US20120228565A1 - Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media - Google Patents

Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media Download PDF

Info

Publication number
US20120228565A1
US20120228565A1 US13/423,055 US201213423055A US2012228565A1 US 20120228565 A1 US20120228565 A1 US 20120228565A1 US 201213423055 A US201213423055 A US 201213423055A US 2012228565 A1 US2012228565 A1 US 2012228565A1
Authority
US
United States
Prior art keywords
population
nanoparticles
hydrophobic
nanoparticle
polymer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
US13/423,055
Other versions
US8691384B2 (en
Inventor
Edward William Adams
Marcel Pierre Bruchez, Jr.
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Life Technologies Corp
Original Assignee
Life Technologies Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Life Technologies Corp filed Critical Life Technologies Corp
Priority to US13/423,055 priority Critical patent/US8691384B2/en
Publication of US20120228565A1 publication Critical patent/US20120228565A1/en
Application granted granted Critical
Publication of US8691384B2 publication Critical patent/US8691384B2/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Classifications

    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • CCHEMISTRY; METALLURGY
    • C09DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09KMATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02Use of particular materials as binders, particle coatings or suspension media therefor
    • CCHEMISTRY; METALLURGY
    • C09DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09KMATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/88Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing selenium, tellurium or unspecified chalcogen elements
    • C09K11/881Chalcogenides
    • C09K11/883Chalcogenides with zinc or cadmium
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588Chemical 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
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00Stock material or miscellaneous articles
    • Y10T428/29Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
    • Y10T428/2982Particulate matter [e.g., sphere, flake, etc.]
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00Stock material or miscellaneous articles
    • Y10T428/29Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
    • Y10T428/2982Particulate matter [e.g., sphere, flake, etc.]
    • Y10T428/2991Coated
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00Stock material or miscellaneous articles
    • Y10T428/29Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
    • Y10T428/2982Particulate matter [e.g., sphere, flake, etc.]
    • Y10T428/2991Coated
    • Y10T428/2998Coated including synthetic resin or polymer
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00Stock material or miscellaneous articles
    • Y10T428/31504Composite [nonstructural laminate]
    • Y10T428/31855Of addition polymer from unsaturated monomers
    • Y10T428/31909Next to second addition polymer from unsaturated monomers
    • Y10T428/31928Ester, halide or nitrile of addition polymer

