Ultrasensitive Homogeneous Electrochemical Detection of Transcription Factor by Coupled Isothermal Cleavage Reaction and Cycling Amplification Based on Exonuclease III.

The assay and monitoring of transcription factors (TFs) has attracted extensive attention due to their important roles in regulation of gene expressions. Herein, a simple, low cost, rapid, and highly sensitive homogeneous electrochemical method utilizing the coupled isothermal cleavage reaction and cycling amplification based on exonuclease III (Exo III) was explored for the analysis of transcription factor NF-κB p50 in aqueous solution. In the assay, a 3'-methylene blue (MB)-labeled hairpin probe is designed, which can be opened up by the single stranded DNA (ssDNA) protected by NF-κB p50 from the Exo III cleavage, to trigger the subsequent Exo III-assisted digestion, thus a large amount of MB-labeled mononucleotides are liberated to result in the greatly amplified electrochemical signal. By virtue of this Exo III-assisted target recycling, the present assay allows the detection of NF-κB p50 at the picomolar level, which is an exciting level for TFs detection. Furthermore, this detection possesses excellent selectivity, demonstrating high application potential in biological system and convenient TFs' inhibitors screening. Comparing with the other reported strategies for TFs detection, this Exo III-assisted homogeneous electrochemical detection platform was just composed of one kind of enzyme and two DNA probes, offered a really simple and low-cost electrochemical detection for TFs assay.