Batch preparation method of graphene electron microscope grid

Abstract

The invention discloses a batch preparation method of a graphene electron microscope grid. By designing a multi-level transfer medium (comprising a PDMS porous template, a high-molecular support layer and a small-molecular buffer layer) and combining with a solvent evaporation induced elastic capillary fitting process, high-quality batch transfer of graphene and an electron microscope grid is realized. Specifically, the surface of graphene is uniformly coated with a micromolecule / macromolecule transfer medium layer, and then the graphene is attached to a PDMS porous array template. Stripping the graphene from the growth substrate, and laminating the graphene supported by the polymeric membrane at the suspended position of the PDMS with an electron microscope grid by using a solvothermal lamination method; and finally, removing the transfer medium to obtain the graphene grid. The method disclosed by the invention has the advantages of simple process, reusability of the PDMS template, good compatibility and the like, and is suitable for large-scale production. Meanwhile, the prepared graphene grid has high cleanliness and integrity, and can meet the application requirements in the fields of freezing electron microscope grids and the like.

Description

Technical Field

[0001] The present invention relates to a method for batch preparation of a super-flat graphene electron microscope support film, belonging to the technical field of graphene materials. Background Art

[0002] Single-particle cryogenic electron microscopy (Cryo-EM) is a core tool for analyzing the high-resolution structures of biological macromolecules. However, its imaging resolution is limited by problems arising from using traditional porous carbon films for sample preparation, such as protein denaturation at the gas-liquid interface, high background noise, and poor ice layer uniformity. Graphene suspended membranes, due to their atomic-level thickness, ultra-low electron scattering, high mechanical strength, and electrical and thermal conductivity, can significantly improve imaging resolution and support atomic-level analysis (<3.0 Å) of small molecular weight proteins (<100 kDa). Chemical vapor deposition (CVD) methods can obtain large-area single-crystal graphene films on metal substrates, providing a prerequisite for preparing high-quality electron microscope sample support films. Currently, graphene cryo-EM grid support films are mainly prepared from graphene grown on single-crystal copper substrates using transfer techniques combined with substrate etching. However, existing graphene transfer techniques have bottlenecks such as contamination residues, high transfer breakage rates, and difficulties in batch preparation, severely restricting their practical applications.

[0003] Currently, the methods for realizing graphene transfer are as follows: (1) Face-to-face bonding and clean transfer technique (Publication No. 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 isopropanol is used to achieve the bonding of graphene to the surface of the grid. After etching away the copper growth substrate, a graphene grid is obtained; this transfer method is affected by the flatness of the graphene substrate and the grid surface, unable to achieve tight bonding, and the resulting holes are likely to cause damage to the later suspended graphene, making it difficult to produce in batches. (2) Polymer-assisted transfer technique (Publication No. CN111847437A): Graphene supported and protected by a polymer film such as polymethyl methacrylate (PMMA) is obtained through an etching method. After the grid picks up the graphene, the glue is removed to obtain a graphene grid; since polymers such as PMMA are directly coated on graphene, forming sp3 hybridization, the transfer cleanliness is poor, and due to the difficulty of the grid picking up and bonding the film, graphene wrinkling problems are likely to occur. (3) Small molecule self-assembly-assisted transfer technique (Publication No. CN 113023718A): Graphene is modified with an organic small molecule self-assembly layer, and after etching, a flexible suspended membrane is formed to achieve clean and complete preparation on the grid. However, the mechanical support performance of the self-assembly molecular layer is poor, and during the bonding process, due to the gravity disturbance problem of the grid, the graphene suspended membrane is prone to random cracking, making large-scale preparation difficult.

