Open AccessHighly Sensitive and Multiple Enzyme Activity Assay Using Reagent-release Capillary-Isoelectric Focusing with Rhodamine 110-based Substrates
Author(s) -
Kenji Sueyoshi,
Yuto Nogawa,
Kasumi Sugawara,
Tatsuro Endo,
Hideaki Hisamoto
Publication year - 2015
Publication title -
analytical sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.392
H-Index - 73
eISSN - 1348-2246
pISSN - 0910-6340
DOI - 10.2116/analsci.31.1155
Subject(s) - chemistry , chromatography , reagent , isoelectric focusing , rhodamine , capillary electrophoresis , capillary action , enzyme , microchemistry , rhodamine b , biochemistry , fluorescence , organic chemistry , physics , materials science , quantum mechanics , composite material , photocatalysis , catalysis
In this study, a simple and highly sensitive enzyme activity assay based on reagent-release capillary-isoelectric focusing is described. Reagent-release capillaries containing a fluorescent substrate, which produces fluorescent products possessing an isoelectric point after reaction with enzymes, provides a simple procedure. This is because it allows to spontaneously inject a sample solution into the capillary by capillary action, mixing reagents, and subsequently concentrating the fluorescent products based on isoelectric focusing. Fluorescent rhodamine 110 and its monoamide derivative, which were generated as a final product and an intermediate, respectively, were then focused and separated by reagent-release capillary-isoelectric focusing. After 30 min of enzyme reactions, two focused fluorescent bands were clearly isolated along the prepared capillaries. Employing the focused band of rhodamine 110 monoamide allowed for highly sensitive detection of enzyme activity in the 10 pg mL(-1) order, while that of the conventional assay using a microplate was in the ng mL(-1) order. Furthermore, arraying reagent-release capillaries of different substrates on a chip allowed for simultaneous multi-assay of enzyme activity with good sensitivity in the pg mL(-1) order for each protein.