A Biosensor Based on Catalytic Hairpin Assembly and Fluorescence Resonance Energy Transfer for Detection of Enterovirus 71

A biosensor based on catalytic hairpin assembly ( CHA) and fluorescence resonance energy transfer (FRET) was established for detection of enterovirus 71 ( EV71 ). A CHA signal amplification strategy was designed to detect the VP1 gene of EV71, which was consisted of a pair of hairpin probes ( H1 and H2). The fluorophores Cy3 and Cy5 were labeled on H1 and H2, respectively. When the VP1 gene was present, the catalytic assembly reaction of H1 and H2 was triggered, Cy3 and Cy5 got closer and FRET occurred, leading to a reduced Cy3 signal and an enhanced Cy5 signal. In the absence of VP1 gene, H1 and H2 existed stably in the reaction system and could not generate FRET. Experimental conditions including reaction buffer concentration, the ratio of H1 to H2, reaction temperature and reaction time were optimized. Under the optimized conditions, the fluorescence intensity ratio of Cy5 to Cy3 showed a good linear relationship with EV71 VP1 gene concentration in the range of 0. 5-20 nmol/ L, and the detection limit was 73 pmol/ L (3 sigma). The spiked recoveries of throat swab samples ranged from 99. 6% to 103. 1% with the relative standard deviations of 0. 3% to 1. 7% , showing good specificity and anti-interference ability. The proposed sensor had a good application prospect in EV71 virus monitoring and early diagnosis of hand, foot and mouth disease.