2.2.1 Preparation of walnut shells biochar

The walnut shells were selected, washed with deionized water to remove any impurities, dried at 105 °C for 24 hrs. to eliminate extra water, and then pulverized with a roller mill to obtain a product with 100–300 µm uniform particle size (Albatrni et al., 2022; Salih et al., 2017). The crushed product was pyrolyzed in a muffle furnace heated to 600 °C for 2 hrs. under vacuum. The obtained biochar was sealed in a hermetic vessel for further use.

2.2.2 Preparation of real samples

For the practical tests of the sensor platform, soil and water samples were collected from Bouregrag River (Rabat-Sale Region). The soil samples were dried (at 105 °C for 24 h) and then pulverized with a crusher to obtain a soil powder of 50 μm sieve. For the extraction of HM ions, one gram of soil was mixed in 40 mL of acetate buffer solution ABS (0.10 M sodium acetate and acetic acid, pH 5.5), sonicated for 10 min., and then centrifuged for 15 min at 3500 rpm and finally filtered to obtain the soil extract solution. The river water sample was filtered (0.45 μm membrane) without any further pretreatment. Before the tests, the river water solution was diluted 2x with 0.10 M acetate buffer solution and adjusted the pH to 5.5. Finally, the soil and water samples were stored in a fridge at 4.0 °C for further use.

The standard solution containing 100 nM of Cd²⁺, Pb²⁺, Cu²⁺, and Hg²⁺ was applied to the samples and diluted to 0 nM of analytes. Then, concentrations of Cd²⁺, Pb²⁺, Cu²⁺, and Hg²⁺ in the soil and water samples were assessed by using the SWV technique with the same as above mentioned optimal conditions.

2.2.3 Characterizations of walnut shells and biochar

The thermal behavior of the samples in the presence of an inert (99.999 % purity nitrogen) and oxidizing (99.999 % purity synthetic air) atmosphere was analyzed by a simultaneous thermo-gravimetric and differential scanning calorimetric analyzer (LabsysEvo, France). The measurements were performed in flowing (90 mL/min) gases, in alumina crucibles, in a 20–600 °C temperature range, with a heating rate of 10 °C/min. The obtained data were baseline corrected and further processed with the thermal analyzer’s software (Calisto Processing, ver. 2.092). The thermal analyzer (both the temperature scale and calorimetric sensitivity) was calibrated by a multipoint calibration method, in which seven different certified reference materials were used to cover the thermal analyzer’s entire operating temperature range.

The Walnut shell powder and its pyrolysis product were studied by XRD (Philips XPert Pro MPD). The Fourier transform infrared spectrometry was used to identify the functional groups that were present in the raw material and its pyrolysis product. FTIR measurements were recorded using Jasco FT/IR-4600 apparatus (Tokyo, Japan), equipped with a Jasco-ATR-Pro-One single reflection diamond ATR accessory (incident angle 45°), and a DLATGS detector operating in the 4000–400 cm⁻¹ interval. A resolution of 4 cm⁻¹ and the co-addiction of 64 individual spectra were applied. Before the evaluation, an ATR correction (Jasco Spectra Manager v.2, Spectra analysis module version 2.15.11) was performed on the raw spectra. An SEM coupled with an EDX apparatus (FEI-Quanta 650) was used to scan the surface morphology and chemical composition of the walnut shell powder before and after pyrolysis.

3.1.1 Thermal measurements

The mass loss and heat flow (TG-DSC) curves of the walnut shell powder and its biochar, measured under N₂ and synthetic air atmosphere, from 20 to 600 °C, are shown in Fig. 1. It is observed that for the walnut shell product under oxidative (Fig. 1a) and inert conditions (Fig. 1b), small mass loss of 7 % (air) and 4 % (N₂) was observed up to 130 °C, accompanied by a small endotherm, due to the loss of physically bound water (moisture).

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Fig. 1

TG-DSC curves of the walnut shell powder (a - oxidative, b - pyrolytic) and its biochar (c - oxidative, d - pyrolytic).

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At temperatures from 200 °C to 500 °C in air, and 400 °C in nitrogen, the oxidative decomposition or pyrolysis of walnut shells occurs, i.e. the removal of chemically bound water, condensation of the remaining carbon content, volatilization of lighter compounds, and, in the case of air-treatment: oxidative burning. In both cases, the mass loss is separated into two steps around 350 °C (more characteristic in the presence of air), which is probably due to the structural inhomogeneity of the shell material, regarding the presence of both the less organized hemicellulose and the denser or more organized cellulose content. While at the end of the second step of mass loss, almost quantitative volatilization took place in the air, under inert conditions a much lower mass loss value was obtained, the rate of the mass loss decreased significantly above 400 °C, and slow pyrolytic degradation of the sample took place.

