WO2004074504A2 - Fluorescent silica-based nanoparticles - Google Patents

Fluorescent silica-based nanoparticles Download PDF

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WO2004074504A2
WO2004074504A2 PCT/US2003/037963 US0337963W WO2004074504A2 WO 2004074504 A2 WO2004074504 A2 WO 2004074504A2 US 0337963 W US0337963 W US 0337963W WO 2004074504 A2 WO2004074504 A2 WO 2004074504A2
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core
nanoparticle
fluorescent
silica
shell
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French (fr)
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WO2004074504A3 (en
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Ulrich Wiesner
Hooisweng Ow
Daniel R. Larson
Watt W. Webb
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Cornell Research Foundation Inc
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Cornell Research Foundation Inc
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Priority to AU2003303290A priority Critical patent/AU2003303290A1/en
Priority to ES03815995T priority patent/ES2709398T3/es
Priority to EP03815995.0A priority patent/EP1572445B1/en
Priority to JP2004568576A priority patent/JP2006514708A/ja
Priority to US10/536,569 priority patent/US8298677B2/en
Publication of WO2004074504A2 publication Critical patent/WO2004074504A2/en
Publication of WO2004074504A3 publication Critical patent/WO2004074504A3/en
Anticipated expiration legal-status Critical
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
    • B82B1/00—Nanostructures formed by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
    • B82B3/00—Manufacture or treatment of nanostructures by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00—Silicon; Compounds thereof
    • C01B33/113—Silicon oxides; Hydrates thereof
    • C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
    • C01B33/18—Preparation of finely divided silica neither in sol nor in gel form; After-treatment thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2002/00—Crystal-structural characteristics
    • C01P2002/80—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/01—Particle morphology depicted by an image
    • C01P2004/03—Particle morphology depicted by an image obtained by SEM
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/60—Particles characterised by their size
    • C01P2004/64—Nanometer sized, i.e. from 1-100 nanometer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • Y—GENERAL 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/24—Structurally defined web or sheet [e.g., overall dimension, etc.]
    • Y10T428/24355—Continuous and nonuniform or irregular surface on layer or component [e.g., roofing, etc.]
    • Y10T428/24372—Particulate matter
    • Y10T428/2438—Coated
    • Y—GENERAL 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/31504—Composite [nonstructural laminate]
    • Y10T428/31652—Of asbestos
    • Y10T428/31663—As siloxane, silicone or silane

Definitions

  • the invention generally relates to fluorescent nanoparticles and more specifically to silica-based fluorescent nanoparticles of less than 30 nm with co alently attached organic dyes.
  • Fluorescent nanoparticles have tremendous promise as indicators and photon sources of biotechnological and information applications such as biological imaging, sensor technology, microarrays, and optical computing. These applications require size-controlled, monodisperse, bright nanoparticles that can be specifically conjugated to biological macromolecules or arranged in higher-order structures.
  • the nanoparticulate material itself is fluorescent (such as semiconductor nanocrystals or metal nanocrystals).
  • the fluorescent nanoparticles are based on the incorporation of organic dye molecules. Given the vast diversity of organic dye molecules and the extraordinar sensitivity of these dye molecules to their local environment, the latter approach raises the possibility of developing nanoparticles with a broad range of precisely controlled fluorescence characteristics.
  • silica nanoparticles with an embedded dye have been synthesized in a range of sizes, colors, and architectures.
  • the dye which is covalently bound inside the silica particle is observed to be quenched in comparison to the free dye.
  • poly(organosiloxane) micro gels in which the dye is non-covalently attached and loaded through diffusion, a slight increase in fluorescence efficiency is observed.
  • the quantum efficiency of the embedded dye is the same or less than the free dye.
  • the quenching of fluorescence is usually attributed to either intraparticle energy transfer or non-radiative decay into the silica matrix. Both of these pathways are likely influenced by the local dye environment within the particle, suggesting that precise control of the architecture within the particle might ameliorate quenching or even lead to fluorescence enhancement.
  • the invention relates to a class of silica nanoparticles in which the architecture within the silica particle has marked, controlled effects on the radiative properties of the constituent dye.
  • the invention provides a fluorescent monodisperse silica nanoparticle comprising fluorophore center core and a silica shell wherein the radiative properties of the nanoparticle are dependent upon the chemistry (composition) of the core and presence of the silica shell.
  • the core- shell architecture provides an enhancement in fluorescence quantum efficiency.
  • the invention generally provides control of photophysical properties of dye molecules encapsulated within silica particles with sizes down to 30 nm and below.
  • the core-shell architecture provides the ability to control the photostability properties of the nanoparticle.
  • the quantum efficiency increase provided by the invention is due to both an increase in the radiative rate and a decrease in the non-radiative rate, with the latter effect being the most variable between architectures.
  • the changes in non- radiative rate correlate well to differences in rotational mobility of the dye within the particle.
  • the fluorescent monodisperse nanoparticle includes a core-shell architecture wherein the core comprises a compact core surrounded by a silica shell.
  • the fluorescent monodisperse nanoparticle includes a core-shell architecture wherein the core comprises an expanded core surrounded by a silica shell.
  • the fluorescent monodisperse nanoparticle includes a core-shell architecture wherein the core comprises a homogenous particle with dyes sparsely embedded within surrounded by a silica shell.
  • the fluorescent monodisperse homogenous nanoparticle is not surrounded by a silica shell.
  • the invention provides a method of making a fluorescent monodisperse nanoparticle with a compact core architecture by mixing a fluorescent compound and an organo-silane compound to form a dye precursor, mixing the resulting dye precursor with an aqueous solution to form a compact fluorescent core, and mixing the resulting compact core with a silica precursor to form a silica shell on the compact core, to provide the fluorescent monodisperse nanoparticle.
  • the invention also provides a method of making a fluorescent monodisperse nanoparticle with an expanded core architecture by mixing a fluorescent compound and an organo-silane compound to form a dye precursor, co-condensing the resulting dye precursor with a silica precursor to form an expanded fluorescent core, and mixing the resulting expanded core with a silica precursor to form a silica shell on the expanded core, to provide the fluorescent monodisperse nanoparticle.
  • the invention provides a method of making a fluorescent monodisperse homogenous nanoparticle by mixing a fluorescent compound and an organo- silane compound to form a dye precursor and co-condensing the resulting dye precursor with a silica precursor to fo ⁇ n a homogenous fluorescent monodisperse nanoparticle.
  • FIG. 1A-C illustrates a schematic of different silica nanoparticle architectures designated as compact core-shell nanoparticle (1A), expanded core- shell nanoparticle (IB), and the homogenous nanoparticle (1C).
  • the silica shell in the compact and expanded core-shell architecture comprises silica without any dye molecules.
  • the homogenous nanoparticle comprises a composite of silica and dye in a matrix.
  • FIG. 2A-C illustrates fluorescence correlation spectroscopy of nanoparticles and synthetic intermediates.
  • FIG. 2D-F illustrates the brightness (counts/particles/second) values for the synthesis stages of the compact core-shell architecture, expanded core-shell architecture and the homogenous architecture based on excitation value of 1.2 mW at 900 nm.
  • FIG. 3 illustrates the steady-state spectroscopy curves showing the quantum efficiency enhancement achieved by the core-shell architecture.
  • Figures 3A-C illustrate the absorbance and fluorescence of the compact core- shell nanoparticle (3 A), expanded core-shell nanoparticle (3B) and homogenous nanoparticle (3C).
  • the absorbance values are in units of optical density.
  • the fluorescence is scaled relative to the organic dye used, TRITC.
  • Figures 3D-F illustrate the comparison between absorbance and excitation for the compact core-shell nanoparticle (3D), expanded core-shell nanoparticle (3E), and homogenous particle (3F).
