Proximity-inducible CRISPR/Cas13a trans-cleavage activity and hydrogel-assisted paper-based distance sensor for the detection of aflatoxin M1.

Accurate and rapid determination of aflatoxin M1 (AFM1) in food is crucial for ensuring the safety of the human diet. In view of this, we proposed a proximity-inducible Cas13a activity and hydrogel-assisted paper-based distance sensor for the detection of aflatoxin M1. The single-stranded DNA (ssDNA) and AFM1 aptamer were split into two parts and linked to the terminus of two separate DNA activator strands, respectively. By introducing AFM1, it stimulated these two DNA activator strands to assemble with CRISPR RNA (crRNA) through spatial proximity mechanism between the target and these two DNA activators to activate the Cas13a trans-cleavage towards the RNA-containing site within hairpin probe to release a DNA initiator. The released DNA initiator triggered the catalytic hairpin assembly (CHA) between magnetic beads (MBs)-DNA capture probe and trypsin-DNA detection probe. After hydrolysis of gelatin hydrogel by trypsin, water released from the hydrogel flowed along the pH indicator strip and exhibited a distinct mark. Depending on this portable method, the limit of detection (LOD) was achieved at 23.6 ng L-1. Furthermore, our developed strategy can be applied to measure AFM1 in ultra-high temperature (UHT) milk with satisfactory effects, and its excellent specificity and reproducibility also demonstrate that our platform has great potential for application in environmental monitoring.