Optics: measuring and testing – By light interference – Having light beams of different frequencies
Reexamination Certificate
2000-07-24
2002-12-10
Turner, Samuel A. (Department: 2877)
Optics: measuring and testing
By light interference
Having light beams of different frequencies
C356S480000
Reexamination Certificate
active
06493090
ABSTRACT:
The present invention relates to a method and an apparatus for sensing the presence, concentration or amount of a substance through detection of a change in refractive index caused by said substance.
In particular, the present invention includes such methods and apparatus for determination of the presence, concentration, or amount of a substance, such as a fluid, in which the refractive index of the fluid is a function of the concentration of one or more chemical species in the fluid. The invention has applicability particularly in the field of clinical analysis, but also, for example, in wet chemical analysis in general, and in gas analysis.
Detection of minute concentrations of chemical species, such as bioactive molecules, is presently typically based on affinity reactions, such as antigen—antibody (immunoassay) reactions, hormone—receptor reactions, DNA/RNA—complimentary string reactions, reactions with cavitands, i.e. cyclic saturated hydrocarbons for selective binding of ions or molecules, such as glucose, or catalytic reactions, such as enzyme- substrate/inhibitor/co-factor reactions.
These reactions facilitate recognition of individual molecules of a substance thereby making identification of substances at very low concentrations, typically at the order of 10
−12
mol/L, possible.
The attractive properties of known detection techniques based on the above-mentioned reactions are high specificity as well as sensitivity to the analyte in the presence of other substances. This means that bioassays based on these methods can be performed, e.g. utilizing biosensors, in samples of body fluids (e.g. whole blood, serum, saliva and urine) whereby time and labor consuming purification steps can be avoided.
Assays based on the above mentioned binding mechanisms, typically employ luminescent detection principles, such as fluorescence, bioluminescence, or chemiluminescence, for detection of the presence of specific chemical species. Luminescent techniques are widely used in assays involving “wet” chemistry, such as assays employing microtitre plates or flow cytometry.
It is known to detect the presence of a specific chemical species by immobilization at a surface of a reagent comprising antibodies or antigens, and bringing an analyte containing the species to be detected into contact with the reagent. The reagent may for example be immobilized at an inner surface of a microflow channel in which the analyte flows. As an affinity or catalytic reaction takes place between the species to be detected and the reagent, the refractive index at the surface bearing the reagent changes and thus, the presence of the species can be detected by detection of the change in refractive index. This change typically ranges from 10
−7
to 10
−4
.
It is also known to detect changes in the refractive index of thin layers of substance by evanescent wave sensing.
A comprehensive review of optical devices for biosensing based on this technique has been disclosed in A. Brecht et al. “Optical Probes and Transducers”, Biosensors & Bioelectronics, 10, pp. 923-936, 1995 (Ref. 1).
Surface plasmon resonance detection as in for instance Ref. 1 relies on the excitation of a surface plasmon in a thin metal film, typically at a glass-liquid interface. This technique uses the evanescent field which has an exponentially decaying field component along the normal to the glass-liquid interface. With an analyte present at the upper boundary of the metal-liquid interface the refractive index in this region is modified causing a change in the resonance surface plasmon excitation angle. This change in angle is a measure for the refractive index change—and consequently the chemical composition of the analyte.
Evanescent waves are intricately associated with propagation of light in waveguides. A wave travelling along the core of a waveguide, be it an optical fibre or planar waveguide, will generate an exponentially decaying field that extends into the cladding of the waveguide. The penetration depth d
ew
of this tail into the cladding depends on the difference between the refractive indices of the core and the cladding such that a large difference in the refractive indices implies a short penetration depth.
The penetration depth ranges from a fraction of the optical wavelength to many wavelengths depending on the specific waveguide parameters.
The evanescent wave, can be used as a means for both excitation and detection of a layer adjacent to the cladding of the waveguide provided that the thickness of the cladding is smaller than the penetration depth
ew
of the corresponding evanescent wave. In the case of excitation, the most well-known method is evanescent wave excitation of fluorescence in capillary waveguides, see e.g. O. S. Wolfbeis in Trends in Analytical Chemistry 15 pp. 225-232, 1996 (Ref. 2). In this application, the use of evanescent waves makes it possible to selectively excite a thin fluorescent labelled detection layer but not the bulk analyte.
Fibre optic biosensors (see e.g. M-P. Marco et al. in Meas. Sci. Tech. 7, pp. 1547-1582, 1996) (Ref. 3) use the evanescent field generated in the cladding of a single-mode optical fibre due to total internal reflection generated at the inner boundary of the fibre core. When molecules with an appropriate excitation energy are located in the evanescent field, they absorb energy leading to an attenuation in the reflected light inside the fibre core. To get a reasonable sensitivity the molecules are typically labelled with e.g. a fluorescent dye so that the molecules are able to re-emit at a longer wavelength (lower energy) as fluorescence after being excited by the evanescent field. Part of the re-emitted light is coupled back into The fibre core and propagates towards the end of the fibre before being detected by a photodetector.
Within the field of integrated optics biosensors may also be implemented. Integrated optics facilitates the possibility of making more device-like biosensors like interferometric sensors (e.g. a Mach Zehnder interferometer) which rely on the detection of an induced phase change between a sensing arm and a reference arm, see Ref. 3. The biological interaction expressed as a phase change between the light beam in the sensing arm and the reference arm is detected by standard interferometric means.
Further, it is known to detect changes in the refractive index of thin layers of substance by intracavity sensing.
In Gourley et al. “Semiconductor micro lasers with intracavity microfluidics for biomedical applications”, Proc. SPIE, Vol. 2987, pp. 186-196 (1997) (Ref. 4) , a method is disclosed in which a cell, e.g. an erythrocyte, is introduced into the cavity of a vertical cavity surface-emitting laser (VCSEL) . Due to the fact, that the cell does not absorb at the wavelength of the GaAs laser (850 nm), the cell in the cavity will act as a Fabry-Perot etalon. Abnormalities in the structure of the cell-membrane, like Sickle-cell anaemia of blood-cells, will cause higher order transverse modes to be generated. These modes can be detected by a suitable solid state imaging device, such as a CCD.
In U.S. Pat. Nos. 5,437,840 and 5,514,596 (Refs. 5,6) an apparatus and method for intracavity sensing of macroscopic properties of chemicals are disclosed. The apparatus comprises an optical resonator that is pumped by a light source that may be positioned inside or outside the optical resonator. A reflective element having a surface is positioned inside the optical resonator so that total reflection of the mode excited within the resonator is provided. A detector for measuring the resonator output is positioned outside the resonator. As the substance to be analyzed is introduced in the evanescent field region at the outer surface of the reflective member, the refractive index at the surface changes causing the resonance frequency of the optical resonator to change accordingly. In some embodiments, this change in resonant frequency is measured. In most embodiments however, fluorescence of a label at the surface is sensed. The change in resonant frequ
Lading Lars
Lindvold Lars
Gonnolly Patrick
Jones Tullar & Cooper PC
Torsana A/S
Turner Samuel A.
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