WO2015082910A1

WO2015082910A1 - Electrochemical sensor

Info

Publication number: WO2015082910A1
Application number: PCT/GB2014/053582
Authority: WO
Inventors: Stephen Green; Charles Peter Winlove; Paul Graham Winyard; Andrew J. Gross; Frank MARKEN; Nigel Benjamin
Original assignee: University Of Exeter; University Of Bath; South Devon Healthcare Nhs Foundation Trust
Priority date: 2013-12-04
Filing date: 2014-12-02
Publication date: 2015-06-11
Also published as: GB201321388D0

Abstract

An electrochemical sensor comprises a first working electrode (10), a second working electrode (16) and a spacer (14) holding the first and second working electrodes (10, 16) in a spaced relationship. At least one of the working electrodes (10, 16) comprises a catalyst or has a layer of catalytic material applied thereto, wherein the spacer (14) is dimensioned such that the spacing of the first and second working electrodes (10, 16) is less than about 40μm. A method of manufacture of such a sensor is also disclosed.

Description

ELECTROCHEMICAL SENSOR

This invention relates to a sensor, and in particular to an electrochemical sensor for use in sensing or detecting the presence of specific materials such as nitrate in a liquid.

Testing for the presence of nitrate within liquids is well known. For example, the presence of nitrate in blood or other biological fluids can be used as a marker for the presence of nitric oxide. Nitric oxide in blood can impact upon neurotransmitter function, and affect the regulation of blood flow which, in turn, can impact upon wound healing. However, the direct detection of the presence and amount of nitric oxide is difficult, in part because the nitric oxide molecule is highly unstable. Rapid measurement of nitric oxide levels in blood would be of assistance to medical practitioners as it would assist in the detection and diagnosis of a number of medical conditions.

Currently, nitric oxide and nitrate levels in blood cannot be measured rapidly at the point of care. Rather, blood samples have to be taken to laboratories for testing, the testing procedures being relatively lengthy and complex, and involving, for example, filtration steps to remove cellular materials therefrom. Other pre-treatment steps such as chemical derivatisation and extraction steps may also be required before the sample is fit for testing. Such pre-treatment methods are used to convert the analyte chemically to another form to make the measurement possible or because the nitrate levels within a sample are typically low compared to the level of interferents within the sample. Once the pre-treatment steps have been undertaken, the nitrate test itself can be undertaken.

A number of techniques for detecting and quantifying nitrate levels in a sample are known. By way of example, assay kits are known in which chemical processing steps are required to reduce nitrate in a sample to nitrite, and in which the presence of nitrite can then be detected. The requirement to reduce the nitrate to nitrite adds additional complexity to the process, increasing the length of time required to undertake the test. Furthermore, the reduction of the nitrate to nitrite is typically achieved using cadmium based reagents or using vanadium chloride, both of which are highly toxic. As an alternative, nitrate reductase may be used but this requires very careful handling in a controlled environment and is of reduced sensitivity, and so its use is not preferred. Other techniques involve high-performance liquid chromatography, gas chromatography-mass spectrometry, ion chromatography and capillary phosphorescence. These techniques provide very accurate results but require the use of specialist equipment and take extended periods of time to perform.

The use of electrochemical sensors in sensing or detecting the presence of a range of analytes in fluids including blood samples is well known. The use of electrochemical sensors generally is advantageous in that they can provide relatively quick test results, are of reasonably good accuracy, and they are suitable for use in a wide range of environments. These sensors include a dual-electrode type consisting of a pair of spaced sensor electrodes, one or both of which may be coated with a catalytic layer. In use, the sample is located between and in contact with the sensor electrodes and a voltage is applied across the electrodes. As a result, reactions occur resulting in the generation of an electrical current. Monitoring for the presence of such an electrical current allows the sensor to be used to detect the presence of the analyte in the sample.

It is known to use electrochemical sensors in testing for the presence of nitrate in, for example, water and aqueous solutions. However, the use of dual-electrode type sensors in this application is not known.

Whilst the use of electrochemical sensors in testing for the presence of nitrate within water or an aqueous solution is known, such sensors have not been suitable for use in testing samples of biological fluids such as blood as the presence of interferents such as oxygen, proteins, cells and micro particulates in the sample inhibits the generation of an accurate output result. Additionally, it is thought that low analyte levels and electrode fouling leading to poor electrode stability and reduced sensitivity would impair performance of such sensors in testing for the presence of nitrate in biological fluids. Whilst the description hereinbefore relates primarily to sensing or detecting the presence of nitrate in a sample, the invention is not restricted in this regard and may be used in detecting the presence of other analytes in a sample.

