Chronocoulometric biosensor for K-ras point mutation detection based on E. coli DNA ligase and AuNPs amplification effects

Abstract

We report a novel method for the amplified detection of K-ras point mutation by using a chronocoulometric DNA sensor based on Escherichia coli DNA ligase and AuNPs amplification effects. Our method is based on a ‘sandwich’ detection strategy, involving capture probe immobilized on a gold electrode (GE) and reporter probe loaded on Au nanoparticles (AuNPs probe). Each probe flanks one of two fragments of the target DNA sequence. Successive hybridization event brings AuNPs, along with hundreds of reporter probes to form the ‘sandwich’ structure. After high-fidelity E. coli DNA ligase reaction and denaturing at an elevated temperature, We then employ chronocoulometry to detect [Ru(NH3)6]3+ electrostatically bounding to the DNA probe strands. If there is an allele mismatch, no ligation takes place. In this way, the novel approach had successfully distinguish the K-ras mutant DNA and the K-ras wild DNA. The charge variable (ΔQ) is proportional to the logarithmic K-ras mutant DNA concentration from 1.0nM to 0.1pM, with the detection limit of 0.01pM. Owing to its ease of operation and low detection limit, it is expected that the proposed approach may hold great promise in both research-based and clinical genomic assays.