Abstract：
Most electroanalytical detection schemes for DNA markers require considerable time and effort from expert personnel to thoroughly follow the analysis and obtain reliable outcomes. This work aims to present an electrochemical assay performed inside a small card-based platform powered by microfluidic manipulation, requiring minimal human intervention and consumables. The assay couples a sample/signal dual amplification and DNA-modified magnetic particles for the detection of DNA amplification products. Particularly, the sul1 and sul4 genes involved in the resistance against sulfonamide antibiotics were analyzed. As recognized by the World Health Organization, antimicrobial resistance threatens global public health by hampering medication efficacy against infections. Consequently, analytical methods for the determination of such genes in environmental and clinical matrices are imperative. Herein, the resistance genes were extracted from E. coli cells and amplified using an enzyme-assisted isothermal amplification at 37 °C. The amplification products were analyzed in an easily-produced, low-cost, card-based set-up implementing a microfluidic system, demanding limited manual work and small sample volumes. The target amplicon was thus captured and isolated using versatile DNA-modified magnetic beads injected into the microchannel and exposed to the various reagents in a continuously controlled microfluidic flow. After the optimization of the efficiency of each phase of the assay, the platform achieved limits of detections of 44.2 pmol L-1 for sul1 and 48.5 pmol L-1 for sul4, and was able to detect down to ≥500-fold diluted amplification products of sul1 extracted from E. coli living cells in around 1 h, thus enabling numerous end-point analyses with a single amplification reaction.
摘要：
用于DNA标记的大多数电分析检测方案需要来自专家人员的相当多的时间和精力来彻底跟踪分析并获得可靠的结果。这项工作的目的是提出一个电化学分析在一个小的基于卡的平台由微流体操作提供动力，需要最少的人为干预和消耗品。该测定偶联样品/信号双重扩增和DNA修饰的磁性颗粒,用于检测DNA扩增产物。特别是,分析了与磺胺类抗生素耐药性有关的sul1和sul4基因。正如世界卫生组织所承认的那样，抗菌素耐药性通过阻碍药物对感染的疗效来威胁全球公共卫生。因此,在环境和临床矩阵中确定此类基因的分析方法是必要的。在这里，从大肠杆菌细胞中提取抗性基因，并在37°C下使用酶辅助等温扩增进行扩增。扩增产物被分析在一个容易产生的，低成本,基于卡的设置实施微流体系统，要求有限的手工工作和小样本量。因此,使用注射到微通道中的通用DNA修饰的磁珠捕获并分离靶扩增子,并在连续控制的微流体流中暴露于各种试剂。在优化分析的每个阶段的效率后，该平台对sul1的检测限为44.2pmolL-1，对sul4的检测限为48.5pmolL-1，并且能够在约1小时内检测到从大肠杆菌活细胞中提取的sul1稀释的扩增产物≥500倍，因此，可以通过单个扩增反应进行许多终点分析。
