Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1999-10-12
2001-04-17
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S527000, C436S164000, C436S805000, C356S128000, C356S136000, C356S328000, C359S566000, C359S569000, C359S573000, C359S885000, C359S888000, C359S637000, C359S640000, C359S890000, C359S896000, C359S572000, C385S010000, C385S012000, C385S030000, C385S036000, C385S037000, C385S129000, C422S082050, C422S082090, C422S082110
Reexamination Certificate
active
06218194
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This Application is a national stage application of PCT application No. PCT/GB97/00346, filed Feb. 10, 1997, which claims priority of Great Britain Application No. 9602542.4, filed Feb. 8,1996.
FIELD OF THE INVENTION
This invention relates to methods and apparatus for the analysis of analyte molecular species in a sample.
BACKGROUND OF THE INVENTION
Devices are known with a surface on which is immobilized a layer of biomolecules having an affinity for other molecules (“the analyte”) in a sample under test. Such devices are commonly referred to as biosensors. The immobilized biomolecules and the analyte may, for example, constitute a specific binding pair such as an antigen-antibody pair. Interaction of the two members of the pair causes a change in the physical properties of the device. This change can be used as an indicator of the presence and/or concentration of the analyte, the strength and/or progress of the interaction etc.
In many biosensors, it is the optical properties of the device which are monitored. One class of optical biosensor comprises a waveguide in the form of a thin layer of relatively high refractive index material coated on a substrate of optically transparent lower refractive index material. Biomolecules are immobilized on the surface of the waveguide and the interface between the substrate and the waveguide is irradiated with a beam of light.
Means are generally provided to facilitate coupling of light into the waveguide. The optical properties of the device will depend on the nature of those means, as well as on other factors including the wavelength of the incident light, the materials used for the waveguide and the substrate, the thickness of the waveguide etc. In general, however incident light is coupled to a greater or lesser extent into the waveguide. Chemical binding events at or in the vicinity of the waveguide surface will cause a localized change in refractive index, which in turn causes a change in the coupling characteristics of the device. This provides a means for monitoring interactions between the immobilized biomolecules and the analyte molecules.
One form of coupling means which has been proposed is a grating structure formed, for instance, in the interface between the substrate and the waveguide. In general, light incident will be reflected, transmitted or scattered into the various diffraction orders of the grating. Further, at certain angles of incidence, where a diffraction order matches the waveguide propagation condition, light will be coupled into the waveguide.
Here, light will propagate in the guide parallel to the substrate surface, where it will continue to interact with the grating. The light will couple back out of the waveguide via the various diffraction orders and into free-space beams. This outcoupled light will include beams in the same direction as the transmitted and reflected, uncoupled beams.
Attempts to measure the coupling condition are hampered by overlap of the waveguide derived beams and the uncoupled, transmitted or reflected components. This leads to measurements of low contrast.
One approach to this problem is to provide a pair of grating structures separated by an unmodulated region. Light incident on one of the gratings is coupled into the waveguide and is then coupled out by the second grating. The coupled-out light is thus spatially separated from the light reflected or transmitted at the first grating. However, the need for the provision of two gratings is a disadvantage.
In another approach, a single grating structure is employed, the grating structure being a superposition of grating elements having two different periodicities. Light incident on the grating structure at a first angle is coupled into the waveguide by the grating element with a first periodicity. It is then coupled out by the grating element having a second periodicity, at a different angle. The coupled-out light is thus angularly separated from the reflected light. Such a bidiffractive grating is relatively difficult to fabricate.
There haste now been devised methods for monitoring the interaction of molecular species, and devices suitable for use in such methods in which light is coupled into a waveguide by a grating structure, which overcome or substantially mitigate the above-mentioned disadvantages.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, a method for monitoring the interaction of a first molecular species in a sample with a second molecular species comprises
providing a sensor device comprising a substrate having a waveguide formed on at least part of the surface thereof, the waveguide having a first major surface which constitutes an interface between the waveguide and the substrate and a second major surface upon which the second molecular species is immobilized, at least a region of the first and/or second major surface being formed with a periodic refractive index modulation;
contacting a sample containing the first molecular species with the second major surface;
irradiating said periodic refractive index modulation with a beam of incident monochromatic light,
varying the angle of incidence of said light or the wavelength of said light through a range of angles or wavelengths respectively, said range including an angle or a wavelength, as the case may be, at which a guided mode is excited in said waveguide;
monitoring the intensity of light reflected from said periodic refractive index modulation as a function of the angle of incidence or wavelength of the incident light; and
determining the angle of incidence or wavelength at which the intensity of the light reflected from the periodic refractive index modulation is a maximum.
In general, the incident light will be partially reflected from the periodic refractive index modulation, partially transmitted and partially coupled into the waveguide. The method according to the invention is advantageous primarily in that, at a certain angle of incidence, a near-total reflection of the incident light beam can be achieved. The incident light excites a guided mode in the waveguide. This guided mode propagates a certain distance and is then coupled out, back into the substrate and into the superstrate adjacent the waveguide. It is possible by appropriate choice of parameters to achieve almost complete destructive interference of the transmitted components at the coupling angle. This interference is between the zeroth order transmitted beam and the beam radiated from the out-coupled guided wave into the superstrate, ie the material beyond the waveguide (in the direction of the incident light beam).
In such a case there is a correspondingly high intensity of the reflected light at the propagation angle, and this is relatively easily monitored. Such high reflection may be termed “anomalous” or “abnormal” reflection. Interaction of the molecular species immobilized on the waveguide surface with analyte molecules in a sample which is contacted with the waveguide causes a local change in refractive index in the vicinity of the waveguide surface. This in turn changes the angle of incidence or wavelength at which the reflection maximum occurs, providing a sensitive indicator of the chemical interaction taking place at the surface.
The method according to the invention utilises a sensor device with only a single periodic refractive index modulation, and may therefore be easier and/or less expensive to fabricate than devices incorporating multiple gratings or bidiffractive gratings.
By the words “reflected” and “reflection” as used herein we mean the return of the incident light beam from the waveguide through the substrate at an angle equal and opposite to the angle of incidence. Although this is superficially similar to conventional specular reflection, the mechanism of “reflection” in the present case includes diffraction effects from the grating.
The periodic refractive index modulation is preferably a surface relief profile or a grating formed in the surface of the substrate to which the waveguide coating is applied
Lyndin Nikolai Mikhailovich
Sychugov Vladimir Alexandrovich
Tishchenko Alexander Valentinovich
Usievich Boris Alexandrovich
Chin Christopher L.
Nixon & Peabody LLP
Pham Minh-Quan K.
Thermo Fast UK Limited
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