A working electrode, a reference electrode, a sensor, and a method of manufacture thereof

Abstract

The present disclosure relates to an ion-selective working electrode which comprises a deposition material, the deposition material comprising at least two metal oxides. The present disclosure also relates to a reference electrode which comprises a stabilising membrane, the stabilising membrane comprising at least a first polymer layer and a second polymer layer. An electrochemical sensor is also described.

Description

[0001] A working electrode, a reference electrode, a sensor, and a method of manufacture thereof

[0002] TECHNICAL FIELD

[0003] This disclosure relates generally to the detection of chemical species dissolved in water, including but not limited to soil moisture, and particularly to a sensor for detecting dissolved nitrates, nitrites and phosphates. The invention has application in particular to the detection of nitrates, nitrites and phosphates in soil or water.

[0004] BACKGROUND

[0005] Nitrogen- and phosphorous-based fertilisers are implemented ubiquitously in agriculture, since these elements are essential nutrients for plant growth and development. To increase the efficiency of fertiliser application, it is necessary to determine the in situ, real-time quantity of nutrients, such as nitrates, nitrites, phosphates and potassium in soil and water. Applying fertiliser when and where it is needed requires the ability to measure nutrient concentrations at different depths and locations across an area. In conjunction, given that excess nitrogen run-off can be damaging to both the surrounding ecosystem (both land and marine-based) and to human health, much attention has been given to developing techniques for measuring nitrate levels in soil. Some of the current methods of measuring soil nutrient levels often include taking a soil sample, extracting, for instance, the nitrogen, and extrapolating the measurements to predict the nitrogen levels in the area from which the sample was taken. These methods present several issues: they are cumbersome, time-consuming, expensive, and often require the use of additional material, such as a reagent, to enable analysis of the sample. Such methods tend to yield inaccuracies, by virtue of the challenge of correlating the lab results to the point of soil collection, and the fact that multiple soil samples must be pooled into a single sample. Further, the results provide only a snap-shot of the nutrient levels in the soil, while a robust procedure would involve weeks of evaluation. It would thus be desirable to construct an ion-selective sensor that robustly and continuously measures at least the concentration of nutrient levels in situ.

[0006] SUMMARY

[0007] A first aspect of the present invention provides an ion-selective working electrode comprising a deposition material, the deposition material comprising at least two metal oxides.

[0008] Preferably, at least one of the metal oxides of the deposition material is cobalt oxide. Typically, the chemical formula of the cobalt oxide is C03O4.

[0009] Preferably, at least one of the metal oxides of the deposition material is copper oxide.

[0010] Optionally, the cobalt oxide comprises at least 5% of the deposition material.

[0011] Preferably, the working electrode is at least selective to nitrate ions and nitrite ions. Optionally, the working electrode may at least be selective to phosphate ions.

[0012] Preferably, the deposition material further comprises a stabilising means for enhancing the stability and lifetime of the sensor.

[0013] Typically, the stabilising means comprises a material, and preferably the material comprises reduced graphene oxide (rGO).

[0014] Preferably, the working electrode further comprising a pH-adjusting means for adjusting the pH.

[0015] Typically, the pH-adjusting means is configured to generate ions.

[0016] Optionally, the pH-adjusting means is configured to generate at least protons to lower the pH.

[0017] Optionally, the pH-adjusting means is configured to generate at least hydroxide (OH-) ions to increase the pH.

[0018] Preferably, the working electrode further comprises an interdigitated electrode, preferably wherein the interdigitated electrode comprises at least two interdigitated combs.

[0019] Preferably, at least one comb, the pH control comb, provides the pH-adjusting means. Typically, the at least one pH control comb is modified with platinum.

[0020] Typically, at least one comb, the ion-selective comb, comprises the deposition material.

[0021] Preferably, the pH control comb and the ion-selective comb are separated by an optimal distance to achieve effective pH control in the vicinity of the ion-selective comb, whilst preventing the deposition material of said ion-selective comb from interacting with the one or more pH-adjustment combs.

[0022] Typically, said optimal distance is between 0.5 and 10 microns.

[0023] Optionally, said optimal distance is 2 microns.

[0024] Typically, the thickness of the deposition material is between 0.1 and 5 microns. Preferably, the thickness of the deposition material is 1 micron.

[0025] A second aspect of the present invention provides a method for manufacturing the working electrode described in any of the statements above, comprising: fabricating, by any suitable microelectrode fabrication process, a conductive surface in the desired shape of the working electrode onto a carrier member; electrodepositing, onto the conductive surface, the deposition material. Typically, depositing the deposition material comprises mixing at least two metal ions with an electrolyte solution.

[0026] Preferably, one of the at least two metal ions comprises copper chloride.

[0027] Preferably, one of the at least two metal ions comprises a cobalt acetate salt.

[0028] Typically, the cobalt acetate salt comprises at least 5% of the electrolyte solution.

[0029] Optionally, the electrolyte comprises sulfuric acid where the working electrode is sensitive to nitrate or nitrite ions.

[0030] Optionally, the electrolyte comprises phosphoric acid where the working electrode is sensitive to phosphate ions.

[0031] Typically, depositing the deposition material comprises mixing at least one organic material with the electrolyte solution.

[0032] Typically, depositing the deposition material comprises mixing reduced graphene oxide (rGO) with the electrolyte solution.

[0033] Optionally, the electrodeposition may be carried out by cyclic voltammetry.

[0034] Optionally, the working electrode is dipped in a solution of phosphate to make the working electrode sensitive to phosphate ions.

[0035] Another aspect of the present invention provides an electrochemical sensor comprising one or more of the working electrode any of the statements above.

[0036] Typically, the measured sensor signal can be both potentiometric and amperometric.

[0037] Preferably, the electrochemical sensor further comprises at least one reference electrode and at least one counter electrode.

[0038] Preferably, the at least one counter electrode is positioned on one side of the one or more working electrodes, and the at least one reference electrodes is positioned on another side of the one or more working electrodes.

[0039] Optionally, the at least one counter electrode and at least one reference electrode run in parallel to the one or more working electrodes. Another aspect of the present invention provides a reference electrode comprising a stabilising membrane, the stabilising membrane comprising at least a first polymer layer and a second polymer layer.

[0040] Preferably, the first polymer layer comprises polypyrrole (Ppy).

[0041] Preferably, the second polymer layer comprises polyvinylchloride (PVC).

[0042] Preferably, the second polymer layer sits atop the first polymer layer on the reference electrode surface.

[0043] Typically, each of the first and second polymer layer is at least 1 micron in thickness.

[0044] Optionally, the reference electrode may be provided in an electrochemical sensor.

[0045] Another aspect of the present invention provides a method for manufacturing the above-mentioned reference electrode, the method comprising: electrodepositing, on an insulating base layer, the first polymer layer of stabilising membrane; depositing the second polymer layer of the stabilising membrane on top of the first polymer layer.

[0046] Typically, the first polymer layer is electrodeposited via cyclic voltammetry.

[0047] Optionally, the second polymer layer is dripped on top of the first polymer layer.

[0048] Typically, prior to depositing the second polymer layer onto the first polymer layer, the second polymer is dissolved in a suitable solvent.

[0049] Optionally, prior to depositing the second polymer layer onto the first polymer layer, the second polymer is mixed with a plasticiser.

[0050] Preferably, the second polymer is PVC and the solvent is tetrahydrofuran.

[0051] Optionally, the plasticiser is dinonylphthalate.

[0052] Another aspect of the present invention provides an electrochemical sensor comprising at least one of the working electrodes and at least one of the reference electrodes described above.

[0053] Typically, the electrochemical sensor further comprises at least one counter electrode.

[0054] Typically, the electrochemical sensor further comprises a carrier member of silicon. Optionally, the electrochemical sensor further comprises an insulating layer of silicon dioxide.

[0055] Typically, the sensor is comprised in a protective housing.

[0056] Optionally, said housing comprises a membrane to aid in moisture absorption from the surrounding area.

[0057] Typically, the membrane comprises at least one polysaccharide.

