Reagentless molecular-imprinted polymer (MIP) electrochemical biosensors can offer the next generation of biosensing platforms for the detection of biomarkers owing to their simplicity, cost-efficacy, tunability, robustness, and accuracy. In this work, a novel combination of Prussian blue (PB), coated as an embedded redox probe on a gold working electrode (GWE), and a signal-off MIP assay has been proposed in an electrochemical format for the detection of troponin I (TnI) in biofluids. TnI is a variant exclusive to heart muscles, and its elevated level in the bloodstream is indicative of acute myocardial infarction (AMI). The proposed lab-manufactured PB/MIP electrochemical biosensor, consisting of a simple signal-off MIP assay and a PB redox probe embedded on the GWE surface, is the first of its kind that allows for reagentless, label-free, and single-step electrochemical biosensing of proteins. The preparation steps of the biosensor were fully characterized by cyclic voltammetry (CV), atomic force microscopy (AFM), and Raman spectroscopy. Finally, the performance of the optimized biosensor was investigated through the determination of various concentrations of TnI, ranging from 10 to 100 pg mL within 5 min, in serum and plasma with limits of detection less than 3.6 pg mL, and evaluation of selectivity towards TnI using some relevant proteins that exist in biofluids with higher concentrations.
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