Voltammetric Determination of Acrylamide Using Coal Tar Pitch Modified Pencil Graphite Electrode by SWV

The adverse effects of acrylamide (AA) on humans are becoming clear, especially after a series of related investigations reported the dependence on consuming foods prepared by exposure to high temperatures for a long-time and cancer risk. Accurate determination of AA in food samples at trace amount is considered the first step to overcome this significant problem. The determination of AA using coal tar pitch modified pencil graphite (PGE/CTP) electrode was reported. The bare PGE and PGE/CTP electrodes were characterized using microscopic imaging technique scanning electron microscopy (SEM). The electrochemical behavior of AA was studied on PGE/CTP electrode in different medium acidities (pH) of phosphate and Briton-Robinson (BR) buffer solutions by employing square wave voltammetry (SWV). Linear sweep voltammetry (LSV) technique was applied to determine the mass transfer mode of AA from bulk solution to the PGE/CTP electrode surface. The optimum conditions were using phosphate buffer solution (PBS) at pH 7.0. The detectability of AA on the surfaces of bare PGE and PGE/CTP electrodes was compared, and the suitability of PGE/CTP electrode usage was determined. The linear relationship between peak current and AA concentration was in the range of 1000.0 to 0.5 nM. The limit of detection of AA was 0.2094 nM, and the limit of quantitation was 0.6912 nM. In addition, the PGE/CTP electrode as a sensor was successfully used for the determination of AA in the instant coffee sample.


Introduction
Acrylamide (AA) is a highly reactive organic compound and one of the well-known potential carcinogen chemicals that was detected in 2002 for the first time in our diet (Lutzow 2002).The molecular structure of acrylamide is shown in Fig. 1.The reactions of flavor compound development that are accompanied by non-enzymatic browning of foods known as Maillard reactions that take place during hightemperature roasting, frying, grilling, baking, and toasting food preparation processes.Maillard reactions start with a reaction among reducing sugars (such as fructose, glucose, and maltose) and the amino acid asparagine before ending with acrylamide formation (Raffan and Halford 2019;Branco 2023).These reactions are admitted as a main way of acrylamide human exposure.Besides its consideration as cancerogenic material, high levels of acrylamide intake can cause significant neurological problems according to studies that were conducted on rats (Tepe and Çebi 2019).
Highly carbohydrate contained vegetables like potato and corn have the highest risk of acrylamide formations during food cooking.In addition, breakfast cereals that already have acrylamide can contribute to the exposure to hazards (Mesias et al. 2022).
Various methods were used to determine AA in food samples.These methods provide accurate quantitative results on AA concentrations due to their sensitivity and reproducibility.Determination of AA using liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) (Alpözen et al. 2013), gas chromatography mass spectrometry (GC/MS) (Kang et al. 2021), ultraperformance liquid chromatography mass spectrometry (UP-LC/MS) (Alyousef et al. 2016), liquid chromatography ultraviolet spectrometry (LC-UV) (Crawford and Wang 2019), ion mobility spectrometric method (Pourmand et al. 1 3 2017) in addition to immuno-enzymatic method (Kılıç and Liman 2022), and digital image colorimetric method (Sáez-Hernández et al. 2022).Despite preceding methods that can determine AA at low concentrations, it needs to derivatize AA to a detectable form which follows an extraction step to isolate AA from its complicated samples.These steps make AA determination time-consuming and not convenient for routine tests.Because the cost of the instruments used is high and expensive, high-quality solvents must be consumed to operate these instruments.On the other hand, electrochemical method which concern to the electrochemical property of the analyte was employed to determine AA in foods by performing different techniques, square wave voltammetry (SWV) (Asnaashari et al. 2019;Sirajuddin et al. 2021;Zargar et al. 2009), osteryoung square wave voltammetry (OSWV) (Stobiecka et al. 2007), differential pulse voltammetry (DPV) (Esokkiya et al. 2021), cyclic voltammetry (CV) (Navarro et al. 2020), continuous fast fourier transform admittance voltammetry (CFFTAV) (Norouzi et al. 2018), and adsorption stripping voltammetry (AdSV) (Veselá and Šucman 2013).
Various electrodes were used in the determination of AA by electrochemical methods, such as hanging mercury drop electrode (HMDE) (Veselá and Šucman 2013) which is not appropriate to use due to its environmental hazards and the risks of mercury exposition; glassy carbon electrode (GCE) (Esokkiya et al. 2021;Sirajuddin et al. 2021); gold electrode (GE) (Norouzi et al. 2018) needs to repolish before use in experiments; carbon paste electrode (CPE) (Navarro et al. 2020) that depends very significantly upon the experimental experience of the user; and screen printed gold electrode (SPGE) (Asnaashari et al. 2019) that is relatively costly.As an advantage of coal tar pitch modified (PGE/CTP) electrodes, they are disposable and can be synthesized in any number.
The present work aims to determine AA using PGE/CTP electrode using SWV technique by applying optimized conditions.For the purpose of finding the best-supporting electrolyte, PBS and BR buffer solutions were tested at different medium acidities (pH).Also, the effect of scan rate on the electrochemical behavior of AA on PGE/CTP electrode was studied to determine the mass transfer mode of AA by linear sweep voltammetry (LSV).As a result of our suggestion, we used diluted aqueous solutions of the analyte in the presence of a supporting electrolyte without using harsh environmental solvents or accumulating steps.This voltammetric approach was applied successfully to determine AA directly in food samples (instant coffee) with no extraction or separation steps.

Materials
Acrylamide 99% was purchased from Sigma.BR buffer solution was made by mixing certain portions of acetic acid, boric acid, and phosphoric acid.PBS was made by mixing sodium dihydrogen phosphate and disodium hydrogen phosphate.0.1 M sodium hydroxide and 0.1 M hydrochloric acid solutions were used to adjust the pH of the prepared buffer solutions.All reagents and materials used in this research were analytical grade.Ultrapure water with 18.2 MΩ resistivity that was obtained via (MP minipure dest up ultra-pure water system) was used to prepare all solutions.

Instruments
Reference 600+ potentiostat/galvanostat/ZRA from (Gamry instruments, USA) equipped with electrochemical three electrode cell system was used in all experiments.The reference and auxiliary electrodes were Ag/AgCl 3M KCl (BASi model MF-2056, USA) and Pt wire (BASi model MW-1032, USA), respectively.pH meter (VWR phenomenal, PH 1100 L, UK) was used to control the pH of the medium.The morphology of bare PGE and PGE/CTP electrode surface was investigated by using SEM (Hitachi-SU 1510, USA.)

Preparation of the PGE/CTP Electrode
PGE/CTP electrode was prepared by modifying a mixture of coal tar pitch (CTP) on the PGE surface.PGE/CTP electrode was prepared with a CTP in acetone-dopped PGE.The amount of CTP was adjusted as 1% (w/w).Ten gram CTP composite was carbonized in an N 2 -atmosphere tube furnace (Protherm, Turkey) with a heating rate of 10 ○ C min −1 to reach 1000 ○ C.This was followed by the annealing of the materials at 1000 ○ C in N 2 atmosphere for 1 h.The black material transformed into metallic gray (Üstündağ and Erkal 2017; İncebay 2018).SEM was applied for characterization of CTP layers deposited on the PGE electrode surface in Fig. 2.

Preparation of the Real Sample
In total, 10.0 g of commercially purchased instant coffee sample was taken and dissolved in 10.0 mL of ultrapure water heated to boiling point.Ten milliliter of PBS 7.0 solution was added to the fully dissolved sample.This prepared sample was used directly in the measurements.

Results and Discussions
The conditions that influence AA peak current were optimized.Consequently, the influence of buffer solution type and the acidity of the medium (pH) on the peak current were studied.

The Influence of Supporting Electrolyte on the AA Peak Current
The oxidation behavior of AA was greatly affected by pH of the supporting electrolyte's solution.So, the determination of the electrochemical oxidation behavior of AA at different pH conditions is necessary.The electrochemical behavior of AA in two different types of buffers was investigated at the PGE/CTP electrode.Measurements of 1 mM AA in BR buffer solution at the pH range 2.0-7.0 were conducted using PGE/CTP electrode by SWV, and the same measurements were repeated in PBS at the pH range 6.2-8.2 as shown in Figs. 3 and 4.
Using PBS at pH 7.0 and BR buffer at pH 6.0 provided the highest AA peak current as observed from Figs. 3 and  4. PBS buffer was selected as the appropriate supporting electrolyte in this study because of its wide buffering capacity, better oxidative peak current value, and due to its similarity to human blood buffer pH.A good electrocatalytic activity of PGE/CTP electrode towards AA was observed in this medium, which also explains the low potential ratio of AA's oxidation peak in PBS buffer.

The Comparison of CTP/PGE and Bare PGE Electrodes
Measurements were accomplished using PGE/CTP and bare PGE electrodes, to recognize the effect of the CTP modification layer on the AA peak current.The results were recorded in Fig. 5.No peak was observed for AA by using bare PGE electrode.

The Effect of Scan Rate on the Electrochemical Behavior of AA on PGE/CTP Electrode
Linear sweep voltammetry (LSV) was applied to realize the effect of scan rate on the electrochemical behavior of AA on PGE/CTP electrode using PBS at pH 7.0 as supporting electrolyte.The linear relationship between peak current and square root of scan rate theoretically obeys Randles-Ševčík equation that's given below: where Ip, peak current (A); n, number of transferred electrons; A, electrode area (cm 2 ); C, analyte concentration (mol L −1 ); D, diffusion coefficient (cm 2 s −1 ); Ʋ, scan rate (1) . According to Fig. 6B, the equation (Ip = 3.1998 Ʋ 1/2 -5.5808) was obtained with the correlation coefficient (R 2 = 0.9886).The good linearity of the relationship between Ip and Ʋ 1/2 resulted.The electrochemical oxidation of AA on PGE/CTP electrode is a diffusion-controlled process.
The linearity of the graph (R 2 = 0.9886) drawn between the square roots of the peak currents and scan rates according to Fig. 6A indicates that AA reaches the PGE/ CTP electrode surface with diffusion control according to the Randles-Ševčík equation.According to Fig. 6C, the linear relationship between log Ip and log Ʋ was observed  (Batra et al. 2013).The relationship between peak potential and log Ʋ was linear as given in Fig. 6E with corresponding equation (Ip = 50.073log Ʋ + 670.58) and correlation coefficient (R 2 = 0.9422).Theoretically, peak potential and log Ʋ are related according to Laviron's theory that is shown below (Li et al. 2019): where E p , peak potential (V); E°, standard potential (V); R, general gas constant (J k −1 mol −1 ); T, temperature (k); F, Faraday constant (C mol −1 ); n, number of transferred ( 2) electrons; Ʋ, scan rate (V s −1 ); α, transfer coefficient; k°, standard heterogeneous reaction rate constant.

