Design and characterization of rGO/PPy/Hb-Ab/BSA-modified pencil graphite electrode for hemoglobin detection

A simple and cost-effective electrochemical biosensor was developed for the determination of free hemoglobin in blood plasma (pfHb) as a biomarker of intravascular hemolysis. The sensing platform was fabricated by modifying a pencil graphite electrode (PGE) with reduced graphene oxide (rGO), polypyrrole (PPy), hemoglobin antibody (Hb-Ab), and bovine serum albumin (BSA). The incorporation of rGO enhanced electron transfer, while PPy doped with ferric chloride (FeCl₃) served as a conductive layer for antibody immobilization. Key experimental parameters, including BSA incorporation, Hb-Ab volume, Hb volume, PPy and FeCl₃ ratio, and Hb incubation time, were systematically optimized to improve analytical performance. Under optimal conditions, the BSA/Hb-Ab/PPy/rGO/PGE biosensor exhibited a linear response in the range of 10–250 µg·mL⁻¹, with a sensitivity of 9.204 µA·µg·mL⁻¹·cm⁻² and a limit of detection of 78.674 µg·mL⁻¹. The sensor demonstrated good repeatability and reproducibility, with relative standard deviation (RSD) values below 5%, and maintained 81.26% of its initial response on the 7th day, remaining functional for up to 12 days. The proposed biosensor showed satisfactory selectivity toward pfHb, indicating its potential applicability for monitoring intravascular hemolysis with further studies directed toward long-term performance enhancement.