A simple, sensitive and cost-effective electrochemical sensor for the determination of N-acetylcysteine

In the present work, we prepared a simple and novel electrochemical sensor based on zeolitic imidazolate framework-67 (ZIF-67) and ionic liquid 1-Butyl-3-methylimidazolium hexa-fluorophosphate (BMIM.PF6) modified carbon paste electrode (CPE), which was effectively used for the determination of N-acetylcysteine. The cyclic voltammetry studies demonstrated the lowest peak potential and the enhanced peak current response for N-acetylcysteine at the surface of ZIF-67/BMIM.PF6-modified CPE compared to the other CPEs due to the significant catalytic effect of ZIF-67 and BMIM.PF6, as well as the combination of them. Under the optimized conditions, the electrochemical response of ZIF-67/BMIM.PF6/CPE sensor provided a good linear relationship with N-acetylcysteine concentration from 0.04 to 435.0 µM. The limit of detection is estimated to be 0.0 1 μM for N -acetylcysteine. In further studies and measurements, the estimation of N-acetylcysteine in tablet samples confirms the usefulness of the ZIF-67/BMIM.PF6/CPE sensor


Introduction
N-acetylcysteine (as one of the thiol-containing drugs) is an acetylated derivative of the nonessential amino acid L-cysteine.N-acetylcysteine has been primarily used as a mucolytic agent for treating chronic bronchitis and other chronic respiratory disorders characterized by the excessive production of thick mucus [1].N-acetylcysteine has the ability to break disulfide bonds, transforming them into two sulfhydryl groups.This process reduces the length of the main chain, resulting in the thinning of mucus and makes it easier to eliminate [2].Also, N-acetylcysteine is widely used as the antidote for hepatoxicity caused by acetaminophen (paracetamol) overdose [3].Furthermore, due to its antioxidant properties, N-acetylcysteine is also known as an antitumor, antiviral, and antiinflammatory agent [4].It also has a potential therapeutic in the treatment of acquired immune deficiency syndrome (AIDS) [5], types of cancer [6], elimination of heavy metals [7], etc.The antioxidant action of N-acetylcysteine can be primarily attributed to two mechanisms [8,9].Firstly, N-acetylcysteine acts as a precursor of reduced glutathione (GSH), which indirectly exerts antioxidant effects.GSH is a potent intracellular antioxidant crucial in neutralizing reactive oxygen species (ROS) and minimizing oxidative damage.Secondly, N-acetylcysteine exhibits direct antioxidant activity by directly scavenging reactive radicals.It can directly react with highly reactive species such as hydroxyl radicals (HO) and hydrogen peroxide (H2O2), reducing their reactivity and forming less reactive species.This scavenging action helps mitigate ROS's harmful effects on cells and tissues.Therefore, due to N-acetylcysteine's medical and biological importance, a highly sensitive and selective method is required for its determination.To this day, various analytical techniques have been used for the determination of N-acetylcysteine, such as chromatography [10], chromatography-tandem mass spectrometry [11,12], fluorescence [13], spectrophotometry [14], chemiluminescence [15], capillary electrophoresis [16], and electrochemistry [17][18][19].
In recent years, electrochemical methods have gained more attention than other methods due to simple and low-cost instrumentation, short analysis time, and portability [20][21][22][23][24][25][26][27][28].The application of chemically modified electrodes in the design and fabrication of electrochemical sensors has a special place in the field of electroanalysis and has brought significant growth.Progress in this field can significantly help improve the efficiency, sensitivity, and selectivity of electrochemical sensors, making them more practical [29][30][31][32][33][34][35][36].Therefore, researchers are continuously discovering and investigating new materials as modifiers to improve the performance of chemically modified electrodes.
Metal-organic frameworks (MOFs) are crystalline materials of metal ions or clusters coordinated to organic ligands.The metal ions and organic ligands in MOFs provide flexibility in designing the structure and properties of these materials.MOFs exhibit a highly porous structure with a large surface area, making them useful in various applications, including energy storage [56], catalysis [57], sensing [58], drug delivery [59], environmental remediation [60], and so on.One of the promising applications of MOFs is in electrochemical sensing, which can be used as sensing elements to enhance the performance of electrochemical sensors [61][62][63].Ionic liquids ) ILs) are compounds that have revolutionized many research fields in recent years due to negligible volatility, high polarity, high thermal stability, high chemical stability, low melting point, non-flammability, high ionic conductivity, large electrochemical window, and structural designability.Focusing on the electrochemical aspects of ionic liquids shows that one of their interesting applications is their use as modifying materials of electrodes for the fabrication of sensors [64,65].Also, the combination of nanomaterials with ILs can play a key role in the efficiency of electrochemical sensors.Several outstanding attributes are observed when incorporating ILs and nanomaterials into electrodes, including long-term stability, higher conductivity, higher sensitivity, improved linearity, superior catalytic ability, and better selectivity [66][67][68].
The main objective of this work is to develop a simple and fast platform for the electrochemical determination of N-acetylcysteine by using a CPE modified with zeolitic imidazolate framework-67 (ZIF-67) and 1-Butyl-3-methylimidazolium hexafluorophosphate (BMIM.PF6) ionic liquid.The electrochemical studies and measurements were performed by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and chronoamperometry (CHA).The developed ZIF-67/BMIM.PF6/CPE sensor exhibited better electrochemical performance for sensitive determination of N-acetylcysteine than the other CPEs with low limit of detection (LOD) and wide linear range.Importantly, this sensor's great potential in quantifying N-acetylcysteine in N-acetylcysteine tablet samples is also confirmed.

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 a reference electrode (RE) (Ag/AgCl/KCl (3.0 M)), counter electrode (CE) (platinum), and working electrode (modified CPE).All solvents and chemicals were commercially available with analytical grade and used directly without further purification.

Synthesis of ZIF-67
For the preparation of ZIF-67, 2 mmol of cobalt(II) nitrate hexahydrate (0.582 g) was dissolved in 10 mL of methanol by stirring for 15 min.Then, 15 mL methanolic solution of 8 mmol 2-methylimidazole (0.656 g) was added slowly into the cobalt solution for 15 min.This prepared solution was stirred at ambient temperature for 24 h.Finally, the prepared precipitate was collected by centrifugation, washed with deionized water and ethanol several times, and dried at 65 °C in a vacuum oven for 14 h.
The morphology of the as-prepared ZIF-67 was observed by the FE-SEM image (Figure 1).It shows the growth of ZIF-67 crystals with regular rhombic dodecahedral morphology and almost uniform size.

Preparation of ZIF-67/BMIM.PF6/CPE
The ZIF-67-BMIM.PF6-modified CPE with a mass of 0.5 g was achieved by hand-mixing 0.47 g of graphite powder and 0.03 g of ZIF-67 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 ZIF-67, which was mixed again for at least 30 min to obtain the ZIF-67/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 (ZIF-67/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 ZIF-67/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.By using the Randles-Ševčik equation, the value of the ESCA for ZIF-67/BMIM.PF6/CPE (0.396 cm 2 ) was 4.4 times greater than unmodified CPE.

Electrocatalytic response of ZIF-67/BMIM.PF6/CPE towards N-acetylcysteine
The effect of pH values (from 2.0 to 9.0) of the supporting electrolyte (0.1 M phosphate buffer solution (PBS)) on the electrochemical oxidation of N-acetylcysteine was studied by using the ZIF-67/BMIM.PF6 modified CPE via the DPV technique.By changing the pH value of PBS, the prepared electrode showed different voltammograms for oxidation of N-acetylcysteine.The peak potential and peak current from the oxidation of N-acetylcysteine showed a strong dependence on pH.By increasing the pH from lower to higher values, the anodic peak potential of N-acetylcysteine was shifted towards the negative potentials.Also, the Ipa of N-acetylcysteine 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 (Figure 2).Therefore, pH 7.0 was used for further electrochemical studies.To assess the electrocatalytic activity of the IL (BMIM.PF6) and as-prepared ZIF-67, the electrochemical responses of N-acetylcysteine on various electrodes were examined by cyclic voltammetry (CV).Figure 3

Effect of scan rate on the oxidation reaction of N-acetylcysteine
To investigate the effect of scan rate, CVs of the ZIF-67/BMIM.PF6/CPE were recorded at different scan rates (10 to 500 mV s -1 ) in 0.1 M PBS containing 100.0 µM N-acetylcysteine (Figure 4).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 N-acetylcysteine and the square root of scan rate ( 1/2 ) (Figure 4 inset).This observation suggests that the oxidation reaction is controlled by the diffusion of N-acetylcysteine species from the bulk solution to the surface of ZIF-67/BMIM.PF6/CPE.

Chronoamperometric measurements of N-acetylcysteine at ZIF-67/BMIM.PF6/CPE
To measure the diffusion coefficient (D) of N-acetylcysteine, the chronoamperometric responses of ZIF-67/BMIM.PF6/CPE was plotted for different concentrations of N-acetylcysteine from 0.1 to 1.6 mM at a fixed potential of 0.71 V (Figure 5).The current-time curves reflect the change in concentration gradient of the electroactive species (N-acetylcysteine) in the vicinity of the electrode surface as time progresses.In order to determine the D, the Cottrell curves (I versus t 1/2 ) were plotted over a certain range of time for different concentrations of N-acetylcysteine (Figure 5A).Then, the slope of the obtained Cottrell curves was plotted vs. the different concentrations of N-acetylcysteine (Figure 5B) and a straight line with a slope of 7.2928 µA s 1/2 / mM was obtained.From the slope of the resulting plot and using Cottrell's equation, the D of N-acetylcysteine on the surface of ZIF-67/BMIM.PF6/CPE was found to be 2.2×10 -6 cm 2 s -1 .

