WO2024115915A1

WO2024115915A1 - Electrochemical electrode assembly

Info

Publication number: WO2024115915A1
Application number: PCT/GB2023/053107
Authority: WO
Inventors: Neville Freeman
Original assignee: E4G Limited
Priority date: 2022-12-01
Filing date: 2023-11-30
Publication date: 2024-06-06
Also published as: GB202218073D0

Abstract

An electrochemical electrode assembly having micro/nano scale characteristics and in particular at least one micro/nano scale working electrode 'active' surface for the selective oxidation/reduction of a target analyte or reaction product thereof. The present working electrode comprises a minimised surface area relative to the surface area size of a surrounding insulating material surface such that a target analyte within a biofluid (e.g. blood, interstitial fluid, sweat, saliva, etc) is required to diffuse over at least a region of the insulating surface towards the working electrode surface.

Description

Electrochemical Electrode Assembly

Field of invention

The present concept relates to an electrochemical electrode assembly and in particular, although not exclusively, to a microelectrode suitable for use as part of an electrochemical sensor.

Background

Electrochemical sensors implemented within an implantable or skin contact device are of great interest in the electroanalytical sector due to the capability for the quantitative and qualitative characterisation of target analytes in complex biological systems. Such pm scale sensors according to existing manufacturing techniques enable high time resolution and time sensitivity analysis.

The first electrochemical sensors for oxygen were reported in the 1960s. In particular, the concept for glucose sensing (in vitro) was suggested by Clark and Lyons in 1962 (based on an oxygen sensor) and the first in vitro commercial electrochemical glucose sensors were made by YSI in 1975 (based on a hydrogen peroxide sensor). The first commercial in vivo glucose sensors were then available in 1999. More recently, due to the development of micro and nano scale manufacturing techniques, mi cro/nano-el ectrode array sensors have emerged as an attractive biofluid enabled sensor offering multiplexing ability and robustness for bioanalysis in a variety of different environments e.g. cells, tissues and organs as implantable and also non-invasive wearable devices.

Micro- or nano-electrode array sensors configured for smart sensing as an invasive or wearable intelligent device typically comprise electrodes with dimensions at the micro- and nano- scale. Various types of mi cro/nano-el ectrode arrays have been developed for different measuring conditions. In general, such electrode arrays have been prepared by layer deposition or layer growth in which a metal, carbon, ceramic etc layer is deposited on a template substrate such as silicon, glass, polymer, ceramic etc. Photolithography, screen printing, membrane formation, laser ablation and 3D printing techniques have been employed to create complex electrode microscale patterns. An example electrode assembly is described in WO 2010/061229 Al in which a laminate structure comprises an insulating capping layer provided on a conducting layer designed to provide exposed electrical contact regions at etched voids extending through the capping layer.

However, existing micro electrochemical electrode assemblies and sensors are disadvantageous for a number of reasons. Typically, existing devices do not provide the separation between individual electrode elements to realise the full benefit of a micro/nano scale electrode. Available structures and sensors involve the use of thin layer techniques that require precise and complex surface patterning to expose regions of the conducting layers. Additionally, existing devices do not appropriately control the diffusion of target analytes (within a carrier biofluid) to the conducting regions leading to oxidation or reduction of excessive amounts of the analytes that may be mitigated only through undesirably long pauses in the applied potential (so as to allow the continued diffusion of fresh analyte to the actives conducting surfaces) or the inclusion of limiting membranes reducing both the signal and temporal response). Accordingly, there is a need for an electrochemical electrode and sensor arrangement that solves the above problems. Summary of the Invention

It is an objective of the present concept to provide an electrode assembly that may be manufactured conveniently and efficiently whilst providing a device suitable for use as an implantable or wearable (skin contacting) electrochemical sensor that offers results with enhanced sensitivity, reliability, speed of response and repeatability.

It is a further objective to provide an electrode assembly and electrochemical electrodebased sensor offering appreciably enhanced analyte diffusion control (non-planar diffusion control) in use. It is a further specific objective to provide an assembly to achieve a diffusion-controlled current which is time independent in operation (typically when polarised to a sufficient potential to oxidise or reduce a target analyte). It is a further specific objective to provide an electrode assembly that controls target analyte diffusion within a biofluid towards a surface of a working electrode such that the rate of diffusion of a target analyte is greater than a rate of oxidation/reduction at the electrode surface. It is a further specific objective to avoid problems of appreciable target analyte volumetric depletion at the vicinity/region of the working electrode surface that would otherwise manifest as an appreciable reduction or zero Faradaic current (in response to the excessive or complete oxidation/reduction of the target analyte or reaction product of the target analyte with a material of a coating at the conducting surface) at the region of the working electrode.

Reference within this specification to a target analyte encompasses a species of interest and/or a species generated as a consequence of the presence of a target analyte. Such a species may be generated by enzymatic reaction as described herein.

It is a further specific objective to provide an electrochemical sensor and in particular an implantable or wearable device capable of interrogating a plurality of target analytes inseries or in-parallel.

The objectives are achieved via the present electrochemical electrode assembly having micro/nano scale characteristics and in particular a micro/nano scale working electrode "active ’ surface for the selective interrogation of a target analyte. The present working electrode comprises a minimised surface area relative to the surface area size of a surrounding insulating material surface. According to the present concept the conducting surface(s) is bound and surrounded by at least one insulating material surface such that a target analyte within a biofluid (e.g. blood, sweat, saliva etc) is required to diffuse over at least a region of the insulating surface towards the working electrode surface. By configuring the relative sizes, geometries and/or relative spatial positioning of the conducting surface(s) relative to the surrounding insulating surface, the target analyte diffusion within the biofluid is dominated by hemispherical diffusion and exceeds the rate of consumption (i.e., reaction, oxidation or reduction) of the target analyte at the conducting surface(s).

The present concept is configured to be responsive to the presence/existence of a target analyte at the active electrode surface so as to effectively determine a concentration or rate of concentration change of the target analyte within a fluid sample e.g., a biofluid.

According to specific implementations, the active electrode surface comprises an analyte responsive layer having a material configured to bind, interact chemically and/or physically with the target analyte. In particular, the analyte responsive layer may comprise an enzymic material to interact or react chemically with the target analyte and to generate an electrochemically active product of the enzymatic reaction with the target analyte. The target analyte may comprise an electrochemically active product of the enzymatic reaction with an initial species. That is, the target analyte may be a chemical product of the enzymatic reaction with the initial target species. For example, where the present concept is configured to identify glucose levels/concentrations, the target analyte may comprise the product of the enzymatic reaction with glucose i.e. hydrogen peroxide. The present concept may be configured to measure the current between the working electrode and an auxiliary electrode resultant from a direct oxidation or reduction of the target analyte. Alternatively, or in addition, the present concept is configurable to measure the current between the working electrode and auxiliary electrode resultant from the oxidation or reduction of an electrochemically active reaction product of the target analyte with the analyte responsive layer. For example, where the target analyte is a glucose, the species for oxidation and reduction at the active electrode surface may comprise hydrogen peroxide (being the electrochemically active reaction product of an enzymatic analyte response layer material coated and/or positioned proximate to the active electrode surface). Reference herein to the oxidation/reduction of a target analyte includes the oxidation/reduction of the analyte directly or indirectly via oxidation/reduction of the reaction product thereof.

Accordingly, the present electrochemical method of interrogating at least one targe analyte within a biofluid comprises applying an oxidising or reducing electrical potential at a working electrode to oxide or reduce the target analyte or rection product thereof coupled with the measuring of a Faradaic current at the working electrode. Optionally, the step of applying the oxidising or reducing potential comprises performing cyclic voltammetry.

