Soil carbon sensing methodology and electrode arrangement

Abstract

Provided is an electrode arrangement configured for detecting humic and fulvic acids in a soil matrix by means of squarewave voltammetry. The electrode arrangement comprises a working electrode with a coating comprising an admixture of ionic liquid 1-butyl-3- methylimidazolium tetrafluoroborate (BMIM-BF4) and sulfonated tetrafluoroethylene-based fluoropolymer-copolymer - sulfur trioxide (SO3) at a molar ratio of 1:1, sulfonated tetrafluoroethylene-based fluoropolymer-copolymer to carbon particles at a mass ratio in a range of between 1:10 and 1:15, sulfonated tetrafluoroethylene-based fluoropolymer-copolymer to polymeric adsorbent non-ionic aliphatic acrylic resin at a mass ratio in a range of between of 1:50 and 1:60, and a cross-linked hydrogel.

Description

SOIL CARBON SENSING METHODOLOGY AND ELECTRODE ARRANGEMENTTECHNICAL FIELD

[0001] This invention relates broadly to the field of soil carbon sensing, and more particularly to a soil carbon sens ing methodology and associated electrode arrangement configured for detecting humic and fulvic acids in a soil matrix by means of squarewave voltammetry .BACKGROUND ART

[0002] The following discussion of the background art is intended to facilitate an understanding of the present invention only . The discus sion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application .

[0003] In general , soil carbon consists of both minerali zed carbonates and organic carbon; soil organic carbon typically represents 2 to 10 wt . % of the total soil mass . In semi-arid and arid environments , soil inorganic carbon can represent a very signi ficant component of overall soil carbon . Soil organic carbon is important for water retention, ion-exchange capabilities and nutrient processing as well as other benefits .

[0004] The pool of organic carbon largely consists of living organic matter and the degradation products of either animal and / or plant matter . Particulate organic carbon is considerably more stable - some forms of humus can persist for decades while organic material like charcoal can persist for hundreds of years . On the other hand, inorganic carbon can persist for thousands of years i f not longer . Microbial activity is correlated with more labile carbon which may degrade over a period of years while mycorrhi zal fungi are associated with more stable carbon particulates ( oraggregates) which may persistent for hundreds or thousands of years .

[0005] Humus plays a role in stabilizing these carbon particulates; a combination of esterif ication / amidation-based cross-linking mechanisms have been proposed as a mechanism for this process. Oxidative coupling and cation-mediated crosslinking have also been proposed. Though there is some debate on their exact origin and chemical structure, humic (soluble under alkaline conditions, medium molecular weight) and fulvic (soluble under all pH conditions, low molecular weight) acids are components of humus and are broadly indicative of the chemical species present in agricultural soils. These chemical species broadly represent the water-soluble components of soil organic carbon are largely responsible for the nutrient retention, moisture capacity and ionexchange capabilities of healthy soils. While not representative of the entire soil carbon pool, the presence of these soluble polar compounds in soil is generally attributed to being productive and healthy .

[0006] Humic / fulvic acid and other water-soluble compounds (e.g. carboxylic acid and sugars) can undergo electrochemical processes in water (i.e. chemical reduction and oxidation of chemical moieties that are characteristic of these compounds) . As a result, chemical sensors predicated on these electrochemical processes can be developed that are capable of in situ detection of these species. Applicant has developed such broad soil carbon sensing technology, as described in International Patent Application no. PCT / AU2023 / 050676.

[0007] Such electrochemical sensors can be relatively inexpensive due to the low-cost nature of the required electrical systems and the associated coated electrodes, potentially enabling cost-effective and long-term continuous deployment at a relativelyhigh spatial density. The coating of such electrodes forms the basis for bringing the relevant chemical species to the electrode surface from the soil such that they can be detected using electrochemical measurement techniques. Alternatively, with the advent of mid-IR lasers and miniaturized detectors, it is possible to undertake Fourier-transform infrared spectroscopy (FTIR) analysis of soil samples in the field, where specific absorbance bands can be attributed to various functional groups that comprise fulvic / humic acids. However, this approach remains quite expensive relative to most electrochemical approaches. Furthermore, in situ detection is difficult due to fouling of sensor optics and the penetration depth of the IR radiation into a soil sample is quite low .

[0008] Additionally, the presence of water complicates soil spectral analysis. Different correction methodologies have been explored to deconvolute the contributions of soil organic carbon and moisture. Near-infrared (NIR) spectroscopy has also been used extensively for the monitoring of soil carbon dioxide, with midinfrared (MIR) technologies also becoming more prevalent for measuring soil methane. While electrochemical sensors are unable to provide a quantitative analysis of total organic / inorganic carbon (which non-in situ combustion analysis can do) , Applicant has identified that the ability to provide a cost-effective method for in situ measurements of soluble organic species in soil can provide insight on the effectiveness of different soil improvement approaches. The current invention was conceived with this goal in mind .SUMMARY OF THE INVENTION

[0009] The skilled addressee is to appreciate that reference herein to an 'electrode arrangement' broadly comprises reference to an electrode, such as a screen-printed electrode, comprising athree-electrode configuration having a working electrode, a counter electrode, and a reference electrode.

[0010] According to a first aspect of the invention there is provided an electrode arrangement configured for detecting humic acids, fulvic acids and water-soluble molecules containing electrochemically active quinone / phenol functional groups in a soil matrix by means of squarewave voltammetry, said electrode arrangement comprising a working electrode with a coating comprising an admixture of: i. ionic liquid l-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) and sulfonated tetrafluoroethylene-based f luoropolymer-copolymer sulfur trioxide (S03) at a molar ratio of 1:1; ii. sulfonated tetrafluoroethylene-based fluoropolymercopolymer to conductive carbon particles at a mass ratio in a range of between 1:10 and 1:15; iii. sulfonated tetrafluoroethylene-based fluoropolymercopolymer to polymeric adsorbent non-ionic aliphatic acrylic resin at a mass ratio in a range of between of 1:50 and 1:60; and iv. a cross-linked hydrogel.

[0011] In an embodiment, a reference electrode of the electrode arrangement is covered.

[0012] In one embodiment, the sulfonated tetrafluoroethylenebased f luoropolymer-copolymer comprises Nafion™.

[0013] In an embodiment, the sulfonated tetrafluoroethylenebased f luoropolymer-copolymer to carbon particles is at a mass ratio of 1:14.9.

[0014] In an embodiment, the sulfonated tetrafluoroethylenebased f luoropolymer-copolymer to polymeric adsorbent non-ionic aliphatic acrylic resin is at a mass ratio of 1:59.8.

[0015] In an embodiment, the conductive carbon particles are selectable from a non-exclusive group consisting of carbon black, activated charcoal and carbon nanotubes.

[0016] In an embodiment, the polymeric adsorbent non-ionic aliphatic acrylic resin comprises DuPont™ AmberLite™ XAD™7HP.

[0017] In an embodiment, the cross-linked hydrogel is applied as a layer over the admixture coating, i.e. a cover layer.

