WO1996002001A1

WO1996002001A1 - Electrochemical immunoassay

Info

Publication number: WO1996002001A1
Application number: PCT/GB1995/001602
Authority: WO
Inventors: Stanislav Victor Kasparov; Dmitri Alexand Farmakovski
Original assignee: Leaver, Jonathan
Priority date: 1994-07-07
Filing date: 1995-07-07
Publication date: 1996-01-25
Also published as: AU2985295A

Abstract

An immunoassay method for detecting a biological analyte forming a binding pair with a bio-receptor uses a three electrode cell comprising a reference electrode (24), an auxiliary electrode (4) and an indicating electrode (3). The indicating electrode comprises a base electrode having an electrically conductive ion exchange polymer coating with bio-receptor incorporated therein. The method comprises determining the potential of the reference electrode (24) with respect to the indicating electrode (3) under conditions of constant current between the indicating and auxiliary electrodes when the three electrodes are placed in a sample-free measuring solution and when the three electrodes are placed in a sample-containing measuring solution. The indicating electrode (3) and auxiliary electrode (4) may be in the form of an integrated electrode assembly. The polymer coating of the indicating electrode is preferably a two polymer film coating with the bio-receptor incorporated in the outer film. The reference electrode (24), such as an Ag/AgCl electrode, is separated from the measuring solution (22) by a membrane (28) permeable to the ions of the measuring solution, but not to macromolecular molecules.

Description

_ ! -

ELECTROCHEMICAL IMMUNOASSAY

FIELD OF INVENTION

The present invention relates to electrochemical immunoassay methods and apparatus for use therein. More particularly the invention relates to a method of assaying a target analyte using a three electrode electrochemical cell comprising an auxiliary electrode, a polymeric indicating electrode incorporating a bio-receptor complementary to the target analyte, and a reference electrode. The invention relates also to apparatus for carrying out the method including electrodes for such a method and methods of manufacture thereof.

BACKGROUND OF THE INVENTION

Immunosensors have been proposed in the art for use to determine relatively large proteins or polypeptides through an antigen-antibody reaction. An immunosensor comprising an electrode having an antigen (or an antibody) immobilized thereon is immersed in a solution containing a corresponding antibody (or antigen) . Formation of an antigen-antibody complex is detected by detecting a change in the electrochemical properties of the sensor. Prior art proposals include use of electrodes comprising an electrically conductive polymer such as polypyrrole or polythiophene. As well as antigen-antibody complexes, other biological molecules forming a binding pair may be considered for detection in this way.

EP-A-0 193 154 discloses an indicating electrode having a polypyrrole film or polythiophene film formed on its surface by electrochemical galvanostatic polymerization of pyrrole or thiophenone, a bio-receptor complimentary to the substance to be tested being immobilized on the surface of the polymer film. EP-A-0 193 154 further discloses formation of a two-electrode electrochemical cell by immersing the indicating electrode and a reference electrode connected by a measuring instrument into a measuring solution, measuring the potential difference between the indicator electrode and reference electrode, placing the sample to be tested into the measuring solution and recording change over time of the potential difference between the indicating electrode and the reference electrode in the measuring solution containing the sample to be tested. WO-89/11649 discloses polymeric electrodes incorporating bio-receptors and their use in determining the presence of molecules binding specifically to those bio-receptors by measuring the change in electrochemical properties of the polymeric electrode which results from binding of the target molecules to the bio-receptors.

The present inventors in WO 92/19959 disclose a three electrode cell comprising a reference electrode, an indicating electrode comprising a polypyrrole layer with immobilized bio-receptor and an auxiliary electrode. The indicating and auxiliary electrode may be provided in integrated form on a chip, and may comprise interlocking comb shapes, with each tooth of the auxiliary electrode in a gap between two neighbouring teeth of the indicating electrode. The degree of binding of target bio-molecules to bio-receptors is determined by determining the change of potential of a reference electrode when a current pulse is applied between the indicating electrode and the auxiliary electrode.

WO 94/20841 discloses an electrically conductive polymer electrode with receptor immobilized therein to which is coupled an alternating voltage waveform that permits a target bio-molecule to reversibly bind to the receptor such that measurement of electrode current provides a measure of such reversible binding.

The above prior art proposals all suffer from poor reproducibility and low sensitivity. It is an object of the present invention to provide an improved electrochemical immunoassay method of good reproducibility and sensitivity.

Immunoassay methods find particular application in the medical field, enabling testing of samples for the presence of biological compounds for the purpose of diagnosis. Immunoassay methods may also be used in other circumstances where the presence of particular biological material needs to be detected. For example, in the detection of organic pollutants, the detection of viruses in vegetation, the detection of harmful biological material in the food industry. It may also be applied generally to antibody/antigen analysis, DNA analysis and drug screening.

SUMMARY OF THE INVENTION

According to the present invention there is provided a method of detecting in a sample a biological analyte forming a binding pair with a bio-receptor by means of a three electrode cell comprising a reference electrode, an auxiliary electrode and an indicating electrode, said indicating electrode comprising a base electrode having an electrically conductive ion exchange polymer coating and a bio-receptor incorporated in said polymer coating. The method comprises determining the potential of the reference electrode with respect to the indicating electrode under conditions of constant current between said indicating and said auxiliary electrodes when the three electrodes are placed in a sample-free measuring solution and when the three electrodes are placed in a sample-containing measuring solution.

