Ratiometric electrochemical biosensor based on Exo III-Assisted recycling amplification for the detection of CAG trinucleotide repeats

Electrochemical detection of specific nucleic acid sequence remains a hot topic in current bioanalytical research. Here, a novel ratiometric electrochemical biosensor based on Exo III-assisted recycling amplification and graphene-modified electrode was fabricated for quantitative detection of trinucleotide repeat sequence d(CAG)(n). The double-signals used are the hairpin DNAs labeled with ferrocene and methylene blue respectively as report DNAs, which can hybridize to target DNA. The hybridized DNA was digested by Exo III, resulting in the release of target and report fragments. The graphene-modified electrode can selectively adsorb the released report fragments to generate double electrochemical signals. The signal ratio (F/M) of ferrocene and methylene blue was used to determine the repeat length accurately: a linear relationship was found between F/M and numbers of repeats (n), F/M = 0.061 n + 1.97, with a correlation coefficient of 0.992. Moreover, any electrochemical signal can be used to test repeat concentration with detection limit of 0.22 pM. Therefore, this novel ratiometric electrochemical biosensor provided a reliable and efficient method for the analysis of d(CAG)(n) trinucleotide repeat and a potential simplified clinical tool for neurodegenerative diseases.