It is an object of the invention to provide a sensor in which at least some of the disadvantages set out hereinbefore are overcome or are of reduced impact. According to the present invention there is provided an electrochemical sensor comprising a first working electrode, a second working electrode and a spacer holding the first and second working electrodes in a spaced relationship, at least one of the working electrodes comprising a catalyst or having a layer of catalytic material applied thereto, wherein the spacer is dimensioned such that the spacing of the first and second working electrodes is less than about 40μηι.

Preferably, the spacing is in the range of about 20ηηι-20μΓΤΐ.

It will be appreciated that by having the working electrodes spaced apart by only a very small distance, upon the introduction of a sample, particles within the sample of dimensions greater than the spacing of the working electrodes will not be able to enter the volume defined between the working electrodes. As such particles are unable to enter the volume defined between the working electrodes, they cannot impact upon the operation of the sensor or the sensitivity thereof. The filtration process thus achieved negates the need to undertake pre-treatment operations such as filtering operations to remove such particles from the sample. Where the sample is a biological sample such as a blood sample, the particles may include, for example, cellular materials, proteins and the like. To restrict or prevent entry of such particles to the volume between the working electrodes, the spacing is preferably towards the lower end of the above mentioned range, being approximately equal to or smaller than the size of the particles to be restricted from entering the volume. However, where the sample is of other forms, and so the nature of the particles to be restricted from entering the volume is different, the spacing may be larger or smaller.

In one example, the working electrodes may comprise layers of gold deposited onto glass or silicon substrates. A thin metal adhesion layer such as titanium may be applied to the glass or silicon substrates to increase adhesion between the substrates and the gold layers. The spacer may comprise, for example, an epoxy resin layer used to bond the working electrodes to one another, part of the epoxy resin layer being etched away to define a volume between the working electrodes, one of the working electrodes having a catalytic material layer in the form of a silver layer applied thereto. In this example, the spacer in the form of the remaining epoxy resin layer holds the working electrodes in a spaced relationship, the epoxy resin layer thickness being such that the spacing falls within the range set out hereinbefore.

In an alternative example, the first working electrode may comprise a gold layer applied to a suitable substrate. A thin metal adhesion layer such as chromium may be applied to the substrate to increase adhesion between the substrate and the gold layer. A spacer in the form of a layer of a photoresist material is applied to the first working electrode and is exposed to ultraviolet radiation to apply a pattern to the photoresist material, for example by direct writing using an appropriately controlled laser writer or through the use of a mask. After such exposing or irradiation, the second working electrode is deposited onto the photoresist material. The second working electrode may comprise, for example, a silver electrode or a gold electrode provided with a catalytic material layer in the form of a silver layer. A developing operation is undertaken to remove the exposed or irradiated part of the photoresist layer, thereby defining a volume between the working electrodes, after which, a photoresist material layer may be applied to the second working electrode to provide support and protection thereto. It will be appreciated that in this arrangement the spacer in the form of the remaining photoresist material layer holds the working electrodes in a spaced relationship, thickness of the spacer being such that the spacing of the electrodes falls within the range set out above. As an alternative, depending upon the nature of the photoresist material, the unexposed part of the photoresist layer may be removed during the developing operation.

It will be appreciated that these two arrangements are merely two examples of arrangements in which a pair of working electrodes of an electrochemical sensor are spaced apart by only a very small distance, and that a number of other arrangements are possible without departing from the scope of the invention.

In any of the above mentioned arrangements, the sensor conveniently further includes a counter electrode and a reference electrode. In such an arrangement, the electrical potentials applied to the working electrodes give rise to reactions of the analyte at and between the electrodes. Current flow arising from these reactions as a result of the potential difference between the counter electrode and one of the working electrodes provides an indication of the presence and amount of the analyte in the sample. The reference electrode is used to ensure that the working electrodes are held at steady potentials.

The invention further relates to a method of manufacture of a sensor comprising applying a first working electrode to a suitable substrate, applying a layer of a photoresist material to the first working electrode, applying a pattern to the layer of photoresist material by exposure thereof to ultraviolet radiation, applying a second working electrode to the photoresist material, and undertaking a developing operation to remove part of the photoresist layer, thereby defining a volume between the working electrodes.

The removed part of the photoresist layer may comprise the part thereof exposed to ultraviolet radiation. Alternatively, the unexposed part thereof may be removed. The application of the pattern may be achieved, for example, by the use of an appropriately controlled laser writer or by the use of a mask or the like.

The first working electrode may comprise a gold layer or silver coated gold layer and the second working electrode may comprise a gold layer. The method may further comprise a step of applying a photoresist material layer to the second working electrode to provide support and protection thereto.

The invention will further be described, by way of example, with reference to the accompanying drawings, in which:

Figure 1 is a diagrammatic illustration of a sensor in accordance with an embodiment of the invention;

Figure 2 illustrates the sensor of Figure 1 , in use;

Figure 3 illustrates steps in the process of the manufacture of the sensor of Figure 1 ; Figure 4 illustrates an alternative design of sensor; and Figure 5 is a graph illustrating the result of a test conducted using a sensor in accordance with an embodiment of the invention.

Referring firstly to Figure 1 , an electrochemical sensor is illustrated, the sensor being intended for use in the detection of nitrate in a liquid sample, for example a biological fluid sample such as blood, urine, saliva or the like. Whilst suitable for use in testing for the presence of nitrate in a biological fluid sample, it will be appreciated that the sensor may be used in a number of other applications, for example in testing for the presence of nitrate in other types of sample, or for use in testing for the presence of other analytes in samples. It is thought that the invention may be used in testing for the presence of analytes in water samples, oil or resin samples, soil samples and the like. It could be used in testing for the presence of environmental pollutants, abused substances, therapeutic compounds and natural products such as amino acids, hormones, electrolytes and the like.

The sensor comprises a first electrode 10 in the form of a layer of deposited gold with a deposited silver or silver containing catalyst material layer 42 deposited thereon, the first working electrode 10 being deposited upon a supporting substrate 12 in the form of a glass substrate. A thin metal adhesion layer such as titanium may be applied to the glass substrate to increase adhesion between the glass and the gold. Upon the first electrode 10 is provided a spacer 14 which in this embodiment comprises a layer of a photoresist material. The spacer 14 does not entirely cover the first electrode 10 but rather only covers part thereof so that a section of the first electrode 10 is exposed. Supported by the spacer 14 is a second electrode 16 in the form of a gold layer, a supporting layer 18 of a photoresist material being provided on the surface of the second electrode 16 remote from the spacer 14 to provide support, rigidity and protection to the second electrode 16. The supporting layer 18 may also define contact pads and a sample reservoir.

As with the first electrode 10, the spacer 14 is only located adjacent a part of the second electrode 16, a section of the second electrode 16 being exposed, the exposed sections of the first and second electrodes 10, 16 being substantially aligned with one another. It will be appreciated that the exposed sections of the first and second electrodes 10, 16 are spaced apart from one another, the spacing thereof being dictated by the thickness of the spacer 14. The thickness of the spacer 14 is such that the first and second electrodes 10, 16 are spaced apart from one another by a distance sufficiently small that particles within a sample to be tested are prevented from entering a volume 20 defined between the first and second electrodes 10, 16, or that such entry is significantly restricted. Accordingly, where the sample is a blood sample, the spacing of the first and second electrodes 10, 16 is conveniently sufficiently small that microparticulates, biological microorganisms, proteins, cells and the like within the sample are unable to enter the volume 20, or that their entry is significantly restricted so that their entry does not significantly impair the operation of the sensor. Diminution of the inter-electrode space serves also to increase the electrical signal for a given analyte concentration. By way of example, it is desirable for the spacing of the electrodes 10, 16 to be less than about 40μηι, and it preferably falls within the range of 20ηηι-20μΓΤΐ. Whilst the spacing of the working electrodes is small, their surface area is relatively large, thereby providing a relatively large area over which reactions can occur in use.

As shown in Figure 3, the sensor is conveniently fabricated by thermal evaporation of gold onto a glass substrate via a thermally evaporated chromium adhesion layer. The gold surface is subsequently modified with a silver or silver containing catalyst material layer 42 to form the first working electrode 10 provided upon a supporting substrate 12. Next, a layer of positive photoresist material 22 is applied to the first working electrode 10, for example by the use of a spin coating technique. Once the photoresist material 22 has been applied, a pattern is transferred to part of the photoresist material 22 by ultraviolet radiation, either using a laser writer or by the use of a mask 24 which serves to shield part of the photoresist material 22 whilst the remainder thereof is irradiated with UV radiation.

