Label-free, non-enzymatic and ultrasensitive electrochemical nucleic acid biosensing by tandem DNA-fueled target recycling and hybridization chain reaction

•A label-free, non-enzymatic and ultrasensitive electrochemical DNA biosensor was developed.•The tandem DNA-fueled target recycling and hybridization chain reaction strategy was proposed.•A superior low detection limit of 20 aM toward target DNA could be achieved.•It creates an opportunity for the effective coupling of the target recycling and post-amplification strategies.

Herein, the DNA-fueled target recycling was effectively married with the hybridization chain reaction (HCR) for the autonomous dual signal amplification, by which an isothermal, non-enzymatic, label-free and ultrasensitive electrochemical DNA biosensor was developed for the first time. The DNA-fueled target recycling was operated onto the three-strand duplex DNA probe immobilized electrode according to the principle of the target DNA-triggered two cascaded toehold-mediated strand displacement reactions, accompanied with the association of plenty of DNA fuel strands on the electrode surface. The introduced DNA fuel strand possesses an emerging DNA fragment, which could be used to further propagate the HCR between two DNA hairpins, inducing the formation of a typical one-dimensional linear DNA concatamer on the electrode surface. The formed DNA concatamers were then bound with Hexaamineruthenium (III) chloride (RuHex) through electrostatic interaction for the significantly enhanced electrochemical response toward target DNA. A very low detection limit of 20 aM with an excellent selectivity toward target DNA could be achieved. The developed biosensor creates an opportunity for the simple, flexible and enzyme-free coupling of the target recycling and post-amplification strategies and thus opens a promising avenue for the electrochemical detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine.