| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | August 2, 2005 |
| PATENT TITLE |
Method for identifying cells |
| PATENT ABSTRACT |
Disclosed are silica-coated nanoparticles and a process for producing silica-coated nanoparticles. Silica-coated nanoparticles are prepared by precipitating nano-sized cores from reagents dissolved in the aqueous compartment of a water-in-oil microemulsion. A reactive silicate is added to coat the cores with silica. Also disclosed are methods for functionalizing silica-coated nanoparticles for use in a variety of applications. -------------------------------------------------------------------------------- |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | November 13, 2001 |
| PATENT REFERENCES CITED |
Liz L et al., "Preparation of Colloidal FE3O4 Ultrafine Particles In Microemulsions" Journal of Materials Science, 1994, pp. 3797-3801, vol. 29, ISSN: 0022-2461, Chapman and Hall Ltd., London, England. Albert P. Philipse, Michel P.B. Van Bruggen and C. Pathmamanoharan, "Magnetic Silica Dispersions: Preparation and Stability of Surface-Modified Silica Particles with a Magnetic Core" LANGMUIR 1994, pp. 92-99, vol. 10, No. 1, XP-002309610, Van Hoff Laboratory for Physical and Colloid Chemistry, Utrecht University, The Netherlands. Song-Yuan Chang, Lei Liu and Sanford A: Asher, "Preparation and Properties of Tailored Morphology, Monodisperse Colloidal Sillca-Cadmium Sulfide Nanocomposites" Journal of American Chemical Society, 1994, pp. 6739-6744, XP-002309608, Department of Chemistry, University of Pittsburgh, Pennsylvania. M.D. Butterworth, S.A. Bell; S.P. Armes, and A.W. Simpson, "Synthesis and Characterization of Polypyrrole-Magnetite-Sillca Particles" Journal of Colloid and Interface Science, pp. 91-99, Article No. 0521, School of Chemistry and Molecular Sciences, and School of Engineering, University of Sussex, Brighton, United Kingdom, © Academic Press, Inc. Sebastien Vaucher, Mei Li, and Stephen Mann, "Synthesis of Prussian Blue Nanoparticles and Nanocrystal Superlattices in Reverse Microemulsions" Agnew. Chem., 2000, pp. 1863-1866, XP-002309611, WILEY-VCH Verlag, Weinheim. |
| PATENT GOVERNMENT INTERESTS |
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH This invention was made with U.S. government support under grant number N00014-98-1-0621 awarded by the Office of Naval Research and grant number NSF BIO-9871880 awarded by the National Science Foundation. The U.S. government may have certain rights in the invention |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
1. A method of identifying cells expressing a preselected molecule comprising the steps of: providing a plurality of cells at least some of which express the preselected molecule; providing a plurality of silica-coated nanoparticles coated with a functional group that binds to the preselected molecule, each of said nanoparticles having a mean size of less than 1 micron and comprising a core comprising a pigment and a silica shell enveloping the core, wherein the pigment is an inorganic salt selected from the group consisting of potassium permanganate, potassium dichromate, nickel sulfate, cobalt-chloride, iron (III) chloride, and copper nitrate; mixing the plurality of silica-coated nanoparticles with the plurality of cells to form a mixture; placing the mixture under conditions that allow the nanoparticles to bind to cells expressing the preselected molecule; and analyzing the cells for bound nanoparticles to identify the cells expressing the preselected molecule. 2. The method of claim 1, wherein the silica-coated nanoparticles are fluorescent. 3. The method of daim 1, wherein the nanoparticles have a mean size between 1 nm and 300 nm. 4. The method of claim 1, wherein the nanoparticles have a mean size between 2 nm and 10 nm. 5. The method of claim 1, wherein the functional group is a protein. 6. The method of claim 5, wherein the protein is an antibody that specifically binds to the preselected molecule. 7. The method of claim 1, wherein the functional group is a nucleic acid. 8. The method of claim 1, wherein the functional group is a substance selected from the group consisting of biotin and streptavidin. 9. The method of claim 1, wherein the silica shell comprises a reactive silicate selected from the group consisting of TEOS (tetraethylorthosilicate) and APTS (aminopropyltrimethoxysilane). 