Quantitative analysis of N-acetylcysteine by DPV
To study the detection efficiency of ZIF-67/BMIM.PF6/CPE, the DPV measurements were performed with the successive addition of N-acetylcysteine (0.04 to 435.0 µM) in 0.1 M PBS (pH 7.0) (Figure 6).From the recorded voltammograms, the increase of the Ipa is proportional to the increase of N-acetylcysteine concentration in a wide range from 0.04 to 435.0 µM.Furthermore, the linear dependence between the enhanced Ipa of N-acetylcysteine and its concentration is presented in the Inset of Figure 6.This dependence can be expressed by I = 0.0683CN-acetylcysteine + 0.9501 with a correlation coefficient 0.9996.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 15 measurements on the blank solution), and m denotes the slope of the corresponding calibration curve, and it was found to be 0.01 µM.The performance comparison of the developed electrode (ZIF-67/BMIM.PF6/CPE) in this study with some previous studies is shown in Table 1.Studies related to the stability of ZIF-67/BMIM.PF6/CPE sensor were performed by recording the current response of the designed sensor towards 60.0 µM N-acetylcysteine every 7 days over 14 days.The obtained results showed that the electrode response retained 97.5 % of its initial value after 7 days and 95.1 % after 14 days.These results indicated that the designed sensor had good stability.
Also, to investigate the repeatability of the ZIF-67/BMIM.PF6/CPE sensor, the measurements were repeated in 0.1 M PBS (pH 7.0) containing 60.0 µM N-acetylcysteine.The acceptable repeatability was obtained with an RSD of 2.9 % after using the same sensor for seven continuous measurements.

Interference studies
The interference studies were also carried out to investigate the selectivity of ZIF-67/BMIM.PF6/CPE sensor towards the determination of N-acetylcysteine in the presence of various species.The DPV responses of ZIF-67/BMIM.PF6/CPE was recorded by adding various species into 0.1 M PBS (pH 7.0) containing 50.0 µM N-acetylcysteine.According to the findings, 700-fold of Na + , K + , Ca 2+ , Al 3+ , NH4 + , F -, Cl -, and Br -; 250-fold of urea, glycine, alanine, and phenylalanine; 8-fold of cysteine did not show significant interference (no signal change more than ± 5 %) for the determination of N-acetylcysteine.

N-acetylcysteine analysis in real samples
To evaluate the practical performance of the developed sensor (ZIF-67/BMIM.PF6/CPE), the determination of N-acetylcysteine in the N-acetylcysteine tablet sample was conducted.The standard addition method was employed for the analysis of N-acetylcysteine by the DPV technique.Measurements were performed by adding the known concentrations of N-acetylcysteine to the N-acetylcysteine tablet sample.The recovery and RSD values are summarized in Table 2.The summarized results in Table 2 show acceptable recovery values (between 96.0 and 103.7 %) and RSD values (n = 5) of ≤ 3.4 %, which confirm that the developed sensor could be used for realtime analysis.

Conclusion
In this work, we demonstrated the application of a high-performance electrochemical sensor (ZIF-67/BMIM.PF6/CPE) for the determination of N-acetylcysteine.The ZIF-67/BMIM.PF6-modified CPE showed more prominent electrocatalytic activity toward N-acetylcysteine oxidation than the other electrodes, with enhanced response current and lowered over-potential.The ZIF-67/ /BMIM.PF6/CPE showed a sensitive peak current response toward N-acetylcysteine in the linear range from 0.04 to 435.0 µM, with a LOD (S/N = 3) of 0.01 µM.Finally, the developed sensor was successfully used for the estimation of N-acetylcysteine in N-acetylcysteine tablet sample with acceptable recoveries (96.0 and 103.7 %) and RSDs values not more than 3.4 %.

Figure 2 .
Figure 2. Plot of the oxidation peak current of 200.0 µM N-acetylcysteine as a function of pH solution at ZIF-67/BMIM.PF6/CPE in 0.1 M PBS at different pH values (2.0 to 9.0).

Table 2 .
Real sample analysis for the determination of N-acetylcysteine spiked into the N-acetylcysteine tablet sample at ZIF-67/BMIM.PF6/CPE.