[0018] In an embodiment, the cross-linked hydrogel comprises part of the admixture coating.

[0019] In an embodiment, the cross-linked hydrogel comprises 2- hydroxyethyl methacrylate (HEMA) to water in a ratio of 5:95.

[0020] In an embodiment, the cross-linked hydrogel comprises ethylene glycol dimethacrylate as a cross-linker at a molar ratio of between 3 % and 7%.

[0021] In an embodiment, the cross-linked hydrogel is polymerised via a photo-induced radical polymerisation technique, such as exposing said hydrogel to ultraviolet radiation for a predetermined amount of time.

[0022] In an embodiment, the cross-linked hydrogel is polymerised via a thermal-induced radical polymerisation technique, such as exposing said hydrogel to a predetermined temperature for a predetermined amount of time.

[0023] In an embodiment, the coating is homogenised prior to application to the working electrode via a drop-cast technique.

[0024] In an embodiment, after application to the working electrode, the coating is annealed at 110 °C to 120 °C for a predetermined period of time, such as 1 hour, to improve sensitivity and mechanical stability.

[0025] According to a second aspect of the invention there is provided a method for detecting humic acids, fulvic acids and water-soluble molecules containing electrochemically active quinone / phenol functional groups in a soil matrix by means of squarewave voltammetry, said method comprising the steps of: arranging, within the soil matrix, an electrode arrangement comprising a working electrode with a coating comprising an admixture of: i. ionic liquid l-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) to sulfonated tetrafluoroethylene-based f luoropolymer-copolymer sulfur trioxide (SO3) at a molar ratio of 1:1; ii. sulfonated tetrafluoroethylene-based fluoropolymercopolymer to conductive carbon particles at a mass ratio in a range of between 1:10 and 1:15; iii. sulfonated tetrafluoroethylene-based fluoropolymercopolymer to polymeric adsorbent non-ionic aliphatic acrylic resin at a mass ratio in a range of between of 1:50 and 1:60; and iv. a cross-linked hydrogel; connecting said electrode arrangement to a potentiostat configured to perform squarewave voltammetry over a voltage range of between -0.2V to 0.5V over a frequency range of between 1 to 100 Hz; andby means of a resulting squarewave voltammogram, monitoring variations in concentrations of humic and fulvic acids in the soil matrix as indicative of soil carbon content and / or soil health.

[0026] According to a further aspect of the invention there is provided an electrode arrangement and an associated method for detecting humic and fulvic acids in a soil matrix by means of squarewave voltammetry, substantially as herein described and / or illustrated .BRIEF DESCRIPTION OF THE DRAWINGSThe description will be made with reference to the accompanying drawings in which:Figure 1 shows squarewave voltammetry results after exposure of coated wafers (reference electrode uncovered - top image, reference electrode covered - bottom image) to solutions containing increasing concentrations fulvic acid and 0.0 wt . % iron (II) sulphate;Figure 2 shows squarewave voltammetry results after exposure of coated wafers (reference electrode uncovered - top image, reference electrode covered - bottom image) to solutions containing decreasing concentrations fulvic acid and 0.0 wt . % iron (II) sulphate;Figure 3 shows squarewave voltammetry results after exposure of reference electrode uncovered coated wafers (0.0 wt . % fulvic acid - top image, 1.0 wt . % fulvic acid - bottom image) to solutions containing increasing levels of 0.0 to 0.5 wt . % iron(II) sulphate;Figure 4 shows squarewave voltammetry results after exposure of reference electrode uncovered coated wafers (0.5 wt . % iron(II) sulphate) to solutions containing increasing levels of 0.0 to 1.0 wt . % fulvic acid;Figure 5 shows voltammetry results after exposure of reference electrode covered coated wafers to varying themeasurement frequency (top, in Hz, at a constant 0.4 wt . % fulvic acid solution) and increasing fulvic acid concentrations (bottom, 0.0 to 1.0 wt.%, at a measurement frequency of 5Hz) ;Figure 6 shows squarewave voltammetry results after exposure of reference electrode uncovered coated wafers to varying fulvic acid concentration (0.04 to 0.20 wt.%) using a 5Hz frequency;Figure 7 shows squarewave voltammetry (top) and forward current (bottom) results comparing earlier experiments and formulation MM-4 coatings on a reference electrode covered wafers to a 0.4 wt.% fulvic acid solution at an operating frequency of 5Hz ;Figure 8 shows squarewave voltammograms where a frequency of 1Hz and amplitude of lOOmV were used (top) for fulvic acid concentrations ranging 0.00 to 0.05 wt.%; these curves were first normalized at -0.2V and then the 0.00 wt.% curve was subtracted from each. At the bottom, the results of an optimized calibration curve approach where a second-order polynomial was fit to the area under the current-voltage curve from -0.2 V to 0.588V. The latter number was allowed to vary and this number was selected as it corresponded to the highest R2 correlation coefficient with this approach;Figure 9 shows squarewave voltammograms where a frequency of 1Hz and amplitude of 100 mV were used for these measurements. At the top, squarewave voltammograms for repeated cycling of a 0.05 wt.% fulvic acid solution, cycle # 1 to 98, and at the bottom, squarewave voltammograms for repeated cycling of a 0.05 wt.% fulvic acid solution, cycle # 105 to 240;Figure 10 shows where a frequency of 1Hz and amplitude of lOOmV were used for these measurements. At the top, squarewave voltammograms for repeated cycling of a 0.05 wt.% fulvic acid solution, cycle # 95 to 110, and at the bottom, a plot of the SWV current at 0.34V (i.e., the peak) over the 240 cycles;Figure 11 shows an example where a frequency of 1Hz and amplitude of 100 mV were used for these measurements. At the top,squarewave voltammograms for water extracts from different commercially available potting mixes, and at the bottom, squarewave voltammograms for fulvic acid solution, water extracts from a potting mix and solution of both the water extract from a potting mix and fulvic acid;Figure 12 shows an example where a frequency of 1Hz and amplitude of lOOmV were used for these measurements. At the top, squarewave voltammograms for 0.05 wt . % FA in 0.1 M phosphate buffer solution at pH values of 4.34, 6.71 and 7.71, and at the bottom, a plot showing the relationship between pH values and the peak voltage for their corresponding squarewave voltammograms;Figure 13 shows an example where a frequency of 1Hz and amplitude of lOOmV were used for these measurements. At the top, squarewave voltammogram (with forward and reverse currents) for 0.05 wt . % FA in 0.1 M phosphate buffer solution at pH 6.71, and at the bottom, squarewave voltammogram (with forward and reverse currents) for 0.05 wt . % FA in 0.1 M phosphate buffer solution at pH 12.23;Figure 14 shows an example where a frequency of 1Hz and amplitude of lOOmV were used for these measurements. The MM-7 coating was coated with a 5 moll cross-linked HEMA hydrogel layer. At the top, squarewave voltammograms for 0.05 wt . % FA in 0.1 M phosphate buffer solution at pH values of 7.09, 7.4 and 7.71, and at the bottom, squarewave voltammograms for 0.05 wt . % FA in 0.1 M phosphate buffer solution at pH values of 4.21, 5.76, 6.04, 6.33, 6.77, 6.87, 7.09, 7.4 and 7.71;Figure 15 shows an example where a frequency of 1 Hz and amplitude of 100 mV were used for these measurements. The MM-7 coating was coated with a 5 moll cross-linked HEMA hydrogel layer. Squarewave voltammograms for 50mL of 0.00 wt . % FA to 0.10 wt . % FA water solutions mixed with 200 grams of a 6 wt . % bentonite: 94 wt . % sand mixture. For this testing, the same wafer was moved from sample to sample showing a degree of resistance against mechanical abrasion;Figure 16 shows at the top, an image of electrode wafer surface before exposure to UV, and at the bottom, and image of electrode wafer surface after exposure to UV lamp (hydrogel and ink layer incorporated together ) ;Figure 17 shows squarewave voltammogram curves ( 100mA amplitude and 1Hz frequency) for hydrogel 45A dry / wet and 45B dry / wet are given . The wet samples have been saturated with a solution of only water to assess surface conductivity;Figure 18 shows squarewave voltammogram curves ( 100mA amplitude and 1Hz frequency) for hydrogel 48 in a dry state and in a water saturated 6 wt . % bentonite : 94 wt . % sand mixture saturated with water solutions of 0 to 0 . 20 wt . % fulvic acid concentration . The wet samples have been saturated with a solution of only water to assess surface conductivity;Figure 19 shows a mixed ink / hydrogel sensor response to 0 . 20 wt . % fulvic acid solution after 90 cycles at 6-hour intervals (bottom) followed by 340 cycles with 20-minute intervals ( top ) ;Figure 20 shows a plot of temperature vs . SWV response ( to a 0 . 20 wt . % fulvic acid solution) over a period of 3 days showing a strong linear correlation between temperature and response ; andFigure 21 shows plots showing the uncorrected response of the sensor to a 0 . 20 wt . % fulvic acid solution over a period of 4 days . The temperature was also recorded during this time showing a strong linear correlation between temperature and response . The ef fect of temperature was normalised to 20 ° C using a ratio method showing a 4 to 5 % decline in the response over a period of 4 days .DETAILED DESCRIPTION