Definitions

  • This invention relates generally to surface-modified nanoparticles, and more particularly relates to surface-modified semiconductor and metal nanoparticles having enhanced dispersibility in aqueous media as well as superior colloidal and photophysical stability.
  • the invention additionally relates to methods for making and using the novel surface-modified nanoparticles.
  • the invention finds utility in a variety of fields, including biology, analytical and combinatorial chemistry, medical diagnostics, and genetic analysis.
  • Semiconductor nanocrystals also known as quantum dot particles
  • Quantum confinement of both the electron and hole in all three dimensions leads to an increase in the effective band gap of the material with decreasing crystallite size. Consequently, both the optical absorption and emission of semiconductor nanocrystals shift to the blue (higher energies) as the size of the nanocrystals gets smaller.
  • Semiconductor nanocrystals are nanoparticles composed of an inorganic, crystalline semiconductive material and have unique photophysical, photochemical and nonlinear optical properties arising from quantum size effects, and have therefore attracted a great deal of attention for their potential applicability in a variety of contexts, e.g., as detectable labels in biological applications, and as useful materials in the areas of photocatalysis, charge transfer devices, and analytical chemistry.
  • semiconductor nanocrystals there is now a fairly substantial body of literature pertaining to methods for manufacturing such nanocrystals. Broadly, these routes may be classified as involving preparation in glasses (see Ekimov et al.
  • aqueous preparation including preparation that involve use of inverse micelles, zeolites, Langmuir-Blodgett films, and chelating polymers; see Fendler et al. (1984) J. Chem. Society, Chemical Communications 90:90, and Henglein et al. (1984) Ber. Bunsenges. Phys. Chem. 88:969), and high temperature pyrolysis of organometallic semiconductor precursor materials (Murray et al. (1993) J. Am. Chem. Soc. 115:8706; Katari et al. (1994) J. Phys. Chem. 98:4109).
  • the two former methods yield particles that have unacceptably low quantum yields for most applications, a high degree of polydispersity, poor colloidal stability, a high degree of internal defects, and poorly passivated surface trap sites.
  • nanocrystals made by the first route are physically confined to a glass matrix and cannot be further processed after synthesis.
  • nanoparticles having enhanced dispersibility in aqueous media, wherein the nanoparticles are comprised of an inner core having a hydrophobic surface and an outer layer of a multiply amphipathic dispersant.
  • a nanoparticle conjugate i.e., a water-dispersible nanoparticle as above
  • a water-dispersible nanoparticle is provided that is comprised of an inner core and an outer layer of a multiply amphipathic dispersant, i.e., a compound having two or more hydrophobic regions and two or more hydrophilic regions.
  • the inner core comprises a semiconductive or metallic material, preferably an inorganic semiconductive material that is in a crystalline state.
  • the inner core also comprises a hydrophobic passivating layer on the semiconductive or metallic material resulting from solvents and/or surfactants used in nanoparticle manufacture.
  • the surface of the inner core is accordingly hydrophobic, and the hydrophobic regions of the dispersant thus have affinity for the core surface and attach thereto, while the hydrophilic regions of the dispersant extend outward from the nanoparticle and provide for dispersibility in water.
  • the dispersant is polymeric and has a plurality of both hydrophobic regions and hydrophilic regions, thus enhancing water dispersibility of the nanoparticle as well as the dispersant's affinity for the core surface.
  • Particularly preferred dispersants are hyperbranched or dendritic polymers, which, relative to prior methods that involve monomeric dispersants, substantially increase the water dispersibility and colloidal stability of the nanoparticles.
  • the nanoparticles are luminescent semiconductive nanocrystals, and include an overcoating “shell” layer between the inner core and the multiply amphipathic outer layer to increase luminescence efficiency.
  • the shell material has a higher bandgap energy than the nanocrystal core, and should also have good conduction and valence band offset with respect to the nanocrystal core.
  • an “affinity molecule,” i.e., one member of a binding pair may be attached to the outer layer of the surface-modified molecule, providing a nanoparticle “conjugate” that is useful in detecting the presence or quantity of target molecules that comprise the second member of the binding pair.
  • the affinity molecule may be, for example, a protein, an oligonucleotide, an enzyme inhibitor, a polysaccharide, or a small molecule having a molecular weight of less than about 1500 grams/Mol.
  • composition is provided that is comprised of the aforementioned nanoparticle conjugate in association with the second member of the binding pair, wherein the association may involve either covalent or noncovalent interaction.
  • a monodisperse population of surface-modified nanoparticles comprising a plurality of water-dispersible nanoparticles each having an inner core comprised of a semiconductive or metallic material and, surrounding the inner core, an outer layer comprised of a multiply amphipathic dispersant as described above, wherein the population is characterized in that the nanoparticles are of substantially the same size and shape, i.e., the population exhibits no more than about a 10% rms deviation in the diameter of the inner core, preferably no more than about a 5% rms deviation in the diameter of the inner core.
  • the narrow size distribution of a monodisperse population increases the “information density” that is obtainable as a result of the particles' luminescence, i.e., the number of discrete luminescence emissions obtainable for a given nanoparticle composition.
  • a method for making the surface-modified nanoparticles described above.
  • the method involves (a) admixing (i) an amphipathic dispersant comprised of a polymer having two or more hydrophobic regions and two or more hydrophilic regions, with (ii) a plurality of hydrophobic nanoparticles, in (iii) a nonaqueous solvent, to provide an admixture of dispersant and nanoparticles in solution; (b) subjecting the admixture to conditions effective to cause adsorption of the dispersant by the nanoparticles; and (c) transferring the dispersant-coated nanoparticles prepared in step (b) to an aqueous medium such as water or an aqueous buffer.
  • an aqueous medium such as water or an aqueous buffer.
  • a dispersant refers to a single dispersant as well as a mixture of two or more dispersants
  • a nanoparticle encompasses not only a single nanoparticle but also two or more nanoparticles, and the like.
  • amphipathic referring to the dispersants employed herein, is used in its conventional sense to indicate a molecular species having a hydrophobic region and a hydrophilic region.
  • the dispersants herein are “multiply amphipathic” in that they contain two or more hydrophobic regions and two or more hydrophilic regions.
  • attachment as in, for example, the “attachment” of a dispersant to a nanoparticle surface, includes covalent binding, adsorption, and physical immobilization.
  • association with binding
  • binding binding
  • bound are identical in meaning to the term “attached.”
  • Attachment of the present multiply amphipathic dispersants to the surface of a metallic or semiconductive nanoparticle will generally involve “adsorption,” wherein “adsorption” refers to the noncovalent retention of a molecule by a substrate surface. That is, adsorption occurs as a result of noncovalent interaction between a substrate surface and adsorbing moieties present on the molecule that is adsorbed. Adsorption may occur through hydrogen bonding, van der Waal's forces, polar attraction or electrostatic forces (i.e., through ionic bonding), and in the present case will typically involve the natural affinity of a hydrophobic region of a molecule for a hydrophobic surface.
  • nanoparticle refers to a particle, generally a semiconductive or metallic particle, having a diameter in the range of about 1 nm to about 1000 nm, preferably in the range of about 2 nm to about 50 nm, more preferably in the range of about 2 nm to about 20 nm.
  • semiconductive, and metallic “nanoparticles” generally include a passivating layer of a water-insoluble organic material that results from the method used to manufacture such nanoparticles.
  • surface-modified nanoparticle and “water-dispersible nanoparticle” as used herein refer to the modified nanoparticles of the invention, while the term “nanoparticle,” without qualification, refers to the hydrophobic nanoparticle that serves as the inner core of the surface-modified, water-dispersible nanoparticle.
  • semiconductor nanoparticle and “semiconductive nanoparticle” refer to a nanoparticle as defined above that is composed of an inorganic semiconductive material, an alloy or other mixture of inorganic semiconductive materials, an organic semiconductive material, or an inorganic or organic semiconductive core contained within one or more semiconductive overcoat layers.
  • metal nanoparticle refers to a nanoparticle as defined above that is composed of a metallic material, an alloy or other mixture of metallic materials, or a metallic core contained within one or more metallic overcoat layers.
  • semiconductor nanocrystal “quantum dot” and “Qdot® nanocrystal” are used interchangeably herein to refer to semiconductor nanoparticles composed of an inorganic crystalline material that is luminescent (i.e., they are capable of emitting electromagnetic radiation upon excitation), and include an inner core of one or more first semiconductor materials that is optionally contained within an overcoating or “shell” of a second semiconductor material.
  • a semiconductor nanocrystal core surrounded by a semiconductor shell is referred to as a “core/shell” semiconductor nanocrystal.
  • the surrounding shell material will preferably have a bandgap energy that is larger than the bandgap energy of the core material and may be chosen to have an atomic spacing close to that of the core substrate.
  • Suitable semiconductor materials for the core and/or shell include, but are not limited to, the following: materials comprised of a first element selected from Groups 2 and 12 of the Periodic Table of the Elements and a second element selected from Group 16 (e.g., ZnS, ZnSe, ZnTe, CDs, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and the like); materials comprised of a first element selected from Group 13 of the Periodic Table of the Elements and a second element selected from Group 15 (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, and the like); materials comprised of a Group 14 element (Ge, Si, and the like); materials such as PbS, PbSe and the like;
  • luminescence is meant the process of emitting electromagnetic radiation (light) from an object. Luminescence results when a system undergoes a transition from an excited state to a lower energy state with a corresponding release of energy in the form of a photon. These energy states can be electronic, vibrational, rotational, or any combination thereof. The transition responsible for luminescence can be stimulated through the release of energy stored in the system chemically or added to the system from an external source.
  • the external source of energy can be of a variety of types including chemical, thermal, electrical, magnetic, electromagnetic, and physical, or any other type of energy source capable of causing a system to be excited into a state higher in energy than the ground state.
  • a system can be excited by absorbing a photon of light, by being placed in an electrical field, or through a chemical oxidation-reduction reaction.
  • the energy of the photons emitted during luminescence can be in a range from low-energy microwave radiation to high-energy x-ray radiation.
  • luminescence refers to photons in the range from UV to IR radiation.
  • a “monodisperse” population of particles means that at least about 60% of the particles, preferably about 75% to about 90% of the particles, fall within a specified particle size range.
  • a population of monodisperse particles deviates less than 10% rms (root-mean-square) in diameter and preferably less than 5% rms.
  • narrow wavelength band or “narrow spectral linewidth” with regard to the electromagnetic radiation emission of the semiconductor nanocrystal is meant a wavelength band of emissions not exceeding about 60 nm, and preferably not exceeding about 30 nm in width, more preferably not exceeding about 20 nm in width, and symmetric about the center. It should be noted that the bandwidths referred to are determined from measurement of the full width of the emissions at half peak height (FWHM), and are appropriate in the range of 200 nm to 2000 nm.
  • FWHM half peak height
  • a broad wavelength band with regard to the excitation of the semiconductor nanocrystal is meant absorption of radiation having a wavelength equal to, or shorter than, the wavelength of the onset radiation (the onset radiation is understood to be the longest wavelength (lowest energy) radiation capable of being absorbed by the semiconductor nanocrystal).
  • the onset radiation is understood to be the longest wavelength (lowest energy) radiation capable of being absorbed by the semiconductor nanocrystal).
  • This onset occurs near to, but at slightly higher energy than the “narrow wavelength band” of the emission.
  • This is in contrast to the “narrow absorption band” of dye molecules, which occurs near the emission peak on the high energy side, but drops off rapidly away from that wavelength and is often negligible at wavelengths further than 100 nm from the emission.
  • emission peak refers to the wavelength of light within the characteristic emission spectra exhibited by a particular semiconductor nanocrystal size distribution that demonstrates the highest relative intensity.
  • excitation wavelength refers to light having a wavelength lower than the emission peak of the semiconductor nanocrystal used in the first detection reagent.
  • a “hydrophobic” compound e.g., a “hydrophobic” monomer
  • a “hydrophobic” monomer is one that will transfer from an aqueous phase to an organic phase, specifically from water to an organic, water-immiscible nonpolar solvent with a dielectric constant ⁇ 5, with a partition coefficient of greater than about 50%.
  • a “hydrophobic monomer unit” refers to a hydrophobic monomer as it exists within a polymer.
  • a “hydrophobic region” refers to a hydrophobic molecular segment, e.g., a molecular segment within a polymer.
  • a “hydrophobic region” may be a single hydrophobic monomer unit or two or more hydrophobic monomer units that may be the same or different and may or may not be adjacent.
  • a “hydrophilic” compound e.g., a “hydrophilic” monomer is one that will transfer from an organic phase to an aqueous phase, specifically from an organic, water-immiscible nonpolar solvent with a dielectric constant ⁇ 5 to water, with a partition coefficient of greater than about 50%.
  • a “hydrophilic monomer unit” refers to a hydrophilic monomer as it exists in a polymeric segment or polymer.
  • a “hydrophilic region” refers to a hydrophilic molecular segment, e.g., a hydrophilic molecular segment within a polymer.
  • a “hydrophilic region” may be a single hydrophilic monomer unit or two or more hydrophilic monomer units that may be the same or different and may or may not be adjacent.
  • ionizable refers to a group that is electronically neutral at a specific pH, but can be ionized and thus rendered positively or negatively charged at higher or lower pH, respectively.
  • alkyl refers to a branched or unbranched saturated hydrocarbon group of 1 to approximately 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl and tetracosyl, as well as cycloalkyl groups such as cyclopentyl and cyclohexyl.
  • lower alkyl intends an alkyl group of 1 to 4 carbon atoms, and thus includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and t-butyl.
  • alkylene refers to a difunctional saturated branched or unbranched hydrocarbon chain containing from 1 to approximately 24 carbon atoms, typically 1 to approximately 12 carbon atoms, and includes, for example, methylene (—CH 2 —), ethylene (—CH 2 —CH 2 —), propylene (—CH 2 —CH 2 —CH 2 —), 2-methylpropylene (—CH 2 —CH(CH 3 )—CH 2 —), hexylene (—(CH 2 ) 6 —), and the like.
  • “Lower alkylene,” as in the lower alkylene linkage of the optional coupling agent herein, refers to an alkylene group of 1 to 4 carbon atoms.
  • alkenyl refers to a branched or unbranched hydrocarbon group typically although not necessarily containing 2 to about 24 carbon atoms and at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, and the like. Generally, although not necessarily, alkenyl groups herein contain 2 to about 12 carbon atoms.
  • lower alkenyl intends an alkenyl group of 2 to 4 carbon atoms
  • alkenylene refers to a difunctional alkenyl group, in the same way that the term “alkylene” refers to a difunctional alkyl group.
  • alkynyl refers to a branched or unbranched hydrocarbon group typically although not necessarily containing 2 to about 24 carbon atoms and at least one triple bond, such as ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, octynyl, decynyl, and the like. Generally, although again not necessarily, alkynyl groups herein contain 2 to about 12 carbon atoms.
  • the term “lower alkynyl” intends an alkynyl group of 2 to 4 carbon atoms, preferably 3 or 4 carbon atoms.
  • heteroatom-containing and the prefix “hetero-,” as in “heteroatom-containing alkyl” and “heteroalkyl,” refer to a molecule or molecular fragment in which one or more carbon atoms is replaced with an atom other carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon.
  • alkoxy refers to a substituent —O—R wherein R is alkyl as defined above.
  • lower alkoxy refers to such a group wherein R is lower alkyl as defined above, e.g., methoxy, ethoxy and the like.
  • aryl refers to an aromatic moiety containing 1 to 3 aromatic rings. For aryl groups containing more than one aromatic ring, the rings may be fused or linked.
  • Aryl groups are optionally substituted with one or more inert, nonhydrogen substituents per ring; suitable “inert, nonhydrogen” substituents include, for example, halo, haloalkyl (preferably halo-substituted lower alkyl), alkyl (preferably lower alkyl), alkenyl (preferably lower alkenyl), alkynyl (preferably lower alkynyl), alkoxy (preferably lower alkoxy), alkoxycarbonyl (preferably lower alkoxycarbonyl), carboxy, nitro, cyano and sulfonyl.
  • aryl is also intended to include heteroaromatic moieties, i.e., aromatic heterocycles. Generally, although not necessarily, the heteroatoms will be nitrogen, oxygen or sulfur.
  • arylene refers to a difunctional aryl moiety in the same way that the term “alkylene” refers to a difunctional alkyl group.
  • aralkyl refers to an alkyl group with an aryl substituent
  • aralkylene refers to an alkylene group with an aryl substituent
  • alkaryl refers to an aryl group that has an alkyl substituent
  • alkarylene refers to an arylene group with an alkyl substituent
  • halo and “halogen” are used in the conventional sense to refer to a chloro, bromo, fluoro, or iodo substituent.
  • haloalkyl refers to an alkyl group in which at least one of the hydrogen atoms in the group has been replaced with a halogen atom.
  • peptide refers to oligomers or polymers of any length wherein the constituent monomers are alpha amino acids linked through amide bonds, and encompasses amino acid dimers as well as polypeptides, peptide fragments, peptide analogs, naturally occurring proteins, mutated, variant, or chemically modified proteins, fusion proteins, and the like.
  • the amino acids of the peptide molecules may be any of the twenty conventional amino acids, stereoisomers (e.g., D-amino acids) of the conventional amino acids, structural variants of the conventional amino acids, e.g., iso-valine, or non-naturally occurring amino acids such as ⁇ , ⁇ -disubstituted amino acids, N-alkyl amino acids, ⁇ -alanine, naphthylalanine, 3-pyridylalanine, 4-hydroxyproline, 0-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and nor-leucine.
  • stereoisomers e.g., D-amino acids
  • structural variants of the conventional amino acids e.g., iso-valine
  • non-naturally occurring amino acids such as ⁇ , ⁇ -disubstituted amino acids, N-alkyl amino acids, ⁇ -alanine, naphth
  • peptide encompasses peptides with posttranslational modifications such as glycosylations, acetylations, phosphorylations, and the like.
  • oligonucleotide is used herein to include a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded DNA, as well as triple-, double- and single-stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the oligonucleotide.
  • the term includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers, providing that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, such as is found in DNA and RNA.
  • PNAs peptide nucleic acids
  • Neugene synthetic sequence-specific nucleic acid polymers
  • polynucleotide oligonucleotide
  • nucleic acid nucleic acid molecule
  • these terms refer only to the primary structure of the molecule.
  • these terms include, for example, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′ P5′ phosphoramidates, 2′-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for, example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoram
  • polymer is used herein in its conventional sense to refer to a compound having two or more monomer units, and is intended to include linear and branched polymers, the term “branched polymers” encompassing simple branched structures as well as hyperbranched and dendritic polymers.
  • branched polymers encompassing simple branched structures as well as hyperbranched and dendritic polymers.
  • monomer is used herein to refer to compounds that are not polymeric. “Polymers” herein may be naturally occurring, chemically modified, or chemically synthesized.
  • water-dispersible refers to an essentially unaggregated dispersion of particles, such that discrete particles of approximately 2 nm to 50 nm can be sustained indefinitely at high concentrations (10-20 ⁇ M).
  • binding pair refers to first and second molecules that specifically bind to each other. “Specific binding” of the first member of the binding pair to the second member of the binding pair in a sample is evidenced by the binding of the first member to the second member, or vice versa, with greater affinity and specificity than to other components in the sample. The binding between the members of the binding pair is typically noncovalent.
  • affinity molecule and “target analyte” are also used herein to refer to the first and second members of a binding pair, respectively.
  • Exemplary binding pairs include any haptenic or antigenic compound in combination with a corresponding antibody or binding portion or fragment thereof (e.g., digoxigenin and anti-digoxigenin; mouse immunoglobulin and goat anti-mouse immunoglobulin) and nonimmunological binding pairs (e.g., biotin-avidin, biotin-streptavidin, hormone [e.g., thyroxine and cortisol]-hormone binding protein, receptor-receptor agonist or antagonist (e.g., acetylcholine receptor-acetylcholine or an analog thereof), IgG-protein A, lectin-carbohydrate, enzyme-enzyme cofactor, enzyme-enzyme inhibitor, and complementary polynucleotide pairs capable of forming nucleic acid duplexes), and the like.
  • biotin-avidin e.g., digoxigenin and anti-digoxigenin; mouse immunoglobulin and goat anti-mouse immunoglobulin
  • a “nanoparticle conjugate” refers to a nanoparticle linked, through an outer layer of an amphipathic dispersant, to a member of a “binding pair” that will selectively bind to a detectable substance present in a sample, e.g., a biological sample.
  • the first member of the binding pair linked to the nanoparticle can comprise any molecule, or portion of any molecule, that is capable of being linked to the nanoparticle and that, when so linked, is capable of specifically recognizing the second member of the binding pair.
  • the nanoparticles of the invention are nanoparticles with hydrophobic surfaces, the particles having a diameter in the range of about 1 nm to about 1000 nm, preferably in the range of about 2 rim to about 50 nm, more preferably in the range of about 2 nm to about 20 nm.
  • the nanoparticles will be comprised of a semiconductive or metallic material, with semiconductive nanoparticles preferred.
  • the semiconductive or metallic material typically has a coating of a hydrophobic passivating layer resulting from the use of solvents and/or surfactants during nanoparticle manufacture.
  • the hydrophobic surfaces of the nanoparticles have affinity for and thus serve to attach the amphipathic dispersant by virtue of the hydrophobic regions within the dispersant.
  • Semiconductive nanoparticles may be composed of an organic semiconductor material or an inorganic semiconductor material.
  • Organic semiconductor materials will generally be conjugated polymers. Suitable conjugated polymers include, for example, cis and trans polyacetylenes, polydiacetylenes, polyparaphenylenes, polypyrroles, polythiophenes, polybithiophenes, polyisothianaphthene, polythienylvinylenes, polyphenylenesulfide, polyaniline, polyphenylenevinylenes, and polyphenylenevinylene derivatives, e.g., poly(2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene vinylene (“MEH-PPV”) (see U.S.
  • Inorganic semiconductive nanoparticles are, however, preferred, and are optimally crystalline in nature; such nanoparticles are termed “semiconductor nanocrystals” herein.
  • Semiconductor nanocrystals are capable of luminescence, generally fluorescence, when excited by light.
  • detection of biological compounds by photoluminescence utilizes fluorescent organic dyes and chemiluminescent compounds.
  • the use of semiconductor nanocrystals as luminescent markers, particularly in biological systems, provides advantages over existing fluorescent dyes. Many of these advantages relate to the spectral properties of nanocrystals, e.g., the ability to control the composition and size of nanocrystals enables one to construct nanocrystals with fluorescent emissions at any wavelength in the UV-visible-IR regions.
  • semiconductor nanocrystals that emit energy in the visible range include, but are not limited to, CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, and GaAs.
  • Semiconductor nanocrystals that emit energy in the near IR range include, but are not limited to, InP, InAs, InSb, PbS, and PbSe.
  • semiconductor nanocrystals that emit energy in the blue to near-ultraviolet include, but are not limited to, ZnS and GaN.
  • 5-20 discrete emissions (five to twenty different size populations or distributions distinguishable from one another), more preferably 10-15 discrete emissions, are obtained for any particular composition, although one of ordinary skill in the art will realize that fewer than five emissions and more than twenty emissions could be obtained depending on the monodispersity of the semiconductor nanocrystal particle population. If high information density is required, and thus a greater number of distinct emissions, the nanocrystals are preferably substantiall y monodisperse within the size range given above.
  • monodisperse refers to a population of particles (e.g., a colloidal system) in which the particles have substantially identical size and shape. In preferred embodiments for high information density applications, monodisperse particles deviate less than 10% rms in diameter, and preferably less than 5% rms. Monodisperse semiconductor nanocrystals have been described in detail in Murray et al. (1993) J. Am. Chem. Soc. 115:8706, and in Murray, “Synthesis and Characterization of II-VI Quantum Dots and Their Assembly into 3-D Quantum Dot Superlattices,” doctoral dissertation, Massachusetts Institute of Technology (1995).
  • the linewidth of the emission may be in the range of 40-60 nm.
  • Semiconductor nanocrystals may be made using techniques known in the art. See, e.g., U.S. Pat. Nos. 6,048,616, 5,990,479, 5,690,807, 5,505,928 and 5,262,357, as well as International Patent Publication No. WO 99/26299, published May 27, 1999.
  • exemplary materials for use as semiconductor nanocrystals in the biological and chemical assays of the present invention include, but are not limited to, those described above, including Group 2-16, 12-16, 13-15 and 14 semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlSb, PbS, PbSe, Ge and Si and ternary and quaternary mixtures thereof.
  • Group 2-16, 12-16, 13-15 and 14 semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaS
  • the surface of the semiconductor nanocrystal is modified to enhance the efficiency of the emissions, prior to surface modification with the multiply amphipathic dispersant, by adding an overcoating layer or shell to the semiconductor nanocrystal.
  • the shell is preferred because at the surface of the semiconductor nanocrystal, surface defects can result in traps for electrons or holes that degrade the electrical and optical properties of the semiconductor nanocrystal.
  • An insulating layer at the surface of the semiconductor nanocrystal provides an atomically abrupt jump in the chemical potential at the interface that eliminates energy states that can serve as traps for the electrons and holes. This results in higher efficiency in the luminescent process.
  • Suitable materials for the shell include semiconductor materials having a higher bandgap energy than the semiconductor nanocrystal core, In addition to having a bandgap energy greater than the semiconductor nanocrystal core, suitable materials for the shell should have good conduction and valence band offset with respect to the core semiconductor nanocrystal.
  • the conduction band is desirably higher and the valence band is desirably lower than those of the core semiconductor nanocrystal.
  • a material that has a bandgap energy in the ultraviolet regions may be used.
  • Exemplary materials include ZnS, GaN, and magnesium chalcogenides, e.g., MgS, MgSe, and MgTe.
  • materials having a bandgap energy in the visible such as CdS or CdSe, may also be used.
  • the preparation of a coated semiconductor nanocrystal may be found in, e.g., Dabbousi et al. (1997) J. Phys. Chem. B 101:9463, Hines et al. (1996) J. Phys. Chem. 100: 468-471, Peng et al. (1997) J. Am, Chem. Soc. 119:7019-7029, and Kuno et al. (1997) J. Phys. Chem. 106:9869.
  • the nanoparticles of the invention may also be metallic. Such particles are useful, for example, in surface enhanced Raman scattering (SERS), which employs nanometer-size particles onto which Raman active moieties (e.g., a dye or pigment, or a functional group exhibiting a characteristic Raman spectrum) are adsorbed or attached.
  • SERS surface enhanced Raman scattering
  • Metallic nanoparticles may be comprised of any metal or metallic alloy or composite, although for use in SERS, a SERS active metal is used, e.g., silver, gold, copper, lithium, aluminum, platinum, palladium, or the like.
  • the particles can be in a core-shell configuration, e.g., a gold core may be encased in a silver shell; see, e.g., Freeman et al. (1996) J. Phys. Chem. 100:718-724, or the particles may form small aggregates in solution. Kneipp et al. (1998) Applied Spectroscopy 52:1493.
  • the unmodified nanoparticles--and thus the inner core of the present surface-modified nanoparticles-- also comprise a hydrophobic coating on the semiconductive or metallic material resulting from solvents and/or surfactants used in nanoparticle manufacture.
  • semiconductive nanoparticles will typically have a water-insoluble organic coating that has affinity for the semiconductive material, the coating comprised of a passivating layer resulting from use of a coordinating solvent such as hexyldecylamine or a trialkyl phosphine or trialkyl phosphine oxide, e.g., trioctylphosphine oxide (TOPO), trioctylphosphine (TOP), or tributylphosphine (TBP).
  • a coordinating solvent such as hexyldecylamine or a trialkyl phosphine or trialkyl phosphine oxide, e.g., trioctylphosphine oxide (TOPO), trioctylphosphine (TOP), or tributylphosphine (TBP).
  • TOPO trioctylphosphine oxide
  • TOP trioctylphosphine
  • TBP tributylphos
  • Hydrophobic surfactants typically used in the manufacture of metallic nanoparticles and forming a coating thereon include, by way of example, octanethiol, dodecanethiol, dodecylamine, and tetraoctylammonium bromide.
  • Metallic inner cores will typically have a surfactant coating that has affinity for the metallic material, the coating similarly deriving from surfactant compounds used in the manufacture of metallic nanoparticles.
  • the surfactant coating is comprised of a hydrophobic surfactant.
  • the dispersant used to modify the hydrophobic surface of the nanoparticles is a multiply amphipathic dispersant, i.e., a compound having two or more hydrophobic regions and two or more hydrophilic regions.
  • the multiply amphipathic dispersant is polymeric, and may be composed of either a linear or branched polymer, whether naturally occurring, chemically modified, or chemically synthesized. Structurally, polymers are classified as either linear or branched wherein the term “branched” generally means that the individual molecular units (i.e., monomer units) of the branches are discrete from the polymer backbone, and may or may not have the same chemical constitution as the polymer backbone.
  • the simplest branched polymers are the “comb branched” polymers wherein a linear backbone bears one or more essentially linear pendant side chains.
  • This simple form of branching may be regular or irregular (in the latter case, the branches are distributed in non-uniform or random fashion on the polymer backbone).
  • regular comb branching is a comb branched polystyrene as described by Altores et al. (1965) J. Polymer Sci., Part A 3:4131-4151, and an example of irregular comb branching is illustrated by the graft copolymers described by Sorenson et al. in Preparative Methods of Polymer Chemistry, 2nd Ed., Interscience Publishers, pp. 213-214 (1968).
  • the amphipathic dispersant may also be a branched polymer in the form of a cross-linked or network polymer, i.e., a polymeric structure wherein individual polymer chains or branches are connected through the use of bifunctional compounds; e.g., acrylic acid monomer units bridged or crosslinked with a diamine linker.
  • a cross-linked or network polymer i.e., a polymeric structure wherein individual polymer chains or branches are connected through the use of bifunctional compounds; e.g., acrylic acid monomer units bridged or crosslinked with a diamine linker.
  • many of the individual branches are not linear in that each branch may itself contain side chains pendant from a linear chain and it is not possible to differentiate between the backbone and the branches.
  • each polymer macromolecule (backbone) is cross-linked at two or more sites to other polymer macromolecules.
  • the chemical constitution of the cross-linkages may vary from that of the polymer macromolecules.
  • the various branches or cross-linkages may be structurally
  • amphipathic dispersant may also have other structural configurations, e.g., it may be a star/comb-branched type polymer, as described in U.S. Pat. Nos. 4,599,400 and 4,690,985, or a rod-shaped dendrimer as disclosed in U.S. Pat. No. 4,694,064.
  • amphipathic dispersants herein are hyperbranched (containing two or more generations of branching) or dendrimeric.
  • dendrimers are regularly branched macromolecules with a branch point at each repeat unit.
  • hyperbranched polymers are obtained via a polymerization reaction, while most regular dendrimers are obtained by a series of stepwise coupling and activation steps.
  • dendrimers include the polyamidoamine (PAMAM) Starburst® dendrimers of Tomalia et al. (1985) Polym. J. 17:117, the convergent dendrimers of Hawker et al. (1990) J. Am. Chem. Soc.
  • the hydrophilic regions represent approximately 30 wt. % to 75 wt. % of the amphipathic dispersant, and are comprised of at least one monomer unit containing an ionizable or polar moiety, preferably an ionizable moiety such as a carboxylic acid, sulfonic acid, phosphonic acid or amine substituent.
  • hydrophilic monomer units include, but are not limited to:
  • water-soluble ethylenically unsaturated C 3 -C 6 carboxylic acids such as acrylic acid, alkyl acrylic acids (particularly methacrylic acid), itaconic acid, maleic acid, fumaric acid, acrylamidomethyl-propanesulfonic acid, vinyl sulfonic acid, vinyl phosphonic acid, vinyllactic acid, and styrene sulfonic acid;
  • allylamine and allylamine salts formed with an inorganic acid e.g., hydrochloric acid
  • di-C 1 -C 3 -alkylamino-C 2 -C 6 -alkyl acrylates and methacrylates such dimethylaminoethyl acrylate, dimethylaminoethyl methacrylate, diethylaminoethyl acrylate, diethylaminoethyl methacrylate, dimethylaminopropyl acrylate, dimethylaminobutyl acrylate, dimethylaminoneopentyl acrylate and dimethylaminoneopentyl methacrylate;
  • olefinically unsaturated nitriles such as acrylonitrile
  • diallylammonium compounds such as dimethyldiallylammonium chloride, dimethyldiallylammonium bromide, diethyldiallylammonium chloride, methyl-t-butyldiallylammonium methosulfate, methyl-n-propyldiallylammonium chloride, dimethyldiallylammonium hydrogensulfate, dimethyldiallylammonium dihydrogenphosphate, di-n-butyldiallylammonium bromide, diallylpiperidinium bromide, diallylpyrrolidinium chloride and diallylmorpholinium bromide;
  • acrylamide and substituted acrylamides such as N-methylolacrylamide and C 1 -C 3 alkyl acrylamides, particularly methacrylamide;
  • the hydrophilic functionality may be directly bound to a carbon atom in the polymer backbone, but will usually be bound through a linkage that provides some degree of spacing between the polymer backbone and the hydrophilic functional group.
  • Suitable linkages include, but are not limited to, branched or unbranched alkylene, branched or unbranched alkenylene, branched or unbranched heteroalkylene (typically alkylene containing one or more ether or —NH— linkages) a branched or unbranched heteroalkenylene (again, typically alkenylene containing one or more ether or —NH— linkages), arylene, heteroarylene, alkarylene, aralkylene, and the like.
  • the linkage will typically contain 2 to 24, more typically 2 to 12, carbon atoms.
  • the hydrophilic regions may also be composed of partially or fully hydrolyzed poly(vinyl alcohol), poly(ethylene glycol), poly(ethylene oxide), highly hydrated poly(alkylene oxides) such as poly(ethylene oxide), cellulosic segments (e.g., comprised of cellulose per se or cellulose derivatives such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, and the like), and polysaccharides such as chitosan or dextran.
  • poly(vinyl alcohol) poly(ethylene glycol), poly(ethylene oxide), highly hydrated poly(alkylene oxides) such as poly(ethylene oxide)
  • cellulosic segments e.g., comprised of cellulose per se or cellulose derivatives such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose,
  • the hydrophobic regions represent approximately 25 wt. % to 90 wt. % of the amphipathic dispersant, and are comprised of at least one non-ionizable, nonpolar monomer unit, facilitating noncovalent association with the hydrophobic surface of the nanoparticle.
  • monomer units include, but are not limited to:
  • acrylates such as methacrylate, methyl methacrylate, ethyl methacrylate, butyl methacrylate, isobutyl methacrylate, hexyl methacrylate, isodecyl methacrylate, lauryl methacrylate, phenyl methacrylate, isopropyl acrylate, isobutyl acrylate and octadecylacrylate,
  • alkylenes such as ethylene and propylene
  • alkyl acrylamides wherein the alkyl group is larger than lower alkyl (particularly alkyl acrylamides wherein the alkyl group has six or more carbon atoms, typically six to twelve carbon atoms, such as hexylacrylamide, octylacrylamide, and the like);
  • styrene and hydrophobically derivatized styrenes i.e., styrene substituted with one or more hydrophobic substituents, e.g., C 5 -C 12 hydrocarbyl groups;
  • vinyl esters such as vinyl acetate
  • vinyl halides such as vinyl chloride.
  • the hydrophobic regions may also be composed of polychloroprene, polybutadiene, polysiloxane, polydimethylsiloxane, polyisobutylene or polyurethane blocks, or they may be polycondensates of 2-poly(hydroxyalkanoic acids) such as 2-hydroxypropanoic acid, 2-hydroxybutanoic acid, 2-hydroxyisobutanoic acid, 2-hydroxyheptanoic acid, 10-hydroxydecanoic acid, 12-hydroxydodecanoic acid, 12-hydroxystearic acid, 16-hydroxyhexadecanoic acid, 2-hydroxystearic acid, 2-hydroxyvaleric acid or the corresponding condensates obtained from lactones, condensates of diols and dicarboxylic acids such as polyethylene adipate, or polylactams such as polycaprolactam.
  • 2-poly(hydroxyalkanoic acids) such as 2-hydroxypropanoic acid, 2-hydroxybutanoic acid, 2-hydroxyisobutanoic
  • any of the aforementioned monomer units and polymer segments can be modified using techniques and reagents routinely used by those of ordinary skill in the art. Such modifications include, for example, routine substitutions, additions of chemical groups such as alkyl groups and alkylene groups, hydroxylations, oxidations, and the like.
  • Such branched polymers, composed of hydrophobic segments and hydrophilic segments are typically comprised of (1) a hydrophobic backbone with hydrophilic branches, (2) a hydrophilic backbone with hydrophobic branches, or (3) a backbone that may be either hydrophobic or hydrophilic, and is substituted with both hydrophilic and hydrophobic branches.
  • Such polymers can be prepared by any suitable method readily known to those of ordinary skill in the art and/or described in the pertinent texts and literature.
  • Polymers of type (1) can be prepared by copolymerization of a hydrophobic monomer with a second monomer that includes suitable reactive groups through which the hydrophilic side chains (branches) can be grafted to the backbone.
  • type (1) polymers can be prepared by polymerizing a single hydrophobic monomer with a suitable reactive side group, and a fraction of those reactive side groups can be modified post-polymerization by grafting hydrophilic side chains.
  • polymers of type (2) can be prepared by copolymerization of a hydrophilic monomer with a second monomer that includes suitable reactive groups through which the hydrophobic side chains (branches) can be grafted to the backbone.
  • type (2) polymers can be prepared by polymerizing a single hydrophilic monomer with a suitable reactive side group, and a fraction of those reactive side groups can be modified post-polymerization by grafting hydrophobic side chains.
  • Type (3) polymers can be prepared by first synthesizing a linear polymer having reactive sites throughout the backbone, and then grafting hydrophilic and hydrophobic side chains onto the backbone in a fashion that may or may not be ordered.
  • amphipathic dispersants include acrylic acid and methacrylic acid polymers modified to include hydrophobic regions, as well as copolymers of acrylic acid and/or methacrylic acid with hydrophobic comonomers such as alkyl acrylamides.
  • examples of such polymers are poly(acrylic acid-co-octylacrylamide), poly(acrylic acid-co-hexylacrylamide), poly(methacrylic acid-co-octylacrylamide), and poly(methacrylic acid-co-hexylacrylamide), with poly(acrylic acid-co-octylacrylamide) most preferred.
  • the specific methodology used to synthesize polymers suitable as the multiply amphipathic dispersant will depend on the particular monomer types that are employed.
  • suitable polymerization techniques include step polymerization, radical chain polymerization, emulsion polymerization, ionic chain polymerization, chain copolymerization, ring-opening polymerization, living polymerization, polycondensation reactions, and graft polymerization.
  • the amphipathic dispersant is formed by addition polymerization of ethylenically unsaturated monomers.
  • Such polymerization reactions are generally catalyzed using metallic catalysts (e.g., transition metal-based metallocenes, Ziegler-Natta catalysts, Brookhart-type catalysts, etc.) and typically involve contacting the monomer(s), catalyst, and a catalyst activator (e.g., methyl aluminoxane, or “MAO”) at a suitable temperature at reduced, elevated or atmospheric pressure, under an inert atmosphere, for a time effective to produce the desired polymer.
  • a catalyst activator e.g., methyl aluminoxane, or “MAO”
  • An added solvent may, if desired, be employed, or the monomeric compounds may serve as solvent.
  • the reaction may be conducted under solution or slurry conditions, in a suspension, or in the gas phase.
  • branched polymers can be prepared using this technique by introducing reactive sites into the polymer backbone during polymerization (e.g., by incorporating some fraction of monomer units having a pendant reactive site), followed by synthesis or grafting of branches at the reactive sites.
  • the amphipathic dispersant is comprised of a hydrophilic backbone that has been modified to contain hydrophobic anchoring groups, i.e., hydrophobic side chains that serve to “anchor” the dispersant to the nanoparticle surface.
  • hydrophilic polymers containing pendant carboxylic acid groups e.g., as in poly(acrylic acid), [—(CH 2 CH(CO 2 H)] n —
  • hydrophilic polymers containing pendant carboxylic acid groups e.g., as in poly(acrylic acid), [—(CH 2 CH(CO 2 H)] n —
  • the pendant carboxylic acid groups of poly(acrylic acid) can be activated with a suitable activating agent, e.g., thionyl chloride or a carbodiimide, followed by reaction with a long chain alkylamine, e.g., a C 4 -C 12 alkylamine such as octylamine, and finally with a hydrolyzing agent such as water.
  • a suitable activating agent e.g., thionyl chloride or a carbodiimide
  • a long chain alkylamine e.g., a C 4 -C 12 alkylamine such as octylamine
  • a hydrolyzing agent such as water.
  • the resulting polymer is an amphipathic polymer with a hydrophilic backbone (by virtue of the carboxylic acid groups present after partial hydrolysis) and hydrophobic side chains (the long chain alkyl group attached to the backbone through an amide linkage).
  • hydrophobically modified hydrophilic polymers are hydrophobically modified peptides, preferably hydrophobically modified synthetic polypeptides.
  • the use of synthetic polypeptides allows for control over a number of factors, including the monodispersity of the molecular weight of the hydrophilic backbone, the number and position of modifiable groups on the backbone, and the regularity of the modification, i.e., whether the hydrophobic groups are randomly distributed throughout the polypeptide chain or present in an ordered, “regular” fashion.
  • Suitable polypeptides are triblock (A-B-A) copolymers, for example, triblock copolymers of aspartate and norleucine, in which case polynorleucine is preferably the central block “B.”
  • A-B-A triblock copolymers of aspartate and norleucine
  • polynorleucine is preferably the central block “B.”
  • Such a triblock copolymer provides a region rich in hydrophobic side chains.
  • the central block “B” can comprise a hydrophilic amino acid, for example, poly(lysine), which can be modified via standard chemistries to include hydrophobic side chains.
  • the carboxylate-rich aspartate side chains (A) provide the polar, ionic groups that not only aid in rendering the nanocrystal water dispersible, but provide reactive sites or functionalizable moieties for further chemistry, such as conjugation to affinity molecules.
  • polypeptide compositions of the present invention may also be monofunctional in nature, e.g., polylysine or polyaspartate, diblock copolymers (A-B) or triblock copolymers of three different amino acids (A-B-C). These compositions are also not restricted to lysine or aspartate, but may make use of any number of combinations of the known amino acids.
  • the hydrophobic regions of a polypeptide are comprised of at least one hydrophobic amino acid and the hydrophilic regions are comprised of at least one hydrophilic amino acid.
  • hydrophobic amino acids include, for example, alanine, glycine, valine, leucine, isoleucine, norleucine, proline, phenylalanine, methionine, tryptophane, cysteine, and includes hydrophilic amino acids modified to include hydrophobic side chains, while hydrophilic amino acids include aspartic acid, glutamic acid, lysine, arginine, histidine, asparagine, glutamine, serine, threonine and tyrosine.
  • the amphipathic dispersant generally although not necessarily has a molecular weight in the range of approximately 500 to 50,000, preferably in the range of approximately 1000 to 10,000, more preferably in the range of approximately 1000 to 5000.
  • the dispersant may be modified so as to contain functionalizable sites useful for covalent or noncovalent attachment to an external molecular moiety.
  • the functionalizable sites may be present in addition to the ionizable groups discussed above, or the ionizable groups may themselves serve as functionalizable sites suitable for binding an external molecular moiety.
  • Functionalizable sites include, for example, any of the conventional functional groups that are modified using simple, conventional chemical techniques, e.g., amino groups, nitriles, carboxylic acids, esters, acid chlorides, and the like.
  • the functionalizable sites are spaced apart from the dispersant structure by an inert linking moiety, e.g., an alkylene or oxyalkylene chain, typically composed of about 2 to 20 carbon atoms, preferably about 4 to 10 carbon atoms, or other linking moieties such as those described above with respect to the spacer linkages that may be present linking hydrophilic functional groups to the polymer backbone.
  • Hydrophobic nanoparticles may be rendered water dispersible by surface modification with the amphipathic dispersant. That is, the hydrophobic regions of the dispersant associate with the hydrophobic nanoparticle surface, and the hydrophilic regions are externally facing and provide water dispersibility. Surface modification of the nanoparticles is carried out as follows.
  • a solution of the amphipathic dispersant is prepared by admixing the selected amphipathic dispersant with a suitable nonaqueous solvent, preferably a nonpolar, water-immiscible solvent such as n-hexane or chloroform.
  • a suitable nonaqueous solvent preferably a nonpolar, water-immiscible solvent such as n-hexane or chloroform.
  • Ionizable groups on the dispersant if present, are then converted to salt form by treatment with an appropriate acid or base, which serves as an ionizing agent.
  • suitable bases are generally inorganic bases, e.g., ammonium hydroxides or hydroxides of alkali metals (e.g., sodium or potassium) or alkaline earth metals (e.g., magnesium or calcium).
  • the hydrophobic nanoparticles are dispersed in the same solvent, either before or after the aforementioned ionization step.
  • the nanoparticles are added after ionization, preferably dropwise, to a stirring solution of the ionized dispersant.
  • the nanoparticles may be dispersed in the solvent at the outset, and the dispersant added thereto.
  • two separate solutions may be prepared and mixed, with one solution containing the dispersant and the other solution containing the nanoparticles, with both solutions preferably containing the same solvent.
  • the admixture is preferably stirred for several minutes to ensure complete mixing of the components.
  • the admixture of nanoparticles, dispersant and solvent is subjected to conditions effective to result in absorption of the dispersant by the nanoparticles.
  • the admixture may be heated or placed under vacuum to remove the solvent, such a drying process resulting in dispersant-coated nanoparticles.
  • the conditions may involve changing the polarity of the solvent and/or changing the ionic state of the polymer.
  • the dispersant-coated nanoparticles are transferred to an aqueous medium such as water, using solvent exchange (if the dispersant-coated nanoparticles are not previously dried) or addition of water or an aqueous buffer (if the dispersant-coated nanoparticles are previously dried).
  • the aqueous buffer if one is used, should be effective to facilitate dispersion of the nanoparticles in the aqueous medium.
  • the water dispersion is then filtered to remove any large micellar structures formed by excess dispersant in solution that is not associated with the particles. These materials may then be used in any applications requiring aqueous-based sols of nanocrystals.
  • the crosslinker used may be tailored to match the properties of the dispersant coating.
  • a diamine could be used to crosslink a dispersant coating containing carboxylic acids.
  • crosslinkers that carry charges or other groups capable of stabilizing the dispersed colloids as described herein.
  • a diamino carboxylate or sulfonate and a diamino polyethylene glycol crosslinkers are especially useful.
  • a similar chemistry would apply for crosslinkers having multiple amine moieties, such as dendrimers, modified dendrimers, and the like.
  • the amount of amphipathic dispersant per unit mass of the “inner core” (i.e., per unit mass of the original, unmodified nanoparticle) in the resulting dispersant-coated nanoparticles is proportional to the size and surface area of the nanoparticles.
  • the number ratio of the dispersant to the inner core will be in the range of approximately 50:1 to approximately 5000:1.
  • the ratio will be closer to 50:1 for smaller nanoparticles, i.e., nanoparticles less than about 5 nm in diameter (e.g., green CdSe quantum dots), and will be closer to 5000:1 for larger nanoparticles, i.e., nanoparticles about 5 nm to 10 nm in diameter (e.g., red CdSe quantum dots).
  • the invention additionally relates to conjugates of the present surface-modified semiconductive nanoparticles and compositions comprising those conjugates in association with a target analyte.
  • the surface-modified semiconductive nanoparticles of the invention may be conjugated to an affinity molecule that serves as the first member of a binding pair.
  • an affinity molecule that serves as the first member of a binding pair.
  • it is the amphipathic dispersant on the nanoparticle surface that provides the means for linkage to the affinity molecule.
  • ionizable groups present within the hydrophilic regions of the amphipathic dispersant may provide the means for linkage to the affinity molecule, and/or other functional groups present within or introduced into the dispersant molecule may provide the means for linkage to the affinity molecule.
  • the linkage will generally be covalent, and suitable linkers are discussed in Section III, above. Suitable methods of conjugating molecules and molecular segments to affinity molecules are described, for example, in Hermanson, Bioconjugate Techniques (Academic Press, NY, 1996).
  • Such semiconductive nanoparticle “conjugates,” by virtue of the affinity molecule, can be used to detect the presence and/or quantity of biological and chemical compounds, interactions in biological systems, biological processes, alterations in biological processes, or alterations in the structure of biological compounds. That is, the affinity molecule, when linked to the semiconductive nanoparticle, can interact with a biological target that serves as the second member of the binding pair, in order to detect biological processes or reactions, or to alter biological molecules or processes.
  • the interaction of the affinity molecule and the biological target involves specific binding, and can involve covalent, noncovalent, hydrophobic, hydrophilic, electrostatic, van der Waal's, or magnetic interaction.
  • the affinity molecule physically interacts with the biological target.
  • the affinity molecule associated with the semiconductive nanoparticles can be naturally occurring or chemically synthesized, and can be selected to have a desired physical, chemical, or biological property.
  • properties include, but are not limited to, covalent and noncovalent association with proteins, nucleic acids, signaling molecules, prokaryotic or eukaryotic cells, viruses, subcellular organelles and any other biological compounds.
  • Other properties of such molecules include, but are not limited to, the ability to affect a biological process (e.g. cell cycle, blood coagulation, cell death, transcription, translation, signal transduction, DNA damage or cleavage, production of radicals, scavenging radicals, etc.), and the ability to alter the structure of a biological compound (e.g. crosslinking, proteolytic cleavage, radical damage, etc
  • the nanoparticle conjugate is comprised of a semiconductive nanoparticle that emits light at a tunable wavelength and is associated with a nucleic acid.
  • the association can be direct or indirect.
  • the nucleic acid can be any ribonucleic acid, deoxyribonucleic acid, dideoxyribonucleic acid, or any derivatives and combinations thereof.
  • the nucleic acid can also be oligonucleotides of any length.
  • the oligonucleotides can be single-stranded, double-stranded, triple-stranded or higher order configurations (e.g. Holliday junctions, circular single-stranded DNA, circular double-stranded DNA, DNA cubes, (see Seeman (1998) Ann. Rev.
  • nanoparticle conjugates can comprise nanocrystals associated with individual nucleotides, deoxynucleotides, dideoxynucleotides or any derivatives and combinations thereof and used in DNA polymerization reactions such as DNA sequencing, reverse transcription of RNA into DNA, and polymerase chain reactions (PCR).
  • Nucleotides also include monophosphate, diphosphate and triphosphates and cyclic derivatives such as cyclic adenine monophosphate (cAMP).
  • FISH fluorescence in situ hybridization
  • nanocrystals are conjugated to oligonucleotides designed to hybridize to a specific sequence in vivo.
  • the fluorescent nanocrystal tags are used to visualize the location of the desired DNA sequence in a cell. For example, the cellular location of a gene whose DNA sequence is partially or completely known can be determined using FISH. Any DNA or RNA whose sequence is partially or completely known can be visually targeted using FISH.
  • messenger RNA (mRNA), DNA telomeres, other highly repeated DNA sequences, and other non-coding DNA sequencing can be targeted by FISH.
  • the nanoparticle conjugate may also comprise a surface-modified semiconductive nanoparticle as provided herein in association with a molecule or reagent for detection of biological compounds such as enzymes, enzyme substrates, enzyme inhibitors, cellular organelles, lipids, phospholipids, fatty acids, sterols, cell membranes, molecules involved in signal transduction, receptors and ion channels.
  • the conjugate also can be used to detect cell morphology and fluid flow; cell viability, proliferation and function; endocytosis and exocytosis (Betz et al. (1996) Curr. Opin. Neurobiol. 6(3):365-71); and reactive oxygen species (e.g., superoxide, nitric oxide, hydroxyl radicals, oxygen radicals).
  • the conjugate can be used to detect hydrophobic or hydrophilic regions of biological systems.
  • Conjugates of semiconductive nanocrystals also find utility in numerous other biological and non-biological applications where luminescent markers, particularly fluorescent markers, are typically used. See, for example, Haugland, R.P. Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, Oreg. Sixth Ed. 1996; Website, www.probes.com.).
  • Examples of areas in which the luminescent nanoparticle conjugates of the invention are useful include, without limitation, fluorescence immunocytochemistry, fluorescence microscopy, DNA sequence analysis, fluorescence in situ hybridization (FISH), fluorescence resonance energy transfer (FRET), flow cytometry (Fluorescence Activated Cell Sorter; FACS) and diagnostic assays for biological systems.
  • FISH fluorescence in situ hybridization
  • FRET fluorescence resonance energy transfer
  • FACS Fluorescence Activated Cell Sorter
  • a modified polyacrylic acid was prepared by diluting 100 g [0.48 mol COONa] of poly(acrylic acid, sodium salt) (obtained from Aldrich, molecular weight 1200) was diluted two-fold in water and acidified in a 1.0 L round bottom flask with 150 ml (1.9 mol) of concentrated HCl. The acidified polymer solution was concentrated to dryness on a rotary evaporator (100 mbar, 80° C.). The dry polymer was evacuated for 12 hours at ⁇ 10 mbar to ensure water removal.
  • the product was dissolved into 400 ml ethyl acetate (Aldrich) with gentle heating, and basified with 200 ml di-H 2 O and 100 g N-N-N-N-tetramethylammonium hydroxide pentahydrate (0.55 mo) for 12 hours.
  • the aqueous layer was removed and precipitated to a gummy white product with 400 ml of 1.27 M HCl.
  • the product was decanted and triturated with 100 ml of di-H 2 O twice more, after which the aqueous washings were back-extracted into 6 ⁇ 100 ml portions of ethyl acetate.
  • the dispersion was then stirred for two minutes to ensure complete mixing of the components and thereafter the chloroform was removed in vacuo with low heat to yield a thin film of the particle-polymer complex on the wall of the flask.
  • Twenty milliliters of distilled water were added to the flask and swirled along the walls of the flask to aid in dispersing the particles in the aqueous medium.
  • the dispersion was then allowed to stir overnight at room temperature.
  • the nanocrystals are freely dispersed in the aqueous medium, possess pendant chemical functionalities and may therefore be linked to affinity molecules of interest using methods well known in the art for biolabeling experiments.
  • the fact that the nanocrystals now have a highly charged surface means they can be readily utilized in polyelectrolyte layering experiments for the formation of thin films and composite materials.
  • the purified dispersion was concentrated to 20 milliliters and 10 milliliters of this nanocrystal dispersion were activated with 79 ⁇ moles (15 mg) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 158 ⁇ moles (34 mg) N-hydroxysulfosuccinimide for 30 minutes at room temperature.
  • EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • 158 ⁇ moles 34 mg
  • N-hydroxysulfosuccinimide for 30 minutes at room temperature.
  • the particle dispersion was then buffer exchanged to pH 8.0 via diafiltration against 50 mM phosphate buffer, pH 8.0.
  • these materials can be stored in any number of biological buffers and used as fluorescent biological labels to detect biotin-labeled analytes of interest.
  • streptavidin was used here as an example, the simplicity and generality of the above coupling chemistries can be efficiently extended to forming functional conjugates with any number of biological molecules of interest, such as antibodies, peptides, and oligonucleotides, for example.
  • Example 2 Ten milliliters of nanocrystals at 3.5 ⁇ M, stabilized as described in Example 2, were purified by tangential flow filtration, as described in Example 3, against 1 liter of distilled water to remove excess polymer.
  • the nanocrystals were concentrated to 10 milliliters and the pH of the aqueous dispersion was decreased to pH 6.5 with 50 ⁇ l additions of 0.1 M HCl. 67 milligrams (315 moles) EDC were added to the stirring nanocrystal dispersion.
  • reaction was allowed to proceed for 10 minutes before 1 milliliter of 0.5M borate buffer (pH 8.5) containing 3.94 moles of the crosslinking reagent Starburst® (PAMAM) Dendrimer, Generation 0, were added to the reaction mixture.
  • the reaction mixture was stirred for 2 hours at room temperature and then transferred to a 50,000 molecular weight cut-off polyethersulfone dialysis bag. Dialysis was performed for 24 hours against 2 changes of 4 liters of water.
  • Example 2 Ten milliliters of nanocrystals at 3.5 ⁇ M, stabilized as described in Example 2, were purified by tangential flow filtration, as described in Example 3, against 1 liter of distilled water to remove excess polymer.
  • the nanocrystals were concentrated to 10 milliliters and the pH of the aqueous dispersion was decreased to pH 6.5 with 50 ⁇ l additions of 0.1 M HCl. 67 milligrams (315 ⁇ moles) EDC were added to the stirring nanocrystal dispersion.
  • reaction was allowed to proceed for 10 minutes before 1 milliliter of 0.5M borate buffer (pH 8.5) containing 3.94 ⁇ moles of the crosslinking reagent lysine (a diamino carboxylic acid) were added to the reaction mixture.
  • the reaction mixture was stirred for 2 hours at room temperature and then transferred to a 50,000 molecular weight cut-off polyethersulfone dialysis bag. Dialysis was performed for 24 hours against 2 changes of 4 liters of water.
  • a triblock polypeptide comprised of (Aspartate) 4 -(Norleucine) 8 -(Aspartate) 4 has been used to stabilize hydrophobic nanocrystals in water by the following method: Five milliliters of a 3.5 ⁇ M nanocrystal solution were washed as described in Example 1 and redispersed in 5 milliliters of chloroform.
  • nanocrystal dispersion in chloroform was then added dropwise to the stirring polypeptide solution and the entire mixture was allowed to stir for an additional 2 minutes before all the solvent was removed in vacuo with low heat (40 degrees Celsius) to yield a thin film of the particle-polymer complex on the wall of the flask.
  • Five milliliters of distilled water were then added to the flask and swirled in order to aid in dispersing the nanocrystals fully in the aqueous medium.
  • these polypeptide stabilized nanocrystals can be efficiently purified away from excess polypeptide by dialysis, tangential flow filtration, or various forms of chromatography known to those skilled in the art.

