Impedimetric Immunosensor for 5-Methylcytosine Detection Based on a Poly(o-phenylenediamine)-Encapsulated Gold Nanoparticle Platform

A label-free impedimetric immunosensor for the detection of 5-methylcytosine (5-mC), a key epigenetic marker, was developed based on a one-step electropolymerized nanocomposite of poly(o-phenylenediamine) (poly(o-PD)) and gold nanoparticles (AuNPs). The nanocomposite film was electropolymerized onto a screen-printed electrode (SPE) with a gold working electrode area of 0.0078 cm². The system was characterized by cyclic voltammetry and electrochemical impedance spectroscopy (EIS). The EIS measurements, including Nyquist, Bode, and complex capacitance plots, confirmed the successful formation of the nanocomposite and its stepwise modification with glutaraldehyde, anti-5-methylcytosine antibody (Ab-5mC), and bovine serum albumin (BSA). The immunosensor utilized the inherent redox activity of the poly(o-PD) toward dissolved oxygen as a transduction mechanism, eliminating the need for external redox mediators. The binding of 5-mC to the immobilized Ab-5mC hindered the access of dissolved oxygen to the redox-active phenazine-like units within the poly(o-PD) matrix, resulting in a measurable increase in the charge transfer resistance. The immunosensor exhibited a linear response to the logarithm of 5-mC concentration in the range of 2.5 to 160 pg mL^–1, with a low limit of detection (LOD) of 1.73 and 1.18 pg mL^–1 when using the imaginary and absolute impedance, respectively. The incorporation of AuNPs significantly enhanced the electrochemically active surface area and improved the electron transfer kinetics, contributing to the high sensitivity of the immunosensor. This work demonstrates the potential of a one-step electropolymerized poly(o-PD)-AuNP nanocomposite for the development of simple, label-free, and sensitive impedimetric immunosensors for epigenetic biomarker detection.