WO2003081244A2

WO2003081244A2 - Method of electrochemical cell analysis

Info

Publication number: WO2003081244A2
Application number: PCT/GB2003/001287
Authority: WO
Inventors: Duncan Ross Purvis
Original assignee: Sensor Tech Ltd
Current assignee: Sensor Tech Ltd
Priority date: 2002-03-26
Filing date: 2003-03-25
Publication date: 2003-10-02
Anticipated expiration: 2004-09-26
Also published as: GB2386950A; WO2003081244A3; GB0207116D0; AU2003214440A1

Abstract

The invention relates to a sensing electrode for use in methods of electrochemical detection of cell metabolic activity. In particular, the sensing electrode comprises an electrically conductive electrode coated with a layer of electroconductive polymer and further coated with immobilised cells.

Description

Method of electrochemical cell analysis

Field of the invention

The invention relates to methods of electrochemical detection of cell metabolic activities and to sensing electrodes for use in such methods of electrochemical detection.

Background to the invention Electrochemical cell analysis, such as measurement of various local or internal changes in pH (acidification) , ionic strength or redox potential due to normal metabolic activities or challenged metabolic activities in biological or synthetic fluids using biosensors is a promising and attractive method of instrument analysis.

It is known in the art to construct biosensor devices based on the use of electroconductive polymer films, such as polypyrrole or polythiophene, which transduce a chemical signal associated with the presence of an analyte into a measurable electrical signal (see [1] and [2]). The published application PCT/GB02/03894 (WO 03/019171) describes methods for preparing highly sensitive potentiometric sensors with an electroconductive polymer film as a sensing element .

The published application PCT/GB98/00548 (WO 98/37409) describes a potentiometric method of electrochemical analysis using an electrochemical sensing electrode comprising a metallic potentiometric electrode coated with a layer of electroconductive polymer containing immobilised bioreceptor molecules which bind specifically to an analyte under test. The presence of analyte is indicated by a change in surface charge of the sensing electrode upon binding of analyte to the immobilised bioreceptors, using an ion-step detection procedure. This same sensor configuration can also be employed for cell analysis by measuring local changes in pH, ionic strength and redox condition outside the cell and/or within the cell itself.

Microphysiometry, developed by Dr. Harden McConnell at Stanford University in 1983, examines the rate at which cells excrete acid during basal or stimulated conditions of energy metabolism. Cells take in nutrients and break them down to produce useful energy and waste products. The primary waste products are lactic acid and carbonic acid (C0₂) . The rate at which cells excrete acids is very closely linked to the rate at which they convert food to energy. By maintaining cells in a low-buffered environment, one can measure the extremely small quantities of excreted acidic byproducts from energy metabolism (for every 2 ATP molecules used during glycolytic metabolism, one hydrogen ion is produced) . As energy metabolism is coupled to cellular ATP usage, any event which perturbs cellular ATP levels (i.e., receptor stimulation and initiation of signal transduction pathways) will cause a change in energy metabolism and therefore an alternation in acid excretion. Membrane bound transport proteins, in particular the Na⁺/H⁺ exchanger, also play an important role in maintaining intracellular pH. The metabolic pathways used by the cell determine the absolute extracellular acidification rate. This rate can be measured using UTS™ technology. UTS™ is a registered trade mark used to refer to technology described in WO 98/37409 and WO 00/11473

The present inventors have now developed methods of electrochemical analysis of cell metabolic activity based on the use of electroconductive polymer coated sensing electrodes.

Description of the invention

Sensing electrodes, assemblies and arrays

In a first aspect the invention provides a sensing electrode for use in methods of electrochemical detection of cell metabolic activity, the sensing electrode comprising an electrically conductive electrode coated with a layer of electroconductive polymer and further coated with cells immobilised in, adsorbed to or attached to the layer of electroconductive polymer.

The electroconductive polymer layer performs a dual function, serving both to bind the cells to the surface of the sensing electrode, and to render the sensing electrode sensitive to variations in the composition of a bathing electrolyte solution (i.e. when the sensing electrode is in use immersed in an electrolyte solution) . In particular, changes in the composition of the electrolyte solution which affect the redox composition of the electroconductive polymer result in a corresponding change in the steady state potential of the sensing electrode.