According to a first aspect of the present concept there is provided an electrochemical electrode assembly comprising: an electrically insulating material having at least one insulating surface; an electrically conducting material having at least one conducting surface with a perimeter bound and surrounded by the insulating surface; wherein a surface area of the conducting surface or a surface area of each conducting surface where the assembly comprises a plurality of conducting surfaces is less than 5% of a surface area of the insulating surface that surrounds the perimeter of the conducting surface or the respective surface area of the insulating surface that surrounds each respective perimeter of each conducting surface, wherein each respective surface area of the insulating surface is defined between an inner boundary at the perimeter of the conducting surface or a respective inner boundary at each respective perimeter of each conducting surface and a respective outer boundary that is separated from the respective inner boundary by a respective distance of at least five times a diameter or smallest width across the conducting surface or across each respective conducting surface where the assembly comprises a plurality of conducting surfaces.

To control the consumption of the target analyte at the working electrode surface, the present assembly is configured such that the active oxidising/reducing electrode surface forms a discontinuity within an insulating material surface so as to represent a much reduced relative ‘active’ surface area within the surrounding insulating material surface. The present concept is further configured to comprise physical characteristics that facilitate the flow and diffusion of target analyte to the active working electrode surface. That is, the present concept is configured to minimise or avoid positioning of the active surface(s) within obscured, restricted or otherwise confined zones or regions at the micro/nano scale structure with such restricted regions hindering a free flow of target analyte to the active surface. In one preferred implementation, the active surface and the surrounding insulating surface are both substantially planar and without grooves, channels, pits, peaks or troughs in the nano/micro scale structure. However, according to certain manufacturing techniques such contouring may be necessary and the active electrode surfaces may be provided at the sidewalls, edges, troughs or valleys of a contoured/profiled nano/micro assembly structure and in particular a laminate structure in which a conducting layer is capped or bound by one or more insulating layers and portions of the laminate structure are ablated or cut (via the creation of channels or cavities within the structure) so as to provide regions of exposed conducting layer surface that provide the region of active oxidising/reducing surfaces. Such arrangements may provide an active electrode surface within a cavity forming part of a 3D array or structure. The present electrochemical electrode assembly may be manufactured according to a variety of techniques not limited to atomic layer deposition (ALD), chemical vapour deposition (CVD), plasma enhanced chemical vapour deposition (PECVD), slot-die coating, screen printing, inkjet printing, spray coating, 3D printing and other existing microprocessor or chip manufacturing techniques within the silicon chip manufacturing sector.

Optionally, a surface area of the individual conducting surface or each surface area of the plurality of conducting surfaces is less than 300 pm², 200 pm², 150 pm², or 100 pm². Optionally, a surface area of the conducting surface or each surface area of the plurality of conducting surfaces is less than 300 pm², 200 pm², 150 pm², 100 pm², 80 pm², 60 pm², 40 pm², 20 pm², 10 pm², 5 pm², 3 pm² or 1 pm².

According to certain embodiments having a working electrode with a minimised active surface area, the total conducting surface of the plurality of active conducting surfaces may be less than 100 pm². Optionally, according to some implementations of electrode configuration and/or electrochemical sensor, the total surface area of the plurality of conducting surfaces may be greater than 100 pm². However, in such configurations, the surface area of each individual conducting surface may be less than 100 pm².

Optionally, the total surface area of the conducting surface or conducting surfaces where the assembly comprises a plurality of conducting surfaces is in a range 0.001 to 500,000 pm²; 0.001 to 250,000 pm²; 0.001 to 100,000 pm²; 0.001 to 10,000 pm²; 0.01 to 8,000 pm²; 0.001 to 6,000 pm²; 0.001 to 4,000 pm²; 0.001 to 2,000 pm²; 0.001 to 1000 pm².

Optionally, the assembly is configured for use with a minimally invasive device such as an implantable or skin contact device for use as an electroanalytical sector for quantitative and qualitative characterisation of target analytes in a complex biological system. Optionally, for such a configuration and use a total surface area of the conducting surfaces, where the assembly comprises a plurality of conducting surfaces, is in a range 0.001 to 1000pm²; 0.001 to 800pm²; 0.001 to 600 pm²; 10 to 600pm²; 100 to 600pm²; 100 to 400pm²; 100 to 200pm²; 0.001 to 500pm²; 0.001 to 300pm²; 0.01 to 300pm²; 0.01 to 300pm²; 0.01 to 200pm²; 1 to 300pm²; 1 to 200pm²; 50 to 300pm²; 50 to 250pm²; 50 to 200pm²; 5 to 200pm² or 10 to 200pm². Such an arrangement contributes to the control of the diffusion of a target analyte towards the conducting surface.

Preferably, the surface area of the single or each conducting surface that where the electrochemical electrode assembly comprises a plurality of individual conducting surfaces, is at least one order of magnitude smaller than the surrounding insulating surface area that surrounds the single conducting surface or each individual conducting surface. In particular, the conducting surface or each conducting surface may be less than 300 pm², 200 pm², 150 pm² or 100 pm² whilst the surrounding insulating material may be of the order of at least 1000 pm². Optionally a surface area of the respective insulating surface that surrounds the conducting surface or each individual conducting surface may be greater than 5000 pm², 10,000 pm², 15,000 pm², 20,000 pm², 50,000 pm², 100,000 pm², 500,000 pm² or 1 mm². Optionally, a width, diameter or smallest distance across the single or each individual conducting surface may be in a range 1 to lOOOnm, 1 to 800nm, 1 to 600nm, 1 to 400nm, 1 to 800nm, 1 to 600nm, 1 to 400nm, 1 to 200nm, 1 to lOOnm, 10 to lOOnm. Optionally, a width, diameter or smallest distance across the single or each individual conducting surface may be in a range 10 to lOOOnm, 50 to 800nm, 50 to 600nm, 50 to 400nm, 50 to 200nm, 100 to lOOOnm, 100 to 800nm, 100 to 600nm, 100 to 400nm, or 100 to 200nm.

Optionally, the width, diameter or smallest distance across each respective conducting surface is less than 100, 80, 60, 40, 20 or 10 nm.

Optionally, the assembly may comprise between 10 to 100,000; 10 to 50,000; 10 to 10,000; 10 to 8,000; 10 to 3,000, 10 to 1000; 10 to 800; 10 to 600; 10 to 400; 10 to 200; 10 to 100; 10 to 80, or 10 to 60 active surfaces.

Optionally, the conducting surface and/or the insulating surface is any one or a combination of substantially planar, convex, concave, profiled so as to comprise peaks or valleys. Preferably the conducting surface is positioned at a region that does not hinder undesirably the diffusion of a target analyte towards the conducting surface.

Optionally, the electrically conducting material is a layer provided on a substrate and the electrically insulating material is a layer provided on the conducting material layer wherein the insulating material layer is discontinuous to expose regions of the conducting material layer that provide the at least one conducting surface. Such exposed regions may be provided by ablating or cutting portions of the laminate structure.

Optionally, the assembly may comprise a single conducting surface. Alternatively, the present assembly may comprise a plurality of conducting surfaces wherein perimeters of each of the conducting surfaces are separated from one another by a distance of at least 5 times a diameter or smallest width across any one of the conducting surfaces. That is, each conducting surface is separated from one another by a distance being greater than a width, diameter or smallest distance across each respective conducting surface. Reference in the specification to a distance across the conducting surface, refers to a straight line bisecting the conducting surface extending from a first point at the perimeter of the at least one conducting surface and a second point at the respective perimeter. Preferably, this distance is of the order of nanometres. Preferably, the corresponding distance between the inner boundary of the insulating surface and the outer boundary of the insulating surface is of the order of micrometres.

