Ultrasensitively electrochemical detection activity of DNA methyltransferase using an autocatalytic and recycling amplification strategy

Herein, we report a novel approach to the ultrasensitive detection of DNA methyltransferase (MTase) in terms of a dual-signal recycling amplification strategy assisted by an autocatalytic and exonuclease III (Exo III). A duplex DNA probe was designed by G-quadruplex-forming oligomer (S1) hybridizing with ferrocene (Fc)-labeled DNA (S2) modified on Au electrode. With DNA adenine methylation (Dam) MTase, the response of the hairpin probe to methylation is methylated. Then it can be cleaved by the methylation-sensitive restriction endonuclease Dpn I, releasing S3. The released S3 sequence hybridizes with the dangling end of S2 sequence, forming a new duplex DNA. Then the Exo III can cleave the S2 sequence step by step, with the release of Fc from the sensing interface and autonomous generation of new secondary S3 for successive hybridization and cleavage. Meanwhile, S1 sequences on the electrode surface are folded into G-quadruplex-hemin complexes by adding K+ and hemin to give a remarkable electrochemical response. Such conformational changes result in the decrease of differential pulse voltammetry (DPV) peak current of Fc and increase of the DPV peak current of G-quadruplex-hemin complexes. The dual-signal changes are linear with the concentration of DNA MTase. This novel, single electrode-based dual-signal assay strategy is superior to previous Dam assays using single electrochemical signal as output.