The sensing_ electrodes of the invention are substantially equivalent to those described in earlier applications (e.g. WO 98/37409), except that the electrode surface is modified with biological cells. The cells can be any type of prokaryotic or eukaryotic cell which it is desired to study.

In one embodiment, cells may be adsorbed directly onto the electroconductive polymer coating on the sensing electrode.

In a further embodiment the surfaces of electroconductive polymer-coated electrodes can be modified by coating with biomolecules or other functional groups which can in turn be used to link cells to the electrode surface. Procedures for the modification of polymer-coated electrodes with biomolecules and other functional groups are known in the art and described, for example in WO 98/37409 and WO 00/11473.

Biological molecule (s) can be immobilised onto a sensing electrode using well known techniques for solid phase coating. Biological molecules may be incorporated into the electroconductive polymer during the polymerisation reaction, or they may be adsorbed onto the surface of a coated sensing electrode in a separate modification step after the polymer coating step, or they may be covalently linked to the polymer coating.

In a particular embodiment the biological molecules may be "adaptor molecules" which enable the attachment of cells, or further molecules (e.g. receptors) capable of binding cells, to the surface of the sensor via a binding interaction. The use of "universal" sensing electrodes containing adaptor molecules allows the immobilisation of a wide variety of cells using specific binding receptors.

The proteins avidin and streptavidin are preferred for use as adaptor molecules. Investigations carried out by the authors of the declared invention have shown that avidin and streptavidin immobilised in an electroconductive polymer film, retain their native properties for an extended period of time (at least one year and possibly longer) and can be used throughout this period to link with biotin conjugated receptors. Techniques which allow the conjugation of biotin to a wide range of different molecules are well known in the art. Thus sensing electrodes with immobilised avidin or streptavidin can easily made specific for the binding of whole cells merely by binding of appropriate biotinylated receptors via biotin/avidin or biotin/streptavidin interactions.

Although avidin and streptavidin are the preferred adaptor molecules it is within the scope of the invention to use alternative adaptor molecules, for example protein A, protein G, lectins and FITC. The incorporation of adaptor molecules enables other biomolecules or whole cells to be attached to the surface of the sensing electrode, for example via protein A/antibody, protein G/antibody, FITC/anti-FITC or lectin/sugar binding interactions.

In a further embodiment cells may be adsorbed to or grown on a secondary substrate, for example a mesh, which is then intimately positioned in close proximity to the surface of a polymer-coated sensing electrode. By "close proximity" is meant that the secondary substrate is sufficiently close for local changes in the internal or external environment of the cells to affect the sensing electrode, thus allowing detection of the changes.

The sensing electrodes of the invention are inexpensive to manufacture and so for convenience can be produced in a disposable format, intended to be used for a single electrochemical detection experiment or a series of detection experiments and then thrown away. The invention further provides an electrode assembly including both a sensing electrode and a reference electrode required for electrochemical detection. As will be discussed below, suitable reference electrodes include silver/silver chloride and calomel electrodes. Conveniently, the electrode assembly could be provided as a disposable unit comprising a housing or holder manufactured from an inexpensive material equipped with electrical contacts for connection of the sensing electrode and reference electrode.

The invention still further comprises a sensing electrode array comprising a plurali-ty of sensing electrodes according to the invention.

Methods of manufacture of sensing electrodes

In a second aspect the invention relates to methods of manufacturing sensing electrodes. Therefore, the invention provides a method of producing a sensing electrode for use in methods of electrochemical detection of cell metabolic activity, the method comprising the steps of:

a) preparing an electrochemical polymerisation solution comprising monomeric units of the electroconductive polymer and background electrolyte; b) immersing a conductive electrode to be coated, an auxiliary electrode and a reference electrode in the electrochemical polymerisation solution; c) applying a controlled galvanic or potential profile between the electrode to be coated and the auxiliary electrode to coat the electrode by electrochemical synthesis of the polymer from the solution; and d) contacting the coated electrode with a cell suspension to capture the cells at the surface of the electrode .

This method may be used in the manufacture of sensing electrodes comprising an electrically conductive electrode coated with an electroconductive polymer, wherein cells are attached or adsorbed directly to the polymer coating.