[0027] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof . This description is included solely for the purposes of exempli fying the present invention to the skilled addressee . It should not be understood as a restriction on thebroad summary, disclosure or description of the invention as set out above. Additionally, features, mechanisms and aspects well- known and understood in the art will not be described in detail, as such features, mechanisms and aspects will be within the understanding of the skilled addressee.

[0028] As broadly described in International Patent Application no. PCT / AU2023 / 050676, Applicant has developed soil carbon sensors for providing in situ soil measurements. The present disclosure continues on the development of such earlier learnings in this field. Broadly, the present invention provides electrode arrangements and methodologies for identifying components of soil organic carbon, such as phenols and quinones functional groups that may be present in different soil matrices, and electrode coatings that are sensitive to these species. As such, a soil carbon sensing methodology and associated electrode arrangement are described that are configured for detecting water-soluble humic / fulvic acids and plant / fungal exudates which contain electrochemically active functional groups (e.g., ketones, quinones, phenols) also found in humic / fulvic acids in a soil matrix by means of squarewave voltammetry.

[0029] As detailed in the laboratory testing and experimental data below, the present invention broadly provides for an electrode arrangement configured for detecting humic acids, fulvic acids and water-soluble molecules containing electrochemically active quinone / phenol functional groups in a soil matrix by means of squarewave voltammetry. The electrode arrangement generally comprises a working electrode with a coating comprising an admixture of: i. ionic liquid l-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) and sulfonated tetrafluoroethylene-based f luoropolymer-copolymer sulfur trioxide (SO3) at a molar ratio of 1:1;ii . sul fonated tetrafluoroethylene-based fluoropolymercopolymer to conductive carbon particles at a mass ratio in a range of between 1 : 10 and 1 : 15 ; iii . sul fonated tetrafluoroethylene-based fluoropolymercopolymer to polymeric adsorbent non-ionic aliphatic acrylic resin at a mass ratio in a range of between of 1 : 50 and 1 : 60 ; and iv . a cross-linked hydrogel .

[0030] In general , the ionic liquid serves as a plastici zer which improves membrane porosity and subsequently permeability to analytes . The 1 : 1 ratio is chosen to optimise coating stability and ionic conductivity . The f luoropolymer-copolymer serves as a binder to keep the carbon particles bound to the electrode surface with the carbon particles acting as a surface for adsorption and detection of fulvic acid . The sul fonate groups have been shown to be strongly coordinated to the carbon particles . The resin is used to improve the electrode coating af finity for fulvic acid . An acrylic resin is used instead of a divinylbenzene-based resin as fulvic / humic acid will be less tightly bound to the resin enabling a reversible sensor response . The hydrogel is necessary for application in soil where the hydrogel gives the surface mechanical protection as well as bring moisture (which contains the analyte of interest ) to the electrode surface . In water with minimal surface abrasion, the incorporation of a hydrogel is optional .

[0031] Additionally, the present disclosure describes an associated method for detecting humic acids , fulvic acids and water-soluble molecules containing electrochemically active quinone / phenol functional groups in a soil matrix by means of squarewave voltammetry . Such a method broadly comprises the steps of arranging, within the soil matrix, a suitable electrode arrangement as described herein; connecting said electrode arrangement to a potentiostat configured to perform squarewavevoltammetry over a voltage range of between -0.2V to 0.5V (amplitudes ranging from 10 mV to 100 mV) over a frequency range of between 1 to 100Hz; and by means of a resulting squarewave voltammogram, monitoring variations in concentrations of humic and fulvic acids in the soil matrix as indicative of soil carbon content and / or soil health.

[0032] The optimum value, i.e. where sensitivity is maximised, for frequency and amplitude was experimentally determined to be around 1Hz and lOOmV, respectively, but variations hereon are possible. For squarewave voltammetry, the sensitivity typically improves with increasing frequency; however, this is not the case as there is not free diffusion at the surface with these membranes and a lower frequency enables a longer reaction time. The frequency and amplitude are varied to optimise the sensitivity primarily; the presence of the hydrogel as well as the moisture level will affect the response time due to changes in the diffusion characteristics in the membrane; however, this response time is expected to be much quicker than any change in the concentration of soluble organic carbon in soil. Changes in pH could also be detected as represented by a change in the peak voltage value for the squarewave voltammogram. The area of the peak could be correlated to the concentration using a calibration equation.

[0033] In one embodiment, a reference electrode of the electrode arrangement is covered. In one embodiment, the sulfonated tetrafluoroethylene-based f luoropolymer-copolymer comprises Nafion™. In one embodiment, the polymeric adsorbent nonionic aliphatic acrylic resin comprises DuPont™ AmberLite™ XAD™7HP. In one embodiment, the carbon particles are selectable from a non-exclusive group consisting of carbon black, e.g. Vulcan™ XC72™ from Cabot™, activated charcoal and carbon nanotubes (e.g. carboxylic acid functionalized multiwalled carbon nanotubes, thecarboxylic acid functional groups help to facilitate dispersion in the deposition solution) .

