Label-Free and Ultrasensitive
Electrochemical Detection of Nucleic Acids Based on Autocatalytic
and Exonuclease III-Assisted Target Recycling Strategy
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Abstract
In this work, a very simple, label-free, isothermal,
and ultrasensitive electrochemical DNA biosensor has been developed
on the basis of an autocatalytic and exonuclease III (Exo III)-assisted
target recycling amplification strategy. A duplex DNA probe constructed
by the hybridization of a quadruplex-forming oligomer with a molecular
beacon is ingeniously designed and assembled on the electrode as recognition
element. Upon sensing of the analyte nucleic acid, the strand of molecular
beacon in the duplex DNA probe could be stepwise removed by Exo III
accompanied by the releasing of target DNA and autonomous generation
of new secondary target DNA fragment for the successive hybridization
and cleavage process. Simultaneously, numerous quadruplex-forming
oligomers are liberated and folded into G-quadruplex–hemin
complexes with the help of K<sup>+</sup> and hemin on the electrode
surface to give a remarkable electrochemical response. Because of
this autocatalytic target recycling amplification and the specifically
catalyzed formation of G-quadruplex–hemin complexes, this newly
designed protocol provides an ultrasensitive electrochemical detection
of DNA down to the 10 fM level, can discriminate mismatched DNA from
perfectly matched target DNA, and holds a great potential for early
diagnosis in gene-related diseases. It further could be developed
as a universal protocol for the detection of various DNA sequences
and may be extended for the detection of aptamer-binding molecules