Preferably the polymer coating comprises a first and second polymer film, the bio-receptor being incorporated in the second, outer, polymer film. The polymer films are preferably partially oxidised, ion exchange polymers.

In a second aspect of the present invention there is provided an electrode assembly including a polymeric electrode incorporating a bio-receptor, said assembly comprising: - a substrate of inert material; an auxiliary electrode comprising a layer of an electrically conductive material on a first part of the external surface of said substrate; an indicating electrode comprising: - a layer of an electrically conductive material on a second part of the external surface of said substrate; a first film of an electrically conductive polymer on the outer surface of the layer of electrically conductive material; a second film of an electrically conductive polymer on the outer surface of said first film, and bio-receptors incorporated in said second film of electrically conductive polymer.

The indicating electrode and the auxiliary electrode may be made by forming a layer of the electrically conductive material on one face of the external surface of the inert material, for example by sputtering, and subsequently locally removing the electric conductive material from parts of the surface of the inert material, for example by photolithography, to form two separated planar electrodes. Electrically conductive polymer and bio-receptors may then be applied to the surface of the indicating electrode. Preferably the first film of electrically conductive polymer is formed by the potentiodynamic oxidation of a suitable monomer solution, and the second film of electrically conductive polymer and bio-receptors immobilized thereon is formed by potentiodynamic oxidation of a solution comprising a suitable monomer and the bio- receptors. The first and/or second electrically conductive polymer films may be processed electro- che ically to obtain preferred electrochemical mechanical properties. The second electrically conductive polymer film should preferably be a thin film, having a thickness no more than about 5 or 10 times the molecular diameter of the immobilized bio-receptor. The first polymer film layer may be electrochemically processed by doping with an ion which is not subsequently easily exchanged, such as by electrochemical doping from a sodium sulphate (Na-,S0₄) solution having a concentration of between 0.05 M and 0.5 M. Where the electrode is intended to be used in saline phosphate buffer solution, which is the preferred measuring solution for the method of the invention, the second polymer layer may be doped with PO^2' and HP0₄ ^' most preferably by electrochemical doping from a saline phosphate buffer solution. Additional positive charges may be introduced particularly to the first polymer film by electrochemical doping with an organic surfactant such as SDS (sodium dodecyl sulphate) . The electrode chip of the present invention enables provision of a compact sensitive element with a large indicating electrode surface and a minimum gap between the indicator and the auxiliary electrodes, for example by forming the indicator and auxiliary electrodes of interlocking comb shapes. In the assay method of the invention which requires current to be passed through a test solution, a high voltage drop resulting from electrical resistance of the test solution can be avoided. Also the volume of test solution can be minimized. Repeatability, accuracy and sensitivity of the electrochemical immunoassay can be improved by the above and by the fixed geometry of the electrodes.

In a third aspect of the present invention there is provided a method of manufacture of an electrode assembly as above described.

In a fourth aspect of the present invention there is provided apparatus for performing an immunoassay. An electrochemical cell comprises a container for measuring solution, a reference electrode for insertion in said measuring solution and an electrode assembly as above described. Preferably the reference electrode is separated from the measuring solution by a membrane permeable to the ions of the measuring solution, but not to macromolecular molecules. The apparatus further comprises means for providing a constant current between the indicating and auxiliary electrodes and means for measuring the potential of the reference electrode relative to the indicating electrode.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention will now be described in more detail by way of example only with reference to the accompanying drawings in which:-

Fig. l is a plan view of an electrode assembly according to the invention;

Fig. 2 is a cross-sectional view of the chip electrode assembly of figure 1; Fig. 3 is a plan view of an alternative embodiment of an electrode assembly according to the present invention;

Fig. 4 is a plan view of an alternative embodiment of an electrode assembly according to the present invention;

Fig. 5 is a schematic representation of apparatus according to the present invention for conducting electrochemical immunoassays;

Fig. 6 schematically illustrates changes of potential across the indicating electrode in a saline phosphate buffer measuring solution when a small constant current is passed between the indicating electrode and the auxiliary electrode in the apparatus of Figure 5; Fig. 7 schematically illustrates the position when a test sample including a bio-molecule which will bind specifically to the bio-receptor of the indicating electrode is added to the measuring solution in the arrangement shown in Figure 6; Fig. 8 is a schematic representation of a sensor with a directly absorbed protein layer;

Fig. 9 is a representation of a sensor with an intermediate hydrophillic membrane, antibody being bound to the outside of the membrane; Fig. 10 is a schematic representation of a sensor having a hydrophillic membrane containing immobilized antibody;

Figure 11 shows the results of carrying out the method of the invention on a negative test sample; Figure 12 shows the results of carrying out the method of the invention on a test sample containing target analyte;

Figure 13 shows the results of carrying out the method of the invention on a test sample which contains target analyte in an amount greater than the test sample of Figure 12;

Figure 14 shows a calibration curve for HBs-Ag; and

Figure 15 is a distribution curve of positive and negative test results.

Figure 16 shows an electrode assembly and reference electrode in a holder.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to Figs. 1 and 2, an electrode assembly or chip 1 comprises a substrate 2 made of an inert dielectric material. This may be for example a ceramic material such as cital or silica oxide.

