Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing – Diagnostic or test agent produces in vivo fluorescence
Reexamination Certificate
2001-02-12
2004-07-13
Acquah, Samuel A. (Department: 1711)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
Diagnostic or test agent produces in vivo fluorescence
C428S402240, C428S403000, C428S404000, C427S213300, C427S215000, C427S220000, C424S009100, C424S009320, C424S009321, C424S009360
Reexamination Certificate
active
06761877
ABSTRACT:
FIELD OF INVENTION
This invention relates generally to novel compositions comprising encapsulated, fluorescent nanocrystals. More particularly, the present invention relates to the use of a vesicle or capsid to encapsulate fluorescent nanocrystals in forming water-soluble fluorescent nanocrystals.
BACKGROUND OF THE INVENTION
Nonisotopic detection systems have become a preferred mode in scientific research and clinical diagnostics for the detection of biomolecules using various assays including, but not limited to, flow cytometry, nucleic acid hybridization, DNA sequencing, nucleic acid amplification, microarrays, immunoassays, histochemistry, and functional assays involving living cells. In particular, while fluorescent organic molecules such as fluorescein and phycoerythrin are used frequently in detection systems, there are disadvantages in using these molecules in combination. For example, each type of fluorescent molecule typically requires excitation with photons of a different wavelength as compared to that required for another type of fluorescent molecule. However, even when a single light source is used to provide a single excitation wavelength (in view of the spectral line width), often there is insufficient spectral spacing between the emission optima of different fluorescent molecules to permit individual and quantitative detection without substantial spectral overlap. Additionally, conventional fluorescent molecules have limited fluorescence intensity. Further, currently available nonisotopic detection systems typically are limited in sensitivity due to the finite number of nonisotopic molecules which can be used to label a biomolecule to be detected.
Doped metal oxide (“dMO”) nanocrystals are nanocrystals that can be excited with a single excitation light source resulting in a detectable fluorescence emission of high quantum yield (e.g., a single quantum dot having at a fluorescence intensity that may be a log or more greater than that a molecule of a conventional fluorescent dye) and with a discrete fluorescence peak. Typically, they have a substantially uniform size of less than 200 Angstroms, and preferably have a substantially uniform size in the range of sizes of from about 1 nm to about 5 nm, or less than 1 nm. In that regard, dMO nanocrystals are preferably comprised of metal oxides doped with one or more rare earth elements, wherein the dopant comprising the rare earth element is capable of being excited (e.g., with ultraviolet light) to produce a narrow spectrum of fluorescence emission (typically more narrow than the spectrum of fluorescence emission emitted by a semiconductor nanocrystal). Such dMO nanocrystals are well known in the art. However, a desirable feature of dMO nanocrystals when used for nonisotopic detection applications is that the nanocrystals be made water-soluble. “Water-soluble” is used herein to mean that the nanocrystals are sufficiently soluble or suspendable in an aqueous-based solution including, but not limited to, water, water-based solutions, and buffer solutions, that are used in detection processes, as known by those skilled in the diagnostic art.
Semiconductor nanocrystals are quantum dots that can be excited with a single excitation light source resulting in a detectable fluorescence emission of high quantum yield (e.g., a single quantum dot having at a fluorescence intensity that may be a log or more greater than that a molecule of a conventional fluorescent dye) and with a discrete fluorescence peak. Typically, they have a substantially uniform size of less than 200 Angstroms, and preferably have a substantially uniform size in the range of sizes of from about 1 nm to about 5 nm, or less than 1 nm. In that regard, quantum dots are preferably comprised of a Group II-VI semiconductor material (of which ZnS, and CdSe are illustrative examples), or a Group III-V semiconductor material (of which GaAs is an illustrative example). Such quantum dots are well known in the art. However, a desirable feature of quantum dots when used for nonisotopic detection applications is that the quantum dots be made water-soluble. Current methods of making semiconductor nanocrystals water-soluble is to add to the semiconductor nanocrystal a layer comprising mercaptocarboxylic acid (Chen and Nie, 1998
, Science
281:2016-2018), or silica (U.S. Pat. No. 5,990,479), or one or more layers of amino acids (U.S. Pat. No. 6,114,038). Depending on which layer composition is used, the treated nanocrystal may have limited stability in an aqueous solution, particularly when exposed to air (oxygen) and/or light. More particularly, oxygen and light can cause the molecules comprising the layer to become oxidized, thereby forming disulfides which destabilize the attachment of the layer molecules to the semiconductor nanocrystals. Thus, oxidation may cause the layer molecules to become detached from the surface of the quantum dots, thereby exposing the surface of the quantum dots which may result in “destabilized quantum dots”. Destabilized quantum dots form aggregates when they interact together, and the formation of such aggregates eventually leads to irreversible flocculation of the quantum dots. Additionally, depending on the layer composition, it can cause non-specific binding, particularly to one or more molecules in a sample other than the target molecule, which is not desirable in a detection assay.
Hence, there is a need to provide alternative forms of water-soluble, fluorescent nanocrystals.
SUMMARY OF THE INVENTION
It is a primary object of the present invention to provide fluorescent nanocrystals which are encapsulated by a vesicle or capsid comprising a liposome.
It is another object of the present invention to provide fluorescent nanocrystals which are encapsulated by or trapped within a vesicle or capsid comprising a liposome, and wherein the surface of the liposome is functionalized with surface groups comprising a reactive functionality that may be used to form a bond with one or more molecules of an affinity molecule which has a reactive functionality which is capable of forming a bond with the surface groups of the liposome.
It is another object of the present invention to provide a fluorescent nanocrystal which comprises one or more fluorescent nanocrystals encapsulated by or trapped within a liposome, and wherein the surface of the liposome is functionalized with surface groups comprising one or more reactive functionalities.
It is another object of the present invention to provide a functionalized, encapsulated fluorescent nanocrystal which comprises one or more fluorescent nanocrystals encapsulated by or trapped within a liposome which is functionalized by the addition of one or more affinity molecules.
It is further object of the present invention to provide a functionalized, encapsulated fluorescent nanocrystal which comprises one or more fluorescent nanocrystals encapsulated by or trapped within a liposome, and wherein the liposome portion may be disrupted to release the fluorescent nanocrystals in a method of “quenching” the fluorescence in a reaction.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
By the term “fluorescent nanocrystals” is meant, for purposes of the specification and claims to refer to fluorescent nanocrystals comprised of doped metal oxide nanocrystals, semiconductor nanocrystals, or a combination thereof.
By the terms “doped metal oxide nanocrystals” or “dMO nanocrystals” is meant, for purposes of the specification and claims to refer to nanocrystals comprised of: a metal oxide, and a dopant comprised of one or more rare earth elements. For example, suitable metal oxides include, but are not limited to, yttrium oxide (Y
2
O
3
), zirconium oxide (ZrO
2
), zinc oxide (ZnO), copper oxide (CuO or Cu
2
O), gadolinium oxide (Gd
2
O
3
), praseodymium oxide (Pr
2
O
3
), lanthanum oxide (La
2
O
3
), and alloys thereof. The rare earth element comprises an element selected from the Lanthanide series and includes, but is not limited to, europium (Eu), cerium (Ce), neodymium (Nd), samarium (Sm), terbium (Tb), gadolinium (Gd),
Acquah Samuel A.
BioCrystal Ltd.
Miller Raymond A.
Pepper Hamilton LLP
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