments, a razor blade 170 may be part of a doctor blade coater or an adjustable thin film applicator.

[0205] FIG. 9 is a graph of current as a function of potential using reduced graphene oxide and reduced graphene oxide with poly(3,4-ethylenedioxythiophene (PEDOT) as conductive fillers in an antifouling coating, according to an illustrative embodiment. In some embodiments, the conductive filler 168 may include conductive fillers 168 in a BSA (BovineSerum Albumin) matrix. In some embodiments, conductive fillers 168 may include reduced graphene oxide (rGO) 174. In some embodiments, conductive fillers 168 may include rGO with PEDOT 176. As illustrated in FIG. 9, the reduced rGO with PEDOT 176 results in a higher potential for a given current than rGO 174 alone. In addition, according to aspects of the present embodiments, both rGO with PEDOT 176 and rGO 174 demonstrate that they are able to be measure uric acid via differential pulsed voltammetry.Antifouling Coating Compositions and Structures

[0206] According to aspects of the present embodiments, antifouling coating materials may include two basic components, one component to provide anti-fouling properties, such as a hydrogel, and another component to provide conductivity, such as a conductive filler like carbon or nanoparticles. In some embodiments, the pore size of the hydrogel matrix as described herein allows for physical immobilization of conductive fillers, and the conductive filler may be loaded above the electrical percolation threshold to form conductive pathway within the matrix. In some embodiments, the coating may include a single component serving as both an anti-fouling hydrogel matrix, and also as a conductive substrate, such as conductive polymer hydrogels (e.g., a Ppy / PEDOT:PSS matrix, gelatin methacrylate (GelMA), poly(N-isopropylacrylamide) (PNIPAM), polyacrylic acid (pAA), etc.). In some embodiments, conductive fillers may include metal nanoparticles (e.g., gold nanoparticles), carbon nanotubes (CNTs), and / or conductive polymers (e.g., polyaniline (PANI)). In some embodiments, additional sensing elements may be added to the matrix based on a specific analyte(s) (e.g., CeOi nanoparticles for H2O2 detection).

[0207] In some embodiments, the antifouling coating of the present embodiments can survive at least 3, 6, 9, and / or 12 months of extensive usage. In some embodiments, the antifouling coating of the present embodiments remains stable in a refrigerated environment. In some embodiments, the antifouling coating of the present embodiments can be restored (i.e., repaired) via cyclic voltammetry (if needed). In some embodiments, the antifouling coating of the present embodiments may include a thickness from a sub-micrometer to about 5 micrometers. In some embodiments, the antifouling coating of the present embodiments may include a thickness from about 0.5 micrometers to about 5 micrometers, or from about 0.5 micrometers to about 3 micrometers, or from about 0.7 micrometers to about 2micrometers, or from about 0.7 micrometers to about 1.5 micrometers, or from about 0.8 micrometers to about 1.2 micrometers, or from about 0.9 micrometers to about 1.1 micrometers, and / or other subranges therebetween. Accordingly, the antifouling coating of the present embodiments provides a versatile coating that enables measurements and the recording of signals (even in a complex matrix) while also preventing or minimizing fouling.Coating Methods

[0208] FIG. 21 is a flow chart diagram of a method of forming a coating, according to an illustrative embodiment. At step 294, the method 292 may include cleaning and activating the electrode surfaces by submerging the electrodes (for example, at least the working electrodes) in piranha solution for a period of about 3 hours (for example, for a period of about 2 to about 4 hours, or for period of about 2.5 to 3.5 hours, and / or for a period of about 2.75 to 3.25 hours). In some embodiments, electrodes are submerged in piranha solution for a period of about 1 to 20 minutes (for example, for a period of about 1 to 15 minutes, for period of about 1-10 minutes, for a period of about 1 to 8 minutes, for a period of about 2 to 8 minutes, for a period of about 2 to 6 minutes, for a period of about 2 to 5 minutes, and / or for a period of about 3 to 5 minutes). In some embodiments, the piranha solution may include 3 parts of 98% concentration sulfuric acid and 1 part of 30% hydrogen peroxide solution, prepared fresh. At step 296, the method 292 may include washing the electrodes (for example, to rinse the piranha solution off) and then storing the electrodes in deionized water. At step 298, the method 292 may include blow drying the electrodes (for example, to remove and / or evaporate the deionized water off of it) with nitrogen gas, thereby maintaining the electrodes in an inert environment. At step 300, the method 292 may include placing masking (for example, a mask, e.g., a silicone mask, a rubber mask, a polymer mask) on the chip to expose only the working electrodes. Once the masking is placed on the chip, only the working electrodes remain exposed, and the other electrodes and surfaces are covered by the masking. The mask may include a thickness approximately equal to a desired thickness of the anti-fouling coating such that once the coating is applied and smoothed out, it will contain an optimal (or at least functional) thickness. At step 302, the method 292 may include preparing a hydrogel precursor by mixing poly (3, 4-ethylenedioxy thiophene) polystyrene sulfonate (PEDOT:PSS), rGO and BSA, thereby forming the hydrogel precursor. At step 304, the method 292 may include adding glutaraldehyde to the hydrogel precursorprior to coating the electrodes. In some embodiments, glutaraldehyde is not added until immediately before coating the electrodes (for example, within about 5, 10, 20, 30, 45, 60 seconds and / or 1, 2, 3, 4, 5, 7, 10, 15, 20, 30 minutes before coating the electrodes). In some embodiments, glutaraldehyde is added from about 5 to about 45 seconds, for example, about 10 to about 30 seconds before coating the electrodes). In some embodiments, a liquid handler may be used in connection with the 96- well high-throughput sensing boards of the present embodiments, to ensure the precursor is added to all electrodes at the same time. In some embodiments, the hydrogel precursor (containing hydrogel monomer, crosslinker and conductive filler) is drop-casted onto working electrode directly. In some embodiments, external thermal or UV sources may be used to initiate hydrogel polymerization (if using thermal crosslinker or UV-sensitive crosslinker and initiator).

[0209] Referring still to FIG. 21, at step 306, the method 292 may include allowing the PEDOT:PSS and rGO to form a conductive filler while BSA crosslinks with the glutaraldehyde. For example, the coated chip may be left undisturbed for a period of time to ensure a consistent degree of polymerization. At step 308, the method 292 may include removing excess precursor using a doctor blade coater (for example, using a razor blade with a mechanism for controlling the thickness) and / or an adjustable thin film applicator (and / or via other suitable methods). Using the doctor blade coater (or equivalent) ensures a homogeneous thickness (the same as the masking) across all electrodes. Accordingly, in some embodiments, a mask (for example, a silicon mask) is used as the masking and comprises a thickness equal to a desired thickness of the coating. At step 310, the method 292 may include storing the electrodes in a humidifying chamber at 4°C overnight at 100% humidity (for example, for about 12 hours, or for a period of about 8 to about 16 hours). At step 312, the method 292 may include washing each chip with water (for example, excess water) to remove unreacted molecules. At step 314, the method 292 may include storing each chip in a buffer (for example, BSA solution (for example, 1-3% BSA solution)) and chilling (for example, in a refrigerator) before being used for measurement. At step 315, and prior to using the sensor 178 for measurement, the method 292 may include scanning the electrode using cyclic voltammetry until the reading is stable. For example, in some embodiments, scanning may include cyclic voltammetry in 5mM ferricyanide and / or ferrocyanide until peak-to-peak separation is stable (for example, no varying by more than 20%, 10%, or in some embodiments 5%). In some embodiments, steps of method 292 may be performed in a different order than the order in which they are presented in FIG. 21. Forexample, preparing the hydrogel precursor may be performed concurrent with or prior to preparation of the electrode surface. In some embodiments, method 292 may be used for coating neuron probes, larger surface electrodes (for example, planar electrodes), and / or in applications where anti-fouling properties are needed. In some embodiments, method 292 may include conjugating additional sensing elements (for example, antibodies, etc.) onto the hydrogel (for example, following step 314, but prior to step 315).III. Multi- Well Sensor Plate Assembly

[0210] FIG. 10A is a photograph of a 96- well sensor board 184, according to aspects of the present embodiments. The 96-well sensor board 184 is sized and configured to be used in connection with 96-well systems and equipment and includes 96 sensors, for example, each sensor 178 having a configuration similar to that shown in FIG. 10B and / or 12B.

[0211] FIG. 10B is an enlarged photograph of a 96-well sensor board, according to an illustrative embodiment. Each well contains a single three-electrode assembly or sensor 178 including a working electrode 194, a counter electrode 196, and a reference electrode 198, which may be configured as concentric circles separated by gaps 143, 145. In some embodiments, the three electrodes are substantially planar and are electrically isolated from each other. In some embodiments, the three electrodes may have non-circular shapes (e.g., rectangles, triangles, etc.). The shape or geometry of the electrodes may vary, but all three electrodes of each set of the three electrodes are configured to fit within a single well in a multi-well plate, i.e., inside the shape defined by a well or multi -hole gasket 182 placed over the sensor board 184. Each set of electrodes in FIG. 10A are separated by a large enough gap such that a multi-hole gasket 182 may be placed over the sensor board 184 with each set of electrodes fitting within a hole of the gasket 182 and a solid portion of the gasket 184 between holes is positioned between adjacent sets of electrodes. In some embodiments, positioning of the sensor board 184 and gasket 182 and sensors 178 ensures each set of sensors or electrodes is fluidly isolated from each other.

