Method, apparatus, and functionalized graphene product for functionalizing graphene.

Abstract

A method for functionalizing graphene on a substrate is provided, the method comprising exposing a substrate having a graphene layer to a plasma comprising helium and at least one functionalizing compound, as well as an apparatus for carrying out the method and the products formed thereby.

Description

[Technical Field]

[0001] The present invention relates to methods for functionalizing graphene, apparatus for carrying out such functionalization, and products characterized by such functionalization, and more particularly, but not exclusively, to their application in the field of electron microscopy, especially cryo-electron microscopy. [Background technology]

[0002] Recent technological advancements have made it possible, in principle, to determine the atomic-resolution structure of purified biomolecular complexes using single-particle cryo-electron microscopy (cryo-EMS). In practice, however, preparing frozen samples suitable for high-resolution imaging remains the biggest obstacle to structural determination.

[0003] High-resolution structural studies using cryo-electron microscopy require embedding biological samples in a thin film of amorphous water ice. 1、2 During preparation and cryoplunging, the sample is exposed to the surface of this thin film of water and may be subject to harmful interactions at the air-water interface just before vitrification. 1、3 These interactions can be improved with the help of a thin film covering the sample support. 4 This provides a contact surface for the sample, but typically involves background noise, motion during imaging, contamination, and the introduction of uncontrolled sample-surface forces.

[0004] Monolayer graphene has been proposed as a possible support film. 5、6、7、8 However, pure graphene is not a suitable substrate for preparing cryo-electron microscopy samples due to its hydrophobicity. Previous studies have achieved hydrophilicity without destroying the graphene lattice by partially hydrogenating it using low-energy plasma, making it suitable for cryo-electron microscopy. 8 . [Overview of the project] [Problems that the invention aims to solve]

[0005] Improvements to the supports and substrates used for cryo-electron microscopy samples, as well as appropriate manufacturing methods for them, are needed.

[0006] This invention was conceived in light of the above considerations. [Means for solving the problem]

[0007] The present invention is based on the insight that by introducing functionalization to graphene or graphene oxide films, surface properties can be tuned for specific applications in the field of electron microscopy or cryo-electron microscopy. Furthermore, the inventors have discovered that using helium plasma as a carrier for functionalizing compounds provides relatively mild conditions that avoid damage to graphene during the plasma treatment / functionalization process.

[0008] Therefore, a novel multifunctional sample support for electron microscopy, such as cryo-electron microscopy, is presented. This support consists of, for example, a single layer of large crystalline graphene or graphene oxide suspended on the surface of a superstable gold sample support. Using a low-energy plasma surface modification system, the surface of this support can be tailored to the sample by patterning a series of covalent functionalizations across the graphene or graphene oxide layers on a single grid. This support design reduces sample movement during imaging, improves image quality, and enables high-resolution structural determination with minimal material and data.

[0009] Therefore, an apparatus and method for performing such functionalization are also presented.

[0010] In one embodiment, a method is provided for functionalizing graphene or graphene oxide on a substrate for use in electron microscopy, for example, cryo-electron microscopy, the method comprising the steps of: (a) exposing a substrate having a layer of graphene or graphene oxide thereon to a plasma containing helium and at least one functionalizing compound.

[0011] In a further aspect, a method for polyfunctionalizing graphene or graphene oxide on a substrate for use in electron microscopy methods, such as cryo-electron microscopy, is provided, the method comprising the following steps: (a) masking a region of the substrate having a layer of graphene or graphene oxide; (b) exposing the unmasked region to a plasma comprising helium and at least one functionalizing compound to form a functionalized zone; optionally (c) removing the mask from the substrate; (d) repeating steps (a) to (c), using a different functionalizing compound in each repetition, to form a substrate having a graphene layer with a plurality of zones having different functionalizations.

[0012] Thus, the method of the present invention may include functionalizing as defined herein and then using the functionalized substrate in an electron microscopy method, such as cryo-electron microscopy.

[0013] In such a method, the exposure time of the substrate to the plasma may be controlled using a shutter that is fixed to cover the substrate and is movable between an open position that exposes the substrate and a closed position that hides the substrate. This allows the user to more finely control factors such as the exposure time and thus the degree of functionalization.

[0014] In the method of the present invention, the functionalizing compound or compounds may be one or more selected from compounds of the following general formula (I): JPEG0007874967000001.jpg27109 where each of R 1 and R 2 is independently selected from the group consisting of H, halo (e.g., -F, -Cl, -Br), thiol (-SH), amino (-NH2), carboxyl (-COOH) and hydroxyphenyl (-C6H4OH); one of R 1 and R 2 is not H; and n is an integer from 1 to 10.

[0015] In the method of the present invention, the functionalized compound or each functionalized compound may be one or more selected from the group consisting of amylamine, hexanoic acid, 1-pentanethiol, and 4-pentylphenol.

[0016] The method described herein may further include a step of exposing a substrate having a graphene layer or graphene oxide layer thereon to a hydrogen-containing plasma to hydrogenate the graphene layer or graphene oxide layer, or a portion of the graphene layer or graphene oxide layer. This step can be performed before or after the functionalization of the graphene or graphene oxide layer to provide or complement the hydrophilicity of the graphene layer or graphene oxide layer.

[0017] In some embodiments, the substrate is a gold cryo-electron microscope grid.

[0018] Apparatus for carrying out the methods described herein is provided.

[0019] In a further embodiment, the present invention optionally provides an apparatus for functionalizing graphene or graphene oxide by the method described herein, the apparatus comprising: a plasma generator; a processing chamber connected to the plasma generator; a substrate holder positioned within the processing chamber to hold a substrate for electron microscopy, such as cryo-electron microscopy, having a graphene layer or graphene oxide layer thereon, and having a mounting area configured for mounting the substrate thereon; an injector for injecting a functionalizing compound into the processing chamber; and a helium source for supplying helium to the plasma generator; and optionally, a shutter movable between an open position in which the mounting area of ​​the substrate holder is exposed into the processing chamber and a closed position in which the mounting area of ​​the substrate holder is concealed.

[0020] The apparatus may be configured such that, during use, helium is supplied to a plasma generator to generate a helium plasma, a functionalized compound is injected into it to form a mixture, and a substrate held in a substrate holder is exposed to the mixture in a processing chamber.

[0021] In some embodiments, the apparatus further includes a mask for masking areas of the substrate. This allows for more complex patterning of the functionalized substrate.

[0022] The mask may, for example, be movable while plasma is present in the processing chamber. This allows for complex patterning, such as generating a "gradation" of functionalization by moving the mask in a predetermined direction at a predetermined speed.

[0023] The mask may be removable from the rest of the device. This allows it to be placed on the substrate outside the rest of the device, for example, in a wider environment.

[0024] The apparatus is preferably configured such that the mask does not come into contact with the graphene layer or graphene oxide layer of the substrate when the substrate is held in the substrate holder. This eliminates the risk of the mask damaging the substrate. For example, the apparatus may be configured such that the mask is located less than 200 μm from the surface of the substrate when the substrate is held in the substrate holder.

