Electrochemical impedance spectroscopy (EIS) is a useful technique for the investigation of electrode surface dependent charge transfer process. Figure 6B shows Nyquist plots recorded at bare and C-dots modified GCE in 0.1 M KCl solution containing 5 mM [Fe(CN)6]3−/4−. Nyquist spectrum consists of a capacitive loop (semicircle part) in higher frequency region and a straight line in low frequency region. The semicircle is associated with electron transfer limited process at the electrode surface and linear portion corresponds to the diffusion process. The electron transfer resistance (Rct), corresponds to the semicircle diameter of the Nyquist plot of –Z' against Z'' (impedance of the interface derived by application of Ohm’s law, consisting of two parts, a real number Z' and imaginary one, Z''). Rct values reveal the electron transfer kinetics of the redox probe at electrode interface (Table 1). It is evident from Fig. 6B that a well-defined semicircle was observed at bare GCE. This suggested that the redox probe has exhibited slow electron transfer kinetics at bare GCE. When it was modified with C-dots, the diameter of the semicircle decreased in comparison with that of bare GCE. Further, C-dots modified GCE showed decreased Rctvalue (15.52Ω cm2) indicating that the conducting C-dots on the electrode surface facilitated the electron transfer kinetics and hence showed less impedance to the electron transfer process compared to that at bare GCE (48.05 Ω cm2). In other words, the presence of carbon dots enhanced the electrical conductivity and accelerated the electron transfer rate.
Optimum determination conditions
Influence of pH.- The pH of a buffer solution influences the electrochemical behaviour of HES. As shown in Fig. 7C and D, oxidation peak potentials of HES were shifted to negative end and peak current decreased gradually with increase in pH (3–9), indicating that the protons have taken part in the electrode process. Linear relationship was noticed between oxidation peak potentials and pH values and the corresponding regression equations are as follows:
Epa1 (V) = 0.0672 + 0.961, R2 = 0.979
Epa2 (V) = 0.068 + 1.531, R2 = 0.989.
The slope value of 67/68 mV pH− 1 is close to the theoretical Nernstian value of 59 mV pH− 1 suggesting that the same number of protons and electrons are involved in the electrode process [45]. Unlike oxidation peaks, redox peaks c1 and a3 yielded straight lines with slope values of 63 and 67 mV/pH respectively, indicating that equal number of protons and electrons have taken part in the electrode process. The corresponding regression equations are shown below:
Epa3 (V) = 0.063 + 0.576, R2 = 0.981
Epc1 (V) = 0.067 + 0.830, R2 = 0.992
It is observed that the anodic peak current increased with increasing pH value until it is reached 4.0; however, anodic peak current decreased remarkably when the pH was greater than 4.0. Therefore, the PBS of pH 4.0 was selected as the electrolyte in the following experiments.
Influence of amount of C-dots and accumulation time,- The amount of modifier on the surface of electrode and accumulation time are important factors that influence the voltammetric response of HES. As shown in Fig. S1, increased peak current was noticed when the volume of C-dots deposited on the surface of GCE was in the range of 1–7 µL. However, beyond 7 µL the peak current decreased dramatically. This was due to the thicker film of C-dots that blocked the electrode surface for electron transfer between the electrode and target. So, 7 µL of C-dots was chosen as the optimum volume to modify GCE.
Accumulation time plays an important role to achieve high sensitivity of the proposed method for analysis. As can be seen from Figure S2, the peak currents of HES increased rapidly with increase in accumulation time from 0 to 150 s. Beyond 150 s, decreased peak current was noticed. This was due to the saturation of HES on the surface of C-dots/GCE. Hence, an accumulation time of 150 s was maintained throughout the study.
Effect of scan rate.- In order to understand the electrochemical reaction mechanism of HES at C-dots/GCE, the effect of potential scan rate on peak current was investigated. FigureS3 shows the CV response of 10 mM HES at C-dots/GCE with the scan rate ranging from 10 to 300 mV/s. It could be observed that the peak currents of HES increased with increase in the scan rate. In addition, the log peak currents showed a linear relationship with log scan rates and the corresponding regression equations are shown below:
log Ipa1 = 0.743 logυ − 4.488, (R2 = 0.994).
log Ipa2 = 0.683 logυ − 4.567, (R2 = 0.997).
log Ipc1 = 0.791 logυ − 3.537, (R2 = 0.988).
log Ipa3 = 0.715 logυ − 3.740, (R2 = 0.928).
