WO2007143076A2 - Nanoparticules et nanoparticules revêtues - Google Patents

Nanoparticules et nanoparticules revêtues Download PDF

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Publication number
WO2007143076A2
WO2007143076A2 PCT/US2007/012908 US2007012908W WO2007143076A2 WO 2007143076 A2 WO2007143076 A2 WO 2007143076A2 US 2007012908 W US2007012908 W US 2007012908W WO 2007143076 A2 WO2007143076 A2 WO 2007143076A2
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nanoparticle
nanoparticles
magnetic
silica
functional group
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PCT/US2007/012908
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English (en)
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WO2007143076A3 (fr
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Jackie Y. Ying
Subramanian T. Selvan
Pranab K. Patra
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Agency For Science, Technology And Research
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Publication of WO2007143076A2 publication Critical patent/WO2007143076A2/fr
Publication of WO2007143076A3 publication Critical patent/WO2007143076A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0065—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle
    • A61K49/0067—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle quantum dots, fluorescent nanocrystals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y25/00—Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
    • C30B29/10—Inorganic compounds or compositions
    • C30B29/46—Sulfur-, selenium- or tellurium-containing compounds
    • C30B29/48—AIIBVI compounds wherein A is Zn, Cd or Hg, and B is S, Se or Te
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
    • C30B29/60—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape characterised by shape
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H01F1/0036—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties showing low dimensional magnetism, i.e. spin rearrangements due to a restriction of dimensions, e.g. showing giant magnetoresistivity
    • H01F1/0045—Zero dimensional, e.g. nanoparticles, soft nanoparticles for medical/biological use
    • H01F1/0054—Coated nanoparticles, e.g. nanoparticles coated with organic surfactant

Definitions

  • the present invention relates to semiconductor nanoparticles and magnetic semiconductor nanoparticles and related methods.
  • Semiconductor nanoparticles are highly emissive materials that may be particularly useful in a variety of applications.
  • semiconductor nanoparticles can have narrow and highly symmetric emission spectra, making them attractive for use as diagnostic tools, such as fluorescent probes in biological labeling and biological imaging.
  • Semiconductor nanoparticles may also exhibit high emission stability and strong resistance to photo-bleaching over long periods of time, providing advantages over known biological probing dyes, such as small molecule organic dyes.
  • Magnetic semiconductor nanoparticles consisting of semiconductor and magnetic nanoparticles are known as magnetic semiconductor nanoparticles or magnetic quantum dots.
  • Magnetic semiconductor nanoparticles contain a combination of optical and magnetic properties in a single material, which may be useful for various applications including biological labeling, magnetic resonance imaging (MRI), and drug targeting.
  • MRI magnetic resonance imaging
  • magnetic semiconductor nanoparticles may enable the simultaneous labeling and sorting of biological species.
  • Magnetic semiconductor nanoparticles have been previously prepared by separately synthesizing semiconductor nanoparticles and magnetic nanoparticles and then linking them together via, for example, a thiol linkage.
  • current methods often do not allow for the controlled tuning of magnetic and/or emissive properties of such nanoparticles, since it is difficult to control the linkage between the particles.
  • current methods for the synthesis of magnetic semiconductor nanoparticles may require the use of ZnS capping, a laborious process employing noxious chemicals, to retain the optical properties of the nanoparticle.
  • many magnetic semiconductor nanoparticles are not compatible with biological systems.
  • the present invention relates to magnetic semiconductor nanoparticles comprising a core comprising a semiconductor nanoparticle region and a magnetic nanoparticle region, wherein a surface of the semiconductor nanoparticle region is contactingly attached to but does not surround a surface of the magnetic nanoparticle region, and a shell at least partially encapsulating the core.
  • the present invention also provides methods of making a magnetic semiconductor nanoparticle comprising forming a semiconductor nanoparticle on a surface of a magnetic nanoparticle to form a nanoparticle core; and forming a silica shell at least partially encapsulating the nanoparticle core.
  • the present invention also provides methods of forming a silica coating on a nanoparticle, comprising forming a first layer on a surface of a nanoparticle, wherein the first layer comprises a species comprising a first functional group and a first silica precursor, wherein the first functional group is bonded to the surface of the nanoparticle and the first silica precursor is exposed at the surface of the first layer; and forming.
  • the second layer in contact with the first layer, the second layer comprising a species comprising a second functional group and a second silica precursor, wherein the second functional group is exposed at the surface, of the second layer and the second silica precursor interacts with the first silica precursor in the first layer to form a silica shell, wherein the silica shell at least partially encapsulates the nanoparticle.
  • the present invention also relates to coatings for nanoparticles comprising a first region comprising a first functional group bonded to a surface of the nanoparticle; a second region comprising a second functional group exposed at a surface of the coating; and a silica shell comprising at least two layers of silicon atoms, wherein the silica shell at least partially encapsulates the nanoparticle, wherein the first region and the second region are covalently linked to the silica shell.
  • FIG. 1 illustrates the synthesis of a magnetic semiconductor nanoparticle, according to one embodiment of the invention.
  • FIG. 2 illustrates the formation of a silica coating for nanoparticles, according to one embodiment of the invention.
  • FIG. 3 A shows a method for functionalization of a nanoparticle surface with a binding partner, according to one embodiment of the invention.
  • FIG. 3 B shows another method for ftmctionalization of a nanoparticle surface with a binding partner, according to one embodiment of the invention.
  • FIG. 4 shows photographs OfFe 2 Os-CdSe nanoparticles (a) under white light in the absence of a magnetic field, (b) under white light in the presence of a magnetic field, (c) under UV excitation at 365 nm and in the presence of a magnetic field, and (d) under UV excitation at 365 nm in the absence of a magnetic field.
  • FIG. 5 shows the (a) absorption spectra and (b) emission spectra of silica- coated magnetic semiconductor nanoparticles I-IV, having varying particle size as described in Table 1.
  • FIGS. 6A-B show TEM micrographs of heterodimers of a Fe 2 O 3 -CdSe nanoparticles.
  • FIG. 7 shows TEM micrographs of a dispersion of semiconductor nanoparticles around magnetic semiconductor nanoparticles at (a) low magnification and (b) high magnification.
