To develop a highly sensitive and selective electrochemical probe based on molecular imprinting polymers (MIPs) technology for the precise detection of 25-hydroxyvitamin D₃ [25-(OH)D₃], aiming to meet the diagnostic needs of vitamin D metabolism-related diseases.

The Co₃O₄@reduced graphene oxide (rGO)@Pt@Au composite substrate was constructed by layer-by-layer modification of screen-printed electrode (SPE) to enhance conductivity. Molecularly imprinted cavities were successfully formed through electropolymerization of polypyrrole and template molecule elution, imparting specific recognition capabilities. By optimizing the scan rate, deposition cycles, incubation time, and temperature, the balance between membrane structure, electron transfer efficiency, and specific binding ability was achieved. The sensor's response performance was evaluated using square wave voltammetry (SWV), and its sensitivity, selectivity, and reproducibility were analyzed.

Scanning electron microscopy (SEM) characterization revealed that after template elution, clear imprinted cavities were formed on the electrode surface, which could detect the target in the range of 100 nmol/L to 1 mmol/L, with a detection limit of 26.76 nmol/L. This detection strategy showed no specific response to interference at 10 times the concentration, and the reproducibility of the electrodes across three batches, with a relative standard deviation of 3.2%, indicated the reliability of the method. The spiked experiments also show its good practical application ability.

This study successfully established a new method with high sensitivity, strong anti-interference ability, and excellent reproducibility, providing a novel approach for the clinical detection of 25-(OH)D₃.