[0034] In one embodiment, the cross-linked hydrogel is applied as a layer over the admixture coating, i.e. a cover layer. In one embodiment, the cross-linked hydrogel comprises part of the admixture coating. In one embodiment, the cross-linked hydrogel comprises 2-hydroxyethyl methacrylate (HEMA) to water in a ratio of 5:95. In one embodiment, the cross-linked hydrogel comprises ethylene glycol dimethacrylate as a cross-linker at a molar ratio of between 3 % and 7%.

[0035] In one embodiment, the coating is homogenised prior to application to the working electrode via a drop-cast technique. In one embodiment, after application to the working electrode, the coating is annealed at 110 °C to 120 °C for a predetermined period of time, such as 1 hour, to improve sensitivity and mechanical stability .

[0036] In one embodiment, the potentiostat configured to perform squarewave voltammetry comprises an EmStat™ Pico™ development board from PalmSens™.Laboratory testing and establishment of sensing principles

[0037] As described in Applicant's above patent application, earlier experimenting and testing have shown that it may be feasible to measure soil organic carbon by means of a sensor using squarewave voltammetry (SWV) with a coated electrode surface. This technology uses a Naf ion™ / ionic liquid-based electrode coating that contains carbon particles and aliphatic cross-linked polymethylmethacrylate (PMMA) resin. Nafion™, being a fluorinated polymer, has exceptional chemical and thermal stability making itan attractive binder to keep the carbon particles and aliphatic cross-linked resin intact on the electrode surface.

[0038] A mixture of Naf ion™ / ionic liquid membrane results in good ionic conductivity - the combination of polar sulfonate groups in the Nafion™ and the aliphatic side chains on the ionic liquid result in a nano / micro-structured films which can easily incorporate the carbon and cross-linked PMMA resin particles. Carbon particles such as activated charcoal, carbon black or biochar can be used to provide a conductive surface as well as providing sites for electrochemical reactions - carbon black is often considered ideal for chemical sensing applications. In this design, cross-linked aliphatic PMMA resins are used as they have a strong and reversible affinity to fulvic / humic acid species. Divinylbenzene resins also have a strong affinity; however, the binding to fulvic / humic acids may not be reversible under realistic sensor conditions found in soil.

[0039] In theory, these surfaces would partition fulvic / humic acid from water (or the water-fraction within soil) where they could be detected using redox electrochemistry. For operation in soil, this sensor is predicated on water (containing the soluble carbon being analysed) being brought to the electrode surface. While the Nafion™ surface is hydrophilic and capable of wetting in soil, the addition of ionic liquid and aliphatic PMMA resin will reduce the wettability likely to a mixed-wettability state.

[0040] An initial laboratory testing of an electrode coating used 100 pL of a 5 wt . % Nafion™ DS2021 (1100 g per mole of sulfonate, assuming 0.90 g / mL solution density, this is equivalent to 4.5 mg of Nafion™ polymer or 4.09 pmoles of sulfonate) was combined with 95 mg of the ionic liquid l-butyl-3- methylimidazolium tetrafluoroborate (BMIM-BF4 which has a molecular weight of 226.02 g / mol, corresponding to 420 pmoles) .This represents a molar ratio of approximately 1:103; a mass ratio of 1:5 Nation™: activated charcoal and 1:2 Amberlite™ XAD7HP : activated charcoal is used. These solids were combined with the liquids, diluted with 70:30 isopropanol (IPA) :water and then subjected to a homogenizer for approximately 1 to 2 minutes. These solutions were immediately drop casted onto Metrohm™ C220BT DropSens™ wafers using a masking technique.

[0041] For testing, 36 different water solutions were prepared representing every combination of iron (II) sulphate (0.0, 0.1, 0.2, 0.3, 0.4 and 0.5 wt.%) and fulvic acid (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 wt.%) . When either iron(II) or iron(III) were added to the higher molecular weight humic acid at these concentrations, precipitation occurred almost immediately. A 0V to IV sweep with an amplitude of 25 mV, step of 5 mV and frequency of 25 Hz was used for squarewave voltammetry measurements after the solutions were allowed to equilibrate with the surface for approximately 45 to 75 minutes. It appeared that in most circumstances, 60 minutes was sufficient to achieve a stable response. For comparison purposes, the current was normalised at 0V for these measurements.

[0042] Figure 1 shows the response to increasing concentrations of fulvic acid - the response seems to be slightly more uniform with increasing fulvic acid concentration with reference electrode covered wafers. It is evident that at 0.0 wt.% that a compliance failure (represented by the step) occurs around 0.8V. It was necessary to equilibrate the wafers in the 0.2 wt.% solution for at least 1 hour before the analyte concentration (and the resulting current from the redox reaction) was sufficient to eliminate this compliance issue. It is important that a chemical sensor exhibits reversibility. Figure 2 shows that the sensor response is reversible. Again, improved response linearity was observed with the reference electrode covered wafers. Based on these results, itwas decided to continue testing only with wafers that have the reference electrodes covered.

[0043] Metal cations and metal cation-fulvic acid chelated complex also exhibits a response when using squarewave voltammetry. It is evident from Figure 3 that these coatings respond to iron(II) sulphate solution in an expected fashion (i.e. the response increases with concentration) . When fulvic acid at a 1.0 wt . % loading is added to these solutions, the response again increases with iron (II) sulphate concentration. Comparing the plots in Figure 3 with each other and comparisons with Figures 1 and 2, the voltammogram generally becomes broader when both iron (II) sulphate and fulvic acid are present. This likely represents redox processes associated with metal cation-fulvic acid chelated complexes. Figure 4 confirms this likelihood as the response is complex when the fulvic concentration is increased in a background of 0.5 wt . % iron (II) sulphate. This suggests that redox processes associated with metal-fulvic complexes are playing a significant role and the degree of complexation is having an appreciable effect on the concentrations of both free iron (II) sulphate and fulvic acid. Regardless, the fulvic acid concentration is quite high and there is a need to improve sensitivity. To do this, the squarewave voltammetry parameters (i.e., frequency and amplitude) as well as the membrane composition were optimised, as detailed below.Optimisation of squarewave voltammetry parameters

[0044] As a result of the diffusion processes governing analyte transport to the surface, the measured current using the squarewave voltammetry is proportional to the square root of the frequency. At higher frequency, the reaction kinetics may not be sufficiently fast so there is an optimum frequency. Furthermore, with a sensing layer as is used here, the diffusion processes are slowed downsubstantially as well; this is further exacerbated when a hydrogel layer is used. As seen in Figure 4, for a 0.4 wt . % fulvic acid solution, the frequency is varied from 1 to 100Hz to find the optimal signal to noise ratio. It is evident that this optimum is approximately 5Hz which is significantly lower than the 25Hz initially used. Furthermore, at 5 Hz, it is possible to optimise for certain slower redox reactions while becoming more insensitive to faster redox reaction - it is hypothesised that this may be the case as the voltage corresponding to peak current shifts from 0.4- 0.6V (see Figure 1, bottom) to 0.8 to 1.0V (see Figure 5, bottom) .

