Effect of incident angle of light on sensitivity and detection limit for layers of antibody with surface plasmon resonance spectroscopy
Introduction
Surface plasmon is charge–density oscillation propagating along the interface of metal and dielectric. Because the wave vector of the surface plasmon is decided by the properties of the metal and the dielectric according to Maxwell's law, changes in the properties of the dielectric are determined by the wave vector (Raether, 1988, Sambles et al., 1991). The wave vector of the surface plasmon can be excited by a light. A method for the excitation was developed by Kretschmann (1971). Because a simple configuration of the metal and the dielectric was employed for the excitation, a surface-plasmon resonance (SPR) instrument was developed for the measurement of chemical and biochemical compounds (Liedberg et al., 1983, Kooyman et al., 1988, Matsubara et al., 1988, Sun et al., 1989).
Basically, two main approaches have been used in SPR sensing. One is an instrument based on the measurement of changes in the resonant angle induced by the SPR (angular SPR); the other one is based on measurements of changes in resonant wavelength induced by the SPR (spectral SPR). For the measurement of chemical or biochemical compounds, angular SPR has been used most widely (Wohlhueter et al., 1994, Gaus and Hall, 1998, Laricchia-Robbio et al., 1998, Vikinge et al., 1998), and there are numerous reports describing its sensitivity (Lukosz, 1991, Kano and Kawata, 1994, Yeatman, 1996, Kolomenskii et al., 1997). However, there are fewer reports about the sensitivity of spectral SPR (Homola, 1997, Salamon et al., 1997). Because spectral SPR has the advantage of using an optical fiber for remote sensing, it would be advantageous to investigate the sensitivity of the spectral SPR in depth.
An instrument using optical fiber based on spectral SPR was first reported by Jorgenson and Yee, 1993, Jorgenson and Yee, 1994. According to the literature, they used a multimode optical fiber and directly deposited silver or gold onto the core of the fiber. The incident angle of the light region was reported to be 75.5–90°, corresponding to an SPR coupling-wavelength region of 560–620 nm. Using the same optical set-up, the sensitivity for immunoreaction was studied and reported to be the same as that of angular SPR (Kunz et al., 1996). However, there was no description of the choice of the incident angle or the SPR coupling wavelength, despite the fact that the sensitivity of the spectral SPR was strongly affected by the incident angle or the SPR coupling wavelength. Because a smaller incident angle or a longer SPR coupling wavelength showed higher sensitivity, it could be said that the appropriate choice of incident angle or SPR coupling wavelength could give spectral SPR higher sensitivity.
The dependence of sensitivity on the SPR coupling wavelength was reported by Homola (1997). According to his research, spectral SPR achieved higher sensitivity by means of longer SPR coupling wavelengths. On the other hand, we studied the incident-angle dependence on the sensitivity (Akimoto et al., 1999). Our results indicated that higher sensitivity was achieved with a small incident angle of light rather than with a large one. For instance, the sensitivity for the thickness of a sensed layer measured with a 66° incident angle was four times higher than that with a 76° incident angle. However, both reports were mainly the result of theoretical calculation. In the case of sensitivity for immunoreaction, the sensitivity can not be studied by theoretical calculation alone because the thickness and refractive index of the protein layer have not been determined clearly (Rahn and Hallock, 1995, Oudshoorn et al., 1996). Therefore, experimental studies on the sensitivity for immunoreaction are necessary to estimate an ability for immunosensor with the spectral SPR.
In this paper, we investigated the sensitivity and detection limit of spectral SPR for immunoreaction. The sensitivity and detection limit were measured experimentally using an antibody as a modeled analyte and an antigen as an immobilized sensor surface. The incident angles were 66, 68, 70, 72, 74 and 76°. The measured sensitivity and detection limit were compared with those of angular SPR to estimate an ability of the spectral SPR. In addition, thicknesses and refractive indices of protein layers were proposed by experimental results and theoretical calculation.
Section snippets
Spectral SPR instrument
A scheme of the spectral SPR instrument is shown in Fig. 1a. All collimated and focused light beams were made using spherical mirrors indicated as M1–M4 in Fig. 1a. A diameter of the incident light was reduced to 2 mm by an aperture (indicated as A2) The incident angle of the light, θ, was defined as shown in Fig. 1a. All the optical components were purchased from Sigmakouki (Saitama, Japan). The reflection light was analyzed spectrographically using a charge-coupled device and a personal
Spectrum of reflection-light intensity of the spectral SPR
Fig. 2 shows typical spectrum of reflection-light intensity for 68 and 76° incident angles. Both the experimentally and theoretically determined reflection curves are shown in Fig. 2 to confirm the accuracy of the spectral SPR. The resonant wavelength was defined as a wavelength that had minimal reflection-light intensity. The experimental reflection curves of BSA layer adsorbed on gold film were obtained with PBS containing Tween 20 as a medium.
The theoretical calculation was performed using
Conclusion
We have demonstrated experimentally the sensitivity and detection limit of the spectral SPR as an irnmunosensor. They were measured by incident angle of a light region of 66–76° using anti-BSA antibody as a modeled analyte. The experimental results demonstrated that a small incident angle has higher sensitivity than a large one. For instance, the sensitivity of a 66° incident angle was three times higher than that with a 76° incident angle. Furthermore, measurements with a 66° incident angle
Acknowledgements
This work was supported by research for the future program of JSPS.
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