Serotonin electrochemical detection in tomatoes at MWCNT-AONP nanocomposite modified electrode

The chemicals listed below were acquired and used as the manufacturers delivered them. Sigma Aldrich (USA) provided antimony trichloride (SbCl₃) (99%), multi-walled carbon nanotubes (MWCNTs) (O.D. × I.D. × L 10 nm ± 1 nm × 4.5 nm ± 0.5 nm × 3− ∼6 μm) (98%), potassium hexacyanoferrate (III) K₃[Fe(CN)₆] (99%), Potassium hexacyanoferrate (IV) K₄ [Fe(CN)₆] (99%), serotonin hydrochloride powder (≥98%), ascorbic acid (99.50%), sodium hydroxide (NaOH) (98%), hydrochloric acid (HCL) (32%), and N, N- dimethylformamide (DMF) (C₃H₇NO) (99%). Toluene (C₇H₈) (99%) was acquired from the SAARCHEM Pty Ltd (RSA). Sodium hydrogen orthophosphate (Na₂HPO₄) (99%) and sodium dihydrogen orthophosphate (NaH₂PO₄) (99%) were purchased from Glassworld Pty Ltd. (RSA) and Labchem company (RSA). Phosphate buffered saline (PBS) (pH 7) was prepared with appropriate amounts of Na₂HPO₄, and NaH₂PO₄ and the pH of the solution was adjusted accordingly using 0.1 M NaOH and 0.1 M HCL. While Nitric acid (HNO₃) (≥55%) was obtained from Merck Pty Ltd (RSA). Distilled water was utilized throughout the study to prepare chemicals. Electrochemical studies were done using a portable dropsense kit from Metrohm Pty Ltd. (RSA) containing biopotentiostat 300 (μstat-i 400 s), 910 potentiostat mini, Dropview 8400 software disc, one cable connector (CABSTAT1), boxed connector (DRP-DSX4MM), screen printed carbon electrodes (SPCE), and power adapter. VWR European company, a German manufacturer, supplied Whatman No 1 filter papers (size: 150 mm, particle retention: 5–13 μm).

Antimony oxide nanoparticles (AONPs) were synthesized using the hydrothermal method published by Chen et al [60]. To get a transparent solution, approximately 2 mM of antimony trichloride (Sb₂Cl₃) was transferred into 20 ml of toluene solution with rapid stirring. To make a lacteous colloid, 20 ml of distilled water was added to the solution. After that, the mixture was stirred for 15 min, and 6 M of NaOH was added to bring the pH to between 8 and 9. After another 20 min of agitation, the solution was placed into a 100 ml Teflon-lined stainless autoclave for 12 h at 120 °C. The resulting white powder was washed repeatedly with 50% ethanol solution and then evaporated at 60 °C for 6 h.

The synthesis of functionalized multi-walled carbon nanotubes (f-MWCNTs) was done via the nitric acid treatment method. Approximately 300 ml of 1 M nitric acid was used to dissolve 300 mg of multi-walled carbon nanotubes (designated as raw multiwall carbon nanotube: r-MWCNTS). Then the mixture was sonicated in ice water at 50 °C for 4 h. The mixture was filtered with deionized water until pH 7 was reached. The product was left overnight in an oven to dry [61].

Carry series 300, UV–vis spectrophotometer (Agilent technologies, Waldbronn, Germany), FTIR (Agilent technologies, Cary 600 series, Billerica, MA, USA), XRD (Bruker-AXS, Madison, USA), SEM (JEOL company, Peabody, MA, USA), and TEM (Tecnai G2 spirit FEI, USA) techniques were used to characterize all prepared nanomaterials, AONPs, f-MWCNTs, and MWCNT-AONP nanocomposite.

Approximately 2 mg of functionalized multi-walled carbon nanotubes (f-MWCNTs) and 6 mg of antimony oxide nanoparticles (AONPs) were suspended in a 2 ml N, N- dimethylformamide (DMF) solution. Then, the suspension mixture was agitated at ambient temperature for 48 h. The resultant nanocomposite was dried in an oven at 25 °C for 24 h [53].

The constructed electrodes were modified using the dried cast method. Roughly 2 mg of each synthesized nanomaterials (AONPs, f-MWCNTs, and MWCNT-AONP nanocomposite) were suspended in 1 ml DMF solution. Each suspension was ultra-sonicated for 30 min at room temperature. Then, 20 μl of each suspended mixture was dropped on the surface of the working electrode (SPCE) with a micropipette, then dried at room temperature to get the modified working electrodes [62].

