Electrochemical detection of point mutation based on surface hybridization assay conjugated allele-specific polymerase chainreaction

In this work, we developed an electrochemical detection method based on allele-specific polymerase chain reaction (AS-PCR) and surface hybridization assay technique for the point mutation detection. A high-fidelity VentR™(exo−) DNA polymerase, which eliminated the 3′→5′ proofreading exonuclease activity by genetical engineering, was used to discriminate and extend the detection probe that perfectly matched with mutant target DNA and generate a redox-active DNA replica which folded into a molecular beacon structure by intramolecular hybridization. After hybridized with capture probe modified on gold electrode by self-assembly reaction, the redox tags can be closed to electrode, resulting in a substantial current with the maximized sensitivity for point mutation analysis. However, when there is an allele mismatch in the wild target DNA, and so no the redox-active replica DNA can be obtained. In this case, no remarkable current signal can be trigged. The proposed approach has been successfully implemented for the identification of single base mutation at the −28 position in human β-globin gene with a detection limit of 0.5 fM, demonstrating that this method provides a highly specific, sensitive and cost-efficient approach for point mutation detection.

► A biphasic architecture of electrochemical point mutation detection technique was developed.
► The redox-active replica DNA can be obtained with allele-specific PCR.
► The redox tags of replica DNA can be closed to electrode surface via surface hybridization.
► The immobilization of capture probes on electrode surface via disulfide anchors.
► This method has been implemented for the detection of single base mutation of human β-globingene.