A novel dual-functional biosensor for fluorometric detection of inorganic pyrophosphate and pyrophosphatase activity...

A novel ultrasensitive dual-functional biosensor for highly sensitive detection of inorganic pyrophosphate (PPi) and pyrophosphatase (PPase) activity was developed based on the fluorescent variation of globulin protected gold nanoclusters (Glo@Au NCs) with the assistance of Cu²⁺. Glo@Au NCs and PPi were used as the fluorescent indicator and substrate for PPase activity evaluation, respectively. In the presence of Cu²⁺, the fluorescence of the Glo@Au NCs will be quenched owing to the formation of Cu²⁺-Glo@Au NCs complex, while PPi can restore the fluorescence of the Cu²⁺-Glo@Au NCs complex because of its higher binding affinity with Cu²⁺. As PPase can catalyze the hydrolysis of PPi, it will lead to the release of Cu²⁺ and re-quench the fluorescence of the Glo@Au NCs. Based on this mechanism, quantitative evaluation of the PPi and PPase activity can be achieved ranging from 0.05 μM to 218.125 μM for PPi and from 0.1 to 8 mU for PPase, with detection limits of 0.02 μM and 0.04 mU, respectively, which is much lower than that of other PPi and PPase assay methods. More importantly, this ultrasensitive dual-functional biosensor can also be successfully applied to evaluate the PPase activity in human serum, showing great promise for practical diagnostic applications.