In one embodiment, whole cells may be adsorbed directly onto the electroconductive polymer coating on the sensing electrode. This may be achieved, for example, by immersing a coated sensing electrode in a suspension of cells, or by placing a drop of cell suspension onto the coated electrode. Cells may also be grown directly on the polymer-coated electrode by immersing the electrode in a suitable suspension of cells in growth medium and incubating under conditions which promote cell growth.

In a further embodiment the method may be adapted for the manufacture of sensing electrodes comprising an electrically conductive electrode coated with an electroconductive^" polymer, wherein adaptor molecules are adsorbed onto the polymer coating, receptor molecules are attached to the adaptor molecules, and cells are bound to the receptor molecules in order to link the cells to the sensing electrode. In this embodiment the method comprises the further steps between step c) and step d) of: contacting the coated electrode with a solution comprising adaptor molecules such that the adaptor molecules are adsorbed onto the electroconductive polymer coating of the electrode; and contacting the coated electrode with a solution containing cell receptor molecules capable of specifically binding to the adaptor molecule and binding to the cells such that receptor molecules are attached to adaptor molecules adsorbed to the electroconductive polymer coating. The electrode may then be contacted with a suspension of cells under conditions which allow the cells to bind to the receptor molecules.

In a further embodiment the method may be adapted for the manufacture of sensing electrodes comprising an electrically conductive electrode coated with an electroconductive polymer, wherein adaptor molecules are incorporated into the polymer coating during the polymerisaton step, receptor molecules are attached to the adaptor molecules, and cells are bound to the receptor molecules in order to link the cells to the sensing electrode. In this embodiment adaptor molecules are included in the electrochemical polymerisation solution in step a) and become immobilised in the growing electroconductive polymer layer. The method also includes the further steps between step c) and step d) of: contacting the coated electrode with a solution containing cell receptor molecules, which receptor molecules are capable of specifically binding to the adaptor molecule and binding to the cells, such that receptor molecules are attached to the adaptor molecules immobilised in the electroconductive polymer coating. The electrode may then be contacted with a suspension of cells under conditions which allow the cells to bind to the receptor molecules.

In the methods of the invention a film of electroconductive polymer is deposited onto the surface of an electrically conductive electrode by electrochemical synthesis from a monomer solution. The electrically conductive electrode is preferably a standard potentiometric electrode possessing metallic or quasi-metallic conductivity which is stable in aqueous media. As will be illustrated in the examples included herein, electrodeposition of the electroconductive polymer film is carried out using a solution containing monomers, a polar solvent and a background electrolyte. Pyrrole, thiophene, furan or aniline are the preferred monomers. Deionised water is preferably used as the polar solvent.

As is well known to persons skilled in the art, electroconductive polymers are often doped at the electrochemical synthesis stage in order to modify the structure and/or conduction properties of the polymer. As reported in a number of papers [4, 5], the ease with which ion exchange takes place and the rapidity with which ion equilibrium is attained for electroconductive polymers immersed in a solution are essentially dependent on the size of the anti-ion introduced at the electrodeposition stage: the larger the ionic radius of the anti-ion, the more readily ion-exchange reactions take place and the more rapidly a state of equilibrium is reached. This is directly linked to the value and rate of change of the potential of the "metal electrode - electroconductive polymer" system in response to variation in the ion composition of the solution [6] . The type of the response (anionic, cationic, redox) and its rate can be determined during the polymerisation [5, 6] .

A typical dopant anion is sulphate (S0₄ ^2_) which is incorporated during the polymerisation process, neutralising the positive charge on the polymer backbone. Sulphate is not readily released by ion exchange and thus helps to maintain the structure of the polymer.

It is possible to provide potentiometric sensitivity of the electroconductive polymer to one particular cation or anion. The ions of background electrolyte are immobile and able to react specifically with the ion of interest, e.g. calcion (cation) , which specifically reacts with calcium and gives precipitated product (salt) .

For redox and pH sensitive sensors it is preferred to use a salt whose anions have a large ionic radius as the background electrolyte when preparing the electrochemical polymerisation solution. In this case ion response is minimised and redox or pH response predominate, potentiometric response is provided by electron exchange between the polymer film and surrounding solution.