[0045] With a covered reference electrode and operation at 5 Hz, the limit of detection was explored by exposing the electrode wafer to solution ranging from 0.04 to 0.2 wt . % (see Figure 6) . It is evident that there is a variation in response to these solutions; however, compliance issues are significant particularly at lower fulvic concentration and higher voltages (corresponding to higher sensitivity with 5Hz operation. It was identified that a more conductive coating would facilitate a reduction in counter electrode voltage and / or using a wafer with a smaller working electrode surface area. As a result, C223BT Dropsens™ wafers with a smaller 1.6mm working electrode surface area were subsequently used. This largely resolved the potentiostat compliance issues allowing for detection of fulvic acid at lower concentrations. The focus then switched to optimising the coating formulation to improve sensitivity and the mechanical robustness of the sensing layer .Electrode coating optimisation and experimentation

[0046] Based on outcomes from previous laboratory experiments, four different formulations were tested:Table 1. Summary of electrode coating formulations tested.

[0047] In a modification to prior experiments on electrode coating formulations, formulations MM-1 to MM-4 were further diluted by doubling the volume with 70 % IPA in water. This last step was done to achieve a more homogeneously dispersed ink - while this does not affect the coating composition, it will potentially influence thickness.

[0048] Formulations MM-2 and MM-4 differ from formulations MM- 1 and MM-3 in that the Amberlite XAD7HP resin (which has been attributed to helping with fulvic / humic acid affinity) was removed to ascertain its effects on sensor response. Formulations MM-1 to MM-2 were tested in the same 0.4 wt . % fulvic acid solution and it became evident that both of these coatings were very conductive (compared to the prior experiments' coating) resulting in a different kind of compliance error where the current exceeded the 5mA maximum current limit of the potentiostat even at voltages less than 0.1V. It became evident that little difference in response could be attributed the Amberlite resin under these conditions .

[0049] Following this, formulations MM-3 and MM-4 were developed which have a lower quantity of carbon black (making the coating less conductive) relative to Nafion. This improved the coating somewhat, but the maximum current limit was still reach at a low voltage of only 0.2V (see Figure 7) . Formulation MM-4 contains a 1.1:1 ratio of carbon black to Amberlite resin which would lower the conductivity presumably as well; however, the high conductivity still presented significant compliance issues for the potentiostat .

[0050] In an attempt to decrease the conductivity while also increasing the relative amount of Amberlite XAD7HP ( to presumably improve the partitioning of fulvic / humic acid) , coating MM-5 looked at decreasing the amount of carbon black relative to Nafion and increasing the amount of Amberlite XAD7HP relative to carbon black - in this case , the conductivity was very low . Furthermore , given the high combined loading of carbon black and XAD7HP relative to Nafion, the amount of binder was very low and the surface was not mechanically stable .

[0051] Following such initial experimentation and development , the following observations were made as a way to improve performance :- The amount of Nafion could be increased considerably to reduce surface conductivity but risks covering the Amberlite resin which would decrease its af finity to fulvic / humic acids and, hence , the sensor sensitivity would be negatively impacted . This would also reduce the surface area available for electrochemical reactions provided by carbon black .- The amount of Amberlite resin could be increased to reduce surface conductivity . This would also have the advantageous ef fect of increasing coating af finity to fulvic / humic acids .- The leeway provided by the higher conductivity of carbon black relative to activated charcoal is essential in being able to increase the amount of Amberlite resin in the coating .In future formulations , the amount of Amberlite relative to carbon black will be increased substantially ( in comparison to MM- 1 to MM-4 ) to modulate the current to a more reasonable value . I f necessary, a portion of activated charcoal may be used in place of the carbon black but this risks a less homogenous ink .

[0052] MM-6 and MM-7 were developed as a compromise between MM- 1 to MM-4 and MM-5. MM-6 was quite sensitive to fulvic acid; however, mechanical stability was a major concern even in nonstirred water solutions. MM-7 was slightly less sensitive to fulvic acid than MM-7 but the mechanical stability was improved. Furthermore, in further testing of MM-6 and MM-7, it was shown that the sensitivity and mechanical stability could be improved by annealing in an oven at 110 to 120 °C for 1 hour. As a result of such preliminary testing, formulation MM-7 was identified as a preferred embodiment for further in-depth of testing.

[0053] In one embodiment, the exact composition details of formulation MM-7 are 9.07g of a 0.165 wt . % Nation solution (with a 1:1 molar ratio of BMIM-BF4 to Nafion-SOs, assuming a sulfonate density of 1100g Nation to 1 mole SO3) was combined with 224. Omg of Vulcan XC72 carbon black and 894.1 mg of Amberlite XAD7HP. This was then diluted to 40mL total volume with a 70:30 isopropanol : water mixture. This mixture was subjected to a homogenizer for 2 minutes before coating onto wafers using a dropcast technique. Before subsequent use of this mixture, the solution was thoroughly stirred and periodically it was sub ected to further homogenization .

[0054] A summary of the experiments conducted with coating formulation MM-7 in water solutions is detailed below. Unfortunately, this coating easily rubs off with mechanical abrasion, which makes application of this coating for soil applications problematic. In an attempt to develop a coating suitable for application in soil, MM-8 through MM-11 were developed which have a higher relative amount of Nation compared to MM-6 and MM-7 - this would give the surface more mechanical stability. The sensor responses of these coatings were measured in 0.20 wt . % fulvic acid solutions while the surface stability was assessed qualitatively (based on prior experience working with MM-6 and MM-7) . It became evident from these experiments that sensor response came at the expense of mechanical stability - there was no ideal balance between these two characteristics which would facilitate application in soil.Test results for optimised coating MM-7 in water solutions

[0055] With the new optimised coating and a smaller working electrode, the practical detection range for fulvic acid to 0.00 to 0.05 wt . % (see Figure 8) was significantly improved. With the prior experiments' formulation, water solutions containing less than 0.20 wt . % typically resulted in compliance issues (see the 0 wt . % result in Figure 2 for an example of this) . While squarewave voltammetry is widely considered to a semi-quantitative method, there are examples where it has been used for quantitative analysis under certain circumstances (see Monatshefte fur Chemie - Chemical Monthly (2023) 154:1225-1233) . With this example, a linear correlation between peak current and concentration for ferulic acid in water is given. Furthermore, the voltage corresponding to the peak current did not change appreciably with concentration. With fulvic acid using the proposed approach, the peak is fairly broad and the voltage representing the peak current varies with concentration .

