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

Lingzhi Zhao; Liu Zhao; Yanqing Miao; Chunye Liu; Chenxiao Zhang

doi:10.1155/2016/4306838

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

J Anal Methods Chem. 2016:2016:4306838. doi: 10.1155/2016/4306838. Epub 2016 Sep 27.

Authors

Lingzhi Zhao¹, Liu Zhao², Yanqing Miao³, Chunye Liu³, Chenxiao Zhang⁴

Affiliations

¹ Department of Pharmacy, Xi'an Medical College, Xi'an 710021, China; Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
² Beijing Research Center of Agricultural Standards and Testing, Beijing 100097, China.
³ Department of Pharmacy, Xi'an Medical College, Xi'an 710021, China.
⁴ Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.

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 (S/N = 3). 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.