10. A method of identifying cells expressing a preselected molecule comprising the steps of: providing a plurality of cells at least some of which express the preselected molecule; providing a plurality of silica-coated nanoparticles coated with a functional group that binds to the preselected molecule, each of said nanoparticles having a mean size of less than 1 micron and comprising a core comprising a dye and a silica shell enveloping the core; mixing the plurality of silica-coated nanoparticles with the plurality of cells to form a mixture; placing the mixture under conditions that allow the nanoparticles to bind to cells expressing the preselected molecule; and analyzing the cells for bound nanoparticles to identify the cells expressing the preselected molecule. 11. The method of claim 10, wherein the silica-coated nanoparticles are fluorescent. 12. The method of claim 10, wherein the nanoparticles have a mean size between 1 nm and 300 nm. 13. The method of claim 10, wherein the nanoparticles have a mean size between 2 nm and 10 nm. 14. The method of claim 10, wherein the dye is selected from the group consisting of Ruthenium-tris(2,2-bipyridyl)dichloride and Europium-bis(2,2-bipyridyl)trichloride. 15. The method of claim 10, wherein the functional group is a protein. 16. The method of claim 15, wherein the protein is an antibody that specifically binds to the preselected molecule. 17. The method of claim 10, wherein the functional group is a nucleic acid. 18. The method of claim 10, wherein the functional group is a substance selected from the group consisting of biotin and streptavidin. 19. The method of claim 10, wherein the silica shell comprises a reactive silicate selected from the group consisting of TEOS (tetraethylorthosilicate) and APTS (aminopropyltrimethoxysilane). 20. A method of identifying cells expressing a preselected molecule comprising the steps of: providing a plurality of cells at least some of which express the preselected molecule; providing a plurality of silica-coated nanoparticles coated with a functional group that binds to the preselected molecule, each of said nanoparticles having a mean size of between 2 nm and 10 nm and comprising a core comprising a metal and a silica shell enveloping the core; mixing the plurality of silica-coated nanoparticles with the plurality of cells to form a mixture; placing the mixture under conditions that allow the nanoparticles to bind to cells expressing the preselected molecule; and analyzing the cells for bound nanoparticles to identify the cells expressing the preselected molecule. 21. The method of claim 20, wherein the core is magnetic. 22. The method of claim 21, wherein the core comprises a metal selected from the group consisting of magnetite, maghemite, and greigite. 23. The method of claim 20, wherein the functional group is a protein. 24. The method of claim 23, wherein the protein is an antibody that specifically binds to the preselected molecule. 25. The method of claim 20, wherein the functional group is a nucleic acid. 26. The method of claim 20, wherein the functional group is a substance selected from the group consisting of biotin and streptavidin. 27. The method of claim 20, wherein the silica shell comprises a reactive silicate selected from the group consisting of TEOS (tetraethylorthosilicate) and APTS (aminopropyltrimethoxysilane). 28. A method of identifying cells expressing a preselected molecule comprising the steps of: providing a plurality of cells at least some of which express the preselected molecule; providing a plurality of silica-coated nanoparticles coated with a functional group that binds to the preselected molecule, each of said nanoparticles having a mean size of less than 1 micron and comprising a core comprising Ag and a silica shell enveloping the core; mixing the plurality of silica-coated nanopartides with the plurality of cells to form a mixture; placing the rruxture under conditions that allow the nanoparticles to bind to cells expressing the preselected molecule; and analyzing the cells for bound nanoparticles to identify the cells expressing the preselected molecule. 29. The method of claim 28, wherein the nanoparticles have a mean size between 1 nm and 300 nm. 30. The method of claim 28, wherein the nanoparticles have a mean size between 2 nm and 20 nm. 31. The method of claim 28, wherein the functional group is a protein. 32. The method of claim 31, wherein the protein is an antibody that specifically binds to the preselected molecule. 