CRISPR/Cas12a-Mediated Rolling Circle Amplification for the Development of Liquid Crystal-Based Sensors

The development of high-performance liquid crystal (LC)-based sensors with remarkable sensitivity and excellent selectivity is of great importance. Herein, a CRISPR/Cas12a-based LC sensor for detecting mycotoxins in food is first reported, and the detection of aflatoxin B₁ (AFB₁) is chosen as a model. AFB₁ is added to magnetic beads (MBs) functionalized with double-stranded DNA (dsDNA) consisting of AFB₁ aptamer and cDNA. As the AFB₁ aptamer specifically recognizes AFB₁, cDNA is released and activates the CRISPR/Cas12a system to cut ligation DNA, thereby preventing the initiation of the rolling circle amplification (RCA) reaction. As the long-chain single-stranded DNA (ssDNA) cannot be produced to capture myristoylcholine (Myr) in the aqueous solution, a dark LC image is obtained because a Myr monolayer forms at the aqueous/LC interface. In contrast, when the RCA reaction is initiated in the absence of AFB₁, Myr in the aqueous solution is captured by long-chain ssDNA generated from the RCA reaction, causing a bright LC image. Notably, the RCA reaction on MBs exponentially amplifies the CRISPR/Cas12a-generated signals, resulting in enhanced sensitivity. The limit of detection (LOD) is about 0.312 ng/mL. Furthermore, the selectivity is greatly enhanced due to the introduction of the AFB₁ aptamer and MBs. Furthermore, a portable device is developed for rapid onsite detection. Therefore, the study provides a sensitive, selective, convenient, and promising assay for detecting mycotoxins in food.