Because the cytotoxic DNA methylated bases are chemically inert and small in size, limited biosensing strategies especially with high sensitivity were established to detect DNA methylation damage. In this work, a label-free electrochemical assay was proposed for the sensitive detection of DNA methylation damage in dsDNA film on indium tin oxide electrode. The human alkyladenine DNA glycosylase was employed to recognize and selectively remove DNA methylated bases, generating apurinic site. Subsequently, exonuclease III not only further converted the methylation sites into strand breaks, but also progressively removed nucleotides from the 3' to 5' end starting from the apurinic sites. These processes could maximum amplify DNA methylation damage, also confirmed by gel electrophoresis and fluorescence measurements. The damaged DNA film bound much less electrochemical indicator, Ru(bpy)2(dppz)2+, and accompanied by an anodic current drop. As a result, DNA methylation damage produced with as low as 10 μM of methyl methanesulfonate can be detected, which was 100-fold lower than the previously reported photoelectrochemical sensing strategy. Other DNA base modification products showed much less decrease of oxidation current, demonstrating its good selectivity. This strategy can be utilized to sensitively and rapidly assess the genotoxicity of environmental pollutants.
Wang Minghui,Yin Huanshun,Zhou Yunlei,Han Jingrui,He Tingqun,Cui Lin,Ai Shiyun
A photoelectrochemical biosensor is described for sensitive detection of microRNA-162a. A multiple amplification strategy is employed that involves (a) isothermal strand displacement polymerase reaction; (b) terminal deoxynucleotidyl transferase-mediated extension, (c) amplification of streptavidin-coated gold nanoparticles, and (d) biotin functionalized alkaline phosphatase. Graphite-like C 3 N 4 (g-C 3 N 4 ) nanosheets were used as photoactive material. By using these amplification strategies, the detection limit is as low as 0.18 fM of microRNA, and the photocurrent increases linearly with the concentration of microRNA-162a in the range from 0.5 fM to 1 pM. The method was successfully applied to quantify the expression level of microRNA-162a in total RNA extracted from the leaves of maize seedlings after incubation with the chemical mutagen ethyl methanesulfonate. The results confirmed the applicability of the method to the analysis of practical biological samples.
Schematic of a photoelectrochemical microRNA assay based on a multiple amplification strategy involving (a) isothermal strand displacement polymerase reaction; (b) terminal deoxynucleotidyl transferase-mediated extension, (c) amplification of streptavidin-coated gold nanoparticles, and (d) biotin functionalized alkaline phosphatase.
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