[0058] BRIEF DESCRIPTION OF DRAWINGS

[0059] The invention will now be further illustrated by the following description of embodiments thereof, given by way of example only with reference to the accompanying drawings, in which:

[0060] Fig. 1 is an exemplary structure of an embodiment of an ion-selective sensor;

[0061] Fig. 2 is an isometric view of an exemplary working electrode structure of the ion-selective sensor of

[0062] Fig. 1 ;

[0063] Fig. 3 is a plan view of an exemplary layout of reference, counter and working electrodes of the ion- selective sensor of Fig. 1 ;

[0064] Fig. 4 is a cross-sectional view of an exemplary reference electrode structure of the ion-selective sensor of Fig. 1 ;

[0065] Fig. 5 is a flow chart illustrating exemplary steps for manufacturing the sensor of Fig. 1 ;

[0066] Figures 6A-6K are cross-sectional views of the sensor at different steps of the manufacturing process of Fig. 5;

[0067] Fig. 7 is a flow chart illustrating exemplary steps for modification of the working electrode;

[0068] Figures 7A-7D are Scanning Electron Microscope images of working electrodes which have been modified to varying degrees;

[0069] Fig. 8A is a cyclic voltammogram of the deposition according to the modification of Fig. 7;

[0070] Fig. 8B is an Elemental X-ray Diffraction image characterising the stoichiometry of the deposition formulation of Fig. 7;

[0071] Fig. 8C is a Raman Spectrograph characterising the stoichiometry of the deposition formulation of Fig. 7;

[0072] Fig. 9A is a plan view of a working electrode before modification (left) and after modification (right) by the exemplary method of Fig. 7;

[0073] Fig. 9B is a cross-sectional view of a working electrode after modification by the exemplary steps of Fig. 7;

[0074] Fig. 9C is an exemplary structure of an embodiment of an ion-selective sensor which is selective to phosphates;

[0075] Fig. 9D is a flowchart showing the exemplary steps for modification of the working electrode, and modification of the reference electrode;

[0076] Fig. 9E is a Scanning Electron Microscope image of the modified reference electrode of Fig. 9D; Fig. 10A is a plan view of an exemplary sensor assembly;

[0077] Fig. 10B is a cross-sectional view of the sensor assembly of Fig. 10A;

[0078] Fig. 11 A is a Differential Pulse Voltammogram graph showing the response of the sensor of Figures 1-7 in a buffer sample, at various concentrations of nitrates;

[0079] Fig. 11 B is a Differential Pulse Voltammogram of the response of the sensor of step 1 of Fig. 7;

[0080] Fig. 11 C is a linear calibration plot of the results of Fig. 11 A;

[0081] Fig. 12A is a Differential Pulse Voltammogram of the response of the sensor of Figures 1-7 in a sample of artificial drinking water;

[0082] Fig. 12B is a linear calibration graph of the results of Fig. 12A;

[0083] Fig. 12C is a Differential Pulse Voltammogram of the response of the sensor of Figures 1-7 in a sample of soil leachate;

[0084] Fig. 12D is a linear calibration graph of the results of Fig. 12C;

[0085] Fig. 12E is another Differential Pulse Voltammogram of the response of the sensor of Figures 1-7 in a sample soil leachate for a range of nitrate concentrations;

[0086] Fig. 12F is a linear calibration plot of the results of Fig. 12E;

[0087] Fig. 13A is a bar chart showing the peak percentage difference in response of the sensor of Figures 1-7 to seven different interference ions;

[0088] Fig. 13B is a bar chart showing the response, in Amps, of the sensor of Figures 1-7 in the presence of a range of chlorine concentrations;

[0089] Fig. 13C is a Differential Pulse Voltammogram of the response of the sensor of Figures 1-7 to nitrate ions and nine interference ions;

[0090] Fig. 13D is a bar chart showing the response, in Amps, of the sensor of Figures 1-7 in the presence of nine interference ions;

[0091] Fig. 14 is a bar chart showing the response of the sensor according to steps 1-3 of Fig. 7 in a range of pH values;

[0092] Figures 15A and 15B are Cyclic Voltammograms of the response of the sensor of Figures 1-7 before and after reference electrode modification, respectively;

[0093] Fig. 16A is a cyclic voltammogram of the response of the sensor in a pH range of 3 to 7 in buffer solutions ranging from pH 3 to pH 7;

[0094] Fig. 16B is a linear fit of the graph of Fig. 16A, showing the measured voltage peaks versus pH;

[0095] Fig. 17 is a Differential Pulse Voltammogram showing the response of the sensor of Figures 1-7 with and without pH control;

[0096] Fig. 18 is a plot of nitrate concentration measured by the sensor of Figures 1-7 compared with that measured via lab samples over 170 days;

[0097] Fig. 19A is the response of a phosphate-selective sensor according to another aspect of the present invention, in a range of pH values of a buffer solution;

[0098] Fig. 19B is a chronoamperometry graph showing the response of the sensor of Fig. 19A in response to a range of phosphate concentrations;

[0099] Fig. 20A is the measured current versus potential of a working electrode configured to measure DO content;

[0100] Fig. 20B is a linear calibration of the results of Fig. 20A. DETAILED DESCRIPTION OF DRAWINGS

[0101] Embodiments of the present disclosure will now be described with reference to some exemplary working electrodes, reference electrodes, sensors and methods of manufacture thereof. It will be understood that the embodiments described are provided to assist in an understanding of the present disclosure and are not to be construed as limiting in any fashion. Furthermore, modules or elements that are described with reference to any one figure may be interchanged and / or combined with those of other figures or other equivalent elements without departing from the spirit of the present disclosure.

[0102] ENTIRE STRUCTURE

[0103] Figure 1 is an exemplary diagram of an ion-selective sensor 12 according to one aspect of the present invention. The sensor of Figure 1 is sensitive to at least nitrates and nitrites, but as described further below, the same sensor configuration can be employed, with slight modification of the electrode manufacture method, to be sensitive to phosphate ions.

[0104] The sensor 12 is sized such that it can be easily implemented in different environments, as well as accommodate the ultramicro / nanoscale dimensions of the sensor components. The exemplary sensor 12 comprises a carrier member 1 , onto which the sensor components are formed. Preferably, said carrier member comprises a solid surface, which may be a silicon substrate. However, those skilled in the art will appreciate that carrier member 1 may comprise any suitable conducting, semiconducting, or insulating material, such as plastic, glass or carbon.

[0105] Preferably, carrier member 1 is of micrometre dimensions, but it will be appreciated by those skilled in the art that any size of sensor may be used that accommodates the sensor components, is adapted for the measuring environment, and provides the most suitable component dimensions for optimal ion sensing, and preferably, pH control.

[0106] The illustrated embodiment shows a four-electrode configuration. In this exemplary embodiment, said four electrodes comprise at least one counter electrode 4, at least one reference electrode 3, and at least one working electrode 2. In the illustrated embodiment, each of the at least one working electrodes 2 in turn comprises at least one ion-selective sensing electrode, and at least one pH control electrode.

[0107] In the exemplary arrangement as illustrated in Figure 1 , a plurality of working electrodes 2 are provided. In the illustrated embodiment, eight working electrodes 2 are shown by way of example. In the illustrated embodiment, the sensor 12 comprises one counter electrode 4 and one reference electrode 3. However, it will be appreciated that any number of working electrodes 2, reference electrodes 3 and counter electrodes 4 may be provided. Typically, carrier member 1 comprises connection means to facilitate electrical connection with control electronics, and enable the sensor 12 to be used in a “plug and play” manner. For example, the connection means may comprise HDMI pinouts 4a, but the connection means may also be in the form of SD, USB, micro-USB-C or any other suitable mode of connection.

[0108] The one or more reference electrodes 3 are a preferred means of providing a fixed potential reference point against which the voltage / current generated by the sensing electrode is measured.

[0109] WORKING ELECTRODE STRUCTURE

[0110] The sensing elements of the sensors disclosed herein are electrochemical. Therefore, the concentration of ions in the soil may be detected using an analyte-specific formulation, which is preferably an ion-selective formulation, typically deposited over a conductive surface. The ions (for example, nitrates) interact with the ion-selective deposition, which produces a current that is proportional to the ion levels.

[0111] Typically, the conductive surface onto which the ion-selective formulation is deposited is a working electrode 2.

[0112] The signal produced by the working electrode 2 may be potentiometric or amperometric.

[0113] Each of the working electrodes 2 is provided as a two-comb interdigitated structure. However, any other suitable interdigitated configuration may be employed, for instance, the electrode arrays may comprise interdigital spirals, arranged in a torus, or as flat concentric circles, or any other suitable design.

[0114] Sensor configurations, particularly biosensor configurations, are often provided as interdigitated electrodes (IDEs). The IDE system comprises two separate electrode arrays, each resembling a comb, which are combined to form a zipper-like arrangement. This arrangement enables quicker ion diffusion by virtue of the reduced spacing between the electrodes, effecting a sensor which is capable of rapid detection with very high sensitivity. As these sensors are small and can be mass- produced, these arrangements are both robust and relatively inexpensive to manufacture, and because they are solid-state, they inherently have an enhanced shelf-life.

[0115] Figure 2 is an exemplary diagram of a layout of eight working electrodes 2, each comprising an interdigitated structure of two combs, 2a and 2b. In preferred embodiments, the ion-selective formulation is deposited on at least one of the two combs of each working electrode 2. In the illustrated embodiment, the ion-selective formulation is deposited on an ion-selective comb, 2b.

[0116] In the illustrated embodiment, each tooth of the interdigitated combs is around 60 microns in length

[0117] L, 1 micron in breadth B and 100 nanometres in height H. It will be appreciated by those skilled in the art that the dimensions described herein are provided by way of example only, and it is not intended to limit the teaching to the exemplary dimensions described. Those skilled in the art will also appreciate that the intensity of the measured signal is proportional to the dimensions of the teeth of interdigitated comb 2b. For example, the length of the combs may be smaller or larger than the example provided, but those skilled in the art will appreciate that, accordingly, the intensity of the measured signal will be lower or greater, respectively.

[0118] In the illustrated embodiments, the ion-selective formulation is nitrate- and nitrite-selective. As will be described further below, the ion-selective formulation can also be phosphate-sensitive. The ion- selective formulation comprises at least two metal oxides. In preferred embodiments, the ion- selective formulation comprises a compound which enhances the electrocatalytic activity of the sensor.

[0119] One of the metal oxides used in this embodiment is a nanocomposite of copper oxide (CuO). Copper oxide has been found to be highly effective in detecting ions, particularly nitrates and nitrites, at least at micro-molar concentrations.

