The arsM gene is a critical biomarker for the potential risk of arsenic exposure in paddy soil. However, on-site screening of arsM is limited by the lack of high-throughput point-of-use (POU) methods. Here, a multiplex CRISPR/Cas12a microfluidic paper-based analytical device (μPAD) was constructed for the high-throughput POU analysis of arsM, with cascade amplification driven by coupling crRNA-enhanced Cas12a and horseradish peroxidase (HRP)-modified probes. First, seven crRNAs were designed to recognize arsM, and their LODs and background signal intensities were evaluated. Next, a step-by-step iterative approach was utilized to develop and optimize coupling systems, which improved the sensitivity 32 times and eliminated background signal interference. Then, ssDNA reporters modified with HRP were introduced to further lower the LOD to 16 fM, and the assay results were visible to the naked eye. A multiplex channel microfluidic paper-based chip was developed for the reaction integration and simultaneous detection of 32 samples and generated a recovery rate between 87.70 and 114.05%, simplifying the pretreatment 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 indicators detected in the study. Thus, a coupling crRNA-based cascade amplification method for analyzing arsM was constructed, and a microfluidic device was developed that contains many more channels than previous paper chips, greatly improving the analytical performance in paddy soil samples and providing a promising tool for the on-site screening of arsM at large scales.

中文翻译：

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高通量 μPAD 通过双酶进行级联信号放大，用于水稻土中的 arsM

arsM基因是水稻土中砷暴露潜在风险的关键生物标志物。然而，由于缺乏高通量使用点（POU）方法，arsM的现场筛选受到限制。在这里，构建了一个多重 CRISPR/Cas12a 微流体纸基分析装置 (μPAD)，用于arsM的高通量 POU 分析，通过耦合 crRNA 增强的 Cas12a 和辣根过氧化物酶 (HRP) 修饰的探针驱动级联放大。首先，设计了 7 个 crRNA 来识别arsM，并评估了它们的 LOD 和背景信号强度。接下来，采用逐步迭代的方法开发和优化耦合系统，将灵敏度提高了32倍，并消除了背景信号干扰。然后，引入用 HRP 修饰的 ssDNA 报告基因，将 LOD 进一步降低至 16 fM，检测结果肉眼可见。开发了多通道微流控纸基芯片，用于反应集成并同时检测32个样品，回收率在87.70%至114.05%之间，简化了预处理程序，实现了高通量POU分析。最后对万山水稻土中的arsM进行了现场筛选，arsM丰度范围为1.05×10 ⁶～6.49×10 ⁷拷贝/g；这一结果不受研究中检测到的环境指标的影响。因此，构建了一种基于耦合crRNA的级联扩增分析arsM的方法，并开发了一种比以前的纸芯片包含更多通道的微流控装置，大大提高了水稻土样品的分析性能，为水稻土样品的分析提供了一种有前途的工具。大规模的arsM现场筛选。