title: Enzyme catalysis-electrophoresis titration for multiplex enzymatic assay via moving reaction boundary chip.
abstract: In this work, we developed the concept of enzyme catalysis-electrophoresis titration (EC-ET) under ideal conditions, the theory of EC-ET for multiplex enzymatic assay (MEA), and a related method based on a moving reaction boundary (MRB) chip with a collateral channel and cell phone imaging. As a proof of principle, the model enzymes horseradish peroxidase (HRP), laccase and myeloperoxidase (MPO) were chosen for the tests of the EC-ET model. The experiments revealed that the EC-ET model could be achieved via coupling EC with ET within a MRB chip; particularly the MEA analyses of catalysis rate, maximum rate, activity, Km and Kcat could be conducted via a single run of the EC-ET chip, systemically demonstrating the validity of the EC-ET theory. Moreover, the developed method had these merits: (i) two orders of magnitude higher sensitivity than a fluorescence microplate reader, (ii) simplicity and low cost, and (iii) fairly rapid (30 min incubation, 20 s imaging) analysis, fair stability (<5.0% RSD) and accuracy, thus validating the EC-ET method. Finally, the developed EC-ET method was used for the clinical assay of MPO activity in blood samples; the values of MPO activity detected via the EC-ET chip were in agreement with those obtained by a traditional fluorescence microplate reader, indicating the applicability of the EC-ET method. The work opens a window for the development of enzymatic research, enzyme assay, immunoassay, and point-of-care testing as well as titration, one of the oldest methods of analysis, based on a simple chip.
authors.affiliation: Laboratory of Bioseparation and Analytical Biochemistry, State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. xiaohua@sjtu.edu.cn, cxcao@sjtu.edu.cn.
authors.forename: Ran
authors.initials: R
authors.lastname: Zhong
authors.forename: Haiyang
authors.initials: H
authors.lastname: Xie
authors.forename: Fanzhi
authors.initials: F
authors.lastname: Kong
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.forename: Sharmin
authors.initials: S
authors.lastname: Jahan
authors.forename: Hua
authors.initials: H
authors.lastname: Xiao
authors.forename: Liuyin
authors.initials: L
authors.lastname: Fan
authors.forename: Chengxi
authors.initials: C
authors.lastname: Cao
mesh_terms: D055162:Biocatalysis; D004586:Electrophoresis; D057075:Enzyme Assays / Q000295:instrumentation*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006801:Humans; D056656:Lab-On-A-Chip Devices; D010088:Oxidoreductases / Q000378:metabolism*
chemical_list: D010088:Oxidoreductases; D006735:Horseradish Peroxidase