CRISPR/Cas12a-based fluorescence assay for the detection of acetylcholinesterase activity

Here we report an ultrasensitive and simple acetylcholinesterase (AChE) detection method by utilizing CRISPR/Cas12a collateral cleavage activity. In this design, the activator ssDNA of CRISPR/Cas12a was absorbed and sealed in the synthesized MnO2 nanosheets. The hydrolysate substrate, acetylthiocholine (ATCh), in the presence of AChE could reduce MnO2 nanosheets to Mn2+, thereby releasing the activator ssDNA, which subsequently activated the collateral cleavage ability of CRISPR/Cas12a to generate a fluorescent signal. As expected, AChE was successfully determined with high sensitivity based on the signal amplification ability of CRISPR/Cas12a collateral cleavage. Under the optimized conditions, the detection limit of AChE could reach as low as 0.0087 U/mL. In addition, for the detection of spiked AChE in human serum samples, our assay demonstrated excellent specificity and good recovery, demonstrating its practical applicability. This work would provide new insight into the construction of novel CRISPR/Cas12a strategies for the detection of enzymes beyond molecular diagnostics.