A Controllable and Separable Nanozyme‐Based Platform for Enhanced Signal‐Off Colorimetric Biosensing

ABSTRACT The “signal‐off” detection mode in nanozyme‐based sensors is widely adopted but inherently suffers from interference due to the “cyclic oxidation” of the chromogenic substrate—namely, the nanozyme continues to catalyze the oxidation of the substrate even as the analyte reduces the colored product during the signal readout step—which compromises sensitivity and accuracy. To address this challenge, this study designed a readily separable nanozyme system for controllable catalysis. Platinum (Pt) nanozymes were immobilized on polydopamine (PDA)‐modified glass beads (GBs) via in‐situ reduction stabilized by sodium citrate (Cis), constructing a peroxidase‐mimic designated GB@PDA@Pt‐Cis. This immobilized nanozyme can be separated from the reaction mixture, enabling precise on‐demand initiation and termination of the catalytic oxidation of 3,3',5,5'‐tetramethylbenzidine (TMB). The system exhibits high peroxidase‐like activity, follows Michaelis‐Menten kinetics, and demonstrates excellent stability and reusability. Leveraging these advantages, a “signal‐off” colorimetric sensor was developed for the sensitive detection of ascorbic acid (AA), uric acid (UA) and alkaline phosphatase (ALP). The strategy's transferability was further demonstrated by transferring the design to a plastic pipette tip platform (P@PDA@Pt‐Cis), achieving similar controllable catalysis and ALP detection. This work provides a simple solution to the “cyclic oxidation” problem in signal‐off sensing, paving the way for the development of more reliable and user‐friendly nanozyme‐based biosensors.