A DNA concatemer-encoded CRISPR/Cas12a fluorescence sensor for sensitive detection of Pb based on DNAzymes.

Lead pollution presents a significant threat to ecological systems and human health, underscoring the urgent need for highly sensitive detection methods. Herein, we introduce a novel DNA concatemer-encoded CRISPR/Cas12a fluorescence sensor (MDD-Cas12a) for sensitive detection of Pb based on DNAzymes. To accomplish this, we designed a substrate strand containing a long DNA concatemer encoding multiple protospacer adjacent motifs (PAMs) and protospacer sequences for activation of the CRISPR/Cas12a system. The DNA concatemer was subsequently anchored to the surface of magnetic beads (MBs) to fabricate a MBs-DNA concatemer nanoprobe. In the presence of Pb, the DNAzyme structure catalyzes the cleavage of the substrate strand, leading to the release of DNA concatemers. Following magnetic separation, the released DNA concatemers significantly activate the non-specific trans-cleavage activity of the Cas12a/crRNA complex. The fluorescence reporter DNA is then completely cleaved by the activated Cas12a/crRNA complex, and the Pb concentration in the sample can be quantified by measuring the fluorescence signal. By harnessing the specific recognition capability of DNAzymes for Pb, the programmability of DNA concatemers, and the self-amplification features of the CRISPR/Cas12a system, the MDD-Cas12a platform demonstrates high sensitivity and specificity for detecting Pb in milk and lake water samples.