Alternatively it may be a polymer material such as polya ide, nylon or polyethylene. The substrate 2 may be shaped for example, as a rectangular plate as shown in Figs. 1 to 4 or a disc. An indicating electrode 3 and an auxiliary electrode 4 may be made by sputtering a layer of electrically conductive material 5 on at least one major surface of the inert substrate. In figures 1 to 4 this layer 5 comprises a sub-layer 5a and an over-layer 5b of an inert electric conductive material for example gold platinum or titanium nitride. Where the substrate 2 is a ceramic material sub-layer 5a may be a metal such as titanium, nickel, cobalt or tungsten. Where the substrate 2 is a polymer material, the sub-layer may be a metal oxide or metal nitrate such as chromium oxide (Cr₂0₅) , titanium oxide (Ti₂0₅) or titanium nitride (Ti₂N₃) . Alternatively, layer 5 may comprise a single layer of titanium nitride. The thickness of the layer 5 is preferably in the range of 5 nm to 2 μm. Figures 3 and 4 show preferred interlocking comb shapes for the auxiliary electrode 4 and the indicating electrode 3. The electrodes 3, 4 may be of arbitrary size. Smaller electrodes enable use of smaller volumes of test solution. The gap 7 between the electrodes 3 , 4 on the substrate is preferable as small as practical, bearing in mind the requirement to coat electrically conductive polymer on the indicating electrode 3, but not on the auxiliary electrode 4, and after coating to leave a gap 7 between the indicating and auxiliary electrodes. Preferably the gap 7 between the electrodes on the substrate is less than 500 μm, more preferably less than 100 μm, say 10 to 100 μm. By carefully controlling manufacture, a preferred separation of 2 to 10 μm may be achieved. Formation of an integrated electrode assembly allows control of the geometry of the electrodes and control of the field effects which arise when passing current between the electrodes in solution. The interlocking comb arrangement of Figure 4, where the teeth of the auxiliary comb electrode are located between neighbouring teeth of the indicating comb electrode, the teeth of the indicating comb being substantially wider than the teeth of the auxiliary electrode, is most preferred. This gives a wide area of indicating electrodes, allowing large numbers of bio-receptors to be immobilized in the second stage of manufacture. In a second stage of manufacture, a layer 8 of an electrically conductive polymer is formed on the surface of the indicating electrode 3 and a bio-receptor 9 is incorporated therein.

The bio-receptor may be any of a biological binding pair. Examples include antibodies, recombinant proteins, enzymes, DNA, RNA, inactivated microorganisms, cells, cell membranes and receptor proteins.

Processes for the deposit of such a polymeric layer 8 and for immobilization of a bio-receptor 9 are known. They are described for example in WO 89/11649, WO 90/02829 and W092/19959. In the embodiments of Figs. 1 to 4 the layer 8 comprises two films 10, 11. The bio- receptor 9 is incorporated in the outer film 11. As further described below this provides advantageous properties for use in the preferred assay method.

The two polymer film indicating electrode of Figs. 1 to 4 may advantageously be made in the following manner.

The first film 10 may be formed by electrochemical polymerization of a monomer solution. Any monomer or combination of monomers which forms a hydrophillic ion exchange, electrically conductive polymer may be used. Such monomers include thiophene, furan and aniline, a preferred monomer being pyrrole. The monomer may be dissolved in a polar solvent together with a base electrolyte. The polar solvent should be such as will dissolve the monomer, but will not dissolve the polymer. Examples of suitable polar solvents include nitriles such as acetonitrile and benzonitrile; amides such as dimethyl formamide; amines such as pyridine; ethers such as tetrahydrofurane; acids such as acetic acid; alcohols such as methanol and ethanol; water; phosphate buffer solution; acetone; dichloromethane; Tris-HCl. Electrolyte concentrations of 0 to 1.0 M are preferred, more preferably 0.02 to 1.0 M.

Polymerization is preferably carried out by potentiodynamic cycling in a three electrode cell comprising the indicating electrode 3 the auxiliary electrode 4 and a reference electrode of arbitrary shape and size such as a standard Ag/AgCl or Hg/HgCl electrode. The polymerization conditions will effect the resultant structure of the polymer film. A preferred structure is provided by potentiodynamic cycling at a positive potential throughout. For polypyrrole the cycling may be from 0 mV to 800 mV at a velocity of 50 to 100 mV per second over 3 to 6 cycles. The polypyrrole first polymer film 10 so formed is in only partially oxidized condition, potentials in excess of 1250 mV being required for full oxidation. The film 10 so formed is of open but even structure. The structure and partial oxidation give the polymer film 10 good ion exchange properties.

The properties of the first polymer film layer 10 can be adjusted by electrochemical doping of the polymer film 10. The indicating electrode 3 with the first polymer film 10 on it, the auxiliary electrode 4 and a standard reference electrode may be placed in a polar solvent and a doping agent added for electrochemical doping of the polymer film. Alternatively, the dopants may be incorporated by including these as the electrolyte in the monomer solution from which the polymer film 10 is formed.

