*3.4. Analytical Performance of Bi2S3/BiVO4/FTO PEC Immunosensor*

Under the optimum immunoassay conditions, the PEC platform response to the AA donor molecule was evaluated after incubation with different cTnI concentrations. Figure 4

exhibits the photocurrent responses for different concentrations. The immunocomplex formation on the platform surface decreased the AA photocurrent, whose inhibition is expressed in ΔI values (ΔI=I0 − In) in which I0 and In correspond to the AA photocurrent before and after interaction with the immunosensor with cTnI, respectively. As can be seen in the inset of Figure 4, an analytical curve was obtained for the concentration range from 1 pg mL−<sup>1</sup> to 1000 ng mL−<sup>1</sup> cTnI, linear equation for which was <sup>Δ</sup>I (μA) = 5.92 (±0.04) + 1.70 (±0.02) log[cTnI] (ng mL−1), with the correlation coefficient of 0.998 (*<sup>n</sup>* = 12). An experimental limit of detection (LOD) of 1 pg mL−<sup>1</sup> was obtained from a signal-to-noise ratio equal to three. The LOD obtained was significantly lower than the maximum limits allowed for a clinical diagnosis of myocardial infarction at approximately 0.1 ng mL−<sup>1</sup> [29]. In combination with the linear range of response, this result was compared to further reported PEC immunosensors for cTnI (Table 1: references [3,15,24–39]). As can be seen, the anti-cTnI/Bi2S3/BiVO4/FTO immunosensor presents some interesting features for the determination of this biomarker in comparison to previously reported PEC sensors.

**Figure 4.** Photoelectrochemical response of the anti-cTnI/Bi2S3/BiVO4/FTO immunosensor after incubation with different cTnI concentrations. [AA]= 0.04 mol L−1. Inset: Analytical curve obtained from of the data of Figure 4. [cTnI] from of 1 pg mL−<sup>1</sup> to 1000 ng mL−1. Eappl = 0 V vs. Ag/AgCl/KClsat.

Considering the anti-cTnI/Bi2S3/BiVO4/FTO PEC immunosensor response to the decrease in the AA molecule with the increase in the cTnI antigen concentration, a schematic representation of the PEC determination of cTnI (Scheme 1) under the incidence of light was proposed. As shown in Scheme 1, the BiVO4 and Bi2S3 harvest photons of energy higher than their band gap, promoting electrons from the valence to the conduction band and giving rising to e<sup>−</sup>/h+ couples. The electron photogenerated at the conduction band of Bi2S3 can be injected into the conduction band of the BiVO4, while the hole photogenerated in the valence band of the Bi2S3 can be transferred to the AA molecule. The AA molecule acts as an ideal electron donor to capture the photogenerated holes in the valence band (VB) of Bi2S3, inhibiting the recombination of electron–hole pairs [30] and generating an anodic photocurrent. The cTnI biomarker can then interact with the immobilized anticTnI/Bi2S3/BiVO4/FTO, decreasing the efficiency of the system to produce a photocurrent since the cTnI biomarker/anti-cTnI interaction reduces the efficiency of the photoactive material to transfer holes to donor molecules, an inhibition that is proportional to the amount of cTnI antigens immobilized on the immunosensor surface.


**Table 1.** Comparison of the analytical parameters of different PEC sensors for detection of cTnI.

<sup>a</sup> N-acetyl-L-cysteine-capped CdAgTe quantum dots and dodecahedral Au nanoparticles; <sup>b</sup> Manganese doped CdS sensitized graphene/Cu2MoS4 composite;**<sup>c</sup>** Carboxymethylated dextran-coated and gold-modified TiO2 nanotube arrays; <sup>d</sup> Nitrogen-doped carbon quantum dots–bismuth oxyiodide–flower-like SnO2; <sup>e</sup> ZnIn2S4/Bi2Se3 Nanocomposite; <sup>f</sup> N,S-GQDs and CdS co-sensitized hierarchical Zn2SnO4 cube; <sup>g</sup> Zr-MOF coated onto TiO2 NRs on fluorine-doped tin oxide; <sup>h</sup> Pd nanoparticles loading on the I-doped bismuth oxybromide with oxygen vacancies sensibilized by superoxide dismutase loaded on gold@polyaniline; <sup>i</sup> Ag2S/ZnO Nanocomposites. **<sup>j</sup>** Silicon nanowire arrays (SiNWs) at Polydiacetylene (PDA); <sup>k</sup> Ag at Cu2O core-shell submicron-particles on CdS QDs sensitized TiO2 nanosheets.

**Scheme 1.** Representation of the proposed mechanism for PEC determination of cTnI by label-free Bi2S3/BiVO4/FTO immunosensor. Ab: anti-cTnI.

For practical application, repeatability, reproducibility, and selectivity are important features of the immunosensor [5,15]. Figure 5A presents the photocurrent response of the Bi2S3/BiVO4/FTO platform to AA. As can be seen, no significative photocurrent changes were observed. The relative standard deviation (RSD) among signals was only 1.04%

(*n* = 15), indicating that the proposed sensing platform had good stability for the interaction with the AA donor molecule.

**Figure 5.** (**A**) Evaluation of repeatability of photocurrent of the Bi2S3/BiVO4/FTO PEC platform. (**B**) Reproducibility of photocurrent of the Bi2S3/BiVO4/FTO PEC platform incubated with 1 ng mL−<sup>1</sup> cTnI antigen solution. (**C**) Evaluation of selectivity of the anti-cTnI/Bi2S3/BiVO4/FTO PEC immunosensor. (**a**) cTnI, (**b**) cTnI + albumin, (**c**) cTnI + C-reactive protein, (**d**) cTnI + glucose, and (**e**) cTnI + myoglobin. [cTnI] = 1 ng mL−1. [Foreign species] = 100 ng mL−1. Measurements performed in 0.1 mol L−<sup>1</sup> phosphate buffer, pH 7.4. [AA]= 0.04 mol L<sup>−</sup>1, Eappl = 0 V vs. Ag/AgCl/KClsat.

The reproducibility of the proposed immunosensor also was evaluated (Figure 5B). This parameter was assessed using five different electrodes for the same concentration of cTnI and under optimal experimental conditions. The RSD value obtained for this study was 2.1%, indicating a satisfactory reproducibility of the immunosensor.

In order to appraise the selectivity of the PEC immunosensor for cTnI detection, some potential interfering substances were investigated. Therefore, a solution containing 1 ng mL−<sup>1</sup> cTnI (a) and solutions containing 1 ng mL−<sup>1</sup> cTnI and 100 ng mL−<sup>1</sup> of potential interfering substances such as albumin (b), C-reactive protein (c), glucose (d), and myoglobin (e) were tested, respectively, under optimized experimental conditions. The results are shown in Figure 5C. As can be seen, there was no significant change in the photocurrent presented by the immunosensor after the addition of those substances, and the RSD of the measurement was within 5%, indicating that the designed immunosensor possesses a remarkable selectivity for detection of cTnI. The data from Figure 5 are presented in Figures S5 and S6 in the supporting information.

#### *3.5. Detection of cTnI in the Artificial Blood Plasma Samples*

To demonstrate the accuracy and the potential of application of the developed immunosensor in clinical samples, the anti-cTnI/Bi2S3/BiVO4/FTO electrode was tested to determine cTnI at different concentrations in artificial blood plasma samples. The samples were spiked with cTnI at three concentration levels (0.05, 2.0, and 50 ng mL−1), and quantification performed using an external calibration method. The recovery values (Table 2) were found between 98.0% and 98.5% with very low values of relative standard deviation, indicating good accuracy and precision. Thus, the results suggest that the immunosensing platform based on Bi2S3/BiVO4/FTO can be used as a promising strategy for the detection of cTnI in clinical blood plasma samples.

**Table 2.** Recovery values obtained from detection of cTnI in artificial blood plasma samples using the proposed immunosensor.