[0056] In cyclic voltammetry, the area under the curve represents the total charge corresponding to the electrochemical process which can be directly attributed to the concentration. A similar process was used here (see Figure 8) , which shows that the area under the curve can be correlated to the concentration using a second-order polynomial (which is consistent with area being a 2nd-order relationship) . With the drop-casting technique used here, there is considerable variability from one sample to another causing slightly different response characteristics between s amp les.

[0057] Electrochemical sensor stability in this context is predicated on two requirements, being:- The electrochemical reactions should ideally be reversible. These sensor coatings (in particular, the Amberlite resin component) partition organic species from the water phase enabling their detection. This partitioning is generally reversible, however electrochemical reactions may change the absorption and water solubility characteristics making this partitioning irreversible (or partially irreversible) . As a result, the sensor response characteristics may be negatively impacted .The chemical and mechanical integrity of the surface coating is important for long-term sensor operation. Nafion is a perfluorinated polymer and is considered to have excellent chemical stability. Carbon black and Amberlite resin are also quite stable, however surface fouling is a more likely outcome in a soil environment. Mechanical integrity is an evident issue for MM-7 with abrasion of the surface easily causing degradation. Despite this, the surface can be in water for measurement over a period of days.

[0058] To assess sensor response stability, the electrode arrangement with the MM-7 coating was placed in a solution of 0.05 wt . % fulvic acid and subjected to repeated measurement cycling every 30 minutes for 240 cycles (over a period of approximately 2 days) . At the 100-cycle mark, the solution was refreshed with a new identical solution of 0.05 wt . % fulvic acid (i.e., the same solution was split into two parts for this test) . If the electrochemical processes were completely reversible, no change in the response is expected when the analyte solution is changed. As seen in Figure 9, there is a continuous gradual decrease in the response from cycles 1 to 98; similar behaviour is seen for cycles 105 to 240 after the solution had been replaced.

[0059] Figure 10 shows the transient that occurs when the solution is refreshed. It is evident that there is initially an increase in the response following the analyte solution being refreshed. This is strong evidence that the electrochemical reactions are not completely reversible and suggests that there is degradation over time. Given that the current climbs markedly above 0.6V which suggests that there might be an irreversible reaction, these experiments were repeated over a shorter voltage range from only -0.2 to 0.5V. A very similar transient was also noted but the degradation was not as severe. In both cases, it was evident that parts of the coating had come off after prolonged continuous operation. It appeared qualitatively that the measurements were contributing to loss. When this coating is placed in fulvic acid solution and no measurements conducted, surface degradation is negligible after several weeks. This suggests that the electrochemical processes may be accelerating sensor degradation.

[0060] In the next test, the responses from different water extracts from commercially available potting mixes were measured. The potting mixes were marketed as 'premium' , 'rose' and 'cacti' mixes. These potting mixes contain a varying degree of organic carbon as well as trace minerals such iron and manganese (which are needed with chlorophyll for photosynthesis) . For each potting mix, 8 grams of potting mix was combined with 40 grams of water and left to soak for 12 hours. These samples were then filtered using a PTFE syringe filter and the sensor response from each water filtrate was determined.

[0061] It is evident from these experiments (see Figure 11 for the results) that there is a significant response to the water extracts with a larger response for the 'premium' and 'rose' mixes as opposed to the 'cacti' mix. With the recognition that the soil sensor is likely able to detect a wide variety of soluble organicmolecules, metal cations and organic molecules that are chelated to metals, the sensor response of the 'premium' potting mix extract is compared with the response from fulvic alone and in combination; the addition of fulvic acid to the soil mix causes broadening of squarewave voltammogram and possible formation of a second distinct peak. These comparisons suggests that the sensor response for the soil extract is distinct from the fulvic acid. It is likely that soluble organic species in the water extracts are similar to fulvic acid but that they have a distinctly different character (i.e., different organic composition and / or presence of metal cations) .

[0062] With the hypothesis that phenols / quinones are the redox active species being detected, there is an expectation of a pH sensitive response. It has been shown previously that the redox behaviour of alizarin (which contains quinone and phenol functional groups) is pH sensitive and that the squarewave voltammogram peak follows an expected Nernstian relationship under varying pH conditions (Electroanalysis, 2015, 27, 917-923 and Micromachines, 2023, 14 (12) , 2188) . Based on this, the sensor response of fulvic acid in phosphate buffer solution at different pH values was measured. It is evident from these results that there is a pH dependent result. Furthermore, changes in pH appear to have an effect on the current as well. The pH dependent effects are largely in agreement with the previous results using alizarin with a near-Nernstian slope of 79mV / decade (compared to the theoretical value of 59.2 mV / decade for a temperature of 25°C) .

[0063] With previous studies with alizarin as an electrochemical pH indicator, at alkaline conditions, complex voltammograms were observed due to oxidized alizarin followed by nucleophilic reactions with hydroxide ions. The structure of fulvic acid is considerably more complex than fulvic acid so the possibility cannot be discounted that the electrochemicalreactions will result in irreversible chemical reactions. To explore this further, the forward and reverse currents associated with the squarewave voltammograms were examined in two different pH regimes: 6.71 and 12.23 (see Figure 13) . Comparing the response characteristics at the two different pH values, it is evident that there are fundamental differences in the behaviour. At pH 6.71, the forward and reverse currents are largely symmetrical indicating that the process is predominantly reversible. At pH 12.23, the situation is markedly different with the forward current being much larger in magnitude compared to the reverse current. This suggests that the process is mostly irreversible - there is some reverse current suggesting that a minor component of the chemical reaction might be reversible. This is consistent with previous studies on alizarin and the expected chemical reactions of fulvic acid.Cross-linked hydrogel

[0064] As a result of the testing of the different ink formulation (MM-1 to MM-11) , it was decided to pursue an approach where a cross-linked hydrogel would be used as a cover layer. Initial hypothesising was that this would improve the mechanical stability of the underlying layer while also helping to bring moisture and dissolved analytes to the sensing surface of the coated working electrode. Both attributes are very important for application of this technology in soil. One downside to this approach is that it could potentially slow down the response time as the water from the soil will have a longer diffusion distance to reach the sensing surface. For long-term in situ operation in the soil, it was concluded that this was reasonable as soil organic carbon is presumed to be a variable that slowly changes over time, i.e. the sensor response time was still much quicker than the expected rate of change in soil organic carbon.

[0065] Initially, it was considered applying a cross-linked HEMA ( 2-hydroxyethyl methacrylate) hydrogel coating on top of the thermally cured MM-7 coating. A photo-polymerisation technique would be used with UV light to initiate the polymerisation in situ. Thermally-initiated polymerisation was not favoured as it is very difficult to practically implement with drop-casted films with solvent evaporation being a major issue. In prior research, a 20 % HEMA / 80% water mixture combined with 2.5 to 5 mol.% of ethylene glycol dimethacrylate (as a cross-linker) and 0.1 mol.% 2,2- dimethoxy-2-phenylacetophenone (as a photo-initiator) was utilized. This ratio of HEMA to water resulted in a material that had good pore interconnectivity. The ratio of cross-linker to HEMA was used as it provided a good compromise between mechanical stability, swellability and pore interconnectivity.

