Coupled plasmon-waveguide resonance spectroscopic device and method for measuring film properties

A conventional SPR spectroscopic device, consisting of a metallic film used with a prism to provide a surface plasmon wave, is modified by coating the film with a dielectric layer. According to one aspect of the invention, such additional layer of dielectric material functions as an optical amplifier that produces an increased sensitivity and enhanced spectroscopic capabilities in SPR. According to another aspect of the invention, the added dielectric layer can be used as a matrix for adsorbing and immobilizing the sensing materials in sensor applications. Furthermore, the dielectric layer provides a shield for both mechanical and chemical protection of the metal layer, thereby preventing the rapid deterioration that commonly accompanies such detectors. In its simplest embodiment, the invention includes only one dielectric layer; in other embodiments, a variety of multi-layer configurations may be implemented for different purposes with diverse dielectric materials.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention pertains in general to the field of surface plasmon 
resonance (SPR) spectroscopy. In particular, the invention relates to a 
novel SPR approach involving the coupling of plasmon resonances in a thin 
metal film and the waveguide modes in a dielectric overcoating. 
2. Description of the Related Art 
Surface plasmon resonance is a technique used in the development of gas 
sensors, measurement of optical properties of metals, degradation 
monitoring of metals, microscopy, and chemical and biochemical sensing. 
Among optical techniques such as ellipsometry, multiple internal 
reflection spectroscopy, and differential reflectivity, SPR is one of the 
most sensitive techniques to surface and interface effects. This inherent 
property makes SPR well suited for nondestructive studies of surfaces, 
interfaces, and very thin layers. SPR also has uses other than surface 
investigations and it has recently been demonstrated as a new optical 
technique for use in immunoassays. 
The SPR phenomenon has been known for over 25 years and the theory is 
fairly well developed. Simply stated, a surface plasmon is an oscillation 
of free electrons that propagates along the surface of a conductor. The 
phenomenon of surface plasmon resonance occurs under total internal 
reflection conditions at the boundary between substances of different 
refractive indices, such as glass and water solutions. When an incident 
light beam is reflected internally within the first medium, its 
electromagnetic field produces an evanescent wave that crosses a short 
distance (in the order of nanometers) beyond the interface with the second 
medium. If a thin metal film is inserted at the interface between the two 
media, surface plasmon resonance occurs when the free electron clouds in 
the metal layer (the plasmons) absorb energy from the evanescent wave and 
cause a measurable drop in the intensity of the reflected light at a 
particular angle of incidence that depends on the refractive index of the 
second medium. 
Typically, the conductor used for SPR spectrometry is a thin film of metal 
such as silver or gold; however, surface plasmons have also been excited 
on semiconductors. The conventional method of exciting surface plasmons is 
to couple the transverse-magnetic (TM) polarized energy contained in an 
evanescent field to the plasmon mode on a metal film. The amount of 
coupling, and thus the intensity of the plasmon, is determined by the 
incident angle of the light beam and is directly affected by the 
refractive indices of the materials on both sides of the metal film. By 
including the sample material to be measured as a layer on one side of the 
metallic film, changes in the refractive index of the sample material can 
be monitored by measuring changes in the surface plasmon coupling 
efficiency in the evanescent field. When changes occur in the refractive 
index of the sample material, the propagation of the evanescent wave and 
the angle of incidence producing resonance are affected. Therefore, by 
monitoring the angle of incidence at a given wavelength and identifying 
changes in the angle that causes resonance, corresponding changes in the 
refractive index and related properties of the material can be readily 
detected. 
As those skilled in the field readily understand, total reflection can only 
occur above a particular critical incidence angle if the refractive index 
of the incident medium (a prism or grating) is greater than that of the 
emerging medium. In practice, total reflection is observed only for 
incidence angles within a range narrower than from the critical angle to 
90 degrees because of the physical limitations inherent with the testing 
apparatus. Similarly, for systems operating with variable wavelengths and 
a given incidence angle, total reflection is also observed only for a 
corresponding range of wavelengths. This range of incidence angles (or 
wavelengths) is referred to as the "observable range" for the purpose of 
this disclosure. Moreover, a metal film with a very small refractive index 
(as small as possible) and a very large extinction coefficient (as large 
as possible) is required to support plasmon resonance. Accordingly, gold 
and silver are appropriate materials for the thin metal films used in SPR; 
in addition, they are very desirable because of their mechanical and 
chemical resistance. 
Thus, once materials are selected for the prism, metal film and emerging 
medium that satisfy the described conditions for total reflection and 
plasmon resonance, the reflection of a monochromatic incident beam is a 
function of its angle of incidence and of the metal's refractive index, 
extinction coefficient, and thickness. The thickness of the film is 
therefore selected such that it produces observable plasmon resonance when 
the monochromatic light is incident at an angle within the observable 
range. 
The classical device by which SPR is carried out is known as the 
Kretschmann prism arrangement, illustrated by the sensor apparatus 10 of 
FIG. 1. A thin film 12 of metal is coated on one face 14 of a prism 16 
which has a high refractive index n (in the 1.4-1.7 range). Gold or silver 
films are most often used due to their refractive and extinction 
properties, as described above, and the relative ease with which these 
metals can be deposited onto a substrate with an accurate thickness. The 
surface chemistry of gold and its resistance to oxidation make it the 
prime choice for SPR experiments, although many other materials can 
support surface plasmon (SP) waves. As well understood by those skilled in 
the art, the main criterion for a material to support SP waves is that it 
have a negative real dielectric component, which results from the 
refractive and extinction properties outlined above. Although materials 
other than metals can support SPR, metals are most commonly used; 
accordingly, metals are used here to denote a support surface for SP 
waves. 