Taking a pyrrole monomer as a representative example, polymerisation may be represented as follows:-

[Equation A]

A preferred dopant ion is sulphate (S0₄ ^2') which is incorporated to neutralise the positive charge of the polymer back bone. It is not readily released from the structure by ion exchange, and helps to retain the polymer structure. A preferred method of manufacture is electrochemical doping from a solution containing Na_∑SO, in a concentration of 0.05 M to 0.5 M.

The polymer, however formed, should preferably be in a form which may be described as weakly polarized, weakly doped or partially oxidized. The polymer should preferably have good ion exchange properties.

Preferably additional positive charges may be introduced by doping with an organic detergent for example SDS (sodium dodecyl sulphate) .

In the preferred two polymer film electrode of Figs. 1 to 4, after formation and doping of a first film polymer film 10, as described above, a second polymer film 11 is deposited. This second polymer film incorporates bio-receptors 9. This can be achieved by potentiodynamic deposit from a monomer solution in a polar solvent, as above, to which the bio-receptors 9 have been added. Preferably the outer film layer of polymer 11 is a thin film, having a thickness of less than 10, preferably less than 5, times the molecular diameter of the immobilized bio-receptor 9, more preferably 2 to 5 molecular diameters. The second polymer film layer 11 may be processed electrochemically to obtain preferred electrochemical mechanical properties. Taking polypyrrole as example with an antibody Ab- as the bio-receptor, the following ariεes:-

[Equation B] o H

A preferred measuring solution for determining the presence of target bio-molecules is saline phosphate buffer solution. Preferably HP0₄ ^" and P0₄ ^2" ions are incorporated as dopant anions in the polymer, in the manner described above. Preferably this may be achieved by depositing the second polymer film 11 by potentiodynamic deposit from a monomer and bio-receptors in a saline phosphate buffer solution. Preferred concentrations of phosphate are 0.03 M to 0.5 M. For polypyrrole film potentiodynamic polymerisation at 0 mV - 800 V at a velocity of 50 mV per second is suitable. As with the first polymer layer 10, the synthesis is directed to producing a weakly polarized, weakly doped, partially oxidized ion exchange polymer layer. The bio-receptor carrying polymer electrode/auxiliary electrode chip of Figs. 1 to 4 may be used in a novel method for determining the presence and/or level of an analyte complimentary to the bio-receptor.

This novel method is based on consideration of what takes place when the indicating electrode 3 and auxiliary electrode 4 chip of Figs. 1 to 4 is placed in an aqueous solution.

The bio-receptor layer 9 in an aqueous measuring solution contains (and orders) a certain amount of water. The polymer layers 10, 11 are hydrophillic ion exchange polymers. These various layers may be considered as aqueous solutions of ions, some of which can be exchanged between the layers. When measurements are established in aqueous solution based on steady-state conditions, a general theoretical approach may start by considering the equilibrium position of two aqueous solutions separated by a thin membrane. Suppose solution A on one side of the membrane contains a dissociable salt, for instance NaCl. Solution B on the other side of the membrane also contains NaCl, but additionally contains a macromolecule P+. The membrane is permeable to the small cations and anions, but not to the macromolecule. In the steady state condition a potential will be established across the membrane, referred to as the Donnan potential.

Figure 5 schematically illustrates apparatus that may be used for the method of the present invention. A reservoir 20 holds a measuring solution 22. An electrode assembly, or chip, 1 according to Figure 4 and comprising indicating electrode 3 and auxiliary electrode 4 is located in the measuring solution. Also located in the measuring solution is a reference electrode 24. This may be a standard Ag/AgCl reference electrode. Preferably the reference electrode 24 comprises a silver wire core with silver chloride coating 26 within a plastic casing 27 which contains agar saturated with silver chloride 29. A permeable membrane 28, such as a nylon membrane, permeable to the measuring solution ions, but not to macromolecules, is preferably provided across an opening of the plastic casing 27 to separate the reference electrode 24 from the measuring solution 22 to avoid contamination of the reference electrode 24. Preferably the electrode chip 1 and reference electrode 24 are mounted in a holder (for example holder 60 shown in Figure 16) so that the indicating auxiliary and reference electrodes can be easily located in the reservoir 20 containing measuring solution 22 to form a three electrode cell. A control unit 30 is electrically connected to the three electrodes. The control unit 30 comprises, consecutively connected, an amplifier 34 connected to the reference electrode 24, an analogue to digital converter 35 and a programmable controller 37. The programmable controller 37 is connected to a CPU and is also connected to a second digital to analogue converter 36 to provide input to a controlled current source 32. The output of the current source 32 is connected to the indicating and auxiliary electrodes 3, 4. This arrangement enables a controlled current to be passed between the indicating and auxiliary electrodes and enables the potential of the reference electrode, with reference to the potential of the indicating electrode, to be determined. Relative potential between the indicating electrode and reference electrode may be determined by much more straightforward circuitry when this potential measuring system is applied, rather than the conventional mechanism of measuring the potential of an electrode with reference to the fixed potential of a reference electrode. Figures 6 and 7 illustrate how changes of potential, caused by the Donnan phenomenon, arise when the apparatus of Figure 5 is employed with a constant current applied between the indicating and the reference electrode. The preset current is preferably in the range of 1 nA to lμA. Figures 6 and 7 illustrate the flow of electrons and ions when such a constant current is supplied, and illustrates the effect of the Donnan potential in the various layers. In Figure 6, the three electrodes are immersed in a pure measuring solution 22 of saline phosphate buffer solution. In Figure 7, an analyte is present with measuring solution which analyte can bind to the bio-receptor 9. Illustrated is antigen 40 and antibody 9 interaction. This leads to a change in surface characteristics, and a change of the Donnan potential, reflected in a change of the potential of the reference electrode with respect to the indicating electrode. The difference between the potentials Ψ, of Figure 6 and Ψ-, of Figure 7 represents the response. Figs. 8 to 10 illustrate the Donnan potential effect in other type of antibody-containing electrodes. Fig. 8 is a sensor where antibody 9 is attached directly to a metal electrode 5. Fig. 9 shows a metal electrode 5 having a polymer coating 8 and bio-receptor 9 bound at the outside of the polymer coating 8 (for example by covalent binding) . Fig. 10 illustrates an electrode comprising a metal layer 5, a polymer layer 8 and bio-receptors 9 distributed throughout and on the surface of the polymer layer 8. The Donnan potentials are schematically illustrated in these Figs. 8 to 10 where Ψ represents the potential and C, C, C ' , C"' are the concentrations of all types of ion in the relevant phases.

