Electrochemistry of Biomimetic Membranes - Applications of Electrochemistry and Nanotechnology in Biology and Medicine - page 164

into a dielectric layer at the metal surface, the reflectivity mini-
mum is shifted to a higher angle. Physical layer thicknesses can be
calculated by quantitative modeling of the reflectivity curves using
Fresnel's equations. This requires knowledge of the refractive in-
dex n and of the film thickness d of each dielectric slab of the mul-
tilayer system under investigation. The exact refractive index of
the compound forming a layer on the metal surface is unknown;
however, reasonable approximations for refractive indices are
n=1.45 for proteins and n=1.5 for lipids. Once the refractive indi-
ces are established, the layer thickness can be extracted from a
comparison between experimental and calculated reflectivity
curves. By monitoring the reflectivity at a particular fixed angle of
incidence close to resonance, the kinetics of adsorption at the inter-
face can be monitored (see Fig. 7c ). The reflectivity at the fixed
angle increases in time from its value prior to the adsorption of the
molecules, attaining a maximum limiting value when the adsorp-
tion process terminates.
At resonance, the interfacial evanescent field is enhanced by a
factor of 16 (for a gold/water interface at O = 633 nm) relative to
the incoming light. Its strength is maximal at the metal surface, and
decays exponentially normal to the surface with a penetration
depth of 150 nm. Surface plasmon fluorescence spectroscopy
(SPFS) exploits this large field enhancement to excite fluorophores
located within the evanescent field. 10 This feature distinguishes
SPFS from total internal reflection fluorescence (TIRF) microsco-
py, which basically utilizes the same optical excitation geometry
but operates without a metal surface and thus with much lower
field enhancement. Nonradiative energy transfer (quenching) from
the excited fluorophore to a planar gold surface decays with the
third power of the distance. In practice, the fluorescence intensity
of the fluorophore increases gradually with increasing distance of
the fluorophore from the gold layer, until it remains completely
unquenched at a distance of about 30 nm. 11 Simultaneous SPR and
SPFS analysis allows an estimate of both the thickness of a film
and its separation from the substrate surface. Thus, when a lipid
bilayer is formed on a gold support by vesicle fusion, this com-
bined analysis may distinguish unambiguously whether vesicles or
planar lipid films are adsorbed on the support surface.
Both SPR and EIS allow an evaluation of film thickness,
based on a reasonable estimate of the refractive index of the film