Rapid Fluorescent Detection of Enterotoxigenic Escherichia coli (ETEC) K88 Based on Graphene Oxide-Dependent Nanoquencher and Klenow Fragment-Triggered Target Cyclic Amplification

Abstract

Based on Klenow fragment (KF)-assisted target recycling amplification and graphene oxide (GO), a novel aptasensor, containing a capture probe (CP) and a signal probe (SP), was constructed and applied for the rapid detection of enterotoxigenic Escherichia coli (ETEC) K88. The CP was constructed of regions I and II, where the region I is aptamer sequence of ETEC K88 and the region II can form a double-stranded DNA structure with the SP. The SP was labeled with carboxyfluorescein (FAM) and acted as the primer sequence of the polymerization reaction. Before the targets were added, the two probes formed a partial double-strand junction (PDSJ) on the surface of the GO and the fluorescence was completely quenched. In the presence of the targets, the fluorescence was recovered due to the formation of the target-aptamer complex and its separation from the surface of the GO. Following this, the target-aptamer complex initiated the polymerization of the DNA strand in the presence of deoxynucleotides (dNTPs) and the KF. The displaced target then combined into another PDSJ, and the cycle started anew, leading to the formation of numerous complementary double-stranded DNAs. Meanwhile, the fluorescence signal was significantly enhanced. The results indicated that the established sensor has higher sensitivity specificity to its target bacteria in a wide range of 1 × 10 2 to 1 × 10 8 colony-forming units (CFU) mL−1. The detection limit based on a signal-to-noise ratio (S/N) of 3 is 1 × 10 2 CFU mL−1. More important, this rapid detection method is superior to other methods, having not only a short detection time but also a low fluorescence background, and is cheaper and has a wider applicability because its probes are easily designed and synthesized. Given these factors, our detection system has great prospects as a potential alternative to conventional ETEC K88 detection.