[0025] The apparatus may further include a hydrogen source for supplying hydrogen to the plasma generator. This allows for not only partial hydrogenation but also functionalization of graphene or graphene oxide in a single apparatus.

[0026] The apparatus may further include a viewport opening into the processing chamber and a UV-vis spectrometer configured to analyze radiation emitted from the processing chamber through the viewport.

[0027] The apparatus may further include one or more functional compound sources, each connected to an injector, for supplying a functional compound or a group of functional compounds to the injector.

[0028] The apparatus may be equipped with multiple injectors, each of which may be connected to a functional compound source in order to supply the functional compound to its respective injector.

[0029] The injector or multiple injectors may be a gas injector or multiple gas injectors.

[0030] Furthermore, the present invention relates to a sample support for electron microscopy, such as cryo-electron microscopy, comprising a metal foil, such as a gold substrate, and a functionalized graphene layer or functionalized graphene oxide layer on the substrate, wherein the graphene or graphene oxide has a plurality of different functionalizations. The graphene or graphene oxide may be partially hydrogenated.

[0031] The present invention includes combinations of the embodiments and configurations described, unless the combination is clearly not permitted or explicitly avoided. [Brief explanation of the drawing]

[0032] Next, embodiments and experiments illustrating the principle of the present invention will be described with reference to the attached drawings.

[0033] [Figure 1a] Figure 1a shows the effects of residual air glow discharge and helium plasma on a single-layer suspended graphene. [Figure 1b] Figure 1b shows the effects of residual air glow discharge and helium plasma on a single layer of suspended graphene. [Figure 1c] Figure 1c shows the effects of residual air glow discharge and helium plasma on a single-layer suspended graphene. [Figure 2a] Figure 2a shows experimental data regarding the wettability of functionalized graphene and the control of the orientation distribution of 20S proteasomes. [Figure 2b] Figure 2b shows experimental data regarding the wettability of functionalized graphene and the control of the orientation distribution of 20S proteasomes. [Figure 2c] Figure 2c shows experimental data regarding the wettability of functionalized graphene and the control of the orientation distribution of 20S proteasomes. [Figure 2d] Figure 2d shows experimental data regarding the wettability of functionalized graphene and the control of the orientation distribution of 20S proteasomes. [Figure 2e] Figure 2e shows experimental data regarding the wettability of functionalized graphene and the control of the orientation distribution of 20S proteasomes. [Figure 2f] Figure 2f shows experimental data regarding the wettability of functionalized graphene and the control of the orientation distribution of 20S proteasomes. [Figure 3] Figure 3 shows the TEM analysis results of polyfunctional graphene on a gold support using a grid square covered with amorphous carbon for electron microscope alignment. [Figure 4] Figure 4 shows the Mollweide projection orientation distribution of 30S ribosomes functionalized with hexanoic acid (Figure 4(a)), 1-pentanethiol (Figure 4(b)) entirely transported by helium plasma, amylamine (Figure 4(c)), and 4-pentylphenol (Figure 4(d)), with partial hydrogenation preceding the functionalization, on partially hydrogenated graphene (Figure 4(e)), and on graphene placed unsupported in ice (Figure 4(f)). [Figure 5] Figure 5 shows (a) a diagram of the sample on graphene on a gold support, and (b) a longitudinal section of a tomographic reconstruction of a pore covered with functionalized graphene containing 30S ribosomes in glassy ice. The ice thickness is 300 ± 10 Å, slightly larger than the particle size. All particles are in a single layer. [Figure 6] Figure 6 shows data regarding improvements in efficiency and resolution by optimizing the orientation distribution of 30S ribosome subunits on functionalized graphene. [Figure 7]Figure 7 shows the high-resolution structural determination of apoferritin on graphene on a gold support. Figure 7(a) is a representative micrograph of apoferritin particles contained in glassy ice on amylamine-functionalized graphene. The scale bar is 500 Å. Figure 7(b) is the Fourier transform of the motion-corrected micrograph from Figure 7(a). [Figure 8] Figure 8 shows the RMS (root mean square) displacement of apoferritin particles during irradiation in unsupported ice on a carbon grid and on graphene on gold. [Figure 9] Figure 9 shows an example of reduced migration of a biological sample on an all-gold grid coated with graphene. The root-mean-square (RMS) displacement of MW2MDa ribosomes is plotted against electron fluence. [Figure 10a] Figure 10a shows the reduced movement of nanoparticles on an all-gold grid coated with graphene. [Figure 10b] Figure 10b shows the reduced movement of nanoparticles on an all-gold grid coated with graphene. [Figure 10c] Figure 10c shows the reduced movement of nanoparticles on an all-gold grid coated with graphene. [Figure 10d] Figure 10d shows the reduced movement of nanoparticles on an all-gold grid coated with graphene. [Figure 10e] Figure 10e shows the reduced movement of nanoparticles on an all-gold grid coated with graphene. [Figure 10f] Figure 10f shows the reduced movement of nanoparticles on an all-gold grid coated with graphene. [Figure 11] Figure 11 schematically shows (a) a gold-on-gold substrate with a graphene layer formed on it as a support, (b) a grid square covered with a suspended graphene film, and (c) multiple batches of graphene-coated all-gold grids. [Figure 12] Figure 12 shows (a) a schematic diagram of the design of an inductively coupled plasma apparatus for covalently functionalizing graphene; (b) a slotted grid holder; and (c) a design of a non-contact knife-edge mask for patterning the plasma-functionalized graphene grid. [Figure 12d] Figure 12d shows the optical spectra when a pure helium carrier plasma (black) and a vapor of a functionalizing chemical are introduced. [Figure 12e] Figure 12e shows the XPS spectrum of functionalized graphene exhibiting chemical modification. [Figure 13] Figure 13 shows (a)–(b) the orientation distribution of 30S ribosome subunits on two different graphene functionalizations from a single patterned grid plotted using the Mollweide projection, and (c)–(f) the surface of 30S ribosome subunits shaded by the Coulomb electrostatic potential. [Figure 14] Figure 14 shows (a) a contour density map of apoferritin showing amino acid side chains within the structure, (b) Fourier shell correlation (FSC) of the apoferritin structure, (c) mean square displacement of unsupported gold nanoparticles in ice (thin circles) or mean square displacement of gold nanoparticles on graphene on an all-gold support in ice (dark cross), and (d) the B factor calculated as a function of electron fluence for the apoferritin dataset. [Modes for carrying out the invention]

[0034] Aspects and embodiments of the present invention will be described with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents referenced herein are incorporated herein by reference.

[0035] (Graphene) Graphene as a material is well known in this field for its manufacturing methods. The main known methods are "top-down" or "bottom-up" synthesis, which broadly involve exfoliating layers from graphite or depositing carbon structures to synthesize graphene structures, for example, by chemical vapor deposition (CVD).

[0036] In this specification, when discussing graphene layers, it is assumed that either a single layer or multiple layers of graphene are included. For example, a graphene layer may be a single layer (mono) of graphene, or it may consist of a graphene structure of two to five layers.

[0037] The graphene is preferably a single layer. Such graphene becomes nearly invisible within the resolution range targeted by cryo-electromagnetism.