[0004] The core of the high-quality transfer technology of graphene to the target substrate lies in enhancing its interfacial interaction. Since the surface of graphene is chemically inert and lacks dangling bonds, the interaction between it and the porous support membrane mainly depends on van der Waals forces. This force follows a decay law inversely proportional to the sixth power of the interfacial distance. Therefore, achieving conformal contact between graphene and the substrate to minimize the interfacial gap is a key factor in maintaining the integrity of the thin film. However, there are micron-scale undulations on the surface of the EM grid. When using the transfer method of directly face-to-face fitting of graphene on a hard growth substrate, due to the large voids, the integrity of the transferred graphene is generally lower than 30%. Transferring graphene to a flexible polymer film or a self-assembled small molecule layer and then fitting helps to achieve conformal contact, but it also faces problems such as polymer contamination or damage caused by insufficient mechanical properties of the small molecule layer. Due to the many challenges in the large-scale high-quality transfer of graphene to the grid, especially problems such as voids, wrinkles, film cracking, and contamination during the fitting process, the existing technology has not achieved the clean, complete, and batch preparation of graphene grids. Therefore, this invention is proposed to solve the problems of difficult fitting, low transfer efficiency, and surface contamination during the batch transfer of graphene. Summary of the Invention

[0005] The object of the present invention is to provide a batch preparation method for graphene grids. By batch transferring graphene onto a porous polydimethylsiloxane (PDMS) / polymer / small molecule support layer and using the solvothermal bonding method, it helps graphene to have conformal contact with the grid under elastic capillary action in the PDMS suspension holes, realizing the high-quality batch transfer of graphene to the grid.

[0006] The batch preparation method for graphene grids provided by the present invention includes the following steps: S1. Sequentially coat a small molecule buffer layer and a polymer medium layer on the surface of the graphene / metal substrate to obtain a composite structure of composite medium layer / graphene / metal substrate; S2. Fit the PDMS porous template to the composite layer structure to obtain a composite structure of PDMS porous template / composite medium layer / graphene / metal substrate; S3. Remove the metal substrate to obtain a suspended support structure of PDMS porous template / composite medium layer / graphene; S4. Face-to-face fit the EM grid to the graphene surface of the suspended support structure, and use a solvent to make the interface between the EM grid and graphene conformally fit; S5. Remove the PDMS porous template and the composite medium layer to realize the transfer of graphene to the EM grid, obtaining a clean graphene EM grid.

[0007] In the method of the present invention, the metal substrate can be single-crystalline copper sputtered on a sapphire wafer; The graphene / metal substrate can be prepared by chemical vapor deposition method.

[0008] 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; In step S1 of the present invention, the small molecule buffer layer contains one or more of menthol, borneol and cyclododecane, and the thickness is 50 nm - 50 μm; The polymer medium layer contains at least one of polypropylene carbonate, polymethyl methacrylate and cellulose acetate, and the thickness is 100 nm - 10 μm.

[0009] In step S2 of the present invention, the PDMS porous template is bonded by roll pressing or vacuum laminating method.

[0010] In step S3 of the present invention, the following method is used to remove the metal substrate; Chemical etching: using ammonium persulfate, ferric chloride or ferric nitrate solution, with a concentration of 0.5 - 3 mol / L; Electrochemical stripping: applying a voltage of 1 - 10 V in an alkaline electrolyte, and the stripping time is 30 seconds - 60 minutes.

[0011] In step S4 of the present invention, the bonding process is as follows: 1) The surface of the porous membrane layer of the electron microscope grid is face - to - face bonded with the graphene surface of the suspended support structure, and ethanol, propanol or isopropanol is drop - coated or sprayed, and the solvent evaporation tension is used to promote the close contact of the bonding surface; 2) After the solvent has evaporated completely, it is baked at 50 - 150 °C for 2 minutes - 5 hours to further promote the close bonding of graphene and the porous membrane; In step S4, the solvent is isopropanol, and the solvent needs to be removed after the conformal bonding at the interface between the electron microscope grid and graphene.

