WO2010007391A2

WO2010007391A2 - Hexacyanoferrate modified electrode

Info

Publication number: WO2010007391A2
Application number: PCT/GB2009/001789
Authority: WO
Inventors: Paul A. Millner; Nikolay A. Pchelintsev; Alexander Vakurov
Original assignee: University of Leeds
Current assignee: University of Leeds
Priority date: 2008-07-18
Filing date: 2009-07-20
Publication date: 2010-01-21
Anticipated expiration: 2011-01-18
Also published as: WO2010007391A9; GB0813200D0; WO2010007391A3

Abstract

An electrode comprising a modified electrically conductive element comprising an electrically conductive element having a surface and a coating of a polyelectrolyte on said surface, and a transition metal hexacyano transition metallate derived material contained within the polyelectrolyte coating.

Description

Hexacvanoferrate Modified Electrode

The present invention relates to an electrode which includes a hexacyanoferrate, in particular Prussian Blue, type material. In particular, although not exclusively, the present invention provides a method of manufacturing an electrode which includes a hexacyanoferrate type material, an electrode which includes a hexacyanoferrate material, an electrode which includes a hexacyanoferrate material and an enzyme, an amperometric biosensor including such an electrode, and a method for the amperometric determination of an analyte, such as glucose.

Amperometric biosensors comprising a biorecognition layer attached to the surface of an electrode have provided an important tool for sensing analytes in medicine, in the food industry and for environmental monitoring. Typically, amperometric biosensors provide relatively fast and reliable screening methods.

Amperometric biosensors typically function by monitoring oxygen consumed or hydrogen peroxide produced during enzymatic oxidation of the enzyme's specific substrate. Suitably, the amount of oxygen consumed or hydrogen peroxide produced provides an assay concentration for the substrate. An artificial electron acceptor (i.e. a redox mediator) may be employed instead of the natural electron acceptor oxygen which may enable the sensor to be operated at lower potentials, thereby reducing interference from unwanted detection of interfering compounds. In this respect, iron hexacyanoferrate, or more commonly known as Prussian Blue, has been employed as a redox mediator for detecting hydrogen peroxide. In particular, amperometric biosensors which include an oxidoreductase enzyme (e.g. glucose oxidase) and Prussian Blue may be used to reduce hydrogen peroxide, thereby enabling detection of hydrogen peroxide at potentials below 0.0 Volts with a high sensitivity and almost eliminating the effects of interfering compounds.

There are a number of issues which need to be addressed when manufacturing electrodes which include an enzyme and a redox mediator, such as Prussian Blue. Firstly, the cost associated with manufacturing the electrode is an important factor, particularly if the electrode is to be employed as a disposable biosensor. Typically the cost associated with manufacturing such an electrode comprises not only the cost of the electrically conductive element and the biorecognition material but also the expense of assembly, which includes the immobilisation of the enzyme and the redox mediator on the electrode's surface. Secondly, the mode of manufacture has to be such that the enzyme is retained on or adjacent the redox mediator. Thirdly, immobilisation of the enzyme should be gentle to reduce consumption of the biological component and to prevent damage to the enzyme. Fourthly, automisation of the procedure is desirable, particularly for manufacturing disposable biosensors on a large scale.

Traditionally, Prussian Blue has been deposited on an electrode's surface by an electrochemical method wherein the electrode is immersed in an iron hexacyanoferrate solution and electrolysis affected. Non-electrochemical procedures for depositing Prussian Blue on an electrode's surface have been reported and include: doping carbon inks or paste with Prussian Blue powder, adsorption of a Prussian Blue polymer or Prussian Blue gold composite nanoparticles, dendrimer-supported Prussian Blue synthesis and chemical deposition of Prussian Blue onto the surface of pretreated screen-printed carbon electrodes. However, such methods do not lend themselves to an automated procedure for producing biosensors on a large scale and at a low cost. Moreover, elaborate procedures may be required to immobilise the enzyme on the non- receptive Prussian Blue material.

The present invention therefore seeks to solve some of the aforementioned problems associated with producing an electrode having a redox mediator, such as Prussian Blue, attached to its surface. In particular the present invention provides a simple and low cost non-electrochemical and non-enzymatic procedure for forming a hexacyanoferrate type material on the surface of an electrode. Conveniently, the procedure typically permits the manufacture of biosensors on a large scale and at a low cost. Moreover, the procedure permits gentle and effective immobilisation of the enzyme on or adjacent to the hexacyanoferrate material in a straight forward manner without the need for any elaborate techniques. In accordance with a first aspect, the present invention provides a method of manufacturing an electrode comprising:

(i) providing a modified electrically conductive element comprising an electrically conductive element having a surface and a coating of a polyelectrolyte on said surface; and,

(ii) contacting the modified electrically conductive element with a hexacyano transition metallate ion containing solution and a separate transition metal ion containing solution, wherein the hexacyano transition metallate ions of the hexacyano transition metallate ion containing solution chemically react with the transition metal ions of the transition metal ion containing solution within the coating of the polyelectrolyte to form a transition metal hexacyano transition metallate derived material within the polyelectrolyte.

According to a second aspect, the present invention provides an electrode obtainable by the method according to the first aspect of the present invention.

According to a third aspect, the present invention provides an electrode comprising: (i) a modified electrically conductive element comprising an electrically conductive element having a surface and a coating of a polyelectrolyte on said surface; and,

(ii) a transition metal hexacyano transition metallate derived material contained within the polyelectrolyte coating.

According to a fourth aspect, the present invention provides an enzyme electrode comprising:

(i) a modified electrically conductive element comprising an electrically conductive element having a surface and a coating of a polyelectrolyte on said surface;

(ii) a transition metal hexacyano transition metallate derived material contained within the polyelectrolyte coating; and,

(iii) an enzyme attached to the polyelectrolyte coating on or adjacent said transition metal hexacyano transition metallate derived material, wherein the enzyme is selected such that a substrate or product thereof is capable of being electrochemically oxidised or reduced at said electrode.

According to a fifth aspect, the present invention provides an amperometric biosensor comprising a cell for receiving an analyte and electrodes for contacting the analyte in the cell, said electrodes comprising a sensing electrode, a standard electrode, and optionally a counter electrode, wherein the sensing electrode comprises an enzyme electrode according to the fourth aspect of the present invention and said enzyme is disposed in relation to the modified electrically conductive element so that, in operation of the biosensor, the enzyme affects the amount of said substrate or product and thereby affects a signal current of said cell.

According to a sixth aspect, the present invention provides a method of determining the amount of an analyte in a sample comprising contacting a solution comprising said analyte with an enzyme electrode according to the fourth aspect of the present invention and with a standard electrode, applying an electrical potential between said electrodes, and monitoring the electrical current.

According to a seventh aspect, the present invention provides a method of determining the amount of an analyte in a sample comprising contacting a solution comprising said analyte with an electrode according to the third aspect of the present invention and with a standard electrode, applying an electrical potential between said electrodes, and monitoring the electrical current. A preferred analyte comprises hydrogen peroxide or a thiol.

In this specification, the following words and expressions, if and when used, shall have the meanings ascribed below: "comprising" or any cognate word specifies the presence of stated features, steps, or integers or components, but does not preclude the presence or addition of one or more other features, steps, integers, components or groups thereof; the expressions "consists of or "consists essentially of or cognates may be embraced within "comprises" or cognates, wherein "consists essentially of permits inclusion of substances not materially affecting the characteristics of the composition to which it applies.

Further, it is understood that any upper and lower quantity, range and ratio limits set forth herein may be independently combined.

The features of the invention relating to each and all aspects of the invention will now be described in more detail as follows:

The Electrically Conductive Element

Suitably, the electrically conductive element comprises a metal or carbon. When the electrically conductive element comprises a metal, preferably the metal comprises gold, platinum, indium-tin oxide, most preferably platinum. When the electrically conductive element comprises carbon, preferably the electrically conductive element comprises graphite, glassy carbon, pyrolitic carbon or surface printed carbon, most preferably screen printed carbon. Screen printed carbon electrodes may be obtained by techniques well known to those skilled in the art, such as described in Analytical Letters, 40 (2007) p1317-1332.

In accordance with a preferred embodiment of the present invention, the electrically conductive element comprises carbon, especially screen printed carbon.

The Hexacvano Transition Metallate Ion Solution

The hexacyano transition metallate ion containing solution comprises a solution of hexacyano transition metallate ions. Preferably, the hexacyano transition metallate ions comprise hexacyanoferrate ions or hexacyanocobaltate ions, especially hexacyanoferrate ions. Suitably, the hexacyano transition metallate ion containing solution comprises a solution of a metal hexacyano transition metallate, preferably an alkali (e.g. lithium, sodium or potassium) or alkaline earth (e.g. calcium, magnesium, barium or strontium) metal hexacyano transition metallate, more preferably an alkali metal hexacyano transition metallate, even more preferably an alkali metal hexacyanoferrate, most preferably potassium hexacyanoferrate.

Suitably, the hexacyano transition metallate ion containing solution comprises one or more ions of the following formulae [TM(CN)₆]^3' , [TM(CN)₆]^4", or a mixture thereof, wherein TM represents a transition metal.

Suitably, the metal hexacyano transition metallate solution comprises one or more compounds of the following formulae N_mM_n[TM(CN)₆]^3' , N_mM_n[TM(CN)₆]^4", or a mixture thereof, wherein N and M represents a metal, TM represents a transition metal, n is an integer of 1 to 4 and m is an integer of 0 to 3 depending on the valences of the metals N and M. Preferably, both of N and M do not represent iron.

Suitably, the alkali or alkaline earth metal hexacyano transition metallate solution comprises one or more compounds of the following formulae M_n[TM(CN)₆]^3" , M_n[TM(CN)₆]^4", or a mixture thereof, wherein M represents an alkali or alkaline earth metal, TM represents a transition metal and n is an integer of 2 to 4 depending on the valence of the metal M.

