Ultrasensitive NIR fluorometric assay for inorganic pyrophosphatase detection via Cu2+-PPi interaction using bimetallic Au-Ag nanoclusters

Abstract

Background: Inorganic pyrophosphatase (PPase) is key enzyme playing a key role in biochemical transformations such as biosynthesis of DNA and RNA, bone formation, metabolic pathways associated with lipid, carbohydrate and phosphorous. It has been reported that lung adenocarcinomas, colorectal cancer, and hyperthyroidism disorders can result from abnormal level of PPase. Therefore, it is of notable significance to develop simple and effective real time assay for PPase enzyme activity monitoring for screening of many metabolic pathways as well as for early disease diagnosis.

Result: The fluorometric detection of PPase enzyme in near infrared region-1 (NIR-1) has been carried out using bimetallic nanoclusters (LA@AuAg NCs). The developed sensing strategy was based on quenching of fluorescence intensity of LA@AuAg NCs upon interaction with copper (Cu²⁺) ions. The off state of LA@AuAg_Cu²⁺ ensemble was turned on upon addition of pyrophosphate anion (PPi) due to strong binding interaction between PPi and Cu²⁺. The catalytic conversion of PPi into phosphate anion (Pi) in the presence of PPase led to liberation of Cu²⁺ ions, and again quenched off state was retrieved due to interaction of free Cu²⁺ with LA@AuAg NCs. The ultrasensitive detection of PPase was observed in the linear range of 0.06-250 mU/mL with LOD as 0.0025 mU/mL. The designed scheme showed good selectivity towards PPase enzyme in comparison to other bio-substrates, along with good percentage recovery for PPase detection in real human serum samples.

Significance: The developed NIR based assay is ultrasensitive, highly selective and robust for PPase enzyme and can be safely employed for other enzymes detection. This highly sensitive nature of biosensor was result of involvement of fluorescence-based technique and synergistic effect of dual metal in NIR based bimetallic NCs. Moreover, owing to the emission in NIR domain, in future, these nanoclusters can be safely employed for many biomedical applications for In vivo studies.

Keywords: AuAg NCs; Fluorometric assay; Good selectivity; NIR emitting; PPase; Ultra-sensitive.