Suitable salts whose anions have large ionic radius include sodium dodecyl sulphate and dextran sulphate. The concentration of these salts in the electrochemical polymerisation solution is varied according to the type of test within the range 0.0001 - 0.05 M.

Redox response can be increased by incorporating into the polymer dopant ions, which can change their redox state due to the changes in the surrounding solution giving the sensor the additional change in redox state. The dopant should be in reduced form if one of the solution components is oxidized and vice versa. K₃ [Fe (CN) ₆] /K₄ [Fe (CN) ₆] can be given as an example for both cases. The concentration of these electrolytes in the electrochemical polymerisation solution can be varied within the range 0.001 - 0.1 M to meet specific requirements of the test.

Use of the electrode

In a further aspect the invention also relates to use of the sensing electrode in methods of electrochemical analysis of cells or analytes.

In particular, the invention provides a method of electrochemical cell analysis and/or detection of an analyte in a test sample, which method comprises the steps of: a) providing a sensing electrode according to the first aspect of the invention, wherein the cells respond, specifically or otherwise, to a desired analyte to be analysed and/or detected; b) treating the sensing electrode by immersion in an electrolyte solution; c) monitoring the electric potential difference between the treated sensing electrode and a reference electrode when both are immersed in an electrolyte; __d) treating the sensing electrode by immersion in a test electrolyte solution comprising the test sample so that said desired analyte interacts with the cells; and e) monitoring the electric potential difference between the sensing electrode and a reference electrode following an induced change in the ionic strength, pH or redox potential of the test electrolyte solution.

An electrochemical measuring cell is assembled by bringing a sensing electrode according to the invention and a reference electrode, connected by a measuring instrument, into contact with an electrolyte solution (also referred to herein as a working solution) and the measuring device is used to record the sensing electrode potential relative to the reference electrode over a fixed time period. Commercially available reference electrodes of suitable size, or electrodes purpose-designed for implementation of the declared invention, may be used as the reference, e.g. calomel or silver/silver chloride electrodes. The measuring instrument is a standard potentiometric measuring instrument or potentiostat . PC-compatible electronic measuring instruments purpose designed for implementation of the declared invention and controlled by custom software can also be used.

For convenience the sensing electrode and reference electrode can be printed on the same device or can be linked to the measuring instrument by means of a special holder equipped with electrical contacts for connection of the sensing electrode and reference electrode and connected to the measuring instrument by a cable or other means. A holder integral with the measuring instrument could also be used, making it possible to miniaturise the measuring system in terms of its overall dimensions.

Aqueous buffer solutions are used as the working solution: phosphate-saline, Tris-HCl, carbonate bicarbonate, acetate, borate, etc. The volume of working solution in the electrochemical cell is typically between 10 and 5000 μl depending on the geometrical dimensions of the sensing electrode. The container for the buffer solution may be any suitably sized vessel in a material with minimal adsorption properties, e.g. the well of a standard microtiter plate. Another embodiment of the declared invention is a variant in which a low-volume (< 1cm³) flow-through cell is used in conjunction with an integral holder for the sensing electrode and reference electrode, through which buffer solution can be pumped by means of a peristaltic pump or other means .

The potential of the sensing electrode relative to the reference electrode potential is recorded for a fixed time period using a chart recorder connected to a potentiometric measuring device or potentiostat, or by means of a special program where PC-compatible electronic instrumentation is used. In the latter case, the program measures the sensing electrode potential relative to the reference electrode potential at pre-determined time intervals (typically every 3-5 seconds for a total of 10-100 seconds) and displays the results in the form of points on the coordinates "sensing electrode signal - time". Recording of sensing electrode potential relative to the reference electrode potential is carried out to determine the background potential value V^ of the sensing electrode, and also to evaluate the background potential drift (y) of the sensing electrode, which is calculated by linearisation of the curve "sensing electrode signal-time" obtained using the least squares method.

