Facile and Sensitive Acetylene Black-Based Electrochemical Sensor for the Detection of Imatinib

A facile and sensitive electrochemical sensor for determining imatinib (IMA) was constructed by modifying a glassy carbon electrode (GCE) with a nanocarbon material, acetylene black (AB). The electrochemical behavior of IMA on the prepared GCE/AB was studied using electrochemical techniques, namely, differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy. The direct determination of IMA by the GCE/AB sensor was accomplished using DPV under optimized conditions. The method verification showed that the oxidation peak current was proportional to the concentrations of IMA in the linear ranges of 0.01–0.5 and 0.5–4 μM, with correlation coefficients of 0.9856 and 0.9946, respectively. The limit of detection of the GCE/AB sensor was 0.15 nM. Moreover, the GCE/AB sensor showed good precision and accuracy. Finally, the GCE/AB sensor was successfully applied to determine IMA in human serum samples, and the recoveries were satisfactory.


Introduction
Imatinib (IMA) is a tyrosine kinase inhibitor for the treatment of cancers, such as chronic myeloid leukemia [1], gastrointestinal stromal tumor [2], and dermatofbrosarcoma protuberans [3].Reportedly, the molecular and cytogenetic response to IMA is related to the low plasma concentrations in patients.Terefore, IMA monitoring is necessary for clinical treatment [4].Tus far, several analytical techniques have been applied to the detection of IMA, such as electrophoresis [5], high-performance liquid chromatography [6], and UV-vis spectroscopy [7].However, due to the expensive equipment, long analysis process, and complicated operation steps of the existing techniques, an economical, fast, simple, and efcient method for IMA determination is urgently needed.Te electrochemical method is a potential analytical technique for the detection of IMA and has attracted great attention because it provides several advantages, such as short analysis time, easy sample pretreatment procedures, and suitability for real-time detection [8].According to previous research, an unmodifed glassy carbon electrode (GCE) fails to provide a satisfactory efect for the detection of IMA, while modifed GCE obtained indispensable adsorption and catalytic properties [9].Acetylene black (AB) is a type of nanocarbon material formed by acetylene combustion in air under pressure [10], which has attracted tremendous attention because of its extraordinary conductivity and chemical stability [11], electrocatalytic properties [12], and adsorption ability [13], thereby promoting its further application in the feld of electrochemical analysis.For instance, AB-modifed electrodes exhibit superior electrochemical properties in measuring various substances, such as erythromycin [14], methotrexate [15], topotecan [16], methimazole [17], and chrysophanol [18].To our knowledge, AB has never been used for the detection of IMA.Tus, AB is applied in this research to modify GCE for the fabrication of an electrochemical sensor (GCE/AB), which is applied for determining IMA in serum.

Instruments and Equipment
An electrochemical workstation (CHI660e) was obtained from Shanghai Chenhua Instrument Co., Ltd.Te three-electrode system consists of the GCE/AB, the saturated calomel electrode, and the platinum electrode (Wuhan Gaoss Union Science and Technology Ltd.).A constant-temperature magnetic stirrer was purchased from Tianjin Sidis Experimental, an analysis instrument manufacturer.A pH meter (PHS-3E) was obtained from Shanghai INESA Scientifc Instrument Co., Ltd.A CNC ultrasonic cleaner (KQ-50DB) was obtained from Kunshan Ultrasound Instrument Co., Ltd.Pipettes and a centrifuge were obtained from the German company Eppendorf.A pure water machine was obtained from Beijing Shuangfeng Zhongbang Technology Development Co., Ltd.A vortex mixer was obtained from Jiangsu Tianli Instrument Co., Ltd.A precision electronic balance was obtained from Sedis, Germany.A hard infrared light bulb was obtained from Shenzhen Anderson Lighting Co., Ltd.A refrigerated freezer (BCD-209 HFA) was obtained from Qingdao Aucma Co., Ltd.

Solution Preparation
Te IMA solution (0.1 mM) was prepared by dissolving IMA in methanol.A phosphate bufer saline (PBS, 0.1 M) of diferent pH values was prepared with Na 2 HPO 4 •12H 2 O and NaH 2- PO 4 •2H 2 O, and the pH of the PBS was adjusted with the HCl and NaOH solutions.Ten, 2.0 mg of AB was added to 2.0 mL of pure water, which was sonicated and dispersed for 1 h to obtain a fnely evenly distributed black suspension (AB suspension).A redox probe containing 5 mM of [Fe(CN) 6 ]3/4(1 :1) and 0.1 M of KCl was prepared by dissolving potassium ferrocyanide (K 4 Fe(CN) 6 ), potassium ferricyanide (K 3 Fe(CN) 6 ), and potassium chloride (KCl) in pure water.

. Electrode Preparation
First, the GCE surface was polished with gamma aluminum polishing powder.Subsequently, the electrode was washed sequentially with an ultrasonic washer in absolute ethanol and pure water for 5 min until the electrode surface was smooth as a mirror.Finally, 4.5 μL of the AB suspension droplet was applied to the GCE surface and dried under an infrared bulb to obtain the GCE/AB.

Sample Preparation
First, 0.5 mL of human serum samples, which were placed at room temperature, was added into Eppendorf tubes, to which 1 mL of methanol was added.After an even oscillation on the vortex mixer, the samples were centrifuged at 10,000 rpm for 15 min.Te supernatant was collected using a syringe and fltered through a 0.22 μm microwell flter to obtain the fltrate, that is, the purifed serum.A 20 μL purifed sample was added to the PBS (10 mL, 0.1 M, and pH 7.0) with specifc IMA concentrations (0.1, 1, and 2 µM) and fnally detected by the GCE/AB electrochemical sensor.

Detection Method
PBS with a pH of 7.0 (10 mL) was selected as the reaction medium, to which 50 μL of IMA solution (0.1 mM) was added.Te PBS was stirred for 4 min, while the GCE/AB was enriched with IMA.After IMA enrichment, the diferential pulse voltammetry (DPV) curves with the potential from 0.1 V to 0.9 V were recorded, and the scan rate, the pulse width, and the pulse amplitude were 20 mVs −1 , 40 ms, and 50 mV, respectively.Electrochemical impedance spectroscopy (EIS) was applied in the redox probe in the frequency range of 0.01 Hz-100,000 Hz.

Fabrication and Characterization of the Sensor.
A series of detection experiments using DPV to select the best electrochemical material for adsorbing and catalyzing IMA were performed.As shown in Figure 1(a), the oxidation peak current of IMA appeared at the potential of 0.560 V, but no reduction peak was observed in the reverse scan, indicating that the reaction of IMA on this electrochemical sensor was pure oxidation.After GCE modifcation, the oxidation peak current of IMA was improved by diferent materials.Te AB-modifed electrode (GCE/AB) performed with the highest oxidation peak current, indicating that the GCE/AB possesses the best adsorption and catalytic properties among the material-modifed electrodes.Tis result demonstrates the feasibility of this research, again proving the high specifc surface area, strong adsorption capacity, and excellent conductivity of AB [10].Terefore, we selected AB as the electrode modifcation material for this experiment.Figure 1(b) presents the interfacial characteristics of bare and diferently modifed GCE via EIS [19].In the Nyquist plot of the EIS, the broader the semicircle curve of the plot is, the greater the resistance is [20].Te electron-transfer resistance at the electrode surface can be quantifed by the diameter of the semicircle [21].Te diameter sizes followed the order GCE > GCE/AB/IMA > GCE/AB.A broad semicircle was monitored in the Nyquist plot for the bare electrode, indicating that the bare GCE faced high resistance.

