Aiming to a Smart Agriculture through the Electrochemical Impedance of a Chitosan-Molybdate Membrane as a Function of Available Phosphate Species

doi:10.21203/rs.3.rs-2982329/v1

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Aiming to a Smart Agriculture through the Electrochemical Impedance of a Chitosan-Molybdate Membrane as a Function of Available Phosphate Species

https://doi.org/10.21203/rs.3.rs-2982329/v1

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Journal Publication

published 02 Nov, 2023

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The identification and quantification of nutrients in the soil is of great importance in agriculture because plants depend on their presence in the soil or their supply as fertilizer to grow properly. However, there is currently no well-defined method for quantifying macronutrients in the field. Therefore, chitosan membranes that have been developed are biocompatible and biodegradable and are applied to stainless steel and carbon inks whose function is to adsorb phosphorus (P) species present in cultivated soils. Studies on Z-potential were carried out on chitosan solutions, varying the pH to measure the surface charge. It was found that as the pH of the solution increased, from 2 to 10, the surface charge decreased until a negative surface charge (from 59.9mV to -5mV). The chitosan/chitosan molybdate membranes were also examined by atomic force microscope (AFM) to observe their roughness and morphology. The chitosan membrane has a smoother appearance than the chitosan-molybdate membrane, confirmed by roughness at the micrometric scale. Electrochemical characterization included electrochemical impedance spectroscopy (EIS) measurements in solutions containing hydrogen phosphide ions HPO₄⁼ to determine the changes in resistance of the chitosan/chitosan-molybdate membranes. As the ion concentration in the solution increased, the charge transfer resistance (R_tc) decreased. However, when ammonium molybdate was added to the chitosan membrane, it acted as a promoter for the uptake of HPO₄⁼ by the membrane. Thus, based on their characterization, chitosan biosensors have shown that they are capable of attracting anionic species, which contributes to the minimization of preference zones on the surface of a sensing electrode.

Available phosphate species

Chitosan molybdate membranes

electrochemical sensing

Electrochemical impedance spectroscopy

Nutrient availability is an essential characteristic to be measured in agricultural applications. The most important nutrients needed for optimal plant growth are the macronutrients: nitrogen (N), phosphorus (P), and potassium (K). Phosphorus in particular is also found in DNA and is responsible for many physiological and biochemical processes. In addition, phosphorus stimulates root growth, is involved in photosynthesis and respiration, and promotes seed formation. Plants absorb phosphorus mainly in the form of phosphate (H₂PO₄⁻ HPO₄⁼) [1, 2]. For the quantification and detection of nutrients in the soil, as is well known, agriculture has begun to use measuring instruments in real-time, which has earned it the name Smart Agriculture [3–5]. In this context, the new technologies that have emerged in agriculture as part of Industry 4.0 are being used to increase the quantity and quality of production, make the best use of resources, and minimize environmental impact [6]. The development of sensors based on natural, environmentally friendly, and low-cost materials is also necessary.

Chitosan is an amino polysaccharide biopolymer composed of randomly distributed units of β-(1–4) D-glucosamine and N-acetyl-D-glucosamine. It is commercially prepared by partial deacetylation of chitin, which is a structural element in the exoskeleton of crustaceans and insects but is also found in the cells of some fungi, green algae, and some protozoa [7, 8]. It is a polyfunctional, biodegradable, and biocompatible material that has been proposed for a variety of very different applications. In agriculture, it has been proposed for the quantification of macronutrients in soil [9, 10]. Chemical species are detected by electrostatic forces and electrical charge capacity in a physical space such as a polymer membrane. Therefore, it is important to determine the electrostatic properties of chitosan in solution [11].

The amount of phosphate in the soil is generally high, but most of it is bound to the soil and only a small portion is directly available to the plant as soluble orthophosphate forms H₂PO₄⁻ and HPO₄⁼. Labile states such as certain phosphate iron and/or aluminum oxides and hydroxides and alkali phosphates serve as replenishers in the solution available to the plant, while nonlabile states such as calcium, iron, and aluminum phosphates or phytates are hardly or mostly unavailable to plants [12]. The availability of phosphate in the soil is highly dependent on pH. The best displacement of P occurs at a pH between 6 and 7. Soil acidification promotes the formation of aluminum and iron phosphates, while as soil pH increases, the risk of phosphorus fixation increases [13]. Typical total phosphorus levels (both soluble and insoluble) in soils range from 150 to 700 µg/g [14].

Traditionally, ammonium molybdate is used to determine phosphate levels in water and/or soil [15] to trigger a spontaneous reaction that produces a blue color complex. This method is characterized as EPA standard colorimetric method [16] for total phosphorus in water, while in the soil a previous step with KCl is required for an anionic exchange. However, this method takes about 2 hours for each sample (depending on the digestion capability). Based on the above statements, this work proposes to prepare a selective biomembrane (chitosan molybdate) that mimics the membrane of mycorrhizal fungi that adsorbs phosphate species to transport them into the root surface [17–19].