Regarding the energetics, under an oxidative atmosphere, overlapping exothermic peaks with peak maxima of 339 °C and 450 °C were found, which confirms that the decomposition and volatilization steps of the WS are accompanied by exothermic burning. Under inert conditions the shape of the heat flow profile is similar, but almost two orders of magnitude lower (Senneca et al., 2018; Xu et al., 2017), which points out that the pyrolyzed WS still contains some oxygen, which intramolecularly oxidizes carbon, leading to the appearance of the small overlapped exotherms.

Up to 500 °C, 97.45 % of the starting mass is lost in the air atmosphere, while under pyrolytic conditions (N₂ atmosphere) the corresponding mass loss was 71.97 %. In the case of the experiment performed in air, the remaining residue consisted of inorganic, mineral matter from the raw walnut shell powder, while in the case of the experiment performed in N₂, carbon was also present besides the above-mentioned inorganic matter.

From Fig. 1c, the mass loss of biochar in the oxidative atmosphere occurred in two stages: a slow mass loss of 15.40 % is visible up to 200 °C, due to the release of water, above this temperature, a second, large step can be seen, when almost all the carbon content was burnt to CO₂. In the case of the N₂ atmosphere (Fig. 1d), the biochar is generally stable in the whole temperature range with a weight loss of only about 10.40 %. The small weight loss could be resulting from the oxidative effect of the residual oxygen content (∼10 ppm) in the nitrogen gas and/or from the intramolecular oxidation by oxygen-containing moieties in the sample. Based on this result, the raw walnut shells can be fully pyrolyzed at 600 °C.

3.1.2 X-ray diffraction analysis

XRD was used to determine the eventual crystalline structure of the raw walnut shell and its biochar, as shown in Fig. 2. In the case of the raw walnut shell, two peaks were detected at a 2θ angle of around 18° and 22° assigned to a crystalline region of cellulose (Gupta et al., 2019). For the biochar, also two peaks are observed at 2θ values of around 23° and 43°, corresponding to (0 0 2) and (1 0 0) crystal planes of graphitic carbon (Salih et al., 2017; Janu et al., 2021). In the case of WS-BC (black curve in Fig. 2), the (0 0 2) diffraction peak located at around 22–26° (2θ) represented the layer spacing and thickness of the aromatic microcrystalline structure. The (1 0 0) diffraction peak at 42–45° (2θ) demonstrated that the microcrystalline structure changed from disorder to order under high-temperature pyrolysis.

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Fig. 2

XRD characterization of raw WS biomass (red) and its biochar (black).

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The dispersion peak was not detected in XRD patterns, which demonstrated that the aliphatic chain in WS-BC was completely split. The parameters of lattice structure such as d₀₀₂ (inter-layer spacing), L_c (crystallite height), and L_α (crystallite diameter) were shown in Table 1. The value of d₀₀₂ was 4.02, revealing that the inter-layer spacing of crystallite decreased and lattice arrangement was more ordered in biochars undergoing high-temperature pyrolysis. The values of L_c and L_α were 0.07 and 0.144 nm, respectively. This could be explained by the increase in the size of crystallite, which resulted in promoted graphite-like carbon structures in WS-BC. During the pyrolysis process, the crystallinity of the walnut shell reduces while its amorphous characteristics increase.

3.1.3 FTIR analysis

To determine the presence of various functional groups on the WS biomass and its biochar, FT-IR spectroscopy was performed on the samples. The FTIR spectrum was measured in the range of 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹ and the co-addiction of 64 individual spectra, as shown in Fig. 3. Major components of the WS biomass are hemicellulose, cellulose, and lignin. Lignin, unlike cellulose, possesses an olefinic carbon–carbon (-C⚌C-) double bond in cyclic as well as side chains and is aromatic (Yang and Qiu, 2010).

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Fig. 3

Identification of surface functional groups by FTIR spectra of the bare and walnut shell biochar.