[0212] FIG. 11A is a schematic diagram of a sensor plate assembly 181 including a bottomless 96-well plate 180, a gasket 182, and a sensor board 184, according to aspects of the present embodiments. The gasket 182 may include a plurality (for example 96) ofcircular holes 183 that are spaced and sized to align with the 96 sensors 178 of the sensor board 184. In some embodiments, the holes in the gasket 182 may be formed by any suitable method (e.g., laser cutting, stamping, molding, 3D printing, Computer Numerical Control (CNC) machining, hand cutting, etc.). When assembled, the sensor plate assembly 181 includes the gasket 182 sandwiched between the sensor board 184 and a bottom surface 185 of the bottomless 96-well plate 180. In some embodiments, the gasket 182 may include or be a double-sided silicone gasket. In some embodiments, gasket 182 may include an adhesive on both sides (i.e., on both the top surface and the bottom surface). The “bottomless” aspect of the bottomless 96-well plate 180 applies to the portions where the wells are. However, there is still a bottom surface 185 in the other areas. The bottomless 96-well plate 180 also includes an exterior lip 187, that extends around the perimeter of the bottomless 96-well plate 180. The area within (i.e., defined by) the exterior lip 187 is sized to be approximately the same size as the area of the gasket 182 such that the gasket 182 may tightly fit within the area defined by the exterior lip 187. The sensor board 184 may have an area that is slightly larger than that of the gasket 182. In some embodiments, the combination of a bottomless well plate and gasket may be replaced by any other means or configuration of components for keeping a volume of target solution above each sensor in each well in a well plate while preventing leaking between wells (e.g., by gluing a multi-well plate to a printed circuit board (PCB) with a watertight seal.

[0213] Referring still to FIG. HA, in some embodiments, the sensor board 184 may include one or more positioning features 189 that interface with corresponding features on the bottomless 96-well plate 180 thereby ensuring that the 96 sensors 178 of the sensor board 184 are aligned with the 96 holes 183 of the gasket 182 and the 96 openings at the bottom of the bottomless 96-well plate 180. In some embodiments, the one or more positioning features 189 may also serve as coupling features to keep the sensor plate assembly 181 tightly sealed. In some embodiments, the one or more positioning features 189 may include, notches, pins, screws, dowels, knobs, compression elements, and / or other suitable mechanisms for coupling the bottomless 96-well plate 180 to the sensor board 184. When the sensor plate assembly 181 is assembled, the sensor board 184 and gasket 182 provide sufficient sealing around the bottom of each well such that the assembly may be fluidly sealed. In some embodiments, side walls of the bottomless 96-well plate 180 are biocompatible such that they do not interfere with reactions inside each well. In some embodiments, the sensor board 184 may include a flat PCB sensor board 184 that allowsfunctionalization of the electrode surfaces before assembly. For example, in some embodiments, each well can be functionalized with a different type of coating, biomolecule, etc., thereby increasing the versatility of potential use cases.

[0214] Still referring to FIG. 11A, in some embodiments, the total reaction volume I capacity within each well may range from 10 pL (i.e., 10 microliters) when just the gasket 182 is used to 350 pL (i.e., 350 microliters) when a standard 96-well plate is used to 2 mL (i.e., 2 milliliters) when a deep well plate is used. The present embodiments may be used in connection with various lab plates (for example, standard lab plates (i.e., polystyrene lab plates), which may be customized to include various coatings (like a protein lo-bind plate) to prevent protein / DNA adhesion, thereby increasing the accuracy of results. The sensor board 184 can be used in connection with other bottomless plate types. For example, in some embodiments a 6- well plate can be attached to the sensor board 184 such that multiple sensors are contained within each well, with each sensor within a well configured for detecting a different biomolecule.

[0215] FIG. 11B is a schematic diagram of a side view of a control board assembly 191 including a sensor board 184 and a control board 188 connected by one or more interconnects 190, according to aspects of the present embodiments. In some embodiments, the interconnects 190 are aligned vertically between the sensor board 184 and the control board 188. In some embodiments, the control board assembly 191 comprises multiple interconnects 190 with one or more spaces 186 being formed between the interconnects 190, which also form a gap between the sensor board 184 and the control board 188. In some embodiments, each of the interconnects 190 may be configured (for example, with quickdisconnect features) such that they may be mechanically and electrically detached thereby allowing the sensor board 184 to be decoupled from the control board 188. Accordingly, the control board assembly 191 may be at least partially disposable allowing the sensor board 184 to be discarded when its useful life has expired, while also allowing the control board to be reused. The bottomless 96-well plate 180 and gasket 182 may also be replaced as needed. The interconnects described herein are one embodiment of making electrical connections between the sensor board and the control board. In some embodiments, other means may be used to electrically connect the sensor board to the control board (e.g., wires, contacts, control electronics directly built into the sensor board, etc.).

[0216] FIG. 12A is a CAD design of a 96-well sensor board 192, according to an illustrative embodiment. In some embodiments, the 96-well sensor board 192 includes 96 sensors 178. FIG. 12B is an enlarged view of a sensor 178, which includes a three-electrode assembly of a working electrode 194, a counter electrode 196, and a reference electrode 198. In some embodiments, the electrodes 194, 196, 198 are configured as concentric circles separated by gaps 143, 145. In some embodiments, the three electrodes may be different non-circular shapes. The electrodes are separated by gaps so that they are electrically isolated from each other.

[0217] FIG. 12C is a CAD design of a 96- well sensor board 192 with board dimensions, according to an illustrative embodiment. In some embodiments, 96-well sensor board 192 includes a center-to-center spacing of about 9.02 mm (or from about 8.8 mm to about 9.2 mm, or from about 8.9 mm to about 9.1 mm, or from about 8.95 mm to about 9.08 mm, or from about 9.0 mm to about 9.05 mm) in both a lateral and a longitudinal direction, in order to align with the spacing of standard 96-well lab equipment. In some embodiments, the 96-well sensor board 192 includes an overall length from about 118 mm to about 122 mm and an overall width from about 75mm to about 80mm. In some embodiments, a sensor board may include different numbers of sensors arranged in an array (e.g., 6, 12, 24, 48, 96, 384, 1536, 3456, 6144, or 9600 sensors) where the positions of the sensors (e.g., the center- to-center spacings between sensors) match the positions of wells in standard 6-, 12-, 24-, 48-, 96-, 384-, 1536-, 3456-, 6144-, or 9600-well plates (e.g., SBS standard multi-well plates).Sensor Board, Control Board and Electronics

[0218] FIG. 14 is a photograph of an illustrative development control board - in this case, the EmStat Pico Development control board manufactured by PalmSens BV of Houten, Netherlands. In some embodiments, the control board may include: a micro-USB port 220 for powering the control board and for connecting the control board to a PC; analog and digital I / O pins 22-2 for connecting to peripheral devices; high end connectors (for example Lemo connectors) and / or screw terminals 224 for connecting the board to the electrochemical cells or sensors 178; Arduino MKR headers 226 disposed at the bottom of the control board; one or more microSD cards 228 for data logging; a AAA battery holder 230 for providing more than 100 hours of continuous measurements; and / or a Bluetooth 4.0 dual mode module 232 for enabling Bluetooth communications. In some embodiments, the control boardincludes a current operating range from about 1 nA to about 100 mA, (i.e., from about 5 nA to about 50 mA, from about 10 nA to about 10 mA, from about 50 nA to about 10 mA, and / or from about 100 nA to about 5 mA) with a current accuracy of about 0.5% of the measured current or 0.1% of the range, whichever is larger. In some embodiments, the control board includes a voltage operating range from about -10 V to about 10 V (i.e., from about -5V to about 5V, from about -3V to about 3V, and / or from about -2V to about 2V), with an applied voltage resolution of about 300 LI V to about 750 p V (i.e., from about 350 p V to about 700 |i V, from about 400 p V to about 650 p V, and / or from about 500 V to about 600 p V). In some embodiments, electronic amplification and / or filtering of the measured current may be performed via additional electronic components and / or via software or firmware.

[0219] FIG. 15 is a schematic diagram of a circuit connecting a control board 234 and a sensor board 240, according to an illustrative embodiment. The control board 234 may include one or more microcontrollers 236, a microchip 244 (e.g., an electrochemical front end), and a demultiplexer 238 (for example, a 1:48 demultiplexer 238 (or a digitally controlled analog switch)). The microchip 244 may be coupled to the demultiplexer 238 via a first EChem channel 246 and a second EChem channel 248. Control board 234 may be coupled (i.e., electrically and communicatively coupled) to the sensor board 240 via a plurality of interconnects 190 (shown in FIG. 14), which may include 96 independent channels.

[0220] FIG. 16 is a CAD diagram of circuitry in a 96-well control board 234, according to an illustrative embodiment. The 96-well control board 234 may include an EChem front end 250 (i.e., an electrochemical front end as described herein), a microcontroller 252, and multiplexers 254. In some embodiments, the control board 234 may also include other devices for connecting the output of the electrochemical front end to the multiple electrochemical systems such as individual wells on the 96-well sensor board 178 instead of, or in addition to, the multiplexers 254. For example, in some embodiments, a device (not shown) that is able to switch outputs on and off with low contact resistance such as relays, digitally controlled switches, serial to parallel converters, and / or shift registers may also be used. According to aspects of the present embodiments, the multiplexers 254 enable the ability to obtain measurements from all 96 wells in a serial fashion as described herein, without any user intervention. In addition, the multiplexers 254 allow a flexible approach in the way this data is sampled, namely that the microcontroller 252 and electrochemical frontend can be configured to take as many samples as needed (or desired) on a single well before switching to another well. In addition, the system can selectively switch back to the previous well or any other well at any time. This arrangement allows users of the described system / platform to create custom protocols for a variety of electrochemical sensing applications, and to define what wells are in use, thereby enabling the system to only take data of relevant wells, without requiring intervention by the user. According to aspects of the present embodiments, the described configuration enables compatibility and communication with the inputs and outputs of commercially available liquid handling systems.

