Facile Synthesis of Enhanced Fluorescent Gold-Silver Bimetallic Nanocluster and Its Application for Highly Sensitive Detection of Inorganic Pyrophosphatase Activity

Herein, gold silver bimetallic nanoclusters (Au-Ag NCs) with the high fluorescent intensity were first synthesized successfully and utilized for the fabrication of sensitive and specific sensing probes toward inorganic pyrophosphatase (PPase) activity with the help of copper ion (Cu2+) and inorganic pyrophosphate ion (PPi). Cu2+ was used as the quencher of fluorescent Au-Ag NC, while PPi was employed as the hydrolytic substrate of PPase. The system consisted of PPi, Cu2+ ion, and bovine serum albumin (BSA)-stabilized Au-Ag NC. The detection was carried out by enzyme-induced hydrolysis of PPi to liberate copper ion from the Cu2+-PPi complex. In the absence of target PPase, free copper ions were initially chelated with inorganic pyrophosphate ions to form the Cu-PPi complexes via the coordination chemistry, thus preserving the natural fluorescent intensity of the Au-Ag NCs. Upon addition of target PPase into the detection system, the analyte hydrolyzed PPi into phosphate ions and released Cu2+ ion from the Cu2+-PPi complex. The dissociated copper ions readily quenched the fluorescent signal of Au-Ag NCs, thereby resulting in the decrease of fluorescent intensity. Under optimal conditions, the detectable fluorescent intensity of the as-prepared Au-Ag NCs was linearly dependent on the activity of PPase within a dynamic linear range of 0.1-30 mU/mL and allowed the detection at a concentration as low as 0.03 mU/mL at the 3s(blank) criterion. Good reproducibility (CV < 8.5% for the intra-assay and interassay), high specificity, and long-term stability (90.1% of the initial signal after a storage period of 48 days) were also received by using our system toward target PPase activity. In addition, good results with the inhibition efficiency of sodium fluoride were obtained in the inhibitor screening research of pyrophosphatase. Importantly, this system based on highly enhanced fluorescent Au-Ag NCs offer promise for simple and cost-effective screening of target PPase activity without the needs of sample separation and multiple washing steps.