Optionally, each conducting surface is separated from one another by a distance that is in a range 10 to 100,000; 10 to 10,000; 10 to 1000; or 10 to 100 times a width, diameter or smallest distance across each respective conducting surface.

Preferably, where the assembly comprises a plurality of conducting surfaces, each surface is separated from one another by a distance that is at least five times a diameter or smallest width across the respective conducting surface. Optionally, this distance may be at least 10, 50, 100 or 1000 times a diameter or smallest width across the respective conducting surface. This arrangement ensures that the conducting surfaces do not interfere with one another and in particular function to draw target analyte from the electrolyte medium in a controlled manner that in turn provides a controlled diffusion and in particular an oxidation/reduction characteristic and an electrode assembly with a time independent response.

Optionally, the assembly may comprise an analyte responsive layer provided on or at the conducting surface. Optionally, the analyte responsive layer comprises any one of: a material configured to bind the target analyte; an enzymic material to interact or react chemically with the target analyte. Optionally, the analyte responsive layer comprises any one or a combination of at least one aptamer; an antibody; a molecularly imprinted polymer. Optionally, the assembly may comprise a plurality of analyte responsive layers each comprising a different material to bind a different respective target analyte.

Optionally, the assembly may comprise a plurality of conducting surfaces each comprising a different material to attract, entrap, adsorb, react chemically and/or bond with a different respective target analyte. Accordingly, a single sensor may be configured to sense a plurality of different target analytes (either directly or indirectly via oxidation or reduction) in parallel and within a single or a plurality of data collection periods. According to a further aspect of the present concept there is provided an electrochemical sensor comprising: at least one working electrode comprising the electrode assembly as claimed and described herein; and at least one auxiliary electrode. As will be appreciated, in use, the working electrode is electrically coupled to a working electrode via the sample fluid (biofluid) within which the target analyte is present. That is, the working electrode and auxiliary electrode are electrically isolated when not in use (for example in "open air ’). Electrical coupling between the working and auxiliary electrode occurs exclusively via the sample fluid (biofluid) to complete the electrical circuit.

Optionally the sensor may comprise an additional reference electrode (relative to the working and/or auxiliary electrode) such that the sensor comprises two, three or more electrodes.

Optionally, the sensor may further comprise a potentiostat electrically connected to the electrodes to apply an electrical potential at least to the working electrode and/or to measure the potential between the working electrode and one of the auxiliary electrodes; and a control utility to receive charge, current and/or charge or current data generated from the oxidation or reduction of the analyte or a reaction product of the target analyte at the working electrode and/or to measure the potential at said electrode. Optionally, the sensor may further comprise an electrochemical potential module to generate and apply the potential to the working electrode. Such a module may be implemented as software and/or firmware stored and run on a suitable operating system/platform (e.g., on a potentiostat device) as will be familiar to those skilled in the art. Optionally, the electrochemical potential module is configured to apply a pulsed potential to the working electrode.

According to a further aspect of the present concept there is provided a sensor as claimed and described herein. According to a further present aspect of the present concept there is provided a wearable device attachable to a body of a human or animal comprising a sensor as claimed and described herein. According to a further aspect of the present concept there is provided a method of manufacturing an electrode assembly as claimed and described herein. According to a further aspect of the present concept there is provided a method of manufacturing an electrochemical sensor as claimed and described herein.

According to a further aspect of the present concept there is provided an electrochemical method of interrogating at least one target analyte within a biofluid comprising: providing an electrochemical sensor as claimed and described herein; contacting the sensor with a body comprising a biofluid containing at least one target analyte; applying an oxidising or reducing electrical potential at the working electrode to oxidise or reduce the target analyte or a reaction product of the target analyte; and measuring current between the working electrode and the auxiliary electrode resultant from the oxidation or reduction of the target analyte or a reaction product of the target analyte.

Optionally, the step of applying the oxidising or reducing potential comprises performing at least one electroanalytical technique such as voltammetry (e.g., cyclic voltammetry), potentiometry or amperometry. Optionally the electroanalytical technique comprises electrochemical impedance spectroscopy (EIS). A large range of electroanalytical techniques are compatible with the present concept as will be appreciated. Optionally, the method comprises applying the electrical potential for a predefined time period.

Optionally, the method comprises applying a pulsed electrical potential at the working electrode having time periods of a first potential and time periods of a second potential being zero or lower than the first potential.

According to a further aspect of the present concept there is provided an electrochemical method of interrogating at least one target analyte within a biofluid comprising: providing an electrochemical sensor having an electrode assembly comprising: an electrically insulating material having at least one insulating surface; an electrically conducting material having at least one conducting surface bound and surrounded by the insulating surface; contacting the sensor with a body comprising a biofluid containing at least one target analyte; applying an oxidising or reducing electrical potential at the working electrode to oxidise or reduce the target analyte or a reaction product of the target analyte; and measuring current between the working electrode and auxiliary electrode resultant from the oxidation or reduction of the target analyte or a reaction product of the target analyte; and wherein a surface area of the conducting surface or a surface area of each conducting surface where the assembly comprises a plurality of conducting surfaces relative to a surface area of the surrounding insulating surface is configured to control a rate of diffusion within the biofluid of the target analyte to the conducting surface such that a rate of consumption (i.e., oxidation or reduction) of a volume of the target analyte at the conducting surface is less than the rate of diffusion within the biofluid of the same volume of target analyte to the conducting surface so as to maintain a supply by diffusion of target analyte at the conducting surface.

The present electrochemical electrode structure comprises at least one active electrode surface being sufficiently small and optionally formed as a discontinuity within a relatively large surface area of an insulating material such that transport of an electro-active species (target analyte) occurs under non-linear diffusion control. Preferably, the inner boundary perimeter of the surrounding insulating surface is at least 10 times smaller than the outer boundary perimeter of the insulating surface. Effectively, the outer boundary determines the minimum distance of any closest or neighbouring conducting surface or edge within the electrode assembly. Preferably, the conducting surface comprises, in at least one dimension (2D or 3D), a width, diameter or other cross surface distance which is less than 10 pm and more preferably is less than 100 nm. For example, for a conducting surface with an area a and a diameter or minimum width x, a perimeter of the conducting surface must be separated by a minimum of 5x in all directions and in a primary plane of the insulating surface and comprises a minimum area of 25a. The relative dimensions of the conducting surface (oxidising/reducing) to the surrounding insulating material surface to provide the desired diffusion control of a target analyte. The present concept provides a sensor arrangement that is responsive under spherical or hemispherical diffusion control so as to provide a time and biofluid volume independent electrochemical sensing arrangement and method.

According to a further aspect of the present concept there is provided an electrochemical electrode assembly comprising: an electrically insulating material having at least one insulating surface; an electrically conducting material having at least one conducting surface bound and surrounded by the insulating surface; wherein a surface area of the conducting surface is less than 5% of a surface area of the insulating surface defined between an inner boundary at the perimeter of the conducting surface and an outer boundary that is separated from the inner boundary by a distance of at least five times a diameter or smallest width across the conducting surface.

