An Innovative Nanobody-Based High-Biocompatibility Gold Interdigitated Microelectrode Electrochemical Bioimpedance Sensor for the Ultrasensitive Detection of Difenacoum in Human Serum

Difenacoum (DIF) is one of the most widely used anticoagulant rodenticides. However, accidental or intentional ingestion of DIF seriously threatens humans and other non-target species. Therefore, a rapid and sensitive detection method to quantify DIF is urgently needed. In this study, one anti-DIF nanobody (Nb) was assembled on the surface of a gold interdigitated microelectrode (IDME) using an Au–S bond to fabricate a bioimpedance sensor. To improve the immobilization amount of Nbs on the electrode, a polycrystalline gold IDME was prepared to provide a larger surface and better biocompatibility. Thus, a novel and ultrasensitive bioimpedance sensor based on electrochemical impedance spectroscopy (EIS) was designed for the determination of DIF, and it displayed good reproducibility and stability in human serum. The proposed bioimpedance sensor displayed a wide working range, between 0.1–1000 pg/mL, with a limit of detection (LOD) of 0.1 pg/mL of DIF. This method exhibited excellent performance, good sensitivity, and reproducibility and achieved the highest sensitivity of all currently existing methods used to quantify DIF. The highly sensitive DIF detection of this proposed bioimpedance sensor indicates its potential as an efficacious approach for DIF monitoring in human serum with high accuracy and precision.