[0120] The second metal oxide of this ion-selective composition is cobalt oxide (C03O4) which substantially enhances the stability of the ion-selective deposition, as it stabilises the copper. It also helps to increase the detection limit of the sensor.

[0121] Both metal oxides in this exemplary embodiment are provided, preferably, as nanoparticles deposited on a metal electrode. By using a metal oxide in a form that has nanoscale dimensions, the material has an inherently high surface area-to-volume ratio which optimises the detection capabilities.

[0122] When compared to traditional macro-electrode sensors, nanoscale electrode sensors have tremendous potential in electrochemical-based applications. Properties of these sensors include enhanced mass transport (by convergent, 3D diffusion) to the electrode, resulting in high current density and hence increased sensitivity, and a low concentration of supporting electrolytes being necessary for accurate detection. Other advantageous properties of nanoscale electrodes include low depletion of target molecules and short RC time constant. All of these characteristics improve signal to noise ratios (S / N) of the sensor, making it a highly effective ion-selective biosensor.

[0123] It has been found that the particular combination of copper and cobalt oxides yields both a greater detection limit (i.e., sensitivity) to ions, and a more uniform deposition of the formulation across the conductive surface, particularly where the two oxides are deposited together in a single deposition step. However, the formulation may be also deposited in a step-wise manner, for example, by using separate solution (one of a copper composite and another of a cobalt composite).

[0124] Alone, deposition of copper oxide is very uniform, as best illustrated in Figures 7A-7D. Figure 7A is a Scanning Electrode Microscope (SEM) image of a working electrode which has been modified with a deposition formulation comprising just copper oxide. However, it was found that modification using cobalt oxide alone results in a sub-optimum deposition, as shown in Figure 7B. Figure 7B shows (i) an SEM image of an unmodified electrode, (ii) an SEM image of an electrode which has been modified solely with cobalt oxide, and (iii) an SEM image of an electrode modified with a deposition formulation comprising both metal oxides. These images illustrate that the ion-selective formulation comprising both metal oxides was both highly sensitive and uniform in deposition.

[0125] Advantageously, the sensor comprises a stabilising means to further enhance the stability of the ion- selective deposition, by preventing the stripping of the deposition mixture from the electrode surface over repeated use. Additionally, the stabilising means enhances the conductivity of the sensor, yielding a greater sensitivity and improved signal. Preferably, the stabilising means is comprised in the deposition formulation along with the metal oxides. In the preferred embodiment described herein, the stabilising means comprises an organic material, typically with a high conductivity. The material used as the stabilising means in this embodiment is reduced graphene oxide (rGO). The inventors, through repeated experimentation, understand that the increased stability upon the addition of rGO, is due to the compound mixing with the deposition formulation. The presence of the rGO increases the adhesion of the alloy to the surface. The rGO may also act as a filler in the deposition formulation, strengthening it. It will, however, be appreciated that the addition of the rGO may also provide further technical effects which achieve enhanced stability.

[0126] Figure 7C is an SEM image of the deposition of the formulation comprising the two metal oxides and rGO on a working electrode tooth surface. Figure 7D is an SEM image of a working electrode tooth modified with this deposition formulation, between two unmodified working electrode 2 teeth.

[0127] In order to optimise the sensor for in-situ analyte level measurements, it is advantageous to locally adjust the pH of the medium being measured (for example, the soil or water), to values suitable for optimised sensing of a target analyte. Soil pH levels generally lie in a range of 6.5 to 8.5. However, for optimal sensing of nitrates, the preferred pH value is around 2-3. Therefore, the preferred embodiment of the sensing device comprises a pH-adjusting means for locally adjusting the pH to a value optimal for the analyte whose concentration is being measured. Preferably, said pH-adjusting means adjusts the pH within the immediate area of the working electrode.

[0128] In the illustrated embodiment, the pH-adjusting means comprises one of the two combs of the working electrode combs being configured to generate ions, to either increase or decrease the acidity of the surrounding environment. In the illustrated embodiment, each working electrode comprises a pH control comb 2a for adjusting the pH of the material or liquid in the vicinity of comb 2b. For instance, if the pH of the measurement environment is too high, pH control comb 2a may be configured to generate positive ions, for example, protons (H+ ions), to acidify the immediate area. If the surrounding pH is too low for optimal nitrate / nitrite detection, the pH control comb 2a may be configured to generate OH- ions to increase the pH value of the immediate area. Typically, the pH control comb comprises the same material regardless of whether it is increasing or decreasing the pH of the immediate environment. This material, preferably, comprises any material which is configured to electrolyse water. Exemplary materials include metals such as gold or platinum, semiconductors, or insulators such as carbon. However, any other suitable material may be employed, as will be appreciated by those skilled in the art.

[0129] Thus, one of the two combs of the interdigitated working electrodes 2 is configured to detect analyte levels (the ion-selective comb, 2b). The other one of the two combs may be configured to locally adjust the pH of the surrounding medium (the pH control comb, 2a).

[0130] In the illustrated embodiment, a bias voltage of around +1 ,6V is applied to the pH control comb 2a of the electrode 2, to induce the production of protons for adjusting the pH value via electrolysis. In the illustrated embodiment, a bias voltage of around -1 ,2V may be applied to the pH control comb 2a to induce the production of hydroxide (OH-) ions. However, those skilled in the art will appreciate that, depending on the reference electrode 3 used, the buffering capacity of, for example, the surrounding soil, and the configuration of the components of sensor 12 employed, any other suitable bias voltage may be applied. For instance, smaller electrodes would require greater bias voltages to induce the production of a sufficient number of ions. In general, greater bias potentials yield increased ion production. Typically, in the illustrated embodiment, at least +0.8V of bias potential may be applied to the pH control comb 2a to produce protons. In the illustrated embodiment, at least -0.55V may be applied to the pH control comb 2a to produce OH- ions. To optimise the efficiency of ion production, at least one of the pH control combs, 2a, may be modified with platinum, typically via electroplating with a platinum electrolyte solution. In preferred embodiments, the electrolyte solution comprises about 0.1 M platinum solution.

[0131] In preferred embodiments, each of the working electrodes 2 has an optimal spacing between the interdigitated combs to facilitate the adjustment of the local pH, whilst preventing the ion-selective deposition of comb 2b from “overspilling” onto (or electrically shorting to) the pH control comb 2a. This spacing accommodates the diffusional distance of ions. Optionally, the spacing between the combs may be in the range of around 0.5 to 10 micrometres. Specifically, a distance of either 1 micrometre, 2 micrometres, or 10 micrometres between the combs may be employed. In preferred embodiments, there is a spacing of 2 micrometres between the two combs, however, any suitable spacing between about 1 and 10 micrometres may be used, without compromising the detection limit of the sensor. In preferred embodiments, the thickness of the deposition layer may be between around 0.1 and 5 microns, accommodating the distance between combs 2a and 2b such that the diffusional distance is suitable. In the exemplary embodiment, the thickness of the deposition formulation is approximately 1 micron.

[0132] This preferred spacing is a compromise between these two requirements - for pH control, a close spacing in a range of 0.5-10 micrometres provides effective pH adjustment within the vicinity of the ion-selective comb; whereas a range of 2-3 micrometres between the combs provides sufficient spacing to prevent interference of the electrodeposition with the pH control comb, which maximises the pH control effect.

[0133] In the illustrated embodiment, the ion-selective comb 2b and the pH control comb 2a are separated by a distance which is greater than or equal to the diffusional distance between the combs 2a and 2b. For instance, in the exemplary embodiment, working electrodes 2 are separated by about 1 mm, but any other suitable separation may be used to allow for at least effective pH control and detection of ions.

[0134] By virtue at least of the working electrodes 2 being ultramicro / nanoscopic in scale, it is not necessary to have much, or even any, supporting electrolyte present for providing electrical contact between the working electrodes or combs thereof. In combination with this, when in use, the moisture and ionic compounds of the soil, water or the like are sufficient. This, in conjunction with the advantageous feature of the pH control comb 2a, with the optimal spacing between the combs 2a and 2b, means that the addition of reagents to the test solution is not required to enable measurement and analysis.

[0135] Those skilled in the art will appreciate that, depending on factors such as the nature of the medium being sensed, the identity of the analytes, and the dimensions and compositions of the electrodes, the spacing may be adjusted to provide a good balance between pH control and analyte sensitivity. Therefore the particular dimensions and spacings recited here may not be optimal for other applications and designs.

[0136] Advantageously, the sensor 12 may comprise detecting means for detecting other parameters of the surrounding environment. For instance, the detecting means may be configured to detect at least the pH value and / or the dissolved oxygen (DO) concentration in the immediate vicinity of the measurement environment. However, other such variables may be measured, such as CO2 concentration. Typically, the detecting means comprises the combs of the working electrodes 2 being configured to detect pH and / or DO concentration. For instance, the combs of the working electrode 2 may comprise gold oxide, or any other suitable material (such as silver, silver chloride, glass etc.). It will be apparent to those skilled in the art the type of working electrode and material thereof which may be used to measure such parameters, and that such configurations are not limited to IDEs.

[0137] The illustrated embodiment shown comprises eight working electrodes. However, any number of working electrodes may be employed. Depending on the preference of the user, all of the working electrodes may comprise an ion-selective comb 2b and pH control comb 2a (hereafter, an ionsensing working electrode). Alternatively, any number of the working electrodes 2 may comprise ionsensing working electrodes, pH detectors, and / or DO sensors.

