Monofunctional 3 to 10 nm Covalent Gold Labels for CryoEM

用于 CryoEM 的单功能 3 至 10 nm 共价金标签

基本信息

  • 批准号:
    8781987
  • 负责人:
  • 金额:
    $ 15.97万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2014
  • 资助国家:
    美国
  • 起止时间:
    2014-09-15 至 2016-03-14
  • 项目状态:
    已结题

项目摘要

DESCRIPTION (provided by applicant): Gold nanoparticles are the label of choice for electron microscopy (EM) since they offer a choice of sizes that provide quantitative, high-resolution identification of targets. Recently, high-resolution EM methods including cryoEM and electron tomography have benefitted from significant advances in instrumentation and a series of breakthroughs in specimen processing and preparative equipment that have made these methods widely accessible. However, conventional colloidal gold labels do not allow the full realization of the potential in these methods, because they require extensive stabilization with large macromolecules that limit penetration and antigen access, and also because their mechanism of conjugation, through adsorption to targeting antibodies or proteins, does not allow direct labeling of specific reactive sites within macromolecular complexes at sufficiently high resolution. Nanoprobes has developed 1.4 nm (Nanogold) and 0.8 nm (Undecagold) gold nanoparticle labels with a single reactive group for selective, covalent labeling of thiols (cysteie residues), amines (N-terminal amines or lysine residues), or other functional groups. However, these are too small to be readily visualized in many types of specimens, and do not allow multiple labeling. Although covalent reactivity has been introduced into larger particles, preparing monofunctional reagents capable of 1: 1 labeling has proved highly challenging. To date, no products are available with native monofunctional reactivity. We will synthesize gold nanoparticles that combine a high degree of monodispersity, controlled surface functionalization and selective reactivity with monofunctional reaction stoichiometry. Preparations will be scaled and simplified to deliver nanoparticles with the following properties in an efficient, reproducible and economical manner on any scale: (1) Monodispersity: populations of gold nanoparticles from 3 nm to 10 nm will be prepared with coefficients of variation of 10% or less using thiocyanate reduction, citrate/tannic acid, or homogeneous reduction. (2) Surface properties: A universal base set of organic ligands will be developed that can confer (a) water-solubility and biocompatibility, (b) desired degree of hydrophobicity, (c) specific covalent reactivity, and (d) controlled shell thickness from 0.6 nm to 3 nm. Enable selection of any combination of these properties for any size. (3) Specific reactivity: amine, thiol, and activated carboxyl reactivity ill be introduced by (a) incorporation of a primary amine during synthesis and later modification, and (b) insertion of protected reactivity then deprotection. (4) Monofunctionality: four strategie will be used to generate particles that react with a single conjugate molecule in solution: (a) isolation from statistical mixture; (b) immobilization using a cleavable cross-linker that generate a unique group upon cleavage; or (c) conjugation of one or very few larger protein tags using cleavable cross- linkers, separation of gold particles by numbers of tags and cleavage to yield precisely functionalized gold particles; and (d) polymeric ligands bridging multifunctional gold coordination with a single cross-linking group. Monofunctionality will be tested in collaboration with Alasdair Steven (NIAMS, NIH) using cryoelectron microscopic labeling and electron tomographic experiments of multi-functional proteins in which site-specific labeling without aggregation will be used to confirm truly monofunctional reactivity. Monofunctional gold nanoparticles would also provide innovation to address a critical challenge in nanoscience: enabling the construction of molecular nanodevices in which multiple nanoparticles with unique and different properties and attachment points perform a coordinated set of actions to complete a task. Construction of such a system requires the assembly of particles with different properties into a construct by attachment at different sites. As a key enabling technology for this, monofunctional gold nanoparticles will help afford a new generation of nanotechnology innovations such as biosensors, indicators, and molecular devices that perform targeted, programmed tasks such as oligonucleotide annealing, temperature sensing, or selectively delivering medication or enhanced local radiation dose to tumor cells.
DESCRIPTION (provided by applicant): Gold nanoparticles are the label of choice for electron microscopy (EM) since they offer a choice of sizes that provide quantitative, high-resolution identification of targets. Recently, high-resolution EM methods including cryoEM and electron tomography have benefitted from significant advances in instrumentation and a series of breakthroughs in specimen processing and preparative equipment that have made these methods widely accessible. However, conventional colloidal gold labels do not allow the full realization of the potential in these methods, because they require extensive stabilization with large macromolecules that limit penetration and antigen access, and also because their mechanism of conjugation, through adsorption to targeting antibodies or proteins, does not allow direct labeling of specific reactive sites within macromolecular complexes at sufficiently high resolution. Nanoprobes has developed 1.4 nm (Nanogold) and 0.8 nm (Undecagold) gold nanoparticle labels with a single reactive group for selective, covalent labeling of thiols (cysteie residues), amines (N-terminal amines or lysine residues), or other functional groups. However, these are too small to be readily visualized in many types of specimens, and do not allow multiple labeling. Although covalent reactivity has been introduced into larger particles, preparing monofunctional reagents capable of 1: 1 labeling has proved highly challenging. To date, no products are available with native monofunctional reactivity. We will synthesize gold nanoparticles that combine a high degree of monodispersity, controlled surface functionalization and selective reactivity with monofunctional reaction stoichiometry. Preparations will be scaled and simplified to deliver nanoparticles with the following properties in an efficient, reproducible and economical manner on any scale: (1) Monodispersity: populations of gold nanoparticles from 3 nm to 10 nm will be prepared with coefficients of variation of 10% or less using thiocyanate reduction, citrate/tannic acid, or homogeneous reduction. (2) Surface properties: A universal base set of organic ligands will be developed that can confer (a) water-solubility and biocompatibility, (b) desired degree of hydrophobicity, (c) specific covalent reactivity, and (d) controlled shell thickness from 0.6 nm to 3 nm. Enable selection of any combination of these properties for any size. (3) Specific reactivity: amine, thiol, and activated carboxyl reactivity ill be introduced by (a) incorporation of a primary amine during synthesis and later modification, and (b) insertion of protected reactivity then deprotection. (4) Monofunctionality: four strategie will be used to generate particles that react with a single conjugate molecule in solution: (a) isolation from statistical mixture; (b) immobilization using a cleavable cross-linker that generate a unique group upon cleavage; or (c) conjugation of one or very few larger protein tags using cleavable cross- linkers, separation of gold particles by numbers of tags and cleavage to yield precisely functionalized gold particles; and (d) polymeric ligands bridging multifunctional gold coordination with a single cross-linking group. Monofunctionality will be tested in collaboration with Alasdair Steven (NIAMS, NIH) using cryoelectron microscopic labeling and electron tomographic experiments of multi-functional proteins in which site-specific labeling without aggregation will be used to confirm truly monofunctional reactivity. Monofunctional gold nanoparticles would also provide innovation to address a critical challenge in nanoscience: enabling the construction of molecular nanodevices in which multiple nanoparticles with unique and different properties and attachment points perform a coordinated set of actions to complete a task. Construction of such a system requires the assembly of particles with different properties into a construct by attachment at different sites. As a key enabling technology for this, monofunctional gold nanoparticles will help afford a new generation of nanotechnology innovations such as biosensors, indicators, and molecular devices that perform targeted, programmed tasks such as oligonucleotide annealing, temperature sensing, or selectively delivering medication or enhanced local radiation dose to tumor cells.