  • FIG. 8 A shows an STEM image of a Fe 2 ⁇ 3 -CdSe nanoparticle and FIG. 8B shows the energy-dispersive X-ray (EDX) analysis of a F ⁇ 2 ⁇ 3 -CdSe nanoparticle.
  • EDX energy-dispersive X-ray
  • FIG. 9 shows confocal laser scanning microscopy (CLSM) images of (a) HepG2 cells labeled with CdSe nanoparticles, (b) NIH-3T3 cells labeled with CdSe nanoparticles, (c) HepG2 cells labeled with CdSe nanoparticles, and (d) 4Tl cell membranes labeled with F ⁇ 2 ⁇ 3 -CdSe nanoparticles.
  • CLSM confocal laser scanning microscopy
  • FIG. 10 shows (a) CLSM and (b) light microscopy images of 4Tl cell membranes labeled with orange-emitting Fe 2 Os-CdSe nanoparticles.
  • FIG. 11 shows (a) CLSM and (b) light microscopy images of NIH-3T3 cell membranes labeled with orange-emitting Fe 2 ⁇ 3 -CdSe nanoparticles.
  • FIG. 12 shows (a) CLSM and (b) light microscopy images of 4Tl cell membranes labeled using green-emitting Fe 2 ⁇ 3 ⁇ CdSe nanoparticles.
  • FIG. 13 shows (a) CLSM and (b) light microscopy images of HepG2 cell membranes labeled using green-emitting CdSe nanoparticles, where the CdSe nanoparticles are co-localized in the cytoplasm.
  • Nanoparticles of the invention may exhibit useful properties (e.g., fluorescent and/or magnetic properties) which can be tailored with a high degree of control using various methods of the invention.
  • the nanoparticles may be formed to exhibit a certain property or combination of properties, such as optical and/or magnetic properties.
  • Another aspect of the invention provides coatings (e.g., silica coatings) and related methods which may enhance various properties of the nanoparticles.
  • Nanoparticles described herein may be useful in applications such as labeling of biological species, drug targeting, magnetic resonance imagine (MRI), among other applications.
  • the nanoparticles may be useful in the labeling of cell membranes.
  • the present invention provides nanoparticles such as semiconductor nanoparticles, magnetic semiconductor nanoparticles, and the like.
  • nanoparticle generally refers to a particle having a maximum cross-sectional dimension of no more than 1 micron.
  • Nanoparticles can be made of material that is, e.g., inorganic or organic, polymeric, ceramic, semiconductor, magnetic, metallic, non-metallic, crystalline (e.g., "nanocrystals”), amorphous, or a combination.
  • “Semiconductor nanoparticles” or “quantum dots” are a class of nanoparticles that can provide unique emission spectra dependent, in part, on the size of the specific particle. “Magnetic nanoparticles” are a class of nanoparticles that exhibit magnetic properties. “Magnetic semiconductor nanoparticles” are nanoparticles that exhibit both luminescent and magnetic properties.
  • the invention provides magnetic semiconductor nanoparticles that comprise a core containing a semiconductor nanoparticle region and a magnetic nanoparticle region, wherein a surface of the semiconductor nanoparticle region is contactingly attached to but does not surround a surface of the magnetic nanoparticle region.
  • the semiconductor nanoparticle region may comprise a semiconductor nanoparticle material that exhibits luminescent properties, such as fluorescence and the magnetic nanoparticle region may comprise a magnetic nanoparticle material that exhibits magnetic properties.
  • the magnetic semiconductor nanoparticles can also comprise a coating or shell that at least partially encapsulates the core.
  • the semiconductor nanoparticle region can be a nanoparticle having a surface that is attached (e.g., contactingly attached) to the surface of the semiconductor nanoparticle region to form a nanoparticle 50 having a heterodimer structure, as shown in FIG. 1.
  • Nanoparticle 50 has a core comprising a magnetic nanoparticle region 10 attached to a semiconductor nanoparticle region 20, forming an interface 35. The core is encapsulated by a coating 40 to form nanoparticle region 50.
  • the heterodimer structure allows the nanoparticle to have a 1:1 ratio between the magnetic nanoparticle region and the semiconductor nanoparticle region.
  • the nanoparticle may have any ratio between the magnetic nanoparticle region and the semiconductor nanoparticle region suitable for use in a particular application.
  • a nanoparticle region that is "contactingly attached" to another nanoparticle region may refer to at least two nanoparticle regions contacting and attached to one another, wherein each nanoparticle region has at least one surface complementary in shape and/or contour to at least one surface on another of the nanoparticle region, forming an interface between the nanoparticle regions wherein the nanoparticle regions are attached to one another via the area formed by the interface.
  • FIG. 1 shows a nanoparticle 50 comprising two regions which contact each other and are attached (e.g. bonded) to each other along the area of interface 35, formed between a surface of magnetic nanoparticle region 10 and a surface of semiconductor nanoparticle 20.
  • "contactingly attached” does not refer to separate and distinct surfaces (e.g., of two nanoparticles) which are not attached to one another but can have transient contact with one another, or to separate and distinct surfaces that are attached to one another at a discrete point or points along the surfaces via a bond or bonds (e.g., covalent bond, non-covalent bond) and can have transient contact with one another.
  • a bond or bonds e.g., covalent bond, non-covalent bond
  • the invention also provides methods for synthesizing magnetic semiconductor nanoparticles.
  • the method comprises forming a semiconductor nanoparticle on a surface of a magnetic nanoparticle to form a nanoparticle core, followed by forming a silica coating or shell around the nanoparticle core to encapsulate or at least partially encapsulate the nanoparticle core.
  • magnetic nanoparticle region 10 is grown to have desired properties (e.g., particle size, magnetic properties, etc.) using methods known in the art.
  • Semiconductor nanoparticle region 20 can then be grown directly on the surface of magnetic nanoparticle region 10 to form nanoparticle core 30 having an interface 35 between the semiconductor nanoparticle region and the magnetic nanoparticle region.
  • the semiconductor nanoparticle region 20 may be grown using methods known in the art, such as flocculation with a non-solvent, to form a semiconductor nanoparticle region having desired properties (e.g., particle size, luminescent properties, etc.). Core 30 may then be treated to form a coating 40 to at least partially encapsulate the core, as described more fully below.
  • desired properties e.g., particle size, luminescent properties, etc.
  • Magnetic semiconductor nanoparticles may contain any combination of • semiconductor nanoparticle region or magnetic nanoparticle region, wherein each region may be selected to exhibit a desired property.
  • Semiconductor nanoparticle region can be synthesized to have a particular emission wavelength, which can be controlled by selection of nanoparticle size (e.g., diameter). For example, a CdSe semiconductor nanoparticle having a 3 nm diameter may produce a 520 nm emission, while a CdSe semiconductor nanoparticle having a 5.5 nm diameter may produce a 630 nm emission upon excitation with light having a frequency of 450 run, corresponding to "blue" light.
  • the magnetic nanoparticle region may be synthesized using known methods and may have any composition suitable for a particular application.
  • the magnetic nanoparticle region may comprise a pure metal (e.g., cobalt or nickel), a metal compound (e.g., Fe 2 Os ) > or alloy.
  • the magnetic nanoparticle region can be selected to provide desired magnetic properties.
  • the magnetic nanoparticles may be selected to be superparamagnetic at certain temperatures.
  • the semiconductor nanoparticle region comprises CdSe and the magnetic nanoparticle region comprises Fe 2 Os.
  • the present invention may be advantageous in that certain properties (e.g., fluorescence, magnetic properties, solubility, etc.) of the nanoparticles may be tailored with a high degree of control.
  • certain properties e.g., fluorescence, magnetic properties, solubility, etc.
  • the optical properties and/or magnetic properties may be easily tuned to suit a particular desired application by simply controlling, for example, the size of the semiconductor nanoparticle region. This may increase the predictability and reproducibility of synthetic methods for magnetic semiconductor nanoparticles, as well as enhance the performance of such nanoparticles.
  • nanoparticles of the invention may have a core at least partially encapsulated by a coating or shell.
  • the core may be fully encapsulated by the coating.
  • the coating may contain a first region comprising a first functional group bonded to a surface of the nanoparticle, a second region comprising a second functional group exposed at a surface of the coating, and a silica shell comprising at least two layers of silicon atoms, wherein the silica shell at least partially encapsulates the nanoparticle, and wherein the first region and the second region are covalently linked to the silica shell.
  • nanoparticle 80 has a silica coating comprising a first set of amine groups attached to the surface of the nanoparticle core and a second set of amine groups exposed at the surface of the coating.
  • the first set of amine groups is covalently linked to the second set of amine groups via the two layers of silicon atoms, which form a shell around the nanoparticle core.
  • the functional groups of the coating may impart desired characteristics (e.g., surface properties) to the nanoparticle. That is, the functional group may include a functionality that, when presented at the surface of the nanoparticle, may be able to confer upon the surface a specific property, such as an affinity for a particular entity or entities.
  • the functional group may include compounds, atoms, or materials that can alter or improve properties such as compatibility with a suspension medium (e.g., water solubility, water stability), photo-stability, and biocompatibility.
  • the functional group may act as a binding partner and may interact or form a bond (e.g., a covalent, ionic, hydrogen, or dative bond, or the like) with an analyte.
  • the functional group may be selected from among amine, carboxylic acid, anhydride, phosphate, hydroxyl, and thiol.
  • the coating comprises a first set of amines bonded to the surface of the nanoparticle and a second set of amines presented at the surface of the coating.
  • Functional groups may be selected to suit a particular application. In some cases, the functional group may be selected based on the ability to have an interaction with a particular analyte.
  • the term "analyte,” may refer to any chemical, biochemical, or biological entity (e.g. a molecule) to be analyzed.
  • the functional groups presented at the surface of the coating may comprising binding partners for biological species, as described more folly below.
  • One screening test for selection of an appropriate functional group may involve placing the functionalized nanoparticle in solution with an analyte and evaluating the ability of the functional group to bind the analyte (e.g., via interaction between pairs of biological molecules or via formation of a covalent bond).
  • the target analyte may be a cell
  • the surface of a magnetic semiconductor nanoparticle may be appropriately functionalized with a fatty acid such as an oleyl group for the purpose of interacting with cells.
  • the appropriate fatty acid may be selected by placing nanoparticles functionalized with various fatty acids in a solution with cells and then observing the fluorescence emission of the cells to determine which fatty acid most effectively binds to cells.
  • Those of ordinary skill in the art would be able to select functional groups which are appropriate for a particular desired application without undue experimentation.
  • a first layer may be formed on the surface of the nanoparticle, where the first layer comprises a species comprising a first functional group and a first silica precursor, wherein the first functional group is bonded to the surface of the nanoparticle and the first silica precursor is exposed at the surface of the first layer.
  • a second layer in contact with the first layer may be formed, where the second layer comprising a species comprising a second functional group and a second silica precursor, wherein the second functional group is exposed at the surface of the second layer and the second silica precursor interacts with the first silica precursor in the first layer to form a silica shell, wherein the silica shell at least partially encapsulates the nanoparticle.
  • the first functional group and the second functional group are independently amine, carboxylic acid, phosphate, hydroxyl, or thiol. In some cases, the first functional group and the second functional group may be the same. In some cases, the first functional group and the second functional group may be different. In one embodiment, the first functional group and the second functional group are amines. Also, in some cases, the first silica precursor and the second silica precursor are the same, while in other cases, the first silica precursor and the second silica precursor are different. In one embodiment, the first silica precursor and the second silica precursor are trimethoxysilane.
  • nanoparticle 50 e.g., semiconductor nanoparticle, magnetic semiconductor nanoparticle, etc.
  • a first silane precursor such as aminopropyl trimethoxysilane
  • nanoparticle 50 may form coated nanoparticle 52, wherein the amine groups of the aminopropyl trimethoxysilane are attached to the surface of the core and the trimethoxysilane groups are exposed at the outer surface of the nanoparticle.
  • Coated nanoparticles 52 may then be suspended in a reverse microemulsion (e.g., an Igepal reverse microemulsion).
  • a second silane precursor such as another aminopropyl trimethoxysilane, may then be added to the mixture such that the exposed trimethoxysilane groups of nanoparticle 52 may be hydrolyzed and condensed with the added aminopropyl trimethoxysilanes to form nanoparticle 54.
  • the resulting silica coating thus has a double layer of silica with amine groups attached to the surface of the core and amine groups exposed at the surface of the silica coating.
