Fabrication of Monolayer Graphene-Coated Grids for Cryoelectron Microscopy

1. Preparation of materials and accessories necessary for the fabrication of graphene grids

NOTE: Graphene readily contaminates, which reduces the efficiency of graphene coating and the quality of the graphene grids; therefore, it is important to thoroughly clean all materials that come into contact with the graphene. Preparation of materials and all steps should be carred out in a fume hood.

1. Gather the necessary materials that will be used for coating the grids with graphene (Figure 2A).
2. Rinse the glassware several times with deionized (DI) water to remove any dust, lint, and oily residues.
3. Use disposable wipes to clean glass coverslips with 75% ethanol and use an air-duster to remove any contaminants.
   ​NOTE: The clamping TEM grid holder block used in this protocol can hold up to 45 grids. For a large batch production of graphene grids, 45 grids or less can be prepared at once. However, it is recommended to start with a small batch production (four to six grids) until the method is established in the lab.

2. Preparation of 0.2 M ammonium persulfate (APS) in water

NOTE: This APS solution is used as an etchant to remove the copper (Cu) support from the graphene/Cu sheet in a later step. Always prepare fresh APS solution. Reused or old solutions will not etch copper effectively and may leave copper residue on the graphene in the later steps.

1. Rinse a 500 mL flask with deionized water (DI) water, then add 200 mL of DI water and microwave using maximum settings for approximately 1 min to degas the water.
2. Add 9 g of ammonium persulfate to 200 mL of DI water to produce a solution of 0.2 M APS.
   CAUTION: APS is toxic, it is advised to wear personal protective equipment (PPE) when handling APS. Dispose of APS waste in an approved waste disposal plant.
3. Stir the solution with a stir bar on a magnetic stirrer while connecting the flask to a vacuum source under a fume hood.
   ​NOTE: Degassing APS solution will help prevent the formation of bubbles, which can reduce the efficiency of Cu etching in step 6.

3. Transfer graphene/copper to a clean coverslip with a piece of blotting paper

NOTE: We use a 15 x 15 cm chemical vapor deposition (CVD) graphene film on Cu from a graphene supplier. Commercially purchased monolayer graphene/Cu sheets should be stored under a vacuum. As graphene is grown on both sides of Cu by the CVD method, graphene suppliers generally conduct quality checks and recommend the better side for use. We refer to this recommended side of the graphene as the "top" side while the other side is the "back" side in this protocol.

1. Cut a piece of blotting paper into a rectangular shape approximately 20 mm x 40 mm. This blotting paper is used as padding for the graphene/Cu sheet and will absorb excess methyl methacrylate (MMA (8.5) MMA EL6) used to coat the graphene/Cu sheet; therefore, be sure to cut it into a size that is bigger than the graphene/Cu sheet to be used.
2. Use polyimide tape to tape the four corners of the blotting paper to the top of a clean coverslip that will fit into a homemade spin coater.
   NOTE: Polyimide tape is used because it is thin and can be removed easily, which makes handling easy.
3. Remove one piece of the graphene/Cu sheet from the vacuum storage.
4. Use clean and dust-free scissors to carefully cut a small square of the Cu-graphene sheet that will be sufficient to completely cover the number of grids to be prepared. For 25 grids arranged in a 5 x 5 array, for example, cut a piece that is 18 mm x 18 mm. Place the graphene/Cu sheet on top of the blotting paper with clean, dry tweezers. Make sure that the back side of the graphene/Cu sheet is facing down and be careful not to accidentally flip the graphene/Cu sheet since it is difficult to discern the top side from the back side.
5. Tape the four corners of the graphene/Cu sheet to the blotting paper/coverslip, minimizing the amount of contact between the tape and the graphene/Cu sheet, since any regions covered with tape will not be covered with MMA in the next step.
   NOTE: Static discharge can damage graphene films, and thus, it is advisable to remain electrically grounded and minimize the accumulation of static charge when handling graphene or graphene grids. This can be accomplished by wearing a wrist grounding strap or touching a grounded metal object immediately prior to handling graphene or graphene grids.

4. Coat the single-layer graphene/Cu sheet with a thin layer of MMA(8.5)MMA EL6 (MMA)

NOTE: After the Cu is etched away, this layer of MMA will support the graphene monolayer to enable the handling of the graphene sheet in future steps. MMA coating also enables the visualization of the graphene film since a monolayer of graphene alone would be transparent.

1. Place the coverslip with the taped graphene/Cu sheet on a homemade spin coater (this can be assembled using a computer fan) inside a fume hood (Figure 2B), as previously described by Han et al.²⁵.
2. Wearing safety goggles, add two drops of MMA using a glass pipette on the graphene sheet and immediately begin spinning at maximum speed.
3. While spinning, add two more drops in the center. Spin for 1 min.
   NOTE: Be sure that sufficient MMA has been applied to fully coat the graphene. If unsure, add a few more drops. If graphene is not fully coated, "holes" appear in the film after Cu is etched away.
4. Let air-dry for 10 min inside a fume hood.

