Characterizing Mixed Polyethylene Glycol Monolayers with Surface-Bound Ferrocene for Label-Free Immunosensing

Label-free electrochemical immunosensors offer a simplified approach to biomolecular detection by directly monitoring antigen-antibody interactions at the electrode surface. However, their sensitivity is often insufficient for detecting low-abundance analytes. To enhance signal response, most strategies rely on nanomaterial-based electrode modifications. In this study, we present a modification strategy to construct a multifunctional monolayer that incorporates surface-bound ferrocene to promote electron transfer. The monolayer is assembled via copper-catalyzed azide-alkyne cycloaddition (CuAAC) using a mixture of polyethylene glycol (PEG)-based spacers: short N₃-PEG3-Fc chains for redox activity, and longer N₃-PEG11-biotin or N₃-PEG24-biotin chains for immobilizing antibodies via streptavidin-biotin conjugation. Thermoplastic electrodes (TPEs) were modified with these mixed monolayers and characterized electrochemically using ferri- and ferrocyanide redox probes, as well as structurally by X-ray photoelectron spectroscopy (XPS). These analyses confirmed successful monolayer formation, integration of ferrocene and biotin functionalities, and minimization of nonspecific adsorption (NSA). As a proof-of-concept, the modified sensors were used to detect inactivated SARS-CoV-2 virus via its nucleocapsid (N) protein in buffer and nasopharyngeal samples using square wave voltammetry (SWV). Sensor performance appeared independent of PEG chain length in terms of electron transfer properties; however, PEG length influenced detection in a redox probe-dependent manner. Specifically, the use of ferrocyanide as the redox probe yielded the lowest and most consistent limits of detection for both PEG11 and PEG24 spacers (21.1 ± 10.6 ng/mL and 21.6 ± 10.8 ng/mL, respectively). These findings demonstrate that strategic design of surface chemistry and redox properties significantly improve the sensitivity of label-free electrochemical immunosensors.