TY - JOUR
T1 - Quantitative measurement of binding kinetics in sandwich assay using a fluorescence detection fiber-optic biosensor
AU - Lin, Chao Hsiung
AU - Chen, Hsaing Yin
AU - Yu, Chih Jen
AU - Lu, Pen Li
AU - Hsieh, Chu Hsin
AU - Hsieh, Bao Yu
AU - Chang, Ying Feng
AU - Chou, Chien
PY - 2009/2/15
Y1 - 2009/2/15
N2 - 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.
AB - 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.
KW - Fiber-optic biosensor
KW - Heart-type fatty acid-binding protein (H-FABP)
UR - http://www.scopus.com/inward/record.url?scp=58149374378&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2008.11.010
DO - 10.1016/j.ab.2008.11.010
M3 - 文章
C2 - 19041630
AN - SCOPUS:58149374378
SN - 0003-2697
VL - 385
SP - 224
EP - 228
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -