Fiber-optic biosensor for antigen/antibody kinetic assays

Biosensors were first developed to address a need for the qualitative and quantitative real-time detection of biomolecular interactions. Fiber-optic biosensors (FOB) soon proved capable of real-time detection with a potential for specific sensitivity. Highly adaptable, they were accordingly developed as important detection and identification tools for a wide variety of assays in biomedical applications.^1,2

Most promising are FOBs based on total internal reflection fluorescence (TIRF).³ The technique is rooted in the fact that a fiber optic consists of a plastic or glass core surrounded by a layer of cladding material. Both components have different densities, hence light can be transmitted based on the principle of total internal reflection, which occurs when light propagating in a dense medium meets an interface with a less dense medium and is reflected. As it does so, some of the incident light propagates a short distance generating an evanescent wave (EW) on the outer side of the fiber interface. This EW can be used to excite fluorophores in the sample medium that are trapped on the fiber.

Since the light only penetrates the sample to a depth of approximately 200nm in a localized region adjacent to the fiber core surface, the EW enables the selective excitation of only the fluorophores that are bound. Hence, the fluorophores of sample molecules located outside the EW sensing volume do not contribute to the fluorescence signal. This feature significantly improves the real-time monitoring of the kinetics of receptors and target analytes in immunoassays.

One FOB implementation uses a ‘sandwich assay’ format in which a primary antibody is immobilized on the surface of the fiber.⁴ Upon the addition of antigen and labeled secondary antibody, a molecular sandwich complex is formed on the fiber surface such that the secondary antibody is within the EW excitation volume. The rate equation describing the antibody-antigen binding process under pseudo-first order kinetics is given by:

where k_a is the association rate constant and k_d is the dissociation rate constant.⁵ [A] is the volume concentration of fluorescence-labeled antibody, [B] is the surface concentration of target antigen bound to the immobilized primary antibody on the uncladded surface of the plastic fiber. R is the concentration of the AB complex at time t, which is proportional to the intensity of the measured fluorescence signal. The surface concentration of B at time t can be expressed by:

where [B]₀ is the maximum surface concentration of B at t =0. From equations (1) and (2), we can calculate both association (k_a) and dissociation constant (k_d) constants using:

We recently demonstrated the use of a FOB to monitor mouse immunoglobulin G (IgG) and anti-mouse IgG interactions.^6,7 In our experiments, a multi-mode plastic fiber was uncladded by immersing it into ethyl acetate and subsequent cleaning with phosphate-buffered saline. We then used the sandwich technique to perform kinetic studies. First, rabbit anti-mouse IgG was immobilized on the uncladded surface of a plastic fiber followed by addition of skim milk to the reaction chamber to avoid unspecific binding events. By injecting different concentrations of mouse IgG into the reaction chamber, an (anti-IgG/IgG) complex was generated for further binding kinetic assays between fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG and mouse IgG. Different concentrations of FITC-conjugated anti-mouse IgG were then added to the chamber to form a sandwich composite (anti-IgG/IgG/FITC anti-IgG) complex on the fiber surface. The sample was excited using a 488nm laser diode and fluorescence was detected with a photomultiplier tube as shown in Figure 1. A lock-in amplifier was incorporated into the FOB to enhance the signal-to-noise ratio.

Figure 1. Setup and configuration of a fiber-optic biosensor. The fluorescence resulting from sample excitation by the evanescent wave generated in the near-field region of the fiber-core surface is detected by a photomultiplier tube placed perpendicularly to the fiber wall axis. This allows the fluorescence signal to be collected more efficiently.

The fluorescence signal generated by the binding of the FITC-conjugated anti-mouse IgG to the mouse IgG is shown in Figure 2. The increase in fluorescence intensity depends on the reaction rate of the binding process; hence, the kinetic rate constants can be determined. In Figure 2, the concentration of protein complex is directly proportional to the fluorescence intensity at the initial stage of the binding reaction. Using Equation (3), we obtained the association and dissociation rate constants, k_a and k_d, from the slope of the experimental data (see Figure 3). The calculated constants for the IgG-anti-IgG binding interactions were k_a = 2.49 × 10⁵ M⁻¹ s⁻¹, k_d= 3 × 10⁻⁴ s⁻¹, and K_D=1.20nM, respectively.

Figure 2. Fluorescence intensity of five different concentrations of FITC-labeled rabbit anti-mouse IgG interacting with mouse IgG detected and measured with a fiber-optic biosensor.

Figure 3. Determination of kinetic constants for mouse IgG/anti-mouse IgG interactions.

These results are in agreement with published IgG-anti IgG kinetic values generated using a conventional method.⁸ Our experimental conditions were also consistent with the diffusion equation for external diffusion in solid-phase immunosensor⁹ since the volume concentration of fluorescence-labeled antibody remained constant under pseudo-first order conditions in our experiments. This implies that the measurement is not limited by diffusion nor mass transport limited.

In summary, we experimentally validated a FOB that can be used not only as a biosensing platform, but also for kinetic real-time measurements.

This research was partially supported by the Department of Health, Taiwan (Grant CCMP93-RD-055) and the National Science Council, Taiwan (grant NSC-93-2323-B-010-004).