Glutathione (GSH) is the most abundant intracellular thiol with the concentration in the millimolar range (1–10 mM) [1-3]. GSH plays a very important role in regulating various physiological and pathological processes [1,4]. It has been reported that GSH serves many cellular functions, including xenobiotic metabolism, intracellular signal transduction, and gene regulation . In addition, the intracellular redox activity of GSH continues with its antioxidant properties[5,6]. Abnormal levels of GSH can lead to diabetes mellitus, Alzheimer’s disease, atherosclerosis and other diseases [6-8]. More importantly, changes in the concentration of GSH in biological tissues are used as a marker for certain disorders . In view of the important biological and clinical significance for GSH, it has thus spurred strong interest in developing useful chemical materials for quantitatively evaluating the level of intracellular GSH [4,8].
Glutathione is a tripeptide which can be synthesized in the liver using no genetic information [9–12]. It is a major antioxidant which protects cells from oxidative damage by reacting with free radicals and peroxides [7,9,10]. Glutathione exists in tissues in two different types which stay in balance in tissues: reduced GSH and oxidized glutathione (GSSG) [13–15]. In the physiological process of GSH, the conversion to GSSG occurs by the glutathione peroxidase enzyme with selenium. Oxidized glutathione reduces the oxidized form of GSH by an NADPH-dependent flavoenzyme GSH reductase . It is important to detect GSH using a reliable method with good sensitivity and selectivity. Due to its electrochemically oxidizable property [16, 17], it has become important to develop an effective electrochemical sensor for the determination of GSH. For this reason, a simple and reliable electroanalytical method and a sensor modified with single-walled carbon nanotube (SWCNT) were developed to determine the reduced-GSH. To the best knowledge of this work, this is the first study of the determination of GSH at SWCNT modified sensor in the literature.
Material and Method
Reagents and Apparatus
Reduced GSH was purchased from Merck, and N, N-dimethylformamide (DMF) was from Sigma. Single-walled Carbon Nanotube (SWCNT) (diameter: <2 nm, EC: >100 S/cm, length: 5-30 µm, purity >95%, surface area: 380 m2g-1) was supplied from © Grafen Inc. Stock GSH solutions were freshly prepared in ultra-pure water for daily. Na2HPO4, KH2PO4(Carlo Erba), KCl (Merck) and NaCl (Merck) were dissolved for 0.10 M phosphate buffer solution (PBS). Milli Q (Millipore, 18.2 MΩ.cm) ultra-pure water was used during the preparation of solutions.
All the electrochemical operations (Cyclic voltammetry (CV) and Differential pulse voltammetry (DPV) were carried out by a BAS (Bioanalytical Systems, Inc.) 100 W electrochemical analyzer. The three electrode system was used consisting of a glassy carbon workingelectrode (GCE) (geometric area: 6.85 mm2, CHI), an Ag/AgCl reference electrode (CHI112) and a Pt disc auxiliary electrode (CHI). The pH was measured with a Jenway 3010 pH meter.
Measurement of GSH in urine samples
Urine samples were diluted 10 times with 0.1 M PBS (pH 7.4) without any additional pretreatment. After that, 100, 200 and 300 µM of reduced GSH added to 10 mL of this solution in an electrochemical cell for electroanalytical measurements. Three multi-run DPV measurements were performed in these samples prepared with three replicates at the modified sensor. The standard addition method was applied to the urine samples by spiking of reduced GSH.
Preparation of Modified Sensors
SWCNT dispersions were prepared with N,N-dimethylformamide (DMF) solution at the concentration of 1.0% (mg/μL). SWCNT/DMF dispersions were sonicated for 4 h before dripping onto the GCE surface. Modification of GCEs was carried out by dripping of 10 μL of SWCNT dispersions on the surface of bare GCE. And then SWCNT/GCE modified sensor was dried overnight.
Electroanalytical determination of 10 µM reduced GSH was done in PBS (pH 7.4) at SWCNT/GCE modified sensor with DPV and CV techniques (Figure 1). In order to obtain a calibration graph, the solutions of reduced GSH were analyzed by the DPV technique at SWCNT/GCE modified sensor. The concentrations of reduced GSH were changed from 1 x 10-8M to 5 x 10-8M. The calibration curve was generated by plotting the current values against the concentrations (Figure 2). The results showed that the anodic peak currents of GSH were linear at the concentration range of 0.01 μM to 0.05 μM. The calibration equation was Ipa (μA) = 0.0956C (μM) + – 0.0166 with correlation coefficients (R2) as 0.9909. Excellent correlation was found between the concentrations of GSH and peak current densities.
