Label-free and ultrasensitive electrochemical detection of nucleic acids based on an exonuclease III-assisted target recycling amplification strategy using a heated gold disk electrode.

The present work demonstrates a label-free, rapid and ultrasensitive electrochemical sensor for specific DNA detection with an exonuclease III (Exo III)-assisted target recycling amplification strategy and elevated electrode temperature at a heated gold disk electrode (HAuDE). The proposed electrochemical DNA (E-DNA) sensor was designed such that in the presence of the target DNA, the electrode self-assembled capture probe hybridizes with the target DNA to form a duplex structure, which triggers Exo III to specifically recognize this structure and selectively digest the capture probe, while the released target DNA underwent recycling to hybridize with a new capture probe, leading to the gradual digestion of a large amount of capture probes. It was found that during the digestion period, the activity of Exo III could be significantly improved by elevating electrode temperature, thus promoting the digestion reaction and improving the sensitivity for target DNA detection. Furthermore, an electrochemical indicator ([Ru(NH₃)₆]³⁺) was electrostatically bound to the capture probe, leading to a significant square wave voltammetry (SWV) response, which directly related to the amount and length of the capture probes remaining in the electrode and provided a quantitative measure for target DNA detection. The proposed strategy realized the highly sensitive detection of the target DNA with a detection limit of 26 aM (S/N = 3) at an electrode temperature of 40 °C during the digestion period, which was about two magnitudes lower than that at 24 °C.