[0012] In step S5 of the present invention, the composite medium layer is removed by organic solvent immersion method, so that the electron microscope grid is separated from the PDMS porous template, and then it is washed. It can be washed with acetone multiple times and then with isopropanol.

[0013] In step S4 of the present invention, the electron microscope grid can be a micro - grid of Au, Cu, Ni, Ni - Ti mesh, and the porous membrane included is a porous metal membrane or a porous carbon membrane.

[0014] For the graphene electron microscope support film prepared by the method of the present invention, the bonding integrity between graphene and the electron microscope grid is ≥99%, and the graphene coverage rate on the suspended pores is ≥95%.

[0015] The graphene electron microscopy support film prepared by the method of the present invention can be used for the preparation of electron microscopy grids, nanoimprinting, cryo-electron microscopy, hydrogen-deuterium separation, etc.

[0016] Compared with the prior art, the present invention has the following beneficial technical effects: (1) Multi-level transfer medium: The present invention designs porous PDMS / polymer / small molecule as the graphene multi-level transfer medium. Among them, the PDMS porous scaffold provides a self-supporting layer for the batch transfer of graphene, facilitating the batch preparation of grids; the uniform and continuous PMMA and other polymer films in the suspended holes of PDMS provide sufficient mechanical support for the adhesion of graphene, and their nano-scale thickness endows elastic freedom and conformal adhesion ability; the low surface energy small molecule layer is conducive to physically isolating the adsorption of the polymer film on the graphene surface, facilitating the clean removal of the transfer medium.

[0017] (2) Elastic capillary action adhesion: Removing the constraint of the hard substrate, using the elastic capillary action generated by solvent evaporation to achieve conformal contact between graphene and the surface-undulating grid. Without relying on the creep of polymers such as PMMA at high temperatures, the graphene can be efficiently adhered to the substrate with high integrity. The method of the present invention significantly improves the transfer quality integrity of graphene to the grid.

[0018] The method of the present invention has the advantages of simple process, reusable PDMS template and good compatibility, etc., 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 fields such as cryo-electron microscopy grids. Description of the Drawings

[0019] Figure 1 is the flow chart for preparing the graphene grid support film of the present invention.

[0020] Figure 2 is the optical photograph (Figure a) and optical microscope photographs (Figures b and c) of the quasi-four-inch graphene / medium layer / porous PDMS sample prepared in Example 1 of the present invention.

[0021] Figure 3 is the optical microscope photograph of the adhesion between the grid and the graphene / medium layer / porous PDMS sample in Example 1 of the present invention.

[0022] Figure 4 is the scanning electron microscope photograph (Figure a), atomic force microscope photograph (Figure b) and transmission optical microscope photograph (Figure c) of the quantifoil graphene grid prepared in Example 1 of the present invention.

[0023] Figure 5 is the cryo-electron microscope photograph of the 20S proteasome loaded on the quantifoil graphene grid prepared in Example 1 of the present invention.

[0024] Figure 6 It is the scanning electron microscope photograph of the quantifoil graphene support grid prepared in Comparative Example 1 of the present invention.

[0025] Figure 7 They are the optical microscope photograph of the fitting of the quantifoil support grid and graphene / growth substrate and the scanning electron micrograph after transfer in Comparative Example 2 of the present invention.

[0026] Figure 8 It is the atomic force microscope photograph of the quantifoil graphene support grid prepared in Comparative Example 3 of the present invention. Detailed implementation manners

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0028] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0029] The mass batch preparation method of the graphene support grid provided by the present invention is to uniformly coat a small molecule / polymer transfer medium layer on the surface of graphene, and then fit it with a polydimethylsiloxane (PDMS) porous array template; after peeling the graphene from the growth substrate, the solvent thermal lamination method is used to laminate the graphene supported by the polymer film at the suspended part of the PDMS with the electron microscope support grid; finally, the transfer medium is removed to obtain the graphene support grid. Compared with the prior art, the present invention can improve the integrity and cleanliness of the transferred graphene film, and can be compatible with graphene raw materials of different sizes for mass preparation of graphene support grids.