The use of two partially doped polymer film layers as shown in Figures 6 and 7 enables best use of the method by providing a system in dynamic balance, where the speed of leaving of electrons and the creation of positive charges in the conductive polymer is balanced by the flow of negative ions (primarily chloride ions) from solution into the polymer matrix. In this way integrity of the electrode may be maintained.

A sample will not comprise solely the target analyte. Other biological material will be present, for example various proteins. These may also have an effect by binding in a non-specific manner. Generally negatively charged proteins will be attracted towards the indicating electrode 3. To take account of this, measurements may be taken in a three stage process. In the first stage, the three electrodes are brought into contact with pure measuring solution to set a background level of potential of the reference electrode with respect to the indicating electrode solution. Three electrodes are then placed in measuring solution containing a sample to be tested. ^' After incubation, the electrodes may be replaced in the pure measuring solution. The change of value of the differential potential between the reference electrode and the indicating electrode before and after incubation in the measuring solution containing the test sample is related to the content of the target analyte. This can be compared against information obtained in the same manner from check samples with known content of the target analyte.

Rather than moving the electrodes from one reservoir to another, the method may be used in the form of flow injection analysis, where measuring solution is continuously pumped through the electrochemical cell, and test samples are introduced directly in the flow of the measuring solution.

Other possibilities may be considered for taking into account non-specific binding. For example, the surface of the polymer electrode where there is no bound antibody may be blocked by, for example, immersing the electrode in non-specific albumin, such as human or bovine serum albumin. Where antibody is incorporated by electrochemical means, it is possible to provide sufficient density of antibody that non-specific binding between antibody moieties becomes unimportant, and blocking is not necessary. It is possible to make the measuring solution approximate to the background composition of the sample to be tested, by inclusion of surface-active agents such as dextran-sulphate, ficoll, polyvinylpyrrolidone, polyethylene glycol, together with albumin such as human or bovine serum albumin to approximate the properties of the measuring solution (density, ion strength, conductivity) to those of the sample to be tested. The composition and concentration of these additional components, to be added to phosphate saline buffer solution, will depend upon the type of sample being tested, for example urine, blood serum, blood plasma and so forth. The preferred method, as described above, where the measuring solution is phosphate buffer saline solution alone, and the three step test procedure is used is preferred for simplicity and accuracy and reproducibility of results.

Ion migration on the composite indicating electrode 3 is assumed to be controlled by 3 properties: the electric field, represented by a potential difference V, the gradient of the redox potential in the conducting polymer, and the ion concentration gradient. The negative ions migrate proportionally to the electric field strength. The redox-potential gradient drives ions to regions with a lower redox potential. The concentration gradient drives ions to regions with a lower ion concentration.

The flow of ions (0) may be represented by the following equation:

ø₍x,t₎ = _b -*^V(*.*> ₊2Vred ⁽y⁽x.t⁾ _C(x,t)-D *^c l* > ^ dx dx ' dx where : - b is electrical mobility of the negative ions;

V is the electric potential; Vred is the redox potential; D is the diffusion coefficient;

C is the ion concentration;

Y is the doping level (number of dopant ions per monomer unit) .

In the preferred assay method 0(x,t) , the flow of ions, is artificially maintained constant by maintaining constant current flow between the indicating electrode 3 and the auxiliary electrode 4. The electric field (and so the electric potential V) , the redox potential Vred and the dopant level Y, are constant. Figs. 6 and 7 illustrate the position where a current is flowing through the measuring solution, electron flow passing from the auxiliary electrode 4 to the indicating electrode 3. By keeping current, I, constant, 0(x,t) may be artificially maintained constant. As illustrated in Figs. 6 and 7 the formation of a bio-receptor/analyte complex on the surface of the indicating electrode 3 leads to a significant change in Donnan potential. This change of Donnan potential represents the response (02 - 01) .