  • the second silane precursor may be further functionalized with, for example, a biological recognition entity, as described more fully below.
  • the coatings as described herein may be used to at least partially encapsulate nanoparticles, such as semiconductor nanoparticles or magnetic semiconductor nanoparticles.
  • the nanoparticle comprises a CdSe core encapsulated with a silica coating.
  • the nanoparticle comprises a F ⁇ 2 ⁇ 3 -CdSe core encapsulated with a silica coating. It should be understood that the methods of the invention may be used for other nanoparticles (e.g., polymer nanoparticles, metal nanoparticles, etc.) as well. Methods of the present invention may be advantageous in that certain steps used in prior art techniques may be eliminated.
  • methods of the invention do not require an additional capping step, such as ZnS capping which can be a laborious process involving the use of noxious reagents, in order to preserve the optical properties (e.g., fluorescence quantum yield) of fluorescent nanoparticles (e.g., semiconductor nanoparticles, magnetic semiconductor nanoparticles).
  • Coated nanoparticles as described herein can exhibit efficient fluorescence quantum yields over a broad wavelength range.
  • some embodiments of the invention comprise relatively thin (e.g., 2-3 nm) silica coatings for nanoparticles, allowing for high fluorescence quantum yields.
  • the present invention advantageously provides magnetic semiconductor nanoparticles that exhibit decreased cytotoxicity and that are compatible with biological conditions (e.g., aqueous conditions), allowing for integration with biological applications.
  • Nanoparticles described herein can also exhibit stability in buffer solutions.
  • Methods of the invention may provide nanoparticles coatings and methods which are simple, efficient, and low in cost.
  • the functional groups presented at the surface of the nanoparticle coating may be further functionalized with a binding partner selected to preferentially bind a target analyte by, for example, binding between two biological molecules or formation of a bond.
  • a binding partner selected to preferentially bind a target analyte by, for example, binding between two biological molecules or formation of a bond.
  • the binding partner may be a chelating group, an affinity tag (e.g., a member of a biotin/avidin or biotin/streptavidin binding pair or the like), an antibody, a fatty acid (e.g., oleyl), a peptide or protein sequence, a nucleic acid sequence, or a moiety that selectively binds various biological, biochemical, or other chemical species.
  • the binding partner may comprise an oleyl group, which can selectively interact with cells to anchor the nanoparticle to the cell membrane.
  • binding can involve any hydrophobic, non-specific, or specific interaction
  • binding between two biological molecules refers to the interaction between a corresponding pair of molecules that exhibit mutual affinity or binding capacity, typically specific or non-specific binding or interaction.
  • the interaction of the nanoparticle and the analyte in some instances, may be facilitated through specific interactions, such as a protein/carbohydrate interaction, a ligand/receptor interaction, or other biological binding partners.
  • binding partner refers to a molecule that can undergo binding with a particular molecule.
  • the binding partner may interact with an analyte to form a bond with the analyte, such as a covalent bond (e.g. carbon-carbon, carbon- oxygen, oxygen-silicon, sulfur-sulfur, phosphorus-nitrogen, carbon-nitrogen, metal- oxygen or other covalent bonds), an ionic bond, a hydrogen bond (e.g., between hydroxyl, amine, carboxyl, thiol and/or similar functional groups, for example), a dative bond (e.g. complexation or chelation between metal ions and monodentate or multidentate ligands), or the like.
  • the interaction may also comprise Van der Waals interactions.
  • the present invention also relates to methods for determining an analyte, such as a biological analyte.
  • Nanoparticles of the invention may be exposed to a sample suspected of containing an analyte and, if the analyte is present, the analyte may become immobilized with respect to the nanoparticle via interaction between the analyte and the coating or shell of the nanoparticle.
  • the analyte may interact with the nanoparticle via binding between two biological molecules or, in some cases, via formation of a bond.
  • the term "determining” generally refers to the analysis of a species or signal, for example, quantitatively or qualitatively, and/or the detection of the presence or absence of the species or signals.
  • Determining may also refer to the analysis of an interaction between two or more species or signals, for example, quantitatively or qualitatively, and/or by detecting the presence or absence of the interaction.
  • a component that is "immobilized with respect to" another component either is fastened to the other component or is indirectly fastened to the other component, e.g., by being fastened to a third component to which the other component also is fastened, or otherwise is translationally associated with the other component.
  • an analyte is immobilized with respect to a nanoparticle if the analyte is fastened to a binding partner attached to the nanoparticle, is fastened to an intermediate binder to which the binding partner attached to the nanoparticle is fastened, etc.
  • the analyte comprises a moiety that is capable of interacting with at least a portion of the nanoparticle.
  • the moiety may interact with the nanoparticle by forming a bond, such as a covalent bond, or by binding (e.g., biological binding) as described herein.
  • the ability of nanoparticles (e.g., luminescent nanoparticles) to preferentially bind analytes may advantageous for a number of applications, such as labeling of biological species, cell imaging, sensors, drug discovery, isolation or purification of compounds, high-throughput screening techniques, magnetic resonance imagine (MRI), drug targeting, and novel optical communications systems (e.g., photonic crystals), among other applications.
  • the binding partner may comprise a biological or a chemical molecule able to bind to another biological or chemical molecule in a medium, e.g. in solution.
  • the binding partner may be a functional group such as an amine, hydroxyl group, thiol, carboxylic acid, or other functional group as described herein.
  • the binding partner may be capable of biologically binding an analyte via an interaction that occurs between pairs of biological molecules including cells, proteins, peptides, antibodies, enzymes, nucleic acids, glycoproteins, carbohydrates, hormones, and the like. It should be understood that biological molecules may also interact with (e.g., bind to) small molecules, ligands, metal ions, and the like.
  • the binding partner may be attached the nanoparticle (e.g., nanoparticle coating) via any suitable linker, such as alkyl, heteroalkyl (e.g, poly(ethylene)glycol), acyl such as carbonyl (e.g, carboxylic ester, amide, ketone, carbamate, carbonate, urea, and the like), aryl, heteroaryl, combinations thereof, and the like.
  • the linker may have any suitable length to allow the binding partner to sufficiently interact with a target analyte. For example, in some cases, a relatively long linker may be required if the binding partner is sterically large or, alternatively, if the analyte is sterically large. In some cases, the linker can be relatively short.