[0066] With this in mind, initially this 20:80 mixture of HEMA and water was used with varied amounts of cross-linker. Such initial hydrogel studies were conducted on glass slides to understand the water swellability and the surface stability. At a 1 mol % loading, the surface was quite stable and it took approximately 30 minutes for the coating to turn from transparent to opaque. At a 2.5 % loading, the surface was also quite stable and the coating turned opaque after only 20 minutes. At a 5 % loading, the surface was again quite stable but the coating turned opaque very quickly after only 5 minutes. At 7% loading, the crosslinked polymer easily came off the glass slide. As a result of these observations, a 5 mol % cross-linker loading was chosen for subsequent study.

[0067] Unfortunately, the MM-7 coating (in particular, the Nafion component) was easily dissolved into this mixture immediately after liquid deposition onto the surface and before polymerisation could be undertaken. It is suspected that the HEMA was acting similarly to the isopropanol component of the Nafionmixture (in which it was initially dissolved) causing the Nafion to be re-dissolved. With the loss of the binder on the surface, the carbon black and resin easily came off. This was resolved by reducing the amount of HEMA relative to water at a 5:95 ratio. With this approach, it was possible to coat the underlying MM-7 layer with a cross-linked HEMA hydrogel layer. This dramatically improved the mechanical stability, and it was possible to place these wafers into both water and soil samples. However, over time, the HEMA layer had different swelling and expansion properties compared to the underlying MM-7 layer, which eventually led to delamination. Nevertheless, some sensing results in both water and soil was possible using this two-layer approach.Testing two-layer hydrogel / MM-7 approach in water solution and preliminary testing in soil matrices

[0068] With the mechanical integrity issues of the MM-7 surface, the possibility of placing a hydrogel overlayer onto the coating was explored to alleviate this issue. In soil applications (unlike water applications) where the moisture level may be limited, the hydrogel would also aid in bringing moisture (and analyte) to the sensing surface. Initially, this opportunity was used to further testing on the pH response of these sensors in water .

[0069] The previous pH variation study was re-conducted using a 2-layer approach but focused on pH values ranging from 4.21 to 7.71 (which are values more typical of soil pH values) - alkaline conditions above pH 11 that were examined previously are not realistic soil pH values. Based on Figure 12, when looking at pH values from 7.09 to 7.71, a reversal in the trend can be seen with increasing pH corresponding to increase in the SWV peak voltage whereas previously we saw a decrease in SWV peak voltage with increasing pH (i.e., from 4.31 to 7.71) . Over a larger pH range,it is evident that there are that there is a complex behaviour likely involving multiple functional groups on the fulvic acid molecules .

[0070] While there is a certain degree of mechanical robustness of the sensing layer, with repeated insertion and removal of these electrode wafers from sand combined with di f ferent levels of hydration of the hydrogel layer, there is a strong propensity for the hydrogel layer to complete de-laminate after approximately 24 to 48 hours of testing . As a result , the development of a singlelayer approach should alleviate this issue to some extent . In another embodiment , the use of suitable adhesion layers to get better adhesion between the wafer and the hydrogel may also be used .Testing single layer MM-7 ink / hydrogel approach

[0071] To prepare the combined hydrogel and ink layer on the surface of an electrode wafer, di f ferent formulations have been used and assessed qualitatively for their conductivity and adhesion properties . Like the studies with the two- layer approach, photo-polymerisation (with a 45-to- 60-minute UV light exposure ) was used here with DPAP as an initiator to form a mixed hydrogelink layer ( see Figure 16 ) .

[0072] In initial experiments , an approximately 20% HEMA: 80% water mixture was used with 5 mol % ethylene glycol dimethacrylate . As anticipated with no ink, a stable layer was formed on both a glass slide and on the wafer . Hydrogels 40B and 40C were not conductive and is likely due to the low level of MM-7 ink . As the amount of ink was increased in 40D and 40E , the surface was unstable on the glass slide and moderately stable on the electrode wafer . Hydrogels 40D and 40E were conductive and their sensitivitytowards fulvic acid in water solution tested. With even more ink in hydrogels 40F and 40G, the surface stability was poor for both glass slides and electrode wafers. As a result, the use of a different crosslinker was identified. At higher ink loadings, it was difficult to accommodate the photo-polymerisation as the surface became very opaque.Table 2: HEMA - 0.121 mL, Water - 0.5 mL, Crosslinker (ethylene glycol dimethacrylate) - 10 pL, Initiator DPAP 14 pL, MM-7 ink.Exposed to UV for 45 min.

[0073] With N, N' -dimethylenebisacrylamide as a crosslinker, the use of HEMA and water at a higher ratio with a 10 mol % crosslinker (the higher amount used due to improved solubility in water) . Both hydrogels 44A and a repeat 44B showed good stability on both glass slides and wafers. The electrical conductivity was also reasonable .Table 3: HEMA - 0.121 mL, Water - 0.5 mL, Crosslinker (water soluble, N, N-dimethylenebisacrylamide ) 15.4 mg, Initiator DPAP - 20 pL, MM-7 ink. Exposed to UV for 60 min.

[0074] In these last two samples 45A and 45B, the water content relative to HEMA is decreased improving the solubility of the ethylene glycol dimathacrylate crosslinker. These surfaces were stable on the wafer, however 60 minutes of UV light exposure was needed for the higher ink loading of 0.3mL . This is presumably due to the opaque nature of the film. From preliminary SWV results for hydrogels 45A and 45B (see Figure 17) , it is evident that the conductivity decreases (i.e., the current decreases) when the samples are swelled in water. Unlike the initial ink, this mixed hydrogel / ink coating can swell in water; under these conditions, the distance between conductive particles increase leading to a change in the bulk surface resistance. For this reason, using carbon nanotubes with the ink formulation may be appropriate. Carbon nanotubes are more conductive than carbon black so a lower loading can be used which will allow for the Nation binder content and Amberlite resin content to be increased. This may improve sensitivity and surface stability.Table 4: HEMA - 0.121mL, Water - 0.35 mL, Crosslinker (ethylene glycol dimethacrylate) - 10 pL, Initiator DPAP 14 pL, MM-7 ink.Exposed to UV for 45 min.

[0075] Samples 47A and 48A (see Table 5 for the compositions) were tested with 0.00 wt . % fulvic acid and 0.20 wt . % fulvic acid solutions; 48A showed a slightly better response and this likelyattributed to a difference in the pore structure of the layer. Increasing the water content to an extent is known to increase the swellability of the hydrogel layer (Journal of Membrane Science, Volume 365, Issues 1-2, 1 December 2010, Pages 248-255) . With swelling in water, it is evident by comparing the dry and wet state (at 0.00 wt . % FA in sand / bentonite ) that the bulk conductivity decreases; this is attributed once again to the hydrogel layer swelling. By varying the concentration of fulvic acid stepwise to 0.20 wt.%, an increase in response is observed. The adhesion of the gold electrode surface could also be improved by functionalizing the surface with cysteamine and then functionalized with ethylene glycol dimathacrylate to bind the hydrogel / ink coating to the surface.Table 5. HEMA and CR stirrer together - then water added - then formulation ink followed by initiator. Exposed to UV light for 50 min .