[0038] As described herein, the functionalized graphene layer is located on a substrate that may have pores itself, for example, on perforated gold foil on a gold mesh or grid. In such cases, it is preferable that each graphene crystal within the layer is larger than one opening or square of the mesh / grid. For example, each graphene crystal may have a minimum two-dimensional dimension (not thickness) of at least about 50 μm. Also, each graphene crystal may have a hexagonal shape.

[0039] If the substrate has pores within it, the graphene is not, of course, directly "sitting" on top of the substrate material of those pores. In such cases, we can say that the graphene is suspended. That is, if the substrate has pores, the graphene layer straddles those pores, making contact with the substrate in some places but not in others.

[0040] Reducing the number of graphene grain boundaries in the graphene layer can be advantageous. For example, this may improve the mechanical and chemical robustness of the graphene layer during the functionalization process (described herein) and cryo-electron microscopy imaging.

[0041] The methods, apparatus, applications, and products of the present invention may utilize a graphene oxide layer instead of graphene. That is, the methods of the present invention may be applied to functionalize a graphene oxide layer on a substrate, and the products of the present invention may include a functionalized graphene oxide layer, and so on; there are various embodiments. All preferred configurations and options discussed with respect to graphene can be applied to embodiments of graphene oxide as appropriate.

[0042] (functionalization) The graphene layer described herein is functionalized. The inventors have found numerous advantages associated with such functionalization.

[0043] Pure graphene, due to its hydrophobicity, is not a suitable substrate for preparing cryo-electron microscopy samples. Previous studies have proposed partial hydrogenation using low-energy plasma as a method to make graphene hydrophilic without damaging the lattice, thus making it suitable for cryo-electron microscopy. However, the inventors have found that partially hydrogenated graphene yields only one type of adhesion surface, which is insufficient for all possible samples. By introducing and controlling functionalization, various samples can be supported in various spatial orientations, significantly improving the efficiency of the cryo-electron microscopy analysis process.

[0044] Furthermore, knowing how purified proteins interact with other well-defined surfaces, such as those present in ion-exchange columns, helps guide appropriate functionalization and buffering conditions for specific cryo-electron microscopy samples. Functionalized graphene is not only a tunable surface for improving sample orientation in cryo-electron microscopy, but also provides a novel method for mapping and quantifying the interaction surfaces of specific biomolecules.

[0045] Functionalized graphene surfaces can be used to orient and disperse biomolecules within thin, glassy ice films, and also reduce sample movement to improve image quality.

[0046] In this specification, "functionalized" graphene refers to graphene in which one or more chemical groups other than hydrogen are covalently bonded to its structure. These groups can be called functionalizations. In many cases, these groups are derived from hydrocarbon-based compounds in which a hydrogen atom has been removed at the site where they attach to / bond to the graphene structure. In other words, functionalized compounds generally correspond to the desired functionalization where H- is present at the binding site of graphene.

[0047] In this specification, the functionalized compounds and their corresponding functionalizations are not particularly limited and can be selected according to the desired effect of the functionalized graphene and the vapor pressure of the functionalized compound.

[0048] When using the plasma functionalization technique described here, in principle, any low-molecular-weight compound can be functionalized, as long as it has sufficient vapor pressure to be injected into the chamber. It should be noted that since the orientation and length of the functional groups are not particularly controlled, functional fragments may be present on the surface. In other words, even when using a single functionalizing compound, the specific functionalization may not be uniform.

[0049] Possible examples are shown in Table 1 below, but of course, variations of these are also conceivable.

[0050] JPEG0007874967000002.jpg79146

[0051] Table 1 shows the binding site to graphene. This is represented by JPEG0007874967000003.jpg3230.

[0052] For example, a functionalized compound is a hydrocarbon in which one or more positions are optionally substituted with functional groups, preferably reactive functional groups.

[0053] The functionalized compound is a C molecule optionally substituted with one or more groups selected from, for example, halos (e.g., -F, -Cl, -Br), thiols (-SH), aminos (-NH2), carboxyls (-COOH), or hydroxyphenyls (-C6H4OH). 1-10 Alkyl, preferably C 3-6 Alternatively, it may be a C5 alkyl group. Furthermore, it may be a compound with a vapor pressure of 0.01 to 50 Torr at 25°C.

[0054] In practice, the vapor pressure of a compound can be increased or decreased to an appropriate level by heating or cooling the compound and / or the injection system. A suitable temperature range for heating or cooling it is from -60°C to +200°C.

[0055] In embodiments focusing on cryo-electron microscopy, suitable functionalized compounds include those having a terminal first functional group with properties similar to amino acid side chains that biomolecules may encounter in vivo. Such functional groups include amines, thiols, carboxyls, phosphates, sugars, and other functional groups present in the cell's native aqueous environment. The compound may also have a second functional group that can react with graphene to form an SP3 (covalent) bond. The second functional group may be unresponsive to the first functional group. The second functional group may also be terminal.

[0056] These two functional groups, if both are present, may be separated by a hydrocarbon chain (preferably saturated), which separates the first functional group from the graphene surface but is otherwise non-reactive. The length of the chain is limited by the vapor pressure. As the chain length increases, the vapor pressure decreases, eventually becoming too low to be injected into the chamber even with heating. Therefore, the optimal chain length is one in which the compound is separated to some extent from the graphene surface, but whose vapor pressure is within a reasonable range for injection into the plasma chamber.

[0057] A functionalized compound or a group of functionalized compounds is defined by the following general formula (I): You can select one or more compounds from JPEG0007874967000004.jpg27109, in the formula, R 1 and R 2 Each of these is independently selected from the group consisting of H, halos (e.g., -F, -Cl, -Br), thiols (-SH), aminos (-NH2), carboxyls (-COOH), and hydroxyphenyls (-C6H4OH); R 1 and R 2 One of them is not H; and n is an integer between 1 and 10.

[0058] In some embodiments, n is an integer between 3 and 6, for example, 5.

[0059] In some embodiments, R 2 H is R 1 The group is selected from the group consisting of halos (e.g., -F, -Cl, -Br), thiols (-SH), aminos (-NH2), carboxyls (-COOH), and hydroxyphenyls (-C6H4OH).

[0060] In some embodiments, R 2 H is H.

[0061] In some embodiments, R 1 R is selected from carboxyl or thiol. In some embodiments, R 1 It is a thiol.

[0062] In functionalized graphene, for example, 0.001 to 10% of the carbon atoms in the graphene structure are bonded to the functionalization, meaning that graphene may be 0.001 to 10% functionalized, or for example, 0.1 to 1% functionalized.

[0063] Functionalization is performed on graphene at 1 nm 2 The functionalization density can be approximately 0.5 to 5 particles per unit, or for example, 1 to 2 particles.

[0064] Functionalization with reactive group-containing compounds, such as thiol-containing compounds, is sometimes preferable because the thiol group undergoes further reactions, making it easier to bond more complex structures to the functionalized graphene. For example, it may be desirable to use the short, flexible hydrocarbon chains mentioned above to bond specific ligands or known bonding groups to the graphene surface.