  • the binding partner may comprise an alkyl or alkenyl chain, such as a fatty acid, which has sufficient length to allow interaction with an analyte, such as a cell, without need for a long linker.
  • nanoparticle 90 is functionalized with a binding partner comprising an oleyl group, selected to preferentially bind cells.
  • Nanoparticle 90 may be, for example, a semiconductor nanoparticle or magnetic semiconductor nanoparticle as described herein.
  • the amine groups presented at the surface of nanoparticle 90 can react with oleyl-O- poly(ethyleneglycol)-succinyl-N-hydroxysuccinimidyl ester 100 to form functionalized nanoparticle 110 such that the oleyl groups are presented at the surface of the coating.
  • the oleyl groups may be used to interact with cells, while the ⁇ oly(ethyleneglycol) groups may enhance water-solubility of the nanoparticle, as well as reduce the non-specific adsorption of biological molecules.
  • functionalized nanoparticle 110 can be used to bind to cells for imaging, labeling, sensing, or other applications.
  • oleyl-0-poly(ethyleneglycol)-succinyl-N- hydroxysuccinimidyl ester may first be attached to a silane precursor, such as aminopropyltrimethoxysilane (APS), to form compound 120, which is then reacted with a nanoparticle 130 comprising a single layer of APS 5 with the silicon groups exposed at the surface of the nanoparticle.
  • APS aminopropyltrimethoxysilane
  • the silicon groups of compound 100 and nanoparticle 90 may then react to form coated nanoparticle 140 comprising a silica coating having at least two layers of silicon atoms, as described herein.
  • the nanoparticles can include a coating that encapsulates, or partially encapsulates, the core. In some embodiments, it is preferable for the coating to encapsulate the majority of the surface area of the core. For example, the coating may encapsulate at least 75% of the surface area of the coating. In some cases, the coating may completely encapsulate the core. In some embodiments, the coating is not chemically bound to the core (e.g., to the magnetic nanoparticles or magnetic semiconductor nanoparticles) and yet may contain the nanoparticle by encapsulation. Thus, the core and coating may be devoid of ionic bonds and/or covalent bonds between the two.
  • the coating may comprise an organic material (e.g., based on carbon and/or polymers of carbon).
  • the coating may comprise a non-organic material (e.g., not based on carbon and/or polymers of carbon, but nonetheless may include carbon atom). It may be preferred for the coating to be non-organic and may be formed of a silicon polymer such as silica.
  • the coating may be porous. For example, the coating may have pores on the mesoscale size. In certain embodiments, the coating may be non-porous.
  • the coating may be composed of a material appropriately chosen to be, for example, electronically insulating (e.g., through augmented redox properties), optically non-interfering, chemically stable, or lattice-matched to the underlying core material (e.g., for epitaxial growth, minimization of defects).
  • the coating can have a thickness great enough to encapsulate the core to the extent desired.
  • the coating has an average thickness of less than 50 nm; and, in some embodiments, the coating has an average thickness of less than 25 nm (e.g., between 2 nm and 20 nm). In other embodiments, the coating has an average thickness of less than 5 nm (e.g., between 2 and 3 nm).
  • the average coating thickness may be determined using standard techniques by measuring the thickness at a representative number of locations using microscopy techniques (e.g., TEM).
  • a hydrophilic species may be associated with the coating (e.g., a silica coating) to provide greater hydrophilicity to the nanoparticle.
  • the hydrophilic species can be, for example, amines, thiols, alcohols, carboxylic acids and carboxylates, sulfates, phosphates, polyethylene glycols (PEGs), or derivatives of polyethylene glycol. Derivatives include, but are not limited to, functionalized PEGs, such as amine, thiol and carboxyl functionalized PEG.
  • the hydrophilic species can be chemically bound to the coating or can be, for example, physically trapped by the coating material. Presence of a hydrophilic species can impart superior water solubility characteristics to the nanoparticles while being biocompatible and nontoxic and can, in some instances, provide for better dispersion of the nanoparticles in solution.
  • the composite may be rendered water soluble at pHs of less than 8.0 or less than or equal to 7.0.
  • these composites may be water soluble at neutral or below neutral pHs and thus may be biocompatible and appropriate for use in biological fluids such as blood and in vivo.
  • water soluble is used herein as it is commonly used in the art to refer to the dispersion of a nanoparticle in an aqueous environment. "Water soluble” does not mean, for instance, that each material is dispersed at a molecular level.
  • a nanoparticle can be composed of several different materials and still be “water soluble” as an integral particle.
  • the presence of PEG or related compounds in the silica coating can provide for a nanoparticle exhibiting a reduced propensity to non-specifically adsorb protein, cells, and other biological materials. This may be advantageous, for example, when used in vivo since the nanoparticles can remain in solution for a longer period of time, allowing for increased circulation and improved deliverability to intended targets.
  • Nanoparticles of the invention generally have a maximum cross-sectional dimension of no more than 1 micron.
  • nanoparticles are of less than 250 mti cross section in any dimension, more typically less than 100 nm cross section in any dimension, and preferably less than 50 nm cross section in any dimension.
  • the nanoparticles may have a diameter of about 2 to about 50 nm.
  • the nanoparticles may have a diameter of about 2 to about 20 nm.
  • the nanoparticles may have diameters of about 2 to about 3 nanometers. Nanoparticles are generally spherical in shape, though other shapes are also possible.
  • the particle size may be determined using standard microscopy techniques, including transmission electron microscopy (TEM) or dynamic light scattering (DLS).
  • TEM transmission electron microscopy
  • DLS dynamic light scattering
  • particle size refers to the diameter of a particle, such as a substantially spherical particle, as determined by microscopy. In the event that a particle of the invention is not absolutely spherical, then size is determined by approximating the shape of the particle in the form of a sphere.
  • TEM can be used to determine particles size.
  • DLS can be used to determine particle size. For example, DLS may be useful in cases where smaller features of the particles, such as a thin silica coating (2-3 run), which may be difficult to observe by TEM.
  • semiconductor nanoparticles may have any suitable semiconductor material composition.
  • semiconductor nanoparticles may be formed of Group H-VI semiconductors such as CdSe, CdTe, CdO, ZnO, ZnS, ZnSe, ZnTe, MgO, MgS, MgSe, MgTe, HgO, HgS 5 HgSe, HgTe, SrS. SrSe, SrTe, BaSe and BaTe.