Preferably, the alkali (e.g. lithium, sodium or potassium) or alkaline earth (e.g. calcium, magnesium, barium or strontium) metal which M represents is an alkali metal and n is an integer of 3 or 4. Most preferably, the alkali metal is sodium or potassium; especially potassium.

Preferably, the transition metal which TM represents in the one or more compounds or one or more ions of formulae [TM(CN)₆]^3' , [TM(CN)₆]^4", NmM_n[TM(CN)₆]^3' , NmM_n[TM(CN)₆]^4", M_n[TM(CN)₆]^3" and M_n[TM(CN)₆]^4" comprises ruthenium, nickel, cobalt, osmium, iridium, copper or iron, more preferably ruthenium or iron, most preferably iron.

Preferably, the hexacyano transition metallate ion containing solution comprises a metal hexacyano transition metallate dissolved in a solvent. Preferably, the solvent is a polar solvent, especially a polar aprotic solvent. More preferably, the solvent comprises water, an alcohol, acetonitrile, dimethyl sulfoxide or dimethyl formamide; especially water (i.e. an aqueous solution of the metal hexacyano transition metallate).

Thus according to a preferred embodiment of the present invention, the hexacyano transition metallate ion containing solution comprises an aqueous solution of an alkali or alkaline earth metal hexacyano transition metallate, preferably an aqueous solution of an alkali metal hexacyanoferrate, most preferably an aqueous solution of potassium hexacyanoferrate. Suitably, according to a preferred embodiment of the present invention the transition metal hexacyano transition metallate derived material contained within the polyelectrolyte coating comprises a hexacyanoferrate derived material.

Suitably, the metal hexacyano transition metallate ion containing solution does not include any further transition metals or transition metal ions apart from TM in the one or more compounds or one or more ions of formulae [TM(CN)₆]^3" , [TM(CN)₆]^4', Nn₁M_n[TM(CN)₆]³- , N_mMn[TM(CN)₆]⁴-, M_n[TM(CN)₆]^3" and M_n[TM(CN)₆]^4".

The Transition Metal Ion Solution

The transition metal ion solution comprises a solution including a transition metal ion. Suitably, the transition metal ion comprises a chromium, zinc, rhenium, ruthenium, rhodium, osmium, palladium, iridium, platinum, nickel, cobalt, manganese, copper or iron ion, preferably an iron ion; especially an iron(ll) or Iron(lll) containing solution.

Thus, according to a preferred embodiment of the present invention the transition metal hexacyano transition metallate derived material contained within the polyelectrolyte coating comprises a hexacyanoferrate or hexacyanocobaltate derived material, preferably a hexacyanoferrate derived material, more preferably an iron (III) hexacyanoferrate or an iron (II) hexacyanoferrate material, especially Prussian Blue. By the term 'Prussian Blue' we mean a compound that includes one or more of the following compounds: MFe(III)[Fe(CN)₆], Fe(I I I)₄[Fe(CN)₆J₃, MFe(II)[Fe(CN)₆], or a mixture thereof, wherein M represents a metal having a valence of 1 , particularly an alkali metal as defined herein, especially potassium. Suitably, the transition metal ion solution comprises a transition metal salt dissolved in a solvent. The transition metal salt comprises the transition metal ion and a counter anion. Preferably, the counter anion comprises a halide (e.g. fluoride, chloride, bromide, iodide), a sulfate, acetate or nitrate. Suitably, the counter ion does not include a hexacyano transition metallate ion comprising one or more ions of the following formulae [TM(CN)₆]^3" or [TM(CN)₆]^4", as defined herein, where TM represents a transition metal particularly iron.

Preferably, the solvent is a polar solvent, especially a polar aprotic solvent. More preferably, the solvent comprises water, an alcohol, acetonitrile, dimethyl sulfoxide or dimethyl formamide; especially water (i.e. an aqueous solution of the transition metal salt).

The Polvelectrolvte

By the term 'polyelectrolyte' we mean a polymer composed of macromolecules in which a substantial portion of the constitutional units contain ionic or ionizable groups, or both.

Suitably the polyelectrolyte comprises an organic polymer. Preferably, the monomeric units of the organic polymer, apart from the ionic or ionizable groups, comprise hydrocarbyl groups. By the term hydrocarbyl, we mean a group consisting essentially of carbon and hydrogen atoms, optionally substituted with oxygen or nitrogen. Preferably, the hydrocarbyl group consists solely of carbon and hydrogen atoms.

Preferably, the polyelectrolyte comprises a hydrophilic polymer, especially a hydrophilic organic polymer. By the term 'hydrophilic polymer' we mean a polymer which has a solubility in water of greater than 0.05 g/l, preferably greater than 0.5 g/l, preferably greater than 1.0 g/l, more preferably greater than 10 g/l.

Preferably, the polyelectrolyte comprises a cationic polymer, cationic exchange polymer, anionic polymer or anionic exchange polymer. By the term 'cationic polymer' we mean a polymer where a substantial fraction of the constitutional units carry a positive charge. By the term 'cationic exchange polymer' we mean a polymer that exchanges cations with the ionic components of an aqueous solution. Suitably, a substantial fraction of the constitutional units of the cationic exchange polymer carry a positive charge in a neutral aqueous solution.

By the term 'anionic polymer' we mean a polymer where a substantial fraction of the constitutional units carry a negative charge. By the term 'anionic exchange polymer' we mean a polymer that exchanges anions with the ionic components of an aqueous solution. Suitably, a substantial fraction of the constitutional units of the anionic exchange polymer carry a negative charge in a neutral aqueous solution.

Preferred cationic and cation exchange polymers comprise amine functional polymers which include amino groups in the main polymer chain and/or polymer side chains. Preferred amine functional polymers include polyamines, polyimines, polyoxazolines, polyammonium compounds and derivatives thereof, especially polyimines and derivatives thereof. Highly preferred amine functional polymers include poly(allylamine), poly(lysine), poly(ethyleneimine), epoxylated poly(ethyleneimine), poly(aniline), polymeric derivatives of imidazole, poly(histidine), polymeric derivatives of pyridine, poly(diallyldialkylammonium chloride), and derivatives thereof, particularly poly(ethyleneimine) or poly(diallyldialkylammonium chloride). An especially preferred amine functional polymer comprises poly(ethyleneimine).

In a preferred embodiment of the present invention, the polyelectrolyte comprises a cationic or cationic exchange polymer, particularly a cationic exchange polymer, especially a poly(ethyleneimine) cationic exchange polymer or derivative thereof.

Preferred anionic and anion exchange polymers comprise carboxylic acid functional polymers, sulfonic acid functional polymers or phosphoric acid functional polymers. Preferred carboxylic acid functional polymers comprise an acrylic acid functional polymer or a (meth)acrylic acid functional polymer. Preferred sulfonic acid functional polymers comprise poly(sulfonates), especially poly(styrene- sulphonate). Preferred phosphoric acid functional polymers include DNA and RNA.

In accordance with a preferred embodiment of the present invention, the modified electrically conductive element is formed by contacting the surface of the electrically conductive element with a solution of the polyelectrolyte. Preferably, the electrically conductive element is immersed in a solution of the polyelectrolyte. Preferably, the solution of the polyelectrolyte comprises the polyelectrolyte dissolved in a suitable solvent. Preferably, the solvent is a polar solvent, especially a polar aprotic solvent. More preferably, the solvent comprises water, an alcohol, acetonitrile, dimethyl sulfoxide or dimethyl formamide; especially water (i.e. an aqueous solution of the polyelectrolyte). Accordingly, the polyelectrolyte may be attached to the surface of the electrically conductive element in a simple and cost- effective manner without the need for elaborate techniques.

Suitably, when the polyelectrolyte comprises a cationic or cationic exchange polymer, the modified electrically conductive element is first contacted with the hexacyano transition metallate ion containing solution and then contacted with the separate transition metal ion containing solution. Preferably, the modified electrode is immersed in the solution of the hexacyano transition metallate ion containing solution and then immersed in the separate solution of the transition metal ion containing solution. Advantageously, in the first step hexacyano transition metallate ions electrostatically bind to and within the coating of the polyelectrolyte. In the second step, the transition metal ions are adsorped by the polyelectrolyte coating and undergo a chemical reaction with the transition metallate ions within the coating to form the transition metal hexacyano transition metallate derived material within the coating.

Suitably, when the polyelectrolyte comprises an anionic or anionic exchange polymer, the modified electrically conductive element is first contacted with the transition metal ion containing solution and then contacted with the separate hexacyano transition metallate ion containing solution. Preferably, the modified electrode is immersed in the solution of the transition metal ion containing solution then immersed in the separate solution of the hexacyano transition metallate ion containing solution. Advantageously, in the first step transition metal ions electrostatically bind to and within the coating of the polyelectrolyte. In the second step, the hexacyano transition metallate ions are adsorped by the polyelectrolyte coating and undergo a chemical reaction with the transition ions within the coating to form the transition metal hexacyano transition metallate derived material within the coating.

Unexpectedly, it has been found that such a simple procedure enables the formation of a transition metal hexacyano transition metallate derived material within the polyelectrolyte. The transition metal hexacyano transition metallate derived material typically comprises one or more compounds of the following formulae M_pX_m[TM(CN)6]n wherein M is the metal derived from the metal hexacyano transition metallate solution as defined herein, X represents the transition metal of the transition metal ion containing solution as defined herein, TM represents the transition metal of the metal hexacyano transition metallate solution as defined herein, p is an integer of 0 to 2, m is an integer of 1 to 4 and n is an integer of 1 to 4. More preferably, the metal M when present is an alkali metal as defined herein, especially potassium, X is an Fe(II) or Fe(III) ion, p is 0 or 1 , m is 1 or 4 and n is 1 or 3. Most preferably, the transition metal hexacyano transition metallate derived material contained within the polyelectrolyte coating comprises a hexacyanoferrate derived material, particularly an iron (III) hexacyanoferrate or an iron (II) hexacyanoferrate material, especially Prussian Blue.

Conveniently, the amount of the transition metal hexacyano transition metallate derived material formed within the polyelectrolyte coating is typically limited by the ion exchange capacity of the polyelectrolyte and the molecular weight of the polyelectrolyte. Preferably, the number average molecular weight of the polyelectrolyte is greater than or equal to 1000 Daltons, more preferably greater than or equal to 2000 Daltons, even more preferably greater than or equal to 10000 Daltons, even more preferably greater than or equal to 20000 Daltons, most preferably greater than or equal to 25000 Daltons. An especially preferred polyelectrolyte, particularly a poly(ethyleneimine), has a number average molecular weight of 25000 to 750000 Daltons. According to a preferred embodiment of the present invention, the modified electrically conductive element is contacted once with the hexacyano transition metallate ion containing solution and once with the separate transition metal ion containing solution.

Enzyme Electrode

Unexpectedly, the formation of the transition metal hexacyano transition metallate derived material within the polyelectrolyte coating by the method of the present invention results in a partial regeneration of the cationic or anionic sites within the polyelectrolyte. Although only theory, it is believed that, for a cationic or cationic exchange polymer, the hexacyano transition metallate ions initially electrostatically bind to the cationic sites within the polyelectrolyte. However, upon contact with the separate transition metal ion containing solution, a more compact crystalline structure of the transition metal hexacyano transition metallate derived material is formed within the polyelectrolyte coating, thereby resulting in the partial regeneration of cationic sites within the electrolyte. The same effect is also observed for anionic and anionic exchange polymers, except the transition metal ions initially electrostatically bind to the anionic sites within the polyelectrolyte and then the resulting material is contacted with a separate solution of the hexacyano transition metallate ion containing solution to form the transition metal hexacyano transition metallate derived material within the polyelectrolyte. In the latter case, this results in a partial regeneration of the anionic sites within the electrolyte. The partial regeneration of the anionic or cationic sites within the electrolyte permits immobilization of an enzyme on the polyelectrolyte adjacent to the transition metal hexacyano transition metallate derived material in a straight forward and gentle manner, as an enzyme including charged sites may also be electrostactically bound to the polyelectrolyte. Suitably, such a procedure typically permits the automated manufacture of biosensors on a large scale and at a low cost.

Thus according to a highly preferred embodiment of the present invention, the method of manufacturing the electrode further includes the step of contacting the modified electrically conductive element with an enzyme containing solution to attach said enzyme to said polyelectrolyte on or adjacent said transition metal hexacyano transition metallate derived material thereby forming an enzyme electrode, wherein the enzyme is selected such that a substrate or product thereof is capable of being electrochemically oxidized or reduced at said electrode.

Preferably, the modified electrically conductive element is contacted once with the enzyme containing solution.

Preferably, the enzyme containing solution comprises an aqueous solution of the enzyme. More preferably, the modified conductive element is immersed in the aqueous solution of the enzyme.

Preferably, the step of contacting the modified electrically conductive element with the enzyme containing solution is performed after the step (ii) of contacting the modified electrically conductive element with said hexacyano transition metallate ion containing solution and said separate transition metal ion containing solution.

Suitably, the enzyme is an oxidoreductase enzyme which produces or consumes hydrogen peroxide, and said electrode is capable of oxidising hydrogen peroxide. Preferred, enzymes include glucose oxidase, acetylcholine esterase or the cascade enzyme cascade acetylcholine esterase plus choline oxidase; especially glucose oxidase.

Alternatively, the enzyme produces or consumes a thiol, and said electrode is capable of oxidizing the thiol. Suitable enzymes include hydrolases.

The enzyme electrode may be employed in an amperometric biosensor according to the fifth aspect of the present invention or used to determine the amount of an analyte in a sample according to the sixth aspect of the invention. The biosensor may include means defining a fluid flow path through the cell, and the enzyme is disposed in the flow path upstream of the cell so as to affect the composition of the fluid flow reaching the cell.

A highly preferred amperometric biosensor according to the fifth aspect of the present invention is adapted for determining glucose levels (i.e. the oxidoreductase enzyme of the enzyme electrode is glucose oxidase), particularly glucose levels in whole blood, serum or plasma. Suitably, the preferred analyte of the method according to the sixth aspect of the present invention is glucose, preferably glucose in whole blood, serum or plasma.

An alternative amperometric biosensor according to the fifth aspect of the present invention is adapted for determining thiocholine levels (i.e. the oxidoreductase enzyme of the enzyme electrode is acetyl choline esterase).

The present invention is illustrated by but in no way limited to the following examples.

Brief Description of Drawings

Figure 1 is a cyclic voltammogram of a Prussian Blue modified electrode. Figure 2 illustrates the different adsorption capacity of a poly(ethyleneimine) for hexacyanoferrate ions with varying molecular weight.

Figure 3 is a calibration plot for a glucose sensor.

Figure 4 is a schematic sectional view of an enzyme electrode.

Figure 5 is schematic sectional view of a working area of the enzyme electrode of Figure 4.

Figure 6 is a schematic sectional view of a biosensor.

Materials

Polyethylene imines with molecular weights of 400 (PEI-400), 2000 (PEI-2K) 25000 (PEI-25K) and 750000 (PEI-750K), K₃Fe(CN)₆, CoCI₂, NiCI₂, CuCI₂, MnCI₂, 30% H₂O₂, Glucose oxidase from Aspergillus niger Type X-S (GOx) were purchased from Sigma-Aldrich. Glucose, KH₂PO4, KOH, KCI were supplied by BDH. FeSO₄ was bought from WVR._Conducting carbon inks type Electrodag PF- 407A and dielectric inks type Electrodag PF-455 were obtained from Acheson (Plymouth, UK), whilst ceramic tiles (96% AI₂O₃ Coorstek Grade ADS96R) were sourced from Laser Cutting Ceramics Ltd (Sheffield, UK). Water deionised with a MiIIi-Q reagent water system (Millipore, USA) was used throughout. Potassium phosphate buffer solution 25 mM containing 100 mM KCI at the pH 7.0 is denoted as PBS. Equipment

A three electrode cell equipped with the saturated calomel electrode as a reference electrode and a platinum rod as an auxiliary electrode was used in all electrochemical experiments. All electrochemical measurements were performed using an Autolab-lll FRA12 system and the general purpose electrochemical software operating system GPES4 from Eco Chemie B.V. (Utrecht, Netherlands). SPCE were employed as working electrodes. All measurements were carried out at room temperature.

Example 1 Formation of Screen-Printed Carbon Electrodes

Screen-printed carbon electrodes (SPCEs) were produced as described in Analytical Letters, 40 (2007) p1317-1332. In this respect, two carbon layers (Electrodag PF-407A) were sequentially printed onto ceramic tiles and each layer was cured for 1 h at 240⁰C in air. The ceramic tiles were then covered with an insulating layer of Electrodag PF-455 to restrict a round shaped working area (7 mm² per electrode). The insulating layer was dried in a vacuum oven for 1 h at 200⁰C at less than 1020 mbar pressure. After fabrication, the electrodes were stored at room temperature in a closed box. A DEK-248 screen printer (DEK Northern Europe, Weymouth, UK) was used for all printing procedures.

Example 2 Preparation of the Modified Electrically Conductive Element

Surface modification of the SPCE of Example 1 was affected with a poly(ethyleneimine) (PEI), having a number average molecular weight of 750K Daltons, at +50⁰C in closed Eppendorf tubes. The surface of the SPCE was cleaned by immersion in ethanol for 3 minutes and then water for 3 minutes. The cleaned SPCE was immersed in an aqueous solution of PEI (10mg/ml) for 30 minutes and subsequently washed with water for 3 minutes to remove any weakly bound polyelectrolyte. No drying was allowed between these steps. After the final step the electrode was thoroughly rinsed with water and incubated in water for another 5 minutes at room temperature before further modification with Prussian Blue. Example 3 Preparation of Prussian Blue Modified Electrode

The modified electrically conductive element of Example 2 was immersed in an aqueous solution of 5mM K₃Fe(CN)_θ (pH 1, 100 mM KCI) for 5 minutes at room temperature. The electrode was quickly rinsed with 100 mM KCI (pH 1), immersed into 5 mM FeSO₄ (pH 1, 100 mM KCI) and kept in the dark for 2 hours. The electrode was then thoroughly washed with the copious amount of water, dried under the nitrogen flow and left in the dark for 1 h before incubation with an enzyme solution as described in Example 8.

Example 4 Cyclic Voltammoqram of Prussian Blue Modified Electrode

A cyclic voltammogram of the Prussian Blue modified electrode of Example 3 in an aqueous solution of 0.1 M potassium chloride (pH 7) at a scan rate of 10 mV/s between +0.7 V to -0.1V is shown as a solid line in Figure 1. Also shown in Figure 1 (dashed line) is a corresponding cyclic voltammogram of a modified electrically conductive element comprising the poly(ethyleneimine) coating containing ferrocyanate ions only i.e. an electrode formed as in Example 3 by immersion of the poly(ethyleneimine) coated electrode in an aqueous solution of 5mM KaFe(CN)₆ (pH 1 , 100 mM KCI) for 5 minutes at room temperature followed by washing in acidic water (pH 1, 100 mM potassium chloride). The cyclic voltammograms indicate that the modified electrode exhibits a high electrocatalytic activity for the reduction of hydrogen peroxide (3.8 μA/mM) unlike the electrode containing ferrocyanate only (0.1 μA/mM).

Examples 5 to 7 Adsorption Capacity of Poly(ethyleneimine)

Modified electrically conductive elements were prepared as in Example 2 with poly(ethyleneimine) polymers having different number average molecular weights (Mn) as detailed in Table 1. Table 1

The ability of each of the PEI coatings to adsorb hexacyanoferrate ions was investigated by immersing each of the respective modified electrically conductive elements in an aqueous solution of 5mM K₃Fe(CN)β (pH 1 , 100 mM KCI) for 3 minutes at room temperature. Then the electrodes were quickly rinsed with an aqueous solution of 100 mM KCI (pH 1). The quantity of ferrocyanate ions adsorbed by the PEI coatings was determined by measuring the area of the oxidation peaks under each respective cyclic voltammogram in an aqueous solution of 0.1 M potassium chloride (pH 7) at a scan rate of 10 mV/s between +0.7

V to -0.1V.

Figure 2 shows the adsorption capacity of the various PEI coatings and demonstrates that a PEI having a higher molecular weight exhibits a stronger adsorption capacity for ferrocyanate ions than a PEI coating having a lower molecular weight. Suitably, a modified electrode having a PEI coating with a higher molecular weight will be expected to produce a higher concentration of Prussian Blue within the polyelectrolyte coating than a corresponding electrode having a lower molecular weight PEI coating and therefore possess higher electrocatalytic activity for the reduction of hydrogen peroxide.

Example 8 Enzyme Electrode

The Prussian Blue modified electrode of Example 3 was incubated for 10 min in an aqueous glucose oxidase solution (2 mg/ml) in PBS (pH 7, 100 mM KCI). After careful rinsing with water an amperometric response for glucose was measured at 0.0 V versus the standard calomel electrode by introducing sequential injections of 1 M glucose (2-20 μl) into the cell containing 5 ml of PBS (pH 7, 100 mM KCI) under intensive stirring. The steady state response after each addition was recorded. A calibration plot for the glucose sensor is shown in Figure 3. Sensitivity to glucose detection was found to be 0.13 μA/mM within the linearity range of 0.15 mM up to 3.5 mM, and a steady state response was typically reached within 20 seconds. The enzyme electrode exhibited a good operational stability, as no current decay was detected within 5 minutes of monitoring an aqueous 13 mM glucose solution which approximately corresponds to a local hydrogen peroxide concentration of 0.2 mM. Such a stable response is indicative of the absence of leaching of the Prussian Blue and the enzyme from the electrode. Suitably, the electrode offers commercially important advantages of operation at low potential and selectivity.

A typical enzyme electrode is shown in Figure 4. The enzyme electrode (2) comprises an alumina support (3) having a screen printed carbon coating (4). The screen printed carbon coating includes a plastic insulating material (6) to define a working area (8) at one end of the electrode (2). The opposite end of the electrode includes an electrical contact pad (10). The working area (8) as shown in Figure 5 comprises the screen printed carbon coating (4) a surface of which includes a poly(ethyleneimine) coating (12). The poly(ethyleneimine) coating contains a Prussian Blue material (14) and a glucose oxidase enzyme (16) adjacent to the Prussian Blue material (14).

Figure 6 shows a typical biosensor (20) including the enzyme electrode (2) of Figure 4. The biosensor (20) comprises a flow cell (22) in which are disposed the enzyme electrode (2) a screen printed carbon counter electrode (24) and a reference electrode (26) comprising screen printed silver inks. Each of the electrodes (2, 24, 26) include a plastic insulating material (28) thereby defining a lower working area (8, 30, 32). The other ends of the electrodes (2, 24, 26) include an electrical contact pad (10, 34, 36) to permit a potential to be applied to the electrodes (2, 24, 26).

Claims

1. A method of manufacturing an electrode comprising:

2. A method as claimed in claim 1 wherein the modified electrically conductive element is formed by contacting said surface of the electrically conductive element with a solution of said polyelectrolyte.

3. A method as claimed in claims 1 or 2 wherein the polyelectrolyte comprises a hydrophilic organic polymer.

4. A method as claimed in any one of the preceding claims wherein the polyelectrolyte comprises a cationic polymer or a cationic exchange polymer.

5. A method as claimed in claim 4 wherein the cationic polymer or cationic exchange polymer comprises an amine functional polymer.

6. A method as claimed in claim 5 wherein the amine functional polymer comprises a polyamine, polyimine or a polyammonium compound.

7. A method as claimed in claim 6 wherein the amine functional polymer comprises a polyimine, preferably poly(ethyleneimine) (PEI) and derivatives thereof.

8. A method as claimed in any one of claims 4 to 7 wherein the modified electrode is first contacted with the hexacyano transition metallate ion containing solution to bind hexacyano transition metallate ions within the polyelectrolyte coating and then the modified electrode is contacted with the separate transition metal ion containing solution.

9. A method as claimed in any one of claims 1 to 3 wherein the polyelectrolyte comprises an anionic polymer or an anionic exchange polymer.

