A sensitive electrochemical aptasensor for ATP detection based on exonuclease III-assisted signal amplification strategy

• Distinctive sequence of hairpin-aptamer was designed.
• Structure-switching of aptamer to reveal the MB labeled terminus.
• Exonuclease-catalyzed target recycling amplified signal significantly.
• Exo III improved the sensitivity of the aptasensor significantly.

A target-induced structure-switching electrochemical aptasensor for sensitive detection of ATP was successfully constructed which was based on exonuclease III-catalyzed target recycling for signal amplification. With the existence of ATP, methylene blue (MB) labeled hairpin DNA formed G-quadruplex with ATP, which led to conformational changes of the hairpin DNA and created catalytic cleavage sites for exonuclease III (Exo III). Then the structure-switching DNA hybridized with capture DNA which made MB close to electrode surface. Meanwhile, Exo III selectively digested aptamer from its 3′-end, thus G-quadruplex structure was destroyed and ATP was released for target recycling. The Exo III-assisted target recycling amplified electrochemical signal significantly. Fluorescence experiment was performed to confirm the structure-switching process of the hairpin DNA. In fluorescence experiment, AuNPs–aptamer conjugates were synthesized, AuNPs quenched fluorescence of MB, the target-induced structure-switching made Exo III digested aptamer, which restored fluorescence. Under optimized conditions, the proposed aptasensor showed a linear range of 0.1–20 nM with a detection limit of 34 pM. In addition, the proposed aptasensor had good stability and selectivity, offered promising choice for the detection of other small molecules.

Figure optionsDownload as PowerPoint slideIllustration of the stepwise aptasensor fabrication based on exonuclease-assisted target recycling for amplification.