Highly Sensitive Fluorescent Aptasensor for Thrombin Detection Based on Competition Triggered Rolling Circle Amplification

Based on the competition reaction of target protein, aptamer probe, padlock probe and the 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. On the contrary, in the presence of target protein, the target molecules bond specifically with its aptamer probe, inducing the displacement of the complementary sequence and hybridization with padlock probe. The padlock probe was circularized with the assistance of Escherichia coli DNA ligase, and the rolling circle amplification process could be accomplished by Phi29 DNA polymerase. The amplification product contained thousands of repeated sequences which could hybridize with the loop of molecular beacons (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 of thrombin was highly sensitively detected by the proposed aptasensing system in a linear range of 0.067–32.4 nM with a detection limit of 0.03 nM (approximately 90 amol target molecules). Moreover, the present sensing method was universal for other target analysis by skillful design of the sequence of aptamer probe and related oligonucleotides.

Target protein binding event induces the displacement of the complementary sequence and hybridization with padlock probe. With the assistance of Escherichia coli DNA ligase and phi29 DNA polymerase, circularization and rolling circle amplification reaction are performed. The amplification products hybridize with molecular beacons for signal generation.Figure optionsDownload as PowerPoint slide