10. A method as claimed in claim 9 wherein the anionic polymer or anionic exchange polymer comprises a carboxylic acid functional polymer, a sulfonic acid functional polymer or a phosphoric acid functional polymer.

11. A method as claimed in claim 9 or 10 wherein the modified electrode is first contacted with the transition metal ion containing solution to bind transition metal ions within the polyelectrolyte coating and then the modified electrode is contacted with the separate hexacyano transition metallate ion containing solution.

12. A method as claimed in any one of the preceding claims wherein the transition metal ion containing solution comprises an iron containing solution, preferably a Fe(II) or Fe(III) ion containing solution.

13. A method as claimed in any one of the preceding claims wherein the hexacyano transition metallate ion containing solution comprises a hexacyanoferrate ion containing solution.

14. A method as claimed in any one of the preceding claims wherein said hexacyano transition metallate ion containing solution comprises a solution of a metal hexacyano transition metallate.

15. A method as claimed in claim 14 wherein said metal hexacyano transition metallate comprises an alkali or alkaline earth metal hexacyano metallate.

16. A method as claimed in claim 15 wherein the alkali or alkaline earth metal hexacyano metallate comprises an alkali metal hexacyanoferrate, preferably potassium hexacyanoferrate.

17. A method as claimed in any one of the preceding claims wherein the electrically conductive element comprises gold, platinum, indium-tin oxide or carbon, preferably carbon.

18. A method as claimed in claim 17 wherein the electrically conductive element comprises graphite, glassy carbon, pyrolitic carbon or surface printed carbon, preferably surface printed carbon.

19. A method as claimed in any one of the preceding claims further including the step of contacting the modified electrically conductive element with an enzyme containing solution to attach said enzyme to said polyelectrolyte on or adjacent said transition metal hexacyano transition metallate derived material, wherein the enzyme is selected such that a substrate or product thereof is capable of being electrochemically oxidized or reduced at said electrode.

20. A method as claimed in claim 19 wherein said enzyme is an oxidoreductase enzyme which produces or consumes hydrogen peroxide, and said electrode is capable of oxidising hydrogen peroxide.

21. A method as claimed in claims 19 or 20 wherein said enzyme is a glucose oxidase.

22. An electrode obtainable by the method as claimed in any one of claims 1 to 21.

23. An electrode comprising:

(i) a modified electrically conductive element comprising an electrically conductive element, as defined in any one of claims 1 to 21 , having a surface and a coating of a polyelectrolyte, as defined in any one of claims 1 to 21 , on said surface; and,

(ii)a transition metal hexacyano transition metallate derived material contained within the polyelectrolyte coating.

24. An electrode as claimed in claim 23 wherein the transition metal hexacyano transition metallate derived material comprises a hexacyanoferrate derived material, preferably Prussian Blue.

25. An electrode as claimed in claim 23 or 24 wherein the polyelectrolyte comprises a cationic or cationic exchange polymer.

26. An electrode as claimed in claim 25 wherein the polyelectrolyte comprises poly(ethyleneimine) or a derivative thereof.

27. An enzyme electrode comprising an electrode as claimed in any one of claims 23 to 26 and an enzyme attached to the polyelectrolyte coating on or adjacent said transition metal hexacyano transition metallate derived material, wherein the enzyme is selected such that a substrate or product thereof is capable of being electrochemically oxidised or reduced at said electrode.

28. An enzyme electrode as claimed in claim 27 wherein said enzyme is an oxidoreductase enzyme which produces or consumes hydrogen peroxide, and said electrode is capable of oxidising hydrogen peroxide.

29. An enzyme electrode as claimed in claim 27 or 28 wherein said enzyme is a glucose oxidase.

30. An amperometric biosensor comprising a cell for receiving an analyte and electrodes for contacting the analyte in the cell, said electrodes comprising a sensing electrode, a standard electrode, and optionally a counter electrode, wherein the sensing electrode comprises an enzyme electrode as defined in any one of claims 27 to 29 and said enzyme is disposed in relation to the modified electrically conductive element so that, in operation of the biosensor, the enzyme affects the amount of said substrate or product and thereby affects a signal current of said cell.

31. An amperometric biosensor according to claim 30 adapted for determining glucose in whole blood, serum or plasma.

32. A method of determining the amount of an analyte in a sample comprising contacting a solution comprising said analyte with an enzyme electrode as defined in any one of claims 27 to 29 and with a standard electrode, applying an electrical potential between said electrodes, and monitoring the electrical current.

33. A method as claimed in claim 32 wherein the analyte is a substrate or an inhibitor for said enzyme.

34. A method as claimed in claim 32 or 33 wherein the analyte gives rise to hydrogen peroxide which is oxidized by the enzyme electrode thereby producing a signal current.

35. A method of determining the amount of an analyte in a sample comprising contacting a solution comprising said analyte with an electrode as defined in any one of claims 23 to 26 and with a standard electrode, applying an electrical potential between said electrodes, and monitoring the electrical current.