[0138] REFERENCE ELECTRODE Low sample volumes, and the need to minimise cell resistances, means that integrated on-chip counter and reference electrodes are desirable components for electrochemical sensors. However, ultramicro / nanosensors provide enough sensitivity to detect the dissociated silver and chloride ions which diffuse from a typical Ag / AgCI reference electrode, resulting in electrochemical peaks that could interfere with the detection of key target analytes. This may preclude their use as a suitable reference material, consequently alternative quasi reference materials may be implemented, based on pure metals that would provide a stable reference voltage and are easily maintained.

[0139] Figure 3 is an exemplary diagram of the working electrodes 2, reference electrode 3, and counter electrode 4 of the carrier member 1 of Figure 1 . The sensor 12 comprises at least one reference 3 and at least one counter electrode 4.

[0140] In preferred embodiments, the reference electrode 3 is parallel with the counter 4 and working electrode(s) 2, where the at least one reference 3 and at least one counter electrode 4 are positioned at either side of the working electrode arrangement, however, any other suitable arrangement may be employed.

[0141] Figure 4 is a cross-sectional view of an exemplary structure of the reference electrode 3 of Figure 3. Typically, the reference base layer comprises quasi- reference material such that the electrode 3 does not engage in reactions but provides a surface or base on which oxidation and reduction reactions may take place. Preferably, said material comprises platinum, but may comprise any other suitable material, for example, gold. However, where a conductive reference material is used, it is preferable to have a non-reactive overcoat to prevent the occurrence of unwanted reactions.

[0142] In typical analyte-selective electrodes, the accuracy of measurements is diminished by the voltage of the reference electrodes drifting. This occurs from chemical reactions taking place at the surface of the reference electrode and the sensing material degrading. Given that the purpose of the reference electrode is to provide a constant and defined potential against which the sensor measurements are compared, this drifting is problematic, as the subsequent readings are unreliable. It would thus be desirable to provide means, in conjunction or as an alternative to the use of quasi reference materials, to prevent such voltage drift of the reference electrode.

[0143] Therefore, the reference electrode preferably comprises a stabilising means to enhance the stability of the sensor, which in turn, enhances the sensor accuracy and lifetime. A preferred stabilising means may be provided by modifying the reference electrode 3 with a stabilising membrane layer 5 which is preferably disposed over the at least one reference electrode 3.

[0144] Preferably, the stabilising membrane 5 is comprised of an inert substance. Preferably, said stabilising layer 5 comprises one or more layers of polymer. This provides improved measurement stability to the sensor, as it prevents oxidation of the reference electrode 3 surface material, for example, the formation of oxidised platinum, Pt-O. This layer also prevents the formation of Pt-H, by blocking H+ ions (protons) from coming into contact with the reference electrode 3 surface, in turn preventing unwanted electrocatalytic activity. Further, this layer, advantageously, prevents chloride ion interference, which may also negatively impact the reliability of the sensor.

[0145] Typically, the stabilising layer 5 comprises a dual-polymer layer. Preferably, the dual layer comprises a layer of polypyrrole (Ppy) 5a and a layer of polyvinyl chloride (PVC) 5b, or any other suitable polymer(s). In preferred embodiments, firstly a layer of Ppy 5a is deposited on the base layer, followed by a layer of PVC 5b.

[0146] Preferably, each polymer layer is at least around 1 micron in thickness. In preferred embodiments, the Ppy layer 5a is of thickness in the range of hundreds of nanometres to a few micrometres. In the illustrated embodiment, the Ppy layer 5a is of 1 micron in thickness. Preferably, the PVC layer 5b has a thickness in the range 1 to 10 pm, and in the illustrated embodiment, has thickness of approximately 3 microns.

[0147] Ppy is a highly effective stabiliser. When a PVC layer 5b is applied on top, this prevents ions, such as protons, from coming into contact with the under-layer of Ppy 5a, yielding a highly stable reference electrode 3, with minimal voltage drift. This is desirable in sensing applications, since voltage drift can result in false positive or false negative readings, and thus significantly diminish the longevity and reliability of the sensor. With the addition of a stabilised reference electrode 3, the sensor 12 of the present disclosure can be employed in any environment with moisture, continuously over periods in excess of 100 days. Further, since reference electrodes are predominately potentiometric in nature, the reference electrode 3 of the present disclosure is applicable to any other electrode-based sensing applications as well.

[0148] As described above, the sensor 12 of the present disclosure may be configured to detect pH values in the immediate area, and adjusts to a pH optimal for ion-selective sensing. By stabilising the reference electrode 3, and thus preventing voltage drift, the pH can be accurately measured and adjusted accordingly.

[0149] In preferred embodiments, the PVC is a low molar weight PVC, dissolved in a suitable solvent and optionally with one or more plasticisers to promote the plasticity and flexibility of the PVC layer 5b.

[0150] One example solvent is tetrahydrofuran (THF), and one example plasticiser is dinonylphthalate.

[0151] In the preferred deposition method used for this embodiment, the low molar weight PVC was dissolved in THF in a concentration of 10-30 micrograms PVC per millilitre THF, with dinonylphthalate added in the range of around 50-100 microlitres per millilitre THF. In preferred embodiments, the concentration at which the PVC was dissolved is 20pg PVC per millilitre of THF. Preferably, the concentration of dinonylphthalate is 77 microliteres per millilitre of THF. However, it will be apparent to those skilled in the art other suitable methods and concentrations for applying the dual-polymer stabilising layer.

[0152] Optionally, the stabilising membrane formulation may contain a charge-selective component, such as an ionomer, which allows the flow of positive charge through the reference membrane but inhibits movement of negative charge, such as chloride ions. Advantageously, this prevents chloride ions interfering with the nitrate-selective membrane, which would case erroneous measurement of nitrate concentrations, particularly when the soil nitrate concentrations are low.

[0153] METHOD OF MANUFACTURE

[0154] Figure 5 is a flow chart of an exemplary method for manufacturing the sensor device, prior to modification of the working electrode 2 via deposition of an ion-selective membrane. Those skilled in the art will appreciate that the following description is merely by way of example only, and that the method of manufacture may comprise any suitable microelectronics fabrication process, or otherwise.

[0155] The steps of the method of Figure 5 will be illustrated also with reference to Figures 6A to 6K which are schematic illustrations of the structure at successive steps.

[0156] Preferably, the sensor device comprises a base layer, i.e., the carrier member 1 , which in turn comprises a solid surface, for example, a silicon wafer. In typical embodiments, the base layer comprises an over-layer of inert substance, to prevent damage to the base which may occur during the sensor manufacturing process, and any unwanted reactions occurring at the surface of the sensor device. Figure 6A is an exemplary diagram of the base layer and inert substance comprising the carrier member 1 . Preferably, this inert substance comprises silicon dioxide, but may be any other suitable material which occurs to the skilled person. Typically, the over-layer comprises silicon dioxide, which may be formed via thermal oxidation, until a suitably thick layer builds. In preferred embodiments, the layer of silicon dioxide is around 300nm, step 501 , however, any other suitable thickness may be used.

[0157] In the exemplary method of Figure 5, a layer of photoresist 1a is then applied to the base layer, step 502, as shown in the exemplary diagram of Figure 6B. Typically, the photoresist layer 1a is applied on top of the layer of silicon dioxide (or other suitable substance). The photoresist 1a may be positive or negative according to the process adopted (see below). Preferably, the photoresist (for example, Microposit (RTM) S1805 (RTM)) is distributed evenly using a spin coater. In typical methods, the photoresist is spin-coated at 3000rpm for approximately 50 seconds. Preferably, the resist film is stabilised, at least by preventing delamination by enhancing adhesion. Typically, this is done by soft- baking the layer, for example, at around 115°Cfor approximately 2 minutes. Those skilled in the art, however, will be aware that any suitable parameters may be employed to apply the photoresist. A mask or template 7a is positioned over the carrier member 1 and used to pattern the areas in which the working electrodes 2 are to be manufactured, continuation of step 502. In the exemplary diagram of Figure 6C a positive photoresist 1a has been applied, and light is shone directly onto the chip, through cut-out portions of the mask or template 7a. A positive photoresist weakens, i.e., becomes soluble, at points which are subjected to (typically UV) light, meaning that the underlying base layer and inert substance (comprising the carrier member 1) are exposed at the cut-out points of the template 7a via UV lithography.

[0158] Following the steps of Figures 6A-6C, the working electrode(s) 2 are then fabricated, step 503. The mask is removed and a first metal coating 2c is blanket-deposited. Said first coating 2c comprises at least one metallic material, deposited onto the preliminary layers of the carrier member 1. In preferred embodiments, at least one metal is used as this first deposition material comprising at least interdigitated combs 2b.

[0159] Typically, the first deposition material comprises two metals, such as titanium and platinum, however, any other suitable metal or metal combination may be used.

[0160] In the exemplary embodiment, this metal deposition layer is deposited via electron beam evaporation, whereby a target material is bombarded by a beam of electrons, such that the target material (i.e., deposition material) evaporates and deposits as a layer or film on top of a substrate. However, any other suitable methods may be used.

[0161] Typically, the target material comprises at least one layer of metal. Typically, said target material comprises a layer of titanium and a layer of platinum. In preferred embodiments, the titanium layer is approximately 10nm thick, and the platinum layer is approximately 100nm thick.