It is noted that another type of sensor used for conventional SPR is the 
Otto device, which consists of the same elements illustrated in FIG. 1 but 
with a very thin air gap between the face 14 of the prism 10 and the metal 
film 12. The principles for the design and operation of these two devices 
are the same. Therefore, this entire disclosure is intended to refer to 
both types of device even though the figures illustrate only Kretschmann 
prism arrangements. 
The surface 18 of the metal film 12 forms the transduction mechanism for 
the sensor 10 and is brought into contact with the sample material 20 to 
be sensed at the interface between the metal film and the emerging medium 
contained in a cell assembly 22. Monochromatic light L is emitted by a 
laser or equivalent light source 24 into the prism or grating 16 and 
reflected off the metal film 12 to an optical photodetector 26 to create 
the sensor output. The light L launched into the prism and coupled into 
the SP mode on the film 12 is p-polarized with respect to the metal 
surface where the reflection takes place. According to these prior-art 
devices and techniques, only p-polarized light is coupled into the plasmon 
mode because this particular polarization has the electric field vector 
oscillating normal to the plane that contains the metal film. This is 
sometimes referred to as transverse-magnetic (TM) polarization. 
As mentioned, the surface plasmon is affected by changes in the dielectric 
value of the material in contact with the metal film. As this value 
changes, the conditions necessary to couple light into the plasmon mode 
also change. For the particular sensing system described in FIG. 1 (and 
for the corresponding Otto configuration), the angle of incidence .alpha. 
for the light beam L with respect to the metal surface 18 and the 
reflected light intensity are the measured parameters of interest. If the 
angle of incidence for the light beam is scanned throughout a range of 
values, a distinct minimum in reflectivity is observed at a discrete angle 
associated with a given refractive index in the sample material 20. This 
angle is commonly known as .alpha..sub.sp, the surface plasmon coupling 
angle. At this particular angle of incidence, set of dielectric values, 
and optical wavelength, the light L is being coupled into the plasmon mode 
and the reflection is attenuated. There is a distinct coupling angle where 
most of the light is attenuated for each sample material. Thus, as 
illustrated schematically in FIG. 2, measurements are carried out by 
mounting the sensor device 10 on a table 28 capable of rotating with 
respect to the fixed light source 24 and by relating .alpha..sub.sp to 
changes in the dielectric values or refractive index of the sample 
material 20. 
SPR is a highly sensitive technique useful for investigating changes that 
occur at the surface 18 of the metal film. Therefore, the basic SPR sensor 
device 10 has been used in a wide variety of SPR research applications. In 
particular, over the last decade there has been a renewed interest in the 
application of surface plasmon resonance spectroscopy to study the optical 
properties of molecules immobilized at an interface between solid and 
liquid phases. This invention was made and is disclosed herein in this 
context. As described in detail in recently published articles, the 
ability of the SPR phenomenon to provide information on the physical 
properties of thin films deposited on a metal layer, including lipid and 
protein molecules forming proteolipid membranes, is based upon two 
principal characteristics of the effect. The first is the fact that the 
evanescent electromagnetic field generated by the free electron 
oscillations decays exponentially with penetration distance into an 
emergent dielectric medium; i.e., the depth of penetration into the 
dielectric material in contact with a metal layer extends only to a 
fraction of the light wavelength used to generate the plasmons. This makes 
the phenomenon sensitive to the optical properties of the metal/dielectric 
interface without any interference from the properties of the bulk volume 
of the dielectric material or any medium that is in contact with it. The 
second characteristic is the fact that the angular (or wavelength) 
position and shape of the resonance curve is very sensitive to the optical 
properties of both the metal film and the emergent dielectric medium 
adjacent to the metal surface. As a consequence of these characteristics, 
SPR is ideally suited for studying both structural and mass changes of 
thin dielectric films, including lipid membranes, lipid membrane-protein 
interactions, and interactions between integral membrane proteins and 
peripheral, water-soluble proteins. See Salamon, Z., H. A. Macleod and G. 
Tollin, "Surface Plasmon Resonance Spectroscopy as a Tool for 
Investigating the Biochemical and Biophysical Properties of Membrane 
Protein Systems. I: Theoretical Principles," Biochim. et Biophys. Acta, 
1331: 117-129 (1997); and Salamon, Z., H. A. Macleod and G. Tollin, 
"Surface Plasmon Resonance Spectroscopy as a Tool for Investigating the 
Biochemical and Biophysical Properties of Membrane Protein Systems. II: 
Applications to Biological Systems," Biochim. et Biophys. Acta, 1331: 
131-152 (1997). 
The present invention is directed at improving these prior-art sensor 
devices and procedures by providing new thin-film interface designs that 
couple surface plasmon and waveguide excitation modes. The resulting 
devices, referred herein as coupled plasmon-waveguide resonators (CPWR), 
exhibit several new properties that constitute material advances in the 
art. 