  • Group H-VI semiconductors such as CdSe, CdTe, CdO, ZnO, ZnS, ZnSe, ZnTe, MgO, MgS, MgSe, MgTe, HgO, HgS 5 HgSe, HgTe, SrS. SrSe, SrTe, BaSe and BaTe.
  • the quantum dots may also be formed of Group III-V compounds such as AlN, AlP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, InN 3 InP, InAs, InSb, TiN, TiP, TiAs and TiSb.
  • the semiconductor nanoparticles may be formed of Group IV semiconductors such as silicon or germanium. It should also be understood that the semiconductor nanoparticles may comprise alloys or mixtures of any of the above-mentioned semiconductors. Other semiconductor nanoparticles compositions known to those of ordinary skill in the art may also be suitable. The specific composition is typically selected, in part, to provide the desired optical properties. In some embodiments, it may be preferred for the semiconductor nanoparticles to have a cadmium-based composition such as CdSe. It is also possible for composites of the invention to include semiconductor nanoparticles having different compositions.
  • the semiconductor nanoparticles generally have particle sizes of less than 100 nanometers. In some cases, the average particle size of the semiconductor nanoparticles in the composite is less than 20 nanometers; in other cases, the average particle size is less than 5 nanometers (e.g., about 3.5 nanometers). In some embodiments, the average particle size of the semiconductor nanoparticles is greater than 0.5 nanometer. In some magnetic semiconductor nanoparticles, the semiconductor nanoparticle region may have a particle size that is less than that of the magnetic nanoparticle region. In other magnetic semiconductor nanoparticles, the semiconductor nanoparticle region may have a particle size that is greater than or equal to that of the magnetic nanoparticle region. As described herein, semiconductor nanoparticles may be grown on the surface of magnetic nanoparticles to produce a magnetic semiconductor nanoparticle.
  • Magnetic nanoparticles (including magnetic nanoparticle regions) of the present invention may have any suitable composition.
  • the magnetic nanoparticles may comprise iron, cobalt and/or nickel, amongst other magnetic materials.
  • the magnetic material is in the form of a metal compound or alloy, such as iron oxide (e.g., F ⁇ 2 ⁇ 3, FeaCu) or iron platinum (FePt).
  • the magnetic material may be a pure metal, such as cobalt or nickel.
  • the composition of the magnetic nanoparticles is selected to provide desired magnetic properties.
  • the magnetic nanoparticles may be superparamagnetic at 5K and 300K. In some embodiments, it may be preferable to use Fe 2 U3 magnetic nanoparticles. Magnetic nanoparticles may be synthesized by methods known in the art.
  • any suitable conventional technique known in the art for forming magnetic nanoparticles and semiconductor nanoparticles may be used.
  • one suitable technique for forming the magnetic nanoparticles has been described in Hyeon et. al., J. Am. Chem. Soc, 2001, 123, 12798, which is incorporated herein by reference.
  • One suitable technique for preparing semiconductor nanoparticles has been described in Peng et. al., J. Am. Chem Soc. 2001, 123, 183, which is incorporated herein by reference.
  • the nanoparticles may include a passivation layer.
  • a "passivation" layer is a material associated with the surface of a nanoparticle (e.g., semiconductor nanoparticle) that serves to eliminate energy levels at the surface of the crystal that may act as traps for electrons and holes that degrade the luminescent properties of the quantum dot.
  • the passivation layer may be formed of a material that is non-conductive and/or non-semiconductive.
  • the passivation layer may be of a material that does not exhibit a higher band gap than a nanoparticle which it surrounds.
  • the passivation layer may be non-ionic and non-metallic.
  • a non-conductive material is a material that does not transport electrons when an electric potential is applied across the material.
  • the passivation layer can be comprised of, or consist essentially of, a compound exhibiting a nitrogen-containing functional group, such as an amine.
  • the amine may be bound directly or indirectly to one or more silicon atoms such as those present in a silane or other silicon polymer.
  • the silanes may include any additional functional group such as, for example, alkyl groups, hydroxyl groups, sulfur- containing groups, or nitrogen-containing groups.
  • Compounds comprising the passivation layer may be of any size but typically have a molecular weight of less than about 500 or less than about 300.
  • the preferred class of compounds are the amino silanes and in some embodiments, aminopropyltrimethoxysilane (APS) can be used.
  • the coating comprises at least one type of silane.
  • Silane conjugation may be carried out with various types of silanes, including those having trimethoxy silyl, methoxy silyl, or silanol groups at one end, which may be hydrolyzed in basic medium to form a silica shell around the nanoparticle.
  • the silanes may also comprise organic functional groups, examples of which include phosphate and phosphonate groups, amine groups, thiol groups, carbonyl groups (e.g., carboxylic acids, and the like), C1-C20 alkyl, C1-C 2 0 alkene, C1-C20 alkyne, azido groups, epoxy groups, or other functional groups described herein.
  • a reverse microemulsion process may be used to form the coating.
  • An “emulsion” is a dispersion of a non-aqueous solvent and an aqueous solvent.
  • a “reverse emulsion” or “aqueous in non-aqueous emulsion” is a dispersion of discrete areas of aqueous solvent (aqueous phase) within a non-aqueous solvent. The reverse microemulsion can be produced using a variety of non-polar solvents.
  • the non-polar solvent is a hydrocarbon and may be an aliphatic hydrocarbon and, in some cases, is a non-aromatic cyclic hydrocarbon such as cyclopentane, cyclohexane or cycloheptane.
  • a surfactant e.g., ionic or non-ionic
  • a "surfactant” is a material exhibiting amphiphilic properties and is used herein as it is commonly used in the art, e.g., for introducing hydrophobic species to hydrophilic environments.
  • surfactants suitable for use in the present invention include, for example, polyphenyl ethers, such as IGEPAL CO-520, dioctyl sulfosuccinate sodium salt (AOT), trioctyl phosphine oxide (TOPO), and the like.
  • a "precursor” is a substance that can be transformed into a second substance that exhibits different properties from the first.
  • a monomer is a polymer precursor if it can be transformed into a polymer.