The Dropview 8400 software programme from Metrohm Pty Ltd. (RSA) was used to conduct electrochemical experiments at 25 ± 1°C using 4 mm diameter screen-printed carbon electrodes (DropSense 110) consisting of the working electrode (A/AgCl), counter electrode, and the reference electrode connected to dropview 8400 910 potentiostat mini. Electrochemical characterization and electrocatalytic experiments at the constructed electrodes (SPCE-bare, SPCE/f-MWCNTs, SPCE-AONPs, and SPCE/MWCNT-AONP) were performed using 5 mM K [Fe(CN)₆]^3−/4− and 0.1 mM 5-HT dissolved in 0.1 M PBS (pH 7) at the scan rate of 25 mVs⁻¹ within −0.2–1.0 V potential window period using cyclic voltammetry (CV) technique. Electro-analytic experiments were conducted utilizing square wave voltammetry (SWV) to determine the proposed electrode's sensitivity, selectivity, and limit of detection. The parameters for the SWV method were set at the frequency (10 Hz), potential window ranging from −0.2–0.8 V, potential amplitude (0.01 V), Estep (0.01 V) and other parameters were left as they were at zero.

The capacity of the designed electrode to detect 5-HT in the real analysis was done using fresh tomatoes samples purchased from a local supermarket. The tomatoes were blended using a domestic electric blender. The resultant blend was then filtered using Whatman No 1 filter paper to obtain a clear, colourless tomato extract solution. A fixed volume of the tomatoes extract (1 ml) was added to different volumes of the stock solution to make up to 10 ml of the sample. The experiment was repeated three times using the SWV technique.

Figure 1 displays the UV–vis for (a) AONPs, (b) r-MWCNTs, f-MWCNTs, and AONP-MWCNT spectra, and (c) the absorption band energy gap of the synthesized nanoparticles. The spectrum in figure 1(a) indicates the formation of AONPs by an absorption peak at 297 nm [62–64]. A shift to a longer wavelength depends significantly on the particle size and aggregation of the particles [65]. The absence of any absorption peak between 400–800 nm region suggests that the synthesized AONPs can be used to manufacture non-linear optical sensors devices [63, 64]. The direct energy band-gap of AONPs was determined from Tauc's plot shown in figure 1(c). To obtain the direct band energy gap, a straight-line portion of (αhν) versus (hν) was extrapolated to zero. The direct band-gap energy was 3.26 eV [63]. The spectrum in figure 1(b) shows the UV spectrum of r-MWCNTs, f-MWCNTs, and MWCNT-AONP nanocomposite. From the spectrum, the absorption peaks at 285 nm and 345 nm observed in the AONP-MWCNT nanocomposite were assigned to f-MWCNTs π–π* transitions [66]. And a peak at 290 nm signifies the presence of AONPs in the AONP-MWCNT nanocomposite.

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3.1.2. FTIR characterization of synthesized materials

Figure 2 shows the FTIR bands of (a) AONPs, (b) raw multi-walled carbon nanotubes (r-MWCNTs), functionalized multi-walled carbon nanotubes (f-MWCNTs), and (c) AONP-MWCNT nanocomposite ranging from 450 cm⁻¹–4000 cm⁻¹ regions. Figure 2(a) illustrates the spectrum of f-MWCNTs and r-MWCNTs. The peaks of interest were the absorption bands at 1569 cm⁻¹, 1742 cm⁻¹, 2326 cm⁻¹, 3410 cm⁻¹, and 3780 cm⁻¹ which were assigned to the vibrational frequency of carboxylate anion (COO−), (C=O) of the (–COOH), (O–H) stretching from the –COOH, (–OH) stretching vibrations of the (−COOH), and free hydroxyl group O–H. [61, 67–69]. The presence of these functional groups in the spectra (figure 2(b)) indicated the presence of the (−COOH) on the surface wall of the MWCNTs, which confirmed the successful acid treatment of MWCNTs.

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The FTIR bands of AONPs and MWCNT-AONP nanocomposite are represented in figure 2(b). From the spectrum, the characteristics bands of all nanomaterials used to synthesis the nanocomposite are visible. The absorption band at 517 cm⁻¹, and 630 cm⁻¹ indicating the stretching of Sb–O and O–Sb–O of the AONPs, respectively, in the nanocomposite [53, 62–64]. The absorption bands at 3399 cm⁻¹ and 37126 cm⁻¹ were attributed to the stretching and vibration of (–OH) from the (–COOH), and the bending vibrations of the OH group in water respectively. Absorption bands at 1408 cm⁻¹, 1569 cm⁻¹, and 2326 cm⁻¹ indicated the vibrational band of C=C stretching benzenoid rings, (C=O) from the (–COOH) and O–H stretching from (–COOH).

3.1.3. X-ray diffraction of synthesized materials

XRD was utilized to determine the crystallinity and crystal structure of the synthesized AONPs. The XRD patterns of (a) AONPs, (b) fMWCNTs, and (c) MWCNT-AONP nanocomposite are shown in figure 3. The spectrum of AONPs in (figure 3(a)) revealed sharp and intense peaks with no diffraction halo, indicating that the prepared AONPs were of high crystallinity and purity [63]. The peaks at 2θ = 19.46° (110), 25.48° (111), 27.73° (222), 28.46° (121), 33.02° (002), 32.73° (131), 34.84° (012), 36.54° (200), 39.63° (032), 44.33° (042), 46.02° (231), 47.11° (240), 50.47° (161), 55.78° (170), 58.90° (242), and 60.90° (261) corresponds to the formation of AONPs and the formed AONPs showed an orthorhombic phase orientation [60]. Figure 3(b) depicts the XRD of f-MWCNTs. The diffraction peak at 2θ = 25.94° (002) and 2θ = 50.66° (004) corresponded to the amorphous nature of f-MWCNTs [53, 70]. Figure 3(c) represents the XRD image of the MWCNT-AONP nanocomposite showing distinctive diffraction peaks of all nanomaterials used to synthesize the nanocomposite. The presence of AONPs in the MWCNT-AONP nanocomposite is indicated by diffraction peaks at 2θ = 19.46° (110), 27.73° (222), 28.46° (121), 32.73° (131), 33.02°(002), 34.84° (012), 35.90° (200), 44.33° (042), 46.00° (231), 50.47° (161), and 55.78° (170). While the diffraction peak at 2θ = 25.96° (400) indicates the formation of f-MWCNTs in the nanocomposite.

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The SEM technique was used to determine the shape of the synthesized nanomaterials and examine the surface area of f-MWCNTs. This is because dry oxidation can cause damages and defects on the surface area of MWCNTs [69]. Shown in figure 4 are the SEM depictions of (a) AONPs, (b) f-MWCNTs, and (c) MWCNT-AONP nanocomposite. In figure 4(a), the morphology of synthesized AONPs was evenly distributed rod-like in shape. Figure 4(b) represents the SEM image of fMWCNTs via the acid treatment method. The morphology of f-MWCNTs was worm-like in shape somewhat similar to this previous study [69]. Moreover, the image showed no signs of any surface damage on the f-MWCNTs surface nor change in their size. SEM depictions in figure 4(c) represent the MWCNT-AONP nanocomposite. The image showed the attachment of AONPs on the surface walls of f-MWCNTs. The presence of AONPs on the surface wall of MWCNTs was taken to mean the successful formation of MWCNT-AONP nanocomposite.

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3.2.1. TEM characterization of synthesized materials

TEM is one of the critical microscopic techniques used in science mainly to elucidate the size of the prepared nanomaterials. Depicted in figure 5 are TEM micrographs of (a) AONPs and (b) MWCNT-AONP nanocomposite each at magnifications of 50 nm, and (c) particles size for AONPs. The average particle size for synthesized AONPs was found to be ≈ 25 nm in size while the largest formed nanoparticle reached the size of 95 nm as shown in figure 5(c).

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Figure 6 represents the comparative cyclic voltammogram for SPCE-bare, SPCE modified with raw MWCNTs (SPCE/raw-MWCNTs), and SPCE modified with functionalized MWCNTs (SPCE/f-MWCNTs) prepared in 5 mM K [Fe (CN) ₆]^3−/4− redox probe at a scan rate of 25 mVs⁻¹. The peak separations between the SPCE-bare, SPCE/r-MWCNTs, and SPCE/f-MWCNTs were 0.48 V, 0.16 V, and 0.15 V, individually. The peak separations exceeded the anticipated value of 0.059 V predicted for a fast one-electron transfer process for all electrodes. The anodic current peak at SPCE/f-MWCNTs was 1.58 and 2.29 times greater than SPCE/r-MWCNTs and SPCE-bare, respectively. This means that the SPCE/f-MWCNTs electrode is fast at electron transfer and has a greater electro-active surface area than the SPCE and SPCE/r-MWCNTs electrodes.