[0065] Graphene in which hydrogen atoms are covalently bonded to the structure is sometimes called hydrogenated graphene. Generally, not all sites bond to hydrogen, so the level of hydrogenation is partial. Partially hydrogenated graphene is known in this field. Here, when discussing the partial hydrogenation of graphene, preferably, about 0.001 to 10% of the carbon atoms in the graphene structure are bonded to hydrogen, i.e., graphene can be hydrogenated to 0.001 to 10%, for example, 0.1 to 1%.

[0066] Under certain conditions, partial hydrogenation is preferable. Partially hydrogenated graphene is more hydrophilic than pure graphene. Such hydrophilic behavior can be desirable. Therefore, if graphene has at least one non-hydrophilic (i.e., hydrophobic) functionality, partial hydrogenation may be preferable.

[0067] For example, when graphene is functionalized with functional groups derived from carboxyl or thiol, it tends to become hydrophobic; therefore, partial hydrogenation helps to make the functionalized graphene hydrophilic.

[0068] This type of hydrophilicity is particularly useful in the field of cryo-electron microscopy, which analyzes samples in water.

[0069] Functionalized graphene may have one or more functionalizations and may optionally be partially hydrogenated. Graphene having more than one functionalization may be called polyfunctional or polyfunctionalized. Functionalized graphene may or may not be partially hydrogenated.

[0070] For example, functionalized graphene may be functionalized with one or more (or exactly one), two or more (or exactly two), three or more (or exactly three), or four or more (or exactly four) functionalizations. These may be selected from the functionalizations shown in Table 1 above.

[0071] It is preferable that the functionalized graphene is divided into several zones or regions, with each zone or region having a different level, type, or combination of functionalization. In this way, a single substrate having a functionalized graphene layer can be used to "hold" biological samples in various configurations, for example, by appropriately selecting different functionalizations and hydrogenations in different zones.

[0072] For example, a substrate having a graphene layer can be divided into four zones or regions: a first zone having a first level and type of functionalization and (partial) hydrogenation, a second zone having a second level and type of functionalization and (partial) hydrogenation, a third zone having a third level and type of functionalization and (partial) hydrogenation, and a fourth zone having a fourth level and type of functionalization and (partial) hydrogenation.

[0073] In this zone configuration, it will be understood that all of the aforementioned functionalization and hydrogenation options and possibilities apply both within each zone and to the functionalized graphene as a whole.

[0074] Such polyfunctionalization, particularly zone functionalization, is advantageous because it means that grids are possible where each square has different functionalization and surface properties or linker chemistry. For example, it means that multiple conditions can be tested simultaneously on the same biological sample on a single grid.

[0075] (substrate) In one aspect, the present invention relates to a substrate having a functionalized graphene (or graphene oxide) layer thereon, for example, a metal, particularly gold, substrate having such a layer. As described herein, the layer and its functionalization can be varied without particular limitation with respect to the substrate.

[0076] [Here, the term “support” is used to refer to the combination of the substrate and the graphene layer; in some embodiments, it refers to the article on which the sample for cryo-electron microscopy analysis is placed.]

[0077] In embodiments of the present invention, the substrate can be, for example, a metallic article such as nickel, copper, or gold, such as foil. In particular, the substrate can be a gold-based substrate.

[0078] As described herein, certain types of substrates known to support cryo-electron microscopy specimens may be particularly preferred. Several examples are known in the art, but here we will discuss gold-based substrates. Such substrates have stability 10 It is preferable in terms of structural integrity and tensile strength, and also in that it provides good electrical contact to graphene even at cryogenic temperatures.

[0079] Such substrates may be of the "gold-on-gold" or "all-gold" type. These substrates contain gold mesh in any (but generally flat) shape, such as a disc or a rectangular plate. Each such mesh is, for example, a disc with a diameter of 1-5 mm, or a plate with sides of 1-5 mm in length. This mesh has, for example, 100-500 lines per inch. This results in, for example, a continuous series of roughly square or hexagonal openings. This is sometimes called a "grid."

[0080] A layer of gold foil is placed on top of the gold mesh (hence the term "gold on gold"). This foil itself spans each opening in the mesh. The foil itself has pores within it, each pore having a size of 1 μm or less, for example, a diameter of 0.01 to 0.5 μm. The foil is thin, for example, with a thickness of 100 to 1000 Å.

[0081] Such substrates are well known in this field. In this invention, a functionalized graphene layer or a functionalized graphene oxide layer can be added on or below the gold foil, and a sample for cryo-electron microscopy analysis can be placed on top of it.

[0082] An example is shown in Figure 11. Figure 11(a) shows an example of a gold-on-gold substrate containing a 3 mm disc-shaped 300-wire / inch gold mesh ("Au grid"), in which a gold foil ("Au foil") approximately 500 Å thick with regularly arranged submicron-sized pores is suspended in the square openings of the mesh. A graphene layer (here a single layer) is deposited on top of the gold foil, covering all the pores ("holes"). As described herein, graphene can be covalently functionalized by exposure to a low-energy helium plasma containing hydrocarbon precursor molecules ("graphene," where dots represent different functional groups). A sample (material for cryo-electron microscopy analysis) is applied to the graphene side of the grid and vitrified to form a continuous ice layer that can be imaged through the pores.

[0083] Section (b) of Figure 11 shows the grid squares covered with a suspended graphene film (secondary electron microscope image). The scale bar is 10 μm. The inset shows a completely covered pore (top) and a pore where the graphene has been destroyed (bottom), both magnified 5 times (scale bar is 1 μm). As shown in Figure 11(c), more than 150 all-gold grids with over 95% graphene coverage are obtained in a single growth-transfer batch (scale bar is 10 mm).

[0084] (Methods and apparatus for functionalization) The inventors have developed a method for creating a substrate having a functionalized graphene (or graphene oxide) layer thereon, and an apparatus for carrying out such a method. The substrate, graphene, and functionalization may, of course, be as described herein.

[0085] In particular, the inventors found that by using helium (an inert gas with a low atomic number) as the primary plasma gas in a remote plasma generator, sputter damage and other alterations to graphene are prevented, while chemical reactions between graphene and chemical precursors (functionalized compounds) are possible. This is in contrast to typical residual air glow discharge systems used in cryo-electron microscopy, which generate plasma with sufficient energy to destroy suspended monolayer graphene even with short exposure times.

[0086] This is shown in Figure 1. Figure 1 shows the effects of residual air glow discharge and helium plasma on a single-layer suspended graphene. It shows scanning electron microscope images of an all-gold grid coated with graphene after exposure to residual air glow discharge (PELCO easiGlow, negative bias, 15 mA, 0.3 Torr) for 45 seconds (Figure 1(a)), after exposure to low-power helium plasma (Fischione 1070, forward power 9 W, 1 Torr) for 45 seconds (Figure 1(b)), and after exposure to the same helium plasma for 4500 seconds (Figure 1(c)). The scale bar is 10 μm.