  • FIG. 4 illustrates the time-resolved fluorescence of nanoparticles.
  • Figure 4A illustrates the normalized fluorescence decay of a homogenous nanoparticle, expanded core-shell nanoparticle, compact core-shell nanoparticle and the TRITC dye.
  • Figure 4B illustrates the fluorescence anisotropy of an expanded core-shell nanoparticle, a compact core-shell nanoparticle, a homogenous nanoparticle, and the TRITC dye.
  • Figure 4C illustrates the normalized fluorescence anisotropy of the nanoparticle curves shown in Figure 4B. Average fit values for the fluorescence lifetime (T/) and rotational lifetime ( ⁇ ) are compiled in Table III.
  • the fluorescent nanoparticles of the present invention include a core comprising a fluorescent silane compound and a silica shell on the core.
  • the core of the nanoparticle can comprise, for example, the reaction product of a reactive fluorescent compound and a co-reactive organo-silane compound
  • the shell can comprise, for example, the reaction product of a silica forming compound.
  • the silica forming compound can produce, for example, one or more layers of silica, such as from 1 to about 20 layers, and various desirable shell characteristics, such as shell layer thickness, the ratio of the shell thickness to the core thickness or diameter, silica shell surface coverage of the core, porosity and carrying capacity of the silica shell, and like considerations.
  • the synthesis of the fluorescent monodisperse core-shell nanoparticles is based on a two-step process.
  • the organic dye molecules tetramethylrhodamine isothiocynate (TRITC)
  • TRITC tetramethylrhodamine isothiocynate
  • the silica gel monomers are added to form a denser silica network around the fluorescent core material, providing shielding from solvent interactions that can be detrimental to photostability.
  • the dye precursor was added to a reaction vessel that contains appropriate amounts of ammonia, water and solvent and allowed to react overnight.
  • the dye precursor was synthesized by addition reaction between TRITC and 3- aminopropyltriethoxysilane in molar ratio of 1:50, in exclusion of moisture.
  • tetraethylorthosilicate (TEOS) was subsequently added to grow the silica shell that surrounded the core.
  • the synthesis of the expanded core-shell nanoparticle was accomplished by co-condensing TEOS with the aforementioned dye precursor and allow the mixture to react overnight. After the synthesis of the expanded core was completed, additional TEOS was added to grow the silica shell that surrounded the core.
  • the synthesis of the homogenous nanoparticles was accomplished by co- condensing all the reagents, the dye precursor and TEOS, at the same time and allow the mixture to react overnight.
  • the FCS curves and monoexponential fits shown in FIG. 2A-C illustrate the monodispersity requirement for each architecture in an aqueous solution.
  • the FCS curve further provides two independent parameters: the diffusion coefficient and the absolute concentration. The diffusion coefficient is directly related to the hydrodynamic radius, and the absolute concentration allows quantification of the particle brightness.
  • FCS curves for each nanoparticle and its respective intermediate are shown in FIG. 2A-C.
  • FIG. 2B A similar relationship between the free TRITC dye, expanded core-shell architecture and complete nanoparticle is shown in FIG. 2B.
  • FIG. 2D-E illustrates the brightness of each architecture and synthesis intermediates.
  • the core is always dimmer than the free dye, despite the fact that multiple dyes are presumably present in the core.
  • the brightness of the complete nanoparticle is always significantly greater than free dye and/or core intermediate.
  • the brightness varies for the different architectures despite their same particle size and same absolute amounts of precursor materials.
  • the fluorescent nanoparticles of the invention include an organic dye, TRITC, which demonstrates quantum efficiency enhancement effects in its luminescent properties.
  • TRITC organic dye
  • the number of TRITC equivalents per particle can be determined through a combination of FCS and absorbance measurements, in which the absolute concentration is determined from FCS and the relative absorbance is measured with respect to TRITC in solution, as shown in FIG. 3A-C. Dilute solutions were used (about 50nM) in FIG. 3A-C so that a sample could be measured by both FCS and absorbance.
