Optical waveguides – Optical waveguide sensor
Reexamination Certificate
1999-07-06
2001-08-07
Naff, David M. (Department: 1651)
Optical waveguides
Optical waveguide sensor
C385S038000, C385S043000, C385S127000, C385S128000, C424S009100, C435S004000, C435S176000, C435S180000, C530S402000, C530S811000, C530S815000
Reexamination Certificate
active
06272262
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to optical sensors, methods of sensor fabrication and uses of such sensors, and more particularly the use of such sensors for the detection if nitric oxide.
BACKGROUND
Recent elucidation of the fact that nitric oxide plays many biological roles has spurred special interest in this molecule. For instance, nitric oxide is believed to play a role in vasodilation. See Marletta et al., “Unraveling the biological significance of nitric oxide”
Biofactors
2:219 (1990). Nitric oxide also appears to inhibit platelet aggregation by elevating intraplatelet levels of cyclic GMP. See Diodati et al., “Complexes of Nitric Oxide with Nucleophiles as Agents for the Controlled Biological Release of Nitric Oxide: Antiplatelet Effect”
Thrombosis and Haemostasis
70:654 (1993)
More recently, nitric oxide is emerging as one of the main neurotransmitters in the central and peripheral nervous systems. See Snyder, “Janus faces of nitric oxide”
Nature
364:577 (1993). It appears to play both neurotoxic roles, such as in AIDS dementia, and neuroprotective roles in degenerative problems such as Parkinson's and Huntington's diseases.
Given the growing importance of the molecule, there have been a number of attempts to develop means to measure cellular levels of nitric oxide. For example, a fiber optic nitric oxide chemiluminescent sensor has been developed. See Zhou and Arnold, “Response Characteristics and Mathematical Modeling for a Nitric Oxide Fiber-Optic Chemical Sensor”
Anal. Chem.
68:1748 (1996). This sensor was constructed by holding a small amount of an internal reagent solution at the tip of a fiber-optic bundle with a piece of gas-permeable membrane. Nitric oxide diffuses across the membrane into this internal solution, where a chemiluminescent reaction between nitric oxide, hydrogen peroxide, and luminol takes place. The drawbacks of this sensor include the following: 1) the response time (approximately 8-17 seconds) is longer than the time needed for nitric oxide in the solution to be converted to nitrite; 2) the detection of nitric oxide is complicated by interferences from dopamine, uric acid, ascorbic acid, and cysteine, 3) the sensor is relatively large in size (greater than 6 mm in diameter) and thus difficult to use for the measurement of cellular nitric oxide levels (and impossible for intracellular measurements); and 4) the sensor has relatively poor sensitivity, i.e. a relatively high limit of detection (approximately 1.3 mM of nitric oxide).
Sensors involving sol-gel technology have also been attempted. The process involves hydrolyzing an alkoxide of silicon to produce a sol, which then undergoes polycondensation to form a gel. Biomolecules are immobilized by being entrapped in the sol-gel. In one case, horse-heart cytochrome c was encapsulated in a sol-gel and absorbance-based spectral shifts were used to monitor the binding of nitric oxide. See Blyth et al., “Sol-Gel Encapsulation of Metalloproteins for the Development of Optical Biosensors for Nitrogen Monoxide and Carbon Monoxide”
Analyst
120:2725 (1995). Unfortunately, the sensor reaction is reported to have taken two hours to reverse, making dynamic measurements impossible.
What is needed is a sensor of relatively small size and good sensitivity that measures nitric oxide with little or no interference from other analytes in a short enough time period to permit dynamic measurements.
SUMMARY OF THE INVENTION
The invention relates generally to optical sensors, methods of sensor fabrication and uses of such sensors, and more particularly the use of such sensors for the detection if nitric oxide. The present invention contemplates both fiber-optic sensors and optical fiberless sensors comprising nitric oxide-binding compounds, such compounds permitting the specific binding of nitric oxide (e.g. non-covalent binding) with little or no interference from other analytes.
With regard to fiber-optic sensors, the present invention contemplates an optical fiber having a fiber tip, said tip comprising an immobilized nitric oxide-binding compound. It is not intended that the present invention be limited by the means by which the nitric oxide-binding compound is immobilized. In one embodiment, the tip of the fiber is treated so as to have reactive groups and the nitric oxide-binding compound is covalently linked directly to the fiber via the reactive groups. In another embodiment, the tip has an inert coating (i.e. inert relative to nitric oxide) such as a metal layer (preferably, a non-linear layer and more preferably, spheres comprising metal) and the nitric oxide-binding compound is immobilized on the metal layer. In a preferred embodiment, the tip is treated to create reactive groups (e.g. thiol groups), spheres of metal colloid are attached to the tip via the reactive groups, and the nitric oxide-binding compound is immobilized on the metal colloid spheres.
It is not intended that the present invention be limited to the nature or dimensions of the metal layer. A variety of metals and metal colloids are contemplated, including but not limited to, colloids of gold, silver, tungsten, thoriasol, antimony pentoxide, carbon, red iron oxide, titanium dioxide and platinum (available commercially from Vector Laboratories, Inc., Burlingame, Calif.; Nanoprobes, Inc., Stony Brook, N.Y.; and Polysciences, Inc., Warrington, Pa.). In a preferred embodiment, the metal layer is a monolayer of spheres comprising gold colloid, said spheres attached to an end of a fiber as a substrate for spontaneous attachment of the nitric-oxide-binding compound. While not limited to particular dimensions, the size of the gold colloid does produce a marked difference in the fluorescence intensity measured. The present invention contemplates colloid sizes (and in particular gold colloid sizes) ranging from very small, 2 nm, to very large, 250 nm (and more preferably, between 5 nm and 100 nm), said colloids immobilized on the end of a fiber to provide a base for protein attachment. While a precise understanding of the mechanism for this phenomenon is not necessary in order to practice the invention, it is surmised the intensity changes seen in the fluorescence emission are not a result of surface coverage, and availability of sites for protein adsorption, but instead a quenching or enhancement by the gold itself. In general, the optimum fluorescence is achieved with particles sizes of approximately 100 nm.
It is also not intended that the present invention be limited by the nature of the particular nitric oxide-binding compound. In one embodiment, the nitric oxide-binding compound is a porphyrin group- or heme group-containing protein. In another embodiment, the nitric oxide-binding compound is a heme-binding protein. Regardless of whether the protein is a heme-group-containing protein or a heme-binding protein, in one embodiment, the present invention contemplates that the protein (or peptide) is dye-labeled (e.g. with dyes which can be used for protein labeling that do not react to nitric oxide, such as Oregon Green dyes). This has been found to increase the signal to noise ratio of the sensors of the present invention.
It is not intended that the present invention be limited to specific heme-group-containing proteins. The heme-group-containing proteins are limited only in the respect that they bind nitric oxide, and more preferably, they bind nitric oxide specifically (i.e. they do not bind interfering substances). The preferred heme-group-containing protein is cytochrome c′ (as distinct from cytochrome c). It is not intended that the present invention be limited to the source of cytochrome c′. Nonetheless, preferred sources include, but are not limited to, microorganisms, more preferably bacterial sources, and more particularly, purple phototropic bacteria, aerobic nitrogen-fixing bacteria, and facultatively denitrifying bacteria, and still more particularly,.sources such as
C. vinosum, R. purpureus,
and
R. gelatinosa.
Insects have been shown to have both heme group-containing prot
Barker Susan
Clark Heather
Kopelman Raoul
Medlen & Carroll LLP
Naff David M.
The Regents of the University of Michigan
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