One-pot synthesis of AuNCs-MnO2 nanoflakes with peroxidase-like characteristics for pyrophosphatase detection based on Exonuclease III and Cu2+-DNAzymes dual-amplified strategy

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A novel dual-amplified electrochemical biosensor based on Exonuclease III (Exo III) and Cu(II)-dependent DNAzymes (Cu2+-DNAzymes) was developed for ultrasensitive electrochemical evaluation of pyrophosphatase (PPase) activity with the help of one-step synthesis of BSA-directed gold nanoclusters-manganese dioxide (AuNCs-MnO2) nanoflakes with peroxidase-like characteristics as a catalytic platform. For biosensor preparation, under the catalytic hydrolysis of the PPase, copper ion (Cu2+) was released from the Cu2+-coordinated pyrophosphate ions (Cu2+–PPi) complex which was formed by the coordination reaction of copper ion (Cu2+) and pyrophosphate ions (PPi). The released Cu2+, acting as an indispensable cofactor, could activate Cu2+-DNAzymes to turn on the amplification process and then generated the single-stranded product (S1) that hybridized to the hairpin-type capture probe (HP1). Subsequently, the addition of Exonuclease III (Exo III) enabled the reuse of S1 and produced the final cleavage fragment, which closely linked the functionalized AuNCs-MnO2 nanoflakes to the electrode surface. The superior peroxidase-like characteristics of nanoflakes provided stable catalytic signal output and realized electrochemical monitoring of PPase activity with a detection limit of 0.0058 mU/mL. Importantly, the biosensor also had a good performance in inhibitors screening of target PPase. The use of AuNCs-MnO2 nanoflakes instead of natural enzymes combined with DNAzymes amplification strategy offered more possibilities for the detection of various biomolecules.