Validation parameters were shown in Table 1. The sensitivity and the detection limit (LOD) of GSH were found from the slope of the calibration curve. The limit of detection (s/m= 3), where sis the standard deviation of the peak currents (for five runs) and mis the slope of the calibration curve, was 0.75 nM and the sensitivity was 0.0956 µA/µM. Three replicate solutions of urine samples containing reduced GSH were analyzed using the DPV technique at SWCNT/GCE modified sensor. GSH concentrations in real samples were calculated from the calibration curve equation by using the obtained current values. The recovery values obtained from the GSH analysis in real samples showed the accuracy and reproducibility of the sensor in Table 2. The proposed method was satisfactorily performed to real samples.
The effect of supporting electrolyte and pH, which are one of the parameters that can affect the voltammetric behavior of GSH, play an important role. Selecting a suitable support electrolyte creates a conductive medium for sensing the analyte in the solution medium to be analyzed and the sensitivity of the sensor increases. The choice of support electrolyte and pH depend on its resolution, dissociation degree, and nucleophilic character [18,19]. For this purpose, the DPV responses of GSH were investigated in PBS at pH 5.0 – 8.0. There was an increase in peak currents up to pH 7.4, but a decrease in peak currents after pH 7.4. It was found that the anodic peak potential of GSH shifted negatively with the increase of pH value, and the anodic peak current decreased due to the participation of two protons in the oxidation process [20, 21]. Consequently, subsequent electroanalytical studies were performed at PBS pH 7.4 as the supporting electrolyte medium. Furthermore, the maximum response at pH 7.4, which is physiological pH, is promising in terms of working with real samples.
The electrochemical determination of GSH was studied at GCE modified with SWCNT and bare GCE by CV and DPV method. The oxidation peak current of DPV response was selected as the analytical signal of GSH. The amperometric sensor displayed excellent electrochemical performance to the oxidation of reduced GSH. The GSH oxidized and reduced at SWCNT/GCE modified sensor by CV techniques at potentials of nearly 247 mV and 177 mV, respectively. GCE could not show any reduction/oxidation peak at bare GCE. GSH oxidized at nearly 212 mV (3.198 µA) by DPV method. The quantification of GSH was successfully achieved with these techniques.
The five successive tests at the same SWCNT/GCE modified sensor and different modified sensors imparted almost the same electroanalytical response with the recoveries of 99.12% and 98.65%, respectively. These results demonstrated that SWCNT/GCE modified sensor has excellent reproducibility and repeatability. Furthermore, ten cycles of the GSH oxidation currents at the SWCNT/GCE modified sensor was exhibited great stabilization by 0.024% RSD. Additionally, very low concentrations of GSH were perfectly determined at the modified sensor and a very low detection limit, such as 0.75 nM, was obtained. In order to determine the analytical applicability and accuracy of the modified sensor, urine samples were analyzed using SWCNT modified sensor. The satisfactory recovery values showed that the proposed sensor could be easily used to identify GSH in physiological fluids.
Several studies have shown the quantification of GSH at different modified sensors. However, the voltammetric response properties obtained in this work (including linear range and detection limit) are better than similar studies from the literature [1,3,5,8,22-32]. As a result, of a comparison between voltammetric response properties of the various modified electrode and proposed modified sensor, higher detection limit values are obtained at the corresponding sensors. This proved that the sensor used in this study could detect lower GSH quantities.
GSH does not present well-known redox response at the bare glassy carbon electrode. Surface modification of the electrode with SWCNT allows analyzing the alteration of the sensor electrochemical response. Voltammetric studies of GSH at the surface of the modified electrode have shown adsorption-like behavior in which, the GSH is linked to the composite. Determination of very low concentrations of GSH in physiological liquids was achieved by the electroanalytical method at the modified sensor with high recovery values. The results suggested that the SWCNT modification of electrodes may provide a new strategy for GSH concentration determination in physiological solutions. Therefore, the modified sensor can be promising to be used in routine analyses.
Scientiﬁc Responsibility Statement
The authors declare that they are responsible for the article’s scientiﬁc content including study design, data collection, analysis and interpretation, writing, some of the main line, or all of the preparation and scientiﬁc review of the contents and approval of the ﬁnal version of the article.
Animal and human rights statement
All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. No animal or human studies were carried out by the authors for this article.
Conflict of interest
None of the authors received any type of financial support that could be considered potential conflict of interest regarding the manuscript or its submission.
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