The variation in sensing electrode potential relative to the reference electrode potential in response to the addition of the test analyte is recorded for a fixed time period using a measuring instrument. Again, the recording is made either using a chart recorder connected to a potentiometric measuring device or potentiostat, or by means of a special program where PC-compatible electronic instrumentation is used. In the latter case, the program measures the sensing electrode potential relative to the reference electrode potential at pre-determined time intervals (typically every 3-5 seconds) and displays the results in the form of points on the coordinates "sensing electrode signal - time". Depending on the particular type of test, the time taken to record the variation in sensing electrode potential relative to reference electrode potential varies between 30 and 600 seconds. On completion of this stage in the procedure, the final value V₂ of sensing electrode potential relative to reference electrode potential is determined. Quantitative characteristics of the change in sensing electrode potential relative to the reference electrode potential can then be calculated:

Based on the quantitative characteristics of the variation in sensing electrode potential in response to a change in pH, ionic strength or redox composition of the working solution, a determination is made as to the quantitative content of target analyte in the test solution, or analysis of the effect of the analyte on the metabolic activity of the cell.

This method of electrochemical detection is of use where the interaction of the target analyte with the cell causes a change in charge in the surface of the sensing electrode which is sufficiently large to be measurable.

Specific applications of the technology

The sensing electrodes of the invention can be used in a wide range of electrochemical cell analysis procedures, including (but not limited to) acidification assays, metabolic rate, toxicity studies, drug screening.

Specific, but non-limiting, examples of applications of the invention are as follows:

GPCRs assays

GPCRs are a super family (>175 identified so far) of seven transmembrane bound protein receptors. They are responsible for initiating a range of cell-signalling pathways mediated via the upregulation of calcium (33%) and cyclic AMP (67%) . These type of receptors are implicated in >50% of all therapeutic indications currently being addressed by the pharmaceutical industry. The industry standard means for probing the antagonism of these receptors is via the measurement of the up-regulation of Calcium using a Fluorescence Imaging Plate Reader (FLIPR) from MDC and a fluorescence indicator dye like Fluo-3 or Fluo-4. This uses LAPS (light activated potentiometric sensor) technology.

cAMP assays cAMP is determined via a number of means but the desired method is using a flash based luminescence reagent like aeqorin. This reagent acts as a substrate for the cAMP and is turned over to give light which can then be read by a luminescence reader like CLIPR for MDC. This turnover can also be read by the change in redox state of the electrochemical sensor. Alternative substrates which are not light producing can also be used.

Extracellular acidification can be a product of general metabolic perturbation. You can ensure that you are looking at specific receptor-ligand interactions, by using specific receptor-mediated responses, in this way, one can identify specific signal transduction pathways and receptor subtypes. E.g. receptor specific antagonists, antibodies, signal transduction probes for evaluating the tyrosine kinase pathway, the NHE and various G-protein mediated pathways such as adenylate cyclase, PKC, PKA, and calcium.

Evaluation of ligand gated ion channels, e.g. for excitatory amino acids (kainate & NMDA) , muscle and neuronal nAChR, P2 Purinoceptors, and GABA-A. The system cannot work with voltage-gated ion channels. Examination of receptors that internalize, e.g.. insulin receptors.

The number of compounds that can be evaluated/hr or /day is obviously dependant on panel size developed. But would also be highly dependent upon the type of compounds screened, e.g. For neurotransmitter type compounds that stimulate cells briefly and, therefore, allow the same population of cells to be used for multiple doses. In this case, the dose rate would be higher than for growth factors and cytokines that stimulate cells for prolonged periods and therefore require new populations of cells for each dose .

In addition the sensing electrodes may also be used as a non-invasive patch clamp, for example to measure the opening and closing of ion channels.

All references mentioned herein are incorporated herein by reference.

The present invention will be further understood with reference to the following non-limiting Examples together with the accompanying Figures, in which:

Fig. 1A: schematically illustrates various sensing electrodes or sensing electrode arrays according to the invention;

Figs. 2A, 2B and 2C illustrate a processes for immobilising cells on sensing electrodes;

Fig. 3A: illustrates an arrangement of the sensing electrode, fluidics and data management for testing analyte on the immobilized cells. Referring to the drawings, Figure 1 schematically illustrates various sensing electrode or sensing electrode arrays. Figure 1(a) shows a single sensing electrode consisting of a potentiometric electrode 1 mounted on an inert substrate 4. A portion of the electrode 1 is coated with a layer of electroconductive polymer 2. The enlarged panel shows a cross-section through the substrate 4 and sensing electrode. Cells 3 are adsorbed onto the polymer coating 2.