#### **4. Conclusions**

In this paper, the feasibility of the sensitization of a BiVO4 semiconductor material with a Bi2S3 electrodeposited film for the development of a PEC immunosensing platform to determine cTnI as a biomarker of myocardial infarction was reported. The proposed immunosensor presented high photoelectrochemical efficiency under visible LED light irradiation, a wide linear range of response to cTnI, high sensitivity, a low limit of detection, and good selectivity and stability. Moreover, the immunosensor demonstrated good accuracy and good precision with excellent application for the termination of the cTnI biomarker in artificial blood plasma samples.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/bios13030379/s1, Figure S1. (A) Photoelectrochemical response of the Bi2S3/BiVO4/FTO platform obtained at different potentials. Amperometric measurements performed in 0.1 mol L−<sup>1</sup> phosphate buffer (pH 7.4) containing 0.03 mol L−<sup>1</sup> AA. (B) Plot of photocurrent vs. Eappl. Data obtained from the Figure S1A; Figure S2. (A) Photoelectrochemical response of the Bi2S3/BiVO4/FTO platform obtained at different buffer solutions. (B) Plot of photocurrent vs. different buffer solutions. Amperometric measurements performed in 0.1 mol L−<sup>1</sup> of buffer (pH 7.4) containing 0.03 mol L−<sup>1</sup> AA. Eappl = 0 V vs. Ag/AgCl/KClsat; Figure S3. (A) Photoelectrochemical response of the Bi2S3/BiVO4/FTO platform obtained at different AA concentrations (0.01–0.06 mol L<sup>−</sup>1). (B) Amperometric measurements performed 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; Figure S4. Photoelectrochemical responses of the anti-cTnI/Bi2S3/BiVO4/FTO PEC immunosensor before (black amperogram) and after incubation with cTnI antigens (red amperograms) at different incubation times. The measurements were performed in 0.1 mol L−<sup>1</sup> phosphate buffer, pH 7.4, containing 0.04 mol L−<sup>1</sup> AA. Eappl =0V vs. Ag/AgCl/KClsat. [anti-cTnI] = 5 μg mL<sup>−</sup>1; [cTnI] = 1 ng mL−1; Figure S5. Photoelectrochemical responses obtained with 5 (five) different anti-cTnI/Bi2S3/BiVO4/FTO PEC immunosensors under optimized conditions before (black amperograms) and after (red amperograms) incubation with cTnI. [cTnI]=1 ng mL−1, tincubation= 25 min; Figure S6. Photoelectrochemical responses obtained with the anti-cTnI/Bi2S3/BiVO4/FTO PEC immunosensor under optimized conditions before (black amperogram) and after (red amperogram) incubation with cTnI (1 ng mL−1) in absence and presence of different species (albumin, C-reactive protein, glucose, and myoglobin). [Foreign specie] = 100 ng ng mL<sup>−</sup>1; tincubation= 25 min.

**Author Contributions:** Conceptualization: T.O.M., A.G.d.S.N., A.S.d.M., F.S.D., R.d.C.S.L. and O.F.-F.; methodology: T.O.M., F.S.D., R.d.C.S.L. and O.F.-F.; formal analysis: T.O.M.; A.G.d.S.N., F.S.D., R.d.C.S.L. and O.F.-F.; investigation: T.O.M., A.G.d.S.N., A.S.d.M., F.S.D., R.d.C.S.L. and O.F.-F.; writing—original draft preparation: T.O.M., R.d.C.S.L. and O.F.-F.; and writing—review and editing: T.O.M., A.G.d.S.N., A.S.d.M., F.S.D., R.d.C.S.L. and O.F.-F.; supervision: F.S.D., R.d.C.S.L. and O.F.-F. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors are grateful to the Instituto Nacional de Ciência e Tecnologia em Bioanalítica (465389/2014-7); FAPESP (Grant: 2020/01050-5); FAPEMA (Grants: INFRA-02021/21; INFRA-02050/21; INFRA-02203/2021; UNIVERSAL-06535/22; POS-GRAD-02432/21), CNPq (Grants: 308204/ 2018-2; 309828/2020-1; 305806/2020-3; 313324/2021-2), and FINEP.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We are also thankful to Multiuser Centre for Research in Materials and Biosystems (CeMatBio), of the Federal University of Maranhão (UFMA), for the support with the XRD and SEM measurement.

**Conflicts of Interest:** The authors declare no conflict of interest.