[0221] FIG. 17 is a schematic diagram of a cross-section of sensor board 184, according to an illustrative embodiment. In some embodiments, the sensor board 184 may include: a top solder mask 650; a nickel, palladium, gold, and / or copper top finish 652; an insulating core 660 (e.g., polyimide, FR-4, standard PCB substrate, or glass); a nickel, palladium, gold, and / or copper bottom layer 654; and a bottom solder mask 656. In some embodiments, the top finish and bottom layer 652, 654 may include a gold finish layer (e.g., gold finish, nickel / palladium adhesion layer, and copper inner layer), while the solder masks 650, 656 are applied to areas that do not have the gold finish layer and provide electrical resistance and protection to traces on the top and bottom layers that are not meant to be exposed. In some embodiments, the bottom layer 654 may be any conductor as it is only used for electrical connections to control electronics and is not part of the electrochemical measurement. In some embodiments the bottom layer is any layer that is not interacting with the electrochemical cell. In some embodiments, the bottom layer (i.e., any layer that is not interacting with the electrochemical cell) does not need to be made of an electrochemically inert material (e.g., gold). In some embodiments, the insulating core 660 makes up (i.e., accounts for) more than 80% of the overall thickness of the sensor board 184. In some embodiments, the sensor board 184 may be fabricated using finishing techniques that include electroless palladium immersion gold and hard gold. In some embodiments, electroless palladium immersion gold (EPIG) may include using a chemical process called autocatalytic plating to deposit / adhere the palladium to the internal copper layers. In some embodiments, a thin layer of gold may then be deposited on top of the palladium. In some embodiments, the gold layer may be about 2 microinches thick (for example, from about 1-2 microinches, or from about 1.5-2.5 microinches) while the palladium may be from about 100 microinches to about 150 microinches thick. In some embodiments, using palladium may be beneficial because palladium is less electrochemically reactive than nickel. In some embodiments,other combinations of materials and components may be used, so long as a non-reactive metal (e.g., gold) is used for the three electrodes in each sensor, and some manner of electrical connectivity is used between the sensors and the control hardware.

[0222] Referring still to FIG. 17, in some embodiments, a hard gold fabrication approach (i.e., a hard gold deposition process) may be used and may include using nickel as the adhesion layer, which is electrochemically active and can affect electrochemical measurements. In some embodiments, the hard gold deposition process results in 50 microinches of denser gold, which may be sufficient to prevent direct contact with the underlying reactive nickel. In some embodiments, the present sensor board 184 may be fabricated via any finishing technique that provides a dense coating of gold that does not allow the reactants to reach any underlying metals under the gold coating. In some embodiments, fabrication of the sensor board 184 may include a finishing process called electroless nickel immersion gold (ENIG) in initial prototypes, as long as the measurement system does not interact with the nickel layer underneath the gold layer. In some embodiments, a physical vapor deposition (PVD) process may be used to deposit a 50 nm to 100 nm thick layer of gold directly onto the sensor board 184. In some embodiments, the gold layer may be less than 50 nm thick (e.g., about 10 nm, 20 nm, 30 nm, 40 nm). In some embodiments, the gold layer may be more than 100 nm thick (e.g., about 110 nm, 120 nm, 150 nm, 200 nm, 300 nm, 500 nm, 1000 nm, or more). In some embodiments, the sensor board 184 may also include a thin adhesive layer (for example, a 20 nm titanium layer). According to aspects of the present embodiments, the sensor board 184 may be scaled up or down to accommodate different plate geometries (for example, in some cases via customized fabrication to accommodate a 384-well plate, a 1536-well plate, a 3456-well plate, a 6144- well plate, or a 9600- well plate).

[0223] FIGS. 36A-36J show different aspects of a control board, according to an illustrative embodiment. FIG. 36A shows a pattern of silkscreened indicators on the bottom of the control board. FIG. 36B shows bottom solder mask 548 along with bottom silkscreen 546. FIG. 36C shows bottom solder paste 550, along with bottom solder mask 548 and bottom silkscreen 546.

[0224] FIG. 36D shows bottom copper layer 552 over laid with the features of FIG. 36C. FIG. 36E shows an inner copper layer 554, corresponding to Interconnects between electronic components on the control board (e.g., between sensor board connections,multiplexers, microcontroller, EChem front end, etc.). FIG. 36F shows another inner copper layer 556, corresponding to additional electronic interconnects, and overlaid with the inner copper layer 554. FIG. 36G shows a top copper layer 558. FIG. 36H shows a top solder paste layer 560, overlaid over the top copper layer 558. FIG. 361 shows a top solder mask 562 overlaid with the top solder paste 560 and top copper layer 558. FIG. 36J shows a top silkscreen pattern 564, overlaid with the features of FIG. 361.

[0225] In some embodiments, referring still to FIGS. 36A-36J, additional functions of the layers may be as follows. The silkscreen layers 546, 564 are decorative layers that add reference indicators and labels to the circuit board. The solder mask layers 548, 562 determine where a nonconductive epoxy is placed so that non-exposed parts of the bottom and top copper layers are protected; a finish layer (e.g., EPIG, ENIG or deep gold) may be applied where not solder mask is specified. The solder paste layers 550, 560 may be used to direct assembly to apply solder paste for component placement and soldering. The top, bottom, and internal copper layers 552, 554, 556, 558 are where the copper is left or deposited to allow for electrical connections to be made. In some embodiments, the solder paste layers 550, 560 are not used.Temperature Control Unit

[0226] In some embodiments, a temperature control unit may be used to facilitate additional control over enzyme activity in conjunction with the sensor board and measurement systems described herein. FIG. 27A is an example of a heater plate 398, according to an illustrative embodiment. The heater plate 398 may include a custom machined aluminum heating block 400 and a resistive heater coil 404 disposed along grooves 401 machined into a bottom surface of the heating block 400. A sensor board may be placed on top of the heater plate 398 such that the heater plate 398 heats up the bottom of the sensor board, which in turn heats up reagents (e.g., enzymes, substrates, etc.) to a desired temperature. The aluminum heating block 400 may include gaps 402 to allow the sensor board to connect to a control board placed at the bottom of the heating block 400. The heater plate 398 may provide a uniform temperature across the aluminum block 400. In other embodiments, the heater coil 404 may include separate portions of heater coils that can independently be controlled to reach different temperatures so that different regions of the aluminum block 400 may reach different temperatures.

[0227] FIG. 27B is an example of a temperature control unit 406, according to an illustrative embodiment. The temperature control unit 406 may include a control board 188, a heater plate 398, and a power supply (not shown). The temperature control unit 406 may be positioned inside a custom holder 407. The heater plate 398 fits into the control board 188 while the gaps 402 allow electronic connections 408 to pass through. Each electronic connection 408 may include a spring-loaded pin that presses against a board to make contact, enabling easy replacement of a sensor board. The heater plate 398 includes a resistive heater coil 404 which provides uniform temperature across the sensor board. In some embodiments, the heater plate 398 may include multiple resistive heater coils to locally control the temperature. For example, the heater plate 398 may include a resistive heater coil for each individual sensor. The power supply passes current through the heater coil 404 for resistive heating.

[0228] In some embodiments, the temperature control unit 406 may include a proportional-integral-derivative controller (PID controller) to maintain a desired temperature setpoint. FIG. 28A is a graph of temperature as a function of time for a sensor board operating at set power input and without a PID controller, according to an illustrative embodiment. The measured temperatures correspond to the middle of the sensor board 412, a comer of the sensor board 414, and the ambient 416. When the input power of the temperature control unit is held constant, the board temperature varies with ambient. FIG. 28B is a graph of temperature as a function of time for a sensor board with a PID controller to maintain a temperature setpoint, according to an illustrative embodiment. In this example, the temperature control unit 406 includes a PID controller which modulates the power supply to maintain a desired board temperature. The temperature stability is demonstrated by plotting average temperature 418, high temperature 420, low temperature 422, and a temperature setpoint 38°C as the board temperature is increased from 25°C to 38°C within 5 minutes and maintained afterwards by the PID. The temperature setpoint may be adjusted over time, to control reactions as they progress. In some embodiments, the temperature control unit 406 may include a cooling module to reach temperatures below ambient. The temperature control unit, for example, may include a Peltier module.Sensor Board with Integrated Heater

[0229] FIG. 29A is a CAD design of a 96-well sensor board 424 with integrated heater, according to an illustrative embodiment. In some embodiments, the 96-well sensor board 192 includes 96 sensors 178, an internal heating layer 426, a heating power connection 428, a temperature sensor 430, and a temperature sensor connector 432. The internal heating layer 426 includes a metal arranged in a serpentine path to provide resistive heating across the sensor board 424. In some embodiments, the internal heating layer 426 includes any conductor, or any metal, or any material that heats when current is passed through it. In some embodiments, the internal heating layer 426 is copper. The temperature sensor 430 monitors the temperature and may be used along a PID controller. In some embodiments, each sensor 178 may include a temperature sensor to enable individual control temperature for each well.

[0230] FIG. 29B is a schematic diagram of a cross-section of sensor board 424, according to an illustrative embodiment. In some embodiments, the sensor board 424 may include the following layers and components: a top solder mask 700 (e.g., a thin insulating layer or epoxy layer); a top copper layer 702 with EPIG, ENIG, or other gold finish as described above; a substrate 704, 708, 712 (e.g., polyimide, FR-4, glass, or other insulating dielectric material); internal copper layers 706, 710 (e.g., any conductive material); bottom copper layer 714 with EPIG, ENIG, or other gold finish as described above, or any other conductive material; and via 718 connecting top, internal, and bottom layers together (e.g., copper, or other conducting material). In some embodiments, the bottom layer 714 may be composed of any conductive material as it is used to make electrical connections to control circuits (e.g., between the sensor board and the control board) and does not participate in electrochemical reactions. In some embodiments the bottom layer is any layer that is not interacting with the electrochemical cell. In some embodiments, the bottom layer (i.e., any layer that is not interacting with the electrochemical cell) does not need to be made of an electrochemically inert material (e.g., gold). The internal copper layer 706 may include a resistive heating path to provide controllable heating. The internal copper layer 710 may include a layer that spans the area of the sensor board 424 to spread heat throughout the board. The top copper layer 702 may be used for sensor electrodes (e.g., three-electrode sensors).

