A label-free chemical bonding strategy mediated by reduced graphene oxide (rGO) basal plane functional groups has been developed for cardiac Troponin I (cTnI) detection. Four different chemical strategies on respective electrode sensing surface were precedingly examined using electrochemical impedance spectroscopy. The impedimetric assessment was carried out by sweeping frequency at the range 0.1–500 kHz perturbated at a small amplitude of AC voltage (25 mV). The chemical strategy-4 denoted as S-4 shows a significant analytical performance on cTnI detection in spiked buffer and human serum, whereby the pre-mixture of rGO and (3-Aminopropyl)triethoxysilane (APTES) creates a large number of amine sites (−NH2), which significantly enhanced the antibody immobilization without excessive functionalization. The as-fabricated immunosensor exhibited an ultra-low limit of detection of 6.3 ag mL−1 and the lowest antigen concentration measured was at 10 ag mL−1. The immunosensor showed a linear and wide range of cTnI detection (10 ag mL−1–100 ng mL−1) in human serum with a regression coefficient of 0.9716, rapid detection (5 min of binding time), and stable and highly reproducible bioelectrode response with RSD < 5%. Hence, the demonstrated S-4 strategy is highly recommended for other downstream biosensors applications. Graphical abstract: [Figure not available: see fulltext.].