The response depends upon: the concentration of negative ions in the solution; the charge on the macromolecules in solution; the concentration gradient of the negative ions in the polymer and in solution. The response can be enhanced in an amplification step by changing one or more of these conditions after formation of the bio-receptor/analyte complex. Possibilities include increasing the charge of the analyte in the solution, by adjusting the pH of the solution; increasing or decreasing the concentration of the negative ions in solution. The latter is preferred and can be achieved by removing the sensor from the test solution and placing it in a new test solution. Because 0(x,t) is artificially maintained constant by maintaining the current constant, the change of conditions is compensated by change in the term dV(x,t) dx in equation 1, that is to say by changing the potential which is proportional to the Donnan potential.

By this process, particularly where a final amplification step is employed, increased accuracy and sensitivity can be achieved for an electrochemical immunoassay.

Example 1

Using apparatus in accordance with Figure 5, samples were tested for presence of the surface antigen of hepatitis B (HBsAg) . The indicating electrode comprised a polypyrrole coated electrode with antibody to HBsAg incorporated as the bioreceptor. Fig. 11 indicates the results for a negative sample. Fig. 12 indicates the results for a sample containing a small amount of HBsAg, and Fig. 13 indicates the results for a sample containing a larger amount of HBsAg. Figs. 11 to 13 each show three sections of graph corresponding to three stages of the test. In the first stage, up to 200 seconds, the curves show the process of stabilization of the biosensor in saline phosphate buffer solution. In the second stage, from approximately 200 to 300 seconds the graphs show incubation in a solution containing samples of human serum diluted forty times. Absorption of the serum components result in shifting the potential of the electrode to a positive zone in Figures 11 and 12. The third stage, from about 300 seconds onwards, represents a stage of washing off, developing of the reaction. Figure 11 in the development stage shows that for the negative sample the potential resets approximately to the initial level. In Figure 12, in the presence of a small amount of HBsAg the potential is set to a lower potential. It should be noted that Figures 11 to 13 indicate the response, that is to say the change in potential with respect to the potential in pure measuring solution. The absolute potential difference between the reference and indicating electrode in the pre-measuring solution may be of the order of 76 mV. Figure 13 illustrates the case when there is a substantial quantity of HBsAg present in the test sample. The effect of specific binding of analyte to bio-receptor, lowering the potential, is considerably greater than the effect of non-specific binding, which raises the potential, such that a negative relative potential (i.e. positive indication of presence of analyte) is immediately obtained. Figure 14 shows a representative calibration curve for HBsAg (ng/ml) using the above-described method applied to standardised test solutions. A qualitative assessment, for example positive, negative or uncertain, can be provided by comparing results of the test against test results accumulated from samples known to be positive or negative, for example as shown in Figure 15. The analysis of the data produced by the test method can be made in any appropriate manner. Conveniently comparison data, algorithms and so forth can be stored and applied by a suitably programmed computer to output the results in any desired format. The integrated electrode assembly makes it possible to ensure that there are only minor variations in performance between different electrode assemblies so that there is no necessity individually to calibrate the electrode assemblies, although the method and apparatus may be used in such a way if required.

While in the preferred embodiment a two polymer film indicating electrode is employed integrated with an auxiliary electrode on an electrode assembly or chip, the method is applicable to indicating and auxiliary electrodes provided other than in such an assembly and to indicating electrodes with a single polymer layer. Thus in an alternative embodiment an electrode assembly comprising an auxiliary electrode and an indicating electrode is made by depositing a sub-layer of a base metal such as titanium nickel, cobalt or tungsten on a substrate and applying an over-layer of an electrically conductive material such as gold or titanium nitride on the outer surface layer thereof with subsequent removal of these layers from the surface to be free from the electrodes. An electrically conductive polymer film is formed by means of electrochemical polymerisation of a monomer dissolved in a polar solvent,. for example in the range of 0.1 to 1.0 M. The indicating electrode is placed in a solution of the monomer in a polar solvent together with a standard reference electrode, for example a silver/silver chloride or calomel metal electrode than the potential of the indicating electrode relative to the reference electrode is varied cyclically for example in the range of -1000 mV to + 2000 mV over a number of cycles. The polymer film is processed electrochemically. Bio-receptors are incorporated on the surface of the polymer film by immersing the substrate with the indicator electrode and the auxiliary electrode together with a reference electrode in a solution of the bio¬ receptor in a suitable solvent, such as water, phosphate buffer solution, acetate, borate, Tris-HCl. A constant current is set up between the indicator electrode and auxiliary electrode and the potential difference between the indicator electrode and reference electrode is measured. Immobilization of the bio-receptor will lead to a change in this potential difference, and the level of that change may be taken as indicative of the extent of absorption, absorption being completed once a target threshold level potential has been reached. The bio- receptors may be fixed on the surface of the indicator electrode by drying. Bi-functional agents such as glutar or malonic aldehyde may be used to fix the bio-receptors. To decrease non-specific responses the free surface of the polymer film after immobilization may be blocked by non¬ specific albumin, for example bovine serum albumin.

An electrochemical cell is formed by immersing the substrate with the indicator and auxiliary electrodes on it and a reference electrode in a measuring solution. Saline phosphate buffer solution may be used as the measuring solution. Surface active agents such as dextran-sulphate, ficoll, polyvinyl pyrrolidone, polyethylene glycol and albumin such as bovine or human serum albumin are added into the measuring solution to approximate the properties of the measuring solution (density, ion, strength conductivity) to those of the sample to be tested. The composition and concentration of the added agents may be varied depending on the type of sample tested (e.g. urine, blood serum, blood plasma etc.). The volume of the measuring solution in the electrochemical cell may be, for example, in the range of 100 to 500 μl .