[0231] FIG. 29C is a schematic diagram of another embodiment of a cross-section of sensor board 750, according to an illustrative embodiment. The sensor board 750 has a substrate 752 made of an insulating dielectric material (e.g., polyimide, FR-4, glass, or otherinsulating dielectric material); a solder mask 764 at the top surface of the board 750; a copper layer 760, with nickel or palladium layer 764 and gold layer 762 to form a top finish layer; conductive via 758 (e g., copper, or other conductive material); solder mask 756 on the bottom surface of the sensor board 750; and copper layer 766, covered by nickel or palladium layer 770 and gold layer 768 to form a bottom layer. In some embodiments, the bottom layer may be composed of any conductive material so that electrical connections can be made between the sensor board 750 and a control board. In some embodiments the bottom layer is any layer that is not interacting with the electrochemical cell. In some embodiments, the bottom layer (i.e., any layer that is not interacting with the electrochemical cell) does not need to be made of an electrochemically inert material (e.g. gold). In some embodiments, the top finish layer may be composed of another inert conductive layer for the sensor electrodes.

[0232] FIGS. 35A-35L show different aspects of a sensor board with a built-in heater, according to an illustrative embodiment. FIG. 35A shows silkscreened indications 526 on a bottom surface of the sensor board. FIG. 35B shows a pattern of bottom solder mask 528, overlaid with silkscreen indications 526. FIG. 35C shows a pattern of bottom solder paste 530, overlaid with the bottom solder mask 528 and bottom silkscreen 526 also visible. These features allow electrical connections to the electrochemical sensors arranged in the 96-well plate to be made.

[0233] FIG. 35D shows a bottom copper 532 layer, which includes connections for the built-in heater 535, connections for the temperature sensor 531, and connections to electrochemical sensors 533. The silkscreened indicators 526, bottom solder mask 528, and bottom solder paste 530 layers are also overlaid and visible. FIG. 35E shows an inner copper layer 534 arranged in a serpentine path to act as a resistive heater. This inner copper layer 534 is equivalent to the heating layer 426 of FIG. 29A. FIG. 35F shows an inner copper layer 536, which helps to spread heat from the heater coil (or inner copper layer 534) throughout the sensor board.

[0234] FIG. 35G shows a top copper layer 538, which corresponds to the electrochemical sensors. Each circular feature in FIG. 35G includes three concentric circles arranged as an electrochemical sensor, or electrochemical sensor pads, similar to the enlarged sensor 178 of a sensor board, as shown in FIG. 12B. FIG. 35H shows a top solder paste pattern 540, overlaid on the electrochemical sensor pads layer 538, for possible use in applying solder paste for component assembly. FIG. 351 shows a top solder mask 542 overlaid on the electrochemical sensors 538 and top solder paste 540 layer. The top andbottom solder masks are a thin polymer layers that insulate the PCB traces that are not to be exposed.

[0235] FIG. 35J shows a top silkscreen pattern 544, with indicators and labels, overlaid with the electrochemical sensors 538, top solder paste 540, and top solder mask 542 visible. FIG. 35K shows the electrochemical sensors 538 and interconnections 532 layers overlaid, showing how each electrode in each sensor is connected (i.e., this image combines features from FIGS. 35D and 35G). FIG. 35L shows the heater coil layer 534 overlaid with the electrochemical sensors 538, showing how the heater coil 534 uniformly distributes heat throughout the 96 sensors 538 (i.e., this image combines features from FIGS. 35E and 35G).

[0236] Referring still to FIGS. 35A-35L, the silkscreen layers 526, 544 are decorative layers that provide that add reference indicators and labels to the circuit board. The solder mask layers 528, 542 determine where a nonconductive epoxy is placed so that non-exposed parts of the bottom and top copper layers are protected; a finish layer (e.g., EPIG, ENIG or deep gold) may be applied where not solder mask is specified. The solder paste layers 540, 530 are used to direct assembly to apply solder paste for component placement and soldering. The top, bottom, and internal copper layers 532, 534, 536 are where the copper is left or deposited to allow for electrical connections to be made.

[0237] FIGS. 37A-37I show different aspects of a sensor board without a built-in heater, according to an illustrative embodiment. The sensor board in FIGS. 37A-37I is very similar to the sensor board in FIGS. 35A-35L except without the heater layer, and is very similar to the sensor board of FIGS. 12A-12C. FIG. 37A shows a bottom silkscreen layer 566. FIG. 37B shows a bottom solder mask 568 along with bottom silkscreen 566. FIG. 37C shows a bottom solder paste layer 570, along with bottom solder mask 568 and bottom silkscreen pattern 566. FIG. 37D shows a bottom copper layer 572, which includes interconnects for the array of 96 electrochemical sensors. FIG. 37E shows a top copper layer 574, which includes the electrochemical sensors arranged in concentric circles. FIG. 37F shows top solder paste layer 576 overlaid on the electrochemical sensors 574.

[0238] FIG. 37G shows a top solder mask layer 578, along with electrochemical sensors 574 and top solder paste layer 576. FIG. 37H shows top silkscreen pattern 580, along with top solder paste layer 576, electrochemical sensors 574, and top solder mask layer 578. FIG. 371 shows electrochemical sensors 574 overlaid with interconnects 572.

[0239] Referring still to FIGS. 37A-37I, the silkscreen layers 566, 580 are decorative layers that add reference indicators and labels to the circuit board. The solder mask layers568, 578 determine where a nonconductive epoxy is placed so that non-exposed parts of the bottom and top copper layers are protected; a finish layer (e.g., EPIG, ENIG or deep gold) may be applied where not solder mask is specified. The solder paste layers 570, 576 may be used to direct assembly to apply solder paste for component placement and soldering. The top and bottom copper layers 572, 574 are where the copper is left or deposited to allow for electrical connections to be made. In some embodiments, the solder paste layers 570, 576 are not used.Integrated heater PCB board

[0240] FIG. 46 is a CAD layout of an integrated heater printed circuit board (PCB) 612, according to an illustrative embodiment. In some embodiments, the integrated heater PCB 612 includes control board connectors 614, an input power connector 616, a power monitor 618, a power supply 620, and a sensor board connection 622. The integrated heater PCB 612 may supply power to the sensor board's inbuilt heater trace and temperature sensor through connector labelled JI (shown in FIG. 47). Temperature data from the sensor board 624 (shown in FIG. 48A) is sent back to the control board through the connectors labelled J2 and J3 (shown in FIG. 47). The control board 612 monitors the voltage, current and power of the power supply 620, and controls the temperature of the sensor board 624 by a pulsewidth modulation (PWM) signal to the power supply 620. In some embodiments, the input power connector 616 is a barrel connector. FIG. 47 is a schematic 662 of integration of the heater PCB 612 with the sensor board 624, according to an illustrative embodiment. The schematic 662 illustrates connector JI electrically coupled to the heater trace circuit 664 of the sensor board, to which it supplies powers. The schematic 662 further illustrates connector J3 communicatively coupled to the sensor circuitry 666 of the sensor board 624, from which the control board receives sensor data. The schematic 662 further illustrates connector J2 also communicatively coupled to the sensor circuitry 666, from which I to which it receives / transmits power, as well as multiple ground connections 668, and one or more interconnects 670 for routing power to / from the sensor circuitry and the heater circuitry.Individual well heater sensor board

[0241] FIG. 48A is a CAD design of an individual well heater sensor board 624, according to an illustrative embodiment. In some embodiments, the individual well heater sensor board 624 includes 96 sensors 626. In some embodiments, the individual well heater sensor board 624 allows each well 626 to be independently heated. FIG. 48B is an enlarged view of a sensor 626 including an individual heater trace 628. By routing a heater trace 628 under each well, they can be individually controlled through connectors on the sides of the sensor board 624. In some embodiments, the heater trace 628 makes multiple passes (for example, in a serpentine arrangement) through the sensor 628. For example, in some embodiments, the heater trace 628 makes 5 full round trips (for a total of 10 passes) from one end of the sensor 626 to the other, as shown in Fig. 48B. In some embodiments, each pass of the heater trace 628 is oriented in a lateral direction. In some embodiments, each pass of the heater trace 628 is oriented in a longitudinal direction. Accordingly, in some embodiments, the sensor 626 includes first and second heater trace contacts 672, 674 positioned along the same edge of the sensor 626 (i.e. , along the bottom edge of the sensor 626 in the illustrative example shown in FIG. 48A). The first and second heater trace contacts 672, 674 are used to connect the heater trace 628 to the rest of the heater circuitry, as described herein. In some embodiments, each sensor 626 includes a separate temperature sensor (not shown) positioned under each well for more precise control. In some embodiments, each temperature sensor is positioned at of the sensor board layer between the heater trace 628 and the top surfaces of the electrode 194, 196, 198.

[0242] Referring still to FIG. 48B, each sensor 626 may be described as including two separate circuits that spatially overlap, but that do not electrically or communicatively interact on the sensor 626. For example, each sensor 626 includes components of both the sensor circuitry and the heater circuitry. The sensor circuitry includes the 3 leads (or conductive contact pads) 205, 207, 209 connected via first, second and third contacts 676, 678, 680 to exposed top surfaces of the working electrode 194, the counter electrode 196, and the reference electrode 198 (each shown in FIG. 48D), respectively. The heater circuitry includes the first and second heater trace contacts 672, 674 and the heater trace 628. The first conductive contact pad 205 connects electrically to the first contact 676 which connects to top surface of the working electrode 194. Similarly, the second conductive contract pad 207 and the second contact 678 electrically couple to the surface of counter electrode 196. Similarly, the third conductive contract pad 209 and the third contact 680 electrically coupleto the surface of reference electrode 198. The heater trace 628 spatially overlaps with the sensor circuitry because each of the first, second, and third conductive contact pads are positioned at a layer of each sensor 626 that is beneath the layer that the heater trace 628 is positioned at. The first, second, and third contacts 676, 678, 680 then connect to the top layer of the sensor enabling electrification of each of the electrodes 194, 196, 198. Accordingly, and as shown in FIG. 48B, the heater trace 628 is configured such that it includes a larger space 684 between two passes of the serpentine arrangement, to make space for the first, second, and third contacts 676, 678, 680 to extent therethrough (i.e., between passes of the heater trace 628). For example, as illustrated in FIG. 48B, spacing 684, through which the first, second, and third contacts 676, 678, 680 extend (i.e., in a direction “out of the page”) is larger than the spacing 682 between other passes of the heater trace 628. In some embodiments, the larger spacing 684 is positioned in the center of the sensor 626 with half of the passes of the heater trace 626 (for example, 5 passes) positioned on one side of the spacing 684, and half of the passes of the heater trace 626 (for example, 5 passes) positioned on the other side of the spacing 684. FIG. 48B also illustrates the approximate footprint 628 or outer periphery of the electrodes 194, 196, 198. In some embodiments, the first, second, and third contacts 676, 678, 680 are oriented in a colinear arrangement with the first contact 676 being positioned concentrically with the working electrode 194, the second contact 678 positioned a first radial distance from the first contact 676, and the third contact 680 being positioned a second radial distance from the first contact such that the second radial distance is larger than the first radial distance.