[0087] During exposure, the grid is covered with a non-contact knife-edge half-mask, and the approximate location of the mask edge is indicated by a dashed line. Graphene exposed to glow discharge is destroyed, and pores not covered by the gold foil appear black (see Figure 1(a) right). Helium plasma irradiation for more than 100 times the typical time required for functionalization does not affect the structural integrity of the graphene film. The shadow of the mask edge is visible in the central square of Figure 1(a), indicating that the patterning resolution of this masking method is less than 50 μm. The inset is a 5x magnified view of the square area. The scale bar in the inset is 2 μm.

[0088] Therefore, the inventors have discovered that helium (He) is an excellent plasma carrier for functionalizing graphene with functionalizing compounds, particularly for the graphene layer of cryo-electron microscope substrates. This plasma is relatively gentle as it does not damage or react with the graphene layer, while simultaneously having sufficient energy to realize functionalization by the functionalizing compound.

[0089] Plasma containing only functionalizing compounds is excessively aggressive and may not only impair the integrity of the graphene layer but also fail to achieve the desired functionalization. Furthermore, using such plasma can lead to the deposition of polymer byproducts within the reaction chamber (depending on the functionalizing compounds present). Similarly, hydrogen plasma is not very suitable because it reacts with the graphene layer and hydrogenates it.

[0090] Therefore, in the functionalization treatment described herein, it is preferable that helium is the main plasma component. That is, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the atmosphere used for treating the graphene layer on the substrate may be helium plasma.

[0091] The output of the plasma used in this functionalization process may be, for example, 0.1 to 100 watts, 1 to 20 watts, or 5 to 15 watts.

[0092] The remaining portion may include, for example, a hydrogen plasma that influences partial hydrogenation, and one or more functionalized compounds as described above. These compounds may be in the form of vapor, gas, or plasma. In this example, the helium plasma effectively dilutes the other reactive components.

[0093] The processing time and conditions can be appropriately selected according to the desired level of functionalization. For example, an exposure time of 10 to 60 seconds to the functionalization plasma (He plasma + one or more functionalized compounds) may be appropriate.

[0094] For partial hydrogenation, hydrogen plasma can be used. 8 Hydrogen plasma may be used alone or in combination with helium plasma. In this case as well, the processing time and conditions should be appropriately selected according to the desired hydrogenation level. For example, to hydrophilize a graphene layer, an exposure time to hydrogen plasma of 30 to 300 seconds, for example, 150 to 210 seconds, may be appropriate.

[0095] Typical plasma pressures are 0.1–10 Torr, for example, 0.5–1 Torr. This applies to any plasma described herein, but the appropriate pressure can be selected depending on the plasma used, the functionalizing compound added, the intended effect on the graphene layer, etc.

[0096] Hydrogenation treatment using such hydrogen-containing plasma can be performed before or after functionalization treatment using helium plasma and one or more functionalized compounds. In some embodiments, it is performed before the helium plasma treatment.

[0097] The inventors have discovered that by selectively masking and unmasking regions of a substrate, multiple functionalizations can be easily achieved, and different zones of functionalization can be precisely controlled. By masking a region before a predetermined plasma treatment, the unmasked regions are affected. Then, the mask can be removed, i.e., the mask can be removed from the substrate, another mask can be applied, and then another plasma treatment can be performed. The same mask may be used, or different masks may be used. Alternatively, additional masks can be added to the previous mask to perform different plasma treatments, and different treatments can be performed depending on the order in which the masks are used.

[0098] Another possibility is that the mask is movable during the functionalization process. Depending on the design of the mask, it may be possible to enable specific functionalization in a limited area or pattern (for example, if the mask is essentially a hole that moves across the substrate, paths on the substrate can be selectively exposed to functionalization). This makes it possible to "write" one or more functionalization paths or patterns onto the substrate.

[0099] Other mask shapes moving across the substrate during functionalization can, for example, induce a gradual change in the degree of functionalization on the substrate. Areas initially uncovered by the mask will be exposed to the functionalization process for a longer period than areas exposed for a shorter time, resulting in a gradient in the degree of functionalization. Many other variations are easily conceivable.

[0100] The mask may be moved continuously during the functionalization process. Alternatively, the mask may be moved in stages to produce a more pronounced change in functionalization.

[0101] Accordingly, this specification describes a method for functionalizing suspended graphene or graphene oxide on a substrate, the method comprising the steps of: (a) masking a region of the substrate having a graphene layer or graphene oxide layer; (b) exposing an unmasked region to a plasma containing helium and at least one functionalizing compound to form a functionalized region; and (c) modifying the masking of the substrate, for example, by moving, adding, or replacing a mask. Step (c) may be performed during or after step (b). This may be followed by a further step of repeating (b) with a different functionalizing compound.

[0102] By repeating this method, a desired series of zones can be constructed using simple or complex masks as needed, while varying the functionalization and / or hydrogenation. Multiple exposures enable multi-axial patterning and sequential patterning with multiple functionalizations applied to the same region.

[0103] Furthermore, using this same patterning method, it becomes possible to directly deposit, for example, amorphous carbon into a 100 μm diameter region in the center of the substrate. This is useful for astigmatism correction and coma adjustment on a multifunctional grid prior to high-resolution data acquisition.

[0104] The mask itself can have any shape chosen for proper masking. For example, the mask may be a half-mask covering 50% of the substrate, for example, one complete half-surface. One mask can be used, removed, and then reapplied in a different orientation; or a new or differently shaped mask can be used.

[0105] The mask may have at least one "knife edge," that is, it is sharpened so that it gradually thins from the main thickness towards the edge. The knife edge is preferably located on or across the substrate. An example is shown in Figure 12(c).

[0106] The mask can be placed slightly above the substrate surface, for example, less than 200 μm from the substrate surface, for example, at approximately 20-100 μm. As the distance increases, the boundary of the plasma treatment becomes less distinct; therefore, increasing the distance from the substrate to the mask allows for smoother surface patterning transitions or gradient functionalization. Of course, if a clearer boundary between the treated and untreated areas is desired, the distance can be shortened.

[0107] With a typical plasma process pressure of approximately 1 Torr and a distance of approximately 50 μm between the mask and the surface, the resolution of this patterning method is approximately 50 μm or 1 grid square.

[0108] Preferably, when applying the mask, the mask does not come into contact with the graphene layer on the substrate. This minimizes the risk of damage.

[0109] The mask itself may be plate-like, with a series of holes or designs cut into or patterned onto it. These holes are placed on the substrate to be used to form the desired masking pattern. Some or each of these holes may have a knife edge.

[0110] For example, in Figures 12(b) and (c), it can be seen that in order to prepare 10 half-masks for 10 disc-shaped substrates, there are 10 masks with crescent-shaped (semicircular) holes.

