Prolidase (PLD) plays a crucial role as a dipeptidase in various physiological processes, specifically involved in the cleavage of proline-containing dipeptides for efficient recycling of proline. The accurate determination of PLD activity holds significant importance in clinical diagnosis. Herein, a solid-state electrochemiluminescence (ECL) biosensor was developed to address the urgent need for PLD assay. The Ru(bpy) was electrophoretically deposited within the nanochannels of vertically-ordered mesoporous silica film (VMSF) on indium tin oxide (ITO) electrodes. The Ru(bpy)-deposited VMSF/ITO (Ru-VMSF/ITO) exhibited a remarkable ECL response towards proline, attributed to the enhanced concentration of the reactants and improved electron transfer resulting from the nanoconfinement effect. As PLD specifically enzymolyzed the Gly-Pro dipeptide to release proline, a proline-mediated biosensor was developed for PLD assay. Increased PLD activity led to enhanced release of proline into the porous solid-state ECL sensors, resulting in a more robust ECL signal. There was a linear relationship between ΔECL intensity and logarithmic concentration of PLD in the range of 10-10000 U/L, with a detection limit of 1.98 U/L. Practical tests demonstrated the reliability and convenience of the proposed bioassay, making it suitable for widespread application in PLD assays.
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