Optical waveguides – Optical waveguide sensor
Reexamination Certificate
1999-11-29
2002-11-26
Ngo, Hung N. (Department: 2874)
Optical waveguides
Optical waveguide sensor
C385S147000, C385S115000, C385S120000, C385S038000, C385S116000, C422S082070, C436S084000, C436S172000
Reexamination Certificate
active
06487326
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The invention relates to combined optical and electrochemical detection, and more particularly, to an apparatus and method for use in the performance of such studies.
BACKGROUND OF THE INVENTION
Without limiting the scope of the invention, its background is described in connection with a combined electrochemical and optical apparatus for luminescence imaging of microenvironments and methods that may be used in the performance of such imaging.
Fluorescence imaging is one of the most valuable methods for analyzing microenvironments, particularly cellular microenvironments, and can be expected to find broader applicability as the rapidly growing computer and video industries provide new tools/hardware for fluorescence imaging. Although promising in theory, fluorescence microscopy is limited by the number of analytes that are amenable to fluorescent detection schemes. While it is straightforward to image analytes with native fluorescence, analytes labeled with a fluorophore, or analytes that can interact with a fluorescent indicator (e.g., H
+
, Ca
2+
, O
2
), imaging other species such as electroactive analytes is problematic. For example, a major intracellular electroactive analyte of particular importance in understanding cellular metabolism, hydrogen peroxide (H
2
O
2
) is presently unamenable to continuous fluorescent analysis on a cellular resolution level.
The motivation for imaging hydrogen peroxide and other reactive oxygen species (ROS) in biological cells and tissue stems from their role in oxidative stress and oxidative burst events. Unfortunately, the in situ monitoring of ROS dynamics on the cellular level is limited by existing technology. Currently, the most common microscopic method for imaging hydrogen peroxide involves loading cells with dichlorofluorescin and quantitating the hydrogen peroxide by following the oxidation of dichlorofluorescin by hydrogen peroxide to produce fluorescent dichlorofluorescein. This technique can be used for detecting ROS liberated in or diffusing in the cytosol of the cell but it is not a direct reporter for ROS generated at the plasma membrane and/or released to the exterior of the cell. Hydrogen peroxide fiber-optic sensors or biosensors that are suitable for single cell analysis by virtue of the ability to acquire a continuous real time measurement with (sub)micrometer spatial resolution have not been demonstrated.
Nicotinamide adenine dinucleotide in its reduced form (NADH), is a biologically important coenzyme that is both fluorescent and electroactive. The quantitation of this molecule is of great interest in chemistry, biology, and medicine. For example, in addition to its use as a metabolic activity marker, NADH is an ideal biosensor reagent since it can modulate the activity of over 200 different dehydrogenases [Pantano, P. & Kuhr, W. G. (1995)
Electroanalysis
7, 405-416]. Unfortunately, both the NADH fluorescence and electrochemical measurements are difficult to perform. For the NADH fluorescence measurement, in addition to its low quantum yield, there is significant biological (auto)fluorescence in the same spectral region as the NADH emission. Furthermore, the throughput of borosilicate glass and silica-based optical fibers is attenuated greatly in the ultraviolet spectral region where the NADH excitation wavelength lies (i.e., 340 nm). Finally, improving a CCD camera's 400-500 nm quantum efficiency (the NADH emission spectral region) by employing back-thinned chips is prohibitively expensive. Also, existing electrochemical fiber-optic NADH-biosensors lack the resolution required for imaging purposes.
What is needed is an apparatus and system that readily permits concurrent luminescence imaging and electrochemical sensing of important analytes with a microscopic level of resolution.
SUMMARY OF THE INVENTION
The present invention is directed to apparatus and methodology for concurrent fluorescence imaging and chemical sensing of an electroactive analyte through a fiber optic electrode with resolution on the microscopic level.
The fabrication and characterization of imaging fiber electrodes (IFEs) is presented, and the use of an electrochemically-modulated, fluorescence-based, imaging-fiber electrode chemical sensor (IFECS) is demonstrated.
In one embodiment, the invention provides a fiber optic electrochemical sensor for detecting an analyte including a fiber optic layer, a electrically conductive translucent metallic layer, and a light energy absorbing dye layer. The fiber optic layer of the fiber optic electrochemical sensor may be a fiber optic bundle that includes one or more of individual optic fibers, wherein each individual optic fiber has a diameter of less than 20 micrometers. In one embodiment, the fiber optic electrochemical sensor includes a fiber optic bundle with individual optic fibers, wherein each individual optic fiber has a diameter of less than 10 micrometers and wherein the bundle has a diameter of less than 2 millimeters. The sensor also has an electrically conductive translucent metallic layer, and a light energy absorbing dye layer wherein the fiber optic electrochemical sensor is capable of electrochemical regeneration. Due to its microscopic resolution capabilities, the apparatus and method of the present invention is particularly applicable to studies of cells and tissues, however, the scope of the invention is not limited to biological microenvironments but rather is applicable to any microenvironment in which fine spatial resolution of imaging concurrent with chemical sensing is needed.
In alternate embodiments, the electrically conductive translucent metallic layer of the fiber optic electrochemical sensor is between 10 and 100 nm thick. The metallic layer may be of any electrically conductive metal or metal oxide that may be applied in a translucent layer wherein “translucent”, used interchangably herein with “transparent”, means susceptible to the through-passage of light energy. In examples provided herein, the fiber optic electrochemical sensor electrically conductive translucent metallic layer is a sputter-coated 20-23 nm layer of gold.
In alternate embodiments, the light energy absorbing dye layer of the fiber optic electrochemical sensor is selected from the group consisting of, e.g. fluorochromes, fluorescent enzyme conjugates, fluorescent substrates and chromophores. In one example, the analyte to be detected is a cellular reactive oxygen species and the light energy absorbing dye layer includes a rhodamine dye.
In another example the analyte to be detected is cellular NADH and the light energy absorbing dye layer includes a ruthenium containing luminophore.
In another embodiment of an imaging system according to the present invention, a fluorescence based imaging fiber electrode chemical sensor system includes a fiber optic electrochemical sensor, a potentiomer or equivalent means for measuring ion flux, a microscope including a light source and an objective lens. The objective lens communicates light from the source to the fiber optic electrochemical sensor and receives light returning from the sensor and provides a means for recording light returning from the sensor though the objective. Potentially useful recording devices include CCD cameras, linear arrays and xy active matrix detectors.
The invention provides in one embodiment a method for preparing a imaging fiber electrode including the steps of; polishing a face of the fiber optic bundle, silanizing the face using a mercapto-trimethyoxysilane, and sputter coating the silanized face to deposit a 10-30 nm thick semi-transparent metal layer.
In another embodiment, a method for preparing a imaging fiber chemical sensor is provided including the steps of; obtaining a fiber optic electrode having a 15-30 nm gold film on a distal end and an electrically conductive aspect leading from the distal end through a lateral dimension of the fiber optic electrode, coating the fiber optic electrode with an ion-exchange polymer and applying a luminescent reporter group.
In one examp
Jin Eunsook S.
Khan Samina S.
Pantano Paul
Stokes, Jr. Harold W.
Board of Regents , The University of Texas System
Flores Edwin S.
Gardere Wynne & Sewell LLP
Ngo Hung N.
Warren, Jr. Sanford E.
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