Point-of-care (POC) testing offers rapid diagnostic results. However, the quantification of current methods is performed using standard curves and external references, and not direct and absolute quantification. This paper describes an integrated multiplex digital recombinase polymerase amplification (ImdRPA) microfluidic chip which combines DNA extraction, multiplex digital RPA and fluorescence detection together in one chip, creating a "sample-in-multiplex-digital-answer-out" system. Multi-layer soft lithography technology was used, with polydimethylsiloxane (PDMS) as the chip material and a glass slide as the substrate. This microfluidic chip has a six-layer structure and screw microvalve control function. The sample preparation for the chip involved magnetic bead-based nucleic acid extraction, which was completed within 15 min without any instrument dependence. The dRPA region was divided into 4 regions (3 positive detection areas and 1 negative control area) and included a total of 12 800 chambers, with each chamber being able to contain a volume of 2.7 nL. The screw valve allowed for the reaction components of each specific goal to be pre-embedded in different regions of the chambers. The reagents were passively driven into the dRPA region using vacuum-based self-priming introduction. Furthermore, we successfully demonstrated that the chip can simultaneously detect three species of pathogenic bacteria within 45 min and give digital quantitative results without the need to establish a standard curve in contaminated milk. Moreover, the detection limit of this ImdRPA microfluidic chip was found to be 10 bacterial cells for each kind of pathogen. These characteristics enhance its applicability for rapid detection of foodborne bacteria at the point-of-care (POC). We envision that the further development of this integrated chip will lead to rapid, multiplex and accurate detection of foodborne bacteria in a feasible manner.

中文翻译：

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一种“样本多重输入数字应答”芯片，用于快速检测病原体。

即时护理（POC）测试可提供快速的诊断结果。但是，当前方法的定量是使用标准曲线和外部参考进行的，而不是直接定量和绝对定量。本文介绍了一种集成的多重数字重组酶聚合酶扩增（ImdRPA）微流控芯片，该芯片将DNA提取，多重数字RPA和荧光检测结合在一个芯片中，从而创建了一个“多重采样数字应答输出”系统。使用了多层软光刻技术，其中以聚二甲基硅氧烷（PDMS）为芯片材料，以载玻片为基材。该微流体芯片具有六层结构和螺杆微阀控制功能。芯片的样品制备涉及基于磁珠的核酸提取，它在15分钟内完成，没有任何仪器依赖性。dRPA区域分为4个区域（3个阳性检测区域和1个阴性对照区域），总共包括12 800个腔室，每个腔室的容积为2.7 nL。螺旋阀允许将每个特定目标的反应成分预先嵌入腔室的不同区域。使用基于真空的自吸引药将试剂被动驱动进入dRPA区域。此外，我们成功地证明了该芯片可以在45分钟内同时检测出三种病原细菌，并给出了数字定量结果，而无需在受污染的牛奶中建立标准曲线。此外，对于每种病原体，该ImdRPA微流控芯片的检出限为10个细菌细胞。这些特性增强了其在即时点（POC）上快速检测食源性细菌的适用性。我们设想，这种集成芯片的进一步发展将导致以可行的方式快速，多重和准确地检测食源性细菌。