  • the number of TRITC equivalents per particle calculated using an extinction coefficient of 42,105 M “1 cm “1 at 514.5 nm are shown in Table II.
  • the relative intensities of the nanoparticle fluorescence and nanoparticle absorbance give the quantum efficiency enhancement over free TRITC, as shown in Table II.
  • the number of TRITC equivalents per particle is indistinguishable at 8.6.
  • the expanded core-shell nanoparticle shows a three-fold quantum efficiency enhancement compared to the free TRITC dye, while the compact core-shell exhibits only a two-fold quantum efficiency enhancement compared to the free TRITC dye, as shown in FIG. 3 A, B and Table II.
  • the largest quantum efficiency enhancement was observed with the homogenous nanoparticle, which has on average 2.3 TRITC equivalents per particle, as shown in FIG. 3C and Table II.
  • the fluorescence lifetime of the particles is shown in FIG. 4 A.
  • Each lifetime, including free TRITC, is multiexponential and the average lifetime values are compiled in Table III.
  • the lifetime increases from the compact core-shell (1.8 ns) to the expanded core-shell (2.9 ns) to the homogeneous particle (3.2 ns) (FIG. 4A).
  • the compact-core shell has a lifetime which is less than free dye, although the dominant contribution to this lifetime is a fast component (FIG 4A). From this lifetime data alone, one might conclude that the dye is quenched inside the compact core-shell particle, but the steady state measurements suggest otherwise.
  • the combination of fluorescence lifetime and quantum efficiency allow for a unique determination of the radiative and non-radiative rate constants, tabulated in both normalized and absolute form in Table III.
  • the enhancement of the radiative rate is constant across the different architectures and is 2.2 fold greater than free TRITC.
  • the non- radiative rate varies across architectures from a relatively high value for the compact core-shell nanoparticles to a value almost 3 fold less for the homogeneous particles.
  • FIG. 4C The scaled anisotropy for each architecture is shown in FIG. 4C.
  • the compact core-shell nanoparticle has the fastest, least hindered rotation (FIG. 4C), followed by the expanded core- shell particle (FIG. 4C), and finally the homogeneous nanoparticle (FIG. 4C).
  • This rotation time has a monotonic, inverse dependence on the non-radiative rate constant (Table III), suggesting that the confined mobility in the silica nanoparticle is responsible for suppression of non-radiative decay.
  • Amounts of water, ammonia and solvent were measured in graduated cylinders.
  • Fluorescent seed particle synthesis was carried out in 1 L Erlenmeyer flasks and stirred with magnetic TEFLON ® coated stir bars at about 600 rpm. De-ionized water and ammonia solution were added to ethanol and stirred. About 2 mL of the reactive dye precursor in either ethanol or THF containing about 425 micromolar APTS, was added to the reaction vessel. Depending on the desired architecture, the resulting mixture was stirred from 1 to 12 hours at room temperature with the reaction vessel covered with aluminum foil to minimize exposure to light to afford a fluorescent seed particle mixture.
  • Tetrahydrofuran Tetrahydrofuran
  • the silica shell coating and growth step was performed in the above mentioned fluorescent seed particle reaction mixture with regular addition of solvent, such as ethanol, methanol, or isopropanol, to prevent drastic changes in solution ionic strength while the silica forming monomer tetraethoxysilane (TEOS) was added.
  • solvent such as ethanol, methanol, or isopropanol
  • the particle size and particle size distribution of the resulting fluorescent nanoparticles were characterized by electron microscopy (SEM) and fluorescence correlation spectroscopy (FCS).
  • Fluorescence Correlation Spectroscopy All FCS measurements were done on a custom FCS microscope based on two-photon excitation.
