Chemistry: analytical and immunological testing – Rate of reaction determination
Patent
1998-08-26
2000-10-03
Snay, Jeffrey
Chemistry: analytical and immunological testing
Rate of reaction determination
436164, 356445, G01N 1371
Patent
active
061271837
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to an improved method of analysing physical or chemical interactions taking place in a film layer at an optical sensor surface by means of refractive index determination through light intensity signal producing techniques.
BACKGROUND OF THE INVENTION
Recently, the interest for surface sensitive measuring techniques has increased markedly as several so-called label-free optical techniques have been developed for measuring and quantifying biomolecular interactions. A so far frequently used such optical technique is based on surface plasmon resonance, hereinafter often referred to as SPR.
When light travels from an optically denser medium (i.e. having a higher refractive index) to a less dense medium (i.e. having a lower refractive index), total internal reflection (TIR) occurs at the interface between the two media if the angle at which the light meets the interface is above a critical angle. When TIR occurs, an electromagnetic "evanescent wave" propagates away from the interface into the lower refractive index medium.
If the interface is coated with a thin layer of certain conducting materials (e.g. gold or silver), the evanescent wave may couple with free electron constellations, called surface plasmons, at the conductor surface. Such a resonant coupling occurs at a specific angle of the incident light, absorbing the light energy and causing a characteristic drop in the reflected light intensity at that angle. The surface electromagnetic wave creates a second evanescent wave with an enhanced electric field penetrating into the less dense medium.
The resonance angle is sensitive to a number of factors including the wavelength of the incident light and the nature and the thickness of the conducting film. Most importantly, however, the angle depends on the refractive index of the medium into which the evanescent wave of the surface plasmon wave propagates. When other factors are kept constant, the resonance angle is thus a direct measure of the refractive index of the less dense medium, the angle being very sensitive to refractive index changes in the medium. For a detailed description of the dielectric equations describing this dependence it may be referred to Kretschmann, E., Z. Phys. B241, 313 (1971).
The SPR evanescent wave decays exponentially with distance from the interface, and effectively penetrates the lower refractive index medium to a depth of approximately one wavelength. Therefore, only changes in refractive index very close to the interface may be detected.
If the metal film is covered with an appropriate sensing layer (e.g. an antibody) capable of specific interaction with a molecule (e.g. an antigen) present in a fluid sample contacted with the sensing layer, SPR based chemical sensors may be constructed, the interaction at the sensor surface changing the solute concentration and bound surface concentration and thus the refractive index within the penetration range of the evanescent wave. A variety of SPR-based chemical sensors have been developed wherein the change of the reflectance curve (reflected light intensity versus angle of incidence or wavelength) with time is measured, which change is correlated to the surface refractive index.
Different techniques may be used for bringing the light to interact with the sensor surface. A commonly used detection system is based on the Kretschmann configuration (Kretschmann and Raether, Z. Naturforsch. Teil A 23: 2135-2136, 1968). In this configuration a thin layer of the reflective metal (gold or silver) is deposited on the base of a prism and TM polarized, monochromatic light is coupled by the prism to the SPR-wave.
An example of a commercial biosensor system constructed on the basis of the Kretschmann configuration is BIAcore.TM., marketed by Pharmacia Biosensor AB (Uppsala, Sweden). This biosensor system incorporates a Kretschmann configuration-based SPR-detection system with a microfluidic system to control the flow of reagents required in the analyses. With this apparatus, biomolecular in
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Ivarsson Bengt
Stenberg Esa
Biacore AB
Snay Jeffrey
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