项目成果

期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)

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RICHARD DENIS POWELL其他文献

RICHARD DENIS POWELL的其他文献

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{{ truncateString('RICHARD DENIS POWELL', 18)}}的其他基金

Smaller, Brighter Probes for Correlative Super-resolution and Electron Microscopy
用于相关超分辨率和电子显微镜的更小、更亮的探头
  • 批准号:
    9049232
  • 财政年份:
    2016
  • 资助金额:
    $ 15.97万
  • 项目类别:
Conductive Metallography for Serial Section Electron Microscopy at Nanometer Resolution
纳米分辨率连续切片电子显微镜的导电金相学
  • 批准号:
    8834483
  • 财政年份:
    2015
  • 资助金额:
    $ 15.97万
  • 项目类别:
Serial Blockface SEM Labels for Assessing Nervous System Plasticity
用于评估神经系统可塑性的串行 Blockface SEM 标签
  • 批准号:
    7746768
  • 财政年份:
    2009
  • 资助金额:
    $ 15.97万
  • 项目类别:
Polymeric Enzyme-Gold Probes for Ultrasensitive Protein Blotting
用于超灵敏蛋白质印迹的聚合酶-金探针
  • 批准号:
    7481912
  • 财政年份:
    2008
  • 资助金额:
    $ 15.97万
  • 项目类别:
Correlative Enzymatic and Gold Probes
相关酶和金探针
  • 批准号:
    6993472
  • 财政年份:
    2005
  • 资助金额:
    $ 15.97万
  • 项目类别:
Correlative Chromogenic Gene and Protein assessment
相关显色基因和蛋白质评估
  • 批准号:
    6834406
  • 财政年份:
    2004
  • 资助金额:
    $ 15.97万
  • 项目类别:
Reiterative Signal Amplification by Gold Deposition
通过金沉积重复信号放大
  • 批准号:
    6647381
  • 财政年份:
    2003
  • 资助金额:
    $ 15.97万
  • 项目类别:
Live Cell Correlative Imaging Probes
活细胞相关成像探针
  • 批准号:
    6549833
  • 财政年份:
    2002
  • 资助金额:
    $ 15.97万
  • 项目类别:
Enzymatic Metallography for Ultrasensitive Biodetection
用于超灵敏生物检测的酶金相学
  • 批准号:
    6883438
  • 财政年份:
    2001
  • 资助金额:
    $ 15.97万
  • 项目类别:
Enzymatic Metallography for Biological Detection
用于生物检测的酶金相学
  • 批准号:
    6403845
  • 财政年份:
    2001
  • 资助金额:
    $ 15.97万
  • 项目类别:

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