Electrochemical DNA biosensor based on proximity-dependent DNA ligation assays with DNAzyme amplification of hairpin substrate signal

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This paper describes a novel electrochemical DNA sensor for the simple, sensitive and specific detection of nucleic acids based on proximity-dependent DNA ligation assays with the DNAzyme amplification of hairpin substrate signal. A long DNA strand contains the catalytic motif of Mg 2+-dependent 10-23 DNAzyme, acting as the recognition probe. When the target DNA was introduced into the system, part of it was complementary to 5′-end of the recognition probe, resulting in the ligation of a stable duplex, the unbinded part of the target DNA was acted as one binding arm for the DNAzyme. This duplex containing a complete 10-23 DNAzyme structure could cleave the purine–pyrimidine cleavage site of the hairpin substrate, which resulted in the fragmentation of the hairpin structure and the release of two single-stranded nucleic acids, one of which was biotinylated and acted as the signal probe. An immobilized thiolated capture probe could bind with the signal probe, using biotin as a tracer in the signal probe, and streptavidin–alkaline phosphatase (SA–ALP) as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 5 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The results revealed that the sensor showed a sensitive response to complementary target sequences of H. pylori in a concentration range from 100 fM to 1 nM, with a detection limit of 50 fM. In addition, the sensing system could discriminate the complementary sequence from mismatched sequences, with high sensitivity and reusability.