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International Journal of Analytical Chemistry A narrow semicircle was observed for the GCE/AB, indicating a low resistance.Te GCE/AB had a lower electrontransfer resistance than the bare GCE, confrming the successful construction of the GCE/AB-based electrochemical sensor.Moreover, the GCE/AB had a larger semicircle after IMA adsorption, indicating that the IMA adsorption on the GCE/AB surface increased the electrontransfer resistance.In addition, the Nyquist plot of the bare GCE showed a relatively large semicircle with an Rct of 1760 Ω.After the modifcation of the GCE with AB, the value of Rct decreased to 199 Ω, which confrmed that the electron-transfer property of AB/GCE improved.Te electrochemically active surface areas of the bare GCE and modifed electrodes were calculated using CV at various scanning rates (40-160 mV s −1 ) in a solution containing 5.0 mM of [Fe(CN)6]3−/4− and 0.1 M of KCl.Te efcacy of the embedded sensors was analyzed according to the Randles-Sevcik equation at 25 °C [22]: where I p is the peak current, n is the electron-transfer number in the process (n � 1), D is the difusion coefcient (7.6 × 10−6 cm 2 /s), A is the electrode surface area (cm 2 ), C is the [Fe(CN)6]3−/4− concentration (mol/cm 3 ), and v is the scanning rate (V/s).As shown in Figure 2, the slopes from the plot of current against the scanning rate square root were used for the calculation of the electroactive surface areas, and A values were 0.05 and 0.11 cm 2 for the surfaces of bare GCE and AB/GCE, respectively.So AB could be reinforced during electroanalysis because of an impressive elevation in its electroactive surface area.International Journal of Analytical Chemistry

Efects of Bufer pH and DPV Parameters.
Te efect of the bufer pH on the peak current of IMA was investigated by changing the pH value from 3.9 to 9.7.As shown in Figure 5(a), the peak current increased signifcantly from 3.9 to 7.0 and decreased in the pH range of 7.0-9.7.Tus, 7.0 is the optimal pH level.In addition to the bufer pH, the efects of DPV parameters on the results should be considered.Te main DPV technique parameters, including the pulse width, scan rate, and pulse amplitude, were studied in the ranges of 20-100 ms, 10-30 mVs −1 , and 20-200 mV, respectively.Te results show that the peak pattern was the best when the pulse width, the scan rate, and the pulse amplitude were 40 ms, 20 mVs −1 , and 50 mV, respectively.

Electrochemical Mechanism of IMA on the AB/GCE.
Te electrochemical response of IMA on the prepared electrode at diferent scanning rates was investigated to further explore its reaction mechanism.Figure 5(a) shows that the peak oxidation potential of IMA moved negatively as pH increased from 3.9 to 9.7.As can be seen in Figure 5(b), the pH value was linearly related to the oxidation peak potential of IMA, and its regression equation was EP (V) � −0.031pH + 0.996 (R 2 � 0.994).Te slope value of −32.13 mV was close to half of the theoretical value of Nernst's equation (−59 mV), which means that the number of protons involved in the IMA oxidation reaction was half of the electrons [23].

International Journal of Analytical Chemistry
In Figure 5(c), the peak current of IMA was linearly correlated with the scanning rate at 60-160 mV s −1 , and the linear equation was I (μA) � 0.279v − 4.864 (R 2 � 0.983).Te results indicated that the reaction system of IMA in this study was controlled by the surface.On this basis, the relationship between the logarithm of the scanning rate (lnv) and oxidation peak potential was discussed, and a linear equation of EP (V) � 0.028lnv + 0.689 (R 2 � 0.997) was made as shown in Figure 5(d).Tis fully confrmed the above conclusion that the oxidation reaction of IMA on the prepared sensor was not reversible.In conclusion, IMA had a surface-controlled irreversible oxidation reaction on AB/GCE, and further analysis required the application of the following relationship [24]: where E0 is the standard potential, T is the temperature, α and F represent the transfer coefcient and Faraday constant, and k0 and n represent the electron-transfer rate constant and electron-transfer number.We took an α value of 0.5 when an irreversible reaction occurred and then obtained n � 1.94.Terefore, the oxidation reaction of MTX at AB/GCE was a two-electron and one-proton electrochemical process, as can be seen in Scheme 1. (μM) + 9.4279 (R 2 � 0.9856) (0.5-4 μM).Te limit of detection (LOD) of the method obtained from LOD � kS b /m was 0.15 nM [25], where k � 3, S b is the standard deviation of the blank signals, and m is the slope of the calibration curve (the lower concentration range).Te performance of the proposed sensor was compared with that of the sensors in the literature, and the results are shown in Table 1.Te fnding shows that the GCE/AB sensor possesses wider linear ranges and a higher sensitivity supported by a lower LOD than the other sensors.Moreover, the GCE/AB sensor requires simple materials and preparation methods.Terefore, the preparation of the GCE/AB electrochemical sensor for IMA detection is necessary.
In addition, the precision of the prepared GCE/AB sensor was evaluated in this study.IMA at 1 μM was repeatedly detected six times within one day, and the coefcient of variation (CV%) of the peak current values was as low as 6.52%.One electrode was prepared daily to detect IMA at 1 μM one week, and the CV% of the peak current values was as low as 8.31%.Te results show that the withinday precision and the day-to-day precision satisfy the IMA detection requirement.Terefore, the GCE/AB sensor has excellent reproducibility and stability for IMA detection.

Accuracy
(1) Interference Test.Anti-interference capacity is an important factor that may afect the accuracy of IMA detection through electrochemical methods [30].In this study, the efects of some organic and inorganic substances on IMA detection were investigated by adding diferent potential interfering substances to PBS containing 1 μM of IMA.If the change in the peak current of IMA induced by the interfering substances is less than ±10%, then noninterference is recorded.Table 2 provides the interference levels of several substances for the 1 μM IMA detection.In the presence of 1000fold of Na + , K + , and Cl − , 200-fold of Fe 3+ , Fe 2+ , Zn 2+ , and SO 4 2− , 100-fold of glucose, 50-fold of vitamin C and citric acid, 10-fold of glycine, and fvefold of folic acid, the potential substances imposed a noninterference efect on IMA detection.Tus, the GCE/AB sensor exhibited a satisfactory anti-interference capacity for IMA detection.
(2) Addition Recovery Test.An addition recovery test is used to assess the ability of experimental methods to accurately determine the pure analytes added to the conventional samples, and the results are expressed as recovery (%) [31].Tree concentration levels of IMA (0.1, 1, and 2 μM) were added to healthy human serum, and then, the peak current values were detected by the GCE/AB sensor.Te recovered concentrations were calculated according to the calibration  International Journal of Analytical Chemistry curve of each added concentration, and the recovery (%) obtained is shown in Table 3.
Te IMA recovery in this work was within the range of 90.1%-98.98%,and the relative standard deviation (RSD) was within the range of 0.3%-8.6%,indicating that the prepared GCE/AB electrochemical sensor can be used in the accurate detection of IMA.

Conclusions
In this research, a novel electrochemical sensor based on ABmodifed GCE was successfully prepared for IMA determination.Within the allowable error limits, the sensor has a good linearity with ranges of 0.01-0.5 μM and 0.5-4 μM.In addition, the sensor exhibited high sensitivity with a low LOD of 0.15 nM and excellent precision.Moreover, the sensor displayed satisfactory anti-interference capacity and accuracy for IMA detection.Terefore, this study provides a practical platform for IMA determination, creating a new possibility for improving the usage safety of clinical drugs.

Figure 5 :
Figure 5: (a) Efects of bufer pH on the peak current of IMA by the GCE/AB sensor.(b) Potential diagram of diferent pH values in the presence of 0.5 μM of IMA in PBS solution.(c) Corresponding plot of current vs. scanning rate (60-160 mV/s) in the presence of 0.5 μM of IMA in PBS solution.(d) Linear relationship between E(V) and the logarithm of the scanning rate.

Table 1 :
Analytical performance of diferent IMA electrochemical sensors.

Table 2 :
Interference levels of several substances for detecting 1 μM of IMA.

Table 3 :
IMA detection in the human serum by the GCE/AB sensor.