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Cyclic voltammograms in figures 7(a) and (c) illustrates the effect of different buffer solution on the anodic peak of 5-HT at the constructed electrode and the inset graphs represent the relationship between anodic peak current versus pH at scan rate (a) 10 mVs⁻¹ and (c) 25 mVs⁻¹ respectively. This experiment was necessary because NTs such as 5-HT and DA are available in bodily fluid at pH 7.40. The experiment was conducted using 0.1 mM 5-HT made in 0.1 M PBS (pH 3, pH 6, and pH 7, pH 9) solutions at scan rates of 10 mVs⁻¹ and 25 mVs⁻¹ respectively. In figures 7(a) and (c), an increase in pH resulted in a peak potential shift towards lower values, suggesting the involvement of protons in the reaction for all scan rates. Similarly, insets in figures 7(a) and (c) revealed that as the pH increased, the peak current also increased until pH 7 at which point it rapidly decreased to pH 9 for all scan rates. This suggests that the SPCE- MWCNT-AONP modified electrode was stable at pH 7 PBS. At pH 7, the current response at a scan rate of 25 mVs⁻¹ was double that at 10 mVs⁻¹. Due to the significant variation in current responses between the two scan rates, the following experiments were conducted using 0.1 mM 5-HT dissolved in 0.1 M PBS (pH 7) at a scan rate of 25 mVs⁻¹.

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The correlation between the peak potential and pH is shown in figures 7(b) and (d), and the relation may be expressed using the following equations: E_pa = 0.034 pH + 0.0597, and E_pa = 0.032 pH + 0.58 with R² values of 0.982 and 0.992 for scan rate at 10 mVs⁻¹, and 25 mVs⁻¹ respectively. The slopes of d_Ep/d_pH plots were 0.034 V pH⁻¹ and 0.032 V pH⁻¹, closer to the Nernstian theoretical value of 0.059 V pH⁻¹ for a one proton per electron stoichiometry for all scan rates. Therefore. The reaction mechanism for5-HT at the surface of the constructed electrode was a two-protons accompanied by a two-electrons reaction mechanism, as seen in scheme 1 [48, 71].

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Cyclic voltammograms for the SPCE-bare, SPCE-AONPs, SPCE/f-MWCNTs, and SPCE-AONP-MWCNT are shown in figure 8. The voltammograms in figure 8 displayed a distinctive and characteristic shape expected for 5 mM K [Fe (CN) ₆]^3−/4− solution at the scan rate of 25 mVs⁻¹ within −0.2–1.0 V potential window. The anodic and cathodic peaks were located at (Ipa = 99.15 μA; Epa = 0.22 V), and (Ipc = −124.18μA; Epc = −0.06 V) at SPCE-MWCNT-AONP nanocomposite modified electrode. A shift in the nanocomposite-modified electrode's peak potential towards negative potentials indicates a decrease in the over-potential of the redox process at the modified electrode due to enhanced electrocatalytic properties. The voltammogram in figure 8 showed a decrease in current response in the following order, SPCE-MWCNT-AONP > SPCE-fMWCNTs > SPCE-AONPs > SPCE-bare. A higher current response at the nanocomposite modified electrode attests to the synergic effect between CNTs and AONPs. According to table 1, all electrodes exhibited Ipa/Ipc values close to one. As a result, all reactions at the surface of modified and unmodified electrodes were reversible. The SPCE-MWCNT-AONP, SPCE-fMWCNTs, SPCE-AONPs, and SPCE-bare, peak to potential separations (ΔEp) of 0.17, 0.19, 0.21, and 0.48 V were observed. All the peak to potential values for all electrodes were greater than the theoretical value of 0.059 V accepted for a quick-one electron transfer mechanism. Each electrode's resulting active surface area was evaluated using the Randle–Sevcik equation (1). A larger active surface area indicates increased chances of the electro-active site exposure to an electrocatalytic reaction [72]. The electrodes showed a decrease in active surface area in this manner, the SPCE-MWCNT-AONP (0.716 cm²) > SPCE-fMWCNTs (0.346 cm²) > SPCE-AONPs (0.123 cm²) > SPCE-bare (0.054 cm²) electrode. The SPCE-MWCNT-AONP modified electrode had thirteen times larger active surface area than the bare electrode. Due to high current response, the SPCE-MWCNT-AONP (Ipa = 76.68 μA, and Ipc = −86.59 μA) modified electrode showed a tiny over-potential compared to other modified and bare electrodes. A high over-potential was noted at the bare SPCE with a low current response of Ipa = 40.84 μA and Ipc = −55.82 μA when compared with the modified electrode. A modified electrode can significantly reduce the electrode's over-potential while increasing the electrode's sensitivity and selectivity for detecting electroactive substances. The above-stated information greatly supports the notion that chemically modified electrodes, especially the SPCE-MWCNT-AONP modified electrodes, exhibit better redox potentials, faster electron transfer kinetics, and stability than unmodified electrode [27]. As a result, the SPCE–MWCNT-AONP modified electrode was selected and used throughout the study.

