A batch preparation method for graphene electron microscope grid

Abstract

The present invention discloses a method for batch preparation of graphene electron microscope grids. The present invention realizes high-quality batch transfer of graphene and electron microscope grids by designing a multi-level transfer medium (including a PDMS porous template, a polymer support layer and a small molecule buffer layer) in combination with a solvent volatilization-induced elastic capillary bonding process. Specifically, the surface of the graphene is uniformly coated with a small molecule / polymer transfer medium layer, and then bonded to the PDMS porous array template. After the graphene is peeled off from the growth substrate, the graphene supported by the polymer film in the suspended part of the PDMS is bonded to the electron microscope grid using a solvent thermal bonding method; finally, the transfer medium is removed to obtain the graphene grid. The method of the present invention has the advantages of simple process, reusable PDMS template and good compatibility, and is suitable for large-scale production. At the same time, the prepared graphene grid has high cleanliness and integrity, and can meet the application requirements in the field of cryo-electron microscope grids.

Description

Technical Field

[0001] The invention relates to a batch preparation method of an ultra-flat graphene electron microscope support film, and belongs to the technical field of graphene materials. Background Art

[0002] Single-particle cryo-electron microscopy (Cryo-EM) is a core tool for high-resolution structural analysis of biomacromolecules, but its imaging resolution is limited by issues associated with using traditional porous carbon membranes for sample preparation, such as protein denaturation at the gas-liquid interface, high background noise, and poor ice uniformity. Graphene freestanding membranes, due to their atomic-level thickness, ultra-low electron scattering, high mechanical strength, and excellent electrical and thermal conductivity, significantly improve imaging resolution and enable atomic-scale resolution (<3.0Å) of small molecular weight proteins (<100 kDa). Chemical vapor deposition (CVD) can produce large-area single-crystalline graphene films on metal substrates, providing a prerequisite for the preparation of high-quality support membranes for EM samples. Currently, graphene cryo-EM grid support membranes are primarily prepared using graphene grown on single-crystalline copper substrates using a transfer technique combined with substrate etching. However, existing graphene transfer techniques suffer from bottlenecks such as residual contamination, high transfer breakage rates, and difficulties in mass production, severely restricting their practical application.

[0003] The current methods for achieving graphene transfer are: (1) Face-to-face bonding clean transfer technology (publication number CN106435727 A): The grid substrate is directly bonded to the graphene attached to the metal growth substrate, and the capillary tension generated by volatile solvents such as isopropyl alcohol is used to achieve bonding between the graphene and the grid surface. After etching and removing the copper growth substrate, the graphene grid is obtained. This transfer method is affected by the flatness of the graphene substrate and the grid surface, and cannot achieve tight bonding. The resulting holes easily cause damage to the suspended graphene in the later stage, making it difficult to mass produce. (2) Polymer-assisted transfer technology (publication number CN111847437A): Graphene supported and protected by a polymer film such as polymethyl methacrylate (PMMA) is obtained by etching. The graphene is removed from the grid and the glue is removed to obtain the graphene grid. Since PMMA and other polymers are directly coated on the graphene, sp3 hybridization is formed, resulting in poor transfer cleanliness. In addition, since the grid is difficult to bond with the film, the graphene wrinkles are likely to occur. (3) Small molecule self-assembly assisted transfer technology (publication number CN 113023718A): Graphene is modified with an organic small molecule self-assembled layer, which is then etched to form a flexible suspension film, achieving clean and complete preparation on the carrier grid. However, the mechanical support performance of the self-assembled molecular layer is poor. During the bonding process, the gravitational disturbance of the carrier grid can easily cause irregular cracking of the graphene suspension film, making large-scale preparation difficult.

