*3.3. Evaluation of Experimental Parameters on the Bi2S3/BiVO4/FTO PEC Platform Response*

Initially, the effect of the applied potential, buffer type, and AA (electron donor molecule) concentration on the response of the Bi2S3/BiVO4/FTO PEC platform were evaluated. The applied potential is an important parameter that can directly influence the analytical performance of a sensor. Thus, the effect of the applied potential on the response of the Bi2S2/BiVO4/FTO PEC platform was investigated, and the results are presented in Figure S1. According to this figure, it can be observed that the photocurrent of the Bi2S2/BiVO4/FTO PEC platform in the presence of 0.03 mol L−<sup>1</sup> AA increased when the applied potential changed from −0.2 V to 0 V vs. Ag/AgCl/KClsat, and it remained almost constant from 0 to 0.1 V vs. Ag/AgCl/KClsat. These results suggest that using a potential of 0 V is enough to obtain a high photocurrent value. Under these conditions, a higher sensitivity to the system was achieved while maintaining low biasing conditions, making it possible to determine the analyte even at very low concentrations that consume a minimum of energy. In addition, it is possible to significantly reduce or eliminate the possible influence of interfering species on the photoelectrochemical processes. At potentials above 0.1 V, it can be observed that the photocurrent tended to decrease. This can be related to a lower stability of the Bi2S2/BiVO4 film, causing a lower efficiency for AA oxidation. In this sense, an applied potential of 0 V was chosen to construct the analytical curve for the determination of the antioxidant; all subsequent measurements were then performed under 0 V.

Posteriorly, the effect of the following buffer solutions: phosphate, McIlvaine, and Britton–Robinson on the response of the Bi2S2/BiVO4/FTO PEC platform in the presence of 0.03 mol L−<sup>1</sup> AA were evaluated (Figure S2). Figure S2 showed no significative difference in the intensity of photocurrent among the different electrolytes studied; however, considering the high stability of the AA photocurrent response and the simplicity of preparing the buffer, a phosphate buffer solution was chosen for all subsequent assays of the PEC sensor. Furthermore, the effect of the donor molecule concentration on the platform response was also investigated by monitoring the photocurrent of the platform for AA in the following concentrations: 0.01, 0.02, 0,03, 0.04, 0.05, and 0.06 mol L−<sup>1</sup> (Figure S3). This study showed that the process of donor molecule oxidation achieves maximum efficiency when the AA concentration reaches 0.04–0.05 mol L−1. Above this concentration range, there is a tendency to obtain a lower photocurrent because the PEC platform hinders the oxidation of the analyte. In this context, the donor molecule concentration was kept at 0.04 mol L−<sup>1</sup> for all subsequent studies. Subsequently, the influence of the concentration of the immobilized anti-cTnI antibodies on the Bi2S3/BiVO4/FTO PEC platform was evaluated, as was the interaction time between the antibody and the antigen (cTnI) immobilized on the PEC platform. Both studies were performed in the presence of 0.04 mol L−<sup>1</sup> AA.

Figure 3A shows the photoelectrochemical response of the Bi2S3/BiVO4/FTO PEC platform in a phosphate buffer containing 0.04 mol L−<sup>1</sup> AA after the incubation of the platform with different concentrations of anti-troponin I (anti-cTnI) (1, 2, 5 and 7 μg mL<sup>−</sup>1). Figure 3B shows the variation of the photocurrents (ΔI=I0 − I, where I0 and I are the photocurrents obtained before and after incubation of the platform with anti-cTnI, respectively) obtained from Figure 3A. According to Figure 3B, it can be observed that the photocurrent presented a high increase from 1 to 2 μg mL<sup>−</sup>1. At concentrations of 2, 5, and 7 μg mL<sup>−</sup>1, the photocurrent presented a percentage of decrease in relation to the initial photocurrent value, I0 (without antibody immobilization) of approximately 50, 40, and 35%, respectively. Based on these results, it was considered that any of the three concentrations (2, 5, or 7 μg mL<sup>−</sup>1) could be used for the preparation of the anti-cTnI/Bi2S3/BiVO4/FTO photoelectrochemical

immunosensor. In this context, an intermediate concentration of 5 μg mL−<sup>1</sup> was chosen for further experiments.

**Figure 3.** (**A**) Photoelectrochemical response of the Bi2S3/BiVO4/FTO PEC platform for different anti-cTnI concentrations and (**B**) plot of the photocurrent variation vs. [anti-cTnI]. Data obtained from Figure 3(**A**). (**C**) Effect of incubation time of the anti-cTnI/Bi2S3/BiVO4/FTO PEC platform with 1 ng mL−<sup>1</sup> cTnI on the variation of the photocurrent. All measurements were carried in 0.1 mol L−<sup>1</sup> phosphate buffer, pH 7.4, containing 0.04 mol L−<sup>1</sup> AA. Eappl = 0 V vs. Ag/AgCl/KClsat.

In order to evaluate the effects of the interaction time between the antibodies (anti-cTnI) immobilized on the platform and the antigens (cTnI) from the incubation solution, the immunosensor platform was incubated with 1 ng mL−<sup>1</sup> of cTnI antigens in a phosphate buffer solution containing 0.04 mol L−<sup>1</sup> AA at several incubation times (10, 15, 20, 25, and 30 min). Figure 3C shows the variation of the photocurrent of the anti-cTnI/Bi2S3/BiVO4/FTO PEC immunosensor before, I0, and after, I, the interaction with the cTnI antigens (ΔI=I0 − I). According to the results shown in Figure 3C, the inhibition of the photocurrent of the anticTnI/Bi2S3/BiVO4/FTO PEC immunosensor to the donor molecule increased significantly when the incubation time increased from 10 min to 20 min, with a low decrease observed for 25 min of interaction. As the standard deviation of the photocurrent was lower at 25 min, this interaction time between the antibodies and the Troponin I antigens was selected for all subsequent assays. The amperograms shown in Figure 3C are presented in Figure S4 in the supporting information. Additionally, it is important to emphasize that the results presented in this figure suggest that it is possible to monitor the cTnI antigen concentrations with the anti-cTnI/Bi2S3/BiVO4/FTO PEC immunosensor from the decrease in the analytical signal. However, in order to propose a possible mechanism for the detection of troponin I, the effects of different concentrations of cTnI on the variation of photocurrent of the immunosensor were evaluated.