BRIEF SUMMARY OF THE INVENTION 
One primary goal of this invention is an SPR spectroscopic tool that 
provides greatly enhanced spectroscopic capabilities and sensitivities 
over conventional SPR sensors and procedures, thereby allowing a broader 
spectrum of applications than has heretofore been possible. 
In particular, a goal of the invention is a technique that affords 
increased spectral resolution and improved sensitivity in SPR 
spectroscopy. 
Another goal is an SPR spectroscopic technique that provides the ability to 
measure anisotropy in both the refractive index and the extinction 
coefficient of a medium of interest. 
Another important objective is a technique that is applicable to a wide 
range of materials, including lipid membranes which have either integral 
membrane proteins incorporated into them, or peripheral membrane proteins 
bound to their surface. 
Another goal of the invention is a tool that is particularly suitable for 
obtaining information about molecular assemblies that can be immobilized 
at a dielectric/water interface. 
Yet another objective is a tool that provides protection of the 
plasmon-generating metallic film against mechanical or chemical 
deterioration during use. 
Another goal is a technique that makes it possible to achieve the 
objectives of the invention with an efficient, practical and economically 
feasible implementation. 
Finally, another objective is a procedure and corresponding apparatus that 
are suitable for direct incorporation with existing SPR spectroscopic 
instruments. 
Therefore, according to these and other objectives, the present invention 
consists of a metallic (or semiconductor) layer (or layers), typically 
either gold or silver, used with either a prism or a grating so as to 
provide a surface plasmon wave, and covered with a solid dielectric layer 
characterized by predetermined optical parameters. According to one aspect 
of the invention, such additional layer of dielectric material functions 
as an optical amplifier that produces an increased sensitivity and 
enhanced spectroscopic capabilities in SPR. According to another aspect of 
the invention, the added dielectric layer can be used as a matrix for 
adsorbing and immobilizing the sensing materials in sensor applications. 
Furthermore, the dielectric layer provides a shield for both mechanical 
and chemical protection of the metal layer, thereby preventing the rapid 
deterioration that commonly accompanies such detectors. In its simplest 
embodiment, the invention includes only one dielectric layer; in other 
embodiments, a variety of multi-layer configurations may be implemented 
for different purposes with diverse dielectric materials. 
Various other purposes and advantages of the invention will become clear 
from its description in the specification that follows and from the novel 
features particularly pointed out in the appended claims. Therefore, to 
the accomplishment of the objectives described above, this invention 
consists of the features hereinafter illustrated in the drawings, fully 
described in the detailed description of the preferred embodiment and 
particularly pointed out in the claims. However, such drawings and 
description disclose but one of the various ways in which the invention 
may be practiced.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION 
The heart of this invention lies in the recognition that the addition of a 
dielectric layer to the metallic layer of a conventional SPR sensor 
provides advantages that constitute a significant advance in the art. It 
is understood that the dielectric layer of the invention is in addition to 
and separate from the sample material or analyte with which the invention 
is used. The sample material at the interface with the emerging medium is 
often itself dielectric in nature, but its properties cannot be used to 
obtain the advantages of the invention without the addition of an 
additional dielectric layer as disclosed herein. Therefore, all references 
to dielectric material pertain only to the additional layer contemplated 
by the invention. 
Referring to the drawings, wherein like reference numerals and symbols are 
used for like parts, FIG. 3 illustrates in schematic form a device 30 
according to one embodiment of the invention. The device 30 contains a 
metallic (or semiconductor) layer (or layers) 12, typically between 45 and 
55 nm thick, formed from either gold or silver deposited on either a glass 
prism or grating 16 for generating a surface plasmon wave. (As mentioned 
above, the same elements could be used in an Otto configuration with an 
air gap between the glass and metal layer.) The silver film is covered 
with a layer 32 of solid dielectric material characterized by an 
appropriate set of values of film thickness, t, refractive index, n, and 
extinction coefficient, k. 
Suitable dielectric materials must have a refraction index nd greater than 
the refractive index n.sub.e of the emerging medium; they must have an 
extinction coefficient k.sub.d as small as possible for a given wavelength 
(for example, .ltoreq.0.1, preferably between 0 and 0.01, for .lambda.=633 
nm); and they must be selected with a thickness that will support a guided 
wave and result in the resonance effects occurring at an angle of 
incidence within the observable range, as defined above. For example, a 
glass prism coated with a 50 nm-thick silver layer protected by a 460 
nm-thick SiO.sub.2 film (n.sub.d =1.4571, k.sub.d =0.0030) is suitable to 
practice the invention with an aqueous analyte (n.sub.e =1.33). A lipid 
bilayer 34 (the material being tested) is deposited from the sample 
solution 20 on the dielectric film 32 and held in place by a TEFLON.RTM. 
spacer 36 according to the teachings of U.S. Pat. No. 5,521,702 (Salamon 
et al.). 
In the SiO.sub.2 embodiment of FIG. 3, with a wavelength of about 633 nm, 
the dielectric material must be at least 50 nm thick to act as a 
waveguide. In addition, the resulting s-resonance will fall within the 
observable range for any thickness larger than 250 nm; on the other hand, 
the p-resonance will be visible for any thickness greater than 400 nm. In 
order to fulfill the conditions of the invention for both types of 
polarization, the dielectric layer must be at least 420 nm thick. 
