Optics: measuring and testing – Refraction testing
Reexamination Certificate
1999-11-12
2002-10-08
Font, Frank G. (Department: 2877)
Optics: measuring and testing
Refraction testing
C356S136000
Reexamination Certificate
active
06462809
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of refractive index based sensing devices. More particularly, the invention relates to a critical angle refractometer and method for sensing and monitoring interactions between an analyte and a binding layer.
BACKGROUND OF THE INVENTION
Analysis of qualitative and quantitative aspects of interactions between analytes and various types of binding layers is important to a wide range of scientific and industrial applications. Consequently, sensors which monitor specific binding of a sample analyte to a particular type of ligands immobilized on the sensing surface have been developed. The term “ligands” here means a type of molecules exhibiting specific binding affinity to another type of molecules. The terms “immobilized binding layer”, “binding layer”, or “sensing layer” here mean a layer formed by ligands immobilized on a sensing surface. The term “sensing surface” means an interface between two media, one of which is the binding layer. The term “contacting phase” here means a fluid phase, which is brought in contact with the binding layer. The term “analyte”, or “sample analyte”, here means the ligands contained in the contacting phase. An analyte in a contacting phase may or may not possess binding affinity to a particular binding layer.
For example, sensors based on the surface plasmon resonance (SPR) phenomenon are known to detect and measure changes in the refractive index of a sample analyte contacting a sensing layer. SPR sensors are often used in such applications as investigation of surface and interface effects, spectroscopy, differential reflectivity, immunoasays. SPR sensors are based on the following principle: when a thin metal layer is illuminated by an incident beam of light, under certain circumstances the energy of the light beam can excite free electrons on the illuminated surface of the metallic film. In particular, the beam will resonate with the surface electrons, which resonance will lead to the creation of an electrical field extending within the range of about 200 nanometers. The resonance occurs at a certain angle of incidence of the incoming light beam and depends on the refractive index of a substance located within the range affected by the generated electrical field. Binding or dissociation of the analyte and an immobilized binding layer at the sensor surface changes the local refractive index at the surface and produces a shift in the resonant angle of incidence, which has been shown to be proportional to the concentration of ligands bonded to an immobilized binding layer up to a predetermined limiting concentration. Thus, by electro-optically monitoring changes of the refractive index at the sensing surface using SPR, qualitative sensing of ligands and quantitative characterization of various binding kinetics and equilibria are possible.
An example of an SPR biosensor is schematically illustrated in FIG.
1
. SPR biosensor
2
includes a prism
4
having a test surface thereof coated by a thin metallic film
6
. A first type of ligands
8
is immobilized on metallic film
6
, and an analyte
10
is introduced into the contacting phase above the test surface. A light source
12
of predetermined wavelength directs an incident beam
14
to metallic film
6
, and a photosensitive detector
16
is arranged to monitor the intensity of reflected beam
14
′. At a certain angle of incidence &agr; of beam
14
, resonant excitation of electrons (surface plasmons) in metallic film
6
results in absorption of incident beam
14
and, consequently, in an energy loss in the reflected beam
14
′, which is observed experimentally as a sharp minimum in the intensity of light received by detector
16
, as illustrated in FIG.
2
.
While SPR sensors exhibit a high degree of sensitivity to changes in refractive indices, which makes them a useful research tool, immobilizing a binding layer on a metallic layer is both difficult and limiting. It is difficult, because the immobilization technique must attach the ligands in a native conformation and in a uniformly reactive and accessible orientation, to a metallic surface that does not allow for a significant amount of non-specific binding. A number of various immobilization techniques have been described in the art, with the choice of a technique being dependent upon particular ligands involved. Because of these and other difficulties associated with manufacturing SPR sensors, such sensors are expensive. Therefore, it would be desirable to come up with a less expensive device capable of measuring changes of the refractive indices caused by interactions between various ligands.
An example of a suitable device for sensitive and quantitative measurements associated with changes in refractive indices is a critical angle refractometer. The operation of a critical angle refractometer is based on the following principle. When light is incident on a surface separating two media, the light is refracted at the interface between the two media in accordance with Snell's law:
n
Sin I=
n
′ Sin I′
where n and n′ are the refractive indices of the two media, and I and I′ are the angles of incidence and refraction, respectively. Light can always pass from a lower refractive index medium to a higher refractive index medium, because in that case angle I′ is smaller than angle I. However, when a beam of light passes from an optically denser medium (having a higher index of refraction n) to an optically rarer medium (having a lower index of refraction n′), the angle of refraction I′ is always greater than the angle of incidence I. As the angle of incidence I increases, the angle of refraction I′ increases at a faster rate. When Sin I=n′
, then Sin I′=1.0 and the angle of refraction I′=90 degrees. Such an angle of incidence is called the critical angle. When the critical angle condition is met, no light propagates into the optically rarer medium. When the angle of incidence is greater than the critical angle, the light is reflected back into the optically denser medium—a phenomenon called total internal reflection (T.I.R.). If the separating boundary of the two media is smooth and clean, 100 percent of the incident light is reflected back. The critical angle phenomenon is used for measurements of refractive indices of various fluid or solid materials.
FIG. 3
a
depicts a critical angle refractometer shown and identified broadly by the reference numeral
22
. Refractometer
22
is shown as including a housing
32
having an inclined top surface portion
34
and a horizontal top surface portion
36
adjacent thereto, an LCD display
38
and a keypad input
40
at inclined top surface portion
34
. A test assembly
24
is situated on horizontal top surface portion
36
. Refractometer
22
is similar to the Leica AR600 automatic refractometer available from Leica Microsystems Inc. The Leica AR600 automatic refractometer is manufactured generally in accordance with the disclosure of commonly-owned U.S. Pat. No. 4,640,616 issued Feb. 3, 1987 and entitled AUTOMATIC REFRACTOMETER. The entire disclosure of U.S. Pat. No. 4,640,616 is incorporated herein by reference as if reprinted in its entirety.
The schematic of
FIG. 4
illustrates the opto-electronic measurement system of refractometer
22
, which is based on the principles of critical angle refractometry described above. The system comprises a photosensitive linear scanned array (LSA)
44
for providing an output signal as a function of the amount and location of light incident thereon. Linear scanned array
44
includes a plurality of closely adjacent and aligned photoelectric cells
46
. The measurement system comprises an optical system for directing light onto linear scanned array
44
, wherein the amount and location of light illuminating the LSA depends on the index of refraction of a test sample
51
. As shown in
FIG. 4
, the optical system includes a light source
48
and a prism
50
for receiving lig
Byrne Michael J.
Ryan Thomas E.
Brown Rudnick Berlack & Israels LLP
Eliseeva, Esq. Maria M.
Font Frank G.
Leica Microsystems Inc.
Stafira Michael P.
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