Hyperbranched rolling circle amplification based electrochemiluminescence aptasensor for ultrasensitive detection of thrombin

An ultrasensitive electrochemiluminescence (ECL) aptamer sensor for protein (thrombin as an example) detection based on hyperbranched rolling circle amplification (HRCA) had been developed. A complementary single-strand DNA (CDNA) of the thrombin aptamer had been modified on the gold electrode firstly, and then hybridized with thrombin aptamer to make the aptamer immobilized on the electrode surface, in the presence of thrombin, aptamer-thrombin bioaffinity complexes formed and made thrombin aptamer leave the electrode surface. Thus, the linear padlock probe hybridized with the free CDNA on the electrode surface and circularized by Escherichia coli DNA ligase. Subsequently, the linear padlock probe was served as a template for the initiation of HRCA reaction, and a lot of dsDNA modified on the electrode surface. Then Ru(phen)32+ (acted as the ECL indicator) intercalates specifically into double-stranded DNA (dsDNA) grooves to generate ECL signal. The ECL intensity of the system has a linear relationship with thrombin concentration in the range of 3.0-300 aM with a detection limit of 1.2 aM (S/N=3). The proposed method combines the high sensitivity of ECL, exponential amplification of HRCA for signal enhancement and high selectivity of aptamer.