According to a further aspect of the present concept there is provided an electrochemical electrode assembly comprising: an electrically insulating material having an insulating surface; an electrically conducting material having a conducting surface with a perimeter bound and surrounded by the insulating surface; wherein a surface area of the conducting surface is less than 5% of a surface area of the insulating surface that surrounds the perimeter of the conducting surface, wherein the insulating surface is defined between an inner boundary at the perimeter of the conducting surface and an outer boundary that is separated from the inner boundary by a distance of at least five times a diameter or width across the conducting surface.

According to a further aspect of the present concept there is provided an electrochemical electrode assembly comprising: an electrically insulating material having an insulating surface; an electrically conducting material having a conducting surface with a perimeter bound and surrounded by the insulating surface; wherein a surface area of the conducting surface is an order of magnitude less than a surface area of the insulating surface that surrounds the perimeter of the conducting surface.

Optionally, the surface area of the insulating surface is defined between an inner boundary at the perimeter of the conducting surface and an outer boundary that is separated from the inner boundary by a distance that is a plurality of times greater than a diameter or width across the conducting surface. Optionally, the distance is in a range at least 5, 10, 50, 100, 500, 1000, 10000 or 100,000 times greater than a diameter or width across the conducting surface. Optionally, the distance is in a range at least 10 to 100,000; 10 to 10,000; 10 to 1000; or 10 to 100 times greater than a diameter or width across the conducting surface. Preferably the width across the conducting surface is the smallest width. The smallest width refers to a distance across the surface that is the shortest where a width or thickness of the surface is variable/non-uniform.

According to a further aspect of the present concept there is provided an electrochemical electrode assembly comprising: an electrically insulating material having at least one insulating surface; an electrically conducting material having a plurality of conducting surfaces each having a perimeter bound and surrounded by the insulating surface; wherein a surface area of each conducting surface is less than 5% of a respective surface area of the insulating surface that surrounds the perimeter of each of the conducting surfaces, wherein each respective surface area of the insulating surface is defined between an inner boundary at the perimeter of each respective conducting surface and a respective outer boundary that is separated from the respective inner boundary by a distance of at least five times a diameter or smallest width across the respective conducting surface.

Brief description of drawings

A specific implementation of the present invention will now be described, by way of example only, and with reference to the accompanying drawings in which:

Figure 1 A is a plan view of a first stage manufacture of an electrode suitable for use as an electrochemical sensor according to a specific implementation;

Figure IB is a plan view of a second stage manufacture of the electrode of figure 1 A;

Figure 1C is a magnified plan view of a distal end of the electrode of figures 1A and B; Figure 2A is a plan view of third stage manufacture of the electrode of figures 1 A to 1C;

Figure 2B is a plan view of final stage manufacture of the electrode of figures 1 A to 2A;

Figure 2C is a magnified side view at the distal end of the electrode of figure 2B; Figure 2D is a magnified side view at the distal end of the electrode of figure 2C;

Figure 3 is a cyclic voltammogram using the electrode of figures 2B and C with a silver chloride reference electrode and Pt wire counter electrode in 1 mmol dm'³ FC A in PBS scan rate 50 mV s'¹;

Figure 4 is a graph of current profile after polarisation of the electrode of figures 2B and C to 0.35 V in 1 mmol dm'³ FCA in PBS;

Figure 5 is a graph of current versus time for 5 repeats at 100 ms 0.35 V pulse in 1 mmol dm'³ FCA in PBS with 10 s rest at Open Circuit Potential between each repeat for the electrode of figures 2B and C;

Figure 6 is a graph of current versus time for 5 repeats at 100 ms 0.35 V pulse in 1 mmol dm'³ FCA in PBS with 10 s rest at Open Circuit Potential between each repeat for an electrode being a nanoband array;

Figure 7 is a graph of current versus time for 5 repeats at 100 ms 0.35 V pulse in 1 mmol dm'³ FCA in PBS with 10 s rest at Open Circuit Potential between each repeat for the electrode being a 100 pm diameter platinum disc;

Figure 8 is a graph of a response to 1 mmol dm'³ FCA in PBS and PBS 0.35 V pulse, 0.1 ms sampling intervals over a 20 ms window with 1 s OCP between repeats for the electrode of figures 2B and C at a widest coherent window;

Figure 9 is a graph of a response to 1 mmol dm'³ FCA in PBS and PBS 0.35 V pulse, 0.1 ms sampling intervals over a 20 ms window with 1 s OCP between repeats for the electrode of figures 2B and C including FCA repeat including earliest data points;

Figure 10A is a graph of 10 repeats of 5 ms 0.35 V pulses in 1 mmol dm'³ FCA in PBS with 50 ms rest at Open Circuit Potential between each repeat using commercial ASIC (ADuCM355) for electrode of Figures 2B and C; Figure 10B is a graph of 10 repeats of 5 ms 0.35 V pulses in 1 mmol dm'³ FCA in PBS with 50 ms rest at Open Circuit Potential between each repeat using commercial ASIC (ADuCM355) for an electrode comprising a nanoband array;

Figure 11 is a graph of 10 repeats of 5 ms 0.35 V pulses in 1 mmol dm'³ FCA in PBS with 50 ms rest at Open Circuit Potential between each repeat using commercial ASIC (ADuCM355) for an electrode comprising a 100 pm platinum disc;

Figure 12A is a side view of a further embodiment of the electrode of figures 2B and C having active electrode surfaces formed as convex protrusions or raised bumps projecting from a substrate;

Figure 12B is a side view of a further embodiment of the electrode of figures 2B and C comprising active electrode surfaces formed as concave, or indented/recessed cavities at a substrate surface;

Figure 13 is a plan view of a dual electrode system comprising a pair of electrodes according to the embodiment of figures 2B and C provided at a top face and bottom face of an intermediate substrate;

Figure 14 is a side elevation view of the distal ends of the electrodes of figure 13 provided at a top face and bottom face of a substrate;

Figure 15A is an initial stage manufacture of a further embodiment of the electrode of figures 2B and C comprising a dual conducting layer;

Figure 15B is a final stage manufacture of the electrode of figure 15A comprising a triconducting layer arrangement, with each layer comprising a plurality of active electrode surfaces according to a further embodiment; Figure 16A is a plan view of a further embodiment of the electrode of figures 2B and C, with the assembly comprising laterally defined multiple individually addressable electrode areas;

Figure 16B is a plan view of a further embodiment of the electrode of figures 2B and C, with the assembly comprising laterally defined multiple individually addressable electrode areas;

Figure 17A is a first stage manufacture of a further embodiment of an electrode assembly;

Figure 17B is a second stage manufacture of the electrode of figure 17A;

Figure 17C is a third stage manufacture of the electrode of figure 17A;

Figure 17D is a fourth stage manufacture of the electrode of figure 17A;

Figure 18A is a further plan view of the electrode of figure 17D;

Figure 18B is a side elevation cross sectional view through C-C of figure 18 A;

Figure 19 is a plan view of a further embodiment of an electrode assembly comprising a plurality of active electrode surfaces defined at an open recess, aperture or open cavity of an electrode assembly having a grid or mesh-like conducting layer structure;

Figure 20A is a first stage manufacture of a further embodiment of an electrode assembly having a grid or mesh-like conducting layer structure;

Figure 20B is a second stage manufacture of a further embodiment of an electrode assembly having a grid or mesh-like conducting layer structure;

Figure 20C is a third stage manufacture of a further embodiment of an electrode assembly having a grid or mesh-like conducting layer structure; Figure 20D is a fourth stage manufacture of a further embodiment of an electrode assembly having a grid or mesh-like conducting layer structure;

Figure 20E is a fifth stage manufacture of a further embodiment of an electrode assembly having a grid or mesh-like conducting layer structure;