[0111] Figure 12 illustrates aspects of the functionalization and patterning of covalently bonded graphene using low-energy helium plasma. Figure 12(a) schematically shows the design of an inductively coupled plasma apparatus for covalently functionalizing graphene. Figure 12(b) schematically shows how a slotted grid holder securely and accurately positions up to 10 grids under a single mask plate. A retractable shutter is used to control the exposure time independently of establishing stable plasma reaction conditions within the chamber. Figure 12(c) shows the design of a non-contact knife-edge mask for patterning graphene grids using plasma-assisted functionalization. Masks of arbitrary shapes are possible. Figure 12(d) shows the optical spectrum of a pure helium carrier plasma (lowest spectrum) and the optical spectrum when a vapor of chemicals is introduced for functionalization. The He spectral line is saturated, and other peaks corresponding to the atomic species of the precursor are visible. The Hα spectral line at 656 nm is used to monitor the decomposition of the hydrocarbon precursor in real time. Figure 12(e) shows the XPS spectrum of functionalized graphene exhibiting chemical modification.

[0112] A preferred functionalization apparatus shown in Figure 12(a) can be further described. In its broadest sense, the apparatus comprises a plasma generator, a processing chamber ("plasma chamber") connected to the plasma generator, a substrate holder ("grid holder") having a mounting area configured to hold a substrate having a graphene layer within the processing chamber and to mount the substrate therein, an injector ("gas injection manifold") for injecting a functionalization compound into the processing chamber, and a helium source for supplying helium to the plasma generator. The present invention also provides the use of the apparatus (as described herein) in the functionalization of graphene (or graphene oxide), in particular the functionalization of a graphene (or graphene oxide) layer on a substrate or support as described herein.

[0113] This specification describes the use of an apparatus for functionalizing graphene. The apparatus comprises a plasma generator, a processing chamber connected to the plasma generator, a substrate holder positioned to hold a substrate having a graphene layer within the processing chamber and having a mounting area configured for mounting the substrate therein, an injector for injecting a functionalization compound into the processing chamber, and a helium source for supplying helium to the plasma generator.

[0114] Such a device is configured such that, during use, helium is introduced into the plasma generator, a helium plasma is formed in the processing chamber, a functionalizing compound is injected into the processing chamber, and the graphene layer on the substrate held in the substrate holder is functionalized.

[0115] The apparatus may have various other configurations. For example, it may be equipped with a hydrogen source to supply hydrogen to the plasma generator as needed. The hydrogen source may be connected to the plasma generator so that the hydrogen flows in through the same path as the helium from the helium source, or separate inlet points may be provided for each. Each hydrogen source or helium source may be controlled by a mass flow controller ("MFC").

[0116] This apparatus may optionally be further equipped with an oxygen source for supplying oxygen to the plasma generator. This can be used to generate oxygen plasma for cleaning the processing chamber and apparatus between functionalization processes. Ideally, there should be no substrate in the processing chamber when oxygen is supplied to the plasma generator.

[0117] The oxygen source may be connected to the plasma generator such that the oxygen flows in through the same pathway as the helium from the helium source and / or the hydrogen from the hydrogen source, if present; alternatively, separate inlet points may be provided for each. Each oxygen source present may be controlled by a mass flow controller ("MFC").

[0118] The plasma generator may be, for example, a known type of inductively coupled plasma generator, such as an RF plasma generator. The injector may be, for example, a gas injector, or other injector suitable for the type of functionalized compound to be injected.

[0119] The apparatus may be configured to inject multiple different functionalization compounds. The apparatus may include a source for each functionalization compound. Each functionalization compound source may use a common injector, or each may have its own injector into the processing chamber. If there is a common injector, each functionalization compound source may have control to regulate the flow of the functionalization compound to the shared injector. This allows for good control of the flow of various functionalization compounds and, consequently, the functionalization by various functionalization compounds.

[0120] In the methods, apparatus, and uses described herein, the appropriate flow rate of each functionalized compound to the processing chamber or to the plasma may be, for example, 1 to 1000 sccm, or optionally 10 to 100 sccm.

[0121] In other words, under conditions of 0°C and 100kPa, the flow rate of each functionalized compound is, for example, 1 to 1000 cm³. 3 / min, arbitrarily 10-100cm 3 / min, or 0.1667cm 3 / sec~16.667cm 3 / second, arbitrarily set to 0.1667cm 3 / sec~1.667cm 3 It is per second.

[0122] The device is equipped with a UV-vis spectrometer, which allows observation of the processing chamber through a viewport, such as a sapphire viewport. This enables monitoring of the plasma state within the processing chamber.

[0123] Furthermore, the processing chamber may have known configurations such as a gate valve and exhaust, for example, pump-driven exhaust. The exhaust of the plasma chamber can be restricted by a "butterfly valve" or the like, and the pump speed can be limited to control the pressure inside the plasma (processing chamber) regardless of the flow rate and composition of the input gas. The apparatus and / or exhaust system may also include a real-time feedback loop for pressure, which can be controlled independently of the input flow rate. That is, the exhaust flow rate can be automatically increased or decreased in response to the flow rate of the plasma source (helium and / or hydrogen, or oxygen) and one or more functionalized compound sources into the processing chamber to maintain a desired pressure inside the processing chamber.

[0124] The apparatus may also have a shutter that is movable relative to the mounting area of ​​the substrate holder between an open position in which the mounting area of ​​the substrate holder is exposed to the processing chamber and a closed position in which the mounting area of ​​the substrate holder is hidden. An example of such a shutter is shown in Figure 12(b).

[0125] In other words, the shutter itself may be moved to expose or conceal the mounting area of ​​the substrate holder, and similarly, the substrate holder itself may be moved to expose or conceal the mounting area of ​​the substrate holder. The movement of the shutter and / or the substrate holder may be controlled from outside the processing chamber.

[0126] The substrate holder itself may be configured to hold multiple substrates, as described herein. In the example in Figure 12(b), for example, it holds 10 substrates. The substrate holder itself may also consist of a portion for receiving the substrates (mounting area), a portion for receiving the mask, and a portion for receiving the shutter mechanism. Of course, as described herein, the substrate holder may be formed integrally with the shutter and / or mask.

[0127] When the shutter is closed to conceal the mounting area (and therefore the substrate or part or all of the substrate), the concealed one or more substrates are no longer affected by the plasma atmosphere. By controllingly opening the shutter to expose one or more substrates (i.e., partially or completely exposing the mounting area), functionalization or hydrogenation at the exposed areas can be initiated as desired. This allows for careful control of the time of functionalization or other plasma treatment. For example, one substrate can be exposed for a longer time than another. This provides further options for rapidly creating a large number of substrates with different degrees or types of functionalization or hydrogenation.

[0128] The apparatus itself may include one or more pre-made masks as described herein. In such cases, by simply setting one or more substrates in the substrate holder or its mounting section, the apparatus's mask and optional shutter can be applied to the substrates and used in the processing chamber.

[0129] If the apparatus includes a mask, it may further include means for moving the mask during or between functionalization processes.

[0130] The configurations disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, are adequately represented in their specific forms, or in terms of means for performing the disclosed functions, or methods or processes for obtaining the disclosed results, and such configurations can be used separately or in any combination to realize the present invention in its various forms.

[0131] The present invention has been described in conjunction with the exemplary embodiments described above, and many equivalent modifications and variations will be apparent to those skilled in the art upon this disclosure. Therefore, the exemplary embodiments of the present invention described above are illustrative and not limiting. Various modifications can be made to the embodiments described without departing from the spirit and scope of the invention.

