Electrochemical measurements address the processes occurring at the electrode surface. It is known that higher electrolyte concentrations reduce the measured resistance.[28] Additionally, some studies describe how the theoretical fit of an electrochemical impedance spectrum can be improved by working with higher conductive solutions[25, 31, 32]. So, increasing concentration could lead to better performance by making the system more accurate and more reliable.
The effect of the redox probe concentration on the electrochemical response was evaluated by varying the concentration of [Fe(CN)6]4−/[Fe(CN)6]3− (1:1 proportion) between 2.5 and 15.0 mmol.L− 1 using bare gold electrodes, Fig. 1. As the concentration increases, the capacitive behavior becomes less dominant.
The data were fitted using a modified Randles equivalent circuit (inset of Fig. 1), using a constant phase element (CPE), instead of a capacitor to account for a nonideal capacitance behavior. The impedance of a CPE is given by Eq. 1, where Q is the parameter related to the electrode capacitance, and α is the dimensionless CPE exponent[33]. The Warburg impedance component (ZW), Eq. 2, accounts for the impedance associated with diffusion processes. Y0 is the real part of admittance calculated at a specific angular frequency (ω), when the phase angle (θ) is zero. Y0 describes the degree of diffusion or mass transport occurring in the system, indicating how easily ions or molecules can diffuse at the electrode interface[25, 33].
$${Z}_{CPE}=\frac{1}{Q{\left(j\omega \right)}^{\alpha }}$$
1
$${Y}_{0}=\frac{1}{{Z}_{W}}={\left(\frac{A}{\sqrt{j\omega }}\right)}^{1}$$
2
As expected, the solution resistance (Rs) and charge transfer resistance (Rct) decreased inversely with redox probe concentration, while the α and Y0 coefficients varied proportionally with increasing concentration (Fig. 2). All values vary linearly, except Rct, which shows an exponentiallike decrease, demonstrating a stronger dependence with the redox probe concentration.
The relative standard deviation (RSD) of the fitted parameters was calculated by dividing the standard deviation by the arithmetic mean value considering three distinct measurements on the same electrode (Table 1). For 10.0 mmol.L− 1 all deviations are already lower than 2.0%, which is below the acceptable limit of 5.0% according to the literature. [34]Therefore, this concentration was chosen as the working concentration for the next analyses. Rct displays the higher RSD values, which suggests that it could not be the best parameter for detection analysis.
Table 1
Calculated RSD for α, Y0, Rs and Rct.
Conc
(mmol.L− 1)

α

Rct
(Ω)

Rs
(Ω)

Y0
(Mho.s1/2)

2.5

1.36%

12.69%

0.96%

1.29%

5.0

0.95%

4.54%

0.46%

0.80%

7.5

0.49%

6.66%

0.47%

1.05%

10.0

0.61%

1.82%

0.39%

0.77%

12.5

0.56%

3.44%

0.53%

0.65%

15.5

0.19%

0.97%

0.29%

0.20%

The potential window determines the limiting potential values to avoid undesired reactions, like electrode corrosion. CV was performed to evaluate the stability of the gold electrodes. Figure 3 shows the electrochemical response of the gold electrode in PBS solution and in the presence of [Fe(CN)6]3−/4− redox couple (10.0 mmol.L− 1). Initially, for the PBS without the redox couple, the scan started at 0.22 V towards positive bias (oxidation) until 1.0 V, then reversed towards negative bias (reduction) until − 0.6 V and back to 0.22 V to complete the cycle. An anodic peak starts at approximately 0.7 V, which indicates the onset of gold oxidation, followed by hydrogen evolution and consequent electrode degradation close to 1.0 V. When the scan changes direction to negative bias, an undesired cathodic peak of peroxide formation is also observed at around − 0.25 V.[35, 36]
By repeating the measurement for a smaller potential window, between − 0.1 V and 0.52 V (blue curve), no appreciable current peaks were observed, which indicates a safe potential window. With the addition of the [Fe(CN)6]3−/4− redox couple the measurements were performed even for a smaller range, between − 0.1 V and 0.52 V. Significant current peaks at 0.276 V and 0.169 V were observed, corresponding to the oxidation of [Fe(CN)6]3− and reduction of [Fe(CN)6]4−, respectively [37, 38].
EIS was performed at various positive potentials within the previously established safe potential window to evaluate the impact on the gain and reproducibility. Nyquist plots of Fig. 4 illustrate the impedance response for an APT/MCH modified electrode.
The values obtained from the equivalent circuit fit for each potential are presented in Fig. 5 for the electrode modified with APT/MCH (orange columns) and APT/MCH/STX (blue columns). Rct is maximized around the OCP value (0.22 V) while CPE showed to be more pronounced at 0.07 V. The smallest value for Warburg impedance was obtained at the OCP.
The calculated RSD (for three distinct electrodes) after the APT/MCH electrode modification remained within the range of 5.0% for all α values. For Rct this range was obtained only for 0.22 V and 0.52 V, and 0.22 V only for Y0. After the additional modification with STX (APT/MCH/STX), less dispersive values were obtained for Rct, where only the − 0.08 V potential resulted in RSD higher than 5.0%. α and Y0 followed the same trend as prior to STX incorporation. Figures S1 and S2 in the supporting information highlight the RSD behavior.
Figure 6 shows the calculated signal gain \(\varDelta S\%=\left\left(S{S}_{0}\right)/{S}_{0}\right\times 100\), where S0 and S represent the values before and after the modification with STX, respectively. It is worth noting that α shows a minimum change and large deviations, therefore it is not a suitable parameter to discriminate STX incorporation at any applied voltage. Rct presented high gain (14.5%) and RSD below 5.0% for 0.22 V; for 0.07 V the gain is reasonable (13.5%) but unfortunately the RSD is close to 20%. Only one applied potential of 0.08V for Y0 displayed acceptable RSD (1.0%) with incredible 40.5% signal gain. It suggests that Y0 at this specific potential may provide valuable information for STX detection.
IES data are often represented using Nyquist plots, where the real (Z') and imaginary (Z") part of impedance are used. Z' provides information about the resistance component of the system and indicates how easily current can flow through it. Z" reflects the capacitive component of the system and indicates the ability to store and release charge carriers. The modulus of Z is a measure of the overall impedance magnitude of the system. Another parameter of interest is the phase angle (Φ), which is the phase difference between the applied voltage and the resulting current in the system. [24, 25] These four parameters, Z’, Z”, Z and Φ were analyzed as a function of the frequency for the five different potentials to assess the overall system response.
Figure 7 shows the selected data for RSD values less than 5.0%, all the others were omitted, where a total of 28 VoltageFrequency combinations were selected for the investigated parameters. Eleven of those combinations (with gain around 18%) were obtained for the OCP potential of 0.22 V and frequencies in the range of 100–25 Hz. Within RSD values above 5.0%, most of the combinations with these parameters could be used to detect STX with a ΔS higher than the obtained for Rct at the OCP potential. It is worth noting that 12 of the total 28 acceptable combinations arise from the impedance modulus Z. Figure S3 of the supporting information shows ΔS values of all the parameter combinations.
These analyses show the importance of carefully selecting the parameters for characterizing the electrochemical behavior of the system and the need of further investigation to identify the most reliable and informative parameters for STX detection.