A triple amplification strategy using GR-5 DNAzyme as a signal medium for ultrasensitive detection of trace Pb2+ based on CRISPR/Cas12a empowered electrochemical biosensor

Abstract

Lead ions (Pb2+) are a well-known toxic heavy metal that poses a significant threat to human health. Therefore, the development of a simple and ultrasensitive technique for detecting Pb2+ is essential. With their trans-cleavage properties, the newly discovered CRISPR-V effectors have become a potential high-precision biometric tool. In this regard, a CRISPR/Cas12a-based electrochemical biosensor (E-CRISPR) has been developed, which is combined with the GR-5 DNAzyme that can specifically recognize Pb2+. In this strategy, the GR-5 DNAzyme acts as a signal-mediated intermediary, which can convert Pb2+ into nucleic acid signals, thereby becoming single-stranded DNA that triggers strand displacement amplification (SDA) reaction. This is coupled with following activated CRISPR/Cas12a cleavage of the electrochemical signal probe, enabling cooperative signal amplification for ultrasensitive Pb2+ detection. The proposed method has a detection limit as low as 0.02 pM. Therefore, we have developed an E-CRISPR detection platform with GR-5 DNAzyme as a signal medium (called SM-E-CRISPR biosensor). This provides a method for the CRISPR system to specifically detect non-nucleic substances by converting the signal using a medium.