Similarly, the same configuration embodied with a TiO.sub.2 dielectric and 
a wavelength of about 633 nm would require a thickness larger than 65 nm 
for the s-resonance and larger than 140 nm for the p-resonance to be 
observable. The conditions of the invention would be met for both types of 
polarization with a TiO.sub.2 layer at least 750 nm thick. 
In another embodiment 40 of the invention, illustrated in FIG. 4, the 
silver-coated glass prism 16 includes two solid dielectric layers. One 50 
nm layer 32 of TiO.sub.2 (n.sub.d =2.2789, k.sub.d =0.000151) protects the 
silver film 12; a second 750 nm layer 38 of a lower density, lower 
refractive index (n=1.35) dielectric material (Na.sub.3 AlF.sub.6) is 
applied over the first layer. In this example this material is selected 
with a lower density and a correspondingly higher porosity so as to 
provide a structural matrix for adsorbing and immobilizing the sensing 
materials 20 (hydrogels are well known materials used for this purpose). 
According to one aspect of the invention, spectroscopic measurements with 
the devices 30 and 40 are based upon the resonant excitation of 
electromagnetic modes of the structure by both TM (p) and TE (s) polarized 
components of a continuous-wave laser light (e.g., He-Ne; .lambda.=632.8 
nm) passing through the glass prism 16 under total internal reflection 
conditions. We found that the addition of the dielectric layer 32, with 
the appropriate set of optical parameters defined above, to the 
conventional SPR arrangement not only provides both mechanical and 
chemical protection for the metal layer 12, but also produces optical 
amplification that results in increased sensitivity and enhanced 
spectroscopic capabilities. 
Using the structures of FIGS. 3 and 4, it was possible to determine that 
the relative bandwidths of the resonances obtained with either p- or 
s-polarized incident light, and therefore the sensitivity of the 
measurement, can be varied by altering the properties of the overcoat film 
32 (or films 32,38). Thus, this discovery makes it possible to both expand 
SPR spectroscopy to the use of s-polarized light and improve the quality 
of the measurements by altering the spectral response of the system. In 
addition, in sensor applications the added dielectric overcoat could also 
be used as a matrix that adsorbs and immobilizes the sensing material 20. 
For example, the DEXTRAN.RTM. layer that is currently used in commercial 
SPR biosensors for fast and efficient immobilization of ligands could be 
manipulated into the dielectric matrix 38 to generate resonances with 
widely varying sensitivities. For a detailed explanation regarding the 
immobilization of membrane proteins on metal or dielectric surfaces, see 
Salamon et al., II: Applications to Biological Systems, supra. 
One way to explain the appearance of an s-polarized resonance component in 
a conventional SPR experiment as a consequence of adding a dielectric 
layer 32 onto the metal surface 18 (FIG. 4) is through the application of 
the electromagnetic field theory to thin-film systems (Macleod, H. A., 
Thin Film Optical Filters, Adam Hilger, Bristol, 1986). According to the 
theory, thin-film materials are characterized by a complex dielectric 
constant that includes the refractive index n and the extinction 
coefficient k (i.e., n-ik). In the optical region of the electromagnetic 
spectrum, this parameter is equal to the ratio of light velocity in vacuo 
(c) to that in a medium (v), and is numerically equivalent to the optical 
admittance, which is defined by the ratio of the amplitudes of the 
electric (B) and magnetic (C) fields of the electromagnetic wave, as 
follows: 
EQU Y=C/B=c/v=n-ik (1) 
where Y is the optical admittance divided by the admittance of free space. 
Using Maxwell's equations, one can describe the propagation of the plane, 
monochromatic, linearly polarized, and homogeneous electromagnetic field 
within a multilayer thin-film system with the following matrix equation: 
##EQU1## 
where s is the number of layers (32,38) deposited on the incident medium 
(the glass prism 16); .beta..sub.j =2.pi.(n.sub.j -ik.sub.j)t.sub.j cos 
.alpha..sub.j /.lambda. gives the phase thickness of layer j at the 
appropriate angle of incidence (.alpha..sub.j) and light wavelength 
(.lambda.); and y.sub.j =(n-ik).sub.j /cos .alpha..sub.j for p-polarized 
light and y.sub.j =(n-ik).sub.j cos .alpha..sub.j for s-polarized light. 
Equation 2 and the relations governing its parameters allow examination of 
the distribution of electromagnetic field amplitudes throughout the 
thin-film system, as well as calculation of the transmittance, absorbance, 
and reflectance. They also allow analysis of the resonance phenomena 
occurring within such thin-multilayer films. 
The reflectance of a multilayer system is given by the following 
relationship involving the optical admittance: 
EQU R=(y.sub.o -Y).sup.2 /(y.sub.o +Y).sup.2 (3) 
where y.sub.o is the admittance of the incident medium (the glass prism 
16). Based on Equation 1, the incident medium should be free from 
absorption, so that y.sub.o is real and equals n.sub.o at normal 
incidence. Equation 3 describes a reflectance spectrum, i.e., reflectance 
as a function of either the incident angle .alpha. using monochromatic 
light of predetermined wavelength .lambda., as used in the present 
description; or of varying .lambda. at a constant value of .alpha.; or of 
varying the thickness of one layer at constant thickness values for the 
other layers and constant .alpha. and .lambda. (an example of the latter 
is presented in FIGS. 5 and 6). Analysis of the optical admittance shows 
that beyond a critical angle for the system the emergent wave in the final 
medium is evanescent and the admittance is imaginary; positive imaginary 
for p-polarized light and negative imaginary for s-polarization. Thus, for 
a surface wave to be confined to the metal surface, the admittance 
exhibited by the adjoining medium must be positive imaginary and of 
magnitude very close to that of the extinction coefficient k of the metal 
(i.e., only materials with a small value of the refractive index n and a 
large value of k, such as silver and gold, will generate a surface wave). 