[0243] FIG. 48C is a CAD design of an individual well heater sensor board 624, according to an illustrative embodiment. In some embodiments, the individual well heater sensor board 624, includes 96 sensors 626. FIG. 48D is an enlarged view of a sensor 626 including an individual heater trace 628, and a three-electrode assembly of a working electrode 194, a counter electrode 196, and a reference electrode 198.

[0244] FIG. 49 panels A and B (FIG. 49A and FIG. 49B) illustrate a quadrant 684 and sub-quadrant 686 of the well heater sensor board 624, according to an illustrative embodiment. As shown in FIG. 48A, each sensor board 624 includes 4 quadrants 684 and is symmetrical both longitudinally and laterally along respective centerlines with respect to the layout of the electrode conductive contacts 205, 207, 209. As further shown in FIG. 49A, each quadrant 684 also includes 4 sub-quadrants 686 such that each sub-quadrant 684 is alsosymmetrical both longitudinally and laterally along respective centerlines with respect to the layout of the electrode conductive contact pads 205, 207, 209. As shown in FIG. 49B, each sub-quadrant 686 comprises 6 sensors 626. For example, along the top row of sensors 626 (from left to right) in FIG. 49A, the conductive contact pads 205, 207, 209 are respectively oriented to the left, left, left (LLL) in the first sensor, then right, left, left (RLL) in the second sensor, then LRL, then RRL, then LRR, then RRR. The second row includes a similar, though mirrored, arrangement as the first row. Rows 3 and 4 are a repeat of rows 1 and 2. The topology of this arrangement enables the concentric electrode configuration at the sensor board 624 surface, while providing the spacing and access for continuous electrical coupling of each of the three electrodes, for each of the 96 sensors, under the top surface. The heater trace first and second contacts 672, 674 are also illustrated in FIG. 49B.

[0245] FIG. 51A is a CAD design of a heater sensor board 690, according to an illustrative embodiment. As shown in FIG. 51A, the heater sensor board 690 includes a single heater trace 628 that extends through the entire heater sensor board 690 in a serpentine fashion. The heater sensor board 690 may include a heater trace lead 688 that is electrically coupled to both the heater trace 628 and a heater board connection 694 (which connects with the sensor board connection 622 of the heater board shown in FIG. 46).

[0246] FIG. 51B is a CAD design of a heater sensor board 690, according to an illustrative embodiment. The heater sensor board 690 is illustrated in FIG. 51B without the heater trace 628. The heater sensor board 690 may also include one or more temperature sensor leads 692 connecting to the temperature sensor 430 (positioned at the center of the board 690, in this embodiment).

[0247] FIG. 52A is a portion of a CAD design of a heater sensor board, according to an illustrative embodiment. Similar to the embodiments with an individual heater trace 628 for each sensor 628, the heater sensor board 690 shown in FIG. 52A includes first, second and third contacts 676, 678, 680 that are positioned between passes of the heater trace 628.

[0248] FIG. 52B is a portion of a CAD design of a heater sensor board (for example, a sensor 626) including the first, second and third contacts 676, 678, 680, and the first, second, and third conductive contact pads 205, 207, 209, according to an illustrative embodiment. FIG. 52C is a portion of a CAD design of a heater sensor board including a group of six (6) sensors 626, according to an illustrative embodiment.Glass Sensor Board Design

[0249] In some embodiments, a buffer may interact with underlying copper of a PCB sensor board and the resulting corrosion may interfere with detection of electrochemical reactions (see D.B. McPhail and B.A. Goodman, “Tris buffer - A case for caution in its use in copper-containing systems”, Biochem. J. (1984), 221(2), 559-560). FIG. 32A is a graph of current as a function of potential obtained by multiple DPV measurements for uric acid in sodium phosphate buffer using a PCB sensor board, according to an illustrative embodiment. The current plots include a smooth peak for all DPV measurements, demonstrating the anticipated uric acid activity. FIG. 32B is a graph of current as a function of potential obtained by multiple DPV measurements for uric acid in Tris-HCl buffer, according to an illustrative embodiment. While the current plots include a smooth peak for the initial measurements, the current plots of the subsequent measurements include a steady current, followed by a sudden drop. The inability to detect the electrochemical reactions is due to corrosion that is caused by the interaction between Tris-HCl buffer and the underlying copper of the sensor board.

[0250] In some embodiments, a sensor board may be fabricated using glass or silicon to prevent destructive interactions between certain buffers and PCB-based sensor boards, resulting in the sensor board’s compatibility with various assays. In addition, a glass sensor board may be used in pH-dependent experiments (i.e., using solutions of varying pH levels) where a traditional board is not suitable (i.e., due to copper, which is electrochemically active, in the PCB boards that may come into contact with liquid and contaminating the results).

[0251] In some embodiments, a fabrication process of a glass sensor board may include applying traditional semiconductor manufacturing processing steps to a suitable support material (e.g. a glass or low resistivity Si wafer), to achieve an architecture of sensor pads, conductive pads and pathways, etc., to avoid any copper content and to allow reactions at a wider range of pH values to be implemented. FIG. 33 shows an exemplary fabrication process 480 of a glass-based sensor board, according to an illustrative embodiment. Step 482 of the fabrication process 480 may include providing a glass wafer 494. The glass wafer 494 may include a diameter of 6 inches and a thickness of 0.5 mm. Step 484 of the fabricationprocess 480 may include laser drilling of via holes 496 into the glass wafer 494. The via holes may include a diameter of 200 pm. Step 486 of the fabrication process 480 may include printing conductive ink 498 into the via holes 496. Step 488 of the fabrication process 480 may include printing conductive contact pads 500 on the backside of the wafer glass 494. Step 490 of the fabrication process 480 may include depositing gold pads using thermal evaporation, to produce sensors 502. Step 492 of the fabrication process 480 may include laser cutting the glass wafer 494 to match the size of a control board. In some embodiments, contact pads, sensor electrodes, and other features of sensor boards may be formed by methods including screen printing, laser etching, photolithography, shadow masking, etc.

[0252] In some embodiments, glass may be used as a substrate for a multilayer circuit board, so a built-in heater may be incorporated into a glass-based sensor board by depositing a resistive heating path (e.g., wire, conductive path, etc.) similar to the built-in heater for a PCB sensor board (e.g., FIG. 35A-35L).Signal Processing and Data Analysis

[0253] In some embodiments, planar electrodes measurements may involve mixing of reagents for proper measurement of enzyme activity. This mixing may create a large ramp waveform overlaid onto an output signal measurement. FIG. 20A is a graph of signal amplitude over time before signal filtering, according to an illustrative embodiment. The amplitude is created by active mixing while the measurement is taken. The measured signal in FIG. 20A displays an oscillating signal due to the mixing. FIG. 20B is a graph of signal amplitude over time after signal filtering, according to an illustrative embodiment. In some embodiments, post-processing filtering techniques to smooth the data may include a rolling average and a fast Fourier transform (FFT). The active mixing of reagents and the subsequent signal filtering, although not necessary, may be useful, for example, if mixing occurs during the time measurement(s) is / are taken, or if there are other sources of external noise such as vibration.

[0254] FIGS. 34A-C illustrates an example of a data acquisition and processing method 510, according to an illustrative embodiment. The data acquisition and processing method 510 may include translating raw output 512 to a readable format, i.e., translated data516, by a data parser 514, plotting translated data 516 to view results 520 by a data visualizer 518 (shown in FIGS. 34A and B), and determining enzyme activity by a data analyzer 522 (shown in FIG. 34C). The raw output 512 may be provided by a Picochip 244. The raw output 512 may include well data, for example voltage, current, and time. The data analyzer 522 may perform automated curve fitting and extraction of enzyme kinetics.IV. Applications of Electrochemical Sensing PlatformExample: Hydrogen Peroxide / Uric Acid Titration

[0255] FIGS. 18A-18D show experimental data from hydrogen peroxide (H2O2) / uric acid titration measurements obtained using the electrochemical platform and methods described herein.

[0256] FIG. 18A is a graph of current as a function of potential in a hydrogen peroxide I uric acid titration experiment using DPV for different hydrogen peroxide concentrations ranging from 0.9 uM to 9 mM, according to an illustrative embodiment. There is a peak in measured current at about 0 V potential, with the peak current increasing with increasing concentrations of H2O2. The inset reactions shows hydrogen peroxide undergoing disproportionation to form water and oxygen (i.e., H2O2 H2O + O2). FIG. 18B is a graph of peak current as a function of hydrogen peroxide concentration obtained from the data in FIG. 18A, according to an illustrative embodiment. The graph in FIG. 18B shows a linear increase in peak current with hydrogen peroxide concentration.