[0132] To avoid any doubt, the theoretical explanations provided here are intended to enhance the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0133] All section headings used in this specification are for organizational purposes only and should not be construed as limiting the subjects described herein.

[0134] Throughout this Spec., including the subsequent claims, unless otherwise required by context, the words “comprise” and “include,” as well as variations such as “comprises,” “comprising,” and “including,” are understood to mean to include the integer or process, or group of integers or processes, described, but not to exclude other integers or processes, or groups of integers or processes.

[0135] It should be noted that the singular forms “a,” “an,” and “the” used herein and in the appended claims include multiple reference words unless the context clearly indicates otherwise. Herein, a range can be expressed from “about” one particular value to and / or “about” another particular value. Where such a range is expressed, in another embodiment, it includes from one particular value to and / or another particular value. Similarly, where a value is expressed as an approximation, the use of the antecedent “about” will be understood to mean that a particular value forms another embodiment. The term “about” in relation to a number is arbitrary and means, for example, ±10%. [Examples]

[0136] In the examples, substrates having a graphene layer as described herein are used. For functionalization, an all-gold support or substrate with a pore size of 800 nm (UltrAuFoil R 0.6 / 1, 300-mesh, Quantifoil) is used, on which a graphene monolayer is provided. These supports or substrates may be referred to as grids.

[0137] For example, a support containing a substrate having a graphene layer can be fabricated using techniques available in this field, such as growing graphene on copper foil using CVD and then transferring the graphene layer to a gold substrate.

[0138] (Example 1) Flufen functionalization (a) Covalent functionalization using low-energy helium carrier plasma Plasma treatment was performed using a modified commercially available plasma cleaner (Fischione 1070) equipped with a custom grid and mask holder, a custom gas injection system, and a custom fiber-coupled sapphire viewport attached to a UV-vis spectrometer (Thorlabs). The modified apparatus thus described is of the present invention and is roughly shown in Figure 12(a).

[0139] The vacuum chamber is powered by a turbo pump at 10 -5 The exhaust was reduced to less than Torr. The N6.0 grade helium carrier plasma was at a flow rate of 28 sccm and 5 × 10⁻¹⁶. -1 It was generated at standard Torr pressure. An 18MHz RF coil was operated at 40% power, yielding a typical forward output of 9W and a reverse output of 0.6W.

[0140] The vapor of the chemical used for functionalization was introduced through a vacuum 5-channel manifold. The chemicals were stored in sealed vacuum 1 mL stainless steel vials, and the vapor of the target chemical was supplied to the plasma chamber via a precision micrometer needle valve and shut-off valve. The effect of the introduced chemical was observed in the real-time spectrum of the plasma. The spectrum was acquired with an exposure time of 6 seconds, which caused the helium line to saturate and additional peaks from the precursor to become visible.

[0141] The most prominent feature in the optical spectrum resulting from the introduction of organic molecules was the Hα peak at 656 nm (Figure 12(d)). This is thought to be because the probability of terminal hydrogens being cleaved from the molecule is high. On the other hand, for larger fragments or non-terminal parts to be cleaved, such as atomic sulfur being cleaved from a thiol, it is necessary to cleave two or more bonds, which is less likely.

[0142] The actual amount of chemicals present in the plasma depends on the vapor pressure of each compound and can be adjusted by controlling the temperature of the liquid container and the settings of the leak valve.

[0143] Once the desired plasma composition was determined, the exposure time was controlled using shutters covering the grid and mask holder. Such shutters and masks are shown in Figures 12(b) and (c). Between the use of different chemicals, the chamber was cleaned with pure He plasma at 70% power for 30–60 minutes until no traces of hydrogen were visible in the optical spectrum.

[0144] These operations were completed using amylamine, hexanoic acid, 1-pentanethiol, and 4-pentylphenol as functionalizing chemicals. Figure 12(e) shows the XPS spectrum of the functionalized graphene exhibiting chemical modification.

[0145] (b) XPS of functionalized graphene Covalent modification of graphene modified according to (a) above was investigated using X-ray photoelectron spectroscopy (XPS). A beam from an Al Kα source (1486.68 eV, ESCALAB 250 Xi) was focused onto the sample with a 900 μm diameter probe. For these measurements, graphene on copper (as grown) and graphene transferred to an UltrAuFoil gold grid (according to Example 2) were used, and charge compensation was applied. Partially hydrogenated graphene and untreated graphene samples were used as controls for comparison with the functionalized graphene. The pressure in the XPS chamber during data acquisition was 5 × 10⁻⁶. -9 It was mBar.

[0146] First, a spectral scan with 1 eV sampling was acquired in the range of 136 eV to 1361 eV with a path energy of 200 eV. Then, 30 to 50 scans with 0.1 eV sampling were acquired around the target region with a path energy of 30 to 50 eV, and the average was used to create the spectrum plotted in Figure 12(e). These peaks are from the NIST XPS database. 27 The data was used for identification. Some of the observed peaks were attributed to signals from the metal substrate supporting the graphene.

[0147] (c) Contact angle measurement Using custom equipment, contact angles were measured on plasma-treated graphene suspended on an all-gold grid. 28 In short, a 1-2 μL droplet of 18 MΩ deionized water was pipetteed onto the center of a clamped grid and photographed parallel to the surface with diffuse illumination as the background. Images where the grid was curved or the droplet was spread asymmetrically were all removed. The standard time from the end of plasma treatment to measurement was 1 minute, and 3-5 repetitions were performed for each data point. The contact angle was measured using ImageJ. 29 This was quantified.

[0148] (d) Patterning of functionalized graphene To pattern grids with multiple functional groups in different regions of each grid, a knife-edge mask was precisely positioned on the top surface of each grid after each plasma treatment step (a) above. This was done using a set of precision-machined masks that fit into predetermined positions on a grid alignment holder. The apparatus was able to pattern 2 × 10 grids at a time.

[0149] The sharpness of the pattern depends on the distance between the mask and the graphene surface, the sharpness of the mask edge, and the mean free path of the chemical species in the plasma, and can be controlled by varying these factors. Under the typical conditions used here (1 Torr, non-thermal remote plasma), the mean free path in the chamber is much larger than other relevant length scales. Therefore, the patterning resolution is considered to be approximately equal to twice the distance between the grid and the mask, i.e., 50 μm. This can also be seen from the width of the transition region when the knife-edge mask is placed 20 μm from the surface of the foil in Figure 1(a).

[0150] (e) Deposition of amorphous carbon onto a graphene-on-gold support 10 -6 Amorphous carbon was directly deposited onto a graphene-coated gold grid using an Edwards 306A evaporator under a vacuum of mbar. The grid was placed in a custom slotted holder similar to that used for plasma treatment and covered with a mask plate. A single-slot opening (EMS GA100-Au) with a pore size of 100 μm was used for the mask, exposing only the central part of the grid to the evaporated carbon. The distance between the grid and the mask was approximately 100 μm. The successful localized deposition of a continuous amorphous carbon film was confirmed by TEM observation (Figure 3).