The next stage in the process involves depositing a gold layer onto the photoresist material to form the second working electrode 16. This is conveniently achieved by the use of a thermal evaporation process. After deposition of the second working electrode 16, developing of the photoresist material results in the UV irradiated section thereof being dissolved in the developer, the remainder of the photoresist material 22 serving to form the spacer 14 which holds the first and second working electrodes 10, 16 in a spaced relationship defining the volume 20 therebetween. Depending upon the nature of the photoresist material, the developing process may remove the unexposed, rather than the exposed, irradiated part of the photoresist material layer. Finally, a protective supporting layer 18 of photoresist material is applied to the second working electrode 16 to provide protection and support thereto.

Whilst not illustrated in Figures 1 and 3, the sensor further comprises a counter electrode 24 and a reference electrode 26, the working electrodes 10, 16 and counter and reference electrodes 24, 26 all being connected to a controller or bipotentiostat 28 operable to control the voltages applied to the electrodes 10, 16, 24, 26 and to measure the flow of an electrical current between the counter electrode 24 and one of the working electrodes 10, 16, for example the first working electrode 10. The controller 28 thus applies appropriate cathodic and anodic voltages to the working electrodes 10, 16 to promote the required chemical reactions in use.

In use, as described hereinbefore, a sensor of the type described hereinbefore may be employed in testing for the presence of nitrate in a sample such as a blood sample. As shown in Figure 2, the sample 30 to be tested is located within a suitable container 32. The sensor is partially introduced into the sample 30 in such a manner that some of the sample 30 is able to flow into the volume 20 between the first and second electrodes 10, 16. As mentioned hereinbefore, the spacing of the first and second electrodes 10, 16 is small, sufficiently small that particles contained within the sample, for example red blood cells and other particles, are unable to flow into the volume 20 between the first and second electrodes 10, 16 but rather are excluded therefrom whilst other parts of the sample are able to enter the volume 20. A voltage is applied to each of the working electrodes 10, 16. The application of the voltage to the first working electrode 10, ie the silver coated electrode, is such that the silver acts as a catalyst reducing nitrate within the sample adjacent to the first working electrode 10 to nitrite. The application of the voltage to the second, gold working electrode 16 oxidises the nitrite back to nitrate. It will be appreciated that the cycle of reducing nitrate to nitrite and oxidising it back to nitrate will repeat whilst the voltages are applied to the working electrodes. The counter electrode 24 has a voltage applied thereto, and the reduction of the nitrate to nitrite at one of the electrodes and oxidation back to nitrate at the other of the electrodes results in the generation of an electrical current between the counter electrode 24 and the second working electrode 16 which can be detected by the controller 28, the size of the electrical current being representative of the level of nitrate within the sample. Whilst in the arrangement illustrated, the presence of nitrate within the sample 30 is detected by sensing the flow of an electrical current between the counter electrode 24 and the second working electrode 16, the detection of an electrical current between the counter electrode 24 and the first working electrode 10 could alternatively be used.

The reference electrode 26 has a voltage applied thereto and is used in regulation of the voltages applied to the first and second working electrodes 10, 16 to ensure that the voltages applied thereto are held substantially constant. This is important as variations in the applied voltage would impact upon the current flow and so impact upon the output of the sensor. The use of a reference electrode in this manner in connection with electrochemical sensors is well known and will not be described herein in further detail.

It will be appreciated that as the electrical current will continue to flow whilst the voltages are applied to the working electrodes 10, 16 and the above described reaction cycle is occurring, sensing for the presence of nitrate within the sample 30 can be undertaken with a good degree of reliability.

It will be appreciated that the small spacing of the working electrodes 10, 16 not only restricts the introduction of particles into the volume 20 therebetween, and so enhances measurement accuracy, but also the small spacing results in the generation of an increased current as the reaction cycle speed is increased. The increased current allows measurement sensitivity to be enhanced. Furthermore, testing can be undertaken on samples of small volume.

The sensing or testing operation can be conducted swiftly, and the process is relatively simple. Accordingly, the procedure can be carried out in a number of locations, for example at the location at which the sample has been obtained, rather than requiring the sample to be transferred to a laboratory with specialist equipment. When used for medical diagnosis, the testing can be undertaken at the point of care of the patient. Whilst in the arrangement described hereinbefore the counter and reference electrodes are separate components, if desired they may be integrated into the sensor, for example by the deposition of additional appropriate electrical conductor layers onto the substrate 12 and/or onto the photoresist layer 22 and by appropriately masking and/or exposing the photoresist layer 22 such that after developing the photoresist layer 22, parts of the additional conductor layers will be exposed and so can contact the sample, in use.

Figure 4 illustrates an alternative to the arrangement described hereinbefore with reference to Figures 1 to 3. In the arrangement of Figure 4, the first and second working electrodes 10, 16 both take the form of gold layers deposited upon suitable supporting substrates 12, 18, for example of silicon or glass form. A thin metal adhesion layer such as titanium may be applied to the glass substrate to increase adhesion between the supporting substrate and the gold. The working electrodes 10, 16 are bonded to one another by the application of a layer of an epoxy resin 40 to one of the electrodes 10, 16 and the application of the other of the electrodes 10, 16 thereto. Pressure is applied to the assembly to ensure good bonding between the electrodes 10, 16 and the epoxy resin 40, and to ensure that the layer of epoxy resin 40 is of a desired thickness approximately equal to the desired spacing of the electrodes 10, 16 whilst the epoxy resin 40 is cured. As mentioned above, the working electrodes 10, 16 are spaced apart by a distance less than about 40μηι, and the thickness of the epoxy resin layer is chosen to achieve this.

After the epoxy resin 40 is cured, part of the assembly is immersed in a suitable acid to etch away the epoxy resin 40 from the immersed part of the assembly. The remaining part of the epoxy resin 40 serves as a spacer 14, holding the working electrodes 10, 16 in a spaced relationship, spaced apart by a small distance and defining a volume 20 between the working electrodes 10, 16. After etching, the assembly is rinsed in water and a layer 42 of a silver or silver containing catalyst material is deposited upon the exposed part of the second electrode 16. By way of example, the silver layer 42 may be deposited by applying a fixed potential to the electrode at -0.2 V vs. SCE for 50s whilst the electrodes are immersed in 1 mM silver nitrate solution containing 0.1 M potassium nitrate. Subsequently, an operation is performed which removes, or strips, silver from the first electrode surface by application of an anodic potential thereto which oxidises the silver. In this manner a catalytic silver layer is deposited onto the second working electrode whilst minimising the possibility that the deposited silver short- circuits the working electrodes. The stripping step also serves as a method of disconnecting the two electrodes which can occur occasionally. This stripping step may be repeated where necessary to disconnect the two working electrodes.

It will be appreciated that this fabrication technique results in the production of a sensor comprising a pair of working electrodes 10, 16 spaced apart from one another by a sufficiently small distance that the entry of particles into the volume 20 between the working electrodes, in use, is restricted.

To demonstrate that the sensor illustrated in Figure 4 is capable of detecting for the presence of nitrate in serum the following test has been conducted. An amount of pure serum was first mixed with a proportion of phosphate buffer solution (pH 5). The tip of the double-electrode sensor with the tips of the reference and counter electrodes 24, 26 were subsequently immersed in the serum:phosphate buffer solution. A current signal was recorded (a cyclic voltammogram) by cycling the potential of the generator electrode (e.g. 0 to -1.45 V vs. SCE) whilst holding the collector electrode at a fixed potential (e.g. 0.8 V vs SCE). Care was taken to avoid the formation of bubbles which may enter the volume 20 between the working electrodes. Next, known amounts of potassium nitrate were added to the solution, and after each addition of the known amount of nitrate, a measurement was recorded. Between measurements, a pre- treatment scan was recorded (a cyclic voltammogram) by cycling the potential of the generator electrode (e.g. 0 to -1.45 V vs SCE) whilst holding the collector electrode at a fixed potential (e.g. 0.5 V vs SCE). In this test, amounts of added nitrate giving final concentrations in the range of 60 to 1440 uM were used. All current signals were measured by measuring the amplitude of the residual current and by measuring the amplitude of the diffusion limited current maximum. Figure 5 illustrates the result of the test, from which it is clear that the measured current provides an indication of the presence and concentration of nitrate in the sample.