Abstract

Water-dispersible nanoparticles are prepared by applying a coating of a multiply amphipathic dispersant to the surface of a hydrophobic nanoparticle comprised of a semiconductive or metallic material. The multiply amphipathic dispersant has two or more hydrophobic regions and two or more hydrophilic regions, and is typically polymeric. Preferred polymeric dispersants are comprised of (1) a hydrophobic backbone with hydrophilic branches, (2) a hydrophilic backbone with hydrophobic branches, or (3) a backbone that may be either hydrophobic or hydrophilic, and substituted with both hydrophilic and hydrophobic branches. Monodisperse populations of water-dispersible nanoparticles are also provided, as are conjugates of the water-dispersible nanoparticles with affinity molecules such as peptides, oligonucleotides, and the like.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a divisional of U.S. patent application Ser. No. 09/841,237, filed Apr. 23, 2001, which claims priority to U.S. Provisional Application No. 60/240,216, filed Oct. 13, 2000. The disclosures of the aforementioned applications are incorporated by reference in their entireties.
  • TECHNICAL FIELD
  • This invention relates generally to surface-modified nanoparticles, and more particularly relates to surface-modified semiconductor and metal nanoparticles having enhanced dispersibility in aqueous media as well as superior colloidal and photophysical stability. The invention additionally relates to methods for making and using the novel surface-modified nanoparticles. The invention finds utility in a variety of fields, including biology, analytical and combinatorial chemistry, medical diagnostics, and genetic analysis.
  • BACKGROUND
  • Semiconductor nanocrystals (also known as quantum dot particles) whose radii are smaller than the bulk exciton Bohr radius constitute a class of materials intermediate between molecular and bulk forms of matter. Quantum confinement of both the electron and hole in all three dimensions leads to an increase in the effective band gap of the material with decreasing crystallite size. Consequently, both the optical absorption and emission of semiconductor nanocrystals shift to the blue (higher energies) as the size of the nanocrystals gets smaller.
  • Semiconductor nanocrystals are nanoparticles composed of an inorganic, crystalline semiconductive material and have unique photophysical, photochemical and nonlinear optical properties arising from quantum size effects, and have therefore attracted a great deal of attention for their potential applicability in a variety of contexts, e.g., as detectable labels in biological applications, and as useful materials in the areas of photocatalysis, charge transfer devices, and analytical chemistry. As a result of the increasing interest in semiconductor nanocrystals, there is now a fairly substantial body of literature pertaining to methods for manufacturing such nanocrystals. Broadly, these routes may be classified as involving preparation in glasses (see Ekimov et al. (1981) JETP Letters 34:345), aqueous preparation (including preparation that involve use of inverse micelles, zeolites, Langmuir-Blodgett films, and chelating polymers; see Fendler et al. (1984) J. Chem. Society, Chemical Communications 90:90, and Henglein et al. (1984) Ber. Bunsenges. Phys. Chem. 88:969), and high temperature pyrolysis of organometallic semiconductor precursor materials (Murray et al. (1993) J. Am. Chem. Soc. 115:8706; Katari et al. (1994) J. Phys. Chem. 98:4109). The two former methods yield particles that have unacceptably low quantum yields for most applications, a high degree of polydispersity, poor colloidal stability, a high degree of internal defects, and poorly passivated surface trap sites. In addition, nanocrystals made by the first route are physically confined to a glass matrix and cannot be further processed after synthesis.
  • To date, only the high temperature pyrolysis of organometallic reagents has yielded semiconductor nanocrystals that are internally defect free, possess high band edge luminescence and no trapped emission, and exhibit near monodispersity. Additionally, this route gives the synthetic chemist a substantial degree of control over the size of the particles prepared. See Murray et al. (1993), supra. One disadvantage of this method, however, is that the particles are sequestered in reverse micelles of coordinated, hydrophobic surfactant molecules. As such, they are only dispersible in organic solvents such as chloroform, dichloromethane, hexane, toluene, and pyridine. This is problematic insofar as many applications that rely on the fluorescence emission of the semiconductor nanocrystals require that the nanocrystals be water soluble or at least water dispersible.
  • Although some methods for rendering semiconductor nanocrystals water dispersible have been reported, they are still problematic insofar as the treated semiconductor nanocrystals suffer from significant disadvantages that limit their wide applicability. For example, Spanhel et al. (1987) J. Am. Chem. Soc. 109:5649, discloses a Cd(OH)2-capped CdS sol; however, the photoluminescent properties of the sol were pH dependent. The sol could be prepared only in a very narrow pH range (pH 8-10) and exhibited a narrow fluorescence band only at a pH of greater than 10. Such pH dependency greatly limits the usefulness of the material; in particular, it is not appropriate for use in biological systems.
  • Other groups have replaced the organic passivating layer of the semiconductor nanocrystal with water-soluble moieties; however, the resultant derivatized semiconductor nanocrystals are not highly luminescent. Short chain thiols such as 2-mercaptoethanol and 1-thio-glycerol have been used as stabilizers in the preparation of water-soluble CdTe nanocrystals. See, Rogach et al. (1996) Ber. Bunsenges. Phys. Chem. 100:1772 and Rajh et al. (1993) J. Phys. Chem. 97:11999. Other more exotic capping compounds have been reported with similar results. See Coffer et al. (1992) Nanotechnology 3:69, which describes the use of deoxyribonucleic acid (DNA) as a capping compound. In all of these systems, the coated semiconductor nanocrystals were not stable and photoluminescent properties degraded with time.
  • Thus, to use these high quantum yield materials in applications that require an aqueous medium, one must find a way of changing the polarity of the organic coating, thereby facilitating the transfer of these particles to water. A great deal of work has been conducted on surface exchange reactions that seek to replace the oleophilic hydrocarbon coating on the nanocrystal surface with a range of bifunctional polar molecules wherein one functional group of the capping molecule bears some affinity for the surface of the nanocrystal, while the other functional group, by virtue of its ionizability or high degree of hydration, renders the nanocrystal water soluble. For example, International Patent Publication No. WO 00/17655 to Bawendi et al. describes a method for rendering semiconductor nanocrystals water dispersible wherein monomeric surfactants are used as dispersing agents, with the hydrophobic region of the surfactants promoting association with the nanocrystals, while the hydrophilic region has affinity for an aqueous medium and stabilizes an aqueous suspension of the nanocrystals. International Patent Publication No. WO 00/17656 to Bawendi et al. describes a similar method wherein monomeric compounds of formula HS-(CH2)n-X, wherein n is preferably ≧10 and X is carboxylate or sulfonate, are used in place of the monomeric surfactants.
  • Kuno et al. (1997) J. Chem. Phys. 106:9869-9882, Mikulec, “Semiconductor Nanocrystal Colloids: Manganese Doped Cadmium Selenide, (Core)Shell Composites for Biological Labeling, and Highly Fluorescent Cadmium Telluride,” doctoral dissertation, Massachusetts Institute of Technology (September 1999), and International Patent Publication No. WO 00/17656 to Bawendi et al., cited supra, give detailed descriptions of surface exchange reactions designed to improve the water dispersibility of hydrophobic nanocrystals. In general, these references indicate that: exchange of the original hydrophobic surfactant layer on the nanocrystal surface is never quite complete, with retention of only about 10% to about 15% of the surfactant (even after multiple exchange reactions); although never quantitatively displaced, exchange of the original phosphine/phosphine oxide surfactant layer with more polar ligands results in a substantial decrease in quantum yield that is never entirely regained; once-dispersed in water, the particles have limited colloidal stability; and attempts to carry out further chemistry with these particles, such as linking them to biomolecules through their pendant carboxyl functionalities, is highly irreproducible and dependent on the size of the nanocrystal.
  • Thus, there remains a need in the art for a reliable, reproducible method for rendering hydrophobic semiconductor nanocrystals dispersible in aqueous media while preserving the quantum efficiencies of the original particles, maintaining colloidal stability, and avoiding or minimizing any change in particle size distribution. Ideally, such a method would be useful not only with semiconductor nanoparticles, but also with other types of nanoparticles having hydrophobic surfaces, e.g., semiconductive nanoparticles that are not necessarily crystalline and metallic nanoparticles that may or may not be surface-modified.
  • SUMMARY OF THE INVENTION
  • It is accordingly a primary object of the invention to address the aforementioned need in the art by providing surface-modified nanoparticles having enhanced dispersibility in aqueous media, wherein the nanoparticles are comprised of an inner core having a hydrophobic surface and an outer layer of a multiply amphipathic dispersant.
  • It is still another object of the invention to provide such surface-modified nanoparticles wherein the inner core is composed of a semiconductive or metallic material.
  • It is yet another object of the invention to provide such nanoparticles wherein the multiply amphipathic dispersant is a polymer having two or more hydrophobic regions and two or more hydrophilic regions.
  • It is a further object of the invention to provide a method for preparing a population of the aforementioned water-dispersible nanoparticles.
  • It is still a further object of the invention to provide a composition composed of a nanoparticle conjugate, i.e., a water-dispersible nanoparticle as above, conjugated to an affinity molecule that serves as the first member of a binding pair.
  • It is yet a further object of the invention to provide such a composition wherein a second member of the binding pair is associated with the first member through either covalent or noncovalent interaction.
  • It is an additional object of the invention to provide a monodisperse population of water-dispersible nanoparticles wherein the population is characterized in that it exhibits no more than about a 10% rms deviation, preferably no more than about a 5% rms deviation, in the diameter of the inner core.
  • Additional objects, advantages, and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.
  • In one aspect of the invention, then, a water-dispersible nanoparticle is provided that is comprised of an inner core and an outer layer of a multiply amphipathic dispersant, i.e., a compound having two or more hydrophobic regions and two or more hydrophilic regions. The inner core comprises a semiconductive or metallic material, preferably an inorganic semiconductive material that is in a crystalline state. Generally, the inner core also comprises a hydrophobic passivating layer on the semiconductive or metallic material resulting from solvents and/or surfactants used in nanoparticle manufacture. The surface of the inner core is accordingly hydrophobic, and the hydrophobic regions of the dispersant thus have affinity for the core surface and attach thereto, while the hydrophilic regions of the dispersant extend outward from the nanoparticle and provide for dispersibility in water. In a preferred embodiment, the dispersant is polymeric and has a plurality of both hydrophobic regions and hydrophilic regions, thus enhancing water dispersibility of the nanoparticle as well as the dispersant's affinity for the core surface. Particularly preferred dispersants are hyperbranched or dendritic polymers, which, relative to prior methods that involve monomeric dispersants, substantially increase the water dispersibility and colloidal stability of the nanoparticles. In a preferred embodiment, the nanoparticles are luminescent semiconductive nanocrystals, and include an overcoating “shell” layer between the inner core and the multiply amphipathic outer layer to increase luminescence efficiency. The shell material has a higher bandgap energy than the nanocrystal core, and should also have good conduction and valence band offset with respect to the nanocrystal core. Further, an “affinity molecule,” i.e., one member of a binding pair, may be attached to the outer layer of the surface-modified molecule, providing a nanoparticle “conjugate” that is useful in detecting the presence or quantity of target molecules that comprise the second member of the binding pair. The affinity molecule may be, for example, a protein, an oligonucleotide, an enzyme inhibitor, a polysaccharide, or a small molecule having a molecular weight of less than about 1500 grams/Mol.
  • In a related aspect of the invention, then, a composition is provided that is comprised of the aforementioned nanoparticle conjugate in association with the second member of the binding pair, wherein the association may involve either covalent or noncovalent interaction.
  • In another aspect of the invention, a monodisperse population of surface-modified nanoparticles is provided, comprising a plurality of water-dispersible nanoparticles each having an inner core comprised of a semiconductive or metallic material and, surrounding the inner core, an outer layer comprised of a multiply amphipathic dispersant as described above, wherein the population is characterized in that the nanoparticles are of substantially the same size and shape, i.e., the population exhibits no more than about a 10% rms deviation in the diameter of the inner core, preferably no more than about a 5% rms deviation in the diameter of the inner core. The narrow size distribution of a monodisperse population increases the “information density” that is obtainable as a result of the particles' luminescence, i.e., the number of discrete luminescence emissions obtainable for a given nanoparticle composition.
  • In another aspect of the invention, a method is provided for making the surface-modified nanoparticles described above. The method involves (a) admixing (i) an amphipathic dispersant comprised of a polymer having two or more hydrophobic regions and two or more hydrophilic regions, with (ii) a plurality of hydrophobic nanoparticles, in (iii) a nonaqueous solvent, to provide an admixture of dispersant and nanoparticles in solution; (b) subjecting the admixture to conditions effective to cause adsorption of the dispersant by the nanoparticles; and (c) transferring the dispersant-coated nanoparticles prepared in step (b) to an aqueous medium such as water or an aqueous buffer.
  • DETAILED DESCRIPTION OF THE INVENTION I. Definitions:
  • Before describing the present invention in detail, it is to be understood that unless otherwise indicated this invention is not limited to specific nanoparticle materials, amphipathic dispersants, or manufacturing processes, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
  • It must be noted that, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, “a dispersant” refers to a single dispersant as well as a mixture of two or more dispersants, “a nanoparticle” encompasses not only a single nanoparticle but also two or more nanoparticles, and the like.
  • In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below,
  • The term “amphipathic,” referring to the dispersants employed herein, is used in its conventional sense to indicate a molecular species having a hydrophobic region and a hydrophilic region. The dispersants herein are “multiply amphipathic” in that they contain two or more hydrophobic regions and two or more hydrophilic regions.
  • The term “attached,” as in, for example, the “attachment” of a dispersant to a nanoparticle surface, includes covalent binding, adsorption, and physical immobilization. The terms “associated with,” “binding” and “bound” are identical in meaning to the term “attached.”
  • Attachment of the present multiply amphipathic dispersants to the surface of a metallic or semiconductive nanoparticle will generally involve “adsorption,” wherein “adsorption” refers to the noncovalent retention of a molecule by a substrate surface. That is, adsorption occurs as a result of noncovalent interaction between a substrate surface and adsorbing moieties present on the molecule that is adsorbed. Adsorption may occur through hydrogen bonding, van der Waal's forces, polar attraction or electrostatic forces (i.e., through ionic bonding), and in the present case will typically involve the natural affinity of a hydrophobic region of a molecule for a hydrophobic surface.
  • The term “nanoparticle” refers to a particle, generally a semiconductive or metallic particle, having a diameter in the range of about 1 nm to about 1000 nm, preferably in the range of about 2 nm to about 50 nm, more preferably in the range of about 2 nm to about 20 nm. As discussed elsewhere herein, semiconductive, and metallic “nanoparticles” generally include a passivating layer of a water-insoluble organic material that results from the method used to manufacture such nanoparticles. The terms “surface-modified nanoparticle” and “water-dispersible nanoparticle” as used herein refer to the modified nanoparticles of the invention, while the term “nanoparticle,” without qualification, refers to the hydrophobic nanoparticle that serves as the inner core of the surface-modified, water-dispersible nanoparticle.
  • The terms “semiconductor nanoparticle” and “semiconductive nanoparticle” refer to a nanoparticle as defined above that is composed of an inorganic semiconductive material, an alloy or other mixture of inorganic semiconductive materials, an organic semiconductive material, or an inorganic or organic semiconductive core contained within one or more semiconductive overcoat layers.
  • The term “metallic nanoparticle” refers to a nanoparticle as defined above that is composed of a metallic material, an alloy or other mixture of metallic materials, or a metallic core contained within one or more metallic overcoat layers.
  • The terms “semiconductor nanocrystal,” “quantum dot” and “Qdot® nanocrystal” are used interchangeably herein to refer to semiconductor nanoparticles composed of an inorganic crystalline material that is luminescent (i.e., they are capable of emitting electromagnetic radiation upon excitation), and include an inner core of one or more first semiconductor materials that is optionally contained within an overcoating or “shell” of a second semiconductor material. A semiconductor nanocrystal core surrounded by a semiconductor shell is referred to as a “core/shell” semiconductor nanocrystal. The surrounding shell material will preferably have a bandgap energy that is larger than the bandgap energy of the core material and may be chosen to have an atomic spacing close to that of the core substrate. Suitable semiconductor materials for the core and/or shell include, but are not limited to, the following: materials comprised of a first element selected from Groups 2 and 12 of the Periodic Table of the Elements and a second element selected from Group 16 (e.g., ZnS, ZnSe, ZnTe, CDs, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and the like); materials comprised of a first element selected from Group 13 of the Periodic Table of the Elements and a second element selected from Group 15 (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, and the like); materials comprised of a Group 14 element (Ge, Si, and the like); materials such as PbS, PbSe and the like; and alloys and mixtures thereof. As used herein, all reference to the Periodic Table of the Elements and groups thereof is to the new IUPAC system for numbering element groups, as set forth in the Handbook of Chemistry and Physics, 81st Edition (CRC Press, 2000).
  • By “luminescence” is meant the process of emitting electromagnetic radiation (light) from an object. Luminescence results when a system undergoes a transition from an excited state to a lower energy state with a corresponding release of energy in the form of a photon. These energy states can be electronic, vibrational, rotational, or any combination thereof. The transition responsible for luminescence can be stimulated through the release of energy stored in the system chemically or added to the system from an external source. The external source of energy can be of a variety of types including chemical, thermal, electrical, magnetic, electromagnetic, and physical, or any other type of energy source capable of causing a system to be excited into a state higher in energy than the ground state. For example, a system can be excited by absorbing a photon of light, by being placed in an electrical field, or through a chemical oxidation-reduction reaction. The energy of the photons emitted during luminescence can be in a range from low-energy microwave radiation to high-energy x-ray radiation. Typically, luminescence refers to photons in the range from UV to IR radiation.
  • The term “monodisperse” refers to a population of particles (e.g., a colloidal system) wherein the particles have substantially identical size and shape. For the purpose of the present invention, a “monodisperse” population of particles means that at least about 60% of the particles, preferably about 75% to about 90% of the particles, fall within a specified particle size range. A population of monodisperse particles deviates less than 10% rms (root-mean-square) in diameter and preferably less than 5% rms.
  • The phrase “one or more sizes of nanoparticles” is used synonymously with the phrase “one or more particle size distributions of nanoparticles.” One of ordinary skill in the art will realize that particular sizes of nanoparticles such as semiconductor nanocrystals are actually obtained as particle size distributions.
  • By use of the term “narrow wavelength band” or “narrow spectral linewidth” with regard to the electromagnetic radiation emission of the semiconductor nanocrystal is meant a wavelength band of emissions not exceeding about 60 nm, and preferably not exceeding about 30 nm in width, more preferably not exceeding about 20 nm in width, and symmetric about the center. It should be noted that the bandwidths referred to are determined from measurement of the full width of the emissions at half peak height (FWHM), and are appropriate in the range of 200 nm to 2000 nm.
  • By use of the term “a broad wavelength band,” with regard to the excitation of the semiconductor nanocrystal is meant absorption of radiation having a wavelength equal to, or shorter than, the wavelength of the onset radiation (the onset radiation is understood to be the longest wavelength (lowest energy) radiation capable of being absorbed by the semiconductor nanocrystal). This onset occurs near to, but at slightly higher energy than the “narrow wavelength band” of the emission. This is in contrast to the “narrow absorption band” of dye molecules, which occurs near the emission peak on the high energy side, but drops off rapidly away from that wavelength and is often negligible at wavelengths further than 100 nm from the emission.
  • The term “emission peak” refers to the wavelength of light within the characteristic emission spectra exhibited by a particular semiconductor nanocrystal size distribution that demonstrates the highest relative intensity.
  • The term “excitation wavelength” refers to light having a wavelength lower than the emission peak of the semiconductor nanocrystal used in the first detection reagent.
  • A “hydrophobic” compound (e.g., a “hydrophobic” monomer) is one that will transfer from an aqueous phase to an organic phase, specifically from water to an organic, water-immiscible nonpolar solvent with a dielectric constant ≦5, with a partition coefficient of greater than about 50%. A “hydrophobic monomer unit” refers to a hydrophobic monomer as it exists within a polymer. A “hydrophobic region” refers to a hydrophobic molecular segment, e.g., a molecular segment within a polymer. A “hydrophobic region” may be a single hydrophobic monomer unit or two or more hydrophobic monomer units that may be the same or different and may or may not be adjacent.
  • A “hydrophilic” compound (e.g., a “hydrophilic” monomer) is one that will transfer from an organic phase to an aqueous phase, specifically from an organic, water-immiscible nonpolar solvent with a dielectric constant <5 to water, with a partition coefficient of greater than about 50%. A “hydrophilic monomer unit” refers to a hydrophilic monomer as it exists in a polymeric segment or polymer. A “hydrophilic region” refers to a hydrophilic molecular segment, e.g., a hydrophilic molecular segment within a polymer. A “hydrophilic region” may be a single hydrophilic monomer unit or two or more hydrophilic monomer units that may be the same or different and may or may not be adjacent.
  • The term “ionizable” refers to a group that is electronically neutral at a specific pH, but can be ionized and thus rendered positively or negatively charged at higher or lower pH, respectively.
  • The term “alkyl” as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to approximately 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl and tetracosyl, as well as cycloalkyl groups such as cyclopentyl and cyclohexyl. The term “lower alkyl” intends an alkyl group of 1 to 4 carbon atoms, and thus includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and t-butyl.
  • The term “alkylene” as used herein refers to a difunctional saturated branched or unbranched hydrocarbon chain containing from 1 to approximately 24 carbon atoms, typically 1 to approximately 12 carbon atoms, and includes, for example, methylene (—CH2—), ethylene (—CH2—CH2—), propylene (—CH2—CH2—CH2—), 2-methylpropylene (—CH2—CH(CH3)—CH2—), hexylene (—(CH2)6—), and the like. “Lower alkylene,” as in the lower alkylene linkage of the optional coupling agent herein, refers to an alkylene group of 1 to 4 carbon atoms.
  • The term “alkenyl” as used herein refers to a branched or unbranched hydrocarbon group typically although not necessarily containing 2 to about 24 carbon atoms and at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, and the like. Generally, although not necessarily, alkenyl groups herein contain 2 to about 12 carbon atoms. The term “lower alkenyl” intends an alkenyl group of 2 to 4 carbon atoms, and the term “alkenylene” refers to a difunctional alkenyl group, in the same way that the term “alkylene” refers to a difunctional alkyl group.
  • The term “alkynyl” as used herein refers to a branched or unbranched hydrocarbon group typically although not necessarily containing 2 to about 24 carbon atoms and at least one triple bond, such as ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, octynyl, decynyl, and the like. Generally, although again not necessarily, alkynyl groups herein contain 2 to about 12 carbon atoms. The term “lower alkynyl” intends an alkynyl group of 2 to 4 carbon atoms, preferably 3 or 4 carbon atoms.
  • The term “heteroatom-containing” and the prefix “hetero-,” as in “heteroatom-containing alkyl” and “heteroalkyl,” refer to a molecule or molecular fragment in which one or more carbon atoms is replaced with an atom other carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon.
  • The term “alkoxy” as used herein refers to a substituent —O—R wherein R is alkyl as defined above. The term “lower alkoxy” refers to such a group wherein R is lower alkyl as defined above, e.g., methoxy, ethoxy and the like. The term “aryl” as used herein, and unless otherwise specified, refers to an aromatic moiety containing 1 to 3 aromatic rings. For aryl groups containing more than one aromatic ring, the rings may be fused or linked. Aryl groups are optionally substituted with one or more inert, nonhydrogen substituents per ring; suitable “inert, nonhydrogen” substituents include, for example, halo, haloalkyl (preferably halo-substituted lower alkyl), alkyl (preferably lower alkyl), alkenyl (preferably lower alkenyl), alkynyl (preferably lower alkynyl), alkoxy (preferably lower alkoxy), alkoxycarbonyl (preferably lower alkoxycarbonyl), carboxy, nitro, cyano and sulfonyl. Unless otherwise indicated, the term “aryl” is also intended to include heteroaromatic moieties, i.e., aromatic heterocycles. Generally, although not necessarily, the heteroatoms will be nitrogen, oxygen or sulfur. The term “arylene” refers to a difunctional aryl moiety in the same way that the term “alkylene” refers to a difunctional alkyl group.
  • The term “aralkyl” refers to an alkyl group with an aryl substituent, and the term “aralkylene” refers to an alkylene group with an aryl substituent; the term “alkaryl” refers to an aryl group that has an alkyl substituent, and the term “alkarylene” refers to an arylene group with an alkyl substituent.
  • The terms “halo” and “halogen” are used in the conventional sense to refer to a chloro, bromo, fluoro, or iodo substituent. The term “haloalkyl” refers to an alkyl group in which at least one of the hydrogen atoms in the group has been replaced with a halogen atom.
  • The term “peptide” refers to oligomers or polymers of any length wherein the constituent monomers are alpha amino acids linked through amide bonds, and encompasses amino acid dimers as well as polypeptides, peptide fragments, peptide analogs, naturally occurring proteins, mutated, variant, or chemically modified proteins, fusion proteins, and the like. The amino acids of the peptide molecules may be any of the twenty conventional amino acids, stereoisomers (e.g., D-amino acids) of the conventional amino acids, structural variants of the conventional amino acids, e.g., iso-valine, or non-naturally occurring amino acids such as α,α-disubstituted amino acids, N-alkyl amino acids, β-alanine, naphthylalanine, 3-pyridylalanine, 4-hydroxyproline, 0-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and nor-leucine. In addition, the term “peptide” encompasses peptides with posttranslational modifications such as glycosylations, acetylations, phosphorylations, and the like. The term “oligonucleotide” is used herein to include a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded DNA, as well as triple-, double- and single-stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the oligonucleotide. More particularly, the term includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers, providing that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, such as is found in DNA and RNA. There is no intended distinction in length between the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and these terms refer only to the primary structure of the molecule. Thus, these terms include, for example, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′ P5′ phosphoramidates, 2′-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for, example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.
  • The term “polymer” is used herein in its conventional sense to refer to a compound having two or more monomer units, and is intended to include linear and branched polymers, the term “branched polymers” encompassing simple branched structures as well as hyperbranched and dendritic polymers. The term “monomer” is used herein to refer to compounds that are not polymeric. “Polymers” herein may be naturally occurring, chemically modified, or chemically synthesized.
  • The term “water-dispersible” as used herein refers to an essentially unaggregated dispersion of particles, such that discrete particles of approximately 2 nm to 50 nm can be sustained indefinitely at high concentrations (10-20 μM).
  • The term “binding pair” refers to first and second molecules that specifically bind to each other. “Specific binding” of the first member of the binding pair to the second member of the binding pair in a sample is evidenced by the binding of the first member to the second member, or vice versa, with greater affinity and specificity than to other components in the sample. The binding between the members of the binding pair is typically noncovalent. The terms “affinity molecule” and “target analyte” are also used herein to refer to the first and second members of a binding pair, respectively. Exemplary binding pairs include any haptenic or antigenic compound in combination with a corresponding antibody or binding portion or fragment thereof (e.g., digoxigenin and anti-digoxigenin; mouse immunoglobulin and goat anti-mouse immunoglobulin) and nonimmunological binding pairs (e.g., biotin-avidin, biotin-streptavidin, hormone [e.g., thyroxine and cortisol]-hormone binding protein, receptor-receptor agonist or antagonist (e.g., acetylcholine receptor-acetylcholine or an analog thereof), IgG-protein A, lectin-carbohydrate, enzyme-enzyme cofactor, enzyme-enzyme inhibitor, and complementary polynucleotide pairs capable of forming nucleic acid duplexes), and the like.
  • A “nanoparticle conjugate” refers to a nanoparticle linked, through an outer layer of an amphipathic dispersant, to a member of a “binding pair” that will selectively bind to a detectable substance present in a sample, e.g., a biological sample. The first member of the binding pair linked to the nanoparticle can comprise any molecule, or portion of any molecule, that is capable of being linked to the nanoparticle and that, when so linked, is capable of specifically recognizing the second member of the binding pair.
  • All molecular weights specified herein are number average molecular weights.
  • H. The Nanoparticles:
  • Prior to surface modification with a multiply amphipathic dispersant, the nanoparticles of the invention are nanoparticles with hydrophobic surfaces, the particles having a diameter in the range of about 1 nm to about 1000 nm, preferably in the range of about 2 rim to about 50 nm, more preferably in the range of about 2 nm to about 20 nm. Generally, the nanoparticles will be comprised of a semiconductive or metallic material, with semiconductive nanoparticles preferred. Also, as will be explained in greater detail below, the semiconductive or metallic material typically has a coating of a hydrophobic passivating layer resulting from the use of solvents and/or surfactants during nanoparticle manufacture. The hydrophobic surfaces of the nanoparticles have affinity for and thus serve to attach the amphipathic dispersant by virtue of the hydrophobic regions within the dispersant.
  • Semiconductive nanoparticles may be composed of an organic semiconductor material or an inorganic semiconductor material. Organic semiconductor materials will generally be conjugated polymers. Suitable conjugated polymers include, for example, cis and trans polyacetylenes, polydiacetylenes, polyparaphenylenes, polypyrroles, polythiophenes, polybithiophenes, polyisothianaphthene, polythienylvinylenes, polyphenylenesulfide, polyaniline, polyphenylenevinylenes, and polyphenylenevinylene derivatives, e.g., poly(2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene vinylene (“MEH-PPV”) (see U.S. Pat. No. 5,189,136 to Wudl et al.), poly (2,5-bischelostanoxy-1,4-phenylene vinylene) (“BCHA-PPV”) (e.g., as described in International Patent Publication No. WO 98/27136), and poly(2-N,N-dimethylamino phenylene vinylene)(described in U.S. Pat. No. 5,604,292 to Stenger-Smith et al.). Inorganic semiconductive nanoparticles are, however, preferred, and are optimally crystalline in nature; such nanoparticles are termed “semiconductor nanocrystals” herein. Semiconductor nanocrystals are capable of luminescence, generally fluorescence, when excited by light. Currently, detection of biological compounds by photoluminescence utilizes fluorescent organic dyes and chemiluminescent compounds. The use of semiconductor nanocrystals as luminescent markers, particularly in biological systems, provides advantages over existing fluorescent dyes. Many of these advantages relate to the spectral properties of nanocrystals, e.g., the ability to control the composition and size of nanocrystals enables one to construct nanocrystals with fluorescent emissions at any wavelength in the UV-visible-IR regions. With respect to composition, for example, semiconductor nanocrystals that emit energy in the visible range include, but are not limited to, CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, and GaAs. Semiconductor nanocrystals that emit energy in the near IR range include, but are not limited to, InP, InAs, InSb, PbS, and PbSe. Finally, semiconductor nanocrystals that emit energy in the blue to near-ultraviolet include, but are not limited to, ZnS and GaN. For any particular nanocrystal composition, it is also possible to tune the emission to a desired wavelength by controlling particle size distribution. In preferred embodiments, 5-20 discrete emissions (five to twenty different size populations or distributions distinguishable from one another), more preferably 10-15 discrete emissions, are obtained for any particular composition, although one of ordinary skill in the art will realize that fewer than five emissions and more than twenty emissions could be obtained depending on the monodispersity of the semiconductor nanocrystal particle population. If high information density is required, and thus a greater number of distinct emissions, the nanocrystals are preferably substantially monodisperse within the size range given above.
  • As explained above, “monodisperse” refers to a population of particles (e.g., a colloidal system) in which the particles have substantially identical size and shape. In preferred embodiments for high information density applications, monodisperse particles deviate less than 10% rms in diameter, and preferably less than 5% rms. Monodisperse semiconductor nanocrystals have been described in detail in Murray et al. (1993) J. Am. Chem. Soc. 115:8706, and in Murray, “Synthesis and Characterization of II-VI Quantum Dots and Their Assembly into 3-D Quantum Dot Superlattices,” doctoral dissertation, Massachusetts Institute of Technology (1995). One of ordinary skill in the art will also realize that the number of discrete emissions that can be distinctly observed for a given composition depends not only upon the monodispersity of the particles, but also on the deconvolution techniques employed. Semiconductor nanocrystals, unlike dye molecules, can be easily modeled as Gaussians and therefore are more easily and more accurately deconvoluted.
  • However, for some applications, high information density will not be required and it may be more economically attractive to use more polydisperse particles. Thus, for applications that do not require high information density, the linewidth of the emission may be in the range of 40-60 nm.
  • Semiconductor nanocrystals may be made using techniques known in the art. See, e.g., U.S. Pat. Nos. 6,048,616, 5,990,479, 5,690,807, 5,505,928 and 5,262,357, as well as International Patent Publication No. WO 99/26299, published May 27, 1999. In particular, exemplary materials for use as semiconductor nanocrystals in the biological and chemical assays of the present invention include, but are not limited to, those described above, including Group 2-16, 12-16, 13-15 and 14 semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlSb, PbS, PbSe, Ge and Si and ternary and quaternary mixtures thereof.
  • In a preferred embodiment, the surface of the semiconductor nanocrystal is modified to enhance the efficiency of the emissions, prior to surface modification with the multiply amphipathic dispersant, by adding an overcoating layer or shell to the semiconductor nanocrystal. The shell is preferred because at the surface of the semiconductor nanocrystal, surface defects can result in traps for electrons or holes that degrade the electrical and optical properties of the semiconductor nanocrystal. An insulating layer at the surface of the semiconductor nanocrystal provides an atomically abrupt jump in the chemical potential at the interface that eliminates energy states that can serve as traps for the electrons and holes. This results in higher efficiency in the luminescent process.
  • Suitable materials for the shell include semiconductor materials having a higher bandgap energy than the semiconductor nanocrystal core, In addition to having a bandgap energy greater than the semiconductor nanocrystal core, suitable materials for the shell should have good conduction and valence band offset with respect to the core semiconductor nanocrystal. Thus, the conduction band is desirably higher and the valence band is desirably lower than those of the core semiconductor nanocrystal. For semiconductor nanocrystal cores that emit energy in the visible (e.g., CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, GaAs) or near IR (e.g., InP, InAs, InSb, PbS, PbSe), a material that has a bandgap energy in the ultraviolet regions may be used. Exemplary materials include ZnS, GaN, and magnesium chalcogenides, e.g., MgS, MgSe, and MgTe. For a semiconductor nanocrystal core that emits in the near IR, materials having a bandgap energy in the visible, such as CdS or CdSe, may also be used. The preparation of a coated semiconductor nanocrystal may be found in, e.g., Dabbousi et al. (1997) J. Phys. Chem. B 101:9463, Hines et al. (1996) J. Phys. Chem. 100: 468-471, Peng et al. (1997) J. Am, Chem. Soc. 119:7019-7029, and Kuno et al. (1997) J. Phys. Chem. 106:9869.
  • The nanoparticles of the invention may also be metallic. Such particles are useful, for example, in surface enhanced Raman scattering (SERS), which employs nanometer-size particles onto which Raman active moieties (e.g., a dye or pigment, or a functional group exhibiting a characteristic Raman spectrum) are adsorbed or attached. Metallic nanoparticles may be comprised of any metal or metallic alloy or composite, although for use in SERS, a SERS active metal is used, e.g., silver, gold, copper, lithium, aluminum, platinum, palladium, or the like. In addition, the particles can be in a core-shell configuration, e.g., a gold core may be encased in a silver shell; see, e.g., Freeman et al. (1996) J. Phys. Chem. 100:718-724, or the particles may form small aggregates in solution. Kneipp et al. (1998) Applied Spectroscopy 52:1493.
  • Generally, and as alluded to above, the unmodified nanoparticles--and thus the inner core of the present surface-modified nanoparticles--also comprise a hydrophobic coating on the semiconductive or metallic material resulting from solvents and/or surfactants used in nanoparticle manufacture. For example, semiconductive nanoparticles, as manufactured, will typically have a water-insoluble organic coating that has affinity for the semiconductive material, the coating comprised of a passivating layer resulting from use of a coordinating solvent such as hexyldecylamine or a trialkyl phosphine or trialkyl phosphine oxide, e.g., trioctylphosphine oxide (TOPO), trioctylphosphine (TOP), or tributylphosphine (TBP). Hydrophobic surfactants typically used in the manufacture of metallic nanoparticles and forming a coating thereon include, by way of example, octanethiol, dodecanethiol, dodecylamine, and tetraoctylammonium bromide. Metallic inner cores will typically have a surfactant coating that has affinity for the metallic material, the coating similarly deriving from surfactant compounds used in the manufacture of metallic nanoparticles. The surfactant coating is comprised of a hydrophobic surfactant.
  • III. The Dispersant:
  • The dispersant used to modify the hydrophobic surface of the nanoparticles is a multiply amphipathic dispersant, i.e., a compound having two or more hydrophobic regions and two or more hydrophilic regions. In a preferred embodiment, the multiply amphipathic dispersant is polymeric, and may be composed of either a linear or branched polymer, whether naturally occurring, chemically modified, or chemically synthesized. Structurally, polymers are classified as either linear or branched wherein the term “branched” generally means that the individual molecular units (i.e., monomer units) of the branches are discrete from the polymer backbone, and may or may not have the same chemical constitution as the polymer backbone.
  • As will be appreciated by those of ordinary skill in the art, the simplest branched polymers are the “comb branched” polymers wherein a linear backbone bears one or more essentially linear pendant side chains. This simple form of branching may be regular or irregular (in the latter case, the branches are distributed in non-uniform or random fashion on the polymer backbone). An example of regular comb branching is a comb branched polystyrene as described by Altores et al. (1965) J. Polymer Sci., Part A 3:4131-4151, and an example of irregular comb branching is illustrated by the graft copolymers described by Sorenson et al. in Preparative Methods of Polymer Chemistry, 2nd Ed., Interscience Publishers, pp. 213-214 (1968).
  • The amphipathic dispersant may also be a branched polymer in the form of a cross-linked or network polymer, i.e., a polymeric structure wherein individual polymer chains or branches are connected through the use of bifunctional compounds; e.g., acrylic acid monomer units bridged or crosslinked with a diamine linker. In this type of branching, many of the individual branches are not linear in that each branch may itself contain side chains pendant from a linear chain and it is not possible to differentiate between the backbone and the branches. More importantly, in network branching, each polymer macromolecule (backbone) is cross-linked at two or more sites to other polymer macromolecules. Also, the chemical constitution of the cross-linkages may vary from that of the polymer macromolecules. In this cross-linked or network branched polymer, the various branches or cross-linkages may be structurally similar (termed “regularly” cross-linked) or they may be structurally dissimilar (termed “irregularly” cross-linked).
  • The amphipathic dispersant may also have other structural configurations, e.g., it may be a star/comb-branched type polymer, as described in U.S. Pat. Nos. 4,599,400 and 4,690,985, or a rod-shaped dendrimer as disclosed in U.S. Pat. No. 4,694,064.
  • Particularly preferred amphipathic dispersants herein are hyperbranched (containing two or more generations of branching) or dendrimeric. In contrast to hyperbranched polymers, dendrimers are regularly branched macromolecules with a branch point at each repeat unit. Also, hyperbranched polymers are obtained via a polymerization reaction, while most regular dendrimers are obtained by a series of stepwise coupling and activation steps. Examples of dendrimers include the polyamidoamine (PAMAM) Starburst® dendrimers of Tomalia et al. (1985) Polym. J. 17:117, the convergent dendrimers of Hawker et al. (1990) J. Am. Chem. Soc. 112:7638, and diaminobutane dendrimers, described in Tomalia et al. (1990) Angew. Chem., Int. Ed. Engl. 29:135-175. With both hyperbranched polymers and dendrimers, however, the increased number of hydrophobic and hydrophilic regions amplifies the effect of the dispersant on the nanoparticle core, with respect to both affinity for the nanoparticle surface (i.e., affinity of the hydrophobic regions of the dispersant for the hydrophobic surface of the nanoparticle) and water dispersibility (as a result of the increased number of hydrophilic regions or segments).
  • The hydrophilic regions represent approximately 30 wt. % to 75 wt. % of the amphipathic dispersant, and are comprised of at least one monomer unit containing an ionizable or polar moiety, preferably an ionizable moiety such as a carboxylic acid, sulfonic acid, phosphonic acid or amine substituent. Examples of hydrophilic monomer units include, but are not limited to:
  • water-soluble ethylenically unsaturated C3-C6 carboxylic acids, such as acrylic acid, alkyl acrylic acids (particularly methacrylic acid), itaconic acid, maleic acid, fumaric acid, acrylamidomethyl-propanesulfonic acid, vinyl sulfonic acid, vinyl phosphonic acid, vinyllactic acid, and styrene sulfonic acid;
  • allylamine and allylamine salts formed with an inorganic acid, e.g., hydrochloric acid;
  • di-C1-C3-alkylamino-C2-C6-alkyl acrylates and methacrylates such dimethylaminoethyl acrylate, dimethylaminoethyl methacrylate, diethylaminoethyl acrylate, diethylaminoethyl methacrylate, dimethylaminopropyl acrylate, dimethylaminobutyl acrylate, dimethylaminoneopentyl acrylate and dimethylaminoneopentyl methacrylate;
  • olefinically unsaturated nitriles, such as acrylonitrile;
  • diolefinically unsaturated monomers, particularly diallylammonium compounds such as dimethyldiallylammonium chloride, dimethyldiallylammonium bromide, diethyldiallylammonium chloride, methyl-t-butyldiallylammonium methosulfate, methyl-n-propyldiallylammonium chloride, dimethyldiallylammonium hydrogensulfate, dimethyldiallylammonium dihydrogenphosphate, di-n-butyldiallylammonium bromide, diallylpiperidinium bromide, diallylpyrrolidinium chloride and diallylmorpholinium bromide;
  • N-vinylpyrrolidone;
  • N-vinylformamide;
  • acrylamide and substituted acrylamides, such as N-methylolacrylamide and C1-C3 alkyl acrylamides, particularly methacrylamide;
  • N-vinylimidazole and N-vinylimidazoline; and
  • other monomers, typically ethylenically unsaturated monomers, preferably vinyl monomers, substituted with at least one hydrophilic functionality such as a carboxylate, a thiocarboxylate, an amide, an imide, a hydrazine, a sulfonate, a sulfoxide, a sulfone, a sulfite, a phosphate, a phosphonate, a phosphonium, an alcohol, a thiol, a nitrate, an amine, an ammonium, or an alkyl ammonium group —[NHR1R2]+, wherein R1 and R2 are alkyl substituents and the group is associated with a negatively charged anion, e.g., a halogen ion, nitrate, etc. The hydrophilic functionality may be directly bound to a carbon atom in the polymer backbone, but will usually be bound through a linkage that provides some degree of spacing between the polymer backbone and the hydrophilic functional group. Suitable linkages include, but are not limited to, branched or unbranched alkylene, branched or unbranched alkenylene, branched or unbranched heteroalkylene (typically alkylene containing one or more ether or —NH— linkages) a branched or unbranched heteroalkenylene (again, typically alkenylene containing one or more ether or —NH— linkages), arylene, heteroarylene, alkarylene, aralkylene, and the like. The linkage will typically contain 2 to 24, more typically 2 to 12, carbon atoms.
  • The hydrophilic regions may also be composed of partially or fully hydrolyzed poly(vinyl alcohol), poly(ethylene glycol), poly(ethylene oxide), highly hydrated poly(alkylene oxides) such as poly(ethylene oxide), cellulosic segments (e.g., comprised of cellulose per se or cellulose derivatives such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, and the like), and polysaccharides such as chitosan or dextran.
  • The hydrophobic regions represent approximately 25 wt. % to 90 wt. % of the amphipathic dispersant, and are comprised of at least one non-ionizable, nonpolar monomer unit, facilitating noncovalent association with the hydrophobic surface of the nanoparticle. Examples of such monomer units include, but are not limited to:
  • acrylates such as methacrylate, methyl methacrylate, ethyl methacrylate, butyl methacrylate, isobutyl methacrylate, hexyl methacrylate, isodecyl methacrylate, lauryl methacrylate, phenyl methacrylate, isopropyl acrylate, isobutyl acrylate and octadecylacrylate,
  • alkylenes such as ethylene and propylene;
  • C4-C12-alkyl-substituted ethyleneimine;
  • alkyl acrylamides wherein the alkyl group is larger than lower alkyl (particularly alkyl acrylamides wherein the alkyl group has six or more carbon atoms, typically six to twelve carbon atoms, such as hexylacrylamide, octylacrylamide, and the like);
  • styrene and hydrophobically derivatized styrenes (i.e., styrene substituted with one or more hydrophobic substituents, e.g., C5-C12 hydrocarbyl groups);
  • vinyl ether;
  • vinyl esters such as vinyl acetate; and
  • vinyl halides such as vinyl chloride.
  • The hydrophobic regions may also be composed of polychloroprene, polybutadiene, polysiloxane, polydimethylsiloxane, polyisobutylene or polyurethane blocks, or they may be polycondensates of 2-poly(hydroxyalkanoic acids) such as 2-hydroxypropanoic acid, 2-hydroxybutanoic acid, 2-hydroxyisobutanoic acid, 2-hydroxyheptanoic acid, 10-hydroxydecanoic acid, 12-hydroxydodecanoic acid, 12-hydroxystearic acid, 16-hydroxyhexadecanoic acid, 2-hydroxystearic acid, 2-hydroxyvaleric acid or the corresponding condensates obtained from lactones, condensates of diols and dicarboxylic acids such as polyethylene adipate, or polylactams such as polycaprolactam.
  • Any of the aforementioned monomer units and polymer segments can be modified using techniques and reagents routinely used by those of ordinary skill in the art. Such modifications include, for example, routine substitutions, additions of chemical groups such as alkyl groups and alkylene groups, hydroxylations, oxidations, and the like. Such branched polymers, composed of hydrophobic segments and hydrophilic segments, are typically comprised of (1) a hydrophobic backbone with hydrophilic branches, (2) a hydrophilic backbone with hydrophobic branches, or (3) a backbone that may be either hydrophobic or hydrophilic, and is substituted with both hydrophilic and hydrophobic branches. Such polymers can be prepared by any suitable method readily known to those of ordinary skill in the art and/or described in the pertinent texts and literature. Polymers of type (1), for example, can be prepared by copolymerization of a hydrophobic monomer with a second monomer that includes suitable reactive groups through which the hydrophilic side chains (branches) can be grafted to the backbone. Alternatively, type (1) polymers can be prepared by polymerizing a single hydrophobic monomer with a suitable reactive side group, and a fraction of those reactive side groups can be modified post-polymerization by grafting hydrophilic side chains. Analogously, polymers of type (2) can be prepared by copolymerization of a hydrophilic monomer with a second monomer that includes suitable reactive groups through which the hydrophobic side chains (branches) can be grafted to the backbone. Alternatively, type (2) polymers can be prepared by polymerizing a single hydrophilic monomer with a suitable reactive side group, and a fraction of those reactive side groups can be modified post-polymerization by grafting hydrophobic side chains. Type (3) polymers can be prepared by first synthesizing a linear polymer having reactive sites throughout the backbone, and then grafting hydrophilic and hydrophobic side chains onto the backbone in a fashion that may or may not be ordered.
  • Particularly preferred amphipathic dispersants include acrylic acid and methacrylic acid polymers modified to include hydrophobic regions, as well as copolymers of acrylic acid and/or methacrylic acid with hydrophobic comonomers such as alkyl acrylamides. Examples of such polymers are poly(acrylic acid-co-octylacrylamide), poly(acrylic acid-co-hexylacrylamide), poly(methacrylic acid-co-octylacrylamide), and poly(methacrylic acid-co-hexylacrylamide), with poly(acrylic acid-co-octylacrylamide) most preferred. The specific methodology used to synthesize polymers suitable as the multiply amphipathic dispersant will depend on the particular monomer types that are employed. As will be appreciated by those of ordinary skill in the art, suitable polymerization techniques include step polymerization, radical chain polymerization, emulsion polymerization, ionic chain polymerization, chain copolymerization, ring-opening polymerization, living polymerization, polycondensation reactions, and graft polymerization. In a preferred embodiment, the amphipathic dispersant is formed by addition polymerization of ethylenically unsaturated monomers. Such polymerization reactions are generally catalyzed using metallic catalysts (e.g., transition metal-based metallocenes, Ziegler-Natta catalysts, Brookhart-type catalysts, etc.) and typically involve contacting the monomer(s), catalyst, and a catalyst activator (e.g., methyl aluminoxane, or “MAO”) at a suitable temperature at reduced, elevated or atmospheric pressure, under an inert atmosphere, for a time effective to produce the desired polymer. An added solvent may, if desired, be employed, or the monomeric compounds may serve as solvent. The reaction may be conducted under solution or slurry conditions, in a suspension, or in the gas phase. As alluded to above, branched polymers can be prepared using this technique by introducing reactive sites into the polymer backbone during polymerization (e.g., by incorporating some fraction of monomer units having a pendant reactive site), followed by synthesis or grafting of branches at the reactive sites.
  • In a preferred embodiment, the amphipathic dispersant is comprised of a hydrophilic backbone that has been modified to contain hydrophobic anchoring groups, i.e., hydrophobic side chains that serve to “anchor” the dispersant to the nanoparticle surface. For example, hydrophilic polymers containing pendant carboxylic acid groups (e.g., as in poly(acrylic acid), [—(CH2CH(CO2H)]n—) can be readily modified to contain a controlled number of branched or unbranched hydrophobic side chains using methods known in the art. In one such method, the pendant carboxylic acid groups of poly(acrylic acid) can be activated with a suitable activating agent, e.g., thionyl chloride or a carbodiimide, followed by reaction with a long chain alkylamine, e.g., a C4-C12 alkylamine such as octylamine, and finally with a hydrolyzing agent such as water. Depending on the relative quantities of the alkylamine and the hydrolyzing agent, the resulting polymer is an amphipathic polymer with a hydrophilic backbone (by virtue of the carboxylic acid groups present after partial hydrolysis) and hydrophobic side chains (the long chain alkyl group attached to the backbone through an amide linkage).
  • Within the aforementioned group of hydrophobically modified hydrophilic polymers are hydrophobically modified peptides, preferably hydrophobically modified synthetic polypeptides. The use of synthetic polypeptides allows for control over a number of factors, including the monodispersity of the molecular weight of the hydrophilic backbone, the number and position of modifiable groups on the backbone, and the regularity of the modification, i.e., whether the hydrophobic groups are randomly distributed throughout the polypeptide chain or present in an ordered, “regular” fashion.
  • Suitable polypeptides are triblock (A-B-A) copolymers, for example, triblock copolymers of aspartate and norleucine, in which case polynorleucine is preferably the central block “B.” Such a triblock copolymer provides a region rich in hydrophobic side chains. In one alternative, the central block “B” can comprise a hydrophilic amino acid, for example, poly(lysine), which can be modified via standard chemistries to include hydrophobic side chains. The carboxylate-rich aspartate side chains (A) provide the polar, ionic groups that not only aid in rendering the nanocrystal water dispersible, but provide reactive sites or functionalizable moieties for further chemistry, such as conjugation to affinity molecules.
  • The polypeptide compositions of the present invention may also be monofunctional in nature, e.g., polylysine or polyaspartate, diblock copolymers (A-B) or triblock copolymers of three different amino acids (A-B-C). These compositions are also not restricted to lysine or aspartate, but may make use of any number of combinations of the known amino acids. Generally, the hydrophobic regions of a polypeptide are comprised of at least one hydrophobic amino acid and the hydrophilic regions are comprised of at least one hydrophilic amino acid. As will be appreciated by those of ordinary skill in the art, hydrophobic amino acids include, for example, alanine, glycine, valine, leucine, isoleucine, norleucine, proline, phenylalanine, methionine, tryptophane, cysteine, and includes hydrophilic amino acids modified to include hydrophobic side chains, while hydrophilic amino acids include aspartic acid, glutamic acid, lysine, arginine, histidine, asparagine, glutamine, serine, threonine and tyrosine.
  • The amphipathic dispersant generally although not necessarily has a molecular weight in the range of approximately 500 to 50,000, preferably in the range of approximately 1000 to 10,000, more preferably in the range of approximately 1000 to 5000. The dispersant may be modified so as to contain functionalizable sites useful for covalent or noncovalent attachment to an external molecular moiety. The functionalizable sites may be present in addition to the ionizable groups discussed above, or the ionizable groups may themselves serve as functionalizable sites suitable for binding an external molecular moiety. Functionalizable sites include, for example, any of the conventional functional groups that are modified using simple, conventional chemical techniques, e.g., amino groups, nitriles, carboxylic acids, esters, acid chlorides, and the like. Preferably, although not necessarily, the functionalizable sites are spaced apart from the dispersant structure by an inert linking moiety, e.g., an alkylene or oxyalkylene chain, typically composed of about 2 to 20 carbon atoms, preferably about 4 to 10 carbon atoms, or other linking moieties such as those described above with respect to the spacer linkages that may be present linking hydrophilic functional groups to the polymer backbone.
  • IV. Preparation of the Surface-Modified Nanoparticles:
  • Hydrophobic nanoparticles may be rendered water dispersible by surface modification with the amphipathic dispersant. That is, the hydrophobic regions of the dispersant associate with the hydrophobic nanoparticle surface, and the hydrophilic regions are externally facing and provide water dispersibility. Surface modification of the nanoparticles is carried out as follows.
  • Initially, a solution of the amphipathic dispersant is prepared by admixing the selected amphipathic dispersant with a suitable nonaqueous solvent, preferably a nonpolar, water-immiscible solvent such as n-hexane or chloroform. Ionizable groups on the dispersant, if present, are then converted to salt form by treatment with an appropriate acid or base, which serves as an ionizing agent. For ionizable acidic groups, suitable bases are generally inorganic bases, e.g., ammonium hydroxides or hydroxides of alkali metals (e.g., sodium or potassium) or alkaline earth metals (e.g., magnesium or calcium). The hydrophobic nanoparticles are dispersed in the same solvent, either before or after the aforementioned ionization step. Typically, however, the nanoparticles are added after ionization, preferably dropwise, to a stirring solution of the ionized dispersant. Alternatively, the nanoparticles may be dispersed in the solvent at the outset, and the dispersant added thereto. As another alternative, two separate solutions may be prepared and mixed, with one solution containing the dispersant and the other solution containing the nanoparticles, with both solutions preferably containing the same solvent. In all cases, after preparation of the nanoparticle-dispersant-solvent admixture, the admixture is preferably stirred for several minutes to ensure complete mixing of the components.
  • In the next step of the process, the admixture of nanoparticles, dispersant and solvent is subjected to conditions effective to result in absorption of the dispersant by the nanoparticles. For example, the admixture may be heated or placed under vacuum to remove the solvent, such a drying process resulting in dispersant-coated nanoparticles. Alternatively, the conditions may involve changing the polarity of the solvent and/or changing the ionic state of the polymer.
  • Next, the dispersant-coated nanoparticles are transferred to an aqueous medium such as water, using solvent exchange (if the dispersant-coated nanoparticles are not previously dried) or addition of water or an aqueous buffer (if the dispersant-coated nanoparticles are previously dried). The aqueous buffer, if one is used, should be effective to facilitate dispersion of the nanoparticles in the aqueous medium. The water dispersion is then filtered to remove any large micellar structures formed by excess dispersant in solution that is not associated with the particles. These materials may then be used in any applications requiring aqueous-based sols of nanocrystals. Prior to using these particles one may further increase the stability of the amphipathic coating by chemically crosslinking the individual polymer chains of the dispersant coating such that each polymer has a potential multiplicity of chemical bonds to other polymer chains on the particle. One of ordinary skill in the art would recognize that the crosslinker used may be tailored to match the properties of the dispersant coating. For example, a diamine could be used to crosslink a dispersant coating containing carboxylic acids. Of particular utility are crosslinkers that carry charges or other groups capable of stabilizing the dispersed colloids as described herein. A diamino carboxylate or sulfonate and a diamino polyethylene glycol crosslinkers are especially useful. A similar chemistry would apply for crosslinkers having multiple amine moieties, such as dendrimers, modified dendrimers, and the like.
  • The amount of amphipathic dispersant per unit mass of the “inner core” (i.e., per unit mass of the original, unmodified nanoparticle) in the resulting dispersant-coated nanoparticles is proportional to the size and surface area of the nanoparticles. Generally, the number ratio of the dispersant to the inner core will be in the range of approximately 50:1 to approximately 5000:1. The ratio will be closer to 50:1 for smaller nanoparticles, i.e., nanoparticles less than about 5 nm in diameter (e.g., green CdSe quantum dots), and will be closer to 5000:1 for larger nanoparticles, i.e., nanoparticles about 5 nm to 10 nm in diameter (e.g., red CdSe quantum dots).
  • V. Nanoparticle Conjugates and Associated Compositions:
  • The invention additionally relates to conjugates of the present surface-modified semiconductive nanoparticles and compositions comprising those conjugates in association with a target analyte.
  • That is, the surface-modified semiconductive nanoparticles of the invention may be conjugated to an affinity molecule that serves as the first member of a binding pair. Generally, although not necessarily, it is the amphipathic dispersant on the nanoparticle surface that provides the means for linkage to the affinity molecule. As noted previously, ionizable groups present within the hydrophilic regions of the amphipathic dispersant may provide the means for linkage to the affinity molecule, and/or other functional groups present within or introduced into the dispersant molecule may provide the means for linkage to the affinity molecule. The linkage will generally be covalent, and suitable linkers are discussed in Section III, above. Suitable methods of conjugating molecules and molecular segments to affinity molecules are described, for example, in Hermanson, Bioconjugate Techniques (Academic Press, NY, 1996).
  • Such semiconductive nanoparticle “conjugates,” by virtue of the affinity molecule, can be used to detect the presence and/or quantity of biological and chemical compounds, interactions in biological systems, biological processes, alterations in biological processes, or alterations in the structure of biological compounds. That is, the affinity molecule, when linked to the semiconductive nanoparticle, can interact with a biological target that serves as the second member of the binding pair, in order to detect biological processes or reactions, or to alter biological molecules or processes. Preferably, the interaction of the affinity molecule and the biological target involves specific binding, and can involve covalent, noncovalent, hydrophobic, hydrophilic, electrostatic, van der Waal's, or magnetic interaction. Preferably, the affinity molecule physically interacts with the biological target.
  • The affinity molecule associated with the semiconductive nanoparticles can be naturally occurring or chemically synthesized, and can be selected to have a desired physical, chemical, or biological property. Such properties include, but are not limited to, covalent and noncovalent association with proteins, nucleic acids, signaling molecules, prokaryotic or eukaryotic cells, viruses, subcellular organelles and any other biological compounds. Other properties of such molecules include, but are not limited to, the ability to affect a biological process (e.g. cell cycle, blood coagulation, cell death, transcription, translation, signal transduction, DNA damage or cleavage, production of radicals, scavenging radicals, etc.), and the ability to alter the structure of a biological compound (e.g. crosslinking, proteolytic cleavage, radical damage, etc
  • In a preferred embodiment, the nanoparticle conjugate is comprised of a semiconductive nanoparticle that emits light at a tunable wavelength and is associated with a nucleic acid. The association can be direct or indirect. The nucleic acid can be any ribonucleic acid, deoxyribonucleic acid, dideoxyribonucleic acid, or any derivatives and combinations thereof. The nucleic acid can also be oligonucleotides of any length. The oligonucleotides can be single-stranded, double-stranded, triple-stranded or higher order configurations (e.g. Holliday junctions, circular single-stranded DNA, circular double-stranded DNA, DNA cubes, (see Seeman (1998) Ann. Rev. Biophys. Biomol. Struct. 27:225-248). Among the preferred uses of the present compositions and methods are detecting and/or quantitating nucleic acids as follows: (a) viral nucleic acids; (b) bacterial nucleic acids; and (c) numerous human sequences of interest, e.g. single nucleotide polymorphisms. Without limiting the scope of the present invention, nanoparticle conjugates can comprise nanocrystals associated with individual nucleotides, deoxynucleotides, dideoxynucleotides or any derivatives and combinations thereof and used in DNA polymerization reactions such as DNA sequencing, reverse transcription of RNA into DNA, and polymerase chain reactions (PCR). Nucleotides also include monophosphate, diphosphate and triphosphates and cyclic derivatives such as cyclic adenine monophosphate (cAMP). Other uses of nanoparticles conjugated to nucleic acids included fluorescence in situ hybridization (FISH). In this preferred embodiment, nanocrystals are conjugated to oligonucleotides designed to hybridize to a specific sequence in vivo. Upon hybridization, the fluorescent nanocrystal tags are used to visualize the location of the desired DNA sequence in a cell. For example, the cellular location of a gene whose DNA sequence is partially or completely known can be determined using FISH. Any DNA or RNA whose sequence is partially or completely known can be visually targeted using FISH. For example without limiting the scope of the present invention, messenger RNA (mRNA), DNA telomeres, other highly repeated DNA sequences, and other non-coding DNA sequencing can be targeted by FISH.
  • The nanoparticle conjugate may also comprise a surface-modified semiconductive nanoparticle as provided herein in association with a molecule or reagent for detection of biological compounds such as enzymes, enzyme substrates, enzyme inhibitors, cellular organelles, lipids, phospholipids, fatty acids, sterols, cell membranes, molecules involved in signal transduction, receptors and ion channels. The conjugate also can be used to detect cell morphology and fluid flow; cell viability, proliferation and function; endocytosis and exocytosis (Betz et al. (1996) Curr. Opin. Neurobiol. 6(3):365-71); and reactive oxygen species (e.g., superoxide, nitric oxide, hydroxyl radicals, oxygen radicals). In addition, the conjugate can be used to detect hydrophobic or hydrophilic regions of biological systems.
  • Conjugates of semiconductive nanocrystals also find utility in numerous other biological and non-biological applications where luminescent markers, particularly fluorescent markers, are typically used. See, for example, Haugland, R.P. Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, Oreg. Sixth Ed. 1996; Website, www.probes.com.). Examples of areas in which the luminescent nanoparticle conjugates of the invention are useful include, without limitation, fluorescence immunocytochemistry, fluorescence microscopy, DNA sequence analysis, fluorescence in situ hybridization (FISH), fluorescence resonance energy transfer (FRET), flow cytometry (Fluorescence Activated Cell Sorter; FACS) and diagnostic assays for biological systems. For further discussion concerning the utility of nanocrystal conjugates in the aforementioned areas, see International Patent Publication No. WO 00/17642 to Bawendi et al.
  • It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
  • The following examples are intended to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the novel compositions of the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc), but some experimental error and deviation should, of course, be allowed for. Unless indicated otherwise, parts are parts by weight, temperatures are in degrees centigrade, and pressure is at or near atmospheric.
  • The practice of the present invention will employ, unless otherwise indicated, conventional techniques of synthetic organic chemistry, biochemistry, molecular biology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Haines & S J. Higgins, eds., 1984); Methods in Enzymology (Academic Press, Inc.); Kirk-Othmer's Encyclopedia of Chemical Technology; and House's Modern Synthetic Reactions. All patents, patent applications, patent publications, journal articles and other references cited herein are incorporated by reference in their entireties.
  • EXAMPLE 1 Synthesis of Hydrophobically Modified Hydrophilic Polymers:
  • A modified polyacrylic acid was prepared by diluting 100 g [0.48 mol COONa] of poly(acrylic acid, sodium salt) (obtained from Aldrich, molecular weight 1200) was diluted two-fold in water and acidified in a 1.0 L round bottom flask with 150 ml (1.9 mol) of concentrated HCl. The acidified polymer solution was concentrated to dryness on a rotary evaporator (100 mbar, 80° C.). The dry polymer was evacuated for 12 hours at <10 mbar to ensure water removal. A stirbar and 47.0 g (0.24 mol) of 1-[3-(dimethyl-amino)-propyl]-ethylcarbodiimide hydrochloride (EDC-Aldrich 98%) were added to the flask, then the flask was sealed and purged with N2, and fit with a balloon. 500 ml of anhydrous N-N,dimethylformamide (Aldrich) was transferred under positive pressure through a cannula to this mixture; and the flask was swirled gently to dissolve the solids. 32 ml (0.19 mol) of octylamine was transferred dropwise under positive pressure through a cannula from a sealed oven-dried graduated cylinder into the stirring polymer/EDC solution, and the stirring continued for 12 hours. This solution was concentrated to <100 ml on a rotary evaporator (30 mbar, 80° C.), and the polymer was precipitated by addition of 200 ml di-H2O to the cooled concentrate, which produced a gummy white material. This material was separated from the supernatant and triturated with 100 ml di-H2O three more times. The product was dissolved into 400 ml ethyl acetate (Aldrich) with gentle heating, and basified with 200 ml di-H2O and 100 g N-N-N-N-tetramethylammonium hydroxide pentahydrate (0.55 mo) for 12 hours. The aqueous layer was removed and precipitated to a gummy white product with 400 ml of 1.27 M HCl. The product was decanted and triturated with 100 ml of di-H2O twice more, after which the aqueous washings were back-extracted into 6×100 ml portions of ethyl acetate. These ethyl acetate solutions were added to the product flask, and concentrated to dryness (100 mbar, 60° C.). The crude polymer was dissolved in 300 ml of methanol and purified in two aliquots over LH-20 (Amersham-Pharmacia-5.5 cm×60 cm column) at a 3 ml/minute flow rate. Fractions were tested by NMR for purity, and the pure fractions were pooled, while the impure fractions were re-purified on the LH-20 column. After pooling all of the pure fractions, the polymer solution was concentrated by rotary evaporation to dryness, and evacuated for 12 hours at <10 mbar. The product was a white powder (25.5 g, 45% of theoretical yield), which showed broad NMR peaks in CD3OD [δ=3.1 b (9.4), 2.3 b (9.7), 1.9 1.7 1.5 1.3 b (63.3) 0.9 bt (11.3)], and clear IR signal for both carboxylic acid (1712 cm−1) and amide groups (1626 cm−1, 1544 cm−1).
  • EXAMPLE 2 Preparation of Surface-Modified Nanocrystals:
  • Twenty milliliters of 3-5 μM (3-5 nmoles) of TOPO/TOP coated CdSe/ZnS nanocrystals (see, Murray et al. (1993) J. Am. Chem. Soc. 115:8706) were precipitated with 20 milliliters of methanol. The flocculate was centrifuged at 3000×g for 3 minutes to form a pellet of the nanocrystals. The supernatant was thereafter removed and 20 milliliters of methanol was again added to the particles. The particles were vortexed to loosely disperse the flocculate throughout the methanol. The flocculate was centrifuged an additional time to form a pellet of the nanocrystals. This precipitation/centrifugation step was repeated an additional time to remove any excess reactants remaining from the nanocrystal synthesis. Twenty milliliters of chloroform were added to the nanocrystal pellet to yield a freely dispersed sol.
  • 300 milligrams of hydrophobically modified poly(acrylic acid) was dissolved in 20 ml of chloroform. Tetrabutylammonium hydroxide (1.0 M in methanol) was added to the polymer solution to raise the solution to pH 10 (pH was measured by spotting a small aliquot of the chloroform solution on pH paper, evaporating the solvent and thereafter wetting the pH paper with distilled water). Thereafter the polymer solution was added to 20 ml of chloroform in a 250 ml round bottom flask equipped with a stir bar. The solution was stirred for 1 minute to ensure complete admixture of the polymer solution. With continued stirring the washed nanocrystal dispersion described above was added dropwise to the polymer solution. The dispersion was then stirred for two minutes to ensure complete mixing of the components and thereafter the chloroform was removed in vacuo with low heat to yield a thin film of the particle-polymer complex on the wall of the flask. Twenty milliliters of distilled water were added to the flask and swirled along the walls of the flask to aid in dispersing the particles in the aqueous medium. The dispersion was then allowed to stir overnight at room temperature. At this point the nanocrystals are freely dispersed in the aqueous medium, possess pendant chemical functionalities and may therefore be linked to affinity molecules of interest using methods well known in the art for biolabeling experiments. In addition, the fact that the nanocrystals now have a highly charged surface means they can be readily utilized in polyelectrolyte layering experiments for the formation of thin films and composite materials.
  • EXAMPLE 3 Preparation of Nanocrystal Conjugates:
  • Functional and specific biological labels have been made with materials of the present invention as follows: The polymer-stabilized particles from Example 1 were purified away from excess (non-absorbed) polymer and tetrabutylammonium hydroxide via tangential flow diafiltration using a 100 K polyethersulfone membrane against one liter of distilled water and one liter of 50 mM morpholinoethanesulfonic acid buffer, pH 5.9. The purified dispersion was concentrated to 20 milliliters and 10 milliliters of this nanocrystal dispersion were activated with 79 μmoles (15 mg) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 158 μmoles (34 mg) N-hydroxysulfosuccinimide for 30 minutes at room temperature. The particle dispersion was then buffer exchanged to pH 8.0 via diafiltration against 50 mM phosphate buffer, pH 8.0. When the particle dispersion reached pH 8.0, streptavidin was added to the particles at a 5:1 protein:particle ratio (175 nmoles, 10.5 mg) and the reaction mixture was incubated overnight at room temperature with stirring. After overnight incubation the conjugated particles were separated from excess, unreacted protein via tangential flow diafiltration using a 100,000 MW polyethersulfone membrane against 2 liters of phosphate buffer, 50mM, pH 7.0.
  • At this point these materials can be stored in any number of biological buffers and used as fluorescent biological labels to detect biotin-labeled analytes of interest. Although streptavidin was used here as an example, the simplicity and generality of the above coupling chemistries can be efficiently extended to forming functional conjugates with any number of biological molecules of interest, such as antibodies, peptides, and oligonucleotides, for example.
  • EXAMPLE 4
  • Crosslinking of Polymer Stabilized Nanocrystals with a Dendrimer:
  • Ten milliliters of nanocrystals at 3.5 μM, stabilized as described in Example 2, were purified by tangential flow filtration, as described in Example 3, against 1 liter of distilled water to remove excess polymer. The nanocrystals were concentrated to 10 milliliters and the pH of the aqueous dispersion was decreased to pH 6.5 with 50 μl additions of 0.1 M HCl. 67 milligrams (315 moles) EDC were added to the stirring nanocrystal dispersion. The reaction was allowed to proceed for 10 minutes before 1 milliliter of 0.5M borate buffer (pH 8.5) containing 3.94 moles of the crosslinking reagent Starburst® (PAMAM) Dendrimer, Generation 0, were added to the reaction mixture. The reaction mixture was stirred for 2 hours at room temperature and then transferred to a 50,000 molecular weight cut-off polyethersulfone dialysis bag. Dialysis was performed for 24 hours against 2 changes of 4 liters of water.
  • EXAMPLE 5
  • Crosslinking of Polymer Stabilized Nanocrystals with a Diamino Crosslinker:
  • Ten milliliters of nanocrystals at 3.5 μM, stabilized as described in Example 2, were purified by tangential flow filtration, as described in Example 3, against 1 liter of distilled water to remove excess polymer. The nanocrystals were concentrated to 10 milliliters and the pH of the aqueous dispersion was decreased to pH 6.5 with 50 μl additions of 0.1 M HCl. 67 milligrams (315 μmoles) EDC were added to the stirring nanocrystal dispersion. The reaction was allowed to proceed for 10 minutes before 1 milliliter of 0.5M borate buffer (pH 8.5) containing 3.94 μmoles of the crosslinking reagent lysine (a diamino carboxylic acid) were added to the reaction mixture. The reaction mixture was stirred for 2 hours at room temperature and then transferred to a 50,000 molecular weight cut-off polyethersulfone dialysis bag. Dialysis was performed for 24 hours against 2 changes of 4 liters of water.
  • EXAMPLE 6
  • Preparation of Surface Modified Nanocrystals with Polypeptides:
  • A triblock polypeptide comprised of (Aspartate)4-(Norleucine)8-(Aspartate)4 has been used to stabilize hydrophobic nanocrystals in water by the following method: Five milliliters of a 3.5 μM nanocrystal solution were washed as described in Example 1 and redispersed in 5 milliliters of chloroform. 75 milligrams of an (Aspartate)4-(Norleucine)8-(Aspartate)4 triblock polypeptide were dissolved in 5 milliliters of a 50:50 mixture of chloroform:methanol and the pH of the polypeptide solution was raised to 10 with aliquots of tetrabutylammonium hydroxide (1.0 M in methanol). This polypeptide solution was then added to 5 milliliters of chloroform in a 50 milliliter round bottom flask. The solution was allowed to stir for 1 minute to ensure complete mixing. The washed nanocrystal dispersion in chloroform was then added dropwise to the stirring polypeptide solution and the entire mixture was allowed to stir for an additional 2 minutes before all the solvent was removed in vacuo with low heat (40 degrees Celsius) to yield a thin film of the particle-polymer complex on the wall of the flask. Five milliliters of distilled water were then added to the flask and swirled in order to aid in dispersing the nanocrystals fully in the aqueous medium. As with the nanocrystals stabilized in Example 1, these polypeptide stabilized nanocrystals can be efficiently purified away from excess polypeptide by dialysis, tangential flow filtration, or various forms of chromatography known to those skilled in the art.