$$\begin{eqnarray}&&ip=2.69\,x\,{10}^{5}{n}^{3/2}A{D}^{1/2}C{\upsilon }^{1/2}\end{eqnarray} \tag{ 1 }$$

Where ip, D, C, and v^1/2 represent the peak currents (Amps), diffusion coefficient (cm²/s), analyte concentration (mol/cm⁻³), and the square root of scan rate (v/mVs⁻¹)^1/2 respectively. Where n and A indicate the number of electrons transported and the active surface area of the modified electrode (cm²), correspondingly.

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Cyclic voltammograms in figure 9(a) show the effect of scan rate study on the anodic peak in 5 mM of K[Fe (CN) ₆]^3−/4− produced in 0.1 M PBS (pH 7) at scan rates of 10–300 mVs⁻¹. The voltammogram in figure 9(a) showed an anodic peak shifted towards a higher potential and the cathodic peaks shifted towards negative potentials as the scan rate increased, suggesting a diffusion-controlled reaction took place. The reduction and oxidation peak currents increase in proportion to the scan rate, indicating a diffusion-controlled reaction took place as shown in figure (b). Additionally, the voltammogram indicated that an ideal reversible reaction occurred, which is confirmed by the almost identical gradient slopes observed at Ipa = 73.215 μA/(mVs¹)^1/2–133.55 (R² = 0.993) and Ipc = −73.549 μA /(mVs¹)^1/2 + 142.87 (R² = 0.995) [73]. The relation between the potential and the scan rate logarithm is shown in figures 9(c) and (d). The Tafel value was determined using a gradient slope of Epa = 0.2031 logv—0.0857 (R2 = 0.994).

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Using the acquired gradient slope, a Tafel value of 406.0 mVdec⁻¹ was determined for the SPCE-MWCNT-AONP modified electrode. The observed Tafel value was greater than the predicted value of 118 mVdec⁻¹, indicating that adsorption or reaction intermediates were involved in the reaction at the surface of the fabricated electrode. The Tafel value was obtained using the Tafel equation (2);

$$\begin{eqnarray}&&Ep=\displaystyle \frac{b}{2}\,\mathrm{log}\,v+c\end{eqnarray} \tag{ 2 }$$

Where Ep, b, and c stands for potential, the gradient slope of the graph, and the constant respectively.

The coefficient of electron transfer (α) and the number of electrons transported (n) at the fabricated electrode were determined to be 0.50 and 0.597 (n) =1 using equations (3) and (4) assigned as the cathodic and anodic peaks by Laviron [74].

$$\begin{eqnarray}&&Slope=\displaystyle \frac{-2.3RT}{\alpha nF}\end{eqnarray} \tag{ 3 }$$

$$\begin{eqnarray}&&slope=\displaystyle \frac{2.3RT}{\left(1-{\alpha }_{a}\right)nF}\end{eqnarray} \tag{ 4 }$$

Whereby R, F, T, and α represents the gas constant (8.314 J mol⁻¹. K), faradays constant (96500 C), temperature (Kelvins), and the coefficient of electron transport at the fabricated electrode respectively.