5. Remove graphene on the back side of the graphene/Cu sheet

NOTE: Graphene grown on the back side of the copper (the side not coated with MMA) must be removed before proceeding to the subsequent steps because this excess graphene will reduce the effectiveness of Cu etching. We remove this graphene by exposing the graphene to plasma, which can be accomplished using any glow discharge device typically used to prepare EM grids for biological sample preparation.

1. Carefully remove the tape and tlift the MMA-coated graphene/Cu sheet from the coverslip with clean, dry tweezers.
2. Tape the piece of the MMA/graphene/Cu sheet with the MMA side down to a clean and dust-free glass coverslip.
3. Place the coverslip with the MMA/graphene/Cu sheet into the glow discharge device and apply settings that would typically be used when preparing grids for negative stain or cryoEM sample preparation.
   ​NOTE: Prolonged glow discharge should be avoided to prevent oxidation of Cu on the back side, which can result in contamination of copper oxide (CuO) (nano)particles on graphene film.

6. Etch away the Cu from the MMA/graphene/Cu sheet in APS solution

1. In a fume hood, pour 200 mL of the freshly made APS solution into a clean and dust-free crystallizing dish (150 x 75 mm).
2. Remove the MMA/graphene/Cu sheet from the glass slide. Keep track of which side contains the MMA.
3. To begin the etching, place the MMA/graphene/Cu sheet with the plasma-treated Cu side down on the surface of the APS solution. Cover the wide glass beaker with a piece of aluminum foil or a lid to prevent dust from entering.
4. Incubate for 3 h. If most of the Cu is not etched after 1 h, repeat the steps in sections 2-6 and then continue to the next step.
   ​NOTE: If most of the Cu is not etched after 1 h, it is likely that the MMA/graphene side of the film has been placed down on the surface of the APS solution instead of the Cu side. Cu should be etched completely after 3 h, and the MMA/graphene film is colorless if Cu is etched completely. Graphene readily contaminates so be sure to cover any containers to avoid the accumulation of lint or any greasy residues on surfaces, as this will negatively impact the quality of the graphene.

7. Rinse the MMA/graphene film in DI water

1. In a fume hood, fill a clean and dust-free crystallizing dish with DI water.
2. Gently scoop up the graphene-MMA film with a clean, dust-free glass coverslip positioned at ~45° angle relative to the APS solution surface.
3. Position the glass slide at a nearly 90° angle to the water, and gently lower the glass coverslip into the water so that the MMA/graphene slowly slides off the slide and floats on the water's surface.
4. Leave the MMA/graphene film on the water surface for 1 h to wash off any APS.

8. Clean the grids to be coated with a monolayer of graphene

NOTE: The grids to which the graphene will be transferred must be as clean as possible to maximize the attachment of the graphene to the grid foil surface. Commercially purchased grids often contain residual contaminants that must be removed prior to graphene transfer.

1. Clean and dry three crystallizing dishes so that they are free of dust.
2. In a fume hood, pour 200 mL of chloroform, acetone, and isopropyl alcohol (IPA) into each of the three crystallizing dishes. Cover the crystallizing dishes with aluminum foil to minimize the evaporation of the organic solvents.
   CAUTION: Chloroform and acetone cause skin irritation and can be toxic if inhaled. Limit exposure to these organic solvents and wear PPE.
3. Place the base of the clamping TEM grid holder block at the bottom of the first crystallizing dish containing 200 mL of chloroform.
4. Transfer each grid individually from the grid box to a well in the grid holder block, ensuring that the fenestrated film side is facing up. Cover the crystallizing dish with aluminum foil, place it on an orbital shaker, and gently shake for 30 min.
5. Place the metal lid on the grid holder block, which will secure the grids during the transfer to the next crystallizing dish. Carefully lift the grid holder block out of the crystallizing dish with a bent fork and long tweezers and place it at the bottom of the second crystallizing dish containing 200 mL of acetone. Remove the lid from the grid holder block and gently shake on an orbital shaker for 30 min.
6. Place the lid on the grid holder block and transfer to a crystallizing dish containing 200 mL of IPA solution to clean off acetone residue. Remove the lid from the grid holder block and gently shake for 20 min.
7. Individually transfer the grids with the fenestrated film side facing up from the grid holder block to a glass Petri dish covered with blotting paper.
8. Dry the grids for at least 30 min under the fume hood, ensuring that the grids are covered to prevent dust from landing on it.

9. Transfer the clean grids to blotting paper placed on a stainless-steel wire mesh or perforated tray under DI water

NOTE: Grids must be submerged under DI water on a flat surface so that the graphene can be floated onto the water and lowered onto the grids. This can be performed using a commercial grid coating trough or with a Petri dish and a peristaltic pump, as used for generating graphene-oxide grids, as described by Palovcak et al.¹⁸.

1. Place the stainless-steel mesh or tray in a grid coating trough and rinse with DI water.
2. Cut the blotting paper that is slightly smaller than the stainless-steel mesh/tray and place it on top of the platform, submerged in the DI water.
   NOTE: The blotting paper is slightly smaller than the platform to enable movement and handling.
3. Gently transfer the cleaned grids with the fenestrated film side facing up on top of the blotting paper. The grids will likely be hydrophobic, so submerge the grids into the water vertically or they may bend due to water tension. Position the grids in a square array so that they are as close to one another as possible, but not overlapping (Figure 2C).
4. The grids are now ready to be coated with a graphene monolayer. Fill the grid coating trough with more DI water so that the surface of the water is at least 5 mm above the grids.

10. Transfer graphene to the grids

1. Carefully scoop out the MMA/graphene film from the crystallizing dish with a clean coverslip by slowly lowering the coverslip into the trough at an angle, some distance away from the graphene film. Position the coverslip underneath the graphene-MMA sheet so that the edges of the sheet and the coverslip are parallel, and then lift the coverslip vertically out of the water, bringing the MMA/graphene film with it.
2. Transfer the MMA/graphene film to the trough by lowering the coverslip into the water at a ~45° angle, so that the MMA/graphene film detaches from the coverslip and floats on the surface of the water.
3. Position the MMA/graphene film directly above the grids before the water level is lowered. Use a glass Pasteur pipette whose tip has been melted to seal the opening to carefully manipulate the position of the MMA/graphene film.
4. Slowly lower the water level with the syringe, at approximately 1.25 mL/min such that the MMA/graphene film fully covers the grid surfaces as it lands on the filter paper
   NOTE: Further manipulation of the graphene-MMA sheet may be necessary to maintain its position above the grids as the water level drops.
5. Use a pair of clean, dry tweezers to lift the blotting paper holding the grids to a clean, dry, dust-free Petri dish, or transfer the entire stainless-steel platform.
6. Air-dry the MMA/graphene grids for at least 30 min under the fume hood. Keep the grids covered with aluminum foil or a lid to prevent any contamination from dust particles.
7. Transfer the grids to an incubator and bake the grids in a 65 °C incubator for 30 min.
8. Remove the grids from the incubator and leave them covered for 5 min at room temperature to cool the grids to the room temperature.

11. Removing MMA and cleaning the grids

NOTE: MMA must be thoroughly washed off in acetone to prevent any residual MMA on the graphene-coated grids.

1. In a fume hood, prepare two crystallizing dishes containing 200 mL of acetone and one crystallizing dish containing 200 mL of isopropanol (IPA) at room temperature. Cover the crystallizing dishes with aluminum foil to minimize the evaporation of the organic solvents.
   CAUTION: Wear PPE when handling acetone as it can cause skin irritation and can be harmful if inhaled.
2. Place the base of the clamping TEM grid holder block at the bottom of the first crystallizing dish containing 200 mL of fresh acetone.
3. Transfer each grid individually from the blotting paper to a well in the grid holder block, ensuring that the MMA side is facing up. Cover the crystallizing dish with foil, place it on an orbital shaker, and gently shake for 30 min.
4. Place the metal lid on the grid holder block, which will secure the grids during the transfer to the next crystallizing dish. Carefully lift the grid holder block out of the crystallizing dish with a bent fork and long tweezers and place it at the bottom of the second crystallizing dish containing 200 mL of fresh acetone. Remove the lid from the grid holder block and gently shake on an orbital shaker for 30 min.
5. Place the lid on the grid holder block and transfer to the crystallizing dish containing 200 mL of IPA solution to clean off acetone residue. Remove the lid from the grid holder block and gently shake for 20 min.
6. Transfer the grids to a small glass Petri dish covered with blotting paper and air-dry the grids for at least 10 min. Keep the grids covered with a lid to prevent any contamination from dust particles.
7. Grids are ready to be used or transferred to a grid box wrapped with aluminum foil inside a vacuum desiccator.
   ​NOTE: Storing graphene grids inside a vacuum desiccator can prevent contamination of hydrophobic particles from ambient conditions. These grids can be stored for up to several months before use²⁷.

12. Render graphene grids hydrophilic with UV/Ozone treatment

NOTE: Graphene is extremely hydrophobic, which is not compatible with cryoEM sample preparation, since blot-plunge approaches require a hydrophilic surface upon which a drop of sample can spread evenly. While traditional glow-discharge devices can be configured to gently pulse plasma to make the graphene surface hydrophilic, these devices tend to destroy the thin graphene monolayer. It was previously shown that a UV/ozone cleaner can be used to partially oxygenate the surface of the graphene²⁵, rendering it hydrophilic for cryoEM sample preparation without damaging the monolayer.

1. If using a UV/Ozone system that requires priming, turn on the system and prime the lamp for 10 min (at this step, ensure that no grids are exposed). While the UV/ozone cleaner is priming, remove grids from the vacuum desiccator and transfer them to a clean coverslip.
2. When the UV/Ozone system is ready, place the coverslip containing the grids with the graphene side facing up into the UV/ozone cleaner, and expose the grids to the ozone gas for 4 min.
3. After exposure to ozone, use the grids immediately for cryoEM sample preparation.
   NOTE: If using a UV/Ozone system that requires priming, grids must be placed in the cleaner immediately after the lamp has been primed for 10 min, or it will be too cool to produce sufficient ozone gas to oxygenate the graphene for sample preparation. Do not expose the grids to the ozone gas for more than 6 min, as it will destroy the graphene layer.