Figures 1(b), 1(c) and 1(d) illustrate various types of arrays of sensing electrodes 1, coated with a layer of electroconductive polymer 2.

Figure 2A illustrates a process for immobilising cells on sensing electrodes, whereby the cells are adsorbed directly to the polymer coating. Sensing electrodes coated with a layer of electroconductive polymer are contacted with a suspension of cells, such that cells are adsorbed directly onto the polymer coating;

Figure 2B illustrates a process for immobilising cells on sensing electrodes, whereby the cells are attached via an adaptor molecule and a specific receptor. Adaptor molecules are adsorbed to or immobilised in the electroconductive polymer coating on the sensing electrodes. Receptor molecules capable of binding -to cells are then attached to the adaptor molecules. Finally, the electrodes are contacted with a cell suspension such that the cells binding to receptor molecules.

Figure 2C illustrates a process for immobilising cells on sensing electrodes, whereby the cells are adsorbed onto a secondary substrate (e.g. a mesh) which is then intimately positioned next to the sensing electrode. In an alternative embodiment the mesh itself may be the sensor.

Example 1

This example describes preparation of a sensing electrode with anionic type of potentiometric response.

A custom-made planar electrode was made comprising PET (polyethyleneterephthalate) support (~125μm) with electro-deposited copper (~17μm) coated with electrochemically-plated gold (~30μm) . The working area was approximately 1.0 sq mm. Electrochemical polymerisation solutions were assembled comprising 0.005M NaBF₄ serving as a background electrolyte and 0.05M pyrrole. The solution was placed in a electrochemical polymerisation cell comprising an auxiliary platinum electrode and a reference electrode (BAS) . The electrode to be coated was placed in the cell, with the working area immersed in the solution. In order to minimise ohmic drop the reference electrode was located at the nearest possible distance from the electrode to be coated.

The electrochemical polymerisation was carried out using μAutolab II potentiostat-galvanostat (EcoChemie) , by applying cycling voltage between electrode to be coated and auxiliary electrode within -0.2 - +1.7 V (vs Ag/AgCl reference electrode) four times with the scan rate 0.05 V/sec. After polymerisation, the coated electrode was removed from the well, and rinsed with deionised water.

The resulting sensing electrode can be used for cell immobilisation and for detection of a change in local anionic strength in a cell sample.

Example 2

This example describes the preparation of a sensing electrode with cationic type of potentiometric response.

Aqueous solutions for electrochemical polymerisation were assembled comprising lOmg/ml of indigo carmine serving as a background electrolyte and 0.1M pyrrole. The solution was placed in a cell for electrochemical polymerisation comprising an auxiliary platinum electrode and a reference electrode (BAS) . The electrode to be coated described in Example 1 was placed in the cell, with the working area immersed in the solution.

Electrochemical polymerisation was carried out using a μAutolab II potentiostat-galvanostat (EcoChemie) , by applying constant current between electrode to be coated and auxiliary electrode. Current density of 0.5mA/cm² for 10 min.

After polymerisation, the coated electrode was removed from the well, and rinsed with deionised water.

The resulting sensing electrode then was used for cell immobilisation and for detection of a change in local cationic strength in a cell sample.

Example 3

This example describes the preparation of the sensing electrode with redox type of potentiometric response and with streptavidin incorporated within the polymer film.

Aqueous solutions for electrochemical polymerisation were assembled comprising 0.005M SDS serving as a background electrolyte, 0.05M pyrrole and 0.2 mg/ml of streptavidin. The solution was placed in a cell for electrochemical polymerisation comprising an auxiliary platinum electrode and a reference electrode (BAS) . The electrode to be coated as described in Example 1 was placed in the cell, with the working area immersed in the solution.

The electrochemical polymerisation was carried out using a μAutolab II potentiostat-galvanostat (EcoChemie) , by applying cycling voltage between electrode to be coated and auxiliary electrode within -0.2 - +1.9 V (vs Ag/AgCl reference electrode) eight times with the scan rate 0.05 V/sec.

