Abstract：
Rapid and sensitive Escherichia coli (E. coli) detection is important in determining environmental contamination, food contamination, as well as bacterial infection. Conventional methods based on bacterial culture suffer from long testing time (24 h), whereas novel nucleic acid-based and immunolabelling approaches are hindered by complicated operation, the need of complex and costly equipment, and the lack of differentiation of live and dead bacteria. Herein, we propose a chemiluminescence digital microwell array chip based on the hydrolysis of 6-Chloro-4-methylumbelliferyl-β-D-glucuronide by the β-D-glucuronidase in E. coli to achieve fast single bacterial fluorescence detection. Taking the advantage of the picoliter microwells, single bacteria are digitally encapsulated in these microwells, thus the accurate quantification of E. coli can be realized by counting the number of positive microwells. We also show that the chemiluminescence digital microwell array chip is not affected by the turbidity of the test samples as well as the temperature. Most importantly, our method can differentiate live and dead bacteria through bacterial proliferation and enzyme expression, which is confirmed by detecting E. coli after pH and chlorination treatment. By comparing with the standard method of plate counting, our method has comparable performance but significantly reduces the testing time from over 24 h-2 h and 4 h for qualitative and quantitative analysis, respectively. In addition, the microfluidic chip is portable and easy to operate without external pump, which is promising as a rapid and on-site platform for single E. coli analysis in water and food monitoring, as well as infection diagnosis.
摘要：
快速灵敏的大肠杆菌(E.大肠杆菌)检测在确定环境污染方面很重要，食品污染,以及细菌感染。基于细菌培养的常规方法具有较长的测试时间（24小时），而新的基于核酸和免疫标记的方法受到复杂操作的阻碍，需要复杂而昂贵的设备，以及活的和死的细菌缺乏分化。在这里，我们提出了一种基于β-D-葡萄糖醛酸酶在大肠杆菌中水解6-氯-4-甲基伞形酰基-β-D-葡萄糖醛酸的化学发光数字微孔阵列芯片，以实现快速的单细菌荧光检测。利用皮升微孔的优势，单个细菌以数字方式封装在这些微孔中，因此,通过计数阳性微孔的数量可以实现大肠杆菌的准确定量。我们还表明，化学发光数字微孔阵列芯片不受测试样品的浊度和温度的影响。最重要的是,我们的方法可以通过细菌增殖和酶表达来区分活的和死的细菌，通过pH和氯化处理后检测大肠杆菌证实。通过与标准的平板计数方法比较,我们的方法具有可比的性能，但显着减少了超过24h-2h和4h的定性和定量分析的测试时间，分别。此外,微流控芯片是便携式的，易于操作，无需外部泵，它有望成为水和食品监测中单一大肠杆菌分析的快速现场平台，以及感染诊断。
