Detection of lead contamination using DNAzyme and split activator-triggered CRISPR/Cas12a

Area of Science:

• Biotechnology
• Environmental Science
• Molecular Biology

Background:

• Lead (Pb²⁺) contamination poses a significant global health risk, especially to children.
• Accurate monitoring of Pb²⁺ is crucial for mitigating its adverse health effects.
• Existing DNAzyme-Cas12a systems for Pb²⁺ detection face challenges with background noise.

Purpose of the Study:

• To develop a novel and reliable DNAzyme-Cas12a assay for ultrasensitive Pb²⁺ detection.
• To overcome the limitations of background signals in conventional DNAzyme-Cas12a assays.
• To create a rapid, specific, and accurate method for monitoring Pb²⁺ in real-world samples.

Main Methods:

• A split activator-based Cas12a switch mechanism was engineered to prevent premature Cas12a activation.
• A flap region in the split activator was utilized to suppress background noise from DNA dynamics.
• Pb²⁺-dependent activation of a DNAzyme triggered flap cleavage, activator reconstitution, and Cas12a trans-cleavage for signal amplification.

Main Results:

• The assay achieved a low detection limit of 615 pM for Pb²⁺.
• High specificity was maintained against various interfering metal ions.
• The method demonstrated rapid detection at ambient temperature without DNA amplification or nanoparticle modification.
• Accurate detection of Pb²⁺ in contaminated tap and drinking water samples was achieved.

Conclusions:

• The developed split activator DNAzyme-Cas12a assay effectively suppresses background noise, enhancing analytical reliability.
• This novel assay provides a sensitive, specific, and rapid method for Pb²⁺ detection.
• The assay shows significant potential as a practical tool for environmental monitoring of lead contamination.