For a metallic film, this condition is fulfilled only for p-polarization 
and a very narrow range of angles of incidence. Coupling of the incident 
light to the surface wave results in the sharp dip in total internal 
reflectance that is characteristic of the resonance effect. For 
s-polarization the admittance is always negative imaginary and, therefore, 
there is normally no corresponding resonance. However, in the coupled 
plasmon-waveguide resonance device of the invention the dielectric 
overcoat layer 32 (or system of layers 32,38) is used to transform the 
admittance of the emergent medium so that the admittance presented to the 
metal is positive imaginary for both s- and p-polarization. Depending on 
the characteristics of the admittance-matching dielectric overcoat (i.e., 
n.sub.d, k.sub.d, and t.sub.d values), the system can produce a narrowing 
or a broadening of the range of angles over which the necessary 
coincidences are achieved, and hence a similar broadening or narrowing of 
the resonances. Examination of the distribution of electric field 
amplitudes through the system shows that the admittance-matching layers 
are important components of the resonant system, rather like cavity layers 
in narrowband filters or thin-film waveguides in optical couplers. The 
term coupled plasmon-waveguide resonance is used here in order to 
distinguish this resonance phenomenon from conventional surface plasmon 
resonance. 
Since the added dielectric layer or layers of the invention make it 
possible to produce resonance with either s- or p-polarized light, it is 
desirable to select the dielectric thickness t.sub.d such that both 
resonance effects fall within the observable range for the system. Thus 
the same device can be utilized to obtain two sets of measurements from 
the same sample. 
A large variety of dielectric overcoat film combinations (32, 38) exists 
that can be used in particular applications. In essence, any one layer of 
dielectric or combination of dielectric layers that satisfy the refractive 
index, extinction coefficient, and thickness requirements for producing 
resonance at incident angles (for a given wavelength) or at wavelengths 
(for a given incident angle) within the observable range is suitable for 
practicing the invention. For example, these materials include MgF.sub.2, 
Al.sub.2 O.sub.3, LaF.sub.3, Na.sub.3 AlF.sub.6, ZnS, ZiO.sub.2, Y.sub.2 
O.sub.3, HfO.sub.3, Ta.sub.2 O.sub.5, ITO, and nitrites or oxy-nitrites of 
silicon and aluminum, which are all normally used in optical applications. 
Measurements using the CPWR devices of the present invention are made in 
the same way as with conventional SPR techniques. As well understood in 
the art, the attenuated total reflection method of coupling the light into 
the deposited thin multilayers is used, thereby exciting resonances that 
result in absorption of the incident radiation as a function of either the 
light incident angle .alpha. (with a monochromatic light source), or light 
wavelength .lambda. (at constant incident angle), with a consequent dip in 
the reflected light intensity. 
Thus, under appropriate experimental conditions, which are determined by 
the system's parameters, the devices 30 and 40 of the present invention 
can be excited by either p- or s-polarized light to resonantly absorb the 
incident light energy. FIG. 5 illustrates such resonances measured as 
reflected light intensity as a function of the thickness of the SiO.sub.2 
dielectric layer 32, obtained with p-polarized light (.lambda.=632.8 nm) 
in the arrangement shown in FIG. 3 and with an incident angle .alpha. (62 
degrees) arbitrarily chosen in the typical observable range for a 
glass-prism/aqueous-emerging-medium system (about 61 to 90 degrees). 
Similarly, FIG. 6 illustrates resonances measured as a function of the 
thickness of the SiO.sub.2 layer with s-polarized light. The two 
resonances are separated and occur at different dielectric thicknesses, 
but these figures demonstrate that it is possible to adjust the thickness 
of the overcoat layer 32 to obtain both s- and p-resonances with the same 
device. The apparatus shown in FIG. 3, with a SiO.sub.2 layer 32 460 nm 
thick applied over a 50 nm silver layer 12, and that of FIG. 4, with a 
combination of a 50 nm TiO.sub.2 layer 32 and a 750 nm layer 38 of 
Na.sub.3 AlF.sub.6, represent two examples of devices that exhibit the 
resonances shown in FIGS. 5 and 6. 
FIG. 7 illustrates surface resonances measured as reflected light intensity 
as a function of the incident angle .alpha. with the apparatus 30 of FIG. 
3, wherein reference symbols p and s identify curves generated with p- and 
s-polarized light, respectively. The dashed curve p.sub.c shows the much 
broader SPR spectrum obtained with the same silver layer of the device in 
FIG. 3 but with a conventional setup without the dielectric overcoat 32. 
FIG. 8 illustrates similar results obtained with the apparatus 40 of FIG. 
4. These spectra show that the dielectric layer or layers add two very 
important features to conventional SPR resonance devices and procedures. 
