Widespread Pbcontamination represents a significant global health threat, particularly to children, highlighting the critical need for accurate monitoring and quantification to mitigate its adverse effects. The integration of DNAzymes with the programmable nuclease Cas12a has emerged as a promising approach for achieving specific and ultrasensitive detection of Pb. However, conventional DNAzyme-Cas12a systems suffer from inevitable background signals caused by dynamic instability between DNAzymes and Cas12a activators, which compromises analytical reliability. Herein, we present a novel DNAzyme-Cas12a assay featuring a split activator-based Cas12a switch. We demonstrate that the split activator containing a flap region effectively prevents Cas12a activation, thereby suppressing background noise induced by "DNA breathing" phenomena. Upon Pb-dependent activation of the GR-5 DNAzyme, the flap is cleaved, enabling reconstitution of the Cas12a activator and triggering trans-cleavage activity for signal amplification. This strategy achieves a detection limit of 615 pM for Pbwhile maintaining high specificity against interfering metal ions. Notably, the assay eliminates requirements for DNA amplification or nanoparticle modification, enabling rapid Pbdetection at ambient temperature. The method demonstrated high accuracy in detecting contaminated tap and drinking water, suggesting its potential as a reliable analytical tool for monitoring Pb2+ contamination in practical samples. 2+ 2+ 2+ 2+ 2+