After polymerisation, the coated electrode was removed from the well, rinsed with deionised water and placed in a reservoir of 0.05M K-phosphate buffer (pH 8.0), where it was stored at +4°C.

The resulting sensing electrode then was used for cell immobilisation and for detection of a change in local pH and redox potential in a cell sample.

References

1. Kasparov S.V., Farmakovsky D.A., Kharlamov A.A. , Damiryan A.U., Remen V.V. Device for detecting biologically active compounds in biological fluids and a method of manufacturing of the sensing element. Patent of the Russian Federation No. 2032908.

2. Kasparov S.V., Farmakovsky D.A. Electrochemical immunoassay. WO 96/02001.

3. Farmakovsky D.A. , Milanovsky E. Yu., Cherkasov V.R., Biryukov Yu. S., Komarov B.V. A method of electrochemical indiction of immuno-chemically active macromolecules in test solutions. Patent of the Russian Federation No. 2107296.

4. Ge Hailin, Wallace G.G. Ion exchange properties of polypyrrole. Reactive polymers, 18, 133-140, 1992.

5. Curtin L.S., Komplin G.C., Pietro W.J., Diffusive Anion Exchange in polypyrrole films. J. of Physical Chemistry, 92, 12-13, 1988.

6. Bobacks J. , Gao Zh. , Ivaska A., Lewenstam. A., Mechanism of ionic and redox sensitivity of p-type conducting polymers. Part 2. Experimental study of polypyrrole. J. of Electrochemical Chemistry, 368, 33-41, 1994.

Claims

Claims :

1. A sensing electrode for use in methods of electrochemical detection of cell metabolic activity, j the sensing electrode comprising an electrically conductive electrode coated with a layer of electroconductive polymer and further coated with cells immobilised in, adsorbed to or attached to the layer of electroconductive polymer.

2. A sensing electrode according to claim 1 wherein the cells are attached to a secondary substrate which is intimately positioned proximal to the surface of the sensing electrode.

3. A sensing electrode according to claim 1 wherein the sensing electrode further comprises adaptor molecules immobilised in, adsorbed to or attached to the layer of electroconductive polymer, and the cells are bound to the adaptor molecules.

4. A sensing electrode according to claim 3 wherein the adaptor molecules are attached to receptor molecules capable of binding to the cells, and the cells are bound to the receptor molecules.

5. A sensing electrode according to claim 4 wherein the adaptor molecules are avidin or streptavidin and biotinylated receptors capable of binding the cells are attached thereto via biotin/avidin or biotin/streptavidin-binding interactions .

6. A sensing electrode according to claim 4 wherein the adaptor molecules are lectins and receptors capable of binding the cells are attached thereto via a lectin/carbohydrate binding interaction.

7. A sensing electrode according to any one of claims 1 to 6 wherein the layer of electroconductive polymer has been doped with mobile anions of large ionic radius. j

8. A sensing electrode according to any one of claims 1 to 6 wherein the layer of electroconductive polymer has been doped with anions which are immobile in the polymer film.

9. A sensing electrode according to any one of claims 1 to 6 wherein the layer of electroconductive polymer has been doped with anions carrying a large amount of negative charge.

10. A sensing electrode according to any one of claims 1 to 6 wherein the layer of electroconductive polymer has been doped with anions capable of specific interaction with cations.

11. A sensing electrode according to any one of claims 1 to 6 wherein the layer of electroconductive polymer has been doped with anions capable of changing their redox state.

12. An electrode assembly comprising a sensing electrode according to any one of claims 1 to 11 and a reference electrode.

13. A sensing electrode array comprising a plurality of sensing electrodes according to any one of claims 1 to 11.

14. A method of producing a sensing electrode for use in methods of electrochemical detection of cell metabolic activity, the method comprising the steps of: a) preparing an electrochemical polymerisation solution comprising monomeric units of the electroconductive polymer and background electrolyte; b) immersing a conductive electrode to be coated, an auxiliary electrode and a reference electrode in the electrochemical polymerisation solution; c) applying a controlled galvanic or potential profile between the electrode to be coated and the auxiliary electrode to coat the electrode by electrochemical synthesis of the polymer from the solution; and d) contacting the coated electrode with a cell suspension to capture the cells at the surface of the electrode.

