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

Chao Hsiung Lin, Hsaing Yin Chen, Chih Jen Yu, Pen Li Lu, Chu Hsin Hsieh, Bao Yu Hsieh, Ying Feng Chang, Chien Chou*

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

22 Scopus citations

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 × 106 M-1 s-1 for ka and 3.15 × 10-3 s-1 for kd. These constants are calculated from the fluorescence signals detected on 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 ka, kd, and KD as 8.48 × 105 M-1 s-1, 1.7 × 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.

Original languageEnglish
Pages (from-to)224-228
Number of pages5
JournalAnalytical Biochemistry
Volume385
Issue number2
DOIs
StatePublished - 15 02 2009
Externally publishedYes

Keywords

  • Fiber-optic biosensor
  • Heart-type fatty acid-binding protein (H-FABP)

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