Claims (22)

1-9. (canceled)
10. A population of water-dispersible nanoparticles, comprising:
a plurality of hydrophobic nanoparticles, wherein each nanoparticle is provided with an outer layer of an amphipathic dispersant, the amphipathic dispersant comprising a polymer having a hydrophobic backbone and a plurality of alkyl acrylamide side chains, wherein the alkyl group of each acrylamide side chain has more than four carbon atoms, and wherein the amphipathic dispersant renders the nanoparticle water-dispersible.
11. The population of claim 10 , wherein the alkyl group of each acrylamide side chain has six or more carbon atoms.
12. The population of claim 10 , wherein the alkyl group of each acrylamide side chain has 6 to 12 carbon atoms.
13. The population of claim 10 , wherein the alkyl group of each acrylamide side chain has eight or more carbon atoms.
14. The population of claim 10 , wherein the plurality of alkyl acrylamide side chains comprises octylacrylamide.
15. The population of claim 10 , wherein the polymer further comprises hydrophilic side chains.
16. The population of claim 10 , wherein the polymer further comprises ionizable groups.
17. The population of claim 10 , wherein the polymer further comprises a group selected from amine, carboxylic acid, sulfonic acid, and phosphonic acid.
18. The population of claim 10 , wherein the polymer comprises at least one ethylenically unsaturated C3-C6 carboxylic acid monomer residue.
19. The population of claim 10 , wherein the polymer comprises an acrylic acid or methacrylic acid monomer residue.
20. The population of claim 10 , wherein the polymer is poly(acrylic acid-co-octylacrylamide), poly(acrylic acid-co-hexylacrylamide), poly(methacrylic acid-co-octylacrylamide), or poly(methacrylic acid-co-hexylacrylamide).
21. The population of claim 10 , wherein the nanoparticle is surrounded with the outer layer of amphipathic dispersant.
22. The population of claim 10 , wherein two or more nanoparticles are surrounded with the outer layer of amphipathic dispersant.
23. The population of claim 10 , wherein the amphipathic dispersant is crosslinked.
24. The population of claim 10 , wherein plurality of hydrophobic nanoparticles comprises semiconductor or metallic nanoparticles.
25. The population of claim 10 , wherein the plurality of hydrophobic nanoparticles comprises semiconductor nanocrystals.
26. The population of claim 10 , wherein the plurality of water-dispersible nanoparticles is dispersed in an aqueous medium.
27. A population of nanoparticle conjugates, comprising:
the population of water-dispersible nanoparticles of claim 10 , wherein each nanoparticle is associated with an affinity molecule.
28. The population of nanoparticle conjugates of claim 27 , wherein the affinity molecule is selected from the group consisting of proteins, nucleic acids, polysaccharides, and molecules having a molecular weight of less than about 1500 grams/mol.
29. The population of nanoparticle conjugates of claim 27 , wherein the affinity molecule is selected from the group consisting of an antigen, hapten, antibody or fragment thereof, biotin, streptavidin, hormone, hormone binding protein, enzyme, enzyme inhibitor, receptor agonist, and receptor antagonist.
30. An aqueous dispersion of nanoparticles, comprising:
the population of water-dispersible nanoparticles of claim 10 ; and
an aqueous medium, wherein the nanoparticles in the population are dispersed in the aqueous medium and exhibit luminescence when excited by an appropriate energy source.
US13/423,055 2000-10-13 2012-03-16 Metallic nanoparticles having enhanced dispersibility in aqueous media comprising a polymer having alkyl acrylamide side chains Expired - Fee Related US8691384B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US13/423,055 US8691384B2 (en) 2000-10-13 2012-03-16 Metallic nanoparticles having enhanced dispersibility in aqueous media comprising a polymer having alkyl acrylamide side chains

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US24021600P 2000-10-13 2000-10-13
US09/841,237 US6649138B2 (en) 2000-10-13 2001-04-23 Surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US10/717,246 US20040101621A1 (en) 2000-10-13 2003-11-18 Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US12/013,371 US20080241375A1 (en) 2000-10-13 2008-01-11 Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US12/624,283 US8158194B2 (en) 2000-10-13 2009-11-23 Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US13/423,055 US8691384B2 (en) 2000-10-13 2012-03-16 Metallic nanoparticles having enhanced dispersibility in aqueous media comprising a polymer having alkyl acrylamide side chains

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US12/624,283 Continuation US8158194B2 (en) 2000-10-13 2009-11-23 Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media

Publications (2)

Publication Number Publication Date
US20120228565A1 true US20120228565A1 (en) 2012-09-13
US8691384B2 US8691384B2 (en) 2014-04-08

Family

ID=26933249

Family Applications (7)

Application Number Title Priority Date Filing Date
US09/841,237 Expired - Lifetime US6649138B2 (en) 2000-10-13 2001-04-23 Surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US10/717,288 Expired - Lifetime US7147917B2 (en) 2000-10-13 2003-11-18 Surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US10/717,246 Abandoned US20040101621A1 (en) 2000-10-13 2003-11-18 Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US10/716,971 Expired - Lifetime US7108915B2 (en) 2000-10-13 2003-11-18 Surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US12/013,371 Abandoned US20080241375A1 (en) 2000-10-13 2008-01-11 Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US12/624,283 Expired - Lifetime US8158194B2 (en) 2000-10-13 2009-11-23 Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US13/423,055 Expired - Fee Related US8691384B2 (en) 2000-10-13 2012-03-16 Metallic nanoparticles having enhanced dispersibility in aqueous media comprising a polymer having alkyl acrylamide side chains

