A strategy for development of electrochemical DNA biosensor based on site-specific DNA cleavage of restriction endonuclease

Abstract

A new strategy for development of electrochemical DNA biosensor based on site-specific DNA cleavage of restriction endonuclease and using quantum dots as reporter was reported in this paper. The biosensor was fabricated by immobilizing a capture hairpin probe, thiolated single strand DNA labeled with biotin group, on a gold electrode. BfuCI nuclease, which is able to specifically cleave only double strand DNA but not single strand DNA, was used to reduce background current and improve the sensitivity. We demonstrated that the capture hairpin probe can be cleaved by BfuCI nuclease in the absence of target DNA, but cannot be cleaved in the presence of target DNA. The difference before and after enzymatic cleavage was then monitored by electrochemical method after the quantum dots were dissolved from the hybrids. Our results suggested that the usage of BfuCI nuclease obviously improved the sensitivity and selectivity of the biosensor. We successfully applied this method to the sequence-selective discrimination between perfectly matched and mismatched target DNA including a single-base mismatched target DNA, and detected as low as 3.3 × 10 −14 M of complementary target DNA. Furthermore, our above strategy was also verified with fluorescent method by designing a fluorescent molecular beacon (MB), which combined the capture hairpin probe and a pair of fluorophore (TAMRA) and quencher (DABCYL). The fluorescent results are consistent with that of electroanalysis, further indicating that the proposed new strategy indeed works as we expected.