  • the instrument consists of a Ti:sapphire oscillator with ⁇ 100 fs pulsewidth and 80 MHz rep. rate pumped by an Ar+ ion laser (Spectra Physics, Palo Alto, CA).
  • the beam was positioned with a Bio-Rad MRC 600 confocal scan box (Hercules, CA) coupled to a Zeiss Axiovert 35 inverted microscope.
  • the fluorescence was separated from the excitation with a 670 DCLP dichroic and passed through a HQ575/150 emission filter to a GaAsP photon-counting PMT.
  • the resulting photocurrent was digitally autocorrelated with an ALV 6010 multiple tau autocorrelator.
  • the autocorrelation function G( ⁇ ) is defined as:
  • the fitting function is the standard function for one-component, three- dimensional diffusion:
  • Fluorescence is collected at right angle to the excitation through an HQ605/90 emission filter and recorded with a bialkali PMT (HC125-02, Hamamatsu).
  • Example 1 Hydrodynamic radii of nanoparticles and nanoparticle cores.
  • the amplitude of the autocorrelation provides the number of the diffusing species and the average count rate is a measure of the photons collected from the optically defined focal volume. From this data, the count rate per molecule for each diffusing species can be obtained, which is a direct measure of the brightness of a probe.
  • the overall enhancement of brightness of the particle over free dye is the product of the number of TRITC equivalents inside the nanoparticle and the relative quantum efficiency enhancement of the dye, as shown in Table II, brightness factor.
  • Table II brightness factor
  • the brightness factor is similar to the independent brightness determination from FCS (Table II, comparing last two columns), and the trend for brightness over the different architectures is the same by both methods.
  • the brightness factor consistently overestimates the values measured by FCS, possibly due to the error in the determination of dye equivalents.
  • the enhancement of the quantum efficiency can be due, in general, to an increase in the radiative rate (k r ), a decrease in the non-radiative rate (k nr ), or both.
  • the relative contributions of these factors are related to the fluorescence lifetime f ⁇ f) and the quantum efficiency ( ⁇ ) by:

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PCT/US2003/037963 2002-11-26 2003-11-26 Fluorescent silica-based nanoparticles Ceased WO2004074504A2 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU2003303290A AU2003303290A1 (en) 2002-11-26 2003-11-26 Fluorescent silica-based nanoparticles
ES03815995T ES2709398T3 (es) 2002-11-26 2003-11-26 Nanopartículas fluorescentes a base de sílice
EP03815995.0A EP1572445B1 (en) 2002-11-26 2003-11-26 Fluorescent silica-based nanoparticles
JP2004568576A JP2006514708A (ja) 2002-11-26 2003-11-26 シリカ系蛍光ナノ粒子
US10/536,569 US8298677B2 (en) 2002-11-26 2003-11-26 Fluorescent silica-based nanoparticles

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US10/306,614 2002-11-26
US10/306,614 US20040101822A1 (en) 2002-11-26 2002-11-26 Fluorescent silica-based nanoparticles

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WO2004074504A3 WO2004074504A3 (en) 2004-12-02

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ES2709398T3 (es) 2019-04-16
CN100443295C (zh) 2008-12-17
EP1604031B1 (en) 2017-05-03
ES2753144T3 (es) 2020-04-07
JP2010070768A (ja) 2010-04-02
JP2006514708A (ja) 2006-05-11
EP2369342A3 (en) 2011-10-19
WO2004074504A3 (en) 2004-12-02
US8298677B2 (en) 2012-10-30
US8409876B2 (en) 2013-04-02
EP1572445B1 (en) 2019-01-09
EP2369342B1 (en) 2019-08-28
EP1604031A4 (en) 2008-08-20
US20100035365A1 (en) 2010-02-11
AU2003303309A1 (en) 2004-08-10
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US20060183246A1 (en) 2006-08-17
EP2369342A2 (en) 2011-09-28
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EP1604031A2 (en) 2005-12-14
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EP1572445A4 (en) 2008-10-22
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