The first is the additional spectroscopic dimension provided by generating 
a second type of resonance with different polarization (the s-polarized 
component). The second is the increased sensitivity resulting from the 
greatly decreased half-width of both s- and p-polarized resonances (as 
clearly seen in FIG. 7). Furthermore, the resonance half-width, and 
therefore the spectral sensitivity of the apparatus, can be adjusted by 
judiciously selecting appropriate overcoating layers and polarization mode 
of operation to meet specific experimental needs, as illustrated by the 
two sets of results shown in FIGS. 7 and 8. For example, these spectra 
show that the two dielectric layer designs of FIGS. 3 and 4 produced 
opposite spectral sensitivity. The device 30 yielded an s-spectrum 
narrower than the p-spectrum, whereas the opposite was true for the design 
of the device 40. 
The overall sensitivity of the devices of the invention includes the 
sensitivity of the shift of the minimum resonance angle, which is 
determined in principle by the refractive index and thickness of the 
sensing layer 34 (for example a lipid bilayer deposited on the surface of 
the dielectric overcoat 32, as shown in FIG. 3). It also includes the 
sensitivity to the change in the shape of the resonance spectrum, which 
depends mainly on the light absorption (and/or scattering) properties of 
the sensing layer 32. Both of these parameters, i.e., the minimum 
resonance angle and the shape of the spectrum as defined by its depth and 
width, are dependent upon the form of the quasi-modes of the 
electromagnetic field generated in the combination of layers designed 
according to the invention. FIGS. 9 and 10 show the electric field 
distributions for p- and s-polarizations, respectively, obtained with the 
interface of the device 30 of FIG. 3. The figures show that the electric 
field at the outer interface between the dielectric 32 and the sensing 
layer 34 is higher by a factor of about 50 for the s-component, and about 
25 for the p-component, in comparison with that at the entrance interface 
between the glass 16 and the metal layer 12. As a result of these 
properties and the corresponding higher sensitivity of the devices of the 
present invention, the three parameters that determine the resonance 
spectrum (thickness t.sub.e, refractive index n.sub.e, and extinction 
coefficient k.sub.e of the sensing layer 34) can be obtained with 
accuracies better than 1 .ANG., 0.001, and 0.002, respectively, for a 
sensing layer whose thickness is only 5 nm, a value comparable with the 
thickness of a lipid membrane (see Salamon, Z., Y. Wang, J. L. Soulages, 
M. F. Brown, and G. Tollin, "Surface Plasmon Resonance Spectroscopy 
Studies of Membrane Proteins: Transducin Binding and Activation by 
Rhodopsin Monitored in Thin Membrane Films," Biophys. J., 71: 283-294, 
1996; Salamon, Z. and G. Tollin, "Surface Plasmon Resonance Studies of 
Complex Formation Between Cytochrome c and Bovine Cytochrome c Oxidase 
Incorporated into a Supported Planar Lipid Bilayer. I: Binding of 
Cytochrome c to Cardiolipin/Phosphatidylcholine Membranes in the Absence 
of Oxidase," Biophys. J., 11:848-857, 1996; and Salamon, Z. and G. Tollin, 
"Surface Plasmon Resonance Studies of Complex Formation Between Cytochrome 
c and Bovine Cytochrome c Oxidase Incorporated into a Supported Lipid 
Bilayer. II: Binding of Cytochrome c to Oxidase-Containing 
Cardiolipin/phosphatidylcholine Membranes," Biophys. J. 71: 858-867, 
1996). In practical terms, this means that in many cases the limitation of 
accuracy in the procedure will result not from the measuring technique 
itself but from the ability to generate a thin sensing film in a 
reproducible manner. 
Because of its characteristics, the present invention provides significant 
advantages over alternative techniques for the detection and measurement 
of small optical changes based on optical waveguides. The coupling 
arrangements are simple and convenient. Moreover, the geometric 
arrangement in CPWR spectroscopy is characterized by a complete isolation 
of the optical probe from the system under investigation, as is also the 
case in conventional SPR spectroscopy. 
The three optical parameters (n.sub.d, k.sub.d, t.sub.d) characterizing a 
deposited dielectric film 32 (or combination of films 32,38) can be 
evaluated for both polarizations, at different angles of light incidence, 
and using different light wavelengths. With these experimental data on 
hand, it is possible to characterize all of the structural parameters of 
thin films 34 under investigation, i.e., thickness, mass distribution 
within the film, orientation of molecules (by measuring the anisotropy in 
n.sub.e), and the orientation of chromophores attached to the molecules 
within the sensing layer (by measuring the anisotropy of k.sub.e). All of 
these characterizations can be obtained using a single device covered with 
a sensing layer 34, and using a measurement method that involves only a 
determination of reflected light intensity under total internal reflection 
conditions. Details of experimental techniques employed to measure the 
resonance spectrum are given in Salamon and Tollin (1996), supra, and 
Salamon et al. (1996), supra. Furthermore, because the electromagnetic 
field decays exponentially within the emerging medium (see FIGS. 9 and 
10), the measurement is sensitive only to the interface region between the 
dielectric overcoat and the emerging medium, and is not affected by the 
bulk properties of the medium. 
