Chemistry: molecular biology and microbiology – High energy substrates
Reexamination Certificate
1999-12-10
2001-02-27
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
High energy substrates
C435S007200, C435S007100, C435S004000, C428S402200
Reexamination Certificate
active
06194213
ABSTRACT:
FIELD OF INVENTION
This invention relates to the field of detection of lipid membranes; and more particularly to functionalized nanocrystals which may be used to contact and incorporate into lipid-containing membranes in fluorescently labeling the membranes.
BACKGROUND OF THE INVENTION
Fluorescent dyes have a wide variety of uses including the labeling of proteins (e.g., antibodies), DNA, carbohydrates, and cells. Fluorescent-labeled substrates have been used for visualization and/or quantitative measurements in various applications including biology, medicine, electronics, biomedicine, and forensics. Typically, conventional fluorescent dyes are used to study biochemical, pharmacological, or pathological changes that occur in tissue by labeling various cell components. Examples of conventional fluorescent dyes which are used to detect cell components comprising lipid membranes include osmium tetraoxide, octadecyl rhodamine B, 2-hydroxyethyl-7,12,17-tris(methoxyethyl)porphycene, and 1,6-diphenyl-1,3,5-hexatriene. However, typically fluorescent dyes have characteristics which interfere with their usefulness. For example, many fluorescent dyes presently used do not have significant absorbance at the desired excitation wave-lengths, or are unstable in aqueous solutions, or are unstable during illumination. More specifically, conventional fluorescent dyes generally suffer from short-lived fluorescence; e.g., undergo photobleaching after minutes of exposure to an excitation light source. Thus, they are not very suitable for applications which requiring a significant length of time needed for ascertaining a staining pattern. Further, conventional fluorescent dyes are sensitive to changes in environment which can decrease their quantum yield. Another disadvantage of conventional fluorescent dyes is that typically the excitation spectrum of a species of fluorescent dye may be quite narrow. However, even when a single light source is used to provide excitation wavelength spectrum, in view of the spectral line width there often is insufficient spectral spacing between the emission optima of different species of fluorescent dyes to permit individual and quantitative detection without substantial spectral overlap. Thus, when using a combination of different fluorescent dyes as labels, multiple filters are typically needed to detect the resultant emission spectra of the combination. Conventional fluorescent dyes are limited in sensitivity and resolution of imaging due to the limitations of intensity, photobleaching, and the finite number of molecules which can be used to label a substrate.
Semiconductor nanocrystals (“quantum dots”) are known in the art. Generally, quantum dots can be prepared which result in relative monodispersity; e.g., the diameter of the core varying approximately less than 10% between quantum dots in the preparation. Examples of quantum dots are known in the art to have a core selected from the group consisting of Group II-VI semiconductor materials, or Group III-V semiconductor materials. Preferred, illustrative examples include CdSe, CdS, or CdTe (collectively referred to as “CdX”). Quantum dots have been passivated with an inorganic coating (“shell”) uniformly deposited thereon. Passivating the surface of the core quantum dot can result in an increase in the quantum yield of the fluorescence emission, depending on the nature of the inorganic coating. The shell which is used to passivate the quantum dot is preferably comprised of YZ wherein Y is Cd or Zn, and Z is S, or Se. Generally, quantum dots (core or core passivated with a shell) have only been soluble in organic, non-polar (or weakly polar) solvents. Thus, the instability of these quantum dots in aqueous media has limited their usefulness in biological applications.
To make quantum dots useful in applications for detection, it is desirable that the quantum dots are water-soluble. “Water-soluble” is used herein to mean sufficiently soluble or suspendable in a aqueous-based solution, such as in water or water-based solutions or physiological solutions, including those used in biological or molecular detection systems as known by those skilled in the art. Several attempts have been made to impart water solubility to nanocrystals such as by treating the water-insoluble quantum dots with a large excess of mercaptocarboxylic acid in CHCl
3
solution (Chan and Nie, 1998, Science 281:2016-2018), or by silicanizing the surface of quantum dots (U.S. Pat. No. 5,990,479). However, depending on the nature of the coating group, quantum dots which have been reported as water-soluble 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 coating to become oxidized, thereby forming disulfides which destabilize the attachment of the coating molecules to the shell. Thus, oxidation may cause the coating molecules to migrate away from the surface of the quantum dots, thereby exposing the surface of the quantum dots in resulting 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 nanocrystals. Additionally, such treated quantum dots are not lipophilic.
Thus, current fluorescent molecules (fluorescent dyes and quantum dots) have characteristics which can limit their usefulness in labeling lipid membranes. In that regard, provided herein are fluorescent molecules that are: (a) functionalized to enhance stability in aqueous environments; (b) functionalized to be lipophilic; (c) extremely sensitive in terms of detection, because of their fluorescent properties (e.g., including, but not limited to, high quantum efficiency, resistance to photobleaching, and stability in complex aqueous environments); and (d) a class of semiconductor nanocrystals that may be excited with a single wavelength of light resulting in detectable fluorescence emissions of high quantum yield and with discrete fluorescence peaks.
SUMMARY OF THE INVENTION
The present invention provides a method for fluorescence labeling of lipid-containing membranes (for purposes of brevity, “lipid membranes”) by contacting the membranes desired to be labeled with an effective amount of nanocrystals functionalized to be lipophilic. The present invention provides a method for fluorescence detection of lipid membranes by contacting the membranes desired to be detected with an effective amount of lipophilic functionalized nanocrystals in labeling the membranes, exposing the labeled membranes to a excitation light source, and then detecting (one or more of visualizing, imaging, measuring, and quantitating) the fluorescence emitted from the excited functionalized nanocrystals in the labeled membranes. The functionalized nanocrystals comprise quantum dots capped with a polar capping compound (multiple molecules of capping compound also referred herein, for ease of reference, as a layer of capping compound), and molecules of an amino acid (diaminocarboxylic acid or monoaminocarboxylic acid) which are operatively linked to the layer of polar capping compound (multiple amino acid molecules also referred to herein for ease of reference as a layer of amino acid). The functionalized nanocrystals may further comprise one or more operatively linked successive amino acid layers operatively linked to the first layer of amino acid.
In a method of fluorescence detection of lipid membranes using functionalized nanocrystals, an effective amount of functionalized nanocrystals may be mixed with a suitable physiologically acceptable carrier (e.g., an aqueous solution); the resultant mixture is then placed in contact with substrate comprising the lipid membranes to be labeled; the labeled membranes are then exposed to a light source comprising an excitation spectrum in the range of from about 190 nanometers (nm) to about 660 nm (the highest functional wavelength for excitation may depend on the wavelength of the maximum peak of the emission
Bio-Pixels Ltd.
Leary Louise N.
Nelson M Bud
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