Electrochemical strategy for pyrophosphatase detection Based on the peroxidase-like activity of G-quadruplex-Cu²⁺...

A new simple and highly sensitive electrochemical method for pyrophosphatase (PPase) activity detection was developed based on the peroxidase-like activity of G-quadruplex-Cu²⁺ DNAzyme. In the absence of PPase, Cu²⁺ could coordinate with pyrophosphate (PPi) to form Cu²⁺-PPi compound. While in the presence of PPase, it could destroy the coordinate compound because PPase catalyzed the hydrolysis of PPi into inorganic phosphate and produced free Cu²⁺, which then could be coupled with G-rich DNA to form G-quadruplex-Cu²⁺ DNAzyme. The formation of a mimic enzyme (G-quadruplex-Cu²⁺ DNAzyme) was immobilized on the surface of screen-printed gold electrode (SPGE). Using 3, 3', 5, 5'-tetramethylbenzidine (TMB) as a redox mediator and H₂O₂ as an enzyme substrate, the DNAzyme catalyzed the reduction of H₂O₂ to generate quantitative chronoamperometric signal. The catalytic activity of G-quadruplex-Cu²⁺ DNAzyme for TMB-H₂O₂ reaction was proportional to the activity of PPase, based on which, a simple and sensitive turn-on electrochemical method for PPase activity was thus developed for the first time. The chronoamperometric intensity of the system had a linear relationship with the PPase activities in the range of 1.0-50.0mU/mL and the detection limit could be down to 0.6mU/mL (S/N = 3). This proposed method was selective, cost-effective and convenient without any labels or complicated operations, which was furthermore applied to screen the inhibitor for PPase with high efficiency.