Application of multifunctional graphene grid in three-dimensional reconstruction of cryoelectron microscope

A three-dimensional reconstruction and multi-functional technology, applied in the direction of material analysis, measuring devices, instruments, etc. using wave/particle radiation, can solve the problem that the molecular direction of target particles cannot be specifically adjusted

Active Publication Date: 2022-01-21
TSINGHUA UNIV
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  • Application Information

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Problems solved by technology

However, these affinity grids cannot specifically adjust the orientation of target particle molecules and may lead to more serious problems of preferred orientations

Method used

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  • Application of multifunctional graphene grid in three-dimensional reconstruction of cryoelectron microscope
  • Application of multifunctional graphene grid in three-dimensional reconstruction of cryoelectron microscope
  • Application of multifunctional graphene grid in three-dimensional reconstruction of cryoelectron microscope

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Experimental program
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Effect test

Embodiment 1

[0053] Embodiment 1, preparation and structural characterization of MFG carrier network

[0054] 1) Preparation of graphene grid

[0055] First, the CVD method is used to grow graphene film on copper foil, and then the copper foil is directly and selectively etched into the micro-grid of the electron microscope grid to obtain an ultra-clean and strong graphene grid, which contains an array of holes ( figure 1 (A) and figure 1 (B)).

[0056] During low-temperature sample preparation, the coverage of graphene and the integrity on the grid depend on the pore diameter and the number of graphene layers, and when the pore diameter of bilayer or multilayer graphene is about 30 μm, it can be well maintained Graphene coverage and integrity on the grid. In this embodiment, a grid covered with 1 to 4 layers of graphene is used as a starting material. Suspended graphene is ultra-clean, and when imaged by high-resolution STEM, the carbon atoms on the etched copper holes can be well ide...

Embodiment 2

[0061] Embodiment 2, the biological application performance research of MFG carrying net

[0062] 1. The MFG grid is hydrophilic and resistant to electron beam radiation

[0063] The present invention checks hydrophilicity by water contact angle (WCA) test. The present invention has measured the WCA of graphene carrier network and multifunctional graphene carrier network, finds that after functionalization, this angle reduces to 39.1 ° from 97.4 ° ( figure 2 (A)), suitable for subsequent bioelectron microscopy specimen preparation. The superior properties of graphene, such as ultrahigh thermal and electrical conductivity and strong mechanical strength, are mainly attributed to the perfect two-dimensional hexagonal lattice formed by sp2 hybridized carbon atoms. Raman spectroscopy shows that there is no D peak (1350cm -1 ), indicating that graphene has almost no defects ( figure 2 (B), upper blue curve). After multiple functionalization, a weak D peak appeared, which was ...

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Abstract

The invention discloses an application of a multifunctional graphene grid in three-dimensional reconstruction of a cryoelectron microscope. The preparation method of the multifunctional graphene grid comprises the following steps: etching a copper foil covered with a graphene film grown by CVD (Chemical Vapor Deposition) to obtain the graphene grid; performing oxygen plasma treatment on the graphene grid; then, adopting an MES buffer solution containing EDC and NHS treat the graphene grid; activating the graphene grid by adopting nitrilotriacetic acid and biotin; and finally reacting with nickel salt. The multifunctional graphene grid can specifically anchor a target biomolecule on the surface of the multifunctional graphene grid, so that the interface problem of air and water is avoided, the orientation distribution of particles can be controlled, and by constructing related protein purification tags to different directions of each biomacromolecule, abundant Euler angles are provided, and the structural analysis of biomacromolecules is more reliable and more efficient, so that the multifunctional graphene grid can promote the universality and repeatability of application of a cryoelectron microscope in structural biological analysis.

Description

technical field [0001] The invention relates to the application of a multifunctional graphene carrier network in the three-dimensional reconstruction of cryo-electron microscope, and belongs to the technical field of biological cryo-electron microscope. Background technique [0002] Due to the continuous development of cryo-EM technology-related hardware and software, cryo-electron microscopy (cryo-EM) is now a routine method for solving atomic-resolution structures of biological macromolecules, which greatly expands the understanding of biological mechanisms at the molecular level . However, this method still faces some key technical challenges, especially for frozen sample preparation. In practice, a small amount of a biomolecule-containing solution is added dropwise onto a freshly glow-discharged grid covered by a support membrane (eg, a porous carbon membrane) and then quenched in liquid ethane. Ideally, biomolecules would be expected to be embedded in a directionally ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N23/046G01N23/20008G01N23/207
CPCG01N23/046G01N23/20008G01N23/207
Inventor 王宏伟陈亚楠刘楠胡翠霞
Owner TSINGHUA UNIV
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