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Journal of Analytical Methods in Chemistry
Volume 2016, Article ID 4306838, 10 pages
http://dx.doi.org/10.1155/2016/4306838
Research Article

Construction of a Turn Off-On-Off Fluorescent System Based on Competitive Coordination of Cu2+ between 6,7-Dihydroxycoumarin and Pyrophosphate Ion for Sensitive Assay of Pyrophosphatase Activity

1Department of Pharmacy, Xi’an Medical College, Xi’an 710021, China
2Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China
3Beijing Research Center of Agricultural Standards and Testing, Beijing 100097, China

Received 12 June 2016; Revised 25 August 2016; Accepted 5 September 2016

Academic Editor: Jaroon Jakmunee

Copyright © 2016 Lingzhi Zhao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The detection of pyrophosphatase (PPase) activity is of great significance in diagnosing diseases and understanding the function of PPase-related biological events. This study constructed a turn off-on-off fluorescent system for PPase activity assay based on PPase-regulated competitive coordination of Cu2+ between a water-soluble fluorescent probe 6,7-dihydroxycoumarin (DHC) and pyrophosphate (PPi). The probe DHC can coordinate with Cu2+ and consequently display on-off type fluorescence response. Furthermore, the in situ formed nonfluorescent Cu2+-DHC complex can act as an effective off-on type fluorescent probe for sensing PPi due to the higher coordination reactivity between Cu2+ and PPi than that between Cu2+ and DHC. The subsequent addition of PPase to the mixture containing Cu2+, DHC, and PPi leads to the fluorescence requenching of the system again (an off state) because PPase catalyzes the hydrolysis of PPi into orthophosphate in the reaction system. Under the optimum conditions, the decrease of the fluorescence intensity of DHC-Cu2+-PPi system was linear with the increase of the PPase activity in the range from 0.1 to 0.3 U. The detection limit was down to 0.028 U PPase (). Moreover, the as-established system was also applied to evaluate PPase inhibitor. This study offers a simple yet effective method for the detection of PPase activity.