title: Fluorescent and Colorimetric Dual-Readout Assay for Inorganic Pyrophosphatase with Cu(2+)-Triggered Oxidation of o-Phenylenediamine.
abstract: 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 Cu(2+), the special inhibitory effects of pyrophosphate (PPi) on the oxidative ability of Cu(2+), and the specific hydrolysis of PPi into orthophosphate by PPase, a convenient small molecule OPD-based analytical system was developed for Cu(2+)/PPi recognition and PPase activity assay. We have confirmed that Cu(2+) 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, (1)H NMR, and XPS spectra analysis. Direct electrochemical insights into the Cu(2+)-triggered and PPi-inhibited mechanism were performed by cyclic voltammetry characterizations of the Cu(2+) 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.
authors.affiliation: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , Changchun, Jilin 130022, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Sun
authors.affiliation: School of Material Science and Engineering, Tianjin University , Tianjin 300072, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , Changchun, Jilin 130022, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , Changchun, Jilin 130022, China.
authors.forename: Zong-Jun
authors.initials: ZJ
authors.lastname: Li
authors.affiliation: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , Changchun, Jilin 130022, China.
authors.forename: Xiurong
authors.initials: X
authors.lastname: Yang
mesh_terms: D003124:Colorimetry / Q000379:methods*; D003300:Copper / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005453:Fluorescence; D043564:Inorganic Pyrophosphatase / Q000032:analysis* / Q000378:metabolism; D015394:Molecular Structure; D010084:Oxidation-Reduction / Q000187:drug effects; D010655:Phenylenediamines / Q000737:chemistry* / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism
chemical_list: D011756:Diphosphates; D010655:Phenylenediamines; D010710:Phosphates; C107241:diphosphoric acid; D003300:Copper; C034193:1,2-diaminobenzene; D043564:Inorganic Pyrophosphatase