  • a "silica precursor” is a substance comprising a silicon atom that can be transformed into a second substance, such as a polymerized silica coating, that exhibits different properties from the first.
  • a and/or B can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase "at least one,” in reference to a list of one. or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements that the phrase "at least one" refers to, whether related or unrelated to those elements specifically identified.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • Example 1 CdSe semiconductor nanoparticles of varying particle sizes were prepared according to the procedures described in Qu, L. et al., J. Am. Chern. Soc.2002, 124, 2049, and Peng, Z. A. et al., J. Am. Chem. Soc. 2001, 123, 183, with slight modification.
  • trioctylphosphine was used to dissolve Se, rather than tributylphosphine (TBP) and/or dioctylamine.
  • Magnetic semiconductor nanoparticles were synthesized by growing CdSe semiconductor nanoparticles onto F ⁇ 2 ⁇ 3 magnetic nanoparticles. First, F ⁇ 2 ⁇ 3 nanoparticles were synthesized in oleic acid and dioctyl ether by the decomposition of iron pentacarbonyl, following known procedures with slight modifications. The magnetic nanoparticles were washed with ethanol (3x) and vacuum-dried at 80 0 C.
  • CdO and stearic acid were combined and heated to 150-200 0 C, and then cooled to room temperature.
  • the Fe 2 ⁇ 3 nanoparticles, trioctyl phosphine oxide (TOPO) and hexadecyl amine (HDA) were added, and the mixture was heated to 300 0 C.
  • a solution of Se dissolved in trioctylphosphine (TOP) was quickly injected in order to begin the growth of CdSe semiconductor nanoparticles on the surface of the TOPO/HDA-passivated Fe 2 ⁇ 3 nanoparticles.
  • the resulting CdSe semiconductor nanoparticles were allowed to grow for different time periods (1—5 min) to yield CdSe nanopaxticles having different sizes, corresponding to green, yellow, orange and red emissions.
  • Example 3 The nanoparticles were shown to exhibit both magnetic and emission properties by application of a magnetic field and electromagnetic radiation (e.g., UV light). Methanol was added to the mixture and a magnet was applied to the vial.
  • FIG. 4 A shows a photograph of magnetic semiconductor nanoparticles under white light before application of a magnet
  • FIG. 4B shows a photograph of magnetic semiconductor nanoparticles under white light after application of a magnet, wherein the particles were attracted to the magnet, leaving behind a clear solution.
  • the nanoparticles were also fluorescent, confirming the successful synthesis of magnetic semiconductor nanoparticles. To obtain different emission colors, aliquots of the growth solution of FCaO 3 -CdSe were taken at different time intervals (1-5 min).
  • FIG. 4 A shows a photograph of magnetic semiconductor nanoparticles under white light before application of a magnet
  • FIG. 4B shows a photograph of magnetic semiconductor nanoparticles under white light after application of a magnet, wherein the particles were attracted to the magnet, leaving behind a clear
  • FIG. 4C shows a photograph of magnetic semiconductor nanoparticles exhibiting both magnetic and luminescence properties (e.g., red emission) under UV excitation at 365 nm.
  • FIG. 4D shows a photograph of magnetic semiconductor nanoparticles, dispersed in chloroform, having green and orange emission under UV excitation at 365 nm.
  • FIG. 6 A and FIG. 6B show TEM micrographs of magnetic semiconductor nanoparticle heterodimers as synthesized in Example 2.
  • the heterodimers were isolated from other semiconductor nanoparticles in the sample upon application of a magnetic field.
  • the heterodimers comprise large regions, which are Fe 2 C>3 magnetic nanoparticles (10 nm), and small regions, which are CdSe semiconductor nanoparticles (4-5 nm).
  • Superparamagnetic characteristics associated with the superparamagnetic Fe 2 Oa nanoparticle region were exhibited by the heterodimer structure.
  • FIG. 7A shows a images of magnetic semiconductor nanoparticles at low magnification, showing the high dispersion of semiconductor nanoparticles around the magnetic semiconductor nanoparticles.
  • FIG. 7B shows a similar dispersion at high magnification.
  • Example 5 Scanning transmission electron microscopy (STEM) was used to confirm the presence of Cd, Se and Fe in the magnetic semiconductor nanoparticles synthesized in Example 2 (e.g., silica-coated FeaCVCdSe particles).
  • FIG. 8A shows the TEM image of the nanoparticles and
  • FIG. 8B shows the energy-dispersive X-ray (EDX) analysis of the nanoparticles.
  • EDX energy-dispersive X-ray
  • Both bare CdSe semiconductor nanoparticles and F ⁇ 2 ⁇ 3 -CdSe magnetic semiconductor nanoparticles were silanized in an Igepal reverse microemulsion to form a silica coating.
  • the nanoparticles e.g., CdSe semiconductor nanoparticles, Fe 2 ⁇ 3 -CdSe magnetic semiconductor nanoparticles
  • passivated with TOPO/HDA were precipitated once with methanol, and the precipitate was dried under normal conditions at room temperature.
  • the precipitated nanoparticles (4 mg) were dispersed in 1 mL of chloroform.
  • Micelles were prepared by dissolving 0.2 g of Igepal-CO520 (polyoxyethylene(5)nonylphenyl ether) in 4 mL of chloroform, and stirring vigorously for 30 minutes. The nanoparticles in chloroform were added to the micelles along with 10—50 ⁇ L of aminopropyl trimethoxysilane (APS), and the mixture was stirred for 1 hour to form a first layer of APS on the surface of the nanoparticle. As shown in FIG. 2, the amine groups of the APS were attached to the surface of the nanoparticles, with the trimethoxysilane groups exposed at the outer surface.
  • Igepal-CO520 polyoxyethylene(5)nonylphenyl ether
  • TMAH TMAH in 2-propanol/methanol
  • 20 ⁇ L of deionized water were added.
  • the mixture was stirred for another 30 min until the bulk organic phase turned colorless, with the formation of orange/red globules on the surface of the glass vial.
  • the colorless organic phase was then discarded, leaving behind the nanoparticles on the surface of the glass vial.
  • the nanoparticles were washed with chloroform 3—5 times to ensure the complete removal of excess surfactants and other unreacted reagents from their surface. In some cases, the nanoparticle was coated with second layer of APS.