The rate constant (k_s) was used to calculate the speed at which the reaction at the SPCE-MWCNT-AONP modified electrode proceeded. For a reversible and quick reaction, the rate constant (k_s) value must be larger than 10^–2 c m s⁻¹, while for a quasi-reversible and a slow reaction, the rate constant value must be greater than 10⁻⁴ but less than 10⁻² cm s⁻¹ [75]. Equation (5) was used to obtain the rate constant value (k_s) of 0.046 cm s⁻¹ for the SPCE-MWCNT-AONP modified electrode.

$$\begin{eqnarray}&&\mathrm{log}\,ks=\alpha \,\mathrm{log}\left(1-\alpha \right)+\left(1-\alpha \right)\mathrm{log}\,\alpha -\,\mathrm{log}\,\displaystyle \frac{RT}{nF\upsilon }-\alpha \left(1-\alpha \right)\displaystyle \frac{nFT}{2.3RT}\end{eqnarray} \tag{ 5 }$$

Figure 10 represents the electrochemical oxidation of 0.1 mM 5-HT at various electrodes made in 0.1 M PBS (pH 7) at a scan rate 25 mVs⁻¹. Figure 10(a) displayed a distinctive anodic peak for 5-HT at Epa = 0.35 V for the SPCE-MWCNT-AONP modified electrode. This is in agreement with other works done [48, 57]. Anodic peak potential for SPCE-fMWCNTs, SPCE-AONPs, and SPCE-bare electrodes were located at E_pa = 0.32 V, 0.27 V, and 0.50 V respectively. Moreover, the voltammogram showed that the electro-oxidation reaction of 5-HT was irreversible for all electrodes. The SPCE-MWCNT-AONP showed the greatest electrocatalytic response to 5-HT when compared to other modified electrodes. The current response decreased in this manner, the SPCE-MWCNT-AONP (84.13 μA) > SPCE-fMWCNTs (33.49 μA) > SPCE-AONPs (24.40 μA) > SPCE-bare (2.89 μA). The current response at the SPCE-MWCNT-AONP electrode was 29 times greater than at the SPCE-bare. A higher current response to 5-HT observed at the SPCE-MWCNT-AONP modified electrode indicated excellent biocompatibility of the nanocomposite with the analyte and improved electrocatalytic activity. These results show a pattern similar to those obtained in figure 8 using redox probe solution. The linear relationship between scan rate and the anodic peak current is shown in figure 11(a). The voltammogram shows a slight shift in oxidation peak current towards higher potential values with increasing scan rate. Figure 11(b) depicts a linear correlation between the current and square root of scan rate for 5-HT, implying a diffusion-controlled reaction took place.

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The proposed sensor's sensitivity and detection limit were carried out using a 0.1 M PBS (pH 7) with various concentrations of 5-HT with the help of SWV. The parameters for the SWV method were set at the frequency (10 Hz), potential window ranging from −0.2–0.8 V, potential amplitude (0.01 V), Estep (0.01 V) and other parameters were left as they were at zero. The objective was to evaluate and analyze the effect of various analyte concentrations on the current response of 5-HT. Figure 12 displays a directly proportional relationship between the concentration of 5-HT and the current response. The linearity of the SPCE-MWCNT-AONP modified electrode ranged from 0.016–0.166 μM, and a linear regression equation of Ipa = 0.2863 [5-HT]/μA + 1.6741 (R² = 0.9851), and a detection limit (LoD) of 24.6 nM, as displayed in figure 12(b). The obtained data show a strong correlation with this work [54]. The resulting limit of detection (LoD) value was calculated from equation (6), and the limit of quantification (LoQ) for the modified electrode was calculated using equation (7). The LoD and LoQ values were calculated to be 0.025 μM and 74 nM, respectively.

$$\begin{eqnarray}&&{\rm{LoD}}=\displaystyle \frac{3.3xSD}{m}\end{eqnarray} \tag{ 6 }$$

$$\begin{eqnarray}&&{\rm{LoQ}}=\displaystyle \frac{10xSD}{m\,}\end{eqnarray} \tag{ 7 }$$

SD stands for standard deviation, while m represents the slope of the calibration plot.

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As demonstrated in table 2, the obtained results, including the LoD and sensitivity of the SPCE-MWCNT-AONP nanocomposite electrode, compared well to other chemically modified electrodes used to detect 5-HT from literature.

