Quantitative measurement of binding kinetics in sandwich assay using a fluorescence detection fiber-optic biosensor

Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/28142

Title:	Quantitative measurement of binding kinetics in sandwich assay using a fluorescence detection fiber-optic biosensor
Authors:	Lin,CH;Chen,HY;Yu,CJ;Lu,PL;Hsieh,CH;Hsieh,BY;Chang,YF;Chou,C
Contributors:	光電科學研究所
Keywords:	POLY(METHYL METHACRYLATE);SURFACE;PROTEIN;SENSORS;IMMUNOSENSORS;ANTIGEN;PLASMA
Date:	2009
Issue Date:	2010-06-29 19:42:24 (UTC+8)
Publisher:	中央大學
Abstract:	Fiber-optic biosensors have been Studied intensively because they are very useful and important tools for monitoring biomolecular interactions. Here we describe a fluorescence detection fiber-optic biosensor (FD-FOB) using a sandwich assay to detect antibody-antigen interaction. In addition, the quantitative measurement of binding kinetics, including the association and dissociation rate constants for immunoglobulin G (IgG)/anti-mouse IgG, is achieved, indicating 0.38 x 10(6), M-1 s(-1) for k(a) and 3.15 x 10(-3) s(-1) for k(d). These constants are calculated from the fluorescence signals detected oil fiber surface only where the excited evanescent wave can be generated. Thus, a confined fluorescence-detecting region is achieved to specifically determine the binding kinetics at the vicinity of the interface between sensing materials and uncladded fiber surface. With this FD-FOB, the mathematical deduction and experimental verification of the binding kinetics in a sandwich immunoassay provide a theoretical basis for measuring rate constants and equilibrium dissociation constants. A further measurement to study the interaction between human heart-type fatty acid-binding protein and its antibody gave the calculated kinetic constants k(a), k(d), and K-D as 8.48 x 10(5) M-1 s(-1), 1.7 x 10(-3) s(-1), and 2.0 nM, respectively. Our study is the first attempt to establish a theoretical basis for the florescence-sensitive immunoassay using a sandwich format. Moreover, we demonstrate that the FD-FOB as a high-throughput biosensor can provide an alternative to the chip-based biosensors to study real-time biomolecular interaction. (c) 2008 Elsevier Inc. All rights reserved.
Relation:	ANALYTICAL BIOCHEMISTRY
Appears in Collections:	[Graduate Institute of Optics and Photonics] journal & Dissertation

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