title: 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.
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.
authors.affiliation: 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.
authors.forename: Lingzhi
authors.identifier: 0000-0002-0121-1614
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Beijing Research Center of Agricultural Standards and Testing, Beijing 100097, China.
authors.forename: Liu
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Department of Pharmacy, Xi'an Medical College, Xi'an 710021, China.
authors.forename: Yanqing
authors.initials: Y
authors.lastname: Miao
authors.affiliation: Department of Pharmacy, Xi'an Medical College, Xi'an 710021, China.
authors.forename: Chunye
authors.initials: C
authors.lastname: Liu
authors.affiliation: Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Chenxiao
authors.initials: C
authors.lastname: Zhang