[0162] Advantageously, the two or more metals mix upon electron-beam bombardment, conferring greater adhesion to the deposition layer. Optionally, a temescal FC-2000 electron beam may be used, at a suitable chamber pressure may be around 5 x 10-7Torr, but other suitable methods and parameters will be apparent to those skilled in the art.

[0163] Figure 6D is an exemplary diagram of a cross section of the sensor, with the first metal coating 2c overlaying the carrier member 1 and the remaining photoresist 1 a.

[0164] Next, step 504, any excess metal deposition and photoresist is removed, leaving just the working electrode structure(s) 8, as shown in the exemplary diagram of Figure 6E. Typically, the excess material is removed via a resist stripper, for example, resist stripper R1165 at around 90°C. However, those skilled in the art will appreciate that any suitable method and corresponding parameters may be used. Figure 6F shows the next step of the fabrication process, wherein, preferably, another layer of photoresist 1 b is applied to the carrier member 1 and first metal coating 2c. Again, as shown in the exemplary diagram of Figure 6G, a mask or template 7b is positioned over the carrier member 1 and a UV light source is directed toward the sensor from above template 7b.

[0165] Again, in the exemplary diagram of Figure 6G, a positive photoresist 1 b is applied, and the mask 7b is configured to allow the photoresist to dissolve from the working electrode depositions 2c, such that they are exposed.

[0166] Typically, masks 7a and 7b comprise patterned chrome on a quartz substrate, but any other suitable material may be used.

[0167] Those skilled in the art will recognize that the method exemplified in Figures 6A-6G can be easily adapted using a negative photoresist and a mask that is adapted accordingly.

[0168] The mask 7b is then removed, and, preferably, a second metal coating 2d is applied to the preceding layers, as shown in the exemplary diagram of Figure 6H. The purpose of this second deposition, step 505, is to produce a counter electrode(s) 4, reference electrode(s) 3 and / or the interconnection tracks between the working electrodes 2 and the other sensor components. For example, between the working electrodes and the counter electrode 4, and reference electrode 3 and pinouts 4a.

[0169] In preferred methods, said second deposition layer comprises at least one metal. Preferably, said second metal deposition layer 2d comprises three metals, namely titanium, nickel and platinum.

[0170] As with the first metal layer 2c, the second deposition coating 2d may be conducted via electron beam evaporation.

[0171] In the illustrated embodiment, the thickness proportions of the target material is around 20nm of titanium, 10Onm of nickel, and 250nm of platinum, but any other suitable proportions of metal(s) may be used. Advantageously, the presence of nickel strengthens the components, including the pinouts 4a, to prevent damage to the components as they are interconnected, and as the sensor 12 is plugged into a connector.

[0172] Typically, the excess metal deposition 2d and photoresist 1 b is again removed, as shown in Figure 6I.

[0173] Optionally, a protective layer may then be applied mainly to prevent unwanted electrochemical reactions and interaction of the sensor components upon interconnection. Figure 6J is an exemplary diagram of the sensor, where said insulating layer 8 has been applied, step 506 of Figure 5.

[0174] In typical methods, this insulating layer 8 comprises a dielectric material. Preferably, said insulating material 8 comprises silicon nitride, as it is non-porous and hydrophobic, meaning that it prevents permeation of water from the surrounding environment which may otherwise cause erroneous measurements. Further, by insulating the interconnection tracks, this significantly reduces the possibility of the appliance short-circuiting.

[0175] Typically, said insulating layer 8 is applied via chemical vapour deposition, preferably plasma- enhanced chemical vapour deposition (PECVD). For instance, 500nm of Si3N4 may be deposited using an STS PECVD system, at mixed frequency, at around 300°C and 10nm of material per minute. However, this method is given merely as an example only and any other suitable method and insulating layer may be used.

[0176] To allow sensing of analyte levels, the working 2, counter 4 and reference 3 electrodes must somehow be in electrochemical contact with the surrounding environment. In preferred methods, said contact is permitted by exposing the electrodes, i.e., by removing the insulating layer 8 from these areas, as shown in Figure 6K, step 507 of Figure 5. Typically, this is done by dry etching, which is the removal of material by plasma treatment, using a mask may to etch the areas desired.

[0177] By way of example, the dry etching may be conducted in an STS Inductively-Coupled Plasma (ICP) system at a rate of approximately 200nm per minute. Then, in order to remove the etch residue, the sensor 12 may be ashed in O2 plasma, at around 50Wfor 2 minutes. Finally, the resist may be stripped via, for instance, an R1165 resist stripper at 90°C for 10 minutes, or by any other suitable method.

[0178] Advantageously, this whole process can be conducted on a larger base layer of silicon wafer, for example, allowing a plurality of sensor devices to be produced simultaneously. The wafer may then be diced in a suitable manner to form a plurality of sensor devices, step 508.

[0179] Those skilled in the art will appreciate that this method of manufacture is given by way of example only, and is in no way limiting to the scope of the invention. For instance, rather than using optical lithography and masks for creating the pattern in the photoresist and insulating layer, the user may instead employ a screen printer, micropipette, an aerosol printer, and / or material such as kapton to pattern the desired areas, or any other suitable method of manufacture.

[0180] MODIFICATION OF THE SENSING ELECTRODE

[0181] Figure 7 is a flow chart showing how the working electrode 2 was progressively modified. The end result is a working electrode with a longer lifetime and improved sensitivity to analyte levels, namely ions, in the surrounding environment. Those skilled in the art will appreciate that the modification of the working electrode sensor 2 is the same, or at least substantially the same, for both nitrate and nitrite detection, and only the resulting measurement values differ by virtue of the differing reaction energies. The detection of nitrates by the sensor 12 is due to the basic reaction comprising the reduction of nitrates to nitrites by the stripping of oxygen, i.e., NO3 is reduced to NO2. The reaction taking place which allows nitrite detection is the reduction of nitrites to nitrogen oxide, i.e., NO2 to NO.

[0182] The following description details an exemplary method for modifying the working electrode 2 such that it is sensitive at least to nitrates and nitrites.

[0183] The first iteration, step 701 , involved the electrodeposition of an ion-selective formulation in the form of a metal oxide nanocomposite, namely copper oxide. This metal oxide is highly sensitive to specific ions.

[0184] Typically, a solution comprising an acidic electrolyte is used for the electrodeposition. Sulfuric acid (H2SO4) is a common electrolyte for the deposition of metal oxides, as it can dissociate in water, forming hydrogen ions and sulphate ions. The hydrogen ions provide the necessary charge to the metal oxide(s) so that it can be deposited on the electrode 2 surface. Further, sulfuric acid helps maintain the pH of the electrolyte solution at a suitable level for electrodeposition. To deposit the metal oxide, metal ions of the electrolyte solution react with the sulphate ions of the dissociated electrolyte to form metal sulphates, which are in turn reduced to form the metal oxides for deposition.

[0185] Preferably, the metal ions comprising the solution are copper chloride ions, yielding the copper oxide nanocomposite deposition material. Typically, in embodiments in which a single metal oxide compound comprises the ion-selective formulation, the concentration of copper chloride in the electrolyte solution is in a range of 0.05 M-0.2 M. Accordingly, the concentration of diluted sulfuric acid of the electrolyte solution is around 0.02 M-0.5 M.

[0186] The copper oxide nanoparticle modified sensor was then used for nitrate detection using differential pulse voltammetry (DPV) from a potential of -0.2V to -1.1V. However, the sensor showed less stability and a lower detection limit. The sensor also showed more chlorine interference, which is not ideal for the in-situ detection of the sensor in typical working environments.

[0187] The sensor was then modified, step 702, with solely cobalt oxide, using the cobalt acetate salt in the range of 0.01 M-0.5 M. Again, in order to perform deposition of cobalt oxide, a metal ion, namely the salt cobalt acetate, is introduced to an electrolyte solution of diluted sulfuric acid. The salt reacts with the dissociated sulphates of the sulfuric acid to form cobalt oxide (C03O4). However, the result of deposition was not desirable, as it was not covering the entire microelectrode surface.

[0188] For a more uniform deposition a solution was used with copper chloride with different concentration of cobalt acetate. The range of 0.01 M-0.5 M of both salts were taken in sulphuric acid, which led to the formation of CuO and C03O4. This resulted with better and uniform deposition on the electrode surface and was selected for further study. The deposition was also done in two ways: by using both copper and cobalt salt together, and using stepwise deposition of separate solutions of copper and cobalt. Later, the depositions were performed using a mixed solution of both salts, as this was time- convenient and yielded desirable deposition results, step 703. Copper oxide is highly sensitive to certain ions. The combination of copper oxide and cobalt oxide in the deposition formulation greatly enhances the stability of the copper, as well as the uniformity of the deposition.

[0189] In the exemplary method of Figure 7, the concentrations of the copper chloride and cobalt acetate are both in the range of 0.01 M-0.5 M in the sulfuric acid solution. Again, in the illustrated embodiment, the concentration of diluted sulfuric acid of the electrolyte solution is around 0.02 M-0.5 M. The relative proportions of the two metallic salts may take any value. Preferably, however, in order to yield a greater detection signal, copper may comprise a significant portion, if not be the predominant constituent, of the deposition formulation. In preferred embodiments, to enhance the stability of the sensor, cobalt comprises at least around 5% of the deposition mixture. By way of example, the deposition mixture may comprise 40% copper oxide, 30% cobalt oxide and 30% rGO. However, any other suitable proportions may be implemented, for instance, the concentration of the two salts may be in the ratio of 1 :1.

