Quantitative measurement of binding kinetics in sandwich assay using a fluorescence detection fiber-optic biosensor
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 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 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.
- SourceAvailable from: Marja van Dieijen-Visser[Show abstract] [Hide abstract]
ABSTRACT: We have developed a new protein microarray (ImmunoFlow Protein Platform, IFPP) that utilizes a porous nitrocellulose (NC) membrane with printed spots of capture probes. The sample is pumped actively through the NC membrane, to enhance binding efficiency and introduce stringency. Compared to protein microarrays assayed with the conventional incubation-shaking method the rate of binding is enhanced on the IFPP by at least a factor of 10, so that the total assay time can be reduced drastically without compromising sensitivity. Similarly, the sensitivity can be improved. We demonstrate the detection of 1 pM of C-reactive protein (CRP) in 70 microL of plasma within a total assay time of 7 min. The small sample and reagent volumes, combined with the speed of the assay, make our IFPP also well-suited for a point-of-care/near-patient setting. The potential clinical application of the IFPP is demonstrated by validating CRP detection both in human plasma and serum samples against standard clinical laboratory methods.Analytical Chemistry 06/2009; 81(13):5165-71. DOI:10.1021/ac801244d · 5.83 Impact Factor
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ABSTRACT: In conventional heterogeneous immunoassays, assay speed is usually limited by the rate of mass transport, i.e., diffusion of antigen to an antibody-coated surface. We previously demonstrated that assay speed can be significantly increased, without losing analytical sensitivity, by rapidly rotating the capture substrate, which decreases the thickness of the diffusion layer. In this work, we raised the rotation speed and observed that the capture of antigens deviates from the mass transport-limited assumption. To examine this issue, a general equation was derived for the rate of immuno-reaction on a rotating capture surface that takes into account both diffusion and the rate of reaction between antigen and antibody, which applies over a wide range of rotation rates. Results show that by vigorously rotating the substrate, the binding of antigens reaches a regime of intermediate binding kinetics, for which mass transport is comparable to the reaction rate. With this general solution, we are able to determine the two important binding kinetics parameters: the diffusion coefficient and the reaction rate constant. Then, using porcine parvovirus as an example, we use these parameters to investigate the limit of the assay speed and the limit of detection achievable on a practical time scale through numerical simulations of the kinetic binding curves for various assay conditions.Analytical Chemistry 08/2009; 81(15):6175-85. DOI:10.1021/ac900704t · 5.83 Impact Factor
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ABSTRACT: With the increased development and use of fluorescence lifetime-based sensors, fiber optic sensors, fluorescence lifetime imaging microscopy (FLIM), and plate and array readers, , calibration standards are essential to ensure the proper function of these devices and accurate results. For many devices that utilize a "front face excitation" geometry where the excitation is nearly coaxial with the direction of emission, scattering-based lifetime standards are problematic and fluorescent lifetime standards are necessary. As more long wavelength (red and near-infrared) fluorophores are used to avoid background autofluorescence, the lack of lifetime standards in this wavelength range has only become more apparent . We describe an approach to developing lifetime standards in any wavelength range, based on Förster resonance energy transfer (FRET). These standards are bright, highly reproducible, have a broad decrease in observed lifetime, and an emission wavelength in the red to near infrared making them well suited for the laboratory and field applications as well. This basic approach can be extended to produce lifetime standards for other wavelength regimes.Journal of Fluorescence 12/2009; 20(2):435-40. DOI:10.1007/s10895-009-0565-9 · 1.67 Impact Factor