There is no limitation on the dielectric materials that can be used in the 
coatings 32,38 of the invention, as long as the optical characteristics 
are favorable, as explained above. Therefore, the dielectric film can be 
formed from any number of layers 32,38 designed and optimized for 
different uses. This feature is especially important in various sensor 
applications, where the dielectric overcoat can also be designed to adsorb 
and immobilize the sensing material either on its surface or within its 
interior. It is noted that the effects of the dielectric overcoat of the 
invention are not diminished by the addition of a very thin (1-5 nm) layer 
of gold or other metal at the interface with the emerging medium for the 
purpose of fixating the analyte to the sensing device, as already done 
with conventional SPR devices. Such a combination of properties in one 
interface permits the construction of a durable sensor device with very 
high sensitivity and an expanded dynamic range of measurements. 
Although the features of the resonance spectrum produced by the present 
invention can be employed in a variety of different ways, one of the most 
fruitful applications lies in biophysical and biochemical studies of the 
structural properties of proteolipid assemblies. Studies of the 
microscopic structure of lipid membranes and interacting lipid-protein 
films represents a technically difficult challenge because they consist of 
very thin layers comprising only one or two monolayers. In addition, they 
contain relatively small amounts of material located at the interface 
between two immiscible phases, and may be labile and structurally 
heterogeneous. As a result, only a limited number of studies have been 
made of lipid and/or protein orientation in molecular films. The following 
example demonstrated the suitability and improved capabilities of the 
invention in obtaining information about structural anisotropy in a 
self-assembled solid-supported lipid bilayer. 
EXAMPLE 
Self-assembled solid supported lipid membranes were used as described by 
Salamon et al. (1996), supra, to illustrate the features of the invention. 
The method of preparation of lipid membranes was based on the principles 
that govern the spontaneous formation of a freely suspended lipid bilayer 
membrane, as described in Mueller, P., D. O. Rudin, H. T. Tien and W. C. 
Wescott, "Reconstitution of Cell Membrane Structure in vitro and its 
Transformation into an Excitable System," Nature, 194: 979-980, 1962. As 
illustrated in the device 30 of FIG. 3, the method involves spreading a 
small amount of lipid bilayer-forming solution 20 (about 24 .mu.L) across 
an orifice (about 4 mm in diameter) in a TEFLON.RTM. sheet (the spacer 36) 
that separates the dielectric thin film 32 of the invention from the 
aqueous phase 20 (for further detail, see Salamon et al., 1997, supra). 
The hydrophilic surface of the dielectric layer 32 (hydrated SiO.sub.2 in 
the present case) attracts the polar groups of the lipid molecules, thus 
forming an adsorbed lipid monolayer with the hydrocarbon chains oriented 
toward the bulk lipid phase. Subsequent to the first step of lipid 
membrane formation, the main body of the sample cell is filled with the 
appropriate aqueous solution. This initiates the second step, which 
involves a thinning process, i.e., the formation of both the second 
monolayer and a plateau-Gibbs border that anchors the bilayer film 34 to 
the TEFLON.RTM. spacer 36, allowing the excess of lipid and solvent to 
move out of the TEFLON.RTM. orifice. Previous work involving both SPR and 
electrochemical measurements with different protein molecules demonstrated 
that this technique generates a membrane that provides a biocompatible 
medium for binding and immobilizing both peripheral and integral membrane 
proteins. See Salamon, Z., J. T. Hazzard and G. Tollin, "Direct 
Measurement of Cyclic Current-Voltage Responses of Integral Membrane 
Proteins at a Self-Assembled Lipid Bilayer-Modified Electrode: Cytochrome 
f and Cytochrome c Oxidase," Proc. Natl. Acad. Sci. USA, 90: 6420-6423 
(1993); Salamon, Z., Y. Wang et al. (1996), supra; and Salamon and Tollin 
(1996), supra. In the experiment of this example, the lipid films were 
formed on the SiO.sub.2 surface from solutions containing 7 mg/mL of egg 
phosphatidylcholine (PC) in squalene (Fluka)/butanol (0.15:10, v/v), using 
thin film coatings prepared at the Tucson Optical Research Co. in Tucson, 
Ariz., by vacuum deposition. The egg PC was obtained in solid form from 
Avanti Polar Lipids (Alabaster, Ala.). 
In general, the experimental procedures for resonance spectra measurement 
and data analysis for the CPWR devices of the invention are the same as 
with conventional SPR studies and are well described in the literature 
(see Salamon and Tollin, 1997, supra; and Salamon et al., 1997, supra). 
The measurements for this example were performed in two different ways. 
First, the resonance spectra generated by both s- and p-polarization were 
measured with the device 30 of FIG. 3 with a single lipid membrane. Then, 
the two different devices in the configuration of FIG. 3 (with varying 
metal and dielectric coating layer parameters, as shown in the figures) 
with two separately formed lipid bilayers 34 were used in measuring the 
resonance spectrum generated by each polarization. This was done in order 
to evaluate the experimental errors produced by the formation of new lipid 
membranes and by using different thin film coatings. FIG. 11 shows a 
typical example of the resonance spectra obtained with the second 
procedure and p-polarized light, using a bare SiO.sub.2 film 32 (curve A) 
and modified by depositing an egg PC bilayer membrane (curve B). FIG. 12 
shows the results of the same measurements generated by s-polarization. 