Figure 20F is a final stage manufacture of a further embodiment of an electrode assembly having a grid or mesh-like conducting layer structure;

Figure 21 A is a further embodiment of an electrode assembly configured with relatively larger active electrode surfaces formed within a conductive layer grid arrangement;

Figure 2 IB is a further embodiment of an electrode assembly configured with relatively larger active electrode surfaces formed within a conductive layer grid arrangement;

Figure 21C is a further embodiment of an electrode assembly configured with relatively larger active electrode surfaces formed within a conductive layer grid arrangement;

Figure 22A is a plan view of a hybrid electrode assembly comprising a nanoband array according to a further implementation;

Figure 22B is a plan view of a hybrid electrode assembly comprising a nanoband array according to a further implementation;

Figure 23 is a plan view of a further embodiment of an electrode assembly comprising a plurality of active electrode surfaces, with each surface provided at a sidewall of a respective channel or groove formed within the multilayer structure;

Figure 24 is a further embodiment of an electrode assembly comprising a plurality of cavities or apertures formed within a multilayer structure to expose a plurality of active electrode surfaces at the open cavities, apertures and/or open recesses. Detailed description of preferred embodiment of the invention

Referring to figures 1 A to C, an electrode assembly 10 comprises a substrate 18 formed from an electrically insulating material. All dimensions indicated for figures 1 A to 2C are in pm. A conducting layer 11 is formed as an elongate strip provided on a surface 19 (figure 2) of substrate 18 having a proximal end connected to electrically conducting tabs 12 and a distal end 13 comprising a plurality of laterally extending conducting fingers 14. Each finger 14 comprises a respective terminal end 15. Conducting layer 11, according to the specific embodiment, comprises a thickness of approximately 0.05 pm, provided on substrate 18 that in turn comprises an approximate thickness of around 250 pm. An approximate width across the electrode at the conducting fingers 14 (between the respective terminal ends 15) is approximately 600 pm. A width of the conducting elongate strip 11 is approximately 60 pm and an approximate overall length of the elongate electrode (comprising strip 11 and fingers 14), is in the region of 40,000 pm. Figure 1 A illustrates a first stage manufacture involving the deposition of conducting strip 11 and fingers 14 onto substrate 18. Figure IB illustrates a second stage manufacture in which a layer of insulating material 16 is deposited on top of both the substrate 18 and the conducting layer 11. Insulating layer 16 comprises a width of approximately 600 pm and a length extending the approximate full length of the conducting strip 11 being also in the region of 40,000 pm.

Figure 2A illustrates a third stage manufacture in which selected regions of the assembly of figure IB are identified for ablation processing using a laser cutter or similar techniques. Following ablation cutting, the finished electrode assembly is illustrated in figure 2B with the structure of figure 2A cut/ablated at ablation lines 17 (illustrated schematically). Accordingly, and referring to the side view of figure 2C, active electrode surfaces 15 are provided at the respective terminal ends of each conducting finger 14 being exposed at the respective sidewall surfaces of the laminate electrode assembly (comprising substrate 18, conducting layer 11 and insulating material 16). Importantly, each electrode active surface 15 is spatially separated from one another so as to provide discreet conducting surface regions for the oxidation/reduction of a target analyte contained within a biofluid (such as sweat, saliva, blood etc). According to the present embodiment as described herein, the insulating layer 16 is a material which is configured and selected to be appropriate for the end use which includes biocompatibility when deployed as a minimally invasive device. Such layer materials offer compatibility, adhesion, etc. for any additional functionalities required to interact chemically i.e., to bind, react, absorb, etc. the target analyte at the region of each of each active surface 15 for oxidation/reduction, reaction or interaction with said surface resulting in a potential change.

According to the present embodiments of the electrode assembly as described herein an active electrode coating may be provided on each exposed active electrode surface 15, such coatings according to existing arrangements are configured to interact chemically with the target analyte so as to bind, react, absorb and the like the target analyte so as to retain the analyte at the active surface 15 for oxidation and/or reduction or effect changes at said surface resulting in a change in potential. In some situations, it may be possible to incorporate the desired characteristics of the active electrode coating in the insulating layer 16 thus eliminating a process step. Such active electrode coatings are well known to those skilled in the art of sensor manufacture and particularly biosensor manufacture. Additional coating may be applied to beneficially modify the performance of the complete sensor including but not limited to the purposes of exclusion of potential interferents. This adds the possibility of using the structures for potentiometry and for the possibility that insulating layer 16 is passive and also for the inclusion of ‘exclusion membranes’ to exclude interferents.

All embodiments described herein are suitable for use within an implantable or wearable electrochemical sensor being a minimally invasive diagnostic sensor.

According to specific electrode assembly of figures 2B and D, a material of substrate 18 comprises polyethylene terephthalate (PET) to provide a first insulating layer. Conducting layer 11 optionally comprises platinum deposited using appropriate techniques. The final electrode structure of figures 2B and D may be achieved using laser ablation or photolithography for example to produce the electrically conducting pattern having the discreet spatially separated regions of active electrode surface for the oxidation/reduction or surface interaction of the target analyte or other species generated as a consequence of the presence of the target analyte such as reaction products of the analyte with an active coating such as an enzymically active coating to react with the analyte.

According to the arrangement of figures 2B and D, the electrode assembly comprises a size and spacing of fingers 14 to ensure that the electrode structure is electrically addressable and comprises twelve active conducting regions 15 which have the appropriate dimensions (each active region being 30 pm long and 50 nm wide) to ensure highly efficient diffusion characteristics (50 nm wide) whilst being sufficiently separated from adjacent active regions 15 (90 pm) to minimise any interactions between neighbouring active regions 15. At the proximal end, the second and third contact pads 12 enable auxiliary electrodes (not shown) to be incorporated (optionally) as required. The dimensions of pads 12 may be varied to accommodate a chosen electrical connection method (pad size, pitch, etc.). These auxiliary electrodes may be incorporated on the reverse face of the structure requiring a through-hole for connection or on the same side as the contact pads 12 adjacent the working electrode structure.

Referring to figure 2D, the insulating surface that surrounds each of the active electrode surfaces 15 is formed by the substrate 18 and layer of insulating material 16. Collectively, the substrate 18 and insulating layer 16 provide the surrounding insulating surface that confines and borders each of the active electrode surfaces 15. As indicated, the relative dimensions of each active electrode surface 15 is significantly smaller than the surrounding surface area at the insulating material (16, 18). In particular, the surface area of each active electrode surface 15 may be approximately 1.2 pm² or is less than 1pm², 0.5pm² or 0.1pm².

Each active electrode surface 15 is confined and surrounded by a region 24 of insulating material. Each region 24 comprises a surface area of the order of microns squared relative to each active electrode surface area 15 being of the order of nanometres squared. In particular, region 24 of insulating material is defined between an inner boundary 21 corresponding to a perimeter 20 of a respective active electrode surface 15 and a respective outer boundary 23. Outer boundary 23 is separated by a distance d being five times a distance e being a diameter or smallest width across a respective active electrode surface 15. Such an arrangement is advantageous to control the diffusion of analyte (target species) to the active conductive surfaces 15 and in particular to avoid undesirably high analyte diffusion rates that in turn provides controlled oxidation/reduction of the target species.

