"Turn-on" aptamer-immune lateral flow assays for the detection of small molecule targets based on CHA-assisted and CRISPR/Cas12a mediated signal transduction and amplification

Abstract

Lateral flow assays (LFAs) have emerged as crucial tools for on-site food safety detection due to their simple operation and intuitive detection results. Nevertheless, LFAs for small molecule targets such as pesticides often present a "Turn-off" signal output, which leads to their low sensitivity and the risk of false positives. In this study, a CRISPR/Cas12a system-mediated strategy was employed to convert aptamer signals into the signals of immune LFAs, achieving a "Turn-on" signal output for highly sensitive detection of small molecule targets. The binding of aptamers to targets released the trigger sequence to initiate the catalytic hairpin assembly (CHA) reaction, generating double-stranded DNA, which subsequently activated the CRISPR/Cas12a system to cleave the FAM-labeled Reporter. Eventually, the "Turn-on" visual output of the signal was realized through an anti-6-FAM immune LFAs. The experiment optimized the sample pool preparation, CHA reaction conditions, CRISPR/Cas12a activation parameters, and the assembly process of the LFAs. The limit of detection for procymidone was as low as 0.015 ng/mL, which was 52.67 times more sensitive than those of conventional aptamer-based LFAs without signal amplification strategies. This method exhibits high specificity for procymidone and a recovery rate ranging from 94.00% to 104.20% in vegetable samples, demonstrating excellent stability and practicability.