To prevent contamination of the inner space of the reference electrode by high molecular weight elements in the measuring solution and in the sample to be tested, the inner space of the reference electrode may be separated from the measuring solution by a membrane permeable to the ions of the measuring solution, but not the larger molecules. The membrane may be made from nylon with pore diameter preferably about 0.25 mm. A constant current is established between the indicator and the auxiliary electrodes. The potential difference between the indicator electrode and the reference electrode is determined. The preset current value may be in the range of 1 nA to 1 μA. The value of the potential difference between the indicator electrode and the reference electrode is registered in pure measuring solution. The electrodes are then brought into contact with measuring solution containing a check sample of known content of the target analyte (the compliment to the bio-receptor) . The potential difference between the indicating electrode and reference electrode is determined. Depending upon the type of assay the check samples may be placed into the measuring solution or alternatively the electrodes may be transferred from the pure measuring solution to measuring solution containing pre-diluted sample. Following incubation of the indicator electrode in the check sample. the electrode is transferred to fresh measuring solution and the potential difference between the reference electrode and the indicating electrode is again determined. The change of the value of the potential difference between the reference electrode and the indicator electrode before and after incubation in the measuring solution containing check sample is related to the content of the target analyte in the check sample. The operations described above are repeated several times using check samples with different contents of target analyte. Calibration curves are built using the results obtained. The above described succession of operations is then carried out using a test sample in which the content of the target analyte is not known. The content of the target analyte in the test sample is calculated from the calibration curves.

Alternatively in this method continuous pumping of measuring solution through the electrochemical cell with check and test samples being introduced directly in the flow of the measuring solution (flow injection analysis) may be used. Example 2

A layer of chrome then a layer of gold were deposited on a rectangular substrate of cital by vacuum cathode sputtering. The total thickness of chrome and gold layers was 0.5 μm. The indicating and auxiliary electrodes were made by photolithography, locally removing the chrome and gold layers where the substrate was to be free of electrode. A polypyrrole film was deposited on the indicating electrode by electrochemical polymerisation of pyrrole in saline phosphate buffer solution at a concentration of 0.1 M (Merck, Federal Republic of Germany) in a three electrode cell comprising the indicating electrode, a teflon/platinum contra-electrode and Ag/AgCl reference electrode. Polymerization was carried out in the potentiodynamic mode, cycling the potential of the indicating electrode in a range of -800 to +800 mV at a speed of 50 mV per second.

At the end of polymerization the polypyrrole film on the indicating electrode was rinsed with deionized water and processed in potentiostatic mode at a potential of +500 mV relative to a Ag/Agcl reference electrode in a cell filled with saline phosphate buffer solution until the background current stabilized. The electrode assembly was dried in air and stored at room temperature.

A reference electrode was made by placing a silver wire coated with a layer of silver chloride in a cylinder of 2 mm diameter filled with a saturated solution of potassium chloride. The bottom end of the cylinder was closed with a nylon membrane with pore diameter 0.25 mm. The substrate with the electrode assembly and the reference electrode were placed in a reservoir filled with 150 μl of a saline phosphate buffer solution containing a suspension of monoclonal antibody HBs-antigen (Biosoft, France) in a concentration of 50 μg/ml. The indicating, auxiliary and reference electrodes were connected to an electronic measurement system incorporating an IBM compatible computer. An electric current of 100 nA was fed, the differential potential between the indicating electrode and the reference electrode being registered with time. The absorption of monoclonal antibodies on the surface of the indicating electrode was continued for 30 minutes until complete stabilization of the potential difference between the indicating electrode and the reference electrode. The substrate with the electrode assembly was taken out of the monoclonal antibody solution and dried in air.

A series of check samples containing 0.07 to 10 ng per ml Hbs-antigen were made by diluting freeze-dried Hbs-antigen with human plasma.

The electrode assembly, comprising the substrate indicating electrode and auxiliary electrode, and the reference electrode were placed in a polystyrene reservoir filled with 150 μl of saline phosphate buffer solution containing 0.1% bovine serum albumin (Sigma), 0.1% polyethyleneglycol 6000 (Serva) and 0.01% of ficoll 400 (Pharmacia) . A constant current of 100 nA was fed between the indicating and auxiliary electrode and the potential difference between the indicating and reference electrode determined.

20 μl of each check sample of known Hbs-antigen content was introduced into a reservoir filled with 150 μl of measuring solution as described above. Successive measurements were taken as described above to establish a calibration curve as shown in Fig. 14 against which results from unknown test samples could be compared.

Claims

C L A I M S

1. An electrode assembly including a polymeric electrode incorporating a bio-receptor, said assembly comprising:- a substrate of inert material; an auxiliary electrode comprising a layer of an electrically conductive material on a first part of the external surface of said substrate; an indicating electrode comprising:- a layer of an electrically conductive material on a second part of the external surface of said substrate; a first film of an electrically conductive polymer on the outer surface of the layer of electrically conductive material; a second film of an electrically conductive polymer on the outer surface of said first film, and bio-receptors incorporated into said second film of electrically conductive polymer.

2. An assembly according to claim 1, wherein the second film has a thickness of less than 10 times the molecular diameter of the bio-receptors.