The auxiliary electrodes have been omitted from figures 1 A to C for clarity. In such an embodiment, the auxiliary electrode was produced using a commercial silver/silver chloride screen printing ink, patterning the reverse side of the substrate 18. Alternate auxiliary electrodes and materials to produce them may be defined and are known to those practiced in the art. The structure was then screen printed with a commercially available dielectric ink to produce the dielectric layer 16 as shown in figure IB on the front side covering the complete structure with the exception of the electrical contact pads 12. The thickness of the dielectric layer was approximately 30 pm (this is more than sufficient to ensure that the active regions exposed in the last step (detailed below) are sufficiently encapsulated in electrochemically inactive material to effect optimal diffusion (sometimes described as hemispherical). Alternatively, a second screen printing step can be incorporated to minimise any areas of exposed conductor (commonly termed called pin holing). The reverse side was also screen printed with the dielectric ink up to the distal 700 pm of the structure, exposing the silver/silver chloride layer at the distal extremity (not shown for clarity). In figure 2A the singulation ablation line 17 of the structure 10 is shown which cuts through the dielectric/electrical conductor/PET substrate at the distal end exposing six electrical conductor end faces 15, each 30 pm long and 50 nm wide on each side. Each surface 15 of the singulated device is separated from adjacent faces by 90 pm (120 pm centre to centre) as shown in figure 2B and figure 2C. The overall working electrode area in this embodiment is 18 square pm which compares to a typical structure in this scenario of ca. 8000 square pm (i.e. approximately 400 times lower surface area). This is advantageous to reduce the inherent capacitance of the electrode. The singulation process was effected with a femto second infrared laser, the final structure having a width of ca. 400 pm. It should be noted that the performance of the device is, to a first approximation, independent of its width, the performance being determined by the number, dimensions, spacing and environment of the active regions (faces 15). This is not the case when considering a conventional electrode structure. This is important as the size of the overall structure has a significant impact on the Foreign Body Response (FBR) which is elicited in situ when the example is used in applications such as minimally invasive implants (such as when applied to continuous glucose monitoring applications). The larger the implant the more rapid the FBR and therefore the shorter the operational lifetime of the implant and therefore the freedom to adopt the optimal width without compromising the performance is an important characteristic.

The electrode embodiment of figures 2B and C was tested with Ferrocene Carboxylic Acid (FCA) and hydrogen peroxide (HP). These two analytes were chosen as surrogates for first generation biosensors which utilise enzymes which generate HP which is subsequently measured at the indicator electrode and second generation biosensors which utilise artificial mediators (FCA). First generation biosensors require the platinum electrode surface to be catalytically active in order to undertake HP electrochemistry.

Unless otherwise stated, the measurements were made in Phosphate Buffered Saline (PBS) solutions at room temperature. The potential range for cyclic voltammetry was 0 - 0.45 V with respect to silver/silver chloride for FCA and -0.2 to 0.75 V for HP (scan rate 50 mV s'¹). Chronoamperometric measurements were run at 0.35 V for FCA and 0.6 V for HP. For comparison, measurements were made using a 100 pm platinum disc electrode as a model for current implantable devices and a conventional 50 nm platinum nanoband array electrode (30 pm apertures on a 60 pm pitch) as an exemplar of current teaching in terms of nanoband electrode structures.

It can be seen from figure 3 that the electrode of figures 2B and C shows typical nanoband capabilities when interrogated using cyclic voltammetry in the presence of 1 mmol dm'³ FCA in that there is no evidence of mass transport limitation (the oxidation wave plateaus above 0.35 V rather than going through a maximum). A similar wave form is observed when interrogating a conventional nanoband array.

When the present embodiment is interrogated using a potential step, in this case a step to 0.35 V with respect to a silver/silver chloride electrode, it can be seen that there is a departure from the behaviour of either a conventional nanoband array or a macro scale platinum electrode area 10 mm² (figure 4). In the case of the nanoband array and the macro electrode the continuing decrease in current over the 60 second measurement window demonstrates that these systems are constrained by mass transport which is unable to replenish the FC A consumed at the electrode surface. In the case of the embodiment, it can be seen that a steady state is achieved within ca. 6 seconds when the decline in current is seen to cease. Such a time independent response is characteristic of the establishment of optimal (hemispherical) diffusion. At this point the rate of mass transport of analyte to the electrode surface is sufficient to replenish the FCA consumed at the electrode. This condition is normally only observed with very careful management of the flow in the region of the electrode surface whilst in this case the solution was quiescent.

The embodiment was challenged with 100 ms 0.35 V pulses with 10 seconds rest (electrode held at open circuit potential) between pulses in 1 mmol dm'³ FCA in PBS and in PBS (‘blank’). The behaviour was compared with the behaviour of a 100 pm Pt disc electrode under the same conditions. The results are shown in figure 5 to 7. It can be seen that in the case of the present embodiment the 5 repeated pulses are randomly ordered whilst the 100 pm Pt disc responses are ordered, reducing from the initial response through repeat 5. This demonstrates that whilst the 100 pm disc consumes more FCA than mass transport in quiescent conditions can replenish even when the consumption period is short (100 ms) and the recovery time long (10 s) this is not the case with the embodiment. For applications where it is not possible or not desirable to manage the solution flow this offers improved measurement accuracy.

The present embodiment was challenged at the shortest time interval available on the instrument used (0.1 ms) over a 20 ms sampling window (with 1 second OCP between repeats) to demonstrate that the present embodiment response remains predominantly faradaic (i.e. dominated by redox processes rather than charging phenomena). Five pulse repeats were measured. There is an instrumental artefact at these high sampling rates with the potentiostat used which manifests itself as a complex waveform. When the waveform for each response is synchronised it can be seen that there is a clear faradaic response (figures 8 and 9). The sample interval is 0.1 ms and as can be seen from FCA repeat 4 which commences from the first data point (t = ‘0’) there is no evidence of charging for either the initial point t = ‘0’ or the first point t = 0.1 ms (figure 9). It is therefore clear that the present embodiment demonstrates a very low non faradaic (capacitive charging) component and could potentially be polled at kilohertz frequencies. Whilst t = ‘0’ will be at a point sometime after the embodiment was polarised the duration will be « 0.1 ms and therefore it is evident that the embodiment might be capable of interrogation at even higher frequencies / shorter sampling intervals with appropriate hardware.

Additionally, the present embodiment was challenged using a commercial Application Specific Integrated Circuit (ASIC) designed for use in mobile / wearable scenarios (using a PalmSens Sensit BT potentiostat which utilises an Analogue Devices ADuCM355 ASIC). The shortest sample interval allowed in software of 1 ms was used for the minimum number of samples (five). It should be noted that the native capability of the ADuCM355 is 400 kilosamples per second. Within the software limitations a 5 ms 0.35 V pulse with 50 ms rest period (electrode held at open circuit potential) was used with 1 mmol dm'³ FCA in PBS and in PBS alone (‘blank’). Responses under these conditions are shown in figures 10A, 10B and 11. Results are presented for the electrode of Figures 2B and C, for the 100 um diameter disc electrodes and for the nanoband array.

The present electrode assembly may be embodied in different forms to suit the application, analyte of interest and its diffusion characteristics in the matrix under consideration. The sensitivity of the device will scale linearly with length thus if the fingers 14 were defined over a 1440 pm length (i.e. twelve fingers 14 on each side rather than six fingers 14, with the same arm width and spacing) the signal current would be doubled. If the number of fingers 14 over the same 720 pm length was doubled a similar increase the signal current would be observed. Accordingly, the structure can be varied from a single monolithic finger 14 (720 pm long in the present example embodiment), to a highly divided finger structure (for example 72 5 pm wide fingers separated by 5 pm spaces), to a sparsely populated finger structure (such as the current embodiment). The appropriate structure can be fabricated (engineered) as the application under consideration demands to provide optimal performance. Furthermore, the present device may be made significantly narrower (400 pm in the present embodiment) with no detriment to the performance. The limitations of the width of the present device are dependent upon the length and resistivity of the conductor track 11 (figure 1 A to 2C) and the manufacturing tolerances of the fabrication methods and tools used. In the case of the present embodiment with the fabrication tools used, embodiments of 60 pm width would be possible. A further practical consideration can be whether the desired mechanical properties are be maintained as the width of the embodiment is reduced. However, it is important to note that the electrochemical performance will not be denuded.

In the embodiment described, the active area faces 15 are flat. According to further embodiments, such active surface 15 they may be concave or convex as shown in figures 12A and 12B and in either 2 or 3 dimensions.

In a further alternative embodiment, the second electrode is defined on the reverse face of the insulating substrate 18 by depositing and patterning an additional conductor layer followed by an insulating layer 16 as described above and is shown in figures 13 and 14. In this embodiment the ‘top face’ and ‘bottom face’ electrodes are coincident. In a further embodiment, the active regions 15 of the two electrodes are offset or staggered. In yet a further embodiment, the active regions 15 and spacings of the two electrode active regions 15 are not identical.

In an alternative embodiment, additional conducting and insulating layers and patterning are added to produce two or more stacked independently addressable active region(s) 15 (collectively described as electrodes) on one face of a single substrate (figures 15A and B). The active regions 15 of the two electrode structures in the embodiment as shown are identical and coincident. In some embodiments, the active region structures may not be identical (for example if configured to oxidise/reduce different target analytes or reaction products thereof) and in further embodiments the active region structures may not be coincident.

Multiple individually addressable active regions 15 may also be defined laterally and some examples are shown in figure 16. Base insulating substrate 18 is occluded by the conducting layer 11 and the dielectric insulating top layer 16. The active areas 15 are identical size spacing and structure but in a further embodiment may differ depending upon the sensing application. A combination of any or all of the above approaches maybe adopted.

In other embodiments of the present assembly, the PET base substrate 18 may be substituted with any appropriate material with the required mechanical, biological, biocompatible and electrical properties (e.g. polyester, polyalkane, etc.). Similarly, the Pt conducting layer 11 may be replaced by any appropriate material with the required mechanical, biological, biocompatible, electrical and electrochemical properties (e.g. platinum group metals, metals, organic conductors, etc.). Also, the thickness of the conductor layer 11 may be varied as required (typically in the 0.1 - 100 nm range). Additionally, the insulating top layer 16 may be substituted with any appropriate material with the required mechanical, biological, biocompatible and electrical properties.

In some cases, it may be necessary to separate the process of creation of the active regions 15 from the process of singulation. An example of this is shown in figures 17A to D and figure 18A and B. The singulation may be made anywhere outside the ablation area 17 or within it, as long as the line is outside the inside line of the ablation.

In this example, the same basic electrode pattern has been used (as figure 2B and C) and comprises six biaxial fingers 14 projecting laterally at a distal end from a central conductor strip. The patterning and the layer structures are also identical to this previous example. In detail, the starting point for manufacture is a PET substrate 18 with a 50 nm Pt layer with a suitable pattern ablated in the Pt layer 11. For this further example, the fingers 14 are 30 pm wide with a 120 pm centre to centre spacing. The pattern and the majority of the remaining Pt/PET area is screen printed with a suitable dielectric ink layer 16 to electrically isolate the Pt layer 11 with a small residual area which defines the electrical contact pad 12 as shown in figure 20B. As with the previous example of figure 2B and C, the contact pad dimensions may be optimised for the electrical connection system used. Figure 20C shows the mark out for the ablation tracks 17 (there may be single track regions if desired) and figure 20D shows the assembly once the ablation track (through the dielectric layer 16, the conductor layer 11 and on to the PET substrate 18, which in this situation acts as an effective ‘etch stop’) has been made. The ablation may be achieved using laser machining or die cutting or similar methodology including methods such as reactive ion etching or chemical etching. Figure 20E shows the cross section which, in this example extends a small way into the insulating substrate 18. The device may be singulated to whatever size and shape is appropriate for the desired application. The materials and structures can be varied as with the first embodiment described.

The two approaches may be combined in order to increase the signal produced. An example is shown in figure 19 where eight additional active surfaces 15 have been defined in the surface to augment the ten active surfaces 15 defined when the structure was singulated. The number, spacing and size of the active regions defined in the surface maybe varied using alternate patterning of the conductor layer and/or the form of the aperture itself to optimise the performance of the structure in the chosen application.

The benefits of the invention have been demonstrated utilising an extreme embodiment which would be suitable for in vivo minimally invasive applications such as for example continuous glucose monitoring. The inventors have identified that the size of the present electrode assembly is important as this dictates the lifetime and performance of the device in use. In applications where this is not a critical issue but where management of the sample flow is not possible/desirable and/or the volume of sample which is addressed is unknown and or small, the basic approach can be extended to assemblies having active surfaces 15 over larger surface areas. An example of the approach is shown in figure 20A to F. For some applications it may be desirable repeat the surface ablation pattern over a wider area. Examples of this approach are given below.

In such a case, the manufacturing starting point is a PET substrate 18 with a 50 nm Pt layer 11 with a suitable pattern ablated in to the Pt layer 11. The pattern and the majority of the remaining Pt/PET layer 11, 18 is screen printed with a suitable dielectric ink 16 to electrically isolate the Pt with a small residual area which defines the electrical contact pad 12. Figure 20C shows the ablation track 17, which alternatively may be a series of five separate tracks 17. Figure 20D shows the assembly once the ablation tracks 17 (through the stack and in or to the PET substrate 18) has been made. Figure 20E shows the mark-out line 17 for the device singulation cutting/lasering 22 and the completed structure is shown in figure 20F. The materials and structures can be varied as with the first embodiment described. In this example the active areas 15 are defined at the side walls of the layered material structure (formed by the ablation cutting) during the singulation process in addition to those created during the surface ablation stage. This is optional and if not required for a given application, the singulation may be conducted in such a way that no additional active areas 15 are created during this process.

The present concept may comprise other electrode structures, for example with nanoband arrays as shown in figure 22 A and figure 22B. In the embodiment of figure 22 A, a nanoband array has been defined and structured as an independently addressable electrode alongside the embodiment as described previously. In this example, the contact pads 12 are on opposite sides of the structure but they could alternatively be arranged to be on the same side of the structure to provide a convenient means to connect to a multiway electrical connector of choice. In the embodiment of figure 22B the structure is addressed such that the present invention and the nanoband array electrode is addressed as a single working/indicator electrode through a single connection pad.

It is also possible to combine the embodiments as described with one or more conventional larger scale electrodes, for example, using screen printing methods if required.

In one further embodiment a series of interdigitated fingers 14 are defined in the conductor layer 11 as shown in figure 23. The active surfaces 15 defined by the ablation/cut as presented at the side walls of the as-formed channels or grooves (formed in the laminate structure). In this example, surfaces 15 on each opposite side wall are not opposite one another (within each respective channel or groove) but are offset or staggered in the longitudinal direction of each channel or groove. This is advantageous to ensure that each active surface 15 is facing a passive region in the structure (i.e. not directly facing another active surface 15), further reducing the capability of active regions 15 denuding opposing active regions 15 in close proximity of oxidisable/reducible analyte. In this example no additional edges or active surfaces 15 are created on singulation (although these could of course be created if additional fingers 14 were defined extending to the perimeter of the construct. The surface patterning approach may be restricted to discrete areas if required and an example is shown in figure 24. The number of discrete ablation apertures (open cavities, recesses or pits) 22 may be varied as requires and the number, size, and spacing of the active areas 15 defined within each ablation aperture 22 may also be varied to provide the desired performance for a chosen application.

