The determination of homocysteine (HCys) has garnered significant interest within the biomedical community in recent years, as it serves as a key biomarker for a variety of diseases. Disruptions in HCys metabolism can lead to elevated blood levels of HCys, which are associated with cardiovascular diseases, Parkinson's disease, and etc. Therefore, the sensitive analysis of HCys levels in biological samples is crucial. An electrochemical method has been successfully developed for the sensitive detection of HCys using carbon paste electrodes (CPE) modified with ZnS nanowires/reduced graphene oxide sheets (ZnS NWs/rGO) nanocomposite and ferrocene monocarboxylic acid (FMC). Cyclic voltammetry (CV) results demonstrate that ZnS NWs/rGO exhibit excellent electrocatalytic activity for the oxidation of HCys at a low potential of 380 mV versus Ag/AgCl. These analytical properties, combined with the low operational potential, facilitate a reliable and sensitive determination of HCys, showing a good dynamic linearity in the range (LDR) of (0.01-10.0 µM and 10.0-90.0 µM) and a detection limit (LOD) of 0.003 µM.

The development of a straightforward and efficient assay for the accurate and long-lasting measurement of HCys is of great importance. Growing evidence from epidemiological studies suggests that individuals with higher blood levels of HCys face an increased risk of cardiovascular diseases. Even moderately elevated levels of HCys have been associated with serious health issues, including heart attacks, strokes, complications during pregnancy, and cognitive decline in middle and older age. HCys, known scientifically as 2-amino-4-mercaptobutyric acid, is an important amino acid that contains a thiol group. It is produced as an intermediate in the metabolic conversion of methionine, an essential amino acid obtained from dietary sources, into cysteine. In healthy individuals, normal plasma levels of HCys typically range from 5 to 16 nmol/ml. However, higher concentrations can lead to conditions such as hyperhomocysteinemia, where levels can reach up to 100 nmol/ml, or homocystinuria, which can present with levels around 500 nmol/ml. These elevated levels are linked to various pathological conditions. Research has shown that HCys levels in plasma are more closely correlated with the risk of atherosclerosis than traditional markers such as cholesterol. This correlation underscores the importance of monitoring HCys levels as a potential cardiac marker. Abnormal increases in HCys concentration can result from genetic defects or, more frequently, from inadequate dietary intake of essential vitamins such as B, B, and folic acid, which are critical for the proper metabolism of HCys. Given the significant health implications associated with elevated HCys levels, the development of reliable monitoring methods is crucial for the medical community. Such assays can aid in early detection and management of cardiovascular risks, ultimately contributing to better health outcomes for individuals at risk.

Various analytical approaches have been developed to achieve this goal, including spectroscopy, high-performance liquid chromatography (HPLC), matrix matching strategies combined with LC-quadrupole-time-of-flight-MS/MS, LC-MS/MS, and UHPLC-Q-Orbitrap high-resolution (HR) mass spectrometry (MS). Recently, electrochemical sensors have emerged as viable alternative analytical tools, enabling the direct sensing of HCys.

Electrochemical sensing technology has swiftly emerged as a favored method for the detection of various oxidizable and reducible organic and inorganic compounds across diverse sectors, including agriculture, food and oil industries, as well as environmental and biomedical applications. The appeal of electrochemical sensors lies in their simplicity, affordability, potential for rapid reactions, and quick response times, making them accessible tools for a wide range of analytical tasks. To enhance the reliability and reproducibility of the signals generated by these sensors, as well as to improve their sensitivity, it is crucial to modify the surfaces of the electrodes, which play a vital role in electrochemical sensing. In recent years, a variety of materials have been explored as electrode modification agents due to their exceptional properties (physical and chemical). This exploration has significantly broadened the scope and effectiveness of electrochemical sensors and biosensors. Currently, the production of nanoscale materials is predominantly achieved through both chemical synthesis and environmentally friendly green processes. The modification of the working electrode (WE) through chemical and electrochemical methods results in substantial alterations in its physical and electrochemical characteristics. These modifications lead to the formation of a modifier layer on the electrode surface, which not only enhances the sensitivity of the WE but also improves its overall performance in electrochemical sensing applications. The integration of advanced materials, such as nanomaterials, conductive polymers, and metal nanoparticles, has further propelled the development of high-performance electrochemical sensors. These materials contribute to increased surface area, improved electron transfer rates, and enhanced catalytic activity, all of which are critical for achieving high sensitivity in the detection of target analytes. As a result, the field of electrochemical sensing continues to evolve, driven by ongoing research and innovation in electrode modification techniques and material science.