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For the walnut shell, FTIR measurement records the intensive peaks at 3334 cm⁻¹ which corresponds to the υO-H stretching vibration (e.g. alcohols, phenols, and carboxylic acids); υ_asCH₂ and υ_sCH₃ stretching vibrations (2925 cm⁻¹) and υ_sCH₂ and υ_sCH₃ stretching vibrations (2850 cm⁻¹) of methylene and methyl groups in the lignin structure; υC = O stretching vibration at 1730 cm⁻¹; υ_asCOO stretching vibration at 1600 cm⁻¹; υC–O (carboxylic and phenolic) and υ_asC—O—C (ester) stretching vibration at 1230 cm⁻¹ and υC–O stretching vibration of primary –OH groups 1029 cm⁻¹. Lastly, the band assigned to the –OH out-of-plane bending vibrations band is located at 666 cm⁻¹ (Yang and Qiu, 2010; Lin et al., 2021).

The FTIR spectrum of the prepared walnut shell biochar is likewise shown in Fig. 3. All of the absorbance peaks corresponding to any ‘O’ content became removed or highly reduced after the pyrolysis processes applied at 600 °C for 2 h to the WS, due to the dehydration/dehydroxylation of the raw materials with the formation of water, and the release of light volatile matters. In the heating process. The BC spectrum reports that the alkane-related stretching vibrations (2925 cm⁻¹ and 2850 cm⁻¹), and the γC–H out of plane vibrations of C—H bonds attached to pillar atoms of C⚌C structures at 850 cm⁻¹ became dominant, as well as γC–H and γC–C deformation vibrations of di- and tri-substituted aromatic structures stand out of the background in the 1500–500 cm⁻¹ region.

A general note is that the FTIR study of biochar was carried out before mixing into the CPE and before it was modified with a −0.5 V - +2.0 V oxidative polymerization of tyrosine. Carbon-based electrodes can be easily oxidized with 0.0–2.0 V oxidative CV scanning (Li et al., 2019). This implies that after the fabrication of tyrosine not only a layer of p-Tyr but numerous –OH and –COOH groups were introduced onto the surface of the electrode.

3.1.4 Morphological study-SEM-EDX analysis

To examine the topography, morphology, and elemental composition SEM-EDX images of WS biomass and its corresponding biochar produced at 600 °C are shown in Fig. 4a, 4b, and 4c.

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Fig. 4

SEM images of the walnut shell: Biomass (a), and the Biochar (b) at 10 and 100 μm resolution, EDX images of Biomass and Biochar (c), (green line indicates the pore diameter, see at 10 μm resolution).

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Fig. 4a shows that the surface of the walnut shell biomass is a completely fused structure with no visible pores at 10 and 100 μm resolutions. In Fig. 4b, the micrographs of the biochar morphology show the presence of voids and more porosity on its surface compared to that of the raw material. Comparing the SEM surfaces of the WS biomass and its WS-BC showed that the morphology became more complex because of aggregation and alteration of mineral compounds. Furthermore, due to the decomposition and volatilization of raw materials, a noticeable number of pores of different sizes (1.147 to 1.825 μm) appeared in WS-BC, as seen in Fig. 4b, at the resolution of 10 μm. This confirms that the produced WS-BC has a larger surface area than the raw WS biomass. This can be attributed to the fact that the heat treatment improves the porous structure of biochar due to the loss of the volatile matter in the walnut shell.

EDX analysis results are shown in Fig. 4c, where the results are presented in the mass percentage of the samples. Qualitative elemental composition analysis of raw walnut shell powder and its biochar using energy dispersive spectroscopy is listed in Table 2. According to Fig. 4c, carbon seemed to be the dominant element (43, 87 %), followed by oxygen (57, 11 %), for biomass and biochar, respectively. Traces of Ca (0.23, 0.94 %), and K (0.83 for the biomass), were also found in all biochars, but in very small amounts. The obtained results show that in the elemental composition of biochar a relatively high carbon content and much lower oxygen content whereas results for WS indicate that C (42.59 %) and O (56.97 %) while the values for the WS-BC are C (87.13 %) and O (11.09 %). Thus O/C ratio decreases from 1.34 to 0.13 with the pyrolysis of the walnut shell, indicating that pyrolysis is a suitable process for enriching carbon in biochar, due to dehydration-carbonization reactions at high temperatures (Xu et al., 2017).

