Surgery – Diagnostic testing – Measuring or detecting nonradioactive constituent of body...
Reexamination Certificate
2001-12-28
2004-07-20
Winakur, Eric F. (Department: 3736)
Surgery
Diagnostic testing
Measuring or detecting nonradioactive constituent of body...
C600S341000, C436S094000, C436S095000, C436S172000, C422S082070, C568S001000, C546S013000
Reexamination Certificate
active
06766183
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to optical biosensor systems that utilize fluorescent molecules in the determination of the concentration of polyhydroxylate analytes, particularly glucose. These fluorescent molecules typically include a boronate moiety and a fluorophore that includes an iminium ion. The invention further relates to implantable, optical biosensor systems that utilize these molecules for the determination of in-vivo glucose concentrations.
BACKGROUND OF THE INVENTION
The accurate monitoring of biological analytes allows for the efficient management and treatment of a number of disease states. Notably, for approximately 16 million people in the United States afflicted with diabetes, detection and quantification of blood glucose levels substantially lessens the disease's symptomatic complications (see, e.g. Kahn, C. R. “Conquering Diabetes, A Strategic Plan for the 21
st
Century”; A report of the Congressionally-Established Diabetes Research Working Group, NIH Publication No. 99-4398; 1999). Current technology generally requires that a blood sample be obtained from a patient for the measurement of blood glucose levels. In order to accurately reflect the body's ever-changing response to meals, activities and even stress, measurement of blood glucose should be on a frequent basis. Due to the discomfort and sterility issues, the frequent sampling of a patient's blood to determine glucose levels is impractical, at best. Thus, devices and methods to frequently monitor blood glucose levels without taking numerous blood samples from a patient are desirable.
Over the years, researchers in the medical devise industry sought new technologies incorporating optochemical methods for detecting and quantifying subcutaneous glucose. Research on optochemical methods for detecting glucose focuses on the development of a long-term, minimally invasive optochemical glucose monitoring devise that, once implanted, is painless to interrogate, amenable to continuous monitoring, and ultimately useful as a component of a closed-loop artificial pancreas.
Fluorescent reporter molecules are useful in optochemical methods for monitoring changing glucose levels in-vivo. However, in order to use fluorescent reporter molecules to detect and measure the concentration of glucose in-vivo, numerous problems need to be surmounted. One particular problem is that fluorescence transmitted through skin is generally poor at visible wavelengths less than about 500 nm. To improve transmission of the signal through the skin, the fluorescent compound should operate at longer wavelengths greater than about 450 nanometers. The transmission through a few millimeters of skin increases logarithmically with wavelength—from 0.1% at about 400 nm to almost 100% at 850 nm. Thus, the longer the wavelength, the greater the transmission through skin. Excitation and emission wavelengths greater than about 600-700 nm are an enormous improvement over those of about 400-450 nm. Because of the significant increase in optical skin transmission at longer wavelengths, a practical glucose sensor can operate more efficiently, more accurately, and with a greater signal-to-noise ratio. In addition, it is advantageous to match the peak excitation wavelength with an existing light source (such as an LED or diode laser). Furthermore, by operating at longer wavelengths, there is a reduction in the tissue autofluorescence background.
Embodiments of this invention address the problem of obtaining adequate optical transmission of fluorescence using fluorescent reporter molecules in-vivo to report on the body's fluctuating glucose concentrations.
SUMMARY OF THE DISCLOSURE
Embodiments of the fluorescent biosensor molecules of the present invention include an oxazine-based fluorophore, a boronate binding moiety which specifically binds polyhydroxlate analyte, and a linker moiety which provides a linkage between the fluorophore and the boronate binding moiety. These biosensor molecules typically emit light in the visible region of the spectrum, preferably above about 600 nm and more preferably above about 650 nm.
Specific embodiments of the biosensor molecules of the invention typically have an excitation spectra that is greater than about 550 nm, preferably above about 600 nm. Further, these biosensor molecules have at least one wavelength in their emission spectra that is greater than about 550 nm, preferably above about 600 nm and more preferably above about 650 nm and exhibits an intensity that is at least about 25 percent of the intensity of a wavelength of maximum intensity in the visible region of the spectrum, more preferably these biosensor molecules have at least one wavelength in their emission spectrum that is greater than about 550 nm, preferably above about 600 nm and more preferably above about 650 nm and exhibits an intensity that is at least 25 percent of the intensity of a wavelength of maximum intensity in the visible region.
An exemplary oxazine-based biosensor molecule of the present invention utilizes Nile blue as the fluorophore as shown in FIG.
2
. The Nile blue-based biosensor molecule has an excitation wavelength of about 625 nm and a maximum emission wavelength of about 675 nm (see, e.g. FIGS.
8
). Similar oxazine-based fluorophores suitable for use in embodiments of the present invention may include one or more functional groups selected from the following: aliphatic, aromatic, haloalkane, alcohol, ether, amine, cyano, nitro group, aldehyde, ketone, ester, carboxylic acid, sulfonic acid and phosphoric acid functional groups.
Other embodiments of the oxazine-based biosensor molecules of the invention can include ditopic molecules, which generally include two boronate substrate recognition sites. These ditopic embodiments of the invention are designed to bind to one or more polyhydroxylate analyte molecules, such as glucose, per ditopic biosensor molecule, and thus, increase the sensitivity of the sensing ability of these biosensor molecules.
Typical embodiments of the invention include oxazine-based biosensor molecules that are either contained within, or attached to, a polymer matrix to form embodiments of the fluorescent biosensor of the invention. Preferably, the polymer matrix materials selected for use with the biosensors of the invention are biocompatible. The use of biocompatible polymeric materials is preferred in contexts where the biosensors are in contact with body fluids and tissue. Thus, embodiments of the biosensors of the invention are especially suitable for implantation beneath the skin where they serve as in-vivo reporters of analyte concentrations, particularly glucose concentrations.
In particular embodiments of the invention disclosed herein, the functional group on the oxazine-based fluorophore that is capable of forming a covalent linkage to the linker moiety is an aldehyde, an amine or a halogen. Also in embodiments of the synthetic method, the linker moiety includes an amino functional group. Still other embodiments of this invention include reacting a linker moiety that further includes a hydroxyl, an amino or a carboxy functional group capable of forming a covalent attachment to a polymer matrix. In an exemplary embodiment, the phenyl group on the polyhydroxylate analyte binding moiety includes a reactive halogen.
Other aspects of the invention include an oxazine-based boronate fluorescent biosensor systems for measuring in-vivo levels of a polyhydroxylate analyte, particularly glucose. Embodiments of these biosensor systems include an oxazine-based fluorescent biosensor molecule attached to, or contained within, a polymer matrix to yield an oxazine-based biosensor of the biosensor system. These biosensor systems also include an optical light source and a detector that detects a fluorescent signal that can be correlated with in-vivo levels of the polyhydroxylate analyte.
Embodiments of the biosensor system of the invention include a biosensor that is implanted subcutaneously beneath a person's skin. These biosensors may also include various agents tha
Heiss Aaron M.
Lane Stephen M.
Mastrototaro John Joseph
Noronha Glenn
Peyser Thomas A.
Gates & Cooper LLP
Medtronic MiniMed, Inc.
Winakur Eric F.
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