环境地球化学国家重点实验室知识库

The arsM gene is a critical biomarker for thepotential risk of arsenic exposure in paddy soil. However, on-sitescreening of arsM is limited by the lack of high-throughput point-of-use (POU) methods. Here, a multiplex CRISPR/Cas12amicrofluidic paper-based analytical device (μPAD) was con-structed for the high-throughput POU analysis of arsM, withcascade amplification driven by coupling crRNA-enhanced Cas12aand horseradish peroxidase (HRP)-modified probes. First, sevencrRNAs were designed to recognize arsM, and their LODs andbackground signal intensities were evaluated. Next, a step-by-stepiterative approach was utilized to develop and optimize couplingsystems, which improved the sensitivity 32 times and eliminatedbackground signal interference. Then, ssDNA reporters modified with HRP were introduced to further lower the LOD to 16 fM, andthe assay results were visible to the naked eye. A multiplex channel microfluidic paper-based chip was developed for the reactionintegration and simultaneous detection of 32 samples and generated a recovery rate between 87.70 and 114.05%, simplifying thepretreatment procedures and achieving high-throughput POU analysis. Finally, arsM in Wanshan paddy soil was screened on site,and the arsM abundance ranged from 1.05 × 106 to 6.49 × 107 copies/g; this result was not affected by the environmental indicatorsdetected in the study. Thus, a coupling crRNA-based cascade amplification method for analyzing arsM was constructed, and amicrofluidic device was developed that contains many more channels than previous paper chips, greatly improving the analyticalperformance in paddy soil samples and providing a promising tool for the on-site screening of arsM at large scales