[0030] Through the design of "multi-level medium design-elastic capillary fitting", the present invention systematically solves the industry problems of low transfer integrity, serious pollution and inability to produce on a large scale of the graphene electron microscope support grid, and provides high-performance standardized consumables for cryo-electron microscopy single particle analysis.

[0031] Example 1. Preparation of a graphene electron microscope support grid using graphene grown on a copper substrate According to Figure 1 The shown process is carried out for preparation.

[0032] 1) Uniformly coat the transfer medium: Dissolve commercially available borneol in isopropanol to prepare a uniform solution with a mass fraction of 25%, and uniformly coat it on the graphene / copper metal substrate once at a rotation speed of 2000 rpm to form a low surface energy small molecule buffer layer. Subsequently, a 4% PMMA photoresist (950K, Microchem) is uniformly coated on the surface of the buffer layer, spin-coated once at a rotation speed of 1500 rpm, and heated and baked at 130 °C for 3 minutes to obtain a composite sample of the medium layer / graphene / copper metal substrate.

[0033] Preparation of PDMS porous template: Use a hole punch to punch holes in PDMS (Gel-Pak) with a size of 10 cm×10 cm to obtain a PDMS porous template with a hole diameter of 3 mm and a hole spacing of 2 mm.

[0034] 2) Adhere the PDMS porous template to the medium layer by the roll pressing method to ensure no bubbles are formed during the adhesion process, thereby obtaining a porous PDMS / medium layer / graphene / copper metal substrate composite sample.

[0035] 3) Immerse the porous PDMS / medium layer / graphene / copper metal substrate composite sample in 1 mol / L ammonium persulfate etching solution to etch the copper substrate. Subsequently, transfer the sample to deionized water for cleaning, then take it out and dry it with the graphene side facing up to obtain porous PDMS / medium layer / graphene ( Figure 2 as shown in Figure a).

[0036] 4) Fit the porous PDMS / medium layer / graphene with a quantifoil porous carbon film carrier grid: Oppose the porous film layer surface of the porous carrier grid to the graphene on the round hole, drop isopropanol, and use the solvent evaporation tension to promote the close contact of the two adhered surfaces ( Figure 3 ). After the solvent has evaporated completely, bake it at 80 °C for 30 minutes to further promote the close adhesion of the graphene and the multi-carbon carbon film, and obtain a porous PDMS / medium layer / graphene / porous film carrier grid.

[0037] 5) Immerse and remove the glue to prepare the graphene carrier grid: Immerse the porous PDMS / medium layer / graphene / porous film carrier grid in acetone at room temperature for half an hour. After removing the PDMS, soak and clean the carrier grid in acetone at 50 °C twice, 5 minutes each time, and finally soak and clean it in isopropanol at 50 °C once. Isopropanol, as a low surface tension solvent, further removes the residues and reduces the wrinkles on the graphene surface. After drying at room temperature, the graphene carrier grid is obtained.

[0038] Figure 2 are the optical photograph (Figure a) and optical microscope photographs (Figure b and Figure c) of the quasi-four-inch graphene / medium layer / porous PDMS sample prepared in Example 1. It can be seen from the figure that the graphene / medium layer can be batch transferred to the PDMS suspended holes, and the film in the holes is clean and intact.

[0039] Figure 3 is the optical microscope photograph of the carrier grid adhered to the graphene / medium layer / porous PDMS sample in this example. It can be seen from the figure that the optical adhesion degree of the graphene / medium layer in the PDMS template holes to the carrier grid is high, and there are almost no voids.

[0040] Figure 4Scanning electron microscope image (Figure a), atomic force microscope image (Figure b), and transmission electron microscope image (Figure c) of the quantifoil graphene grid prepared in this example. As can be seen from the figures, the transfer of graphene to the quantifoil grid has a high integrity (>95%) and a clean surface.