[0190] Preferably, the electrodeposition is carried out using cyclic voltammetry, but any other suitable deposition method may be employed. In preferred methods, for the chosen concentrations and volumes, cyclic voltammetry was conducted from -0.1 V to -0.9 V, with a scan rate of around 50mV / s.

[0191] The nitrate detection using this electrode was first done, step 704, in buffer samples of 0.1 M sodium acetate solution with a nitrate concentration range of 10 pM -100 pM. The sensor was also tested for stability by repeating the detection test with same nitrate concentration multiple times. It was found that the sensor showed a decrease in current after 30 scans, which was understood to be a result of the material stripping from the electrode surface.

[0192] If the lifetime of the sensor can be prolonged, it can be used repeatedly and still render accurate results, without the need for replacement of the sensor of component parts thereof.

[0193] Therefore, a further iteration was conducted, step 705 wherein the deposition material was improved with means for enhancing the stability of the sensor, a preferred stabilising means being reduced graphene oxide (rGO). Reduced graphene oxide was introduced into the deposition mixture prior to electrodeposition in a range of 0.1 mg / ml to 1 mg / ml. The introduction of rGO not only increased the stability of the sensor, but also its sensitivity to analyte levels, mainly by increasing the conductivity of the deposition material. Again, cyclic voltammetry was used to electrodeposit the deposition mixture, but at a range of -0.1 V to -1 .2 V, as a more negative voltage range improved the deposition of the rGO.

[0194] Figure 8A is a cyclic voltammogram showing the response of the sensor 12 in 5 mM of nitrate ions, NO3. It is useful to confirm the composition of the deposition formulation by characterisation techniques. Figures 8B and 8C show, respectively, an Elemental X-ray Diffraction (EDX) image and a Raman Spectrograph illustrating the stoichiometry of the deposition mixture. Figure 8C is composed of a Raman Spectrograph of the deposition mixture both with and without the inclusion of rGO.

[0195] Figure 9A is an exemplary diagram of a working electrode 2 prior to modification (left) and post modification (right) whereby one comb 2a of each interdigitated electrode 2 is modified with platinum for pH control, and the other 2b is modified with the ion-selective formulation for nitrate sensing. Figure 9B is a cross-sectional view of a modified working electrode.

[0196] Those skilled in the art will appreciate that this method of electrode modification is given by way of example only, and is in no way limiting the scope of the present invention.

[0197] PHOSPHATE DETECTION

[0198] Figure 9C is an exemplary diagram of a sensor 120, which is configured to sense, in particular, phosphate ions. The components and method of manufacture of sensor 120 are substantially the same as those for sensor 12, and similar elements are labelled with the same reference numerals.

[0199] Preferably, as with the nitrate-selective embodiment, 12, the ion-selective component is applied to electrode 2 via electrodeposition. Preferably, the deposition formulation comprises two or more metal oxides.

[0200] In preferred embodiments, the electrolyte solution used for electrodeposition comprises phosphoric acid. Preferably, the concentration of phosphoric acid is within the range of 0.1 M -1 M. The metal oxide deposition is produced by the reaction of one or more metal ions with the disassociated components of the phosphoric acid.

[0201] In preferred embodiments, one of the metal ions comprises copper chloride, and the other cobalt acetate. As indicated previously, the copper component of the deposition confers a particularly high sensitivity to ions. The cobalt component enhances the stability of the deposition. Any suitable ratio of these components may be implemented. However, those skilled in the art will appreciate that a greater proportion of copper yields a greater detection signal. Thus, preferably, the copper component comprises a significant portion of the deposition formulation, and may be the predominant constituent. In preferred embodiments, cobalt comprises at least around 5% of the deposition formulation. In the exemplary method, the concentration range of each metal ion is 0.01 M-0.5 M. In the illustrated embodiment, the ratio of the metal oxides is 1 :1 . However, those skilled in the art will appreciate that these values are used merely by way of example only, and are in no way intended to limit the scope of the invention. Other ratios may be employed, for instance 3:2 copper oxide to cobalt oxide. In the illustrated embodiment, cyclic voltammetry is conducted from -0.2 V to -1 .2 V. As an additional step, the sensor is dipped into a solution of phosphate, of approximate concentration 0.1 M, for around 1 hour before being implemented for phosphate sensing. This step induces reactions in the deposition mixture which confer a high sensitivity to phosphate ions.

[0202] Like the deposition formulation used for the nitrate / nitrite sensor, said mixture for the phosphate sensor is a composite of CuO and C03O4. Said deposition formulation for the phosphate sensor may optionally also comprise rGO.

[0203] As with the working electrodes 2 of the nitrate-Znitrite-selective sensor, the phosphate-selective working electrode 2 also, preferably, comprises two combs - one for sensing phosphates, (ion- selective comb 2b) and one for adjusting the pH of the immediate environment, (pH control comb 2a). Comb 2a preferably adjusts the pH to around 4, which is optimal for phosphate sensing.

[0204] Optionally, sensor 12 may be configured to detect both nitrates / nitrites and phosphate ions simultaneously. For example, one or more of the working electrodes 2 may be configured to detect phosphates, and one or more of the remaining working electrodes 2 may be configured to detect nitrates / nitrites.

[0205] MODIFICATION OF REFERENCE ELECTRODE

[0206] Referring again to Figure 4, as described previously, the reference electrode 3 typically comprises a dual-polymer layer 5 for enhancing the accuracy, stability and lifetime of the sensor. Typically, the aforementioned two or more polymers comprise at least poly-pyrrole (Ppy). Typically, the two or more polymers comprise at least the polyvinyl chloride (PVC). In preferred methods, firstly a layer 5a of Ppy is deposited, followed by a layer 5b of PVC. Preferably, each polymer layer is at least around 1 micron in thickness. In preferred embodiments, the Ppy layer 5a is of thickness in the range of hundreds of nanometres to a few micrometres. In the illustrated embodiment, the Ppy layer 5a is of 1 micron in thickness. Typically, the PVC layer 5b has a thickness in the range 1 to 10 pm, and in the illustrated embodiment its thickness is approximately 3 microns. In typical methods, the Ppy deposition was conducted using cyclic voltammetry, usually in a range of 0V to 1 ,2V and a scan rate of around 50mV / s for one cycle. As detailed above, preferably, the PVC is mixed in a solution of THF and dripped onto the carrier member 1 . Figure 9D is a flowchart showing the exemplary steps of modification of the working electrode(s) 2 and modification of the reference electrode 3. Figure 9E is an SEM image showing the morphology of the modified reference electrode.

[0207] SYSTEM AND IMPLEMENTATION

[0208] The sensing assembly typically comprises an optimising means to optimise the sensor for in-situ measurements. Preferably, the optimising means is configured to absorb moisture from the surrounding area, for example, soil, and transfer the moisture to the sensing component. Typically, said means comprises a hygroscopic material. Preferably, said hygroscopic material comprises one or more polysaccharides, but may comprise any other material suitable for moisture absorption. In preferred embodiments, said hygroscopic comprises chitosan.

[0209] Preferably, said hygroscopic material further comprises polymeric carbohydrates agarose, optionally of around 1-2% weight per volume.

[0210] Alternatively, said membrane may comprise a polymer like polyvinyl alcohol, cellulose acetate, graphene aerogel, at any suitable concentration.

[0211] In preferred embodiments, the ratio of polymeric carbohydrate agarose to chitosan is approximately 1 :1 to 1 :2.

[0212] In a preferred method, said one or more polysaccharides is stirred in acetic acid, preferably at around 80°C, forming a gel-like substance. Those skilled in the art will appreciate, however, that any other suitable methods of manufacture may be used. The gel is then poured into, for example, a cuboidal template or the like, and allowed to dry, forming a membrane 9 (Figures 10A and 10B) which accompanies the sensor 12 when the sensor assembly is in use.

[0213] By using this hygroscopic material, not only do soil nutrient measurements become more efficient and accurate, but the sensor components are also safeguarded against abrasions caused by stones and other objects in the surrounding area.

[0214] Typically, the sensor assembly comprises additional means of protecting the sensor components when in use. In preferred embodiments, the sensor is contained within a housing assembly 10 before implementation. Preferably, said housing 10 is cuboidal, but may take any other suitable shape, and encapsulates the sensor 12 and hygroscopic membrane.

[0215] Figures 10A and 10B show, respectively, plan and cross-sectional views of the housing assembly 10 with the sensor contained therein. Preferably, said housing comprises insulating material. The housing 10 typically is a 3D-printed enclosure and may comprise for example, polymer coatings such as polypropylene or epoxy resin, or rubber, or any other suitable insulating and moisture-proof material.

[0216] Typically, said housing 10 comprises a receiving means for providing electrochemical contact between the soil and the sensing electrode. Preferably, the receiving means comprises a recess for receiving and holding a soil sample. Typically, the receiving means comprises a soil port 11 located directly above the hygroscopic membrane, which in turn is positioned over the sensing electrode. The sensor may be inserted into the soil at any depth suitable for the direct measurement of soil nutrient concentrations. One sensor assembly may be repeatedly inserted into the soil at different depths to build a profile of nutrient concentration with depth and time. Alternatively, several different sensor assemblies may be inserted into the soil at different depths in the same measurement window.