Whilst two specific embodiments have been described, it will be appreciated that a wide range of modifications and alterations may be made thereto without departing from the scope of the invention. Whilst the description hereinbefore is of the use of the sensor in detecting for the presence of nitrate within a sample, it will be appreciated that the sensor may be modified for use in detecting for the presence of other analytes, for example by appropriate modification of the materials of the working electrodes or catalysts applied thereto. For example, modification of the electrodes with a porous or conducting polymeric material, graphene or carbon nanotube materials is envisaged. In this manner, the sensor could be modified to detect the presence of, for example, nitrite, hydrogen sulphide and redox-active proteins or other analytes.

By appropriate materials section, it may be possible to use electrodes of transparent or substantially transparent form in the sensor. Consequently, a user may be able visually inspect a sample whilst testing is ongoing. This may permit testing to be undertaken in conjunction with other tests, for example those in which a result in indicated by a change of colour.

Other modifications may be made without departing from the scope of the invention as defined by the appended claims.

Claims

CLAIMS:

1 . An electrochemical sensor comprising a first working electrode, a second working electrode and a spacer holding the first and second working electrodes in a spaced relationship, at least one of the working electrodes comprising a catalyst or having a layer of catalytic material applied thereto, wherein the spacer is dimensioned such that the spacing of the first and second working electrodes is less than about 40μηι.

2. A sensor according to Claim 1 , wherein the spacing is in the range of about 20ηηι-20μΓΤΐ.

3. A sensor according to Claim 1 or Claim 2, wherein the spacing is smaller than the average cell diameter in a biological fluid sample being tested using the sensor.

4. A sensor according to Claim 3, wherein the spacing is smaller than the average red blood cell diameter in a blood sample being tested.

5. A sensor according to any of the preceding claims, wherein the working electrodes comprise layers of gold deposited onto substrates, the spacer comprising a resin material layer serving to bond the working electrodes to one another, part of the resin material layer being etched away to define a volume between the working electrodes.

6. A sensor according to Claim 5, further comprising an adhesion layer between the substrates and the gold layers.

7. A sensor according to any of Claims 5 and 6, wherein the resin material layer comprises an epoxy resin layer.

8. A sensor according to any of Claims 1 to 4, wherein the first working electrode comprises a gold layer applied to a suitable substrate, the spacer comprising a layer of a photoresist material applied to the gold layer and supporting the second working electrode in a position spaced from the first working electrode.

9. A sensor according to Claim 8, wherein, during manufacture, the photoresist material layer is selectively exposed to ultraviolet radiation, and subsequently a developing operation is undertaken to remove part of the photoresist layer, thereby defining a volume between the working electrodes.

10. A sensor according to Claim 9, wherein the selective irradiation is achieved by the use of a laser writer or by the use of a mask.

1 1. A sensor according to Claim 9 or Claim 10, wherein the exposed part of the photoresist material layer is removed in the developing operation.

12. A sensor according to Claim 9 or Claim 10, wherein the unexposed part of the photoresist material later is removed in the developing operation.

13. A sensor according to any of Claims 8 to 12, further comprising a photoresist material layer applied to the second working electrode to provide support and protection thereto.

14. A sensor according to any of the preceding claims, wherein at least one of the working electrodes has a catalytic material layer applied thereto.

15. A sensor according to Claim 14, wherein the catalytic material layer comprises a silver or silver containing layer.

16. A sensor according to any of the preceding claims, further comprising a counter electrode and a reference electrode.

17. A method of manufacture of a sensor comprising applying a first working electrode to a suitable substrate, applying a layer of a photoresist material to the first working electrode, selectively exposing the photoresist material to ultraviolet radiation, applying a second working electrode to the photoresist material, and undertaking a developing operation to remove part of the photoresist layer, thereby defining a volume between the working electrodes.

18. A method according to Claim 17, wherein the first working electrode comprises a gold or silver coated gold layer and the second working electrode comprises a gold layer.

19. A sensor according to Claim 17 or Claim 18, wherein the exposed part of the photoresist material layer is removed in the developing operation.

20. A sensor according to Claim 17 or Claim 18, wherein the unexposed part of the photoresist material later is removed in the developing operation.

21. A method according to any of Claims 17 to 20, further comprising applying a photoresist material layer to the second working electrode to provide support and protection thereto.