Since a membrane capable of accumulating ionic species can change its chemical capacity, electrochemical impedance spectroscopy (EIS) is a suitable technique to study the change of an electrical element as a function of ion concentration in a membrane. Assuming that available phosphate species can be dissolved by mixing a certain amount of soil in water, this article investigates the electric charge attributed to phosphate species by changing the chemical capacity of a chitosan-molybdate membrane. This extraordinary proposal is the first step of our group to use the most powerful part of each component of a selective biopolymer membrane sensor to contribute to smart agriculture.

2.1 Materials

Chitosan powder was purchased from Chemsavers at 90% deacetylation, glacial acetic acid (CH₃COOH 99.97% pure) and potassium dihydrogen phosphate (KH₂PO₄) were purchased from J.T. Baker, ammonium molybdate ((NH₄)₆Mo₇O₂₄ 4H₂O) was purchased from Fermont, stainless steel sheet was purchased from Precision Brand, and deionized water (H2O) was purchased from Meyer.

2.2 Methods

The chitosan precursor solution was prepared to obtain a 0.1% w/v chitosan in a 1% v/v aqueous solution of acetic acid.

The other precursor solution containing chitosan and ammonium molybdate tetra hydrated was prepared using the above-mentioned chitosan solution and adding the required amount to complete 5 mg/L (4 mM) of the molybdenum salt.

From these solutions, the chitosan and chitosan-molybdate membranes were cast on stainless steel and stainless steel/carbon electrodes, adding 100 µL of the precursor solution over an area of 1 cm², and drying in an oven at 40°C, this step was repeated five times.

Two different standard solutions of phosphate (1 and 10 mg/L) were prepared to dissolve adequate quantities of potassium dihydrogen phosphate in deionized water and were used as synthetic sources of phosphate for electrochemical determinations.

The zeta potential of chitosan solutions was obtained in a Litesizer 500 of Anton Paar using the electrophoretic mobility principle. AFM images of chitosan and chitosan-molybdate membranes on the stainless-steel electrode were obtained using the Easyscan 2 from Nanosurf. FTIR-ATR of both membranes were collected in a Perkin Elmer Spectrum Two. Electrochemical characterization; cyclic voltammetry and Impedance Spectroscopy was traced in a Princeton Applied Research PARSTAT2273 using a three-electrode cell employing Ag/AgCl as a reference electrode and a counter electrode of Pt wire.

3.1 Determination of Surface Charge of Chitosan in Solution

The zeta potential (ζ)) was measured to investigate the surface charge of a 0.1% w/v chitosan solution at different pH values. Figure 1 shows that at a low pH (pH = 3), the amino groups present in the chitosan are protonated, resulting in a positive surface charge. As the pH increases, the proton is gradually released from the amino groups, leading to a decrease in surface charge. The continuous decrease in surface charge with increasing pH indicates further deprotonation of the amino groups and the acquisition of negative surface charges on chitosan. The isoelectric point is found at pH = 8, where the surface charge of chitosan stabilizes and approaches zero. This means that at this pH, the number of protonated amino groups is equal to the number of deprotonated groups. Moreover, the negative surface charge observed at pH above 8 can be attributed to the presence of deprotonated amino groups and other charged species in the chitosan structure. Since chitosan has a positive surface charge at pH less than 7, favorable electrostatic interactions may occur with negatively charged ions as phosphate species. These interactions may determine the adsorption of phosphate species in chitosan membranes through electrostatic complex formation [11].

3.2 Morphological Characterization of Chitosan Membranes

3.2.1 Atomic Force Microscopy

Characterization of the morphology and roughness of the chitosan and chitosan-molybdate membranes was performed by atomic force microscopy (AFM) in contact mode. The thickness of the membranes deposited on the stainless-steel electrode was analyzed to avoid interferences caused by the presence of a carbon film during the casting process.

The morphological characterization of the stainless steel/chitosan membrane is shown in Fig. 2(a)-(b). Figure 2(a) shows a general view of the membrane morphology, which is homogeneous with no aggregates and no perceptible particle shape at a window size of 100 micrometers. To ensure the presence of the membrane, an indentation in the membrane can be seen on the lower right side of the image to distinguish it from the stainless-steel substrate; in this image, the roughness of the membrane surface was measured and an average value (Sa) of 74.79 nm was obtained. Figure 2(b) shows a 3D projection of the film with an image size of 2.5 micrometers. This image shows a rough film with some protrusions on the order of 10 nm and an average roughness (Sa) of 1.776 nm, confirming that the film looks practically smooth at lower magnification. To determine the thickness of the chitosan membrane, a scalpel was used to scratch the surface. Figure 2(c) shows the image of the interface between steel and membrane. By measuring a line roughness in this area (Fig. 2(d)), it is possible to determine the height difference and thus the thickness of the applied chitosan film. A film thickness of 1.5 µm was determined.