15. A method according to claim 14 which comprises the further steps between step c) and step d) of: contacting the coated electrode with a solution comprising adaptor molecules such that the adaptor molecules are adsorbed onto the electroconductive polymer coating of the electrode; and contacting the coated electrode with a solution containing cell receptor molecules capable of specifically binding to the adaptor molecule and binding to the cells such that receptor molecules are attached to adaptor molecules adsorbed to the electroconductive polymer coating.

16. A method according to claim 14 wherein the electrochemical polymerisation solution in step a) further includes adaptor molecules, and which comprises the further step between step c) and step d) of: contacting the coated electrode with a solution containing cell receptor molecules capable of specifically binding to the adaptor molecule and binding to the cells such that receptor molecules are attached to the adaptor molecules immobilised in the electroconductive polymer coating.

17. A method according to claim 15 or claim 16 wherein the adaptor molecules are avidin or streptavidin and the receptor molecules are conjugated with biotin such that the biotinylated receptors bind to molecules of avidin or streptavidin immobilised in or adsorbed to the electroconductive polymer coating of the electrode via biotin/avidin or biotin/streptavidin binding interactions.

18. A method according to any one of claims 14 to 17 in which a salt whose anions have a large ionic radius is added to the electrochemical polymerisation solution.

19. A method according to claim 18 wherein the salt is sodium NaC10₄ or NaBF₄.

20. A method according to any one of claims 14 to 17 in which a salt whose anions are immobile in polymer film is added to the electrochemical polymerisation solution.

21. A method according to claim 20 wherein the salt is sodium dodecylsulphate or sodium dextran sulphate.

22. A method according to any one of claims 14 to 17 in which an organic electrolyte carrying the large amount of negative charge is added to . the electrochemical polymerisation solution.

23. A method according to claim 22 wherein the organic electrolyte is indigo carmine, methylene blue or sodium dodecylsulphate or naphthalenesulphonate .

24. A method according to any one of claims 14 to 17 in which an electrolyte whose anions are capable i of specific interaction with cations is added to the electrochemical polymerisation solution.

25. A method according to claim 24 wherein the electrolyte is calcion.

26. A method according to any one of claims 14 to 17 in which a salt whose anions can change their redox state is added to the electrochemical polymerisation solution.

27. A method according to claim 26 wherein the salt is [Fe(CN)₆] or K₄[Fe(CN)₅].

28. A method according to claim 26 or claim 27 in after step (c) a step of electrochemical treatment of the sensing electrode is performed by applying a constant voltage or constant current between sensing electrode and auxiliary electrode when both are immersed in electrolyte solution without monomeric units of the electroconductive polymer.

29. A method according to any one of claims 14 to 17 in which a salt whose anions can change their redox state is added to^" the electrochemical polymerisation solution jointly with a salt whose anions are immobile in the polymer film.

30. A method according to any one of claims 14 to 29 wherein the monomeric units of the electroconductive polymer are aniline, thiophene, furan or pyrrole.

31. A method according to any one of claims 14 to 30 wherein the electric potential is controlled potentiostatically.

32. A method according to any one of claims 14 to 30 wherein the electric potential is controlled galvanostatically .

33. A method according to any one of claims 14 to 30 wherein the electric potential is controlled galvanodynamically.

34. A method of electrochemical cell analysis and/or detection of an analyte in a test sample, which method comprises the steps of: a) providing a sensing electrode according to any one of claims 1 to 11, wherein the cells respond, specifically or otherwise, to a desired analyte to be analysed and/or detected; b) treating the sensing electrode by immersion in an electrolyte solution; c) monitoring the electric potential difference between the treated sensing electrode and a reference electrode when both are immersed in an electrolyte; d) treating the sensing electrode by immersion in a test electrolyte solution comprising the test sample so that said desired analyte interacts with the cells; and e) monitoring the electric potential difference between the sensing electrode and a reference electrode following an induced change in the ionic strength, pH or redox potential of the test electrolyte solution.

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