Family Applications Before (6)

Application Number Title Priority Date Filing Date
US09/841,237 Expired - Lifetime US6649138B2 (en) 2000-10-13 2001-04-23 Surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US10/717,288 Expired - Lifetime US7147917B2 (en) 2000-10-13 2003-11-18 Surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US10/717,246 Abandoned US20040101621A1 (en) 2000-10-13 2003-11-18 Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US10/716,971 Expired - Lifetime US7108915B2 (en) 2000-10-13 2003-11-18 Surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US12/013,371 Abandoned US20080241375A1 (en) 2000-10-13 2008-01-11 Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US12/624,283 Expired - Lifetime US8158194B2 (en) 2000-10-13 2009-11-23 Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media

Country Status (6)

Country Link
US (7) US6649138B2 (en)
EP (2) EP2233202A3 (en)
JP (2) JP4638128B2 (en)
AU (1) AU2002243207A1 (en)
CA (1) CA2424082C (en)
WO (1) WO2002055186A2 (en)

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013151666A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
WO2013151736A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics In vivo production of proteins
WO2014152211A1 (en) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
CN104181141A (en) * 2014-08-30 2014-12-03 西安电子科技大学 Genetic algorithm based method for analyzing data of luminescent material combination sample library
WO2015034928A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Chimeric polynucleotides
WO2015034925A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Circular polynucleotides
WO2015051214A1 (en) 2013-10-03 2015-04-09 Moderna Therapeutics, Inc. Polynucleotides encoding low density lipoprotein receptor
WO2016014846A1 (en) 2014-07-23 2016-01-28 Moderna Therapeutics, Inc. Modified polynucleotides for the production of intrabodies
WO2017112943A1 (en) 2015-12-23 2017-06-29 Modernatx, Inc. Methods of using ox40 ligand encoding polynucleotides
CN104749147B (en) * 2015-03-09 2017-07-04 西安电子科技大学 A kind of method for optimizing analysis luminescent material performance and component
WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
WO2018213731A1 (en) 2017-05-18 2018-11-22 Modernatx, Inc. Polynucleotides encoding tethered interleukin-12 (il12) polypeptides and uses thereof
WO2018226861A1 (en) 2017-06-07 2018-12-13 Regeneron Pharmaceuticals, Inc. Compositions and methods for internalizing enzymes
WO2019157224A1 (en) 2018-02-07 2019-08-15 Regeneron Pharmaceuticals, Inc. Methods and compositions for therapeutic protein delivery
WO2020161342A1 (en) 2019-02-08 2020-08-13 Curevac Ag Coding rna administered into the suprachoroidal space in the treatment of ophtalmic diseases
WO2022023559A1 (en) 2020-07-31 2022-02-03 Curevac Ag Nucleic acid encoded antibody mixtures
WO2022059736A1 (en) 2020-09-17 2022-03-24 株式会社レストアビジョン Composition for treating or preventing diseases, disorders, or conditions associated with endoplasmic reticulum stress or all-trans-retinal, protecting retinal thickness, or suppressing reduction in retinal thickness or progress of reduction in retinal thickness
EP4144378A1 (en) 2011-12-16 2023-03-08 ModernaTX, Inc. Modified nucleoside, nucleotide, and nucleic acid compositions
EP4159741A1 (en) 2014-07-16 2023-04-05 ModernaTX, Inc. Method for producing a chimeric polynucleotide encoding a polypeptide having a triazole-containing internucleotide linkage
WO2023133579A1 (en) 2022-01-10 2023-07-13 Regeneron Pharmaceuticals, Inc. Bbb-targeted gaa delivered as gene therapy treats cns and muscle in pompe disease model mice
WO2023220603A1 (en) 2022-05-09 2023-11-16 Regeneron Pharmaceuticals, Inc. Vectors and methods for in vivo antibody production
WO2024026474A1 (en) 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Compositions and methods for transferrin receptor (tfr)-mediated delivery to the brain and muscle