Interference Effects
Food samples generally have proteins, fats, carbohydrates, and inorganic salts.Amino acids contained in proteins theoretically can interfere with the detection of AA by voltammetry due to their electrochemical properties.As mentioned in some studies, no significant effect of amino acids as interferences on the determination of AA by voltammetry (Esokkiya et al. 2021), as well as the influence of some metal ions on the determination of AA was not observed (Mersal et al. 2021).

Standard Calibration Curve
The voltammetric determination of AA at PGE/CTP electrode using SWV was performed in PBS buffer (pH 7.0).Figure 7 shows the SWVs of AA at PGE/CTP electrode in different concentrations.The peak current belongs to AA oxidation is observed at about −0.3 V on PGE/CTP electrode.The anodic peak current of AA systematically increased with increasing concentration in the range 1000.0-0.5 nM (Fig. 7).
The calibration plot shows a linear relationship with a correlation coefficient of 0.9905 in Fig. 7B, obeying the equation: The calibration plot shows a linear relationship with a correlation coefficient of 0.9975 in Fig. 7C, obeying the equation: The formula of the limit of detection LOD= 3.3 × (SD of intercept/slope) and limit of quantitation LOQ = 10 × (SD of intercept/slope) were used to calculate the detection limit and the limit of quantification.Under the optimized conditions, the detection limit of this method is 0.2094 nM, and the limit of quantification is 0.6912 nM.
Table 1 lists some AA electrochemical sensors, in which the electrode composition, which technique, the linear range and LOD are summarized.The developed sensor in the present work exhibited a wide linear range and a competitive LOD value by comparison with the others.It shows the high sensitivity of the as-fabricated sensor, which should be related to the good electric conductivity and catalytic ability of the PGE/CTP electrode.In general, the synthesis of coating compounds is complex.Most of them purchased are expensive.Furthermore, preparing the modified electrodes is taking too much time, while the preparation of PGE/CTP electrode is simple and quick.

Determination of AA in Real Sample
The proposed method was successfully used for the determination of AA using PGE/CTP electrode for instant coffee sample.The voltammogram of PGE/CTP electrode was taken by SWV technique in PBS solution at pH 7.0 for the measurement of AA in the real sample in Fig. 8.After the peak current was measured, the obtained peak was used to find the AA concentration in the instant coffee sample from the calibration curve by interpolating the peak current obtained from the voltammogram.The peak current value read for AA in the instant coffee sample from the voltammogram was measured as 7.31 μA.When the necessary calculations were made by using this value from the calibration graphic drawn, the amount of AA in the sample was calculated as 783.96 nM that is 55.723 μg kg −1 .

Conclusion
The demand for determination of acrylamide in foods is increasing due to the significant hazards accompanied by its exposure.This research provides a simple, low cost, and rapid method to determine acrylamide in food samples using PGE/CTP electrode by performing SWV technique.The optimization of supporting electrolyte and pH measurements was carried out.Phosphate buffer solution at pH 7.0 was utilized.The scan rate investigations stated that mass transport mode of AA on PGE/CTP electrode was diffusion-controlled process.The linear relationship between AA concentrations and peak current was observed within the range of 1000.0 to 0.5 nM.The methodology applied in the present investigation can be suitably extended to other natural samples.
Authors' Contribution ŞK: She took part in all experimental procedures for this study, writing and reading the manuscript.
WB: He took part in all experimental procedures for this study, writing and reading the manuscript.
İEM: He carried out the operations of directing, controlling and interpreting the results of the experiments for the study.He also carried out the final reading of the manuscript.
ADM: She carried out the operations of directing and controlling the experiments for the study.She also carried out the final reading of the manuscript.

Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
Ethics Approval This article does not contain any studies with human or animal subjects.

Fig. 6 A
Fig. 6 A LSV voltammograms of 1.0 mM AA in 0.1 M PBS at pH 7.0 with different scan rates using PGE/CTP electrode.B Plot of peak current versus scan rate.C Plot of log peak current versus log scan

Table 1
Comparison among preceding relevant works of AA determination with the current workHb hemoglobin, Fe Mag NP iron magnetic nanoparticle, PMB poly (methylene blue), dsDNA double stranded DNA, MWCNT multi-walled carbon nanotube, CuNP copper nanoparticle, PANI poly(aniline) Fig. 8 PGE/CTP electrode voltammogram of AA in instant coffee sample