[0217] Further, the sensor of this disclosure may be easily implemented as an array of sensors distributed across a large area, each having the ability to continuously monitor nitrate concentrations. This may be done by, for example, a multisystem sensor and Analog Front-End (AFE) electronics. This implementation is highly desirable since fertiliser is typically applied to fields by spraying, and upon seeping into the soil, there is a high degree of unpredictability as to the soil’s nutrient profile both spatially and temporally - the ions in the fertilisers may undergo binding with other soil nutrients, and may be unevenly absorbed by the random distribution of roots etc.. Typically, the sensor 12 measures the nitrate / nitrite concentration in molar / L, and by using the measured moisture content, these values can be converted into, for example, kg per hectare.

[0218] Optionally, the sensor assembly disclosed herein, or the system in which it is comprised may further comprise means for measuring the dissolved oxygen content and / or the pH of the soil or surrounding environment. This may involve, for example, one or more of the working electrodes being configured to detect such parameters, said configuration being known by those skilled in the art.

[0219] It will be appreciated by those skilled in the art that the sensor described heretofore has been described for detecting ions in soil moisture. It is not intended to limit the present disclosure to detecting ions in soil, and there are alternative applications of the sensor, namely in any environment which retains or consists of moisture, for example blood, and monitor the ion levels in said environments.

[0220] RESULT ANALYSIS

[0221] In preferred methods, the sensing of nitrate ions is done by Differential Pulse Voltammetry (DPV). DPV is an effective electroanalytical method as it minimises the effect of the charging current (non- Faradaic current) such that only the Faradaic current is measured, yielding more accurate measurements of very low concentrations.

[0222] Figure 11 A is a DPV graph illustrating the measured current in a buffer sample across a spectrum of nitrate ion concentrations (ranging from 0 to 1 mM) in response to a potential difference applied within the range of -0.2V to -1.1V.

[0223] To highlight the improved detection limit of the sensor upon addition of the second metal oxide (cobalt oxide), Figure 11 B shows a DPV graph of the detection of nitrates of concentration range 1 pM to 100pM, where the working electrode(s) 2 has been modified with a layer of copper oxide only. It is clear that, for example, for a 100pM concentration of NO3, the detection limit is improved by around two orders of magnitude with the addition of cobalt oxide to the deposition mixture.

[0224] Figure 11 C is a linear calibration plot of the measured current versus concentration of nitrate ion NO3 of Figure 11 A, from which the detection limit (i.e., the lowest concentration of a target analyte that can be detected) of the sensor can be determined using the following formula:

[0225] Detection limit = 3.3(standard deviation of the response / gradient of the slope)

[0226] In the concentration range of 10pM to 10OpM, the detection limit was calculated to be about 0.8pM. Plots such as that shown in figure 11 C can be used in subsequent readings to determine the concentration of nitrates / nitrites from the measured current. For example, the gradient of this plot can be measured, and using the equation y = mx + c, where y is the current, x is the nitrate / nitrite concentration, c is the intercept and m is the gradient, one can rearrange and solve for x (the concentration). Those skilled in the art will note however that the signal produced by the working electrode 2 may be potentiometric or amperometric, and the readings interpreted accordingly.

[0227] Figure 12A is a DPV graph illustrating the response of the sensor in samples of artificial drinking water. Figure 12B is a linear calibration graph of the graph of Figure 12A, showing current versus the NO3 concentration.

[0228] 12C is a DPV graph illustrating the response of the sensor 12 in a sample of 2mM soil leachate, and Figure 12D is a linear calibration of Figure 12C.

[0229] Figure 12E again shows the response of the sensor to various concentrations of nitrate ions ranging from 2 - 62 ppm (or 0.025 - 0.2mM), and Figure 12F is a linear calibration of the graph of Figure 12E.

[0230] To further test the capability of the sensor, and to simulate the in-situ conditions of a typical working environment, an interference study was conducted. Figure 13A illustrates the results of placing the sensor in water samples which contained different “interference” ions (i.e., ions which may interfere with the nitrate measurements). The “percentage peak difference” on the y-axis is the difference in the measured concentration of nitrate ions between each interference ion. The second peak of Figure 13A is a reference point, 100%, set by the detection of purely nitrate ions for comparison.

[0231] The ions chosen were Na+, Ca+, Cu+, Fe2+, K+, Mg2+, and SO4-. Each interfere nt was mixed in a water sample containing nitrate ions, where both the nitrate ions and interferent are each at a concentration of 1 mM, which is the upper limit of the typical concentration in agricultural or natural environments As shown in Figure 13A, there is little deviation between the optimal detection limit, with the calcium ions causing the most interference, but still yielding a detection limit of over 80% of the optimal result expected for a solution of nitrate ions only. Evidently, the sensor remains highly selective even in high concentrations of interferents.

[0232] Chlorine is one of the main constituents of soil and natural bodies of water. Thus, the interference of chlorine ions in the measurement of nitrate / phosphate concentration is also a point of interest, and a particular source of inaccuracies with many nitrate sensors. Figure 13B is a plot of the measured current, corresponding to nitrate levels in water, against varying concentrations of chlorine in a range of OmM to 20mM. This plot demonstrates that up to around 5mM of chlorine, the sensor maintained its stability of measurement, and was largely insensitive to the presence of chlorine. Beyond about 10mM, the interference of chlorine in the measurements becomes more prominent for a solution of water with 100pM of nitrate ions.

[0233] Figures 13C and 13D further illustrate the performance of the sensor in the presence of interference ions. Here the sensor tested comprised working electrodes which were modified with copper oxide and cobalt oxide only (i.e., excluding reduced graphene oxide). Figure 13C is a DPV graph of the response of the sensor in the presence of nitrate ions and interference ions, where it is clear that the most prominent response is that to nitrate ions NO3. This is further elucidated in the bar chart of Figure 13D, which shows the response of the sensor for the nitrate ions is much stronger than that for each interfering ion. The interferents used for this experiment are Na+, Ca+, Cu+, Fe2+, K+, Mg2+, PO-, CI-, NO2-, and NH3, each at a concentration of 1 mM.

[0234] Figure 14 exemplifies the importance of optimising the pH of the surrounding environment for nitrate detection. As described above, at least one comb of each of the at least one working electrodes comprises pH-adjusting means for adjusting the pH in the immediate area. Usually, in soil, the pH is around 6.5-8.5, but a more acidic environment is ideal for nitrate ion detection. Figure 14 shows a plot of the measured current, indicative of nitrate concentration, versus the pH of a 0.1 M solution of sodium sulphate (Na2SC>4) containing 25mM of nitrate ions. The working electrode 2 of the sensor employed for this study was modified with the copper oxide and cobalt oxide composite described previously (i.e., excluding rGO). Evidently, a pH of 2 is most conducive to the accurate detection of nitrates.

[0235] Figures 15A and 15B are cyclic voltammograms showing the response of the sensor in a 1 mM ferrocene monocarboxylic acid solution, before and after reference electrode 3 modification, respectively. These plots clearly illustrate the effect of a stabilising, dual-polymer layer on the stability of the reference electrode, whereby over 500 scans, there is a significant reduction in peak drift after modification of the reference electrode (Figure 15B). This is a highly desirable effect, as the results of ion sensitivity are much more reliable.

[0236] Figure 16A is a cyclic voltammetry graph of the response of the sensor 12 in a buffer solution ranging from pH 3 to pH 7, which simply highlights the stability of the reference electrode, meaning the sensor is able to read the pH in a very similar manner to a sensor which incorporates a commercial reference electrode. Figure 16B is a linear calibration graph of the response, which plots the voltage value of the minima peaks of Figure 16A versus the buffer solution pH values, demonstrating that the measured voltage decreases with increasing pH, and hence the benefit of the pH-adjustment comb 2a.

[0237] Finally, Figure 17 is a DPV graph illustrating the response of the sensor 12 with and without pH control. For these readings, the sensor was inserted into a soil pot and the resulting signal continuously measured. While the concentration of nitrates did not vary significantly, this figure demonstrates that in the absence of pH control, whereby the pH of the soil is around neutral, the sensor signal is weak. However, with pH control, which lowers the pH to 2-3, the peaks are clearly evident and the nitrate concentration can be deduced.

[0238] It is desirable that the reference electrode serves as a reliable, stable potential reference point against which the potential of the working electrode(s) 2 is measured. If the reference electrode is not stable, it can cause the cyclic voltammetry peaks to drift as the potential of the reference electrode changes overtime. This in turn leads to changes in potential of the working electrode(s) 2, and means the electrochemical behaviour of the system under study cannot be accurately analysed.

[0239] A common cause of electrode instability is the presence of dissolved oxygen in the electrolyte solution or surrounding environment (for example, the soil). The dissolved oxygen can react with the electrode surface and cause changes in potential.

[0240] Another factor which can cause instability is the degradation of the reference electrode overtime. As described above, the stability of the reference electrode, which preferably comprises platinum, may be improved by the deposition of a dual-polymer layer, preferably a layer of Ppy 5a followed by a layer of PVC 5b. These layers prevent both the formation of Pt-O and Pt-H, and the degradation of the electrode.