3.2.1 Fabrication of the p-Tyr-BC-CPE modified electrode

The poly-tyrosine film was electrochemically deposited on the surface of BC-CPE using cyclic voltammetry from 2.0 mM l-tyrosine monomer dissolved in 0.2 M PBS at pH 7.0 in an electrochemical cell over the potential range of −0.6 V to 2.0 V at a scan rate of 100 mV.s⁻¹ for 20 successive cycles (Zbair et al., 2019). From Fig. 5, the first oxidation peak potential of the l-tyrosine monomer was observed at 1.01 V and the reduction peak appeared at −0.26 V. On continuous scanning, the anodic peak decreased gradually with the increase of the cycle number, indicating that the electro-polymerization of tyrosine was successful. The cathodic peak increased with the increase in cycling time, indicating that the poly-tyrosine film was growing on the modified electrode surface. After 20 cycles, a blank-blue film could be observed on the surface of BC-CPE, confirming the poly-tyrosine electrodeposition.

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Fig. 5

Cyclic voltammograms of p-Tyr-BC-CPE preparation in PBS (0.2 M, pH 7) containing 2.0 mM of Tyrosine at V = 100 mV.s⁻¹ from 1 to 20 cycles.

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3.2.2 Electrochemical characterization of p-Tyr-BC-CPE

The electrochemical properties of the modified electrodes were studied by CV and EIS using [Fe(CN)₆]^-3/-4 as a redox probe. Fig. 6a shows the CV responses of unmodified CPE and WS-CPE, BC-CPE, p-Tyr-CPE, and p-Tyr-BC-CPE modified electrodes in 1.0 mM [Fe(CN)₆]^-3/-4 electrolyte solution containing 5.0 mM KCl at a potential scan rate = 100 mV.s⁻¹. The bare CPE is characterized by a reversible redox reaction with a ΔE of 295 mV, indicating slow electron transfer on the CPE.

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Fig. 6

CV voltammograms (a) and EIS Nyquist plot (b) for bare CPE, and WS-CPE, BC- CPE, p-Tyr-CPE, p-Tyr-BC-CPE coated electrodes in 1.0 mM [Fe(CN)₆]^3-/4- containing 0.5 mM KCl.

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For the WS-CPE, the potential difference and the peak current show no significant difference between this response and that of CPE. This result is due to the poor conductivity of walnut shell biomass. After its modification with pyrolyzed biochar, the charge transfer was ameliorated, leading to a ΔE of 228 mV and a current increase to 0.064 µA, possibly due to the presence of the hydroxyl and carboxylic functional groups in the biochar surface, providing a large active surface and high adsorption capacity (Leftheriotis et al., 2007).

The p-Tyr-CPE showed significant improvement in redox behavior and current intensities. The potential difference ΔE observed is 95 mV. The intensity redox peaks are well defined and their intensity increased to 0.233 µA. These results indicate that the p-tyrosine film was successfully deposited on the electrode surface and can effectively promote electron transfer on the electrode surface (Wang et al., 2021).

The p-Tyr-BC-CPE showed a lower redox peak separation of 57 mV and a current increase to 0.556 µA. This is due to the synergetic effect between p-tyrosine film and biochar by increasing the electroactive surface area and improving the electrical properties of the electrode. It is important to note that p-Tyr-BC-CPE has much better conductive characteristics than p-Tyr-CPE and BC-CPE alone. This would indicate a serious synergistic effect between p-Tyr film and the BC additive, however, one should keep in mind that the CV polarization between −0.5 V and + 2.0 V can cause oxidative stress for the carbon materials, thus it cannot be excluded that the pyrolyzed biochar (with almost no O-content, due to IR report) suffered oxidation as well, and this might have contributed to the enhanced electro-activity of the p-Tyr-BC-CPE.

The electrochemically active surface area (A) of the electrodes was estimated using the Randles-Sevcik Eqs. (1) (Loaiza et al., 2011):

Where I_p, n, A, D, υ, and C represent the peak current, the number of electrons changed (n = 1), the electrochemical surface area of the electrode, the diffusion coefficient (7.60 × 10^-6 cm².s⁻¹), the scan rate, and the concentration of K₃[Fe(CN)₆], respectively.

The electroactive surface areas for CPE, WS-CPE, BC-CPE, p-Tyr-CPE and p-Tyr-BC-CPE were 0.033, 0.060, 0.091, 0.33, and 0.75 mm², respectively. This shows that the p-Tyr-BC-CPE (A = 0.75 mm²) had better electrochemical reactivity and, as such, was provided for simultaneous detection of Cd²⁺, Pb²⁺, Cu^2+, and Hg²⁺.

