*2.5. Optimization of the Platform Response for the AA before and after the Immobilization of the Biological Materials*

Some experimental parameters were optimized to achieve a higher sensitivity of the Bi2S3/BiVO4/FTO PEC platform to the AA donor molecule. Thus, the effect of the applied potential on the sensor response was initially investigated by evaluating the photocurrents in 0.03 mol L−<sup>1</sup> AA in a 0.1 mol L−<sup>1</sup> phosphate buffer, at pH 7.4 under different potentials (from −0.2 V to 0.2 V vs. Ag/AgCl/KClsat). The influence of the nature of the buffer solution on the response of the platform was then evaluated by monitoring the photocurrents obtained from 0.03 mol L−<sup>1</sup> AA under an applied potential of 0 V vs. Ag/AgCl/KClsat in three different buffer solutions: phosphate, McIlvaine, and Britton– Robinson. The study of the effects of the buffer solutions was performed by maintaining the buffer concentration at 0.1 mol L−<sup>1</sup> and pH 7.4. The effect of the concentration of the AA donor molecule on the response of the platform was also investigated for AA concentrations from 0.01 to 0.06 mol L<sup>−</sup>1.

After the optimization of the Bi2S3/BiVO4/FTO PEC platform response for the concentration of the AA molecule, the platforms were modified with the antibody of troponin (anti-cTnI). Initially, the electrode was incubated in an aqueous solution of 0.003 mol L−<sup>1</sup> thioglycolic acid (TGA) at room temperature for 15 min to introduce carboxylic groups at the surface of the Bi2S3/BiVO4/FTO PEC platform. Following this, the excess TGA was removed with water and 15 μL of an EDC/NHS mixed solution (0.15 mol L−<sup>1</sup> EDC and 0.10 mol L−<sup>1</sup> NHS) was added and incubated for 1 h on the functionalized surface to activate the -COOH groups. The activated platform was then incubated with 10 μL of different concentrations of anti-cTnI (from 1 to 5 μg mL−1) at room temperature for a determinate time. The platform modified with the antibody was gently washed to remove the weakly adsorbed anti-cTnI. Furthermore, the EDC/NHS excess was removed from surface with purified water. Next, 10 μL of 1 % (*m*/*v*) BSA solution was added to block

nonspecific binding sites. Posteriorly, the effect of the interaction time between the antibody and the antigen on the response of AA (10, 15, 20, 25, and 30 min) was studied using a concentration of 1 ng mL−<sup>1</sup> cTnI. After evaluating all the experimental parameters, the conditions that provided the highest photocurrent value were fixed to finally obtain the analytical curve for the determination of cTnI. The analytical curve was obtained after the incubation of the anti-cTnI/Bi2S3/BiVO4/FTO PEC immunosensor platform with different cTnI concentrations (from 1 pg mL−<sup>1</sup> to 1000 ng mL−<sup>1</sup> cTnI).

#### *2.6. Preparation and Analysis of Artificial Blood Plasma Samples*

The performance of the anti-cTnI/Bi2S3/BiVO4/FTO PEC immunosensor was evaluated by the determination of cTnI in artificial blood plasma samples using an external calibration method. The artificial samples were composed of 0.138 mol L−<sup>1</sup> NaCl, 0.004 mol L−<sup>1</sup> NaHCO3, 0.003 mol L−<sup>1</sup> KCl, 0.001 mol L−<sup>1</sup> Na2HPO4 · 3H2O, 0.002 mol L−<sup>1</sup> MgCl2 · 6H2O, 0.003 mol L−<sup>1</sup> CaCl2, and 0.507 mmol L−<sup>1</sup> Na2SO4 [24]. The samples were spiked with different concentrations of cTnI (0.05, 2.0 and 50 ng mL−1), and aliquots of 10 μL of each sample were added directly onto the immunosensor surface at the optimal conditions and analyzed in presence of the AA molecule using the developed sensor in three replicates.