The morphological characterization of the stainless steel/chitosan molybdate membrane is shown in Fig. 3 (a)-(b). Figure 3 (a) shows a general overview of the membrane morphology. It is less smooth than the chitosan membrane, but also homogeneous and exhibits some porosity. The roughness of the membrane surface was determined and gave an average value (Sa) of 81.73 nm. The 3D projection of the film with an image size of 2.5 micrometers (Fig. 3 (b)) shows the presence of a less rough film with projections in the order of 20–45 nm and an average roughness (Sa) of 13.07 nm. To determine the thickness of the chitosan-molybdate membrane, a scratch was made on the surface with a needle. Figure 3(c) shows the image of the interface between steel and membrane. By measuring a line roughness in this area (Fig. 3(d)), it is possible to determine the height difference and thus the thickness of the deposited chitosan film. A film thickness of 2.59 µm was determined. The change in morphology and roughness of the chitosan-molybdate membrane indicates that the presence of the molybdate ion during the casting process of the membrane does not increase the relative size of the particles, but it does increase their porosity, which is certainly due to the interaction of the molybdate ion with the chitosan causing segregation of the micelles during the drying process.

3.2.2 FTIR spectroscopy

The FTIR spectra of the chitosan powder (Ch_P), the chitosan membrane (Ch_M), and the chitosan-molybdate membrane (Ch/M_M) are shown in Fig. 4. In the three spectra, a peak at 3400 cm^− 1 is observed, which corresponds to the O-H stretch, and low-intensity signals at 2920 and 2880 cm^− 1 are assigned to the C-H stretch, which is particular to –CH2– groups. At 1650 cm-¹ a stretching peak is observed that corresponds to C = O, at 1420, 1380, and 1075 cm^− 1 the N-H, C-H, and C-O stretching signals are observed respectively. The FTIR spectra correspond which those reported for chitosan. In the case of the chitosan membrane, it is observed that the signals are intensified, this is attributed to greater compaction in the chitosan membrane concerning chitosan powder and chitosan-molybdate membrane, corroborating what is observed in the morphological characterization. Additionally, the chitosan-molybdate membrane does not show any important modification in its surface [20, 21].

3.3 Electrochemical Characterization

The first electrochemical characterization of the prepared chitosan membranes was performed using cyclic voltammetry at a scan rate of 25 mV/s to determine the oxidation-reduction signals of the molybdate ion MoO₄⁼ in the presence of orthophosphate ions, reported in previous electrochemical studies [12]. Electrodes with membranes of different thicknesses were analyzed to observe their electrochemical performance

Figure 6 (a)-(b) shows the cyclic voltammetry results of 2 different electrodes. Figure 6 (a) shows the voltammetric response of a thin film of chitosan (150 nm) deposited on a stainless-steel substrate immersed in a solution of 640 mg/L (NH₄)₆Mo₇O₂₄ at pH = 1. The positive scan without the presence of phosphate ions shows two peaks, the first at -0.17 V vs Ag/AgCl is related to the electrochemical oxidation of Mo(II) → Mo(IV) and the second for the oxidation of Mo(IV) → Mo(VI), for the negative scan the two signals corresponding to the molybdenum reduction processes are detected at -0.22 V and − 0.07 V vs. Ag/AgCl, which are associated with the electrochemical reduction of Mo(VI) → Mo(IV) and Mo(IV) → Mo(II), respectively [23]. However, the presence of the chitosan membrane reduces the intensity of the signals associated with the molybdenum oxidation-reduction processes by gradually increasing the phosphate concentration. This can be attributed to the interaction between the amino groups in the chitosan membrane and the orthophosphate ion in the analyte, which reach equilibrium when adsorbed on the membrane surface, limiting the interaction of the molybdate ion with the surface. As the thickness of the chitosan membrane increases, the voltammetric analysis shows a capacitive response in all cases, with only the signals related to the oxidation-reduction processes of Mo(II) ↔ Mo(IV) being minimally observed. Moreover, the reduction of the capacitive current confirms the favoring of the adsorption of the HPO₄⁼ species on the surface of the chitosan membrane, avoiding the interaction of the MoO₄⁼ ion, for which the voltammetric analysis is not the most appropriate way to quantify available (dissolved) phosphate species.

At this point, the polarization of either chitosan and chitosan-molybdate membranes have shown their sensitivity to phosphate species; although a proper quantification is still a challenge. Considering that the biopolymeric membrane tends to accumulate phosphate species, it is natural to investigate if an electrochemical impedance analysis could give a quantitative evaluation of chitosan membranes capacity to accumulate the available phosphorous species.