Electrochemical impedance spectroscopy (EIS) is applied to investigate the charge transfer processes on the surfaces of different coated electrodes. The impedance responses of unmodified and modified electrodes, measured at a 0.35 V bias potential (open circuit potential (OCP)) and frequency range from 10^-1 to 105 Hz, were registered. EIS Nyquist plots for bare CPE, and WS-CPE, BC-CPE, p-Tyr-CPE, and p-Tyr-BC-CPE coated electrodes in 1.0 mM [Fe(CN)6]^3-/4- containing 0.5 mM KCl, are represented in Fig. 6b. The resulting impedance diagrams are composed of a semicircle part at the high-frequency range, characterized by the parallel connection of the charge transfer resistance (R_ct) and double-layer capacitance (C_dl), while the linear part at low frequencies is related to the Warburg-diffusion process (Xiong et al., 2021). The Nyquist diagrams data were fitted by the standard Randle’s equivalent circuit, having a chi-squared coefficient of 10^-3.

The solution resistance (R_s) is determined by the conductivity of the electrolyte (which is determined by its composition) and the distance between the working electrode and the reference electrode. Both are specific to the conditions of the measurement, not to the tested system. The main parameters of the standard Randle’s equivalent circuit affected by different coatings are the charge-transfer resistance (R_ct) and double layer capacitance (C_dl) as shown in Table 3. The R_ct values of the bare CPE and the WS-CPE, BC-CPE, p-Tyr-CPE, and p-Try-BC-CPE coated electrodes, are tabulated in Table 3. The R_ct value of the bare CPE electrode was obtained to be 613.0 Ω.cm². The R_ct values of WS-CPE and BC-CPE electrodes were lower, 511.7 Ω.cm² and 417.7 Ω.cm², respectively. The lower charge transfer resistance of modified electrodes is most probably the result of the increase of the effective surface area and the presence of surface active functional groups in the biomass layer. A much more significant decrease of R_ct was obtained for poly-tyrosine modified electrodes: 309 Ω.cm² for p-Tyr-CPE, and 158.8 Ω.cm² for p-Tyr-BC-CPE. This means of 50 % decrease in the case of p-Tyr-CPE compared to bare CPE and a 62 % decrease in the case of p-Tyr-BC-CPE compared to BC-CPE electrodes.

The R_p decrease is related to the presence of p-Tyr-biochar which could effectively accelerate the electron transfer, which is consistent with the CV results. The double-layer capacitance values characterize the interfacial properties of electrodes. There was only a minor difference obtained between C_dl of bare CPE and WS-CPE electrodes, 4.9 μF.cm⁻² and 5.3 μF.cm⁻², respectively, because of the similar surface condition (Table 3). The BC-modified CPE electrode has increased C_dl (19.1 μF.cm⁻²), which points out the presence of surface-active functional groups in the porous BC-layer. A much larger increase in C_dl is attained with p-Tyr-modified electrodes, 993 μF.cm⁻² for p-Tyr-CPE and 1817 μF.cm⁻² for p-Tyr-BC-CPE electrodes. This significant increase in the double layer capacitance can be attributed to various reasons, including the increase in the effective surface area and the presence of surface active functional groups in the thick porous layers.

3.2.3 Effect of scan rate on the sensor efficiency

To determine the electrochemical mechanism of the Fe²⁺/Fe³⁺ redox process on the prepared p-Tyr-BC-CPE, the influence of scan rate variation was investigated. Fig. 7a shows the CV measurements in 1.0 mM of [Fe(CN)₆]^3-/4- at sweep rates ranging from 10 to 200 mV.s⁻¹. The results show a linear relationship between the redox peak currents and the square root of the scan rate (υ^1/2), possibly indicating that the reaction process was controlled by diffusion (Yang et al., 2014; Pamuk et al., 2013). The relationship between the redox peak currents and (υ^1/2), the dependence between the redox peak currents and the scan rate (υ), and the relationship between the logarithm of the redox peak currents and the logarithm of the scan rates (log(υ)) are all linear as shown by Fig. 7b, 7c, and 7d, respectively.

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Fig. 7

Scan rate effect of the modified electrode from 10 to 200 mV.s⁻¹ (a), the effect of the variation of υ^1/2 on the redox current intensity (b), the effect of the variation of υ on the redox current intensity (c), and the effect of the variation of Log(υ) on log(I) (d) (potentials are referred to SCE).

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Based on the literature review, when the slope of the logarithm of peak current versus the logarithm of scan rate has a value between 0.5 and 1.0, that means the detection mechanism is diffusion/adsorption controlled. As shown in Fig. 7d, the slope was 0.55 which is categorized as a mixed diffusion-adsorption controlled mechanism (Wang et al., 2011).