  • the deposition of a second layer of APS was achieved by adding 10-50 ⁇ L aminopropyl trimethoxysilane, which resulted in hydrolysis of the methoxy groups of the bound APS moieties and condensation with a second APS molecule, resulting in a silica coating with amine groups exposes at the surface, as shown in FIG. 2.
  • the second APS layer was observed to improve the stability of the coated nanoparticles in buffer solution.
  • coated nanoparticles were then dispersed in 1 mL of Ix PBS and used immediately in the next reaction to prevent precipitation.
  • ⁇ std integrated area under the emission spectrum of dye
  • FIG. 5 A shows the absorption spectra and FIG. 5B shows the emission spectra of silica-coated Fe 2 Os-CdSe nanoparticles I-IV, having particles sizes as described in Table 1, in phosphate buffer saline (PBS). Nanoparticles I-IV have total particle sizes of approximately 15-20 nm. TEM was used to determine the size of the particles in most of the cases. The silica coating was often difficult to observe by TEM due to its smaller size (2-3 nm), relative to the total nanoparticle. Dynamic light scattering
  • the absorption and emission wavelengths are characteristic of the CdSe region size.
  • the spectra closely resemble the absorption and emission spectra of the Fe 2 Oj-CdSe nanoparticles ⁇ vithout the silica coating, indicating that the thin, silica coating preserves the optical properties of the underlying FeaOa-CdSe nanoparticle.
  • the emission spectra were narrow, with a full-width-half-maximum (FWHM) of ⁇ 40 nm.
  • the photoluminescence quantum yield of the Fe 2 ⁇ 3 -CdSe nanoparticles before and after silica coating were 13—18% and 8—10%, respectively. The quantum yields increased with increasing particle size and emission wavelength.
  • FIG. 3A shows the conjugation of a binding partner with a nanoparticle.
  • the binding partner employed in this embodiment was a bioanchoring membrane (BAM), comprising a PEG group to impart biocompatibility and an oleyl group to target cell membranes.
  • BAM bioanchoring membrane
  • the BAM group was attached to the surface of the nanoparticles using two methods.
  • 10 mg of oleyl-O- poly(ethyleneglycol)-succinyl-N-hydroxysuccinimidyl ester obtained from NOF Corporation, Tokyo
  • the N-hydroxysuccinimidyl ester readily reacted with the surface amine groups of the nanoparticles to form an amide linkage.
  • FIG. 3B illustrates a second method, wherein nanoparticles coated with one layer of APS are reacted with a BAM group functionalized with an APS moiety.
  • 10 mg of oleyl-O-poly(ethyleneglycol)-succinyl-N- hydroxysuccinimidyl ester were dissolved in 1 mL of anhydrous dichloromethane and 10—50 ⁇ L of APS was added. The mixture was stirred for one hour, wherein the N- hydroxysuccinimidyl ester reacted with the amine group of the aminopropyl trimethoxysilane to form an amide.
  • the solvent was then evaporated and 1 mL of aqueous dispersion of the nanoparticles with one layer of APS was added.
  • the surface silanol groups on the silica coating reacted with the APS conjugated to BAM.
  • the final aqueous solution was filtered through a 0.2- ⁇ m filter to remove any large aggregates.
  • Nanoparticles e.g., semiconductor nanoparticles, magnetic semiconductor nanoparticles
  • oleyl groups presented at the surface of the nanoparticle were used as labels for mammalian cells.
  • Three particular adherent cell lines NIH-3T3 mouse fibroblast cells, HepG2 human liver cancer cells, and 4Tl mouse breast cancer cells, were used for bioimaging.
  • the NIH-3T3 cells and HepG2 cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin, while the 4Tl cells were cultured in Roswell Park Memorial Institute's medium (RPMI-1630).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • RPMI-1630 Roswell Park Memorial Institute's medium
  • All cells were precultured overnight at the density of 0.2 million cells/mL on a 10-mm cover slip at 37°C and 5% CO 2 .
  • the oleyl-functionalized nanoparticles (50—100 ⁇ L) were loaded onto the cells and incubated for at least 5 minutes.
  • FIGS. 9A-D show the CLSM images of different cell membranes labeled with nanoparticles.
  • FIG. 9A shows the labeling of HepG2 cells with oleyl-functionalized CdSe nanoparticles.
  • FIG. 9B shows the labeling of NIH- 3T3 cells with oleyl-functionalized CdSe nanoparticles.
  • FIG. 9C shows the labeling of HepG2 cells with oleyl-functionalized CdSe nanoparticles.
  • FIG. 9D shows the labeling of 4Tl cells with an oleyl-functionalized Fe 2 Oa-CdSe nanoparticle.
  • FIG. 10 shows CLSM (left) and light (right) microscopy images of 4Tl cell membranes labeled with orange-emitting oleyl-functionalized Fe 2 ⁇ 3 -CdSe nanoparticles.
  • FIG. 11 shows CLSM (left) and light (right) microscopy images of NIH-3T3 cell membranes labeled with orange-emitting oleyl-ftinctionalized Fe2C «3-CdSe nanoparticles.
  • FIG. 12 CLSM (left) and light (right) microscopy images of 4Tl cell membranes labeled using green-emitting oleyl-functionalized CdSe nanoparticles.
  • FIG. 13 CLSM (left) and light (right) microscopy images of HepG2 cell membranes labeled using green-emitting oleyl-functionalized CdSe nanoparticles. The nanoparticles were found to be co-localized in the cytoplasm.

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Abstract

La présente invention concerne des matériaux et des procédés comprenant des nanoparticules, comme des nanoparticules semi-conductrices et des particules semi-conductrices magnétiques. Les nanoparticules selon l'invention peuvent présenter des propriétés utiles qui peuvent être affinées à un degré de contrôle élevé au moyen des divers procédés de l'invention. Dans certains modes de réalisation, les nanoparticules peuvent être réalisées pour présenter une certaine propriété ou combinaison de propriétés, comme des propriétés optiques et magnétiques. La présente invention concerne également des revêtements (par ex. des revêtements de silice) et des procédés associés qui peuvent améliorer diverses propriétés des nanoparticules. Les nanoparticules ci-décrites peuvent être utiles dans des applications comme l'étiquetage d'espèces biologiques, le marquage de médicaments, l'imagerie par résonance magnétique (IRM), entre autres applications.
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