The determination of the phosphate sensor capacitance of the fabricated electrodes was then performed using electrochemical impedance in a frequency range from 100 kHz to 1 Hz. For this purpose, the electrodes were analyzed in a 3-electrode cell with a reference electrode of Ag/AgCl and a platinum wire as a counter electrode.

The Nyquist plots of eS_Ch, eSC_Ch, eS_Ch/M, and eSC_Ch/M are shown in Fig. 7(a)-(d). The characteristic shape of the spectra corresponds to a process where at low frequencies the diffusion of the analyte into the solution is not evident, while at high and medium frequencies the formation of the characteristic semicircle can be observed, which results from the parallel response of the charge transfer resistance (R_ct) and the chemical capacitance (Cµ) corresponding to an equivalent circuit R1 + CPC2/R2. Analysis of the Nyquist diagrams shows in all cases a decrease in R_ct with increasing concentration of HPO₄⁼ ion in the solution. This effect can be attributed to ion absorption by the chitosan membrane. In addition, an increase in the Cµ of the electrodes is observed as the concentration of HPO₄⁼ in the solution increases, which is related to the increase in the number of ions adsorbed on the membrane surface.

The Nyquist plots for the chitosan membranes deposited on both electrodes (steel and steel/carbon) show a higher R_ct resistance than the membranes containing molybdate, which reach values close to 50 kΩ and 70 kΩ at a concentration of 1 mg/L, while a decrease in R_ct is observed as the concentration increases, promoted by the interaction of the HPO₄⁼ ion with the membrane surface, increasing the conductivity. The change in R_ct value shows a rather linear trend for the eS_Ch electrode, while in the case of the chitosan-molybdate membranes, lower R_ct values are observed at low concentrations, with values between 40 kΩ and 30 kΩ for membranes deposited on steel and Steel/carbon electrodes are determined, in this case the linear trend in terms of decrease of R_ct with the increase of concentration of HPO₄⁼ ion is more evident for the carbon surface electrode, indicating that the presence of molybdate in the film improves the conductivity and that this ion also has a better interaction with the carbon film. This suggests that ammonium molybdate acts as a promoter for the uptake of HPO₄⁼ by the membrane and facilitates the charge transfer at the electrodes [22].

Figure 8 (a)-(d) show the values of R_ct and Cµ obtained by fitting the equivalent circuit (R1 + CPC2/R2) according to the shape of the impedance spectrum. It can be observed that the best linear fit for R_ct is obtained from eS_Ch and eSC_Ch/M, while the eSC_Ch electrode shows a semi-logarithmic trend. The chemical capacitance values in the case of the chitosan membranes show opposite trends, while for the chitosan-molybdate membranes they show a "more "linear" behavior. This indicates that the HPO₄⁼ ions are more homogeneously distributed on the surface of the membrane and this phenomenon can be related to its porosity. Therefore, the eSC_Ch/M electrode is considered to have better performance as a means of detecting phosphate in agricultural soils.

AFM characterization of chitosan and chitosan-molybdate membranes indicated that the inclusion of ammonium molybdate made a rough surface and a thicker layer (with the same mass) suggesting that molybdate may generate a partial segregation of chitosan micelles. However, the sensing capacity was improved as shown in the electrochemical impedance analysis due to a better distribution of the HPO₄⁼ ions on the membrane surface, relating this phenomenon with a larger porosity. Therefore, the stainless-steel/carbon/chitosan-molybdate electrode is considered to have better performance as a means of detecting phosphate in agricultural soils.

Chitosan and chitosan-molybdate membranes were also tested as adsorbent material of orthophosphate species, finding that voltametric measurements are strongly influenced by the capacitance of the membrane.

Electrochemical impedance spectroscopy and their use as a quantification technique showed to be a more suitable form to measure the changes of available phosphates in solution, giving a good will for a smart agriculture methodology, minimizing quantification times and optimizing chemical and organic fertilization processes.

Conflicts of Interest:

The authors declare no conflicts of interest.

Acknowledgments

The Authors thank the Tecnologico Nacional de México/I.T. Tijuana for providing the facilities for this research. M.A. Beltrán-Ortega would like to thank to CONAHCyT for the scholarship provided for her doctorate science research.

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You are reading this latest preprint version

Aiming to a Smart Agriculture through the Electrochemical Impedance of a Chitosan-Molybdate Membrane as a Function of Available Phosphate Species

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Experimental details

2.1 Materials

2.2 Methods

3. Results and discussion

3.1 Determination of Surface Charge of Chitosan in Solution

3.2 Morphological Characterization of Chitosan Membranes

3.2.1 Atomic Force Microscopy

3.2.2 FTIR spectroscopy

3.3 Electrochemical Characterization

4. Conclusions

Declarations

Conflicts of Interest:

Acknowledgments

References

Additional Declarations

Status:

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