An electrochemical gene detection assay utilising T7 exonuclease activity on complementary probe–target oligonucleotide sequences

Abstract

This communication describes the synthesis of an electrochemically active oligonucleotide probe and its application in sensing complementary oligonucleotides sequences using a T7 exonuclease enzyme. Target oligonucleotides are detected by hybridisation with a ferrocene labelled probe oligonucleotide followed by addition of T7 exonuclease. The T7 enzyme is a double strand specific exonuclease that removes the terminal 5′ nucleotide of the probe sequence. The 5′ nucleotide is attached to a ferrocene label, which is subsequently detected at an electrode using differential pulse voltammetry. Time and temperature resolved measurements were performed and an associated study using dual labelled fluorophore–quencher labelled probes was performed to confirm the validity of the electrochemical assay.