Calculations of the electric fields throughout the thin-film system 
consisting of the CWPR device and the deposited lipid bilayer show that 
for p-polarized light the amplitude of the component of the electric field 
normal to the film boundaries is an order of magnitude larger than that 
parallel to the surface (as illustrated in FIGS. 9 and 10). In s-polarized 
excitation the field is completely parallel to the boundaries. Therefore, 
a good first approximation is to assume that in the p-polarization case 
the electric vector is completely normal to the surface. As a consequence, 
the optical parameters obtained with these two modes of excitation refer 
to the parallel and perpendicular directions within the lipid film. 
Table 1 below shows average values of thickness, refractive index, and 
extinction coefficient of a self-assembled solid-supported egg 
phosphatidylcholine bilayer obtained by measuring CPWR resonance spectra 
excited either by p- or s-polarized light in this example. The table also 
shows the experimental errors resulting from the different measurement 
procedures described above. These results clearly indicate that, although 
the thickness values obtained with both polarizations are the same within 
the experimental error, and compare well with those obtained previously 
with conventional SPR techniques (see Salamon et al., 1996, supra), there 
are significant differences in both the n and k parameter values. 
TABLE 1 
______________________________________ 
parameter p s 
______________________________________ 
t (nm) 5.2 .+-. 0.1 
5.2 .+-. 0.1 
n 1.52 .+-. 0.01 
1.47 .+-. 0.01 
k 0.10 .+-. 0.01 
0.020 .+-. 0.002 
______________________________________ 
In this type of measurement, the anisotropy in n may derive from the 
two-dimensional nature of the film 34 and from the ordering of anisotropic 
molecules within the two-dimensional structure. Simple calculations show 
that the difference in the index of refraction between the n- and 
p-polarization results is too large to be explained by a form 
birefringence. Thus, the source of the anisotropy in n must be the 
anisotropic character of the lipid molecules comprising the bilayer 
structure. Because in this experiment the lipid did not absorb the 
exciting light (the wavelength of the laser excitation, 632.8 nm, is far 
from the absorption band of PC), a k value different than zero reflects a 
diminution of measured light intensity due only to scattering processes 
which result from imperfections in the lipid film. It is expected that in 
anisotropic films the two polarized components of light will be scattered 
differently, thereby producing a scattering anisotropy. 
The value obtained for the refractive index with p-polarization agrees 
rather well with those obtained by previous measurements using SPR with a 
bare silver layer (Salamon et al., 1996, supra). Although with a complex 
mixture such as egg PC it was not possible to calculate theoretical 
polarizabilities and refractive indices for comparison, the average 
experimental values of refractive index shown in Table 1 are in very good 
agreement with theoretical values of these indices calculated for five 
different lipid molecules containing saturated fatty acid side chains (the 
average values are: n=1.559 with p-polarization; and n=1.483 with 
s-polarization), assuming the additivity principle in tetrahedral 
aliphatic hydrocarbons (see Den Engelsen, "Optical Anisotropy in Ordered 
Systems of Lipids," Surf. Sci., 56: 272-280, 1976) and assuming 
ellipsometrically measured values of various phospatidylcholines (see 
Ducharme, D., J. Max, C. Saleese and R. M. Leblanc, "Ellipsometric Study 
of the Physical States of Phosphatidylcholines at the Air-Water 
Interface," J. Phys. Chem., 94: 1925-1932, 1990). This similarity clearly 
indicates a high degree of ordering of the egg PC molecules in the 
solid-supported self-assembled lipid bilayer system used in the example. 
The refractive index anisotropy obtained in these measurements seems to be 
larger than that calculated from ellipsometric determinations on freely 
suspended black lipid membranes of lecithin, where values of n=1.47 and 
n=1.45 (for p- and s-polarizaton, respectively) have been reported in the 
literature (Den Engelsen, 1976, supra). This indicates a higher degree of 
ordering of the PC molecules in the solid-supported membrane, which is not 
unexpected. 
These results clearly demonstrate that CPWR spectroscopy provides a useful 
new technique for obtaining information about molecular assemblies which 
can be immobilized at a dielectric/water interface. Three major 
improvements over conventional SPR methodologies have been documented: 
increased spectral resolution, improved sensitivity, and the ability to 
measure anisotropy in both n and k. Furthermore, the method is applicable 
to a wide range of materials, including, without limitation, lipid 
membranes that have either integral membrane proteins incorporated into 
them or peripheral membrane proteins bound to their surface. 
Various changes in the details, steps and components that have been 
described may be made by those skilled in the art within the principles 
and scope of the invention herein illustrated and defined in the appended 
claims. For example, other dielectric materials with n and k parameters 
suitable for the invention could be used. For a given material and other 
system parameters, a range of thicknesses could be used with equivalent 
results. For example, the system of FIG. 3 can be implemented with any 
SiO.sub.2 layer greater than 420 nm; the same system can be implemented 
with any TiO.sub.2 layer greater than 750 nm. Similarly, the observable 
range can be increased or decreased by changing the properties of the 
prism and/or the emerging medium. For example, changing the prism to a 
material with n=2.2 would essentially double the observable range from 
about 61-90 degrees to 35-90 degrees in a system with an aqueous emerging 
medium. 
Therefore, while the present invention has been shown and described herein 
in what is believed to be the most practical and preferred embodiments, it 
is recognized that departures can be made therefrom within the scope of 
the invention, which is not to be limited to the details disclosed herein 
but is to be accorded the full scope of the claims so as to embrace any 
and all equivalent processes and devices.