Families Citing this family (431)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7192778B2 (en) * 1999-10-06 2007-03-20 Natan Michael J Surface enhanced spectroscopy-active composite nanoparticles
US8497131B2 (en) * 1999-10-06 2013-07-30 Becton, Dickinson And Company Surface enhanced spectroscopy-active composite nanoparticles comprising Raman-active reporter molecules
US6734420B2 (en) * 2000-04-06 2004-05-11 Quantum Dot Corporation Differentiable spectral bar code methods and systems
TW572965B (en) * 2000-08-04 2004-01-21 Sued Chemie Ag Use of amphiphilic polymers or copolymers for the surface modification of reactive inorganic fillers
US20050059031A1 (en) 2000-10-06 2005-03-17 Quantum Dot Corporation Method for enhancing transport of semiconductor nanocrystals across biological membranes
US6649138B2 (en) * 2000-10-13 2003-11-18 Quantum Dot Corporation Surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US6861263B2 (en) * 2001-01-26 2005-03-01 Surromed, Inc. Surface-enhanced spectroscopy-active sandwich nanoparticles
JP2002316350A (en) * 2001-04-19 2002-10-29 Fuji Photo Film Co Ltd Method and device for manufacturing laminated object
FR2824563B1 (en) * 2001-05-10 2004-12-03 Bio Merieux COMPOSITE PARTICLES, DERIVATIVES, PREPARATION METHOD AND APPLICATIONS
US6956083B2 (en) * 2001-05-31 2005-10-18 The Regents Of The University Of California Single ion conductor cross-linked polymeric networks
US6819845B2 (en) * 2001-08-02 2004-11-16 Ultradots, Inc. Optical devices with engineered nonlinear nanocomposite materials
US6794265B2 (en) * 2001-08-02 2004-09-21 Ultradots, Inc. Methods of forming quantum dots of Group IV semiconductor materials
US6710366B1 (en) 2001-08-02 2004-03-23 Ultradots, Inc. Nanocomposite materials with engineered properties
US20030066998A1 (en) * 2001-08-02 2003-04-10 Lee Howard Wing Hoon Quantum dots of Group IV semiconductor materials
WO2003021635A2 (en) * 2001-09-05 2003-03-13 Rensselaer Polytechnic Institute Passivated nanoparticles, method of fabrication thereof, and devices incorporating nanoparticles
JP3701622B2 (en) * 2002-03-27 2005-10-05 日立ソフトウエアエンジニアリング株式会社 Semiconductor nanoparticle fluorescent reagent and fluorescence measuring method
JP2006508095A (en) * 2002-05-07 2006-03-09 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Bioactivation of particles
DE10221010A1 (en) * 2002-05-11 2003-11-27 Basf Coatings Ag Aqueous dispersion of inorganic nanoparticles, process for their preparation and their use
DE10221009B4 (en) 2002-05-11 2016-10-13 Basf Coatings Gmbh Coating materials, their use, methods for producing coatings and transparent coatings
DE10221007B4 (en) 2002-05-11 2016-10-13 Basf Coatings Gmbh Aqueous dispersion of inorganic nanoparticles, process for their preparation and their use
US7670623B2 (en) * 2002-05-31 2010-03-02 Materials Modification, Inc. Hemostatic composition
JP4005850B2 (en) * 2002-06-10 2007-11-14 日立ソフトウエアエンジニアリング株式会社 Semiconductor nanoparticle manufacturing method
EP1371696B1 (en) * 2002-06-14 2006-08-09 Canon Kabushiki Kaisha Particle composition, recording method, and recording apparatus using the particle composition
EP1527090B1 (en) * 2002-06-21 2006-01-11 Applied NanoSystems B.V. Method of binding a compound to a surface
JP3847677B2 (en) * 2002-07-23 2006-11-22 日立ソフトウエアエンジニアリング株式会社 Semiconductor nanoparticle, method for producing the same, and semiconductor nanoparticle fluorescent reagent
EP1530453A1 (en) * 2002-07-25 2005-05-18 Amcol International Corporation Viscous compositions containing hydrophobic liquids
US7338711B1 (en) * 2002-08-12 2008-03-04 Quantum Logic Devices, Inc. Enhanced nanocomposite combustion accelerant and methods for making the same
WO2004065362A2 (en) * 2002-08-16 2004-08-05 University Of Massachusetts Pyridine and related ligand compounds, functionalized nanoparticulate composites and methods of preparation
JP2004077389A (en) * 2002-08-21 2004-03-11 Hitachi Software Eng Co Ltd Functional fluorescent reagent containing semiconductor nanoparticle
JP4230741B2 (en) * 2002-08-30 2009-02-25 日立ソフトウエアエンジニアリング株式会社 Purification method of semiconductor nanoparticles
EP1534831A2 (en) * 2002-09-04 2005-06-01 Board Of Regents The University Of Texas System Composition, method and use of bi-functional biomaterials
US7572393B2 (en) * 2002-09-05 2009-08-11 Nanosys Inc. Organic species that facilitate charge transfer to or from nanostructures
EP1537187B1 (en) * 2002-09-05 2012-08-15 Nanosys, Inc. Organic species that facilitate charge transfer to or from nanostructures
DE10247359A1 (en) * 2002-10-10 2004-04-29 Basf Coatings Ag Nanoparticles, processes for modifying their surface, dispersion of the nanoparticles, processes for their production and their use
US20040101822A1 (en) * 2002-11-26 2004-05-27 Ulrich Wiesner Fluorescent silica-based nanoparticles
JP2004243507A (en) * 2002-12-19 2004-09-02 Hitachi Software Eng Co Ltd Semiconductor nanoparticles and method of manufacture
JP4903555B2 (en) * 2003-02-20 2012-03-28 ウィルソン−クック・メディカル・インコーポレーテッド Medical device with adhesive coating and method for manufacturing the same
EP1606103A4 (en) * 2003-03-06 2007-01-10 Rensselaer Polytech Inst Rapid generation of nanoparticles from bulk solids at room temperature
EP1606844A2 (en) * 2003-03-17 2005-12-21 Philips Intellectual Property & Standards GmbH Semiconductor device with isolation layer
JP4181435B2 (en) * 2003-03-31 2008-11-12 日油株式会社 Polyethylene glycol modified semiconductor fine particles, production method thereof, and biological diagnostic materials
WO2004087339A1 (en) * 2003-03-31 2004-10-14 Behr Gmbh & Co. Kg Heat exchanger and method for treating the surface of said heat exchanger
US20040252488A1 (en) * 2003-04-01 2004-12-16 Innovalight Light-emitting ceiling tile
US7279832B2 (en) * 2003-04-01 2007-10-09 Innovalight, Inc. Phosphor materials and illumination devices made therefrom
JP2004327229A (en) * 2003-04-24 2004-11-18 Konica Minolta Holdings Inc Composition for forming conductive pattern, forming method for conductive pattern, and manufacturing method of composition
US8859000B2 (en) * 2003-05-05 2014-10-14 The Research Foundation Of State University Of New York Synthesis of nanoparticles by an emulsion-gas contacting process
US20050250094A1 (en) * 2003-05-30 2005-11-10 Nanosphere, Inc. Method for detecting analytes based on evanescent illumination and scatter-based detection of nanoparticle probe complexes
WO2005003730A2 (en) * 2003-06-27 2005-01-13 Trustees Of Princeton University Sensors
JP2007530397A (en) * 2003-07-16 2007-11-01 ザ ユニヴァーシティー オブ リーディング Composite nanoparticles
WO2005029539A2 (en) * 2003-07-21 2005-03-31 Dendritic Nanotechnologies, Inc. Stabilized and chemically functionalized nanoparticles
US20060240573A1 (en) * 2003-07-29 2006-10-26 Lamdagen, Llc Optical system including nanostructures for biological or chemical sensing
JP4418220B2 (en) * 2003-09-09 2010-02-17 日立ソフトウエアエンジニアリング株式会社 Nanoparticles with excellent durability and method for producing the same
US7219017B2 (en) 2003-09-11 2007-05-15 Franco Vitaliano Quantum information processing elements and quantum information processing platforms using such elements
US7219018B2 (en) * 2003-09-11 2007-05-15 Franco Vitaliano Quantum information processing elements and quantum information processing platforms using such elements
US7216038B2 (en) 2003-09-11 2007-05-08 Franco Vitaliano Quantum information processing elements and quantum information processing platforms using such elements
WO2005034205A2 (en) * 2003-10-06 2005-04-14 Dow Corning Corporation Self assembling nanoparticle-polymer hybrids
CN101300026A (en) 2003-10-15 2008-11-05 得克萨斯系统大学评议会 Multifunctional biomaterials as scaffolds for electronic, optical, magnetic, semiconducting, and biotechnological applications
KR100697511B1 (en) * 2003-10-21 2007-03-20 삼성전자주식회사 Photocurable Semiconductor Nanocrystal, Photocurable Composition for Pattern Formation of Semiconductor Nanocrystal and Method of Patterning Nanocrystal using the same
WO2005053649A1 (en) * 2003-11-05 2005-06-16 The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services Biofunctionalized quantum dots for biological imaging
AU2004289210B2 (en) * 2003-11-05 2010-07-15 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Carbohydrate antigen-nanoparticle conjugates and uses thereof as antimetastatic agents in treating cancer
FR2862955B1 (en) * 2003-12-02 2006-03-10 Commissariat Energie Atomique INORGANIC NANOCRYSTALS WITH ORGANIC COATING LAYER, PROCESS FOR THEIR PREPARATION, AND MATERIALS THEREOF
US7695642B2 (en) * 2003-12-12 2010-04-13 Life Technologies Corporation Preparation of stable, bright luminescent nanoparticles having compositionally engineered properties
US7846412B2 (en) * 2003-12-22 2010-12-07 Emory University Bioconjugated nanostructures, methods of fabrication thereof, and methods of use thereof
US7361410B2 (en) * 2003-12-29 2008-04-22 Intel Corporation External modification of composite organic inorganic nanoclusters comprising raman active organic compound
WO2005075132A1 (en) * 2004-02-04 2005-08-18 Ebara Corporation Composite nanoparticle and process for producing the same
US8048477B2 (en) * 2004-02-19 2011-11-01 Nanosolar, Inc. Chalcogenide solar cells
US20060057180A1 (en) * 2004-02-20 2006-03-16 Ashutosh Chilkoti Tunable nonfouling surface of oligoethylene glycol
US20070053845A1 (en) * 2004-03-02 2007-03-08 Shiladitya Sengupta Nanocell drug delivery system
CA2558263A1 (en) * 2004-03-02 2005-09-15 Massachusetts Institute Of Technology Nanocell drug delivery system
CN1969190A (en) * 2004-04-20 2007-05-23 爱默蕾大学 Multimodality nanostructures, methods of fabrication thereof, and methods of use thereof
US8128908B2 (en) * 2004-04-30 2012-03-06 University Of Florida Research Foundation, Inc. Nanoparticles and their use for multifunctional bioimaging
US7943396B2 (en) * 2004-06-22 2011-05-17 The Regents Of The University Of California Peptide-coated nanoparticles with graded shell compositions
US7405002B2 (en) 2004-08-04 2008-07-29 Agency For Science, Technology And Research Coated water-soluble nanoparticles comprising semiconductor core and silica coating
WO2006017746A2 (en) * 2004-08-06 2006-02-16 Heller Adam Ph D Devices and methods of screening for neoplastic and inflammatory disease
US7750352B2 (en) 2004-08-10 2010-07-06 Pinion Technologies, Inc. Light strips for lighting and backlighting applications
KR100604976B1 (en) * 2004-09-03 2006-07-28 학교법인연세대학교 Water-Soluble Nanoparticles Stabilized with Multi-Functional Group Ligands
US7288134B2 (en) * 2004-09-10 2007-10-30 International Business Machines Corporation Dumbbell-like nanoparticles and a process of forming the same
US7534489B2 (en) * 2004-09-24 2009-05-19 Agency For Science, Technology And Research Coated composites of magnetic material and quantum dots
US20060073337A1 (en) * 2004-10-01 2006-04-06 Krzysztof Nauka Conductive path made of metallic nanoparticles and conductive organic material
US20080009568A1 (en) * 2004-10-18 2008-01-10 Nitin Kumar Methods to disperse and exfoliate nanoparticles
US20060090692A1 (en) * 2004-10-29 2006-05-04 Dominguez Juan E Generating nano-particles for chemical mechanical planarization
WO2006050257A2 (en) * 2004-10-29 2006-05-11 Massachusetts Institute Of Tecchnology Detection of ion channel or receptor activity
FR2877571B1 (en) * 2004-11-05 2007-04-13 Nanobiotix Sarl NANOPARTICLES WITH INTRACELLULAR TARGETING ELEMENT, PREPARATION AND USES
US20060240590A1 (en) * 2004-11-09 2006-10-26 The Research Foundation Of State University Of New York Controlled synthesis of nanowires, nanodiscs, and nanostructured materials using liquid crystalline templates
JP4555055B2 (en) * 2004-11-12 2010-09-29 日立ソフトウエアエンジニアリング株式会社 Semiconductor nanoparticles with high luminescent properties
US7524776B2 (en) * 2004-11-30 2009-04-28 Spire Corporation Surface-activation of semiconductor nanostructures for biological applications
US7514725B2 (en) * 2004-11-30 2009-04-07 Spire Corporation Nanophotovoltaic devices
EP1836497B1 (en) * 2004-12-16 2013-04-03 Life Technologies Corporation Quantum dot-encoded bead set for calibration and quantification of multiplexed assays, and methods for their use
US20060148103A1 (en) * 2004-12-30 2006-07-06 Yin-Peng Chen Highly sensitive biological assays
EP1679359B1 (en) 2005-01-06 2010-05-26 Hitachi Software Engineering Co., Ltd. Semiconductor nanoparticle surface modification method
JP4928775B2 (en) * 2005-01-06 2012-05-09 株式会社日立ソリューションズ Semiconductor nanoparticle surface modification method
US7824466B2 (en) 2005-01-14 2010-11-02 Cabot Corporation Production of metal nanoparticles
TW200640596A (en) 2005-01-14 2006-12-01 Cabot Corp Production of metal nanoparticles
US8383014B2 (en) 2010-06-15 2013-02-26 Cabot Corporation Metal nanoparticle compositions
US8334464B2 (en) 2005-01-14 2012-12-18 Cabot Corporation Optimized multi-layer printing of electronics and displays
WO2006076609A2 (en) 2005-01-14 2006-07-20 Cabot Corporation Printable electronic features on non-uniform substrate and processes for making same
WO2006084013A2 (en) * 2005-02-01 2006-08-10 Evident Technologies Semiconductor nanocrystal complexes and methods of detecting molecular interactions using same
JP4820981B2 (en) * 2005-03-03 2011-11-24 国立大学法人山口大学 Polymer compound for water-solubilizing nanoparticles and nanoparticles solubilized using the same
JP2008532522A (en) 2005-03-08 2008-08-21 モレキュラー プローブス, インコーポレイテッド Monitoring and manipulating cell membrane potential differences using nanostructures
WO2006099445A2 (en) * 2005-03-14 2006-09-21 Massachusetts Institute Of Technology Nanocells for diagnosis and treatment of diseases and disorders
GB0505569D0 (en) * 2005-03-18 2005-04-27 Syngenta Ltd Formulations
US7405001B2 (en) 2005-03-24 2008-07-29 3M Innovative Properties Company Surface modified nanoparticle and method of preparing same
US7172811B2 (en) * 2005-03-24 2007-02-06 3M Innovative Properties Company Methods of preparing polymer nanocomposite having surface modified nanoparticles
WO2006105102A2 (en) * 2005-03-28 2006-10-05 The Research Foundation Of State University Of New York Synthesis of nanostructured materials using liquid crystalline templates
US20080246006A1 (en) * 2005-03-31 2008-10-09 Agency For Science, Technology And Research Cdte/Gsh Core-Shell Quantum Dots
US7897257B2 (en) * 2005-04-18 2011-03-01 Ge Healthcare Bio-Sciences Ab Magnetic beads comprising an outer coating of hydrophilic porous polymer and method of making thereof
US20060246524A1 (en) * 2005-04-28 2006-11-02 Christina Bauer Nanoparticle conjugates
EP1877101B1 (en) 2005-04-28 2016-11-16 Ventana Medical Systems, Inc. Enzymes conjugated to antibodies via a peg heterobifuctional linker
US8084001B2 (en) * 2005-05-02 2011-12-27 Cornell Research Foundation, Inc. Photoluminescent silica-based sensors and methods of use
EP1883820A4 (en) * 2005-05-04 2010-06-16 Agency Science Tech & Res Novel water-soluble nanocrystals comprising a low molecular weight coating reagent, and methods of preparing the same
DE102005047807A1 (en) * 2005-06-04 2006-12-07 Solvay Infra Bad Hönningen GmbH Modified nanoparticles
US8845927B2 (en) 2006-06-02 2014-09-30 Qd Vision, Inc. Functionalized nanoparticles and method
US9297092B2 (en) 2005-06-05 2016-03-29 Qd Vision, Inc. Compositions, optical component, system including an optical component, devices, and other products
DE102005026485A1 (en) 2005-06-09 2006-12-14 Bayer Technology Services Gmbh Hydrophilic nanoparticles with functional surface groups, their preparation and use
DK2463386T3 (en) 2005-06-15 2017-07-31 Complete Genomics Inc Nucleic acid analysis using random mixtures of non-overlapping fragments
EP1895976A2 (en) * 2005-06-22 2008-03-12 L'Oréal Make-up compositions for keratinous materials
WO2007002539A2 (en) * 2005-06-24 2007-01-04 Applied Nanoworks, Inc. Nanoparticles and method of making thereof
US8421231B2 (en) 2005-07-01 2013-04-16 National University Of Singapore Electrically conductive composite
GB0517382D0 (en) * 2005-08-26 2005-10-05 Plasticell Ltd Cell culture
EP1929300B1 (en) * 2005-08-30 2017-12-13 Agency for Science, Technology and Research Water-soluble fluorescent particle comprising entangled fluorescent polymer and amphiphilic molecule
JP2007079857A (en) * 2005-09-13 2007-03-29 Canon Inc Server apparatus, client apparatuses and those control methods, computer program, storage medium
US7713689B2 (en) 2005-09-15 2010-05-11 Duke University Non-fouling polymeric surface modification and signal amplification method for biomolecular detection
US20070072309A1 (en) * 2005-09-29 2007-03-29 General Electric Company Analytical compositions including nanometer-sized transducers, methods to make thereof, and devices therefrom
JP2007101498A (en) * 2005-10-07 2007-04-19 Fujifilm Corp Fluorescent probe and fluorescent detection method
CA2624896C (en) 2005-10-07 2017-11-07 Callida Genomics, Inc. Self-assembled single molecule arrays and uses thereof
CN102294207B (en) * 2005-10-14 2014-06-04 维乌作物保护有限公司 Composite nanoparticles, nanoparticles and methods for producing same
WO2007050984A2 (en) * 2005-10-27 2007-05-03 Clemson University Fluorescent carbon nanoparticles
EP1948829A4 (en) * 2005-11-15 2009-08-26 Becton Dickinson Co Sers-based methods for detection of bioagents
KR101167733B1 (en) * 2005-11-16 2012-07-23 삼성전기주식회사 Dispersant for nanoparticles having surfaces to which capping-ligands are bound, Method for dispersing the nanoparticles using the same and Nanoparticle thin film comprising the same
WO2007060591A2 (en) * 2005-11-22 2007-05-31 Koninklijke Philips Electronics N. V. Luminescent particle and method of detecting a biological entity using a luminescent particle
US7465497B2 (en) * 2005-11-23 2008-12-16 General Electric Company High dielectric constant nanocomposites, methods of manufacture thereof, and articles comprising the same
ES2677555T3 (en) 2005-11-23 2018-08-03 Ventana Medical Systems, Inc. Molecular conjugate
US20090305247A1 (en) * 2005-11-30 2009-12-10 Zhiqiang Gao Nanoparticle and methods therefor
DE602006008254D1 (en) * 2005-12-06 2009-09-17 Hitachi Software Eng Method for modifying the surface of semiconductor nanoparticles
US20090317802A1 (en) * 2005-12-09 2009-12-24 Bhatia Sangeeta N Compositions and Methods to Monitor RNA Delivery to Cells
US8409863B2 (en) 2005-12-14 2013-04-02 Becton, Dickinson And Company Nanoparticulate chemical sensors using SERS
DE102005060302A1 (en) * 2005-12-16 2007-06-28 Basf Coatings Ag Aqueous coating material, useful for preparing e.g. thermoplastic and duroplastic materials, comprises an olefin saturated grafted ionically or non-ionically stabilized polyurethane, a surfactant or dispersion agent and an organic solvent
CA2635896A1 (en) * 2005-12-30 2007-09-13 Applera Corporation Synthesis and use of cross-linked hydrophilic hollow spheres for encapsulating hydrophilic cargo
US7723100B2 (en) 2006-01-13 2010-05-25 Becton, Dickinson And Company Polymer coated SERS nanotag
US7226752B1 (en) 2006-01-19 2007-06-05 Avago Technologies General Ip (Singapore) Pte. Ltd. Methods for detecting an analyte in a sample
EP1977242B1 (en) * 2006-01-27 2016-08-03 Becton Dickinson and Company Lateral flow immunoassay with encapsulated detection modality
WO2008057127A2 (en) * 2006-02-06 2008-05-15 Massachusetts Institute Of Technology Self-assembly of macromolecules on multilayered polymer surfaces
CA2571904A1 (en) * 2006-02-15 2007-08-15 Fio Corporation System and method of detecting pathogens
SG10201405158QA (en) 2006-02-24 2014-10-30 Callida Genomics Inc High throughput genome sequencing on dna arrays
EP1994180A4 (en) 2006-02-24 2009-11-25 Callida Genomics Inc High throughput genome sequencing on dna arrays
KR100713745B1 (en) * 2006-02-27 2007-05-07 연세대학교 산학협력단 Water-soluble magnetic or metal oxide nanoparticles coated with ligands and preparation method thereof
US8849087B2 (en) 2006-03-07 2014-09-30 Qd Vision, Inc. Compositions, optical component, system including an optical component, devices, and other products
WO2007120441A2 (en) * 2006-03-27 2007-10-25 Los Alamos National Security, Llc Nanophosphors for large area radiation detectors
WO2007120762A2 (en) * 2006-04-14 2007-10-25 Cambrios Technologies Corporation Fluorescent particles bound to multifunctional scaffolds and their uses
US8858832B2 (en) * 2006-05-23 2014-10-14 The University Of Akron Construction of quantum dots via a regioselective dendritic functionalized cellulose template
KR20070113762A (en) * 2006-05-26 2007-11-29 삼성전자주식회사 Method for forming nanoparticle array using capillarity and nanopartlce array preparaed by the same
US7625637B2 (en) * 2006-05-31 2009-12-01 Cabot Corporation Production of metal nanoparticles from precursors having low reduction potentials
US9212056B2 (en) 2006-06-02 2015-12-15 Qd Vision, Inc. Nanoparticle including multi-functional ligand and method
US8354160B2 (en) * 2006-06-23 2013-01-15 3M Innovative Properties Company Articles having durable hydrophobic surfaces
US7696122B2 (en) * 2006-07-05 2010-04-13 Cabot Corporation Electrocatalyst inks for fuel cell applications
US20080245769A1 (en) * 2006-07-17 2008-10-09 Applied Nanoworks, Inc. Nanoparticles and method of making thereof
ES2649986T3 (en) * 2006-07-24 2018-01-16 Becton Dickinson And Company Apparatus and procedure for the concentration of test particles
KR101556798B1 (en) 2006-10-05 2015-10-01 메사츄세츠 인스티튜트 어브 테크놀로지 Multifunctional Encoded Particles for High-Throughput Analysis
ES2544238T5 (en) 2006-10-11 2023-11-08 Agfa Nv Methods for preparing curable pigment inkjet ink sets
KR100830871B1 (en) * 2006-10-11 2008-05-21 삼성전기주식회사 Method for surface modification of nondispersible metal nanoparticles and modified metal nanoparticles for inkjet by the same method
US7914664B2 (en) * 2006-10-23 2011-03-29 Cleveland State University Nitric oxide sensor
WO2008070352A2 (en) 2006-10-27 2008-06-12 Complete Genomics, Inc. Efficient arrays of amplified polynucleotides
KR100768632B1 (en) * 2006-10-30 2007-10-18 삼성전자주식회사 Method for dispersing nanoparticles and method for producing nanoparticles thin film using the same
EP3564674B1 (en) 2006-11-01 2021-08-25 Ventana Medical Systems, Inc. Haptens, hapten conjugates, compositions thereof and method for their preparation and use
US7754329B2 (en) * 2006-11-06 2010-07-13 Evident Technologies, Inc. Water-stable semiconductor nanocrystal complexes and methods of making same
US20090075343A1 (en) 2006-11-09 2009-03-19 Complete Genomics, Inc. Selection of dna adaptor orientation by nicking
WO2008063653A1 (en) 2006-11-21 2008-05-29 Qd Vision, Inc. Semiconductor nanocrystals and compositions and devices including same
WO2008063652A1 (en) 2006-11-21 2008-05-29 Qd Vision, Inc. Blue emitting semiconductor nanocrystals and compositions and devices including same
WO2008063658A2 (en) 2006-11-21 2008-05-29 Qd Vision, Inc. Semiconductor nanocrystals and compositions and devices including same
KR101207124B1 (en) 2006-11-24 2012-11-30 주식회사 엘지화학 Inorganic nano particle coating solution containing hydrophilic group-modified acrylic resin, and inorganic nano particle film
US20090093551A1 (en) * 2006-12-08 2009-04-09 Bhatia Sangeeta N Remotely triggered release from heatable surfaces
CA2580589C (en) 2006-12-19 2016-08-09 Fio Corporation Microfluidic detection system
EP1935434A1 (en) * 2006-12-19 2008-06-25 Novosom AG Construction and use of transfection enhancer elements
EP2109900A1 (en) * 2007-01-08 2009-10-21 Plextronics, Inc. Quantum dot photovoltaic device
US20080193766A1 (en) * 2007-02-13 2008-08-14 Northern Nanotechnologies Control of Transport to and from Nanoparticle Surfaces
KR101434325B1 (en) 2007-03-05 2014-08-27 비브 나노, 인코포레이티드 Control of transport properties to and from nanoparticle surfaces
US7758961B2 (en) * 2007-03-22 2010-07-20 Milliken & Company Functionalized nanoparticles and their use in particle/bulk material systems
WO2008119184A1 (en) 2007-04-02 2008-10-09 Fio Corporation System and method of deconvolving multiplexed fluorescence spectral signals generated by quantum dot optical coding technology
US7682789B2 (en) * 2007-05-04 2010-03-23 Ventana Medical Systems, Inc. Method for quantifying biomolecules conjugated to a nanoparticle
CA2687178C (en) 2007-05-23 2014-02-04 Ventana Medical Systems, Inc. Polymeric carriers for immunohistochemistry and in situ hybridization
FR2916660B1 (en) * 2007-05-28 2010-10-15 Commissariat Energie Atomique THIN LAYERS OF CONJUGATED POLYMERS CONTAINING INORGANIC NANOPARTICLES AND METHOD FOR THE PRODUCTION THEREOF
CN101821322B (en) 2007-06-22 2012-12-05 Fio公司 Systems and methods for manufacturing quantum dot-doped polymer microbeads
US7816135B2 (en) 2007-07-05 2010-10-19 Becton, Dickinson And Company Method of analyzing lymphocytes
WO2009006739A1 (en) 2007-07-09 2009-01-15 Fio Corporation Systems and methods for enhancing fluorescent detection of target molecules in a test sample
KR100949398B1 (en) * 2007-07-19 2010-03-25 부산대학교 산학협력단 Multifunctional microcarrier for water dispersive nanoparticles and encapsulation method for the same
JP2010534322A (en) * 2007-07-23 2010-11-04 フィオ コーポレイション Methods and systems for collating, storing, analyzing, and accessing data collected and analyzed for biological and environmental analytes
EP2185598A4 (en) * 2007-08-23 2011-02-09 Agency Science Tech & Res Polymerization on particle surface with reverse micelle
US7732533B2 (en) * 2007-08-31 2010-06-08 Micron Technology, Inc. Zwitterionic block copolymers and methods
WO2009079061A2 (en) * 2007-09-25 2009-06-25 The Texas A & M University System Water-soluble nanoparticles with controlled aggregate sizes
CN101861203B (en) 2007-10-12 2014-01-22 Fio公司 Flow focusing method and system for forming concentrated volumes of microbeads, and microbeads formed further thereto
KR100943839B1 (en) * 2007-10-31 2010-02-24 한국과학기술연구원 Method for the production of bio-imaging nanoparticles with high yield by early introduction of irregular structure
US9943490B2 (en) * 2007-11-05 2018-04-17 The Trustees Of Princeton University Composite flash-precipitated nanoparticles
US9662389B2 (en) * 2008-03-31 2017-05-30 Duke University Functionalized metal-coated energy converting nanoparticles, methods for production thereof and methods for use
RU2367513C2 (en) 2007-11-21 2009-09-20 Учреждение Российской Академии Наук Институт Биохимической Физики Им. Н.М. Эмануэля Ран (Ибхф Ран) Method for preparation of polymer coating on particles surface
US9551026B2 (en) 2007-12-03 2017-01-24 Complete Genomincs, Inc. Method for nucleic acid detection using voltage enhancement
WO2009085092A2 (en) * 2007-12-04 2009-07-09 National Institute Of Aerospace Associates Fabrication of metallic hollow nanoparticles
WO2009085342A1 (en) 2007-12-27 2009-07-09 Lockheed Martin Corporation Nano-structured refractory metals, metal carbides, and coatings and parts fabricated therefrom
US7999025B2 (en) * 2008-01-28 2011-08-16 University Of Utah Research Foundation Asymmetrically-functionalized nanoparticles organized on one-dimensional chains
US7785998B2 (en) * 2008-02-21 2010-08-31 Micron Technology, Inc. Methods of forming dispersions of nanoparticles, and methods of forming flash memory cells
US8021517B2 (en) * 2008-02-28 2011-09-20 Honeywell Asca Inc. Use of fluorescent nanoparticles to make on-line measurements of cross-web and machine-direction component and property variations in paper and continuous web products
WO2009117646A2 (en) 2008-03-20 2009-09-24 Drexel University Method for the formation of sers substrates
US9027633B2 (en) * 2008-03-24 2015-05-12 Auburn University Nanoparticle-enhanced phase change materials (NEPCM) with improved thermal energy storage
EP2260081B1 (en) * 2008-03-25 2014-11-26 Xerox Corporation Silica encapsulated organic nanopigments and method of making same
US8796184B2 (en) 2008-03-28 2014-08-05 Sentilus, Inc. Detection assay devices and methods of making and using the same
US9525148B2 (en) 2008-04-03 2016-12-20 Qd Vision, Inc. Device including quantum dots
EP2283342B1 (en) 2008-04-03 2018-07-11 Samsung Research America, Inc. Method for preparing a light-emitting device including quantum dots
US10724903B2 (en) * 2008-05-23 2020-07-28 Nanyang Technological University Polymer encapsulated particles as surface enhanced Raman scattering probes
US7858953B2 (en) * 2008-05-23 2010-12-28 Honeywell Asca Inc. Use of fluorescent nanoparticles to measure individual layer thicknesses or composition in multi-layer films and to calibrate secondary measurement devices
WO2009142763A1 (en) * 2008-05-23 2009-11-26 Swaminathan Ramesh Hybrid photovoltaic cell module
US8703490B2 (en) 2008-06-05 2014-04-22 Ventana Medical Systems, Inc. Compositions comprising nanomaterials and method for using such compositions for histochemical processes
WO2010002540A2 (en) 2008-06-05 2010-01-07 Life Technologies Corporation Activation and monitoring of cellular transmembrane potentials
WO2009155704A1 (en) 2008-06-25 2009-12-30 Fio Corporation Bio-threat alert system
US8747517B2 (en) * 2008-06-30 2014-06-10 Life Technologies Corporation Methods for isolating and purifying nanoparticles from a complex medium
EP2307309B1 (en) 2008-07-02 2015-11-11 Life Technologies Corporation METHOD FOR PRODUCING STABLE InP/ZnS CORE/SHELL SEMICONDUCTOR NANOCRYSTALS AND PRODUCT OBTAINED
PL216549B1 (en) * 2008-08-19 2014-04-30 Univ Jagielloński Method of manufacturing of conductive carbon layers on the powder carriers
EP2334827B1 (en) 2008-08-22 2015-03-11 Ventana Medical Systems, Inc. Method for chromogenic detection of two or more target molecules in a single sample
CN101343540B (en) * 2008-08-28 2011-02-16 上海交通大学 Method for preparing quantum point with hyperbranched polymer supermolecule nano-reactor
MX2011002235A (en) 2008-08-29 2011-04-05 Fio Corp A single-use handheld diagnostic test device, and an associated system and method for testing biological and environmental test samples.
WO2010028217A1 (en) * 2008-09-05 2010-03-11 The Board Of Trustees Of The University Of Illinois Poly(ethylene glycol) methyl ether carbodiimide coupling reagents for the biological and chemical functionalization of water soluble nanoparticles
WO2010040111A2 (en) * 2008-10-03 2010-04-08 Life Technologies Corporation Sulfonate modified nanocrystals
WO2010040032A2 (en) 2008-10-03 2010-04-08 Life Technologies Corporation Methods for preparation of znte nanocrystals
US20110229397A1 (en) * 2008-10-03 2011-09-22 Life Technologies Corporation Process and apparatus for continuous flow synthesis of nanocrystals
EP2337763A4 (en) 2008-10-03 2013-09-04 Life Technologies Corp Methods for preparation of nanocrystals using a weak electron transfer agent and mismatched shell precursors
EP2342162A4 (en) * 2008-10-03 2013-01-09 Life Technologies Corp Compositions and methods for functionalizing or crosslinking ligands on nanoparticle surfaces
WO2010057114A2 (en) 2008-11-14 2010-05-20 Dune Sciences Inc. Functionalized nanoparticles and methods of forming and using same
WO2010062267A1 (en) * 2008-11-25 2010-06-03 Agency For Science, Technology And Research Method of forming a rare earth metal doped nanoparticle
KR101525523B1 (en) * 2008-12-22 2015-06-03 삼성전자 주식회사 Semiconductor Nanocrystal Composite
CA2749660C (en) 2009-01-13 2017-10-31 Fio Corporation A handheld diagnostic test device and method for use with an electronic device and a test cartridge in a rapid diagnostic test
SG173426A1 (en) * 2009-02-26 2011-09-29 Ppt Res Inc Corrosion inhibiting compositions
US11235062B2 (en) * 2009-03-06 2022-02-01 Metaqor Llc Dynamic bio-nanoparticle elements
US11096901B2 (en) 2009-03-06 2021-08-24 Metaqor Llc Dynamic bio-nanoparticle platforms
US8986836B2 (en) * 2009-03-19 2015-03-24 Ohio University Microspheres and their methods of preparation
EA201171195A8 (en) * 2009-03-30 2014-08-29 Серулин Фарма Инк. CONJUGATES, PARTICLES, POLYMER-AGENT COMPOSITIONS AND METHODS OF THEIR APPLICATION
WO2010114770A1 (en) * 2009-03-30 2010-10-07 Cerulean Pharma Inc. Polymer-agent conjugates, particles, compositions, and related methods of use
WO2010114768A1 (en) * 2009-03-30 2010-10-07 Cerulean Pharma Inc. Polymer-epothilone conjugates, particles, compositions, and related methods of use
DE102009017607B4 (en) * 2009-04-08 2011-11-17 Namos Gmbh Process for the functionalization of semiconductor nanoparticles with a detection molecule, the semiconductor nanoparticles produced thereby and their use
GB0914195D0 (en) 2009-08-13 2009-09-16 Plasticell Ltd Vessel for culturing cells
CN102667473B (en) 2009-10-12 2016-06-08 文塔纳医疗系统公司 The pathology of the enhancing for organizing measure and multi-modal contrast and the light field background of multiple analyte detection reproduce
CA2777748C (en) 2009-10-20 2017-09-19 Soane Energy Llc Proppants for hydraulic fracturing technologies
GB0918564D0 (en) 2009-10-22 2009-12-09 Plasticell Ltd Nested cell encapsulation
US8378075B2 (en) * 2009-10-27 2013-02-19 The United States Of America, As Represented By The Secretary Of The Navy Covalent attachment of peptides and biological molecules to luminescent semiconductor nanocrystals
WO2011057295A2 (en) 2009-11-09 2011-05-12 University of Washington Center for Commercialization Functionalized chromophoric polymer dots and bioconjugates thereof
US8920681B2 (en) 2009-12-30 2014-12-30 Korea University Research And Business Foundation Electrically conductive polymers with enhanced conductivity
WO2011082293A1 (en) 2009-12-31 2011-07-07 Ventana Medical Systems, Inc. Methods for producing uniquely specific nucleic acid probes
EP2531569B1 (en) 2010-02-02 2017-01-25 Ventana Medical Systems, Inc. Composition and method for stabilizing fluorescent particles
USPP22463P3 (en) * 2010-02-16 2012-01-17 Menachem Bornstein Gypsophila plant named ‘Pearl Blossom’
ES2606012T3 (en) 2010-02-26 2017-03-17 Ventana Medical Systems, Inc. In situ hybridization with PolyTag probes
WO2011106495A1 (en) 2010-02-26 2011-09-01 Ventana Medical Systems, Inc. Cytogenic analysis of metaphase chromosomes
CN105858600A (en) 2010-04-23 2016-08-17 皮瑟莱根特科技有限责任公司 Synthesis, capping and dispersion of nanocrystals
EP2563865B1 (en) 2010-04-28 2016-06-01 3M Innovative Properties Company Articles including nanosilica-based primers for polymer coatings and methods
EP2563848B1 (en) 2010-04-28 2020-08-26 3M Innovative Properties Company Silicone-based material
DK2569425T3 (en) 2010-05-10 2016-10-03 Univ California Endoribonuclease COMPOSITIONS AND METHODS OF USE THEREOF
CN106691462B (en) 2010-05-17 2020-11-10 申提留斯控股有限公司 Detection device and related method of use
EP2576839B1 (en) 2010-06-07 2017-05-10 Firefly Bioworks, Inc. Nucleic acid detection and quantification by post-hybridization labeling and universal encoding
CA2800936A1 (en) 2010-07-02 2012-01-05 Ventana Medical Systems, Inc. Hapten conjugates for target detection
WO2012007725A2 (en) 2010-07-16 2012-01-19 Plasticell Ltd Method of reprogramming a cell
CA2806127C (en) 2010-07-23 2021-12-21 Advanced Cell Technology, Inc. Methods for detection of rare subpopulations of cells and highly purified compositions of cells
US20130261003A1 (en) 2010-08-06 2013-10-03 Ariosa Diagnostics, In. Ligation-based detection of genetic variants
US20120034603A1 (en) 2010-08-06 2012-02-09 Tandem Diagnostics, Inc. Ligation-based detection of genetic variants
JP6016797B2 (en) * 2010-10-04 2016-10-26 スリーエム イノベイティブ プロパティズ カンパニー Method for changing particle dissolution rate by adding hydrophobic nanoparticles
US9285584B2 (en) 2010-10-06 2016-03-15 3M Innovative Properties Company Anti-reflective articles with nanosilica-based coatings and barrier layer
JP6279902B2 (en) 2010-10-18 2018-02-14 ユニバーシティ オブ ワシントン センター フォー コマーシャライゼーション Chromophore polymer dot
JP2014503446A (en) 2010-10-27 2014-02-13 ピクセリジェント・テクノロジーズ,エルエルシー Nanocrystal synthesis, capping and dispersion
CN102557112A (en) * 2010-12-08 2012-07-11 江南大学 Method for preparing nano CdS particles by using switchable surfactant micelle
CN102531041A (en) * 2010-12-10 2012-07-04 江南大学 Method for preparing nano cadmium dating sulphide (CdS) particles with switching mode surfactant micro-emulsion
US8695618B2 (en) 2010-12-22 2014-04-15 Carnegie Mellon University 3D chemical pattern control in 2D fluidics devices
CN107916448B (en) 2010-12-28 2021-03-12 生命科技公司 Preparation of nanocrystals using mixtures of organic ligands
EP2659000B1 (en) 2010-12-30 2016-08-10 Ventana Medical Systems, Inc. Enhanced deposition of chromogens utilizing pyrimidine analogs
US8679858B2 (en) 2011-01-11 2014-03-25 The Board Of Trustees Of The Leland Stanford Junior University Lanthanide mass dots: nanoparticle isotope tags
US10131947B2 (en) 2011-01-25 2018-11-20 Ariosa Diagnostics, Inc. Noninvasive detection of fetal aneuploidy in egg donor pregnancies
WO2012103031A2 (en) 2011-01-25 2012-08-02 Ariosa Diagnostics, Inc. Detection of genetic abnormalities
US9448231B2 (en) 2011-02-28 2016-09-20 Ventana Medical Systems, Inc. Application of quantum dots for nuclear staining
WO2012118745A1 (en) 2011-02-28 2012-09-07 Arnold Oliphant Assay systems for detection of aneuploidy and sex determination
WO2012123387A1 (en) 2011-03-14 2012-09-20 F. Hoffmann-La Roche Ag A method of analyzing chromosomal translocations and a system therefore
CA2834976C (en) 2011-05-04 2016-03-15 Htg Molecular Diagnostics, Inc. Quantitative nuclease protection assay (qnpa) and sequencing (qnps) improvements
ES2578370T3 (en) 2011-07-01 2016-07-26 HTG Molecular Diagnostics, Inc METHODS TO DETECT GENEUS FUSIONS
US20140000891A1 (en) 2012-06-21 2014-01-02 Self-Suspending Proppant Llc Self-suspending proppants for hydraulic fracturing
US9297244B2 (en) 2011-08-31 2016-03-29 Self-Suspending Proppant Llc Self-suspending proppants for hydraulic fracturing comprising a coating of hydrogel-forming polymer
US9868896B2 (en) 2011-08-31 2018-01-16 Self-Suspending Proppant Llc Self-suspending proppants for hydraulic fracturing
EP2751387B1 (en) 2011-08-31 2019-06-19 Self-Suspending Proppant LLC Self-suspending proppants for hydraulic fracturing
WO2013055995A2 (en) 2011-10-14 2013-04-18 President And Fellows Of Harvard College Sequencing by structure assembly
WO2013057586A1 (en) 2011-10-19 2013-04-25 Oslo Universitetssykehus Hf Compositions and methods for producing soluble t - cell receptors
US9359689B2 (en) 2011-10-26 2016-06-07 Pixelligent Technologies, Llc Synthesis, capping and dispersion of nanocrystals
KR101739576B1 (en) * 2011-10-28 2017-05-25 삼성전자주식회사 Semiconductor nanocrystal-polymer micronized composite, method of preparing the same, and optoelectronic device
US10837879B2 (en) 2011-11-02 2020-11-17 Complete Genomics, Inc. Treatment for stabilizing nucleic acid arrays
ES2675307T3 (en) 2011-12-22 2018-07-10 Centre Hospitalier Universitaire Vaudois (Chuv) Selective plasma activation for medical implants and wound healing devices
WO2013093631A2 (en) * 2011-12-22 2013-06-27 Nanoco Technologies, Inc. Surface modified nanoparticles
CA2859048C (en) 2011-12-30 2020-08-18 Daniel T. Chiu Chromophoric polymer dots with narrow-band emission
WO2013108126A2 (en) 2012-01-16 2013-07-25 University Of Oslo Methyltransferases and uses thereof
CN102585801A (en) * 2012-01-29 2012-07-18 上海交通大学 Preparation method of quantum dot-hyperbranched polyether nanocomposite-nitrogen oxide fluorescent probe
EP2809510B1 (en) * 2012-02-03 2021-03-31 University of Washington through its Center for Commercialization Polyelectrolyte-coated polymer dots and related methods
US20130236986A1 (en) * 2012-02-20 2013-09-12 Cerulean Pharma Inc. Methods of separating nucleic acid polymer conjugates
EP2823285A1 (en) 2012-03-09 2015-01-14 Firefly Bioworks, Inc. Methods and apparatus for classification and quantification of multifunctional objects
US9664667B2 (en) 2012-04-30 2017-05-30 Trustees Of Tufts College Digital quantification of single molecules
WO2013167387A1 (en) 2012-05-10 2013-11-14 Ventana Medical Systems, Inc. Uniquely specific probes for pten, pik3ca, met, top2a, and mdm2
CA2874413A1 (en) 2012-05-21 2013-11-28 The Scripps Research Institute Methods of sample preparation
US9914967B2 (en) 2012-06-05 2018-03-13 President And Fellows Of Harvard College Spatial sequencing of nucleic acids using DNA origami probes
US9628676B2 (en) 2012-06-07 2017-04-18 Complete Genomics, Inc. Imaging systems with movable scan mirrors
US9488823B2 (en) 2012-06-07 2016-11-08 Complete Genomics, Inc. Techniques for scanned illumination
US8476206B1 (en) * 2012-07-02 2013-07-02 Ajay P. Malshe Nanoparticle macro-compositions
WO2014009535A2 (en) 2012-07-12 2014-01-16 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting the survival time and treatment responsiveness of a patient suffering from a solid cancer with a signature of at least 7 genes
HUE036166T2 (en) 2012-07-20 2018-06-28 Harvard College Cell based quality control bioassays for nutriceutical and medicinal products
KR20140032811A (en) 2012-09-07 2014-03-17 삼성전자주식회사 Backlight unit and liquid crystal display having the same
WO2014048942A1 (en) 2012-09-25 2014-04-03 Ventana Medical Systems, Inc. Probes for pten, pik3ca, met, and top2a, and method for using the probes
BR112015006873A2 (en) 2012-09-27 2017-07-04 Rhodia Operations process to produce silver and copolymer nanostructures useful in such a process
US9476089B2 (en) 2012-10-18 2016-10-25 President And Fellows Of Harvard College Methods of making oligonucleotide probes
US8883239B2 (en) 2013-02-26 2014-11-11 Universidad De Talca Clarification and selective binding of phenolic compounds from liquid foodstuff or beverages using smart polymers
WO2014139979A1 (en) 2013-03-12 2014-09-18 Ventana Medical Systems, Inc. Quantum dot in situ hybridization
CN105209916A (en) 2013-03-14 2015-12-30 华盛顿大学商业中心 Polymer dot compositions and related methods
US9540685B2 (en) 2013-03-15 2017-01-10 President And Fellows Of Harvard College Methods of identifying homologous genes using FISH
GB201306589D0 (en) 2013-04-11 2013-05-29 Abeterno Ltd Live cell imaging
ITRM20130269A1 (en) 2013-05-07 2014-11-08 Univ Bologna Alma Mater METHOD FOR THE CONTROL OF QUANTUM DOTS SOLUBILITY
AU2014277045B2 (en) 2013-06-03 2017-07-13 Ventana Medical Systems, Inc. Fluorescence imaging system for tissue detection
US9925478B2 (en) * 2013-06-28 2018-03-27 University Of South Carolina Purification of nanocrystals by gel permeation chromatography and the effect of excess ligands on shell growth and ligand exchange
WO2015019297A1 (en) * 2013-08-07 2015-02-12 University Of Zululand The synthesis of core-shell metal-semiconductor nanomaterials
WO2015036405A1 (en) 2013-09-10 2015-03-19 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for diagnosing and treating basal cell carcinoma
WO2015069787A1 (en) 2013-11-05 2015-05-14 Htg Molecular Diagnostics, Inc. Methods for detecting nucleic acids
EP3617322A1 (en) 2014-02-24 2020-03-04 Ventana Medical Systems, Inc. Automated rna detection using labeled 2 -o-methyl rna oligonucleotide probes and signal amplification systems
US9932521B2 (en) 2014-03-05 2018-04-03 Self-Suspending Proppant, Llc Calcium ion tolerant self-suspending proppants
AU2015267312B2 (en) * 2014-05-27 2018-06-28 Artificial Cell Technologies, Inc. Automated layer by layer construction of multilayer coated cores by TFF
DK3152577T3 (en) 2014-06-06 2018-10-01 Ventana Med Syst Inc SIGNIFICANCE OF INTRATUMORAL HER2 HETEROGENITY IN BREAST CANCER AND APPLICATIONS HEFAR
US20180320226A1 (en) 2014-08-19 2018-11-08 President And Fellows Of Harvard College RNA-Guided Systems For Probing And Mapping Of Nucleic Acids
US10450562B2 (en) 2014-09-09 2019-10-22 Igenomx International Genomics Corporation Methods and compositions for rapid nucleic acid library preparation
EP4306636A2 (en) 2014-09-24 2024-01-17 Exscientia GmbH Monolayer of pbmcs or bone-marrow cells and uses thereof
EP3009147A1 (en) 2014-10-16 2016-04-20 INSERM (Institut National de la Santé et de la Recherche Médicale) Method for treating resistant glioblastoma
JP6482828B2 (en) * 2014-11-17 2019-03-13 国立大学法人 筑波大学 Dispersant for inorganic nanomaterials
AU2015349870B2 (en) 2014-11-21 2019-09-26 Nanostring Technologies, Inc. Enzyme- and amplification-free sequencing
JP2018502567A (en) 2015-01-12 2018-02-01 アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル Diagnosis method of pancreatic cancer
DK3254110T3 (en) 2015-02-03 2020-05-18 Ventana Med Syst Inc Histochemical test to assess programmed death ligand 1 expression (pd-l1)
AU2016232223A1 (en) 2015-03-16 2017-08-31 University Of Washington Materials and methods for detecting androgen receptor splice variants and uses thereof
CA2982536C (en) 2015-04-17 2023-04-18 Spring Bioscience Corporation Antibodies, compositions, and immunohistochemistry methods for detecting c4.4a
WO2016189065A1 (en) 2015-05-26 2016-12-01 Ventana Medical Systems, Inc. Method and system for assessing stain quality for in-situ hybridization and immunohistochemistry
JP6962914B2 (en) 2015-07-20 2021-11-05 センティルス ホールディングカンパニー エルエルシーSentilus Holdco, Llc Chips, detectors, and how they are manufactured and used
JP6882782B2 (en) 2015-08-04 2021-06-02 デューク ユニバーシティ Genetically encoded, essentially chaotic delivery stealth polymers and how to use them
WO2017029391A1 (en) 2015-08-20 2017-02-23 INSERM (Institut National de la Santé et de la Recherche Médicale) New method for treating cancer
WO2017055319A1 (en) 2015-09-29 2017-04-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for quantifying the population of b cells in a tissue sample
WO2017055327A1 (en) 2015-09-29 2017-04-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for quantifying the population of endothelial cells in a tissue sample
WO2017055326A1 (en) 2015-09-29 2017-04-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for quantifying the population of myeloid dendritic cells in a tissue sample
WO2017055325A1 (en) 2015-09-29 2017-04-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for quantifying the population of nk cells in a tissue sample
WO2017055321A1 (en) 2015-09-29 2017-04-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for quantifying the population of fibroblasts in a tissue sample
WO2017055322A1 (en) 2015-09-29 2017-04-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for quantifying the population of neutrophils in a tissue sample
WO2017055320A1 (en) 2015-09-29 2017-04-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for quantifying the population of cytotoxic lymphocytes in a tissue sample
WO2017055324A1 (en) 2015-09-29 2017-04-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for quantifying the population of cells of monocytic origin in a tissue sample
JP6784485B2 (en) * 2015-09-30 2020-11-11 ニチハ株式会社 Manufacturing method of building materials
WO2017060397A1 (en) 2015-10-09 2017-04-13 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting the survival time of subjects suffering from melanoma metastases
WO2017067944A1 (en) 2015-10-19 2017-04-27 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting the survival time of subjects suffering from triple negative breast cancer
RU2743169C2 (en) 2015-11-06 2021-02-15 Вентана Медикал Системз, Инк. Representative diagnosis
EP3374518B1 (en) 2015-11-10 2019-11-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting the survival time of patients with decompensated alcoholic cirrhosis
US11752213B2 (en) 2015-12-21 2023-09-12 Duke University Surfaces having reduced non-specific binding and antigenicity
WO2017122039A1 (en) 2016-01-13 2017-07-20 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting pancreatic cancer treatment response
WO2017182834A1 (en) 2016-04-19 2017-10-26 INSERM (Institut National de la Santé et de la Recherche Médicale) New method for treating resistant glioblastoma
CN109715825B (en) 2016-05-16 2023-01-06 纳米线科技公司 Method for detecting target nucleic acid in sample
WO2017202962A1 (en) 2016-05-24 2017-11-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and pharmaceutical compositions for the treatment of non small cell lung cancer (nsclc) that coexists with chronic obstructive pulmonary disease (copd)
WO2017210476A1 (en) 2016-06-01 2017-12-07 Duke University Nonfouling biosensors
JP2019520071A (en) 2016-06-14 2019-07-18 アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル Method for predicting treatment response of acute severe colitis
WO2018011107A1 (en) 2016-07-11 2018-01-18 INSERM (Institut National de la Santé et de la Recherche Médicale) Use of er-alpha 46 in methods and kits for assessing the status of breast cancer
WO2018011166A2 (en) 2016-07-12 2018-01-18 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for quantifying the population of myeloid dendritic cells in a tissue sample
WO2018046736A1 (en) 2016-09-12 2018-03-15 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting the survival time of patients suffering from cancer
WO2018046738A1 (en) 2016-09-12 2018-03-15 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting the survival time of patients suffering from cancer
WO2018054960A1 (en) 2016-09-21 2018-03-29 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting and treating resistance to chemotherapy in npm-alk(+) alcl
US20200016177A1 (en) 2016-09-22 2020-01-16 Inserm (Institut National De La Sante Et De La Recherche Medicale) Methods and pharmaceutical compositions for reprograming immune environment in a subject in need thereof
EP3541958A1 (en) 2016-11-15 2019-09-25 Ventana Medical Systems, Inc. Compositions and methods for prognosing and treating colorectal cancer
KR102476709B1 (en) 2016-11-21 2022-12-09 나노스트링 테크놀로지스, 인크. Chemical compositions and methods of using same
WO2018104891A1 (en) * 2016-12-09 2018-06-14 Sabic Global Technologies B.V. Quantum dot film and applications thereof
EP4220164A3 (en) 2016-12-19 2023-08-09 Ventana Medical Systems, Inc. Methods and systems for quantitative immunohistochemistry
WO2018122245A1 (en) 2016-12-28 2018-07-05 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods of predicting the survival time of patients suffering from cms3 colorectal cancer
WO2018122249A1 (en) 2016-12-28 2018-07-05 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting the survival time of patients suffering from a microsatellite stable colorectal cancer
US11648200B2 (en) 2017-01-12 2023-05-16 Duke University Genetically encoded lipid-polypeptide hybrid biomaterials that exhibit temperature triggered hierarchical self-assembly
WO2018136201A1 (en) * 2017-01-20 2018-07-26 National Technology & Engineering Solutions Of Sandia, Llc (Ntess) Self-assembled nanoparticle film for nanostructure-initiator mass spectrometry (nims)
WO2018146239A1 (en) 2017-02-10 2018-08-16 INSERM (Institut National de la Santé et de la Recherche Médicale) Biomarker for outcome in aml patients
EP3367098A1 (en) 2017-02-24 2018-08-29 CeMM - Forschungszentrum für Molekulare Medizin GmbH Methods for determining interaction between biological cells
WO2018162404A1 (en) 2017-03-06 2018-09-13 INSERM (Institut National de la Santé et de la Recherche Médicale) Biomarker for outcome in aml patients
WO2018172540A1 (en) 2017-03-24 2018-09-27 INSERM (Institut National de la Santé et de la Recherche Médicale) Method to predict the progression of alzheimer's disease
EP3601613B1 (en) 2017-03-29 2021-12-01 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for assessing pregnancy outcome
WO2018189215A1 (en) 2017-04-12 2018-10-18 INSERM (Institut National de la Santé et de la Recherche Médicale) Method for predicting the survival time of a patient suffering from hepatocellular carcinoma
WO2018213320A1 (en) 2017-05-15 2018-11-22 Duke University Recombinant production of hybrid lipid-biopolymer materials that self-assemble and encapsulate agents
WO2018218284A1 (en) * 2017-05-30 2018-12-06 Ellume Pty Ltd Nanoparticle aggregates
EP3658168A4 (en) 2017-06-30 2021-07-14 Duke University Order and disorder as a design principle for stimuli-responsive biopolymer networks
WO2019038219A1 (en) 2017-08-21 2019-02-28 INSERM (Institut National de la Santé et de la Recherche Médicale) New prognostic method of pancreatic cancer
WO2019043138A1 (en) 2017-09-01 2019-03-07 INSERM (Institut National de la Santé et de la Recherche Médicale) Method for predicting the outcome of a cancer
EP3692368A1 (en) 2017-09-25 2020-08-12 Institut National de la Sante et de la Recherche Medicale (INSERM) Use of vnn1 as a biomarker and a therapeutic target in sarcomas
WO2019086476A1 (en) 2017-10-31 2019-05-09 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh Methods for determining selectivity of test compounds
US11691141B2 (en) 2017-11-13 2023-07-04 Roche Sequencing Solutions, Inc. Devices for sample analysis using epitachophoresis
WO2019104070A1 (en) 2017-11-21 2019-05-31 Nanostring Technologies, Inc. O-nitrobenzyl photocleavable bifunctional linker
WO2019207030A1 (en) 2018-04-26 2019-10-31 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting a response with an immune checkpoint inhibitor in a patient suffering from a lung cancer
KR20210061962A (en) 2018-05-14 2021-05-28 나노스트링 테크놀로지스, 인크. Chemical composition and method of use thereof
EP3797296A1 (en) 2018-05-21 2021-03-31 Genentech, Inc. Her2 heterogeneity as a biomarker in cancer
WO2019229489A1 (en) 2018-05-31 2019-12-05 INSERM (Institut National de la Santé et de la Recherche Médicale) Use of mir-146a-5p and mir-186 as biomarkers of osteoarthritis
TW202016151A (en) 2018-06-09 2020-05-01 德商百靈佳殷格翰國際股份有限公司 Multi-specific binding proteins for cancer treatment
US11649275B2 (en) 2018-08-02 2023-05-16 Duke University Dual agonist fusion proteins
CN109401731B (en) * 2018-09-28 2020-07-17 中国矿业大学 Amphiphilic polymer-loaded nanofluid and preparation method thereof
WO2020106655A1 (en) 2018-11-21 2020-05-28 Self-Suspending Proppant Llc Salt-tolerant self-suspending proppants made without extrusion
EP4059569A1 (en) 2019-01-03 2022-09-21 Institut National De La Sante Et De La Recherche Medicale (Inserm) Methods and pharmaceutical compositions for enhancing cd8+ t cell-dependent immune responses in subjects suffering from cancer
US20220119516A1 (en) 2019-01-16 2022-04-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Variants of erythroferrone and their use
EP3924520A1 (en) 2019-02-13 2021-12-22 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and compositions for selecting a cancer treatment in a subject suffering from cancer
WO2020182932A1 (en) 2019-03-13 2020-09-17 INSERM (Institut National de la Santé et de la Recherche Médicale) New gene signatures for predicting survival time in patients suffering from renal cell carcinoma
WO2020212586A1 (en) 2019-04-18 2020-10-22 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for the treatment and prognosis of cancer
JP2022530390A (en) 2019-04-24 2022-06-29 アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル Methods for Predicting Antipsychotic Responses
CN114269916A (en) 2019-05-14 2022-04-01 豪夫迈·罗氏有限公司 Device and method for sample analysis
WO2020229521A1 (en) 2019-05-14 2020-11-19 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for inhibiting or reducing bacterial biofilms on a surface
WO2021001539A1 (en) 2019-07-04 2021-01-07 INSERM (Institut National de la Santé et de la Recherche Médicale) New strategy to detect and treat eosinophilic fasciitis
US11512314B2 (en) 2019-07-12 2022-11-29 Duke University Amphiphilic polynucleotides
EP4025712A1 (en) 2019-09-05 2022-07-13 Institut National de la Santé et de la Recherche Médicale (INSERM) Method of treatment and pronostic of acute myeloid leukemia
WO2021063968A1 (en) 2019-09-30 2021-04-08 INSERM (Institut National de la Santé et de la Recherche Médicale) Method and composition for diagnosing chronic obstructive pulmonary disease
EP4110823A1 (en) 2020-02-26 2023-01-04 A2 Biotherapeutics, Inc. Polypeptides targeting mage-a3 peptide-mhc complexes and methods of use thereof
WO2021170777A1 (en) 2020-02-28 2021-09-02 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for diagnosing, prognosing and managing treatment of breast cancer
WO2021186014A1 (en) 2020-03-20 2021-09-23 INSERM (Institut National de la Santé et de la Recherche Médicale) Method for predicting the survival time of a patient suffering from a cancer
US20230218608A1 (en) 2020-06-18 2023-07-13 INSERM (Institut National de la Santé et de la Recherche Médicale) New strategy for treating pancreatic cancer
WO2022018163A1 (en) 2020-07-22 2022-01-27 INSERM (Institut National de la Santé et de la Recherche Médicale) Method for predicting survival time in patients suffering from cancer
KR20230096993A (en) 2020-09-16 2023-06-30 나노스트링 테크놀로지스, 인크. Chemical compositions and methods of use thereof
WO2022135753A1 (en) 2020-12-21 2022-06-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for prognosis the humoral response of a subject prior to vaccination
US20220228200A1 (en) 2021-01-19 2022-07-21 10X Genomics, Inc. Methods and compositions for internally controlled in situ assays
WO2022207566A1 (en) 2021-03-29 2022-10-06 INSERM (Institut National de la Santé et de la Recherche Médicale) New method to evaluate pancreatic cancer prognosis
EP4326903A1 (en) 2021-04-23 2024-02-28 Inserm (Institut National De La Sante Et De La Recherche Medicale) Methods and compositions for treating cell senescence accumulation related disease
CN117396613A (en) 2021-06-01 2024-01-12 10X基因组学有限公司 Methods and compositions for analyte detection and probe resolution
WO2022256422A1 (en) 2021-06-02 2022-12-08 10X Genomics, Inc. Sample analysis using asymmetric circularizable probes
WO2023288225A1 (en) 2021-07-13 2023-01-19 10X Genomics, Inc. Methods for preparing polymerized matrix with controllable thickness
US20230057571A1 (en) 2021-08-03 2023-02-23 10X Genomics, Inc. Nucleic acid concatemers and methods for stabilizing and/or compacting the same
WO2023023484A1 (en) 2021-08-16 2023-02-23 10X Genomics, Inc. Probes comprising a split barcode region and methods of use
WO2023129898A2 (en) 2021-12-27 2023-07-06 10X Genomics, Inc. Methods and compositions for rolling circle amplification
WO2023141588A1 (en) 2022-01-21 2023-07-27 10X Genomics, Inc. Multiple readout signals for analyzing a sample
WO2023144303A1 (en) 2022-01-31 2023-08-03 INSERM (Institut National de la Santé et de la Recherche Médicale) Cd38 as a biomarker and biotarget in t-cell lymphomas
WO2023152133A1 (en) 2022-02-08 2023-08-17 INSERM (Institut National de la Santé et de la Recherche Médicale) Method for diagnosing colorectal cancer
WO2023164570A1 (en) 2022-02-23 2023-08-31 Insitro, Inc. Pooled optical screening and transcriptional measurements of cells comprising barcoded genetic perturbations
WO2023181158A1 (en) * 2022-03-23 2023-09-28 ソニーグループ株式会社 Structure and manufacturing method of structure
WO2023192616A1 (en) 2022-04-01 2023-10-05 10X Genomics, Inc. Compositions and methods for targeted masking of autofluorescence
US20240026427A1 (en) 2022-05-06 2024-01-25 10X Genomics, Inc. Methods and compositions for in situ analysis of v(d)j sequences
WO2023215612A1 (en) 2022-05-06 2023-11-09 10X Genomics, Inc. Analysis of antigen and antigen receptor interactions
US20240084378A1 (en) 2022-05-11 2024-03-14 10X Genomics, Inc. Compositions and methods for in situ sequencing
US20240035071A1 (en) 2022-06-17 2024-02-01 10X Genomics, Inc. Catalytic de-crosslinking of samples for in situ analysis
WO2024036304A1 (en) 2022-08-12 2024-02-15 10X Genomics, Inc. Puma1 polymerases and uses thereof
US20240084373A1 (en) 2022-08-16 2024-03-14 10X Genomics, Inc. Ap50 polymerases and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040101621A1 (en) * 2000-10-13 2004-05-27 Adams Edward William Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media