The results suggested that the electrode process was adsorption controlled for all the peaks as evident from their linear dependence of peak current on square root of scan rate (figure not shown). According to kinetic theory of an electrode reaction, the slope of log Ip vs. log υ shall be close to unity for a purely adsorption controlled process and close to 0.5 for a purely diffusion controlled process [45]. Hence, the observed slope values confirmed the presence of adsorption controlled electrode process for electrochemical behaviour of HES at C-dots/GCE. Electrochemical kinetic parameters were evaluated using Laviron equations [46, 47]:
Epa = E0' + RT/αnF[ln(RTks)/(αnF) - lnυ] for anirreversible system (1)
Epa = E0'+2.3RT/(1-α)nF logυ (2)
Epc = E0'- 2.3RT/αnF logυ for a reversible system (3)
Log ks = α log(1 - α) + (1 - α ) log α - log RT/nFυ - (1 - α) αnF∆Ep/2.3RT (4)
where n, α and ks are the number of electrons transferred, the charge transfer coefficient and heterogeneous electron transfer rate constant respectively, E0 is the formal potential and other symbols have their usual significance. In the totally irreversible reaction, the values of αn and ks were calculated to be 1.08 and 1.05 s− 1 for peak a1 and, 0.63 and 1.02 s− 1 for peak a2. Generally, α is assumed to be 0.5 in a totally irreversible electrode process. So, the value of n was calculated to be 2 and 1 for peak a1 and peak a2, respectively. For the redox reaction, the values of α and n were evaluated to be 0.56 and 4 respectively. By knowing the values of α and n, ks was evaluated to be 1.96 s− 1. From these results, we propose that two and one electrons are involved in the irreversible oxidation that corresponds to peaks a1 and a2 respectively. For the redox peak, four electrons have taken part in the electrode process.
The surface concentration (Γ) of HES at C-dots/GCE was further calculated from the slope of the plot of peak current vs. scan rate according to equation shown below [48]:
Ip = n2F2 ΓAυ/4RT
where Ip is the peak current, n is the number of electrons transferred and A is the surface area of the electrode. The value of Γ was calculated to be 2.16 X 10− 10 mol cm− 2 at the scan rate of 0.1 V/s. Smaller Γ value indicated that the electrode reaction of HES at C-dots modified GCE was controlled by diffusion process.
Electrochemical determination of HES on C-dots/GCE.- The electrochemical sensing performance of C-dots/GCE towards the sensing of HES was investigated by DPV, SWV and adsorptive stripping differential pulse voltammetric (AdSDPV) techniques under optimized conditions. Among these three methods, SWV was noticed to be sensitive compared to that of DPV and AdSDPV as evident from their low LOD values. Voltammograms of increasing concentrations of HES showed that the peak current increased linearly with concentration and the results are shown in Fig. 8A-C. HES showed the good linear relationship between peak current and concentration in the range of 0.05–62.5 µM for DPV, 0.01–125 µM for SWV and 10–62 µM for AdSDPV respectively. Corresponding regression equations are shown below:
Ip = 0.35 + 3 X 10− 9, R2 = 0.994 for DPV method
Ip = 0.46 + 4 X 10− 9, R2 = 0.986 for SWV method
Ip = 0.043 + 3 X 10− 9, R2 = 0.986 for AdSDPV method
The values of limit of detection and limit of quantification were calculated to be 3.2 x 10− 8 M and 0.4 x 10− 7 M for DPV, 0.18 x 10− 8 M and 0.15 x 10− 8 M for SWV, and 2.09 x 10− 7 M and 6.97 x 10− 7 M for AdSDPV (Table 2) respectively. Low values of these confirmed the sensitivity of the proposed methods. The inter-day and intra-day reproducibility of the proposed sensor was examined and RSD values were found to be less than 2.5% (Table 2). Low values of RSD revealed good precision of proposed methods for the assay of HES.
Table 2
Characteristics of calibration plot for HES in DPV, SWV and AdSDPV methods
| DPV | SWV | AdSDPV |
Linearity range, µM | 0.05–62.5 | 0.01–125 | 10–62 |
LOD, µM | 0.032 | 0.0018 | 0.21 |
LOQ, µM | 0.04 | 0.015 | 0.69 |
Inter-day assay RSD*, % | 1.5 | 2.1 | 1.8 |
Intra-day assay RSD*, % | 2.3 | 1.9 | 2.4 |
*For 5 replicates |
The stability and reproducibility of C-dots/GCE.- The reproducibility and stability of the modified electrode was evaluated by differential pulse voltammetric method under optimum conditions. Five C-dots modified electrodes were made and their current responses for 10 mM HES were investigated. The relative standard deviation (RSD) was found to be 2.02% indicating excellent reproducibility of the prepared sensor. The stability of C-dots/GCE was evaluated over a period of three weeks. During this period five parallel experiments were performed. In three weeks, the peak current of HES was decreased by less than 5% of the initial response, indicating that the C-dots/GCE was stable for electrochemical application.
