A DNA polymerase-powered self-propelled DNA walking strategy for one-step, amplified and dual-signal electrochemical target detection.

Developing a simple and easy-to-operate sensing platform for sensitive and reliable target analysis would provide enormous opportunities to boost the applications in clinical biomedicine and disease diagnosis. Herein, a DNA polymerase-powered self-propelled DNA walking strategy was developed to achieve one-step, dual-signal and amplified nucleic acid detection. The sensing platform was fabricated easily by immobilizing two hybrid probes on an electrode surface. Each hybrid probe consisted of a DNA hairpin and a redox reporter-labelled signal strand. The HIV-1 DNA fragment was used as a model target. It could trigger the DNA polymerization cascade between two hairpins assisted by DNA polymerase, accompanying the release of two signal strands from the electrode surface for the concurrent responses of two electrochemical signals of methylene blue and ferrocene. The simultaneous dual-signal and amplified responses facilitated the sensitive and reliable analysis of the target. The low detection limit toward the target nucleic acid could reach 0.1 fM whether by methylene blue or ferrocene responses. It could also achieve the selective discrimination toward mismatched sequences and the application for target detection in a serum sample. The distinct features of the current sensing strategy also include its autonomous one-step operation and no extra DNA reagent requirement for signal amplification except for a DNA polymerase. Thus, it provides an attractive means for biosensor fabrication directed toward the reliable and sensitive analysis of nucleic acids or more analytes.

An enzyme-responsive electrochemical DNA biosensor achieving various dynamic range by using only-one immobilization probe.

Developing a simple and easy-to-operate biosensor with tunable dynamic range would provide enormous opportunities to promote the diagnostic applications. Herein, an enzyme-responsive electrochemical DNA biosensor is developed by using only-one immobilization probe. The immobilization probe was designed with a two-loop hairpin-like structure that contained the mutually independent target recognition and enzyme (EcoRI restriction endonuclease) responsive domains. The target recognition was based on a toehold-mediated strand displacement reaction strategy. The toehold region was initially caged in the loop of the immobilization probe and showed a relatively low binding affinity with target, which was improved via EcoRI cleavage of immobilization probe to liberate the toehold region. The EcoRI cleavage operation for immobilization probe demonstrated the well regulation ability in detection performance. It showed a largely extended dynamic range, a significantly lowered detection limit and better discrimination ability toward the mismatched sequences whether in two buffers (with high or low salt concentrations) or in the serum system. The advantages also includes simplicity in probe design, and facile biosensor fabrication and operation. It thus opens a new avenue for the development of the modulated DNA biosensor and hold a great potential for the diagnostic applications and drug monitoring.