**1. Introduction**

Cardiovascular diseases, especially those that present with acute morbidity, are considered one of the leading causes of mortality worldwide. Acute myocardial infarction (AMI) is the main concern, defined by pathology as myocardial necrosis due to a prolonged reduction of blood supply to the heart. It is estimated that by 2030, approximately 23.3 million people globally will die annually from AMI [1,2]. In this sense, AMI requires an early, rapid, and effective diagnosis to improve the survival rate and ensure the health quality of patients. Among the known biochemical markers of early AMI, cardiac troponin I (cTnI) is considered the golden standard in medical diagnosis [3]. In addition to being a protein specifically related to myocardial damage, it can also remain in the myocardial tissue for a long time and is released from the cells in levels of a very low concentration within 3–4 h after the onset of AMI symptoms [4,5]. For these reasons, a sensitive method for detecting cTnI is reasonable. Several reliable, sensitive, and robust methods for detecting cTnI have been proposed, including methods based on fluorescence microscopy [6], 2D-chromatography [7], colorimetry [8], surface plasmon resonance (SPR) [9,10], liquid chromatography–tandem mass spectrometry [11], and SERS-based immunoassays [12]. However, some previously proposed methods may be time-consuming, exploit labeled probes, require trained personnel, or require expensive facilities to implement, making their use in point-of-care testing difficult [6,8–12].

**Citation:** Monteiro, T.O.; Neto, A.G.d.S.; de Menezes, A.S.; Damos, F.S.; Luz, R.d.C.S.; Fatibello-Filho, O. Photoelectrochemical Determination of Cardiac Troponin I as a Biomarker of Myocardial Infarction Using a Bi2S3 Film Electrodeposited on a BiVO4-Coated Fluorine-Doped Tin Oxide Electrode. *Biosensors* **2023**, *13*, 379. https://doi.org/10.3390/ bios13030379

Received: 11 February 2023 Revised: 6 March 2023 Accepted: 9 March 2023 Published: 13 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Photoelectrochemical (PEC) immunoassay has attracted growing attention in recent years [13–15]. This approach exploits a combination of the high sensitivity of the PEC bioanalysis and the affinity of the antigen/antibody molecules [14]. In principle, a PEC immunosensor can be easily designed using a photoelectroactive material such as a semiconductor as the signal-producing transducer and an antibody as the biological recognition element [14]. PEC measurements can be performed by employing the signal-off strategy in which the photocurrent is decreased while the formation of antibody–antigen conjugates occurs, blocking the transport of redox probes to the sensing surface (such as a label-free immunosensor) [3]. Thus, the high photoresponsive sensitivity of the semiconductor materials on the electrode is an essential aspect for the successful application of these devices.

Some photoactive semiconductor materials present a good biocompatibility, rapid reactivity, and the rapid generation and separation of electron–hole pairs [15]. Among these semiconductors, bismuth vanadate (BiVO4) is a promising material. Bismuth vanadate is an n-type semiconductor that presents a commonly monoclinic crystalline structure with good photocatalytic activity and a bandgap energy of 2.4 eV. It is appropriate to production of charge carriers under visible light irradiation [16]. In order to improve its photoelectrochemical efficiency and reduce the recombination processes of BiVO4, heterojunctions commonly based on semiconductors with a narrower bandgap could be employed, such as bismuth sulfide (Bi2S3). Bismuth sulfide also is an n-type semiconductor and has a bandgap of 1.3 eV. Bi2S3 presents a reasonable efficiency of photocurrent conversion under visible light [17] and has become attractive for many PEC applications [18–21]. In this paper, based on the properties of the BiVO4 and Bi2S3 materials described herein, we report a label-free PEC immunosensor designed with a junction of these two semiconductors to determine the cTnI biomarker in clinical samples in real time, exploiting the effects of the immunoreaction upon the response of the PEC platform to the ascorbic acid (AA) donor molecule.

## **2. Materials and Methods**

#### *2.1. Reagents and Chemicals*

Human cardiac troponin I (cTnI), monoclonal cTnI antibody (anti-cTnI), N'-ethylcarbo diimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), bovine serum albumin (BSA), bismuth nitrate (Bi(NO3)3), ammonium metavanadate (NH4VO3), ethylene glycol, and thioglycolic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium thiosulfate, ethylenediaminetetraacetic acid disodium salt dihydrate (C10H14N2Na2O8 · 2H2O), sodium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, ascorbic acid, citric acid, acetic acid, boric acid, phosphoric acid, sodium chloride, potassium chloride, calcium chloride, ammonium chloride, disodium sulfate, potassium dihydrogen phosphate, and urea were acquired from ISOFAR (Duque de Caxias, RJ, Brazil). All aqueous solutions were prepared with water purified in a OS10LXE Gehaka osmose system (São Paulo, SP, Brazil).

#### *2.2. Experimental Apparatus*

Photoelectrochemical experiments were performed using an Autolab potentiostat/ galvanostat model PGSTAT 128N (Metrohm Autolab B. V., Netherlands) equipped with a Frequency Response Analyzer module, controlled by NOVA software, and coupled to a three-electrode electrochemical cell confined in a box to control the illumination on the photoelectrodes. A commercial 36 W LED lamp was used as a visible light source. A FTO glass photoelectrode (5 cm length × 1 cm width, with a modified area of 0.7 × 1.0 cm2), modified with Bi2S3/BiVO4, was used as the working electrode. Ag/AgCl/KClsat) was used as the reference electrode, and a Pt wire was used as the counter electrode. Electrochemical impedance spectroscopy experiments were carried out in a 0.1 mol L−<sup>1</sup> KCl solution containing 5 mmol L−<sup>1</sup> K3[Fe(CN)6] in the frequency range of 10<sup>−</sup>1–105 Hz under an AC amplitude of 10 mV.
