Highly Sensitive Fluorescent Aptasensor for Thrombin Detection Based on Competition Triggered Rolling Circle Amplification / 分析化学

ABSTRACT

Based on the competition reaction of target protein, aptamer probe, padlock probe and complementary sequence, a highly sensitive fluorescent aptasensor was developed in this study in combination with rolling circle amplification. In the absence of target protein, the ligation-rolling circle amplification reaction was repressed because the complementary sequence hybridized with aptamer probe to form double-stranded duplex. While in the presence of target protein, the target molecules bound specifically with aptamer probe, inducing displacement of the complementary sequence and hybridization with padlock probe. The padlock probe was circularized with the assistance of E. coli DNA ligase, and the rolling circle amplification process could be accomplished by Phi 29 DNA polymerase. The amplification product contained thousands of repeated sequences which could hybridize with the loop of molecular beacon ( the detection probes) , resulting in a significant fluorescence signal. The effects of length of complementary DNA ( CDNA ) sequence and concentration of padlock probe were investigated. Under the optimized experimental conditions, the model target protein thrombin could be highly sensitively detected by the proposed aptasensing system in a linear range of 0 . 067-32 . 4 nmol/L with a detection limit of 0 . 03 nmol/L ( approximately 90 amol target molecules). Moreover, the presented sensing method was universal for other target analysis by skillfully design of the sequence of aptamer probe and related oligonucleotides.