Coupling DNA with enzyme activity: A complex electrochemical sensor with enhanced specificity

• A complex electrochemical assay couples the detection of target DNAs with LDH.
• A positive response can only be gained in the presence of both target DNAs and LDH.
• The sensor detects dual-target DNA with broad range and low detection limit.

We report here an electrochemical sensor which couples the detection of target DNAs with enzyme activity. A hairpin probe DNA complementary to both target DNAs was dually labeled with 5′-thiol and 3′-methylene blue (MB), and covalently immobilized to gold electrode. In the presence of both target DNAs, the hairpin structure shifted from “close” state to “open” state with the help of Escherichia coli (E. coli) DNA ligase, which can be characterized by alternating current voltammetry (ACV). For target DNA 1 and target DNA 2, the detection limits were 1 nM and 0.25 nM, respectively. Furthermore, the assay was coupled with the activity of lactate dehydrogenase (LDH), which catalyzes the conversion of NADH to NAD+. The latter acted as the co-substrate of DNA ligase and produced current change in the presence of both target DNAs. The sensor can analyze the co-existence of multi-components, which efficiently improves the accuracy in the applications such as species identification.

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