Facile preparation of a sensitive electrochemical sensor with good performance for determination of methionine

In this work, a novel voltammetric sensor for the detection of methionine was designed and prepared by using a carbon paste electrode (CPE) modified with ZnO hollow quasi-spheres (ZnO hollow QSs) and 1-butyl-3-methylimidazolium hexafluorophosphate ( BMIM.PF 6 ). The results by cyclic voltammetry showed that the prepared electrode (ZnO-BMIM.PF 6 /CPE) effectively increased the oxidation peak current and reduced the oxidation peak potential of methionine and had a suitable electrocatalytic activity for the oxidation of methionine. Notably, the ZnO-BMIM.PF 6 /CPE exhibited high detection capability towards the quantification of methionine in 0.1 M PBS (pH 7.0) over a concentration range from 0.04 to 330.0 µM with a limit of detection of 0.02 µM. More importantly, the effectiveness of the ZnO-BMIM.PF 6 /CPE sensor was also confirmed in real samples (urine detection with acceptable recoveries (98.0 to 102.7 %) and relative standard deviation values ≤ 3.3 %.


Introduction
Methionine is classified as a sulfur-containing amino acid because it contains a sulfur atom in this chemical structure.Methionine is a primary source of sulfur in the diet, playing a vital role in maintaining the health and integrity of various tissues, including the hair, skin, and nails [1].Also, methionine plays a crucial role in various biological processes, including protein synthesis, synthesis of amino acids, such as cysteine, taurine, homocysteine, and glycine, transmethylation reaction, and other physiological processes [2].By increasing lecithin production in the liver, methionine can indirectly reduce cholesterol levels [3].In addition, methionine acts as a chelator for heavy metals and functions as a powerful antioxidant for free radicals scavenging [3].Methionine deficiency has been studied in relation to various diseases, including toxaemia, Parkinson's disease, and acquired Herein, we developed a high-performance modified CPE based on ZnO hollow QSs-BMIM.PF6 for detection of methionine.The ZnO-BMIM.PF6 modified CPE reduces the overpotential and enhances the oxidation peak current for the effective electrochemical detection of methionine.Furthermore, the modified CPE provided acceptable results for the detection of methionine in real samples.

Instruments and materials
All electrochemical studies and measurements were done using a potentiostat/galvanostat device (Metrohm Autolab -PGSTAT302N (Utrecht, The Netherlands)), controlled by the GPES 4.9004 software.The electrochemical tests were performed in a typical three-electrode setup by using reference electrode (RE) (Ag/AgCl/KCl (3 M)), counter electrode (CE) (platinum), and working electrode (modified CPE).All solvents and chemicals were commercially available (Merck and Sigma-Aldrich companies) with analytical grade and used directly without further purification.
The synthesis and characterization of ZnO hollow QSs were reported in our previous work [58].Figure 1 shows the FE-SEM image of ZnO hollow QSs.

Preparation of ZnO-BMIM.PF6/CPE
The ZnO-BMIM.PF6 modified CPE with a mass of 0.5 g was achieved by hand-mixing 0.48 g of graphite powder and 0.02 g of ZnO hollow QSs for 5 min until a homogeneous blend was formed.Then, paraffin oil and BMIM.PF6 in the ratio 3:1 was added to the blend of graphite and ZnO hollow QSs, which was mixed again for at least 30 min to obtain the ZnO-BMIM.PF6 modified carbon paste.Finally, the modified paste was packed into the glass tube cavity.The electrical contact was established through a conductive copper wire.Also, the surface of the prepared electrode (ZnO-BMIM.PF6/CPE) was polished on a smooth paper to obtain a shiny and smooth appearance.
To calculate the electrochemically active surface area (EASA) of the unmodified CPE and ZnO-BMIM.PF6/CPE, the CVs were recorded at different scan rates in 0.1 M KCl solution containing 1.0 mM K3[Fe(CN)6] as a redox probe.Using the Randles-Ševčik equation, the value of the ESCA for and ZnO-BMIM.PF6/CPE (0.297 cm 2 ) was 3.3 times greater than unmodified CPE.

Electrocatalytic response of ZnO-BMIM.PF6/CPE towards methionine
The effect of pH values (from 2.0 to 9.0) of the supporting electrolyte (0.1 M PBS) on methionine's electrochemical oxidation was studied using the ZnO-BMIM.PF6 modified CPE via the DPV technique.It was observed that by changing the pH value of PBS, the prepared electrode showed different voltammograms for oxidation of methionine.The peak potential and peak current from the oxidation of methionine showed a strong dependence on pH.By increasing the pH from lower to higher values, the anodic peak potential of methionine was shifted towards the negative potentials.Also, the Ipa of methionine gradually increased with the increase of pH from 2.0 to 7.0 and then decreased.The maximum Ipa was obtained at pH 7.0.Therefore, pH 7 .0 was used for further electrochemical studies.
To assess the electrocatalytic activity of the IL (BMIM.PF6) and as-prepared ZnO, the electrochemical responses of methionine on unmodified CPE and modified CPE were examined by cyclic voltammetry (CV).Figure 2  As can be seen, a broad oxidation peak with a low anodic peak current (Ipa) was shown in unmodified CPE.The ZnO-BMIM.PF6/CPE clearly improves the oxidation of methionine, as evident from the increase of the Ipa (from 3.5 to 13.0 µA) and decrease of the overpotential (from 950 to 850 mV) when compared with unmodified CPE.This result could be related to the electrocatalytic effect of the IL and ZnO NPs.In addition, the absence of any reduction peak on the reverse scan revealed the irreversible oxidation of methionine over unmodified and modified CPE.

Effect of scan rate on the oxidation reaction of methionine
To investigate the effect of scan rate, CVs of the ZnO-BMIM.PF6/CPE were recorded at different scan rates (10 to 250 mV/s) in 0.1 M PBS containing 100.0 µM methionine (Figure 3).An increase in the anodic peak current (Ipa) with an increase in scan rate can be observed.Also, from the obtained voltammograms, it was possible to observe a linear dependence between Ipa of methionine and the square root of scan rate ( 1/2 ) (Ipa = 1.8901 1/2 -2.9739) (Figure 3 Inset).This observation suggests that the oxidation reaction is controlled by the diffusion of methionine species from the bulk solution to the surface of ZnO-BMIM.PF6/CPE.E / mV vs. Ag/AgCl/KCl

Chronoamperometric measurements of methionine at ZnO-BMIM.PF6/CPE
To measure the diffusion coefficient (D) of methionine, the chronoamperometric responses of ZnO-BMIM.PF6/CPE were plotted in different concentrations of methionine from 0.1 to 1.7 mM in a fixed potential of 0.9 V (Figure 4).The current-time (I-t) curves reflect the change in concentration gradient of the electroactive species (methionine) in the vicinity of the electrode surface as time progresses.To determine the D, the Cottrell curves (I versus t -1/2 ) were plotted over a certain range of time for different concentrations of methionine (Figure 4A).Then, the slope of the obtained Cottrell curves was plotted vs the different concentrations of methionine (Figure 4B) and a straight line with a slope of 19.7 µA s 1/2 mM -1 was obtained.From the slope of the resulting plot and using Cottrell's equation, the D of methionine on the surface of ZnO-BMIM.PF6/CPE was found to be 1.6×10 -5 cm 2 s -1 .

Quantitative analysis of methionine by DPV
To study the detection efficiency of ZnO-BMIM.PF6/CPE, the DPV measurements were performed with the successive addition of methionine (0.04 to 330.0 µM) in 0.1 M PBS (pH 7.0) in the following conditions: step potential 0.01 V and pulse amplitude 0.025 V (Figure 5).From the recorded voltammograms, the increase of the Ipa is proportional to the increase of methionine concentration in a wide range from 0.04 to 330.0 µM.Furthermore, the linear dependence between the enhanced Ipa of methionine and its concentration is presented in the Inset of Figure 5.This dependence can be expressed by I = 0.0812CMethionine + 0.8557 with a correlation coefficient of 0.999.The LOD was calculated according to the ensuing formula 3Sb/m, where Sb denotes the standard deviation of the blank (PBS) signal (obtained based on 12 measurements on the blank solution), and m denotes the slope of the corresponding calibration curve, and it was found to be 0.02 µM.The limit of quantification was found to be 0.04 µM.Table 1 lists the comparative characteristics of the as-prepared sensor with those of previously reported sensors for the determination of methionine.

Stability and reproducibility studies of ZnO-BMIM.PF6/CPE sensor towards the determination of methionine
Studies related to the stability of ZnO-BMIM.PF6/CPE sensors were performed by recording the current response of the designed sensor towards 75.0 µM methionine over 20 days.The results showed that the electrode response retained 95.9 % of its initial value after 20 days.These results indicated that the designed sensor had good stability.
Also, the reproducibility of the prepared sensor (ZnO-BMIM.PF6/CPE) was evaluated by recording the current response of five electrodes prepared independently under the same conditions.All five prepared electrodes showed almost the same responses and the relative standard deviation (RSD) was 2.7 % in the determination of 75.0 µM methionine.

Interferences studies
The effect of the possible interferences from some species such as Na + , Ca 2+ , Mg 2+ , NH4 + , Al 3+ , Cl -, SO4 2-, S 2-, glucose, acetaminophen, epinephrine, norepinephrine, uric acid, tryptophan, glycine, phenylalanine, and L-serine on the electrochemical response of methionine was evaluated at the surface of ZnO-BMIM.PF6/CPE sensor.It was observed that these species did not show significant interference for the determination of methionine (no signal change more than ± 5 %).These results confirmed that the developed sensor has good selectivity for the determination of methionine.

Methionine analysis in real samples
To evaluate the practical performance of the developed sensor (ZnO-BMIM.PF6/CPE), the determination of methionine in the urine sample was conducted.The standard addition method was employed for the analysis of methionine by the DPV technique.By adding the known concentrations of methionine to the urine sample, measurements were performed.The recovery and RSD values are summarized in Table 2.The summarized results in Table 1 show acceptable recovery values (between 98.0 and 102.7 %) and RSD values (n = 5) of ≤3.3 %, which confirm that the developed sensor could be used for real-time analysis.

Conclusions
In this study, the efficient and accurate detection of methionine was reported based on ZnO hollow QSs-BMIM.PF6 modified CPE.The obtained results demonstrated that the ZnO-BMIM.PF6/CPE sensor was well developed and showed an enhanced electrochemical response towards methionine oxidation.The ZnO-BMIM.PF6/CPE can be used to determine methionine in the concentration from 0.04 to 330.0 M with an LOD of 0.02 µM.Finally, excellent precision (RSD ≤3.3 %) and accuracy (recovery for spiked samples ranging from 98.0 to 102.7 %) were obtained.

Table 2 .
Real sample analysis for the determination of methionine spiked into the urine samples.