Broadband plasmon waveguide resonance spectroscopy for probing biological thin films

Abstract

A commercially available spectrometer has been modified to perform plasmon waveguide resonance (PWR) spectroscopy over a broad spectral bandwidth. When compared to surface plasmon resonance (SPR), PWR has the advantage of allowing measurements in both s- and p-polarizations on a waveguide surface that is silica or glass rather than a noble metal. Here the waveguide is a BK7 glass slide coated with silver and silica layers. The resonance wavelength is sensitive to the optical thickness of the medium adjacent to the silica layer. The sensitivity of this technique is characterized and compared with broadband SPR both experimentally and theoretically. The sensitivity of spectral PWR is comparable to that of spectral SPR for samples with refractive indices close to that of water. The hydrophilic surface of the waveguide allows supported lipid bilayers to be formed spontaneously by vesicle fusion; in contrast, the surface of an SPR chip requires chemical modification to create a supported lipid membrane. Broadband PWR spectroscopy should be a useful technique to study biointerfaces, including ligand binding to transmembrane receptors and adsorption of peripheral proteins on ligand-bearing membranes.

Fig. 1

Schematic diagram of (top) the spectral PWR instrument and (bottom) the waveguide structure (not to scale). The optical fibers used to direct light to the prism and collect reflected light are not shown. The polychromator used to spectrally disperse light onto the CCD is also not shown.

Fig. 2

(a) Experimental PWR spectra for 2% sucrose (n = 1.3359, curve 1) and 4% sucrose (n = 1.3388, curve 2) in p-polarization. (b) Sensitivity curve in p-polarization (y = 3860.4x − 4499.4, R² = 0.9953). In (c) and (d) are shown the corresponding plots in s-polarization (y = 998.39x − 694.8, R² = 0.9454). The s-polarized spectra exhibit smaller changes in reflectivity across the spectral region of interest, which makes the noise (i.e., the fluctuations in the spectral regions of higher reflectivity) appear greater than that in the p-polarized spectra; however, the noise levels in s and p are actually equivalent.

Fig. 3

Calculated sensitivity curves for spectral PWR in (a) p-polarization and (b) s-polarization.

Fig. 4

AFM image of the surface of a SiO₂ waveguide. The image area is 5 μm by 5 μm and the scale bar is 20 nm.

Fig. 5

(a) Experimental p-polarized PWR spectra before (curve 1) and after (curve 2) fusion of a planar supported lipid membrane on the waveguide. (b) Theoretical spectra corresponding to formation of a 5 nm thick lipid bilayer (n = 1.45) on the waveguide.

Fig. 6

Typical FRAP curves for planar supported lipid bilayers of EggPC containing 0.5 mol % NBD-PC on a waveguide (◆) and glass (▲). The solid lines indicate least squares fits of the data to Eq. 1.