[0257] FIG. 18C is a graph of peak current as a function of potential in a hydrogen peroxide / uric acid titration experiment for different uric acid concentrations ranging from 2.34 uM to 300 uM, according to an illustrative embodiment. The inset reaction shows the oxidation reaction of uric acid to form allantoin and carbon dioxide. The curves show a peak occurring at about 0.3 to 0.35 V potential, peak current increasing for increasing uric acid concentration. FIG. 18D includes two graphs of peak current as a function of uric acid concentration obtained from the data in FIG. 18C, shown on a linear scale (left) and logarithmic scale (right), according to an illustrative embodiment. The data in FIG. 18D shows there is a linear increase in peak current as a function of uric acid concentration. The results in FIGS. 18A-18D show good linearity across a broad range of concentrations for detection.

[0258] FIG. 19 is a graph of current as a function of potential for a differential pulse voltammetry (DPV) measurement with ascorbic acid alone 288 and with ascorbic acid and uric acid together 290, according to an illustrative embodiment. The two curves have a common peak just above 0.4 V corresponding to ascorbic acid, while the curve for ascorbic acid and uric acid together 290 shows an additional peak at about 0.75 V corresponding to uric acid. The data in FIG. 19 demonstrates that the presence of ascorbic acid does not interfere with the detection of uric acid, as the uric acid peak still appears at a similar potential and similar intensity. The described platform may be used in connection with a single coated chip for measuring both hydrogen peroxide and uric acid titration, and has demonstrated both linearity and repeatability with regard to each. In some embodiments, the platform is capable of making measurements (i.e., via PAD method) about every 5 ms (for example, from about 1 ms to about 10 ms, or from about 2 ms to about 8 ms, or from about 3 ms to about 7 ms, or from about 4 ms to about 6 ms). According to aspects of the present embodiments, different analytes can be measured at a sensitivity of about 2.0 pM to about 3.0 pM, or about 2.0 pM to about 2.5 pM, or about 2.2 pM to about 2.45 pM, or about 2.24 uM to about 2.4 pM, or about 2.3 pM to about 2.35 pM for uric acid. According to aspects of the present embodiments, different analytes can be measured at a sensitivity of about 0.6 pM to about 1.2 pM, or about 0.65 pM to about 1.15 pM, or about 0.7 pM to about 1.1 pM, or about 0.75 pM to about 1.05 pM, or about 0.8 pM to about 1.0 pM, or about 0.85 pM to about 0.95 pM, or about 0.87 pM to about 0.92 pM for hydrogen peroxide. In some embodiments, making high accuracy and / or high sensitivity measurements may include using differential pulse voltammetry (DPV). In some embodiments, using DPV results in, for example, ascorbic acid not interfering with uric acid detection, thereby allowing the uric acid peak to still appear at the expected potential and intensity.Electrochemical Measurement Methods

[0259] The platform, systems, and methodologies of the present embodiments can also measure other amperometry, voltammetry and / or impedance measurements. For example, the platform, systems, and methodologies can be used to perform multi-step amperometry to measure two compounds at the same time (for example, by first measuring depletion of the substrate and accumulation of product, followed by measuring activities of two enzymes when they co-exist. In another example, the platform, systems, andmethodologies described herein can be used for voltammetry to 1) detect trace amounts of biomolecules in complex fluid, and / or 2) measure affinity-based binding on / off rate, etc. In another example, the platform, systems, and methodologies described herein can be used for impedance measurements to measure label-free biomolecule binding and cell binding, among other possible applications. In some embodiments, the platform, systems, and methodologies described herein can include a miniaturized form capable of all the measurement types and measurement accuracies of a conventional 3 -electrode system. For example, the platform, systems, and methodologies described herein can be used to quickly and cost effectively measure bioreactions (that is, to directly evaluate the activity of lytic polysaccharide monooxygenase in one example), thereby enabling direct measurement of an electrochemical response of an enzyme in a high-throughput manner.

[0260] In some embodiments, the platform, systems, and methodologies described herein can be used to measure analytes with high sensitivity (e.g., low detection limit). For example, using differential pulse voltammetry (DPV) a low detection limit of uric acid is 2.34 uM and that of hydrogen peroxide is 0.9 uM.

[0261] In some embodiments, the platform, systems, and methodologies described herein can be used to measure reactions with high speed. For example, using pulsed amperometric detection (PAD) measurements can be taken every 5 ms.

[0262] The electrochemical measurement system described herein allows the integration of additional control units (e.g., temperature control units) between the sensing board and control board (e.g., for precise control of temperature).

[0263] The electrochemical measurement system described herein allows the modification or functionalization of electrode surfaces in order to perform measurements such as affinity, binding kinetics, etc. In some embodiments, sensing biomolecules may be conjugated on electrode surfaces by either forming covalent bonds or through strong non- covalent interactions (such as streptavidin-biotin binding). In some embodiments, biomolecules (e.g., antibodies, proteins, etc.) may be conjugated to the electrode surface using EDC / NHS coupling. In some embodiments, bio / streptavidin may be embedded in a coating matrix, followed by exposure of the electrode surface to a streptavidin-labelled sensing element, the surface may be functionalized.

[0264] The platform and system described herein can be used to conduct experiments with repeated or varying conditions. For example, some wells in a multi-well plate may contain exactly the same reactants so that replicates can be run for the same measurement. In another example, enzymatic reactions at different concentrations can be run in different wells on the same multi-well plate in parallel, or each well may contain a different substrate, so that kinetics may be measured in a shorter amount of overall time. In another example, each well in a multi- well plate may contain an entirely different set of reactants or conditions (for example, the titer of enzyme can be measured in one set of wells, and the kinetics of an enzyme can be measured in another set of wells, etc.).

[0265] FIG. 23 is a flow chart diagram of a method 342 of performing a sensing test, according to an illustrative embodiment. At step 344, the method 342 may include using a pulsed amperometric detection (PAD) technique at the oxidation potential of a substrate. At step 346, the method 342 may include determining if a measurement is being performed. If a measurement is being performed, the method 342 advances to step 352. If no measurement is performed, the method of 342 advances to step 348. At step 348, the method 342 may include setting a potential suitable for cleaning the electrode, to prevent possible fouling on the electrode surface. At step 350, the method 342 may return to step 346. At step 352, the method 342 may include setting the potential to an oxidation potential. At step 354, the method 342 may include performing a standard calibration, which may include step 356, step 358, step 360, step 362, step 364, and step 366. At step 356, the method 342 may include adding a substrate of a known concentration to the electrode. At step 358, the method 342 may include waiting for the diffusion to reach equilibrium. In some embodiments, the equilibrium is reached once the current has stabilized (max-min current change is smaller than 5%, for example). At step 360, the method 342 may include measuring the stable current baseline. At step 362, the method 342 may include changing the concentration of the substrate. At step 364, the method 342 may return to step 356 to repeat the measurement for the new concentration of the substrate 362. At step 366, the method 342 may include calculating the substrate constant, for example, by using a linear fitting method. At step 368, the method 342 may include adding a diluted enzyme to the electrode. At step 370, the method 342 may include waiting for the equilibrium current to be reestablished. At step 372, the method 342 may include measuring the current decay over time (for example, dl / dt). At step 374, the method 342 may include measuring the concentration change over time. At step 376, the method 342 may revert to step 346.Automated Data Collection

[0266] FIG. 22 is a flow chart diagram of a method 316 of performing automated data collection, according to an illustrative embodiment. At step 318, the method 316 may include dispensing substrate, by the liquid handler, into required wells on the 96-well sensor plate. At step 320, the method 316 may include receiving a signal, by the microcontroller, from the liquid handler to start the measurement routine. At step 322, the method 316 may include determining, by a programmed routine of the microcontroller, the next well to be measured. At step 324, the method 316 may include communicating, by the microcontroller, with the multiplexers. At step 326, the method 316 may include (1) connecting, by the multiplexers, each element of the electrochemical front end (i.e., working electrode, reference electrode and counter electrode) to the correct well and (2) disconnecting all other wells. At step 328, the method 316 may include receiving, by the liquid handler, a signal from the microcontroller to add reactant, enzyme and / or small molecule(s). At step 330, the method 316 may include sending a signal, by the microcontroller, to the electrochemical front end to start taking measurements for preprogrammed amount of time and / or number of datapoints. At step 332, the method 316 may include receiving data, by the microcontroller, from the electrochemical front end. At step 336, the method 316 may include storing data on the microcontroller or offloading data to external storage space. At step 334, the method 316 may include receiving a signal, by the microcontroller from the electrochemical front end indicating the measurement is done. At step 338, the method 316 may return to step 330 to repeat the measurement for a preprogrammed number of times. At step 340, the method 316 may return to step 322 to continue the measurement for the next well in the program routine.

[0267] Referring still to FIG. 22, according to aspects of the present embodiments, the flexibility of the control board 188 allows a large variety of routines to be run in many formats. Each well may be measured serially, where the enzyme or analyte may be added one well at a time and measure each reaction to completion before moving to the next well. In addition, the reactant may also be added to all 96 wells at the same time, and then a single high-speed measurement may be taken on each well, measuring all 96-wells in “parallel” until all the necessary data is collected. In addition, various combinations (or hybrid methods including combinations of parallel and in series methodologies) can be employed asdescribed herein due to the flexibility of the design, which provides the user the ability to design experiments in the most appropriate way.Single well progression curve fitting

[0268] FIG. 50 illustrates an example of a single well progression curve fitting method 650, according to an illustrative embodiment. The data acquisition and processing method 650 may include determining amperometry potential of all 96 wells 652, and applying potential to each well 654. Each well can achieve millisecond (ms) intervals (i.e., millisecond granularity) between measurements 656.Example: Automated Procedure to Test Enzymatic Activity

[0269] In some embodiments, the multi-well sensor plate assembly may be integrated with a standard liquid handler. For example, a customized holder may be used to hold multiwell sensor plate assembly, and liquid samples and / or reagents may be added to the wells in parallel by multiple pipette tips attached to an automated pipetting head. FIGS. 30A-30I illustrate an example of an automatic procedure using a multi-well sensor plate assembly and an automated liquid handling system or automated liquid handler, according to an illustrative embodiment. In this example, the liquid handler is a Biomek liquid handler. FIG. 30A shows the original position of the automated pipetting head 462 including 8 pipette tips 464, automated transfer arms 460 and a custom holder 407 including a bottomless 96-well plate 180 that is placed over a multi-well sensor plate assembly (including sensor board 181 and control board 183) inside the custom holder 407.

[0270] FIG. 30B shows the automated pipetting head 462 raising up, toward the 96- well plate 180. FIG. 30C shows the automated pipetting head 462 translating over the multiwell sensor plate assembly such that the pipette tips 464 are positioned above the wells.FIG. 30D shows the automated pipetting head 462 lowering the pipette tips 464 into the wells. FIG. 30E shows the automated pipetting head 462 raising the pipette tips 464 out of the wells. FIG. 30F shows the automated pipetting head 462 translating away from the 96- well plate 180, and the automated transfer arms 460 carrying a transparent cover 468 toward the 96-well plate 180. FIG. 30G shows the transparent cover 468 that is placed over the 96- well plate 180 and the automated transfer arms 460 translating away. FIG. 30H shows the automated pipetting head 462 translating over the multi- well sensor plate assembly such thatthe pipette tips 464 are positioned above the wells again. FIG. 301 shows the automated pipetting head 462 lowering the pipette tips 464 into the wells. The fluid is suctioned from the wells into the pipette tips 464 and subsequently discharged from the pipette tips 464 back into the wells. The suctioning and discharge of fluid is repeated five times, which results in mixing of the fluids, concluding with the fluid being discharged into the wells. In some embodiments, data acquisition and processing routines are used for the automatic procedures. An example data acquisition and processing method is further described herein in connection with FIGs. 34A-C.

[0271] In some embodiments, to achieve liquid homogeneity, the fluid may be mixed constantly without using a liquid handler. The mixing method may include stirring, shaking, agitating, and / or titling of the 96-well plate 180. The mixing may be performed by a device that functions as a constant mixer, such as a motor. In some embodiments, the measurement may be performed rapidly, allowing testing multiple concentrations using a single well.

[0272] In some embodiments, the multi-well sensor plate assembly may be used on bench top. In the case of a bench top use, similar steps are followed through human interactions, for example adding fluid, gentle shaking, etc., or through use of mechanical agitation or shaking.

[0273] FIG. 31A shows an example of data acquired by a 96-well sensor board, according to an illustrative embodiment. The acquired data may include 96 graphs 470 of current as a function voltage for different concentrations of uric acid, each graph 470 corresponding to one individual well, and each curve corresponding to a particular concentration of uric acid. Each graph 470 is further analyzed by measuring the peak current at different concentrations and performing a linear fitting. FIG. 31B shows calibration curves 472 (current as a function of concentration) calculated from data acquired by the 96- well sensor board, according to an illustrative embodiment. Each calibration curve 472 corresponds to enzyme activity within one individual well.

[0274] In connection with the present embodiments, the circular concentric planar electrode configuration as described herein may help to enable automation compatible electrochemical measurements in a high throughput (i.e., multi- well) environment. By providing an electrode configuration with a footprint that approximately matches that of (i.e., the area of) each well, and by using coatings as described herein, measured signals may be amplified and / or obtained in higher resolution. The electrochemical biosensors of the presentembodiments may be used in connection with measurements of biological and chemical signals including (but not limited to) biological signals from antibodies, DNA, enzymes, and cells (including binding activities of peptides and / or aptamers, etc.); measurement activities that include a redox response; and / or enzymatic reactions involving any of: glucose oxidase, peroxidase, lactate dehydrogenase, superoxide dismutase, cytochromes, carbonic anhydrase, and / or any enzymes involved in the Krebs cycle; and / or biological signals from specific enzymes described herein.EQUIVALENTS

[0275] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMSWhat is claimed:

1. An automation-compatible electrochemical sensing platform, the platform comprising: a sensor plate assembly comprising a sensor board, a multi- well plate, and a gasket securing the sensor board to the multi-well plate, wherein the sensor board comprises an electrode assembly corresponding to each well of the multi- well plate; a control board comprising hardware for detecting signals from the sensor board; and an interconnect detachably connecting the sensor board to the control board.

2. The platform of claim 1, wherein the control board has dimensions for compatibility with commercial lab automation systems.

3. The platform of claim 1 or 2, wherein the control board comprises: an electrochemical (EChem) front end to control the sensor board to obtain electrochemical measurements; a multiplexer to control temporal connection between the EChem front end and the electrode assemblies of the sensor board corresponding to the wells of the multi- well plate; and a microcontroller to manage the EChem front end and the multiplexer.

4. The platform of claim 3, wherein the control board further comprises one or more members selected from the group consisting of a battery holder, a battery, and a memory card for data logging.

5. The platform of any one of the preceding claims, further comprising one or more additional control units positioned between the sensor board and the control board.

6. The platform of any one of the preceding claims, wherein the sensor board is a printed circuit board (PCB) and the gasket secures the PCB to the bottomless multi-well plate with a watertight seal.

7. The platform of any one of the preceding claims, wherein the electrode assembly on the sensor board comprises electroless palladium immersion gold (EPIG).

8. The platform of any one of the preceding claims, wherein the electrode assembly on the sensor board comprises hard gold.

9. The platform of any one of the preceding claims, wherein the electrode assembly on the sensor board comprises polyimide between metal electrodes of the multiple electrode assemblies corresponding to each well of the multi-well plate.

10. The platform of any one of the preceding claims, wherein the electrode assembly is planar.

11. The platform of any one of the preceding claims, wherein the electrode assembly is cylindrical.

12. The platform of any one of the preceding claims, wherein the sensor plate assembly accommodates a well volume that ranges from about 10 microliters to greater than 200 microliters.

13. The platform of any one of the preceding claims, wherein the multi-well plate is made of a polymer and / or wherein the multi- well plate has a coating to prevent or reduce protein adhesion.

14. The platform of any one of the preceding claims, wherein the sensor board is a printed circuit board (PCB) that is compatible for use with multiple types of bottomless multi-well plates to facilitate performing multiplexing measurements.

15. The platform of any one of the preceding claims, further comprising a liquid handler, wherein the platform is programmed to perform the following steps:(i) the liquid handler dispenses substrate into required wells of the multi- well sensor plate for a reaction from which electrochemical measurements are to be obtained;(ii) the microcontroller is sent a signal from the liquid handler to begin a programmed measurement sequence;(iii) the microcontroller communicates with the multiplexer to connect a working electrode, reference electrode, and counter electrode of the EChem front end to a correct corresponding well on the multi- well sensor board and disconnects all other wells;(iv) the liquid handler is sent a signal from the microcontroller such that the liquid handler dispenses a composition to produce a reaction with the substrate and generate an electrical signal to be measured;(v) the microcontroller communicates with the EChem front end and directs it to begin taking measurements for a programmed amount of time and / or datapoints, then the microcontroller receives the data from the EChem front end and stores the data on the microcontroller or offloads the data to an external storage space; and(vi) when the EChem front end signals it has finished a measurement, the microcontroller proceeds with at least step (iii) and (v) for a next well in the programmed sequence and repeats this until all of the wells in the programmed sequence have been measured.

16. The platform of claim 15, wherein the platform is programmed such that the microcontroller repeats step (v) as many times as it is programmed to obtain measurements over a time period during which the reaction progresses.

17. The platform of claim 15 or 16, wherein the platform is programmed such that the liquid handler dispenses the composition into (or onto) a single well at a time to produce the reaction with the substrate in each well, serially.

18. The platform of claim 15 or 16, wherein the platform is programmed such that the liquid handler dispenses the composition into multiple wells at a time such that measurements are obtained from the multiple wells “in parallel” until all the data is collected.

19. The platform of any one of claims 15 to 18, wherein output signals are smoothed via a post processing filtering technique to produce the measurements.

20. The platform of any one of claims 15 to 19, wherein the electrochemical measurements are obtained by performing pulsed amperometric detection (PAD) to produce enzymatic reaction kinetic data.

21. The platform of claim 20, wherein the enzymatic reaction kinetic data are uricase activity measurement.

22. The platform of claim 20, wherein the product / measured species is / comprises hydrogen peroxide and wherein the liquid handler dispenses lytic polysaccharide monooxygenase (LPMO) to produce the reaction with the substrate.

23. The platform of any one of claims 15 to 19, wherein obtaining the electrochemical measurements comprises performing differential pulse voltammetry (DPV) to obtain enzymatic reaction kinetic data.

24. The platform of claim 23, wherein the substrate / measured species is / comprises ascorbic acid, uric acid and / or hydrogen peroxide.

25. The platform of any one of the preceding claims, wherein the electrode assembly has a conductive, anti-fouling coating.

26. The platform of claim 25, wherein the conductive, anti-fouling coating comprises a first component to provide anti-fouling and a second component to provide conductivity.

27. The platform of claim 25 or 26, wherein the conductive, anti-fouling coating comprises a hydrogel matrix that allows for physical immobilization of a conductive filler, wherein the conductive filler is loaded above an electrical percolation threshold to form a conductive pathway within the hydrogel matrix.

28. The platform of any one of claims 25 to 27, wherein the anti-fouling coating comprises a hydrogel matrix, and wherein the hydrogen matrix comprises one or more sensing elements to facilitate detection of a desired analyte.

29. The platform of any one of claims 25 to 28, wherein the anti-fouling coating comprises a conductive filler in a bovine serum albumin (BSA) matrix.

