Polybithiophene‐Based Molecularly Imprinted Polymers: A Comparative Mechanistic Study on Electrochemical Signal Transduction Approaches for Protein Detection

Abstract Molecularly imprinted polymers (MIPs) are synthetic alternatives to biological recognition elements like antibodies and are commonly used in developing electrochemical biosensors. However, comprehensive mechanistic studies of electrochemical detection methods on identical platforms remain scarce. This study fills this gap by creating an electropolymerized MIP (e‐MIP) on a single electrode (e‐MIP/SE) and systematically comparing differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) for protein sensing. Cystatin‐C (Cys‐C) is used as a model protein for both molecular imprinting and sensing. Polybithiophene‐based recognition layers are directly formed on gold electrodes using a carboxylic acid‐derivatized bithiophene crosslinker and a bithiophene monomer under potentiostatic conditions. Morphological and spectroscopic analyses confirm the uniformity of the functional cavity‐enriched e‐MIP films, which are ≈2.5 µm thick. The electrochemical comparison shows that DPV outperforms EIS, with detection and quantification limits of 0.19 µg mL −1 (≈15 nM) and 0.64 µg mL −1 (≈49 nM), respectively, and excellent linearity (R 2 = 0.99). The superior performance of DPV results from an enhanced signal‐to‐noise ratio due to suppression of capacitive current. Overall, this work provides a mechanistic understanding of electrochemical protein detection strategies using e‐MIP/SEs, employing a methodology adaptable for detecting various protein targets.