Selectivity and interference.- Electrochemical sensors are sensitive which can be used for the assay of wide range analytes. However, this is sometimes restricted due to the interference from other molecules (electroactive) present in the analyte solution that may undergo oxidation/reduction at similar potentials as the analyte. In the present study, we have examined the selectivity of the proposed sensor by recording SWV of a mixture containing HES and genestein that has structure similar to HES on bare GCE and C-dots/GCE (Fig. S4). Only two oxidation peaks were noticed at bare GCE. However, the peak due to oxidation of genestein was not noticed. But, three oxidation peaks (a', b' and c) were observed on C-dots/GCE at 0.581, 0.632 and 1.03 V, respectively. The peaks, a’ and b’ were due to the oxidation of HES while peak c was due to oxidation of genestein. So, genestein did not interfere in the proposed electrochemical determination of HES.
Interference study was conducted under optimal conditions for the determination of HES in the presence of six interferents viz., urea, glucose, ascorbic acid, starch, thiourea and uric acid by SWV method. Oxidation peak current of HES was barely affected in the presence of several folds of above mentioned inteferents (Table 3). This indicated that the proposed sensor could be used for selective determination of HES.
Table 3
Tolerance of interferents in the determination of 10 µM HES by SWV method
Interfering substance (IS) | Concentration of IS (µg mL− 1) | Fold excess | Recovery (%) |
Glucose | 20 | 18.3 | 97.48 |
Starch | 20 | 13.1 | 96.43 |
Ascorbic acid | 40 | 26.1 | 95.79 |
Uric acid | 15 | 28.5 | 95.49 |
Urea | 15 | 25.2 | 94.65 |
Thiourea | 20 | 30.7 | 98.52 |
Analysis of biological samples.- The practical analytical application of the method was further established by determining HES in human urine samples without any preliminary treatment. The recovery of HES from the urine sample was examined by spiking drug free urine with known amounts of HES and by recording voltammograms. The calibration graph was used to determine the concentrations of HES in urine samples. Higher average recovery (97.66%) (Table 4) revealed the accuracy of proposed methods. Low RSD values (below 3.33%) suggested good precision of proposed methods.
Table 4
Results of analysis of HES in spiked human urine samples
| Amount of HES added, µM | n | Amount found, µM | Average recovery, % |
DPV method | 0.5 | 5 | 0.486 | 97.20 |
1.0 | 5 | 0.971 | 97.10 |
1.5 | 5 | 1.478 | 98.53 |
SWV method | 0.5 | 5 | 0.482 | 96.40 |
1.0 | 5 | 0.968 | 96.80 |
1.5 | 5 | 1.466 | 97.73 |
AdSDPV method | 0.5 | 5 | 0.487 | 97.40 |
1.0 | 5 | 0.972 | 97.20 |
1.5 | 5 | 1.451 | 96.73 |
Studies on the interaction between HES and BSA.- In order to understand the binding of HES to BSA, cyclic and differential pulse voltammograms of 20 µM HES were recorded at C-dots/GCE in the absence and presence of BSA. The corresponding voltammograms are shown in Fig. 9. After the addition of BSA, the oxidation peak current (Peak a1) of HES decreased with a slight positive shift. However, no new peak was noticed in the same potential range. Two factors may be considered for the decreased oxidation peak current; firstly, the competitive adsorption between HES and BSA on C-dots/GCE and secondly, formation of an electrochemically inactive complex. In the present study, competitive adsorption factor can be excluded by considering the electrochemical kinetic parameters of HES (peak a1) in the presence and absence of BSA. The values of αn and ks were calculated to be 1.02 and 1.046 s− 1 in the presence of BSA and 1.04 and 1.05 s− 1 in the absence of BSA, respectively. These results indicated that there was no competitive adsorption. Thus, the decreased peak current indicated the formation of an electrochemically inactive complex, BSA-HES on the electrode surface.
Determination of binding constant and binding number.- As per the reported method [49], it was assumed that HES has interacted with BSA and formed a single complex, BSA-mHES. The binding number, m and the binding constant, βs could be calculated using the following equation:
Log[ΔI/ΔImax – ΔI] = log βs + m log [HES]
whereΔI is the change in peak current (of HES) in the absence and presence of BSA, ΔImax is the maximum peak current change and βs is the binding constant and m is the binding number. From the plot of log (ΔI/ΔImax – ΔI) versus log [HES], the values of m and log βs were calculated to be 1.21 and 1.25 X 105 M− 1 respectively. The value of m = 1.21 indicated the formation of a1:1 complex of HES-BSA. The binding constant of the order of 105 revealed the stronger binding between HES and BSA.
Interaction of HES with BSA/C-dots/GCE.- Modification of the surface of C-dots with BSA plays a crucial role in developing biosensors for monitoring the drug since it dictates the accessibility of BSA to drug solution and inturn influences the drug binding. So, we have carried out interaction studies at BSA-C-dots/GCE that was prepared by immobilization technique. Cyclic voltammograms of HES at C-dots/GCE and BSA-C-dots/GCE are shown in Fig. 9C. The oxidation peak potential of peak a1 was shifted towards positive potential at BSA-C-dots/GCE when compared to that at C-dots/GCE indicating the interaction between HES and BSA to be hydrophobic [49].