30. The platform of any one of claims 25 to 29, wherein the anti-fouling coating comprises a conductive hydrogel matrix.

31. The platform of any one of claims 25 to 30, wherein the anti-fouling coating is created as follows: a hydrogel precursor is dropcasted directly onto the working electrode; optionally, the electrode surface is activated to allow attachment of the hydrogel; optionally, external thermal and / or UV sources are used to initiate hydrogel polymerization; optionally, a blade is used to ensure homogeneous precursor application (e.g., wherein the sensor board comprises a 96-well plate); a sensor chip is quenched after reaction for a period of time to ensure consistent degree of polymerization and washed to remove unreacted monomer; the sensor chip is stored in buffer and cooled to prevent coating degradation; and optionally, an additional sensing element is conjugated onto the hydrogel.

32. An electrode assembly of an automation-compatible electrochemical sensing platform, said electrode assembly corresponding to a given well of a multi-well plate, wherein said electrode assembly has a conductive, anti-fouling coating.

33. The electrode assembly of claim 32, wherein the conductive, anti-fouling coating comprises a first component to provide anti-fouling and a second component to provide conductivity.

34. The electrode assembly of claim 32 or 33, wherein the conductive, anti-fouling coating comprises a hydrogel matrix that allows for physical immobilization of a conductive filler, wherein the conductive filler is loaded above an electrical percolation threshold to form a conductive pathway within the hydrogel matrix.

35. The electrode assembly of any one of claims 32 to 34, wherein the anti-fouling coating comprises a hydrogel matrix, and wherein the hydrogen matrix comprises one or more sensing elements to facilitate detection of a desired analyte.

36. The electrode assembly of any one of claims 32 to 35, wherein the anti-fouling coating comprises a conductive filler in a bovine serum albumin (BSA) matrix.

37. The electrode assembly of any one of claims 32 to 36, wherein the anti-fouling coating comprises a conductive hydrogel matrix.

38. The electrode assembly of any one of claims 32 to 37, wherein the anti-fouling coating is created as follows: a hydrogel precursor is dropcasted directly onto the working electrode; optionally, the electrode surface is activated to allow attachment of the hydrogel; optionally, external thermal and / or UV sources are used to initiate hydrogel polymerization; optionally, a blade is used to ensure homogeneous precursor application (e.g., wherein the sensor board comprises a 96-well plate); a sensor chip is quenched after reaction for a period of time to ensure consistent degree of polymerization and washed to remove unreacted monomer; the sensor chip is stored in buffer and cooled to prevent coating degradation; and optionally, an additional sensing element is conjugated onto the hydrogel.

39. A method for measurement of enzymatic activity, the method comprising: obtaining electrochemical measurements in a well of a multi-well plate, said well containing a solution comprising a substrate and a composition dispensed into the well by a liquid handler, wherein said composition produces a reaction with the substrate thereby generating an electrical signal to be measured.

40. The method of claim 39, wherein obtaining the electrochemical measurements comprises performing pulsed amperometric detection (PAD) to produce enzymatic reaction kinetic data.

41. The method of claim 40, wherein the enzymatic reaction kinetic data are uricase activity measurement.

42. The method of claim 40, wherein the substrate / measured species comprises hydrogen peroxide and wherein the liquid handler dispenses lytic polysaccharide monooxygenase (LPMO) to produce the reaction with the substrate.

43. The method of any one of claims 40 to 42, wherein obtaining the electrochemical measurements comprises performing differential pulse voltammetry (DPV) to produce enzymatic reaction kinetic data.

44. The method of claim 43, wherein the substrate / measured species comprises ascorbic acid.

45. A system for performing high-throughput electrochemical sensing for measurement of enzymatic activity, the system comprising:(i) a liquid handler programmed to dispense a composition into wells of a multiwell sensor plate to produce a reaction with a substrate and generate an electrical signal to be measured, from which enzymatic reaction kinetic data is determined;(ii) a microcontroller that (a) receives a signal sent from the liquid handler to begin a programmed measurement sequence, (b) communicates with an electrochemical (EChem) front end and directs it to begin taking measurements for a programmed amount of time and / or datapoints in a given well, (c) receives data from the EChem front end and stores the data on the microcontroller or offloads the data to an external storage space, and (d) sends a signal to the multiplexer to proceed to the next well in the sequence upon completion of measurements for a given well; and(iii) a multiplexer that receives a signal sent from the microcontroller to connect a working electrode, a reference electrode, and a counter electrode of the EChem front end to a correct corresponding well on the multi- well sensor plate and disconnects all other wells.

46. The platform of claim 1, wherein the sensor board has dimensions for compatibility with commercial lab automation systems.M. The platform of claim 1, wherein the gasket has dimensions for compatibility with commercial lab automation systems.

48. The platform of any of claims 1-31 wherein the antifouling coating is coated only on regions of the sensor board directly overlapping with the regions of the sensor assemblies.

49. The platform of claim 31, wherein a masking gasket is placed on the sensor board prior to dropcasting of the hydrogen precursor, wherein the masking gasket comprises a thin flexible layer with a plurality of holes disposed through the masking gasket, and wherein the plurality of holes overlaps with the sensor assemblies on the sensor board.

50. The platform of claim 49, wherein the masking gasket and the gasket are the same.

51. The platform of any of claims 1-31 and claims 46-50, wherein the number of sensor assemblies on the sensor board exceeds the number of holes in the gasket and the number of wells in the multi- well plate, such that more than one sensor assembly is disposed within each hole of the gasket and within each well of the multi- well plate.

52. The platform of any of claims 1-31 and claims 46-51, wherein the volumes of each of the wells of the multi- well plate are fluidly isolated from each other, and wherein each sensor assembly of the sensor board is electrically isolated from each other.

53. The platform of any of claims 1-31 and claims 46-52, wherein the sensor assemblies comprise center-to-center spacings that correspond to center-to-center spacings of wells in standard well-plates.

54. The platform of any one of claims 1-31 and claims 46-53, wherein the sensor board further comprises a built-in temperature control unit.

55. The platform of claim 54, wherein the built-in temperature control unit is a built-in heating layer.

56. The platform of claim 55, wherein the built-in heating layer comprises metal arranged in a serpentine pattern through the interior of the sensor board.

57. The platform of any one of claims 54-56, wherein the built-in temperature control unit provides a substantially uniform temperature across the sensor board.

58. The platform of any one of claims 54-57, wherein the sensor board further comprises a temperature sensor.

59. The platform of claim 58, wherein the sensor board further comprises a proportionalintegral-derivative (PID) controller.

60. The platform of any one of claims 54-56, wherein the built-in temperature control unit provides an individual temperature control to each well of the multi-well plate.

61. The platform of claim 60, wherein the sensor board further comprises a plurality of temperature sensors, wherein each temperature sensor corresponds to each well of the multiwell plate.

62. The platform of any one of claims 54-61, wherein the built-in temperature control unit further comprises a cooling module.

63. The platform of claim 62, wherein the cooling unit comprises a Peltier module.

64. The platform of any one of claims 1-31 and claims 46-63, wherein the sensor board comprises a printed circuit board (PCB), a glass wafer, a low-conductivity silicon wafer, or an insulating substrate.

65. The platform of any one of claims 1-31 and claims 46-53, wherein the platform further comprises an external temperature control unit comprising:a heater block comprising a metal block with external dimensions matching a standard well-plate, the heater block machined to have openings to accommodate electrical connections, and a plurality of grooves; a heater coil comprising wiring for resistive heating, disposed within the plurality of grooves; a power supply for passing current through the heater coil; and a temperature sensor disposed on a surface of the heater block.

66. The platform of claim 65, wherein the external temperature control unit further comprises a cooler.

67. The platform of claim 66, wherein the cooler comprises a Peltier cooler.

68. The platform of claims 65-67, wherein the external temperature control unit further comprises a PID controller electrically connected to the power supply, the temperature sensor, and the cooler, and wherein the PID controller is configured to maintain a setpoint temperature.

69. A data processing method for high-throughput electrochemical sensing using a plurality of electrochemical sensors, the method comprising: acquiring raw data from the plurality of electrochemical sensors; translating the raw data to a readable format by a data parser; ploting the translated data by a data visualizer; and performing automated curve fitting by a data analyzer.

70. The platform of claim 21, wherein the uricase activity measurement comprises uricase in serum.

71. The platform of claim 65, wherein the electrical connections comprise a plurality of metallic, spring-loaded prongs.

72. The platform of any one of claims 1-5, wherein the sensor board is a printed circuit board (PCB) that is glued to the bottomless multi-well plate with a watertight seal.

73. The platform of claim 20, wherein the enzymatic reaction kinetic data comprise L- ascorbate oxidase activity measurement and / or horseradish peroxide activity measurement.

74. The platform of claim 20, wherein the enzymatic reaction kinetic data comprise biological signals from enzymatic reactions that include or are involved with glucose oxidase, peroxidase, lactate dehydrogenase, superoxide dismutase, cytochromes, and / or carbonic anhydrase.

75. The platform of claim 20, wherein the enzymatic reaction kinetic data comprise biological signals from enzymatic reactions that include or are involved with a redox response.

76. The platform of any one of claims 15 to 19, wherein the electrochemical measurements are obtained by measuring biological signals of antibodies, DNA, enzymes, and cells and / or biological signals from binding activities of peptides and / or aptamers.

77. The platform of claim 21, wherein the uricase activity measurement comprises uricase in cell culture media.

78. The platform of claim 77, wherein cell culture media is Fetal bovine serum (FBS).

79. A sensor for measuring electrochemical activity, the sensor comprising: a three-electrode circuit comprising a working electrode, a counter electrode, and a reference electrode, each electrode of each three-electrode circuit comprising: an electrode top surface; a conductive contact pad; and a contact electrically coupling the electrode top surface to the conductive contact pad; wherein the electrode top surfaces of each electrode of the three-electrode circuit are arranged in a concentric configuration.

80. The sensor of claim 79, wherein the contacts of each of the three electrode circuits are positioned in a linear configuration.

81. The sensor of claim 79, further comprising a heater circuit positioned at least partially between the electrode top surfaces and the conductive contact pads, wherein the heater circuit is not electrically coupled to the electrode circuit, and wherein the heater circuit comprises a heater trace arranged in a serpentine configuration.

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