Cyclic voltammogram was utilized to establish the selective of the proposed electrode by simultaneously detecting 0.1 mM 5-HT and 0.1 mM AA prepared in 0.1 M PBS (pH 7) scan rate ranging from 10–300 mVs⁻¹. Figure 13(a) shows the results of the experiment. An increase in scan rate resulted in a positive peak potential shift for all analytes. However, the electrode showed a higher current response towards AA than 5-HT. At scan rate of 25 mVs⁻¹, AA and 5-HT peak potentials were located at −0.014 V and 0.302 V, respectively. Figures 13(b) and (c) shows the relationship between current response versus square root of scan rate for AA and 5-HT. A linear relationship between current response and the square root of scan rate for both analytes was observed, suggesting that the reaction at the proposed electrode was diffusion controlled. The linear regression equation for AA and 5-HT were Ipa = 6.615 v^1/2–23.969 (R² = 0.991) and Ipa = 5.493 v^1/2–17.893 (R² = 0.992). The R² value of 5-HT improved significantly compared to those in figure 11.

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The interference study for the proposed electrochemical sensor was done by simultaneously detecting varying concentrations of 5-HT in 1 mM AA solution as an interfering molecule using square wave voltammetry. A higher AA concentration was utilized because interfering compounds such as AA and UA are present in the body fluids at higher concentrations than NTs. From figure 14(a), the peak potentials for AA and 5-HT were located at 0.044 V and 0.325 versus As the 5-HT concentration increased from 100–333 μM AA, the 5-HT current response increased, but 5-HT current response was higher than that of AA. However, as 5-HT concentration increased from 500–545 μM, the AA current response began to stabilize and drop while the 5-HT current response kept on increasing, as illustrated in figure 14(a). As the 5-HT concentration increased from 100–333 μM, the solution became saturated with 5-HT cationic molecules, resulting in a decrease in the AA peak current afterwards.

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Furthermore, compared to anionic AA molecules, there was a better π – π* interaction between the f-MWCNTs and the cationic 5-HT molecules, resulting in better cationic 5-HT molecules attachment on the surface of the electrode. Figure 14(b) demonstrated that the 5-HT current peak increased linearly as concentration increased. Figure 15 depicts the simultaneous detection of 1 mM 5-HT and 1 mM AA at the modified electrode. The fabricated electrode exhibited excellent anti-interference behaviour even at fixed concentrations, as demonstrated by distinctive visible peaks of each analyte and a broad peak-to-peak separation of 367.90 mV between two analytes.

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Stability, reproducibility, and shelve-life study

To analysis the effectiveness of the constructed electrochemical sensors, the stability, repeatability, and shelve studies were undertaken utilizing CV at a scan rate of 25 mVs⁻¹. The stability study was achieved by repeatedly scanning the electrode 20 times in 5 mM of K [Fe (CN) ₆]^3−/4− made in 0.1 M PBS (pH 7). The repeated scans showed an increase in the anodic peak current of 26.39% and a decrease in the cathodic current peak by 17.71%, as shown in figure 16(a). An increase in anodic current peak could be ascribed to the increased electro-active interactions at the modified electrode's surface area with time. The results indicated that the constructed electrochemical sensor was not prone to biofouling during the voltammetry experiment and had excellent stability.

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Similarly, the SPCE-MWCNT-AONP modified electrode was scanned repeatedly 20 times in 0.1 M PBS containing 0.1 mM 5-HT as an analytical probe. The anodic peak current of 5-HT decreased by 68% and demonstrated an RSD value of 34.42% towards 5-HT detection, as displayed in figure 16(b). The shelve-life study of the modified electrode was carried out every week in 0.1 mM 5-HT prepared in 0.1 M PBS (pH 7). The results of the experimentation are represented in figure 16(c). When the SPCE-MWCNT-AONP nanocomposite electrode was not in use, the electrode was kept in a dry environment. Figure 16(c) reveals that the current response decreased by 18% after 21 days, then increased by 1% after the 28 days, and then plummeted by 54% after the 35 days. The results confirm that the modified electrode has a long shelf life towards the detection of 5-HT.

The applicability of the fabricated sensor towards the determination of 5-HT in tomatoes was undertaken to utilize the SWV. The results from the experiment are summarized in table 3. For detection of 5HT in tomatoes, the SPCE-MWCNT-AONP modified electrode showed good recoveries ranging from 91.32%–108.28% with an average RSD (%) value of 2.57 (n = 3). This study suggests that the modified electrode is suitable for 5-HT detection in real samples.