An electrochemical biosensor based on hairpin-DNA aptamer probe and restriction endonuclease for ochratoxin A detection

A new electrochemical DNA biosensor based on hairpin anti-ochratoxin A (OTA) aptamer and site-specific DNA cleavage of restriction endonuclease TaqaI was reported in this paper. TaqaI is able to specifically cleave only double strand DNA, but not single strand DNA. The hairpin-DNA aptamer probe (Hap), thiolated single strand DNA labeled with biotin group, was immobilized on a gold electrode. In the presence of OTA, Hap bind with OTA to form Hap-OTA G-quadruplex. Meanwhile, the stem of Hap, which contains a TaqaI recognition site, is opened to form single strand and resistant to the cleavage of TaqaI. After reaction with the streptavidin-HRP, the resulting HRP-tagged Hap-OTA can effectively catalyze the hydrogen peroxide (H2O2)-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine sulfate (TMB), accompanied by a change from colorless to blue in solution color and an increased electrochemical current signal. However, in the absence of OTA, the biotin group of the Hap can be easily cleaved by TaqaI due to the double-stranded stem, which makes the streptavidin-HRP cannot bind with the Hap and increase electrochemical current signal. By employing the above strategy, this DNA biosensor can detect as low as 0.4pg/mL OTA with ultrahigh selectivity.