Stock material or miscellaneous articles – Coated or structually defined flake – particle – cell – strand,... – Particulate matter
Reexamination Certificate
2000-06-08
2001-11-20
Nutter, Nathan M. (Department: 1711)
Stock material or miscellaneous articles
Coated or structually defined flake, particle, cell, strand,...
Particulate matter
C428S404000, C257S065000, C257S614000, C257S642000, C427S213300, C427S214000, C427S215000, C427S220000, C424S009100, C424S009320, C424S009360, C424S009420, C424S009600
Reexamination Certificate
active
06319607
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to fluorescent nanocrystals; and more particularly, to a novel method for purifying, or synthesizing and purifying, functionalized fluorescent nanocrystals having affinity ligand operably bound thereto using a solid support system.
BACKGROUND OF THE INVENTION
Typically, conventional fluorescent dyes (e.g., fluorescein, rhodamine, phycoerythrin, and the like) are used for labeling microspheres. These conventional fluorescent dyes typically have an excitation spectrum that may be quite narrow; hence, it is often difficult to find a wave-length spectrum of light suitable for simultaneously exciting several different fluorescent labels (e.g., differing in color of fluorescence emission). However, even when a single light source is used to provide a excitation wave-length spectrum (in view of the spectral line width), often there is insufficient spectral spacing between the emission optima of different species (e.g., differing in color) of fluorescent dyes to permit individual and quantitative detection without substantial spectral overlap. Additionally, conventional fluorescent dyes are susceptible to photobleaching which limits the time in which a fluorescent signal can be detected, and limits time-resolved fluorescence (fluorescent signal integration over time). Additional limitations of fluorescent dyes include fluorescence quenching, and shifts in fluorescence emission spectra, depending on the environment in which dyes are excited.
Fluorescent nanocrystals comprising either semi-conductor nanocrystals or doped metal oxide nanocrystals have been reported to resist photobleaching, share an excitation wavelength spectrum, and are capable of emitting fluorescence of high quantum yield and with discrete peak emission spectra. However, these nanocrystals lack sufficient solubility in aqueous-based environments required in fluorescence-based biological assays; i.e., in aqueous-based environments, the nanocrystals interact together in forming aggregates, which leads to irreversible flocculation of the nanocrystals. As disclosed in detail in U.S. application Ser. Nos. 09/372,729 and 09/577,761 (the disclosures of which are herein incorporated by reference), functionalized fluorescent nanocrystals comprise fluorescent nanocrystals which have been functionalized by the addition of a plurality of molecules; and preferably, the molecules are selected from an amino acid, a carboxylic acid, and a combination thereof. A plurality of these molecules, when operably bound to a fluorescent nanocrystal, functionalizes the fluorescent nanocrystal to become water-soluble, as well as provides reactive functionalities which may be used to operably bind one or more molecules of affinity ligand. Functionalized fluorescent nanocrystals, comprising affinity ligand operably bound thereto, may be placed in contact with a sample being analyzed in a biological assay for the presence or absence of a substrate for which the affinity ligand has binding specificity. Contact, and subsequent binding, between affinity ligand of the functionalized fluorescent nanocrystals and the substrate, if present in the sample, results in a complex comprising the functionalized fluorescent nanocrystals-substrate which can emit a detectable fluorescence signal for quantitation, visualization, or other form of detection.
However, current methods of functionalizing fluorescent nanocrystals with affinity ligand suffer from a serious drawback. Even with a high efficiency of operably binding functionalized fluorescent nanocrystals with affinity ligand, the coupling reaction may result in a significant number of functionalized fluorescent nanocrystals which do not become operably bound to affinity ligand, and molecules of affinity ligand which do not become operably bound to functionalized fluorescent nanocrystals (“free affinity ligand”); a phenomenon referred to as “failed coupling”. In a biological assay in which an amount of substrate is to be detected using a detection reagent which contains both functionalized fluorescent nanocrystals having affinity ligand operably bound thereto, and free affinity ligand, free affinity ligand can compete with the affinity ligand of functionalized fluorescent nanocrystals for binding the substrate. Particularly in instances when a minute amount of substrate is present, the effect of binding substrate by free affinity ligand in the detection reagent can lead to an undesirable and significant loss of sensitivity in the assay. In contrast, functionalized fluorescent nanocrystals which are not operably bound to affinity ligand will be washed away from a detection system, and hence, do not pose a significant problem if present in a detection reagent.
Thus, there remains a need for a process of purifying functionalized fluorescent nanocrystals substantially free of free affinity ligand that may be present after a step of operably binding affinity ligand to the functionalized fluorescent nanocrystals. Also needed is a purification method which is simple, uses relatively few reagents, and provides functionalized fluorescent nanocrystals having affinity ligand operably bound thereto at a high level of purity for use in fluorescence-based detection systems.
SUMMARY OF THE INVENTION
The present invention overcomes the problems outlined above by providing methods for producing functionalized fluorescent nanocrystals having affinity ligand operably bound thereto which are purified from free affinity ligand. Thus, it is a primary object of the present invention to provide a process for preparing functionalized fluorescent nanocrystals using a solid support in a reactor, such as in the form of a column, through which solutions are circulated in a continuous flow process of purification.
It is another object of the present invention to provide a system in which a solid support matrix, such as contained in a reactor, is used to operably bind and immobilize functionalized fluorescent nanocrystals in forming a solid phase, and then free affinity ligand that may be present is flowed out of the reactor in a solution. In a subsequent step, the functionalized fluorescent nanocrystals are separated from the solid phase, and then flowed out of the reactor.
It is another object of the present invention to provide a method and system for synthesizing functionalized fluorescent nanocrystals, immobilized as part of a solid phase, to be operably bound to affinity ligand; and then purifying the resultant functionalized fluorescent nanocrystals to be substantially free of free affinity ligand by flowing the free affinity ligand through the solid phase and out of the system.
It is another object of the present invention to provide a method and system for purifying functionalized fluorescent nanocrystals operably bound to affinity ligand by flowing free affinity ligand that may be present through the solid phase and out of the system so that the resultant functionalized fluorescent nanocrystals are substantially free of free affinity ligand.
The above and other objects, features, and advantages of the present invention will be apparent in the following Detailed Description of the Invention.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
By the term “affinity ligand” is meant, for purposes of the specification and claims, to mean a molecule which has binding specificity and avidity for a molecular component of, or associated with, an analyte. In general, affinity ligands are known to those skilled in the art to include, but are not limited to, lectins or fragments (or derivatives) thereof which retain binding function; monoclonal antibodies (“mAb”, including chimeric or genetically modified monoclonal antibodies (e.g., “humanized”)); peptides; aptamers; nucleic acid molecules (including, but not limited to, single stranded RNA or single-stranded DNA, single-stranded nucleic acid hybrids, or nucleobases); avidin, or streptavidin, or avidin derivatives; and the like. The invention may be practiced using a preferred affinity ligand (e.g., a mAb) to the exclusion of affinity
Barbera-Guillem Emilio
Castro Stephanie L.
Bio-Pixels Ltd.
Nelson M. Bud
Nutter Nathan M.
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