[0004] The key to high-quality graphene transfer technology onto target substrates lies in enhancing interfacial interactions. Because graphene's surface is chemically inert and lacks dangling bonds, its interaction with porous support membranes relies primarily on van der Waals forces. This force decays inversely with the sixth power of the interfacial distance, making conformal contact between the graphene and substrate to minimize interfacial gaps crucial for maintaining film integrity. However, electron microscope grids exhibit micron-scale surface undulations. When transferring graphene directly face-to-face with a rigid growth substrate, the integrity of the transferred graphene is generally less than 30% due to the large gaps. Transferring graphene to flexible polymer films or self-assembled small molecule layers followed by bonding can help achieve conformal contact, but this also presents challenges such as polymer contamination or breakage due to insufficient mechanical properties of the small molecule layer. Due to the numerous challenges associated with large-scale, high-quality graphene transfer onto support grids, particularly issues such as gaps, wrinkles, film cracking, and contamination during the bonding process, existing technologies have yet to achieve clean, complete, and scalable production of graphene grids. The present invention is proposed to solve the problems of difficult lamination, low transfer efficiency and surface contamination in the batch transfer process of graphene. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for batch preparation of graphene carrier meshes, by transferring graphene in batches to a porous polydimethylsiloxane (PDMS) / polymer / small molecule support layer, and utilizing a solvent thermal bonding method to assist the graphene in conformal contact with the carrier mesh under elastic capillary action in the suspended pores of PDMS, thereby achieving high-quality batch transfer of graphene to the carrier mesh.

[0006] The method for batch preparation of graphene carrier grids provided by the present invention comprises the following steps:

[0007] S1. sequentially coating a small molecule buffer layer and a polymer medium layer on the surface of a graphene / metal substrate to obtain a composite structure of a composite medium layer / graphene / metal substrate;

[0008] S2. Laminating the PDMS porous template to the composite layer structure to obtain a composite structure of PDMS porous template / composite medium layer / graphene / metal substrate;

[0009] S3, removing the metal substrate to obtain a suspended support structure of PDMS porous template / composite medium layer / graphene;

[0010] S4, placing an electron microscope grid on the graphene surface of the suspended support structure, and using a solvent to conformally bond the interface between the electron microscope grid and the graphene;

[0011] S5. Remove the PDMS porous template and the composite medium layer to transfer the graphene to the electron microscope grid, and obtain a clean graphene electron microscope grid.

[0012] In the method of the present invention, the metal substrate may be single crystal copper sputtered on a sapphire wafer;

[0013] The graphene / metal substrate can be prepared by a chemical vapor method.

[0014] In the method of the present invention, the pore diameter of the PDMS porous template is 3 mm-10 cm, the pore spacing is 1 mm-10 cm, and the pore shape is circular or polygonal;

[0015] In step S1 of the present invention, the small molecule buffer layer comprises one or more of menthol, borneol and cyclododecane, and has a thickness of 50 nm to 50 μm;

[0016] The polymer medium layer comprises at least one of polypropylene carbonate, polymethyl methacrylate and cellulose acetate, and has a thickness of 100 nm-10 μm.

[0017] In step S2 of the present invention, the PDMS porous template is bonded by roller pressing or vacuum bonding.

[0018] In step S3 of the present invention, the metal substrate is removed by the following method;

[0019] Chemical etching: using ammonium persulfate, ferric chloride or ferric nitrate solution with a concentration of 0.5-3 mol / L;

[0020] Electrochemical stripping: Apply a voltage of 1-10 V in an alkaline electrolyte for 30 seconds to 60 minutes.

[0021] In step S4 of the present invention, the laminating process is as follows:

[0022] 1) placing the porous membrane surface of the electron microscope grid on the graphene surface of the suspended support structure, and applying ethanol, propanol, or isopropanol by drop coating or spraying, and utilizing the solvent evaporation tension to promote close contact between the laminating surfaces;

[0023] 2) After the solvent evaporates, bake at 50-150°C for 2 minutes to 5 hours to further promote close adhesion between graphene and the porous membrane;

[0024] In step S4, the solvent is isopropyl alcohol, and the solvent needs to be removed after the interface between the electron microscope grid and the graphene is conformally bonded.

[0025] In step S5 of the present invention, the composite medium layer is removed by soaking in an organic solvent, thereby separating the electron microscope grid from the PDMS porous template, and then cleaning is performed. The cleaning can be performed multiple times with acetone and then with isopropyl alcohol.

[0026] In step S4 of the present invention, the electron microscope grid can be a microgrid of Au, Cu, Ni, or Ni-Ti mesh, and the porous membrane included is a porous metal membrane or a porous carbon membrane.

[0027] The graphene electron microscope support film prepared by the method of the present invention has a bonding completeness between the graphene and the electron microscope grid of ≥99%, and a graphene coverage rate on the suspended holes of ≥95%.