[0076] For hydrogel 48, Figure 19 shows little degradation in the sensor response over repeated cycles with a 6-hour interval over a period of 23 days. Subsequent testing over a period of 6 days with a more frequent 20-minute interval shows a time-dependent relationship with response. The approximately 24-hour cyclical nature of the response suggested that temperature might beinfluencing the response (i.e. the temperature of the laboratory where testing occurred was warmer during the day and colder at night) . In latter experiments, this variation was shown to have a strong linear correlation between temperature and sensor response over a test period of 3 days when exposed to a solution with a constant concentration 0.20 wt . % (see Figure 20) . Using a ratio method with this linear correlation, the data was corrected to a constant temperature of 20 °C in Figure 21 and an 8.7% standard deviation in the response over a period of 4 days was measured.

[0077] Applicant believes that the above-described electrode arrangements and sensing coatings, along with the associated methods for detecting humic and fulvic acids in a soil matrix by means of squarewave voltammetry, are able to provide a cost- effective method for in situ measurements of soluble organic species in soil in order to provide insight on the effectiveness of different soil improvement approaches.

[0078] In the example embodiments, well-known processes, well- known device structures, and well-known technologies are not described in detail, as such will be readily understood by the skilled addressee. Optional embodiments of the present invention may also be said to broadly consist in the parts, elements and features referred to or indicated herein, individually or collectively, in any or all combinations of two or more of the parts, elements or features. Where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0079] It is to be appreciated that reference to "one example" or "an example" of the invention, or similar exemplary language (e.g., "such as") herein, is not made in an exclusive sense.Various substantially and specifically practical and usefulexemplary embodiments of the claimed subject matter are described herein, textually and / or graphically, for carrying out the claimed subject matter. Accordingly, one example may exemplify certain aspects of the invention, whilst other aspects are exemplified in a different example. These examples are intended to assist the skilled person in performing the invention and are not intended to limit the overall scope of the invention in any way unless the context clearly indicates otherwise.

[0080] Variations (e.g. modifications and / or enhancements) of one or more embodiments described herein might become apparent to those of ordinary skill in the art upon reading this application. The inventor (s) expects skilled artisans to employ such variations as appropriate, and the inventor (s) intends for the claimed subject matter to be practiced other than as specifically described herein.

[0081] The use of the terms "a", "an", "said", "the", and / or similar referents in the context of describing various embodiments (especially in the context of the claimed subject matter) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising, " "having, " "including, " and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0082] Any method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed .

Claims

CLAIMS1. An electrode arrangement configured for detecting humic acids, fulvic acids and water-soluble molecules containing electrochemically active quinone / phenol functional groups in a soil matrix by means of squarewave voltammetry, said electrode arrangement comprising a working electrode with a coating comprising an admixture of: i. ionic liquid l-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) and sulfonated tetrafluoroethylene-based f luoropolymer-copolymer sulfur trioxide (S03) at a molar ratio of 1:1; ii. sulfonated tetrafluoroethylene-based fluoropolymercopolymer to conductive carbon particles at a mass ratio in a range of between 1:10 and 1:15; iii. sulfonated tetrafluoroethylene-based fluoropolymercopolymer to polymeric adsorbent non-ionic aliphatic acrylic resin at a mass ratio in a range of between of 1:50 and 1:60; and iv. a cross-linked hydrogel.

2. The electrode arrangement of claim 1, wherein a reference electrode thereof is covered.

3. The electrode arrangement of either of claims 1 or 2, wherein the sulfonated tetrafluoroethylene-based f luoropolymer-copolymer to carbon particles is at a mass ratio of 1:14.9.

4. The electrode arrangement of any of claims 1 to 3, wherein the sulfonated tetrafluoroethylene-based f luoropolymer-copolymer to polymeric adsorbent non-ionic aliphatic acrylic resin is at a mass ratio of 1:59.8.

5. The electrode arrangement of any of claims 1 to 4, wherein the conductive carbon particles are selectable from a nonexclusive group consisting of carbon black, activated charcoal and carbon nanotubes.

6. The electrode arrangement of any of claims 1 to 5, wherein the cross-linked hydrogel is applied as a layer over the admixture coating, i.e. a cover layer.

7. The electrode arrangement of any of claims 1 to 6, wherein the cross-linked hydrogel comprises part of the admixture coating.

8. The electrode arrangement of any of claims 1 to 7, wherein the cross-linked hydrogel comprises 2-hydroxyethyl methacrylate (HEMA) to water in a ratio of 5:95.

9. The electrode arrangement of any of claims 1 to 8, wherein the cross-linked hydrogel comprises ethylene glycol dimethacrylate as a cross-linker at a molar ratio of between 3 % and 7%.

10. The electrode arrangement of any of claims 1 to 9, wherein the cross-linked hydrogel is polymerised via a photo-induced radical polymerisation technique, such as exposing said hydrogel to ultraviolet radiation for a predetermined amount of time.

11. The electrode arrangement of any of claims 1 to 10, wherein the cross-linked hydrogel is polymerised via a thermal-induced radical polymerisation technique, such as exposing said hydrogel to a predetermined temperature for a predetermined amount of time.

12. The electrode arrangement of any of claims 1 to 11, wherein the coating is homogenised prior to application to the working electrode via a drop-cast technique.

13. The electrode arrangement of any of claims 1 to 12, wherein after application to the working electrode, the coating is annealed at 110 °C to 120 °C for a predetermined period of time, such as 1 hour, to improve sensitivity and mechanical stability.

14. A method for detecting humic acids, fulvic acids and water-soluble molecules containing electrochemically active quinone / phenol functional groups in a soil matrix by means of squarewave voltammetry, said method comprising the steps of: arranging, within the soil matrix, an electrode arrangement comprising a working electrode with a coating comprising an admixture of: i. ionic liquid l-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) to sulfonated tetrafluoroethylene-based f luoropolymer-copolymer sulfur trioxide (SO3) at a molar ratio of 1:1; ii. sulfonated tetrafluoroethylene-based fluoropolymercopolymer to conductive carbon particles at a mass ratio in a range of between 1:10 and 1:15; iii. sulfonated tetrafluoroethylene-based fluoropolymercopolymer to polymeric adsorbent non-ionic aliphatic acrylic resin at a mass ratio in a range of between of 1:50 and 1:60; and iv. a cross-linked hydrogel; connecting said electrode arrangement to a potentiostat configured to perform squarewave voltammetry over a voltage range of between -0.2V to 0.5V over a frequency range of between 1 to 100 Hz; and by means of a resulting squarewave voltammogram, monitoring variations in concentrations of humic and fulvic acids in the soil matrix as indicative of soil carbon content and / or soil health.

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