3. An assembly according to claim 2 , wherein the second film has a thickness of 3 to 5 times the molecular diameter of the immobilized bio-receptors.

4. An assembly according to any preceding claim, where the indicating electrode and auxiliary electrode are separated from each other by a distance of less than 500 μm.

5. An assembly according to claim 4, where the separation is less than 100 μm.

6. An assembly according to claim 5, where the separation is 2 to lOμm.

7. An assembly according to any preceding claim, wherein the indicating and auxiliary electrodes comprise interlocking comb shapes.

8. An assembly according to any preceding claim, wherein the electrically conductive layer of the auxiliary electrode and/or indicating electrode comprises a sub-layer of a base metal, metal oxide or metal nitride and an over layer of an inert conductive material.

9. An assembly according to claim 8, wherein the sub-layer comprises titanium, nickel, cobalt, tungsten, chromium oxide, titanium oxide or titanium nitride.

10. An assembly according to claim 9, wherein the inert conductive material comprises gold, platinum or titanium nitride.

11. An assembly according to any preceding claim, wherein the electrically conductive polymer of the first and/or second film comprises polypyrrole, polythiophene polyfuran or polyaniline.

12. An assembly according to any preceding claim, wherein the electrically conductive polymer of the first and/or second film is partially oxidised.

13. An assembly according to any preceding claim, wherein the electrically conductive polymer of the first layer comprises a dopant anion which is not readily exchanged.

14. An assembly according to claim 13, wherein the dopant anion is sulphate.

15. An assembly according to any preceding claim, wherein the electrically conductive polymer of the second film comprises the dopant anions P0₄ ^2* and HP0₄ ^'.

16. A method of forming an immunosensor electrode assembly according to any preceding claim, said method comprising:- applying a layer of an electrically conductive material to the external surface of a substrate; locally removing said layer of electrically conductive material to provide an indicating electrode portion and an auxiliary electrode; applying a first film of an electrically conductive polymer to the outer surface of said indicating electrode portion by potentiodynamic polymerization of a monomer dissolved in a polar solvent; electrochemically doping said first film of electrically conductive polymer; applying a second film of an electrically conductive polymer to the outer surface of said first film by potentiodynamic polymerization from a solution comprising a second monomer and a bio-receptor to produce a film of said electrically conductive polymer and immobilized bio-receptor; electrochemically doping said second film of electrically conductive polymer.

17. A method according to claim 16, wherein the first film is formed by a potentiodynamic polymerisation of a monomer in Na₂ S0₄ solution at a concentration of 0.01 to 1 M cycling from 0 mV to 800 mV at a velocity of 50 to 100 mV/sec over 3 to 6 cycles, and electrically doped with sodium sulphate in a concentration of 0.05 M to 0.5 M and an organic detergent such as DDS; the second film is applied by a potentiodynamic polymerisation of solution comprising monomer and a bio- receptor at a potential of 0 to 800 mV at a velocity of 20 to 100 mV/sec over 2 to 4 cycles and electrochemically doped with saline phosphate buffer solution (comprising NaHP0₄, NaP0₄ and NaCl) in a concentration of 0.03 M to 0.5 M.

18. A method of detecting in a sample a biological analyte forming a binding pair with a bio- receptor by means of a three electrode cell comprising a reference electrode, an auxiliary electrode and an indicating electrode, said indicating electrode comprising a base electrode having an electrically conductive ion exchange polymer coating and bio-receptor incorporated in said polymer coating; said method comprising determining the potential of the reference electrode with respect to the indicating electrode under conditions of constant current between said indicating and said auxiliary electrodes when the three electrodes are placed in a sample-free measuring solution and when the three electrodes are placed in a sample-containing measuring solution.

19. A method according to claim 18, wherein the polymer coating comprises a first partially oxidised polymer film and a second outer partially oxidised bio- receptor containing film.

20. A method according to claim 1 or claim 2, wherein said reference electrode is separated from the measuring solution by a membrane permeable to the ions of the measuring solution, but not to macromolecular molecules.

21. A method according to any one of claims 18 to 20, wherein the potential of the reference electrode with respect to the indicating electrode in the sample- free measuring solution is determined both before and after determination of the potential of the reference electrode with respect to the indicating electrode in a sample-containing measuring solution.

22. A method according to any preceding claim, wherein after determination of the potential of the reference electrode with respect to the indicating electrode when placed in a sample-containing measuring solution, the potential of the reference electrode with respect to the indicating electrode is determined in a measuring solution comprising a lower or a higher ion concentration of ions of the measuring solutions than that of the sample-containing measuring solution, or in a measuring solution having a pH lower or higher than that of the sample-containing measuring solution.

23. A method according to any one of claims 18 to 22, wherein the auxiliary electrode and indicating electrode comprise an electrode assembly according to any one of claims 1 to 15.

24. Apparatus for carrying out the method of claim 23, comprising:- electrode assembly according to any one of claims 1 to 15; a reference electrode; means for providing constant current between the indicating electrode and the auxiliary electrode of said electrode assembly when placed in a test solution; means for determining the potential of a reference electrode placed in said solution with reference to the potential of the indicating electrode of said electrode assembly placed in said solution while a constant current is passed between said indicating electrode and said auxiliary electrode.