[0028] The graphene electron microscope support film prepared by the method of the present invention can be used for preparing electron microscope grids, nanoimprinting, low-temperature cryo-electron microscopes, hydrogen-deuterium separation, etc.

[0029] Compared with the prior art, the present invention has the following beneficial technical effects:

[0030] (1) Multi-level transfer medium: The present invention designs porous PDMS / polymer / small molecule as a multi-level transfer medium for graphene, wherein the PDMS porous support provides a self-supporting layer for batch transfer of graphene, which is convenient for batch preparation of carrier network; the uniform and continuous polymer film such as PMMA in the suspended pores of PDMS provides sufficient mechanical support for the bonding of graphene, and its nanoscale thickness gives elastic freedom and conformal bonding ability; the low surface energy small molecule layer is conducive to the physical isolation of the adsorption of the polymer film on the graphene surface, which facilitates the clean removal of the transfer medium.

[0031] (2) Elastic capillary bonding: By removing the constraints of the hard substrate and utilizing the elastic capillary action generated by solvent evaporation, conformal contact between the graphene and the undulating surface of the carrier mesh is achieved. Without relying on the creep of polymers such as PMMA at high temperatures, the graphene can be efficiently bonded to the substrate with high integrity. The method of the present invention significantly improves the quality and integrity of the transfer of graphene to the carrier mesh.

[0032] The method of the present invention has the advantages of simple process, reusable PDMS template, and good compatibility, making it suitable for large-scale production. Furthermore, the resulting graphene grid has high cleanliness and integrity, meeting the application requirements of cryo-electron microscopy grids and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The present invention is a flow chart of preparing a graphene grid support film.

[0034] Figure 2 These are an optical photograph (Figure a) and optical microscope photographs (Figures b and c) of the quasi-four-inch graphene / intermediary layer / porous PDMS sample prepared in Example 1 of the present invention.

[0035] Figure 3 This is an optical microscope photograph of the bonding between the carrier grid and the graphene / intermediary layer / porous PDMS sample in Example 1 of the present invention.

[0036] Figure 4 These are a scanning electron microscope photograph (Figure a), an atomic force microscope photograph (Figure b), and a transmission optical microscope photograph (Figure c) of the quantifoil graphene grid prepared in Example 1 of the present invention.

[0037] Figure 5 This is a cryo-electron microscopy photograph of the quantifoil graphene grid loaded with 20S proteasome prepared in Example 1 of the present invention.

[0038] Figure 6 This is a scanning electron microscope photograph of the quantifoil graphene grid prepared in Comparative Example 1 of the present invention.

[0039] Figure 7 These are optical microscope photos of the quantifoil grid and graphene / growth substrate bonded together in Comparative Example 2 of the present invention and scanning electron microscope photos after transfer.

[0040] Figure 8 This is an atomic force microscope photograph of the quantifoil graphene grid prepared in Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] The method for mass-producing graphene grids provided by the present invention involves uniformly coating the graphene surface with a small molecule / polymer transfer medium layer, then laminating the graphene surface to a polydimethylsiloxane (PDMS) porous array template. After the graphene is peeled from the growth substrate, the graphene, supported by a polymer film suspended in the PDMS, is then laminated to an electron microscope grid using a solvent thermal bonding method. Finally, the transfer medium is removed to obtain the graphene grid. Compared to existing technologies, this method improves the integrity and cleanliness of the transferred graphene film and is compatible with graphene raw materials of varying sizes, enabling the mass production of graphene grids.

[0044] Through the design of "multi-level media design-elastic capillary bonding", the present invention systematically solves the industry problems of low graphene electron microscope grid transfer integrity, severe pollution, and inability to scale production, providing high-performance standardized consumables for single-particle analysis in cryo-electron microscopy.

[0045] Example 1: Preparation of a graphene electron microscope grid using graphene grown on a copper substrate

[0046] according to Figure 1 Prepared according to the process shown.

[0047] 1) Transfer Medium Coating: Commercially available borneol was dissolved in isopropyl alcohol to prepare a 25% (mass fraction) uniform solution. This solution was then uniformly coated once on the graphene / copper substrate at 2000 rpm to form a low-surface-energy, small-molecule buffer layer. Subsequently, a 4% (mass fraction) PMMA photoresist (950K, Microchem) was uniformly coated on the buffer layer surface at 1500 rpm. The solution was then baked at 130°C for 3 minutes to obtain a transfer medium / graphene / copper substrate composite sample.

[0048] Preparation of PDMS porous template: Use a hole puncher to punch holes in 10 cm × 10 cm PDMS (Gel-Pak) to obtain a PDMS porous template with a hole diameter of 3 mm and a hole spacing of 2 mm.

[0049] 2) The PDMS porous template was laminated onto the dielectric layer using a roller pressing method, ensuring that no bubbles were formed during the lamination process, thereby obtaining a porous PDMS / dielectric layer / graphene / copper metal substrate composite sample.

[0050] 3) The porous PDMS / intermediary layer / graphene / copper metal substrate composite sample was immersed in 1 mol / L ammonium persulfate etching solution to etch the copper substrate. Subsequently, the sample was transferred to deionized water for cleaning, and after being fished out, the graphene surface was faced upward and dried to obtain a porous PDMS / intermediary layer / graphene ( Figure 2 (Figure a in the middle).

[0051] 4) Porous PDMS / intermediary layer / graphene and quantifoil porous carbon film grid bonding: Align the porous membrane surface of the porous grid with the graphene on the circular hole, drop isopropyl alcohol, and use the solvent evaporation tension to promote close contact between the two bonding surfaces ( Figure 3 After the solvent evaporates and is dried, the film is baked at 80°C for 30 minutes to further promote close adhesion between the graphene and the multi-carbon carbon film, thereby obtaining a porous PDMS / intermediary layer / graphene / porous film support grid.

[0052] 5) Soaking to Remove Resin and Prepare the Graphene Grid: Soak the porous PDMS / intermediate layer / graphene / porous membrane grid in acetone at room temperature for half an hour. After removing the PDMS, rinse the grid twice in acetone at 50°C for 5 minutes each, followed by a final rinse in isopropyl alcohol at 50°C. Isopropyl alcohol, as a low-surface-tension solvent, further removes residue and reduces wrinkles on the graphene surface. Dry at room temperature to obtain the graphene grid.

[0053] Figure 2The following are optical photographs (Figure a) and optical microscope photographs (Figures b and c) of the quasi-four-inch graphene / intermediary layer / porous PDMS sample prepared in Example 1. As can be seen from the figures, the graphene / intermediary layer can be transferred to the PDMS suspended holes in batches, and the film inside the holes is clean and intact.

[0054] Figure 3 This is an optical microscope photograph of the bonding between the carrier grid and the graphene / intermediary layer / porous PDMS sample in this embodiment. As can be seen from the figure, the optical bonding between the graphene / intermediary layer and the carrier grid in the PDMS template holes is high, with almost no gaps.

[0055] Figure 4 Figures 1 and 2 show scanning electron microscope (SEM) images (Figure a), atomic force microscope (AFM) images (Figure b), and transmission electron microscope (TEM) images (Figure c) of the quantifoil graphene grid prepared in this example. As can be seen from the figures, the graphene transferred to the quantifoil grid has a high degree of integrity (>95%) and a clean surface.

[0056] Figure 5 This is a cryo-electron micrograph of the quantifoil graphene grid loaded with 20S proteasome prepared in this example. As can be seen from the figure, the amorphous ice layer prepared by the present invention is uniform, the protein is evenly distributed, and the imaging is clear.

[0057] Example 2

[0058] This example differs from Example 1 in that step 3 utilizes electrochemical exfoliation of the metal growth substrate, while all other conditions remain unchanged. The specific conditions for electrochemical exfoliation are: a 3 V DC potential is applied between two electrodes in a 1 M sodium hydroxide electrolyte for 5 minutes. Uniform hydrogen bubbles are generated to weaken the interaction between the graphene and the growth substrate, while an external force is applied to separate the graphene from the substrate.

[0059] Comparative Example 1

[0060] Except that the PDMS support membrane was not perforated, other steps were exactly the same as those in Example 1.

[0061] Figure 6 This is a scanning electron microscope photo of PDMS transferred without punching. The results show that the graphene on PDMS is not sufficiently conformal to the carrier grid, and the coverage of the graphene film on the quantifoil carrier grid is 0.

[0062] Comparative Example 2

[0063] Using a conventional adhesive-free clean transfer method, the porous membrane surface of the porous mesh was clasped against the graphene / copper substrate and directly bonded with isopropyl alcohol. After the solvent evaporated, the porous membrane mesh / graphene / copper substrate composite sample was immersed in a 1 mol / L ammonium persulfate etchant to etch the copper substrate. The resulting mesh was rinsed three times with deionized water for 15 minutes each, followed by a final rinse with isopropyl alcohol for 3 minutes and dried at room temperature to obtain a clean graphene mesh.

[0064] Figure 7 These are optical microscope photos of the quantifoil grid and graphene / growth substrate bonded together in this comparative example, and scanning electron microscope photos after transfer. As can be seen from the figures, due to the constraints of the growth substrate, the graphene and the grid cannot achieve a high degree of bonding, and the integrity of the graphene transferred to the grid is <30%.

[0065] Comparative Example 3

[0066] The difference between this embodiment and embodiment 1 is that the small molecule buffer layer is not evenly coated in step 1, and other conditions remain unchanged.

[0067] Figure 8 This is an atomic force microscope photo of the transfer without small molecule spin coating. The results show that due to the lack of a small molecule buffer layer, there is residual glue contamination on the surface of the graphene / quantifoil carrier grid prepared by PMMA / porous PDMS transfer.

Claims

1. A method for batch preparation of graphene carrier meshes, comprising the following steps: S1. sequentially coating a small molecule buffer layer and a polymer medium layer on the surface of a graphene / metal substrate to obtain a composite structure of a composite medium layer / graphene / metal substrate; S2. Laminating the PDMS porous template to the composite structure to obtain a composite structure of PDMS porous template / composite medium layer / graphene / metal substrate; The PDMS porous template has a pore diameter of 3 mm-10 cm, a pore spacing of 1 mm-10 cm, and a circular or polygonal pore shape; S3, removing the metal substrate to obtain a suspended support structure of PDMS porous template / composite medium layer / graphene; S4, placing an electron microscope grid on the graphene surface of the suspended support structure, and using a solvent to conformally bond the interface between the electron microscope grid and the graphene; S5. Remove the PDMS porous template and the composite medium layer to obtain a clean graphene electron microscope grid.

2. The preparation method according to claim 1, wherein: In step S1, the small molecule buffer layer comprises one or more of menthol, borneol and cyclododecane, and has a thickness of 50 nm to 50 μm; The polymer medium layer comprises at least one of polypropylene carbonate, polymethyl methacrylate and cellulose acetate, and has a thickness of 100 nm-10 μm.

3. The preparation method according to claim 1 or 2, characterized in that: In step S2, the PDMS porous template is bonded by roller pressing or vacuum bonding.

4. The preparation method according to claim 1 or 2, characterized in that: In step S3, the metal substrate is removed by the following method: Chemical etching: using ammonium persulfate, ferric chloride or ferric nitrate solution; Electrochemical stripping: A voltage of 1–10 V was applied in an alkaline electrolyte.

5. The preparation method according to claim 1 or 2, characterized in that: In step S4, the solvent is ethanol, propanol or isopropanol, and the solvent needs to be removed after the interface between the electron microscope grid and the graphene is conformally bonded.

6. The preparation method according to claim 1 or 2, characterized in that: In step S5, the composite medium layer is removed by an organic solvent soaking method, so that the electron microscope grid and the PDMS porous template are separated to obtain a graphene electron microscope grid.

7. The batch preparation method according to claim 1 or 2, characterized in that: In step S4, the electron microscope grid is a microgrid of Au, Cu, Ni, or Ni-Ti mesh, and the porous membrane contained therein is a porous metal membrane or a porous carbon membrane.

8. A graphene electron microscope support film, prepared by the method according to any one of claims 1 to 7, wherein: The bonding integrity between graphene and electron microscope grid is ≥99%, and the graphene coverage on the suspended holes is ≥95%.

9. Use of the graphene electron microscope support film according to claim 8 in the preparation of electron microscope grids, nanoimprinting, cryo-electron microscopy, and hydrogen-deuterium separation.

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