[0041] Figure 5 Cryo-electron microscope image 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 distribution is uniform, and the imaging is clear.

[0042] Example 2

[0043] The difference between this example and Example 1 is that in step 3, an electrochemical stripping of the metal growth substrate is adopted, and other conditions remain unchanged. The specific implementation conditions for the electrochemical stripping are as follows: in a 1 M sodium hydroxide electrolyte solution, a DC potential of 3 V is applied between two electrodes for a duration of 5 minutes. The interaction between graphene and the growth substrate is weakened by generating uniform hydrogen bubbles, and at the same time, an external force is applied to separate graphene from the substrate.

[0044] Comparative Example 1 Except that the PDMS support film is not perforated, other steps are exactly the same as in Example 1.

[0045] Figure 6 Scanning electron microscope image of the transfer without perforating the PDMS. The results show that the conformal coating of graphene on the PDMS with the grid is insufficient, and the coverage of the graphene film on the quantifoil grid is 0.

[0046] Comparative Example 2 According to the traditional adhesive-free clean transfer method, the surface of the porous membrane layer of the porous grid is buckled with the graphene / copper substrate, and isopropanol is directly dropped for bonding. After the solvent evaporates, the composite sample of the porous membrane grid / graphene / copper substrate is immersed in a 1 mol / L ammonium persulfate etching solution to etch the copper substrate. The obtained grid is washed 3 times with deionized water for 15 minutes each time, and finally washed with isopropanol for 3 minutes and dried at room temperature to obtain a clean graphene grid.

[0047] Figure 7 Optical microscope image of the bonding of the quantifoil grid with the graphene / growth substrate and scanning electron microscope image after transfer in this comparative example. As can be seen from the figure, restricted by the growth substrate, graphene cannot be highly bonded with the grid, and the integrity of the graphene transferred to the grid is <30%.

[0048] Comparative Example 3 The difference between this example and Example 1 is that in step 1, a small molecule buffer layer is not evenly coated, and other conditions remain unchanged.

[0049] Figure 8 It is an atomic force microscope photograph of the transfer without a small molecule spin coating layer. The results show that there is no small molecule buffer layer, and there is residual glue contamination on the surface of the graphene / quantifoil grid prepared by the transfer of PMMA / porous PDMS.

Claims

1. A batch preparation method of a graphene-supported grid, comprising the following steps: S1. Coating a small molecule buffer layer and a polymer medium layer on the surface of the graphene / metal substrate in sequence to obtain a composite structure of a composite medium layer / graphene / metal substrate; S2. Bonding the PDMS porous template to the composite layer structure to obtain a composite structure of a PDMS porous template / composite medium layer / graphene / metal substrate; S3. Removing the metal substrate to obtain a suspended support structure of a PDMS porous template / composite medium layer / graphene; S4. Oppositely attaching an electron microscope grid to 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. Removing 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, characterized in that: 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.

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

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

5. The preparation method according to claim 1 or 2, characterized in that: In step S3, the following method is used to remove the metal substrate: Chemical etching: using ammonium persulfate, ferric chloride, or ferric nitrate solution; Electrochemical stripping: applying a voltage of 1 - 10 V in an alkaline electrolyte.

6. 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.

7. 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 immersion method to separate the electron microscope grid from the PDMS porous template, and a graphene electron microscope grid is obtained.

8. 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 grid, and the porous membrane included is a porous metal membrane or a porous carbon membrane.

9. A graphene electron microscope support film prepared by the method according to any one of claims 1-8, wherein, The fitting integrity between the graphene and the electron microscope grid is ≥99%, and the graphene coverage rate on the suspended pores is ≥95%.

10. Use of the graphene electron microscope support film according to claim 9 in the preparation of an electron microscope grid, nanoimprinting, cryogenic electron microscopy, and hydrogen - deuterium separation.

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