Consequently, significant efforts have been directed toward selecting optimal functional materials for the WE and modifying its surface. In this regard, numerous studies have explored the role of nanotechnology and nanomaterials engineering in enhancing electrode materials and their modifications. It is widely recognized that functional nanostructures can improve catalytic activity, facilitate electron transfer on the electrode surface, and enhance conductivity. Among the various materials investigated, carbon-based nanomaterials, nanowires (NWs), nanofibers (NFs), metal nanoparticles (NPs), hybrid nanostructures, and their nanocomposites have garnered particular attention in the development of electrochemical sensors. These materials possess distinctive properties that enhance sensor performance, such as a larger surface area, better charge transport, and improved interaction with target analytes, resulting in greater sensitivity in electrochemical detection.

The functionalization and modification of the WE significantly influence the surface response of electrochemical sensors by reducing overpotential, enhancing the output signal, expanding the LDR, and improving key sensor characteristics such as LOD and sensitivity. As a result, a diverse array of functional nanomaterials, including semiconductor and carbon-based materials, has been explored for the cost-effective and large-scale production of innovative electrochemical sensors. Metal nanostructures, such as copper, silver, palladium, platinum, gold and etc., as well as bimetallic variants, have been extensively utilized to fabricate electrochemical sensors due to their exceptional catalytic activity, unique structures, conductivity, and high specific surface areas, which contribute to increased sensitivity. Within the semiconductor family, II-VI group compounds hold significant technological relevance across various scientific and technological applications. Notably, materials such as ZnS, CdS, ZnO, and CdTe are valued for their outstanding electronic and optical characteristics, making them essential for optoelectronic applications. Among these materials, ZnS stands out as a prominent II-VI semiconductor with a bandgap of approximately 3.7 eV. Consequently, ZnS nanoparticles find numerous applications in solar cells, gas sensors, antiviral coatings, and electroluminescent devices. The nonlinear properties of ZnS are particularly intriguing for the development of optical devices. Various synthesis methods have been employed for ZnS, including homogeneous precipitation, microwave techniques, thermal evaporation, pulsed laser deposition, spray pyrolysis, and sol-gel processes. ZnS exhibits favorable electrocatalytic behavior due to its redox chemistry and crystalline structure. Furthermore, combining ZnS semiconductors with carbon-based materials has been shown to enhance their electrical properties, thereby broadening their applicability in sensor technologies.

Carbon-based materials have been utilized as electrochemical sensing interfaces due to their exceptional electrochemical properties, extensively. Forms such as graphene (GR), carbon nanofibers (CNF), carbon nanotubes (CNT), and carbon dots (CND) are prevalent in electroanalytical research because of their chemical inertness, low background current, and relatively wide potential window. Graphene oxide (GO) can be synthesized from GR through oxidation and exfoliation using the Hummers method. GO consists of a two-dimensional sheet of carbon atoms arranged in a hexagonal lattice structure, featuring various oxygen functional groups. The reduction of GO to form rGO enhances its surface area and conductivity, making it even more suitable for electrochemical applications. Both carbon nanomaterials and metal nanoparticles exhibit promising physical and chemical characteristics that improve the conductivity of the WE surface and enhance electrocatalytic activity toward target analytes. Furthermore, the integration of two or more nanomaterials to create hybrid composites can serve as an alternative sensitive layer for sensor modification. This approach often results in improved catalytic performance of the composite material due to the synergistic effects that arise from the combination of different materials.

Redox-active materials play a crucial role as common electron-transfer mediators and electrochemical tags in electrochemical sensors. There is a diverse selection of materials that can enhance electron transfer, such as iron complexes, hydroquinones, anthraquinones, and organic dyes, each contributing to the efficiency and sensitivity of electrochemical detection in various applications. Among these, FC and its derivatives are particularly favored in biosensor fabrication due to its high stability in redox reactions and the ease of synthesizing derivatives. FC is a stable metal complex characterized by an iron (II) atom that is sandwiched between two cyclopentadienyl ligands. This unique structure imparts remarkable stability and distinctive electrochemical properties, making FC a valuable compound in various applications, including catalysis and as an electron transfer mediator in electrochemical sensors. The synthetic chemistry of FC has revolutionized the domain of molecular sensing and recognition, establishing it as a pivotal element in the development of electrochemical molecular receptors for diverse analytes. The redox responses of FC can be significantly altered upon the binding of guest molecules, allowing for the design of FC-based molecular receptors that can selectively bind cationic, anionic, or neutral guest species. This capability enhances the functionality of biosensors, making FC an invaluable component in the field of electrochemical sensing.

This article focuses on the development of a ZnS NWs/rGO/FMC/CPE sensor for the detection of HCys in real samples. The study provides an in-depth discussion on the role of the ZnS NWs/rGO nanocomposite and FMC in the electrochemical detection of HCys. The primary motivation behind this research is to explore a straightforward sensor that enables fast and reliable electrochemical detection of HCys in real samples.