Thermal stability and micrdose-based coupling CRISPR/Cas12a biosensor for amplification-free detection of hgcA gene...

The lack of point-of-use (POU) methods hinders the utilization of the hgcA gene to rapidly evaluate methylmercury risks. CRISPR/Cas12a is a promising technology, but shortcomings such as low sensitivity, a strict reaction temperature and high background signal limit its further utilization. Here, a thermally stable microsystem-based CRISPR/Cas12a biosensor was constructed to achieve POU analysis for hgcA. First, three target gRNAs were designed to recognize hgcA. Then, a microsystem was developed to eliminate the background signal. Next, the effect of temperature on the activity of the Cas12a-gRNA complex was explored and its thermal stability was discovered. After that, coupling gRNA assay was introduced to improve sensitivity, exhibiting a limit of detection as low as 0.49 pM with a linear range of 0.98-125 pM, and a recovery rate between 90 and 110 % for hgcA. The biosensor was finally utilized to assess hgcA abundance in paddy soil, and high abundance of hgcA was found in these paddy soil samples. This study not only systematically explored the influence of temperature and microsystem on CRISPR/Cas12a, providing vital references for other novel CRISPR-based detection methods, but also applied the CRISPR-based analytical method to the field of environmental geochemistry for the first time, demonstrating enormous potential for POU detection in this field.