Family Cites Families (44)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US633110A (en) * 1898-12-06 1899-09-19 Fred H Bagg Detachable awning for windows or doors.
US4138381A (en) 1975-04-14 1979-02-06 E. I. Du Pont De Nemours And Company Polymeric thickeners, processes for their preparation and uses thereof
DE3115532A1 (en) * 1980-04-17 1982-01-28 Canon K.K., Tokyo INK-JET RECORDING METHOD AND RECORDING INK FOR RECORDING ON AN IMAGE RECEIVER
NZ204799A (en) 1982-07-12 1986-01-24 Dulux Australia Ltd Aqueous dispersion of particles of film-forming addition polymer,at least one comonomer of which is amphipathic
US4504518A (en) * 1982-09-24 1985-03-12 Energy Conversion Devices, Inc. Method of making amorphous semiconductor alloys and devices using microwave energy
DE3411759C1 (en) * 1984-03-30 1985-04-25 Th. Goldschmidt Ag, 4300 Essen Particles modified on their surface by hydrophilic and hydrophobic groups
US4599400A (en) 1984-12-18 1986-07-08 The Dow Chemical Company Star/comb-branched polyamide
US4694064A (en) 1986-02-28 1987-09-15 The Dow Chemical Company Rod-shaped dendrimer
US5162445A (en) * 1988-05-27 1992-11-10 Exxon Chemical Patents Inc. Para-alkylstyrene/isoolefin copolymers and functionalized copolymers thereof
US5110505A (en) 1989-02-24 1992-05-05 E. I. Du Pont De Nemours And Company Small-particle semiconductors in rigid matrices
US5221334A (en) * 1990-04-11 1993-06-22 E. I. Du Pont De Nemours And Company Aqueous pigmented inks for ink jet printers
US5189136A (en) 1990-12-12 1993-02-23 The Regents Of The University Of California Conducting polymer formed of poly(2-methoxy,5-(2'-ethyl-hexyloxy)-p-phenylenevinylene)
US5262357A (en) 1991-11-22 1993-11-16 The Regents Of The University Of California Low temperature thin films formed from nanocrystal precursors
US5505928A (en) 1991-11-22 1996-04-09 The Regents Of University Of California Preparation of III-V semiconductor nanocrystals
JPH05320276A (en) * 1992-05-20 1993-12-03 Nippon Paint Co Ltd Method for microencapsulating hydrophobic powder by using amphipathic graft polymer
JPH05320726A (en) 1992-05-26 1993-12-03 Sumitomo Metal Ind Ltd Device for preventing clogging with fine coal injection pipe into blast furnace
US6162456A (en) 1992-09-24 2000-12-19 Ortho-Mcneil Pharmaceutical, Inc. Adhesive transdermal drug delivery matrix of a physical blend of hydrophilic and hydrophobic polymers
US5874701A (en) * 1992-10-11 1999-02-23 Toto Co., Ltd. Photocatalytic air treatment process under room light
US6048616A (en) 1993-04-21 2000-04-11 Philips Electronics N.A. Corp. Encapsulated quantum sized doped semiconductor particles and method of manufacturing same
US5604292A (en) 1994-05-31 1997-02-18 The Untied States Of America As Represented By The Secretary Of The Navy Polymers with electrical and non-linear optical properties
US6007845A (en) * 1994-07-22 1999-12-28 Massachusetts Institute Of Technology Nanoparticles and microparticles of non-linear hydrophilic-hydrophobic multiblock copolymers
US5587441A (en) 1994-11-08 1996-12-24 Cornell Research Foundation, Inc. Hyperbranched polymers from AB monomers
DE4440328A1 (en) * 1994-11-11 1996-05-15 Huels Chemische Werke Ag Amphiphilic compounds with at least two hydrophilic and at least two hydrophobic groups based on amides
US5690807A (en) 1995-08-03 1997-11-25 Massachusetts Institute Of Technology Method for producing semiconductor particles
DE69621045T2 (en) * 1995-12-29 2002-12-12 Ciba Spec Chem Water Treat Ltd PARTICLES WITH A POLYMER SHELL AND THEIR PRODUCTION
US6433039B1 (en) * 1996-05-20 2002-08-13 Xerox Corporation Ink jet printing with inks containing comb polymer dispersants
JP2967112B2 (en) * 1996-09-05 1999-10-25 工業技術院長 Organic thin film manufacturing method
DE19652261A1 (en) 1996-12-16 1998-06-18 Hoechst Ag Aryl-substituted poly (p-arylenevinylenes), process for their preparation and their use in electroluminescent devices
GB2341864B (en) 1997-06-14 2001-11-07 Secr Defence Surface coatings
DE69842217D1 (en) 1997-11-07 2011-05-19 State University Radiopaque polymeric biomaterials
US6602497B1 (en) * 1997-11-07 2003-08-05 Rutgers, The State University Strictly alternating poly(alkylene oxide ether) copolymers
US6322901B1 (en) 1997-11-13 2001-11-27 Massachusetts Institute Of Technology Highly luminescent color-selective nano-crystalline materials
US5990479A (en) * 1997-11-25 1999-11-23 Regents Of The University Of California Organo Luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes
US6501091B1 (en) 1998-04-01 2002-12-31 Massachusetts Institute Of Technology Quantum dot white and colored light emitting diodes
ATE275977T1 (en) 1998-04-13 2004-10-15 Massachusetts Inst Technology COMB POLYMERS FOR REGULATING CELL SURFACE INTERACTION
US6326144B1 (en) * 1998-09-18 2001-12-04 Massachusetts Institute Of Technology Biological applications of quantum dots
US6306610B1 (en) * 1998-09-18 2001-10-23 Massachusetts Institute Of Technology Biological applications of quantum dots
EP1113986A2 (en) 1998-09-18 2001-07-11 Massachusetts Institute Of Technology Inventory control
US6251303B1 (en) * 1998-09-18 2001-06-26 Massachusetts Institute Of Technology Water-soluble fluorescent nanocrystals
US6426513B1 (en) 1998-09-18 2002-07-30 Massachusetts Institute Of Technology Water-soluble thiol-capped nanocrystals
JP4630459B2 (en) 1998-09-24 2011-02-09 インディアナ・ユニバーシティ・リサーチ・アンド・テクノロジー・コーポレーション Water-soluble luminescent quantum dots and biomolecular conjugates thereof
US6333110B1 (en) * 1998-11-10 2001-12-25 Bio-Pixels Ltd. Functionalized nanocrystals as visual tissue-specific imaging agents, and methods for fluorescence imaging
US6114038A (en) * 1998-11-10 2000-09-05 Biocrystal Ltd. Functionalized nanocrystals and their use in detection systems
US6716949B2 (en) * 2001-09-20 2004-04-06 Hewlett-Packard Development Company, L.P. Amphipathic polymer particles and methods of manufacturing the same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040101621A1 (en) * 2000-10-13 2004-05-27 Adams Edward William Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US20080241375A1 (en) * 2000-10-13 2008-10-02 Invitrogen Corporation Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media
US20100068380A1 (en) * 2000-10-13 2010-03-18 Life Technologies Corporation Method for preparing surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4144378A1 (en) 2011-12-16 2023-03-08 ModernaTX, Inc. Modified nucleoside, nucleotide, and nucleic acid compositions
WO2013151666A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics Modified polynucleotides for the production of biologics and proteins associated with human disease
WO2013151736A2 (en) 2012-04-02 2013-10-10 modeRNA Therapeutics In vivo production of proteins
WO2014152211A1 (en) 2013-03-14 2014-09-25 Moderna Therapeutics, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
WO2015034928A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Chimeric polynucleotides
WO2015034925A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Circular polynucleotides
WO2015051214A1 (en) 2013-10-03 2015-04-09 Moderna Therapeutics, Inc. Polynucleotides encoding low density lipoprotein receptor
EP4159741A1 (en) 2014-07-16 2023-04-05 ModernaTX, Inc. Method for producing a chimeric polynucleotide encoding a polypeptide having a triazole-containing internucleotide linkage
WO2016014846A1 (en) 2014-07-23 2016-01-28 Moderna Therapeutics, Inc. Modified polynucleotides for the production of intrabodies
CN104181141A (en) * 2014-08-30 2014-12-03 西安电子科技大学 Genetic algorithm based method for analyzing data of luminescent material combination sample library
CN104749147B (en) * 2015-03-09 2017-07-04 西安电子科技大学 A kind of method for optimizing analysis luminescent material performance and component
EP4039699A1 (en) 2015-12-23 2022-08-10 ModernaTX, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017112943A1 (en) 2015-12-23 2017-06-29 Modernatx, Inc. Methods of using ox40 ligand encoding polynucleotides
WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
WO2018213731A1 (en) 2017-05-18 2018-11-22 Modernatx, Inc. Polynucleotides encoding tethered interleukin-12 (il12) polypeptides and uses thereof
WO2018226861A1 (en) 2017-06-07 2018-12-13 Regeneron Pharmaceuticals, Inc. Compositions and methods for internalizing enzymes
US11208458B2 (en) 2017-06-07 2021-12-28 Regeneron Pharmaceuticals, Inc. Compositions and methods for internalizing enzymes
WO2019157224A1 (en) 2018-02-07 2019-08-15 Regeneron Pharmaceuticals, Inc. Methods and compositions for therapeutic protein delivery
WO2020161342A1 (en) 2019-02-08 2020-08-13 Curevac Ag Coding rna administered into the suprachoroidal space in the treatment of ophtalmic diseases
WO2022023559A1 (en) 2020-07-31 2022-02-03 Curevac Ag Nucleic acid encoded antibody mixtures
WO2022059736A1 (en) 2020-09-17 2022-03-24 株式会社レストアビジョン Composition for treating or preventing diseases, disorders, or conditions associated with endoplasmic reticulum stress or all-trans-retinal, protecting retinal thickness, or suppressing reduction in retinal thickness or progress of reduction in retinal thickness
WO2023133579A1 (en) 2022-01-10 2023-07-13 Regeneron Pharmaceuticals, Inc. Bbb-targeted gaa delivered as gene therapy treats cns and muscle in pompe disease model mice
WO2023220603A1 (en) 2022-05-09 2023-11-16 Regeneron Pharmaceuticals, Inc. Vectors and methods for in vivo antibody production
WO2024026474A1 (en) 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Compositions and methods for transferrin receptor (tfr)-mediated delivery to the brain and muscle

Also Published As

Publication number Publication date
US20080241375A1 (en) 2008-10-02
EP1326704B1 (en) 2015-11-18
EP2233202A3 (en) 2012-07-04
AU2002243207A1 (en) 2002-07-24
JP2011073136A (en) 2011-04-14
JP4638128B2 (en) 2011-02-23
US20100068380A1 (en) 2010-03-18
US6649138B2 (en) 2003-11-18
WO2002055186A2 (en) 2002-07-18
US7108915B2 (en) 2006-09-19
US20050003187A1 (en) 2005-01-06
EP2233202A2 (en) 2010-09-29
US20020045045A1 (en) 2002-04-18
US20040101621A1 (en) 2004-05-27
US8691384B2 (en) 2014-04-08
US7147917B2 (en) 2006-12-12
WO2002055186A3 (en) 2003-03-20
JP2004517712A (en) 2004-06-17
CA2424082A1 (en) 2002-07-18
CA2424082C (en) 2007-04-03
US20040101465A1 (en) 2004-05-27
US8158194B2 (en) 2012-04-17
EP1326704A2 (en) 2003-07-16

Similar Documents

Publication Publication Date Title
US8691384B2 (en) Metallic nanoparticles having enhanced dispersibility in aqueous media comprising a polymer having alkyl acrylamide side chains
Susumu et al. Purple-, blue-, and green-emitting multishell alloyed quantum dots: synthesis, characterization, and application for ratiometric extracellular pH sensing
Karakoti et al. Surface functionalization of quantum dots for biological applications
Costa-Fernández et al. The use of luminescent quantum dots for optical sensing
CN109233810B (en) Chromophoric polymer dots
US7368086B2 (en) Functionalized fluorescent nanocrystals, and methods for their preparation and use
Li et al. Fluorescence properties of gold nanorods and their application for DNA biosensing
US20060014315A1 (en) Stable, water-soluble quantum dot, method of preparation and conjugates thereof
EP1490691B1 (en) Luminescent, spheroid, non-autofluorescent silica gel particles having variable emission intensities and frequencies
US20090098663A1 (en) Novel water-soluble nanocrystals comprising a polymeric coating reagent, and methods of preparing the same
JP2002536285A (en) Emission spectral characteristics of CdS nanoparticles
Murcia et al. Fluorescence correlation spectroscopy of CdSe/ZnS quantum dot optical bioimaging probes with ultra-thin biocompatible coatings
Wang et al. Enhancing the photoluminescence of polymer-stabilized CdSe/CdS/ZnS core/shell/shell and CdSe/ZnS core/shell quantum dots in water through a chemical-activation approach
Zhang et al. A multifunctional polypeptide via ugi reaction for compact and biocompatible quantum dots with efficient bioconjugation
Giri et al. Preparation of water soluble L-arginine capped CdSe/ZnS QDs and their interaction with synthetic DNA: Picosecond-resolved FRET study
Shi et al. Preparation of CdS nanocrystals within supramolecular self-assembled nanoreactors and their phase transfer behavior
Hou et al. Temperature-modulated photoluminescence of quantum dots
KR20110122320A (en) Fret-based nano-sensor system and detection method using the same
Wang et al. A robust ligand exchange approach for preparing hydrophilic, biocompatible photoluminescent quantum dots
Brkić Biocompatibility of cadmium selenide quantum dots
Ghosh et al. Surface charge tunability and size dependent luminescence anisotropy of aqueous synthesized ZnS/dendrimer nanocomposites
Sperling Surface modification and functionalization of colloidal nanoparticles
Algar et al. Quantum dots for the development of optical biosensors based on fluorescence
Brijitta et al. A confocal laser scanning microscopic study on thermoresponsive binary microgel dispersions incorporated with CdTe quantum dots
Aguila et al. Polymer–Quantum Dot Hybrid Materials

Legal Events

Date Code Title Description
STCF Information on status: patent grant

Free format text: PATENTED CASE

MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 4TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1551)

Year of fee payment: 4

FEPP Fee payment procedure

Free format text: MAINTENANCE FEE REMINDER MAILED (ORIGINAL EVENT CODE: REM.); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

LAPS Lapse for failure to pay maintenance fees

Free format text: PATENT EXPIRED FOR FAILURE TO PAY MAINTENANCE FEES (ORIGINAL EVENT CODE: EXP.); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

STCH Information on status: patent discontinuation

Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362

FP Lapsed due to failure to pay maintenance fee

Effective date: 20220408