龙峰等：Simultaneous and sensitive determination of Escherichia coli O157:H7 and Salmonella Typhimurium using evanescent wave dual-color fluorescence aptasensor based on micro/nano size effect
Abstract: The simultaneous and sensitive determination of two common pathogenic bacteria, Escherichia coli O157:H7 (E. coli O157:H7) and Salmonella Typhimurium (S. Typhimurium) was achieved using evanescent wave dual-color fluorescence aptasensor and the fiber nanoprobe through combining the micro/nano size and time-resolved effect. Two fluorescence labeled aptasensors, Cy3-apt-E and Cy5.5-apt-S, were regarded as biorecognition elements and signal reporters for E. coli O157:H7 and S. Typhimurium, which were alternatively excited by evanescent waves originated from 520 nm and 635 nm excitation lights, respectively. The fiber nanoprobe with in-situ etched nanopores was used for distinguishing free aptasensors and aptasensors bound to pathogenic bacteria based on the limited penetrated depth of evanescent wave and the significant size difference of bacteria and nanopore. The E. coli O157:H7 and S. Typhimurium were directly and simultaneously quantitated in less than 35 mins without the requirement of the complex immobilization of biorecognition molecules and bacteria enrichment/separation processes. The limits of detection of E. coli O157:H7 and S. Typhimurium were 340 CFU/mL and 180 CFU/mL, respectively. The satisfied recovery rate of real samples testing verified the feasibility and accuracy of the proposed method. Our strategy not only greatly simplifies the detection and identification process of multiple pathogenic bacteria, but also is easy to extend as a universal technology for sensitive determination of other bacteria using their respective biorecognition molecules.
Keywords: Evanescent wave dual-color fluorescence; Aptasensor; Escherichia coli O157:H7; Salmonella Typhimurium; fiber nanoprobe
摘要：基于微/纳米尺寸和时间分辨效应，利用倏逝波双色荧光核酸适配体传感器和光纤纳米探针实现了大肠杆菌O157:H7（E.coli O157:H7）和鼠伤寒沙门氏菌（S. Typhimurium）同时灵敏检测。荧光标记核酸适配体Cy3-apt-E和Cy5.5-apt-S分别用作E.coli O157:H7和S. Typhimurium 生物识别元件和信号报告分子，其被520nm和635nm激发光交替激发。利用倏逝波穿透深度有限和细菌与纳米孔尺寸差异显著的特点，采用原位刻蚀纳米孔的纳米光纤探针区分游离的和与病原菌结合的核酸适配体传感器，实现了E.coli O1 57:H7和S. Typhimurium直接同时定量检测，检测时间少于35min。该方法无需复杂的生物识别分子固定化和细菌富集/分离过程。E.coli O1 57:H7和S. Typhimurium的检测限分别为340cfu/mL和180cfu/mL。实际样品测试的回收率验证了该方法的可行性和准确性。该方法不仅大大简化了多种病原菌的检测和鉴定过程，而且可作为一种病原菌通用检测的技术。也就是说，只要不同病原菌的生物识别分子即可实现其快速定量检测。