Fluorescent and Colorimetric Dual-Readout Assay for Inorganic Pyrophosphatase with Cu2+-Triggered Oxidation of o-Phenylenediamine

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• 作者：Jian Sun ; Bin Wang ; Xue Zhao ; Zong-Jun Li ; Xiurong Yang
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：January 19, 2016
• 年：2016
• 卷：88
• 期：2
• 页码：1355-1361
• 全文大小：471K
• ISSN：1520-6882

文摘

We demonstrate a rationally designed fluorescent and colorimetric dual-readout strategy for the highly sensitive, quantitative determination of inorganic pyrophosphatase (PPase) activity, a key hydrolytic enzyme involved in a variety of metabolic processes. Inspired by the selective oxidative and chromogenic reaction of o-phenylenediamine (OPD) with Cu²⁺, the special inhibitory effects of pyrophosphate (PPi) on the oxidative ability of Cu²⁺, and the specific hydrolysis of PPi into orthophosphate by PPase, a convenient small molecule OPD-based analytical system was developed for Cu²⁺/PPi recognition and PPase activity assay. We have confirmed that Cu²⁺ acts as the oxidant in the reaction and the main chromogenic product of OPD is 2,3-diaminophenazine (usually called OPDox) in the assay by combining the ESI-MS, ¹H NMR, and XPS spectra analysis. Direct electrochemical insights into the Cu²⁺-triggered and PPi-inhibited mechanism were performed by cyclic voltammetry characterizations of the Cu²⁺ in the absence and presence of PPi for the first time. Furthermore, the proposed analytical system with clear response mechanism exhibits a promising outlook for the PPase activity assay in real biological samples and inhibitor screening.