An ultrasensitive Cd2+ detection biosensor based on DNAzyme and CRISPR/Cas12a coupled with hybridization chain reaction

The detection of cadmium is essential because it poses a significant threat to human health and the environment. Recent advancements in biosensors that detect nonnucleic-acid targets using CRISPR/Cas12a in combination with aptamers or DNAzymes show promising performance. Herein, we integrated DNAzyme, hybridization chain reaction (HCR) and CRISPR/Cas12a into a single biosensor for the first time and realized the ultrasensitive detection of Cd2+. A single phosphorothioate ribonucleobase (rA)-containing oligonucleotide (PS substrate) and a Cd2+-specific DNAzyme (Cdzyme) are used for Cd2+ recognition, releasing short single-stranded DNA. Then, the HCR is triggered by the cleavage products for signal transduction and amplification. Next, the trans-cleavage activity of Cas12a is activated due to the presence of crRNA complementary strands and PAM sites in the HCR products. As a result, FQ-reporters are cleaved, and the fluorescence values can be easily read using a fluorometer, allowing Cd2+ quantification by measuring the fluorescent signal. The Cd2+ detection biosensor is ultrasensitive with a detection limit of 1.25 pM. Moreover, the biosensor shows great stability under different pH and various anion conditions. The proposed sensor was utilized for environmental water sample detection, demonstrating the dependability of the detection system. Considering the high sensitivity and reliable performance of the assay, it could be further used in environmental monitoring. In addition, the design strategy reported in this study could extend the application of CRISPR/Cas12a in heavy metal detection.