[0241] Figure 18 is a plot of nitrate concentration as measured continuously by the sensor 12 of the present disclosure, and that measured when sampling from the same area periodically and analysing the results in a lab. Evidently, the nitrate sensor of the present disclosure can measure the fluctuations in ion levels robustly and continuously, capturing key stages of agricultural processes and providing live data feedback of the results. For instance, large troughs and peaks for example, as a result of fertilising the soil, or irrigation, may be observed which would otherwise go undetected. This presents a notable advantage over the measurements of a standard ion-selective sensor, which must be inserted, removed and re-inserted several times periodically over the sampling period, and fails to pick up on the agricultural processes which significantly affect ion concentration levels. Further, as shown in Figure 18, the concentration of nitrates may be continuously measured over around 170 days without the need for replacement, significantly reducing the time and energy associated with period removal and replacement of a typical sensor. As described above, embodiments of the sensor comprise modification of the working electrode(s) 2 to be sensitive to phosphates. Figure 19A shows the response of the sensor in a range of pH values from 2-6 in a sodium acetate buffer solution. The phosphate sensor described herein responds optimally in an environment of pH 4. In preferred methods, the detection of phosphates is done by an analytical technique known as chronoamperometry, whereby the electric potential of the working electrode(s) is stepped, and the resulting current from faradaic processes occurring at the electrode is recorded as a function of time. Figure 19B shows the measured current as a function of time in response to phosphate concentrations ranging from 10nM to 300nM of pH 4, upon application of a potential of 0.3V for 200 seconds. The last 10 seconds of application were extracted for the plot, as this is when the reading stabilised, and from this it is clear the sensor may be easily adapted to the detection of phosphates as well.

[0242] Figure 20A is the measured current versus potential of a working electrode 2 configured to measure DO content. Figure 20B is a linear calibration of the results of Figure 20A.

[0243] The invention is not limited to the embodiment(s) described herein but can be amended or modified without departing from the scope of the present disclosure. Additionally, it will be appreciated that in embodiments of the present disclosure some of the above-described steps may be omitted and / or performed in an order other than that described.

[0244] Similarly the words comprises / comprising when used in the specification are used to specify the presence of stated features, integers, steps or components but do not preclude the presence or addition of one or more additional features, integers, steps, components or groups thereof.

Claims

CLAIMS1 . An ion-selective working electrode comprising a deposition material, the deposition material comprising at least two metal oxides.

2. The working electrode of claim 1 , wherein at least one of the metal oxides comprises copper oxide.

3. The working electrode of claim 1 or 2, wherein at least one of the metal oxides comprises cobalt oxide.

4. The working electrode of claim 3, wherein the chemical formula of cobalt oxide is C03O4.

5. The working electrode of claims 3 or 4, wherein the cobalt oxide comprises at least 5% of the deposition material.

6. The working electrode of any preceding claim, wherein the working electrode is at least selective to nitrate ions.

7. The working electrode of any preceding claim, wherein the working electrode is at least selective to nitrite ions.

8. The working electrode of any preceding claim, wherein the working electrode is at least selective to phosphate ions.

9. The working electrode of any preceding claim, wherein the deposition material further comprises a stabilising means for enhancing the stability and lifetime of the sensor.

10. The working electrode of claim 9, wherein the stabilising means comprises a material.11 . The working electrode of claim 10, wherein the material comprises reduced graphene oxide (rGO).

12. The working electrode of any preceding claim, further comprising a pH-adjusting means for locally adjusting the pH of a medium.

13. The working electrode of claim 12, wherein the pH-adjusting means is configured to generate ions.

14. The working electrode of any of claims 12 and 13, wherein the pH-adjusting means is configured to generate at least protons to lower the pH of the medium.

15. The working electrode of any of claims 12 to 14, wherein the pH-adjusting means is configured to generate at least hydroxide (OH-) ions to increase the pH of the medium.

16. The working electrode of any preceding claim, further comprising an interdigitated electrode.

17. The working electrode of claim 16, wherein the interdigitated electrode comprises at least two interdigitated combs.

18. The working electrode of claim 17, when dependent on any of claims 12 to 15, wherein at least one of the interdigitated combs is a pH control comb configured to provide the pH- adjusting means.

19. The working electrode of any of claims 17 or 18, wherein at least one of the interdigitated combs comprises an ion-selective comb comprising the deposition material.

20. The working electrode of claim 18, or of claim 19 when dependent on claim 18, wherein the at least one pH control comb is modified with platinum.21 . The working electrode of any of claims 18 to 20, wherein the at least one pH control comb and the at least one ion-selective comb are separated by an optimal distance to achieve effective pH control in the vicinity of the ion-selective comb, whilst preventing the deposition material of said ion-selective comb from interacting with the one or more pH-adjustment combs.

22. The working electrode of claim 21 , wherein said optimal distance is between 0.5 microns and 10 microns.

23. The working electrode of any of claims 21 and 22, wherein said optimal distance is 2 microns.

24. The working electrode of any preceding claim, wherein the thickness of the deposition material is between 0.1 microns and 5 microns.

25. The working electrode of claim 24, when dependent on claim 23, wherein the thickness of the deposition material is 1 micron.

26. A method for manufacturing the working electrode as claimed in any one of claims 1 to 25, the method comprising: fabricating, by any suitable microelectrode fabrication process, a conductive surface in the desired shape of the working electrode onto a carrier member; electrodepositing, onto the conductive surface, the deposition material.

27. The method of claim 26, wherein depositing the deposition material comprises mixing at least two metal ions with an electrolyte solution.

28. The method of claim 27, wherein one of the at least two metal ions comprises copper chloride.

29. The method of any of claims 27 and 28, wherein one of the at least two metal ions comprises a cobalt acetate salt.

30. The method of claim 29, wherein the cobalt acetate salt comprises at least 5% of the electrolyte solution.31 . The method of any of claims 27 to 30, wherein the electrolyte comprises sulfuric acid where the working electrode is sensitive to nitrate or nitrite ions.

32. The method of any of claims 27 to 30, wherein the electrolyte comprises phosphoric acid where the working electrode is sensitive to phosphate ions.

33. The method of any of claims 27 to 31 , wherein depositing the deposition material comprises mixing at least one organic material with the electrolyte solution.

34. The method of claim 33, wherein depositing the deposition material comprises mixing reduced graphene oxide (rGO) with the electrolyte solution.

35. The method of any of claims 26 to 34, wherein the electrodeposition is carried out by cyclic voltammetry.

36. The method of any of claims 26 to 35, wherein the working electrode is dipped in a solution of phosphate to make the working electrode sensitive to phosphate ions.

37. An electrochemical sensor comprising one or more of the working electrode of claim 1 .

38. The electrochemical sensor of claim 37, wherein the sensor signal can be both potentiometric and amperometric.

39. The electrochemical sensor of any of claims 37 and 38, further comprising at least one reference electrode and at least one counter electrode.

40. The electrochemical sensor of claim 39, wherein the at least one counter electrode is positioned on one side of the one or more working electrodes, and the at least one reference electrodes is positioned on another side of the one or more working electrodes.41 . The electrochemical sensor of any of claims 39 and 40, wherein the at least one counter electrode and at least one reference electrode run in parallel to the one or more working electrodes.

42. A reference electrode comprising a stabilising membrane, the stabilising membrane comprising at least a first polymer layer and a second polymer layer.

43. The reference electrode of claim 42, wherein the first polymer layer comprises polypyrrole (Ppy).

44. The reference electrode of any of claims 42 and 43, wherein the second polymer layer comprises polyvinylchloride (PVC).

45. The reference electrode of any of claims 42 to 44, wherein the second polymer layer sits atop the first polymer layer on the reference electrode surface.

46. The reference electrode of any of claims 42 to 45, wherein each of the first and second polymer layer is at least 1 micron in thickness.

47. The reference electrode of any of claims 42 to 46, wherein the reference electrode is provided in an electrochemical sensor.

48. A method for manufacturing the reference electrode of any of claims 42 to 47, the method comprising: electrodepositing, on an insulating base layer, the first polymer layer of stabilising membrane; depositing the second polymer layer of the stabilising membrane on top of the first polymer layer.

49. The method of claim 48, wherein the first polymer layer is electrodeposited via cyclic voltammetry.

50. The method of any of claims 48 to 49, wherein the second polymer layer is dripped on top of the first polymer layer.51 . The method of any of claims 48 to 50, wherein, prior to depositing the second polymer layer onto the first polymer layer, the second polymer is dissolved in a suitable solvent.

52. The method of any of claims 48 to 51 , wherein, prior to depositing the second polymer layer onto the first polymer layer, the second polymer is mixed with a plasticiser.

53. The method of claim 51 , wherein the second polymer is PVC and the solvent is tetrahydro furan.

54. The method of claim 52, wherein the plasticiser is dinonylphthalate.

55. An electrochemical sensor comprising at least one of the working electrodes of any of claims 1 to 25 and at least one of the reference electrodes of any of claims 42 to 47.

56. The electrochemical sensor of claim 55, further comprising at least one counter electrode.

57. The electrochemical sensor of any of claims 55 and 56, further comprising a carrier member of silicon.

58. The electrochemical sensor of any of claims 55 to 57, further comprising an insulating layer of silicon dioxide.

59. The electrochemical sensor of any of claims 55 to 58, wherein the sensor is comprised in a protective housing.

60. The electrochemical sensor of claim 59, wherein said housing comprises a membrane to aid in moisture absorption from the surrounding area.61 . The electrochemical sensor of claim 60, wherein the membrane comprises at least one polysaccharide.

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