33. The method of claim 28, wherein the functional group is a nucleic acid. 34. The method of claim 28, wherein the functional group is a substance selected from the group consisting of biotin and streptavidin. 35. The method of claim 28, wherein the silica shell comprises a reactive silicate selected from the group consisting of TEOS (tetraethylorthosilicate) and APTS (aminopropyltrimethoxysilane). -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The invention relates generally to the field of nanoparticles and methods of making nanoparticles. More particularly, the invention relates to silica-coated nanoparticles prepared by using microemulsions. BACKGROUND OF THE INVENTION Nanoparticles are very small particles typically ranging in size from as small as one nanometer to as large as several hundred nanometers in diameter. Their small size allows nanoparticles to be exploited to produce a variety of products such as dyes and pigments; aesthetic or functional coatings; tools for biological discovery, medical imaging, and therapeutics; magnetic recording media; quantum dots; and even uniform and nanosize semiconductors. Nanoparticles can be simple aggregations of molecules or they can be structured into two or more layers of different substances. For example, simple nanoparticles consisting of magnetite or maghemite can be used in magnetic applications (e.g., MRI contrast agents, cell separation tools, or data storage). See, e.g., Scientific and Clinical Applications of Magnetic Microspheres, U. Häfeli, W. Schütt, J. Teller, and M. Zborowski (eds.) Plenum Press, New York, 1997; Sjøgren et al., Magn.Reson. Med. 31: 268, 1994; and Tiefenauer et al., Bioconjugate Chem. 4:347, 1993. More complex nanoparticles can consist of a core made of one substance and a shell made of another. Many different type of small particles (nanoparticles or micron-sized particles) are commercially available from several different manufacturers including: Bangs Laboratories (Fishers, Ind.); Promega (Madison, Wis.); Dynal Inc.(Lake Success, N.Y.); Advanced Magnetics Inc.(Surrey, U.K.); CPG Inc.(Lincoln Park, N.J.); Cortex Biochem (San Leandro, Calif.); European Institute of Science (Lund, Sweden); Ferrofluidics Corp. (Nashua, N.H.); FeRx Inc.; (San Diego, Calif.); Immunicon Corp.; (Huntingdon Valley, Pa.); Magnetically Delivered Therapeutics Inc. (San Diego, Calif.); Miltenyi Biotec GmbH (USA); Microcaps GmbH (Rostock, Germany); PolyMicrospheres Inc. (Indianapolis, Ind.); Scigen Ltd.(Kent, U.K.); Seradyn Inc.; (Indianapolis, Ind.); and Spherotech Inc. (Libertyville, Ill.). Most of these particles are made using conventional techniques, such as grinding and milling, emulsion polymerization, block copolymerization, and microemulsion. Methods of making silica nanoparticles have also been reported. The processes involve crystallite core aggregation (Philipse et al., Langmuir, 10:92, 1994); fortification of superparamagnetic polymer nanoparticles with intercalated silica (Gruttner, C and J Teller, Journal of Magnetism and Magnetic Materials, 194:8, 1999); and microwave-mediated self-assembly (Correa-Duarte et al., Langmuir, 14:6430, 1998). Unfortunately, these techniques have not proven to be particularly efficient for consistently fabricating nanoparticles with a particular size, shape and size distribution. SUMMARY OF THE INVENTION The invention relates to a new method for preparing nanoparticles having a core enveloped by a silica (SiO2) shell. Such silica-coated nanoparticles can be used, for example, as dye-doped particles, "pigmentless" pigment particles, metal particles, semiconductor particles, magnetic particles, and drug molecule particles. The method employs a microemulsion, i.e., isotropic and thermodynamically stable single-phase system, to produce nanoparticles cores of a predetermined, very uniform size and shape. Cores produced using the microemuslion are then coated with silica using a silicating agent. The nanoparticles thus formed can be customized for a particular application by derivatizing various chemical groups onto the silica coating. Accordingly, the invention features nanoparticles having a core and a silica shell enveloping the core. The nanoparticles can have a mean size of less than 1 micron (e.g., between 1 nm and 300 nm, or between 2 nm and 10 nm). In some variations, the nanoparticle cores can be magnetic and can include a metal selected from the group consisting of magnetite, maghemite, and greigite. In other variations, the core includes a pigment which can be an inorganic salt such as potassium permanganate, potassium dichromate, nickel sulfate, cobalt-chloride, iron(III) chloride, or copper nitrate. Similarly, the core can include a dye such as Ru/Bpy, Eu/Bpy, or the like; or a metal such as Ag and Cd. The invention also features nanoparticles with a silica shell that is derivatized with a functional group such as a protein (e.g., an antibody); a nucleic acid (e.g., an oligonucleotide); biotin; or streptavidin. Also within the invention is a method of making coated nanoparticles. This method includes the steps of: providing a microemulsion; providing a first aqueous solution of a first reactant and a second aqueous solution of a second reactant (the first reactant and second reactant being selected such that a solid precipitate forms upon mixing the first and second reactants together in an aqueous environment); adding the first aqueous solution to a first aliquot of the microemulsion and the second aqueous solution to a second aliquot of the microemulsion; mixing together the first and second aliquots to form a reaction mixture that reacts to form nanoparticle cores; and adding a coating agent to the cores to form coated nanoparticles. The microemulsion can be a water-in-oil microemulsion that can be made by mixing together water; a relatively polar liquid such as isooctane, n-hexane, or cyclohexane; a surfactant such as AOT, TX-100, and CTAB; and, in some cases, a cosurfactant such as n-hexanol. The coating agent can be a reactive silicate such as TEOS and APTS. In some variations, the method includes a step of derivatizing the silica shell with a functional group such as a protein (e.g., an antibody); a nucleic acid (e.g., an oligonucleotide); biotin; or streptavidin. Thus, the method of the invention can be used to make protein-derivatized, silica-coated nanoparticles having cores including a metal such as magnetite, maghemite, or greigite. In another aspect, the invention features a method of identifying cells expressing a preselected molecule. This method includes the steps of: providing a plurality of silica-coated nanoparticles coated with a functional group that binds to a preselected molecule; providing a plurality of cells at least some of which express the preselected molecule; mixing the plurality of silica-coated nanoparticles with the plurality of cells to form a mixture; placing the mixture under conditions that allow the nanoparticles to bind to cells expressing the preselected molecule; and analyzing the cells for bound nanoparticles. In one variation of this method, the functional group is an antibody that specifically binds to the preselected molecule. In another variation, the silica-coated nanoparticles are fluorescent. As used herein, the word "nanoparticle" means a particle having a diameter of between about 1 and 1000 nm. Similarly, by the term "nanoparticles" is meant a plurality of particles having an average diameter of between about 1 and 1000 nm. For the purposes herein, a microemulsion is defined as a thermodynamically stable, optically isotropic dispersion of two immiscible liquids consisting of nanosize domains of one or both liquids in the other, stabilized by an interfacial film of surface-active molecules. By reference to the "size" of a nanoparticle is meant the length of the largest straight dimension of the nanoparticle. For example, the size of a perfectly spherical nanoparticle is its diameter. By the phrase "specifically binds" means that one molecule recognizes and adheres to a particular second molecule in a sample, but does not substantially recognize or adhere to other molecules in the sample. Generally, an antibody that "specifically binds" a preselected antigen is one that binds the antigen with a binding affinity greater than about 105 to 106 liters/mole. As used herein, the phrase "functional group" means a chemical group that imparts a particular function to an article (e.g., nanoparticle) bearing the chemical group. For example, functional groups can include substances such as antibodies, oligonucleotides, biotin, or streptavidin that are known to bind particular molecules; or small chemical groups such as amines, carboxylates, and the like. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions will control. In addition, the particular embodiments discussed below are illustrative only and not intended to be limiting. |
| PATENT EXAMPLES | available on request |
| PATENT PHOTOCOPY | available on request |
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