Preparation method and application of novel two-dimensional transition metal sulfide (TMDs) material cryoelectron microscope grid

Abstract

The invention discloses a novel MoS2 two-dimensional material cryoelectron microscope grid as well as a preparation method and application of the novel MoS2 two-dimensional material cryoelectron microscope grid. The preparation method of the MoS2 support membrane comprises the following steps: S1, preparing a single-layer MoS2 continuous membrane; s2, transferring the MoS2 continuous film to a carrying net; and S3, carrying out plasma treatment on the continuous film obtained in the step S2. The MoS2 support membrane provided by the invention can be used as or used for preparing a cryoelectron microscope carrier net, is applied to cryoelectron microscope structure analysis and tomography analysis of 20S protease, ribosome and MEF cells, and shows excellent performance in single-particle cryoelectron microscopes and cell cryotomography.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterials and cryo-electron microscopy, and in particular to a cryo-electron microscopy grid for a novel two-dimensional transition metal sulfide (TMD) material, its preparation method, and its application in structural analysis. Background Technology

[0002] Two-dimensional transition metal sulfides (TMDs), such as molybdenum disulfide (MoS2), are typically composed of transition metal atoms (such as molybdenum, tungsten, and chromium) and chalcogen atoms, forming a layered structure that exhibits two-dimensional properties similar to graphene. This unique structure endows TMDs with excellent optoelectronic, electrical, and mechanical properties, making them promising for a wide range of applications in energy, electronic devices, optoelectronics, and nanotechnology.

[0003] MoS2 is a typical TMD material, consisting of a molybdenum atomic layer and two sulfur atomic layers, exhibiting stable hexagonal (2H) and trigonal (3R) crystal phases. Monolayer 2H-MoS2 possesses excellent electrical properties, including conductivity and carrier mobility, making it suitable for fabricating electronic devices such as field-effect transistors. Furthermore, monolayer MoS2 exhibits high flexibility and elastic modulus, making it suitable for catalytic hydrolysis, electrocatalysis, and hydrogenation reactions.

[0004] In recent years, the application of MoS2 in electron microscopy methodologies has gradually attracted attention. For example, liquid-sealed cells can be constructed using monolayer MoS2 films combined with graphene films, enabling dynamic real-time monitoring of local liquid environments under in-situ transmission electron microscopy. However, the application of MoS2 in cryo-electron microscopy grids has not been fully studied. Summary of the Invention

[0005] The purpose of this invention is to provide a novel cryo-electron microscopy grid for MoS2 two-dimensional materials and to provide its application in cryo-electron microscopy structure analysis.

[0006] The method for preparing the MoS2 supported film provided by the present invention includes the following steps: S1. Preparation of a monolayer MoS2 continuous film; S2. Transfer the MoS2 continuous film onto a carrier wire; S3. The continuous membrane obtained in step S2 is subjected to plasma treatment to obtain the final product.

[0007] In step S1, the MoS2 continuous film is prepared by chemical vapor deposition. Preferably, mica is used as a substrate, and a clean surface is peeled off with tape before chemical vapor deposition is carried out in a CVD reaction chamber. The precursor used is K2MoS4.

[0008] The conditions for the chemical vapor deposition method are as follows: Temperature: 500-1200°C; Pressure: atmospheric pressure. The process was carried out in an atmosphere of argon and oxygen: after cleaning the quartz tube with 200 sccm (standard cubic centimeters / minute) of argon for 5 minutes, the furnace was heated to 840 °C over 41 minutes in an argon atmosphere of 100 sccm. To reduce the nucleation density, 0.2 sccm of oxygen was introduced into the system while the temperature was between 600 °C and 840 °C; subsequently, the mica substrate was held at 840 °C for more than 20 minutes to obtain large-area, continuous MoS2 monolayer films (centimeter-scale dimensions).

[0009] The thickness of the MoS2 film can be adjusted by controlling the reaction time and the flow rate of the precursor gas.

[0010] Preferably, in step S2, the MoS2 continuous film is transferred according to the following method: The MoS2 continuous film was placed in water and automatically peeled off from the substrate using van der Waals forces, then floated on the liquid surface. The carrier screen was picked up with tweezers and inserted obliquely below the liquid surface. Under the action of the interfacial tension between the carrier screen and MoS2, the MoS2 film was smoothly transferred to the carrier screen and fixed after drying.

[0011] Preferably, in step S3, the plasma treatment is a low-oxygen plasma treatment to construct S vacancy defects and improve the hydrophilicity and electrostatic adsorption capacity of the membrane surface. The conditions for the low-oxygen plasma treatment are as follows: Under normal temperature conditions, a mixture of argon and oxygen is selected, wherein the volume content of oxygen is 0.001 to 0.1, and the gas flow rate is 10 to 100 sccm.

[0012] The MoS2 support membrane provided by this invention can be used as or used to prepare cryo-electron microscopy grids, and applied to cryo-electron microscopy structural analysis and tomographic analysis of 20S proteases, ribosomes, and MEF cells, demonstrating its excellent performance in single-particle cryo-electron microscopy and cell cryo-tomography. 20S protease structure resolution: The structure of the 20S protease was resolved by single-particle cryo-electron microscopy characterization using a MoS2-supported membrane mesh, which showed excellent protein particle uniformity and a high number of protein particles, ultimately achieving high-resolution structure reconstruction.

[0013] Ribosome structure analysis: In the ribosome structure analysis, the MoS2-supported membrane network exhibited a uniform monodisperse distribution and high-resolution three-dimensional reconstruction capability.

[0014] Cryo-electron tomography analysis of MEF cells: Cryo-electron tomography of cell samples using a monolayer MoS2 support membrane revealed the fine structure of cells and their pseudopodia, and improved cell density and electrostatic adsorption.

[0015] In this invention, the cells in the cell sample can be model cells for studying membrane proteins (e.g., MEF cells), model cells for studying cell proliferation and differentiation (e.g., embryonic stem cells, induced pluripotent stem cells, adult stem cells, etc.), or model cells for studying cell cycle and cell death (e.g., various cancer cell lines). Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the structure of a single-layer MoS2 thin film and its transmission electron microscopy characterization.

[0017] Figure 2 High-resolution scanning transmission electron microscopy characterization of monolayer MoS2 thin films before and after plasma treatment.

[0018] Figure 3 Characterization of the structure and morphology of monolayer MoS2 thin films.

[0019] Figure 4 To determine the structure of the 20S protease using a monolayer MoS2-supported membrane mesh.

[0020] Figure 5 Cryo-electron microscopy images and three-dimensional reconstructed structure density maps of ribosomes supported by a single-layer MoS2 membrane mesh.

[0021] Figure 6 Cryo-electron microscopy images of MEF cell samples supported by a monolayer MoS2 membrane mesh.

[0022] Figure 7 Analysis of cryo-electron tomography data of MEF cells on a MoS2-supported membrane network.

[0023] Figure 8 Fluorescence microscopy analysis of MEF cell samples on MoS2, GO support membranes and blank cell slides.

[0024] Figure 9 High-resolution transmission electron microscopy image of MoS2 in an argon-oxygen mixture atmosphere containing 1.5% oxygen during plasma treatment.

[0025] Figure 10 High-resolution transmission electron microscopy image of MoS2 in an argon-oxygen mixture atmosphere containing 8% oxygen, used for plasma treatment. Detailed Implementation

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] Example 1: Preparation of MoS2 Supported Membrane Network 1. Preparation of monolayer MoS2 continuous film Ultra-smooth mica was selected as the substrate, and a clean surface was created by peeling off with adhesive tape. Chemical vapor deposition (CVD) was performed in the CVD reaction chamber using K₂MoS₄ as the precursor, placed on the clean mica substrate in a quartz boat, which was then positioned at the center of the CVD furnace. Temperature and precursor gas flow rate were controlled: after cleaning the quartz tube with 200 sccm (standard cubic centimeters / min) of argon gas for 5 minutes, the furnace was heated to 840 °C over 41 minutes at 100 sccm of argon gas. To reduce the nucleation density, 0.2 sccm of oxygen was introduced into the system while the temperature remained between 600 °C and 840 °C. The mica substrate was then held at 840 °C for at least 20 minutes to obtain large-area, continuous MoS₂ monolayer films (centimeter-scale dimensions).

[0029] 2. MoS2 thin film transfer The MoS2 film was placed in water and automatically detached from the mica substrate using van der Waals forces, then floated on the surface. A carrier mesh was then held by tweezers and inserted obliquely below the liquid surface. Under the interfacial tension between the carrier mesh and MoS2, the MoS2 film was smoothly transferred to the carrier mesh and fixed after drying. Figure 1 ).

[0030] The MoS2 thin film was characterized using high-resolution scanning transmission electron microscopy (HAADF-STEM) and Raman spectroscopy. The monolayer atomic structure of MoS2 was clearly observed, with Mo and S atoms arranged regularly and a lattice spacing of 1.6 Å. Low-magnification micrographs and TEM images showed extremely high coverage of the MoS2 support film transferred to the grid. The consistent diffraction pattern angles in the four pores at different locations within a single square further confirm the large-size monolayer single-crystal structure of the continuous film prepared in this invention.

[0031] 3. Plasma treatment MoS2 supported membrane substrates were subjected to low-oxygen plasma treatment under the following conditions: at room temperature, a mixture of argon and oxygen was used, with an oxygen volume content of 0.002 and a gas flow rate of 100 sccm. This process created abundant S vacancy defects, which led to changes in the local electrical properties of MoS2, thereby constructing high-energy active sites that facilitated the adsorption of oxygen-containing functional groups. Figure 2This process enhances the hydrophilicity and electrostatic adsorption capacity of the membrane surface. Impurities adsorbed on the surface of the plasma-treated MoS2 supported membrane are effectively removed, and the contact angle is reduced to 47.5°, improving its performance in cryo-electron microscopy grids, such as... Figure 3 As shown.

[0032] In addition, the oxygen content in the mixed gas affects the vacancy defects in the support membrane, such as... Figure 9 and Figure 10 The images shown are high-resolution transmission electron microscopy (TEM) images of MoS2 in an argon-oxygen mixture containing 1.5% and 8% oxygen in an plasma treatment atmosphere (at a gas flow rate of 20 sccm). It can be seen that the number of vacancies in the support film increases with increasing oxygen content.

[0033] Example 2: Application of Cryo-Electron Microscopy The MoS2 support membrane carrier was applied to cryo-electron microscopy structural analysis and tomographic analysis of 20S protease, ribosomes and MEF cells, demonstrating its excellent performance in single-particle cryo-electron microscopy and cell cryo-tomography.

[0034] The conditions for single-particle cryo-electron microscopy structural resolution were as follows: a Titan Krios cryo-electron microscope equipped with a Gatan K3 camera and an accelerating voltage of 300 kV was used; the number of frames taken was 32; and the total cumulative exposure dose was 50 eE. - / Å 2 The collected photos were corrected using MotionCorr2 software and reconstructed using cryoSPARC software. The settings were: 1.2 s, 29,000× magnification, 0.97 Å pixel size, and -1.2 to -1.5 μm defocus range.

[0035] The electron tomography conditions were as follows: Cryo-electron tomography (cryo-ET) tilt series data of 20S protease, ribosomes and MEF cell margins were collected on a 300 kV Titan Krios cryo-electron microscope (Thermo Fisher Scientific) equipped with a GIF Quantum energy filter (Gatan) and a K3 direct detection camera (Gatan).

[0036] For 20S protease and ribosomes, data were collected using SerialEM software with a dose-symmetric strategy. The tilt series calibration pixel size was 1.36 Å, the defocus range was -4.5 to -6.5 μm, the tilt increment was 3°, and the imaging dose for all tilts was 3 e - / Å 2 / s. The tilt range is from +60° to -60°, starting from 0°, with a total dose of approximately 123 e. - / Å2 The tilted series was imported into Warp software for motion correction and CTF estimation. The corrected tilted series was then aligned in IMOD software using block tracking, followed by final alignment using linear interpolation. The aligned image was resampled to a pixel size of 5.44 Å, and the final tomographic image was reconstructed using weighted back projection, followed by noise reduction using a similar filter based on algebraic iteration.

[0037] For MEF cells, the electron irradiation dose per image is approximately 3 e. - / Å 2 / s, total dose approximately 120 e - / Å 2 The magnification was 42,000×. For each deflection series, images were collected in 3° increments from +60 to −60° at an underfocus value of approximately -5.0 μm. Each image was calibrated using MotionCor2, and all deflection series were calibrated and reconstructed using IMOD at a binning factor of 6, corresponding to a pixel size of 10.18 Å.

[0038] 1. Structural analysis of 20S protease The monolayer MoS2 support membrane mesh exhibits excellent performance in single-particle cryo-electron microscopy characterization.

[0039] Taking 20S protease as an example ( Figure 4 The protein concentration and ice layer distribution were relatively uniform. Statistical analysis of protein particle distribution showed that the average number of protein particles in cryo-electron microscopy images of the 20S protease using a MoS2-supported membrane mesh reached as high as 524 per image. At the same protein concentration, this value is significantly higher than the average number of protein particles (~240) in electron microscopy images without a support membrane mesh. Furthermore, the structure of the 20S protease could be reconstructed using over five hundred particles in a single cryo-electron microscopy image on the MoS2-supported membrane, with a calculated resolution of 3.4 Å. Figure 4 As shown, the protein contrast is high, and good two-dimensional classification, complete three-dimensional structure and fine side chain details can be calculated.

[0040] 2. Ribosome structure analysis When using a monolayer MoS2-supported membrane grid for ribosome structural analysis, a higher particle density can be obtained compared to GO and rGO grids. The average number of protein particles per cryo-electron microscopy image on the MoS2 grid reaches as high as 725. For example... Figure 5As shown, ribosomes exhibit a uniform monodisperse distribution and two-dimensional structures with multiple angles on the MoS2 supported membrane. Calculations using cryoSPARC software show that the resolution of the ribosomes can reach 2.3 Å, and they exhibit a fine side-chain structure consistent with the corresponding high-quality three-dimensional structure density map.

[0041] 3. Cryo-electron microscopy tomography analysis of MEF cells Due to the abundant high-energy active sites on the surface of monolayer MoS2 films, they exhibit stronger interactions with cell membrane surfaces. Experiments show that when a monolayer MoS2 support membrane mesh is placed in cell culture medium, MEF cells can attach to the mesh surface, and their needle-like and sheet-like pseudopodia can spread relatively smoothly on the mesh. Figure 6 This phenomenon is crucial for studying the intrinsic state of cells and helps to more accurately simulate cellular behavior within organisms. By observing and analyzing the behavior of cells on a monolayer MoS2 support membrane, we can gain a deeper understanding of their interaction with the support, providing a more profound understanding for the development of cryo-tomography technology for biomolecules.

[0042] Furthermore, by comparing the cell distribution on the GO support mesh, it was found that under the same cell culture and frozen sample preparation conditions, the cell density on the monolayer MoS2 support membrane support mesh was higher. This may be due to the increase in defect-induced sulfur vacancy active sites, which enhances the electrostatic adsorption and van der Waals forces between biomass and the support membrane. This was further confirmed by cryo-tomography analysis. Figure 7 The fine structures within MEF cells and pseudopodia can be clearly observed. Tomographic cross-sections of the ice layer show that cells and their pseudopodia are mainly distributed on the MoS2 support membrane, and intracellular information such as ribosomes and microfilaments can be observed.

[0043] To visually compare the effects of different two-dimensional materials on cell morphology, fluorescence microscopy was used to observe the morphology of MEF cells on cell slides supported by MoS2 and GO membranes. Blank slides served as a control group, and all other treatment conditions were kept identical. After 2 hours in MEF cell culture medium, the MoS2 and GO supported membrane slides showed a significantly higher cell number than the control group. Furthermore, on the MoS2 supported membrane, the natural spreading of sheet-like and needle-like pseudopodia was clearly visible. After 16 hours of cell culture, the cell number increased on all three slides. Moreover, the cells on the MoS2 supported membrane slides still exhibited the natural spreading of different pseudopodia. Figure 8 Based on the two-position transition metal sulfide support membrane provided by this invention, the role of two-dimensional materials in cryo-electron microscopy bioimaging will be analyzed in detail and in depth, further expanding the resolution of biological macromolecules or cell boundaries using single-particle and tomographic cryo-electron microscopy techniques.

[0044] The monolayer MoS2 support membrane prepared in this invention has excellent performance and can be used in cryo-electron microscopy (cryo-EM) grids. The MoS2 support membrane grid not only performs exceptionally well in the single-particle cryo-EM structural analysis of 20S proteases and ribosomes, but also has unique advantages in observing natural cell morphology and performing tomographic analysis, thus promoting the development of cryo-EM methodology.

Claims

1. A method for preparing a MoS2 supported film, comprising the following steps: S1, preparing a single-layer MoS2 continuous film; S2, transferring the MoS2 continuous film onto a support net; S3, subjecting the continuous film obtained in step S2 to plasma treatment to obtain the MoS2 supported film.

2. The method of claim 1, wherein: In step S1, the MoS2 continuous film is prepared by chemical vapor deposition.

3. The method of claim 2, wherein: Mica is used as the substrate, and adhesive tape is used to peel off the clean surface. The precursor used is K2MoS4.

4. The method of claim 3, wherein: The conditions of the chemical vapor deposition method are as follows: The temperature is 500-1200°C, and the pressure is normal pressure.

5. The production method according to any one of claims 1 to 4, characterized by: In step S3, the plasma treatment is low-oxygen-content plasma treatment to obtain a hydrophilic MoS2 supported film with abundant vacancy defects. The conditions of the low-oxygen-content plasma treatment are as follows: Under normal temperature conditions, a mixed gas of argon and oxygen is selected, wherein the volume content of oxygen is 0.001-0.1, and the gas flow rate is 10-100 sccm.

6. The MoS2 supported film prepared by the method of any one of claims 1-6.

7. The use of the MoS2 supported film of claim 6 as or for preparing a cryo-EM support net.

8. Use according to claim 7, characterized in that: The MoS2 supported film is used for single-particle cryo-EM characterization.

9. Use according to claim 7, characterized in that: The MoS2 supported film is used for observing the natural morphology of cells and for tomographic analysis thereof.

10. Use according to claim 9, characterized in that: The cells are model cells for studying membrane proteins, model cells for studying cell proliferation and differentiation, or model cells for studying cell cycle and cell death.

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