Claims

1.

An electrochemical electrode assembly comprising: an electrically insulating material having at least one insulating surface; an electrically conducting material having at least one conducting surface with a perimeter bound and surrounded by the insulating surface; wherein a surface area of the conducting surface or a surface area of each conducting surface where the assembly comprises a plurality of conducting surfaces is less than 5% of a surface area of the insulating surface that surrounds the perimeter of the conducting surface or the respective surface area of the insulating surface that surrounds each respective perimeter of each conducting surface, wherein each respective surface area of the insulating surface is defined between an inner boundary at the perimeter of the conducting surface or a respective inner boundary at each respective perimeter of each conducting surface and a respective outer boundary that is separated from the respective inner boundary by a respective distance of at least five times a diameter or smallest width across the conducting surface or across each respective conducting surface where the assembly comprises a plurality of conducting surfaces.

2. The assembly as claimed in claim 1 wherein a surface area of the individual conducting surface or each surface area of the plurality of conducting surfaces is less than 300 pm², 200 pm², 150 pm², 100 pm², 80 pm², 60 pm², 40 pm², 20 pm², 10 pm², 5 pm², 3 pm² or 1 pm².

3. The assembly as claimed in claim 1 wherein a total surface area of the conducting surfaces where the assembly comprises a plurality of conducting surfaces is in a range 0.001 to 500,000 pm²; 0.001 to 250,000 pm²; 0.001 to 100,000 pm²; 0.001 to 10,000 pm²; 0.01 to 8,000 pm²; 0.001 to 6,000 pm²; 0.001 to 4,000 pm²; 0.001 to 2,000 pm²; 0.001 to 1000 pm².

4. The assembly as claimed in claim 1 wherein a total surface area of the conducting surfaces where the assembly comprises a plurality of conducting surfaces is in a range 0.001 to 1000pm²; 0.001 to 800pm²; 0.001 to 600 pm²; 10 to 600pm²; 100 to 600pm²; 100 to 400pm²; 100 to 200pm²; 0.001 to 500pm²; 0.001 to 300pm²; 0.01 to 300pm²; 0.01 to 300pm²; 0.01 to 200pm²; 1 to 300pm²; 1 to 200pm²; 50 to 300pm²; 50 to 250pm²; 50 to 200pm²; 5 to 200pm² or 10 to 200pm².

5. The assembly as claimed in claim 1 wherein a width, diameter or smallest distance across the single or each individual conducting surface is in a range 10 to lOOOnm, 50 to 800nm, 50 to 600nm, 50 to 400nm, 50 to 200nm, 100 to lOOOnm, 100 to 800nm, 100 to 600nm, 100 to 400nm, or 100 to 200nm.

6. The assembly as claimed in any preceding claim comprising in a range 10 to 100,000; 10 to 50,000; 10 to 10,000;10 to 10,000; 10 to 8,000; 10 to 3,000, 10 to 1000; 10 to 800; 10 to 600; 10 to 400; 10 to 200; 10 to 100; 10 to 80, or 10 to 60 conducting surfaces.

7. The assembly as claimed in any preceding claim wherein the conducting surface and/or the insulating surface is any one or a combination of substantially planar, convex, concave, profiled so as to comprise peaks or valleys.

8. The assembly as claimed in any preceding claim wherein the electrically conducting material is a layer provided on a substrate and the electrically insulating material is a layer provided on the conducting material layer wherein the insulating material layer is discontinuous to expose regions of the conducting material layer that provide the at least one conducting surface.

9. The assembly as claimed in any preceding claim comprising a plurality of conducting surfaces wherein the perimeters of each of the conducting surfaces are separated from one another by a distance of at least 5 times a diameter or smallest width across any one of the conducting surfaces.

10. The assembly as claimed in any preceding claim further comprising an analyte responsive layer provided on the conducting surface.

11. The assembly as claimed in claim 10 wherein the analyte responsive layer comprises any one of:

• a material configured to bind the target analyte;

• an enzymatic material to interact or react chemically with the target analyte.

12 The assembly as claimed in claims 10 or 11 comprising a plurality of analyte responsive layers each comprising a different material to bind a different respective target analyte.

13. The assembly as claimed in claim 12 comprising a plurality of conducting surfaces each comprising one of the different materials.

14. An electrochemical sensor comprising: at least one working electrode comprising the electrode assembly as claimed in any one of claims 1 to 13; and at least one auxiliary electrode.

15. The sensor as claimed in claim 14 further comprising:

• a potentiostat electrically connected to the working and auxiliary electrodes to apply an electrical potential to the working electrode and/or to measure the potential between the working electrode and one of the auxiliary electrodes; and

• a control utility to receive charge, current and/or charge or current data generated from the oxidation or reduction of the analyte or a reaction product of the target analyte at the working electrode and/or to measure the potential at said electrode.

16. The sensor as claimed in claims 14 or 15 further comprising an electrochemical potential module to generate and apply the potential to the working electrode.

17. The sensor as claimed in claim 16 wherein the electrochemical potential module is configured to apply a pulsed potential to the working electrode.

18. An implantable device comprising a sensor as claimed in any one of claims 14 to 17.

19. A wearable device attachable to a body of a human or animal comprising a sensor as claimed in any one of claims 14 to 17.

20. A method of manufacturing an electrode assembly as claimed in any one of claims 1 to 13.

21. A method of manufacturing an electrochemical sensor as claimed in any one of claims 14 to 17.

22. An electrochemical method of interrogating at least one target analyte within a biofluid comprising: providing an electrochemical sensor according to any one of claims 14 to 17; contacting the sensor with a body comprising a biofluid containing at least one target analyte; applying an oxidising or reducing electrical potential at the working electrode to oxidise or reduce the target analyte or a reaction product of the target analyte; and measuring current between the working electrode and the auxiliary electrode resultant from the oxidation or reduction of the target analyte or a reaction product of the target analyte.

23. The method as claimed in claim 22 wherein the step of applying the oxidising or reducing potential comprises performing any one of voltammetry, potentiometry or amperometry.

24. The method as claimed in claims 22 or 23 comprising applying an electrical potential for a predefined time period.

25. The method as claimed in claims 22 or 23 comprising applying a pulsed electrical potential at the working electrode having time periods of a first potential and time periods of a second potential being zero or lower than the first potential.

26. An electrochemical method of interrogating at least one target analyte within a biofluid comprising: providing an electrochemical sensor having an electrode assembly comprising: an electrically insulating material having at least one insulating surface; an electrically conducting material having at least one conducting surface bound and surrounded by the insulating surface; contacting the sensor with a body comprising a biofluid containing at least one target analyte; applying an oxidising or reducing electrical potential at the working electrode to oxide or reduce the target analyte or a reaction product of the target analyte; and measuring current between the working electrode and auxiliary electrode resultant from the oxidation or reduction of the target analyte or a reaction product of the target analyte; and wherein a surface area of the conducting surface or a surface area of each conducting surface where the assembly comprises a plurality of conducting surfaces relative to a surface area of the surrounding insulating surface is configured to control a rate of diffusion within the biofluid of the target analyte to the conducting surface such that a rate of consumption of a volume of the target analyte at the conducting surface is less than the rate of diffusion within the biofluid of the same volume of target analyte to the conducting surface so as to maintain a supply by diffusion of target analyte at the conducting surface.