[0151] Figure 3 shows a polyfunctional graphene-on-gold support with a grid square covered with amorphous carbon for electron microscope alignment. Figure 3(a) is a diagram of the aperture mask used during carbon deposition to cover only about one grid square of the graphene-on-gold grid with a thin film of amorphous carbon. Deposition is performed before plasma treatment. Figure 3(b) is a low-magnification transmission electron microscope image of the modified polyfunctional grid, showing an asymmetrical center mark and the dark area surrounding it covered with an amorphous carbon film approximately 300 Å thick. The scale bar is 80 μm. Figure 3(c) shows that the Thon ring from the carbon-coated region (Fourier transform of a transmission electron microscope image with nominal magnification 155,000x, defocus 0.4 μm, and intentionally stigmatized at 700 Å) can be used to correct astigmatism and coma. The 2.13 Å graphene lattice reflection is still visible. In contrast, clean monolayer graphene in the masked area does not exhibit Thon ring formation.

[0152] This patterning method allows amorphous carbon to be directly deposited onto a 100 μm diameter region in the center of a graphene-coated gold grid, enabling astigmatism correction and coma alignment on the same multifunctional grid prior to high-resolution data acquisition.

[0153] (Example 2) Motion tracking in glassy ice using gold nanoparticles (a) Preparing the grid Samples were prepared by manual plunge freezing at 4°C. All-gold supports with a pore diameter of 800 nm (UltrAuFoil R 0.6 / 1, 300-mesh, Quantifoil) were used, with or without graphene. To ensure sufficient hydrophilicity before vitrification, the sample supports were plasma-treated as follows: an all-gold grid was treated with a 9:1 Ar:O2 plasma for 60 seconds, and a graphene-coated grid was treated with hydrogen plasma for 60 seconds. Subsequently, 3 μL of a 10 nm non-conjugated gold colloid solution (BBI Solutions) with an OD of 100 was applied to the foil side of the grid, blotted from the same side for 15 seconds, and then immersed in liquid ethane held at 93 K. 30 .

[0154] (b) Imaging Data from graphene-supported ice was collected using Titan Krios, and data from non-graphene-supported ice were collected using Tecnai Polara, both using the Falcon 3 detector in integration mode under typical single-particle data acquisition conditions (300 keV, 1e). - Data was collected at / pix / frame (sample temperature 80K). Illumination was positioned in the center of each exposed hole, and the beam diameter was set to be slightly larger than the hole to minimize beam motion. 31 The magnification was chosen to include the edge of the pore in the micrograph for drift tracking. This corresponds to 1.74 Å / pix (nominal 47,000x) for Krios and 1.72 Å / pix (nominal 59,000x) for Polara. The video has a cumulative fluence of 60 e - / Å 2 The image was acquired for 5-8 seconds until the desired result was achieved. Only holes from flawless squares in the gold leaf were imaged.

[0155] (c) Motion analysis The entire video of the microscope images was motion-corrected using MotionCorr. 20 Stage drift was removed. The motion-corrected stack is 1.5e per frame. - / Å 2 The particles were binned to achieve a fluence.21 They were manually selected and extracted from a motion-corrected stack of 128x128 pixel frames. Then, the motion-corrected video of each particle was again applied using MotionCorr. The corrected particle videos were visually inspected. (First 20e) - / Å 2 Using the trajectories of all particles during irradiation, the mean square displacement and root mean square displacement of the particles in the ensemble were calculated.

[0156] (Example 3) Cryo-electron microscopy of T. thermophilus 30S ribosome subunit In this example, 30S ribosome subunits are used as test specimens to demonstrate the potential of a polyfunctional graphene support for optimizing the orientation distribution of a sample. 12,13 Graphene functionalized with amylamine, hexanoic acid, 1-pentanethiol, and 4-pentylphenol was prepared by applying these chemicals to half of a gold grid coated with partially hydrogenated graphene. The orientation distribution of 30S particles on each of these surfaces was analyzed (Figures 13(a)-(b), 4(a)-(d)). These distributions are for the distribution on partially hydrogenated graphene and in unsupported ice (Figures 4(e)-(f)). Furthermore, combinations of functionalized surfaces that increased the efficiency of the orientation distribution to 0.8 were identified. 14 This enabled high-resolution reconstruction calculations of only the 30S subunit (Figure 6). Clear differences in all observed distributions indicate that the particles are interacting with the functionalized graphene. This was confirmed by tomographic reconstruction, which showed the particles were in a single layer on the graphene surface, within an ice layer slightly thicker than the particle diameter. 15 (Figure 5).

[0157] (a) Preparing the grid Purified T. thermophilus 30S ribosome subunits (5 mM HEPES, 50 mM KCl, 10 mM NH4Cl, 10 mM Mg(OAc2)) were provided (by the Ramakrishnan laboratory at the MRC Laboratory of Molecular Biology). Such samples are commercially available, for example, from Sigma.

[0158] The concentration was adjusted to 8 mg / mL, except for samples on partially hydrogenated graphene, which were at 1.7 mg / mL. All graphene-coated grids were first exposed to H2 plasma for 180 seconds to make the entire surface hydrophilic, and then half of the grid was treated for 30 seconds and the other half for another 30 seconds with He plasma carrying the vapor of the corresponding functionalizing chemical (amylamine, hexanoic acid, 1-pentanethiol, or 4-pentylphenol). A plain UltrAuFoil grid for control experiments without graphene was treated for 60 seconds with a 9:1 Ar:O2 plasma mixture. The grids were plunged using an FEI Vitrobot equilibrated to 4°C and 100% relative humidity, and the liquid ethane was maintained at a fixed temperature of 93 K. 3-4 μL of protein solution was pipetteed onto the graphene-coated surface of the grid, double-blotted for 5 seconds, and immediately immersed in ethane. The time from plasma treatment to vitrification was typically less than 10 minutes. The grid was stored in liquid nitrogen until it was transferred to an electron microscope for imaging.

[0159] (b) Imaging and data acquisition Microscopic images of 30S ribosomes in ice on functionalized graphene were acquired using a Tecnai Polara microscope operating at 300kV with a Falcon 3 detector in integral mode. The nominal magnification was 93,000x, corresponding to 1.17 Å / pix (calibrated using graphene reflectance of 2.13 Å). Microscopic images of 30S ribosomes in unsupported ice and in ice on partially hydrogenated graphene were acquired using a Titan Krios microscope operating at 300kV in integral mode with a Falcon 3 detector at a nominal magnification of 59,000x, corresponding to 1.34 Å / pix.

[0160] (c) Single-particle data analysis The sectors of the functionalized grid were identified using asymmetrical center marks (see Figure 2(e)). Microscope images were motion-corrected using Relion 3. 17 Fit the CTF with Gctf 22 Particles were manually picked up using EMAN2, and 2D classification and 3D refinement were performed using Relion 3. All particles in the functionalized grid were processed together, and the final orientation angles were categorized for each surface based on the name in the micrograph and the corresponding coordinates on the grid. Particles in the control experiment were processed separately due to their different pixel sizes. The orientation distribution of the particles was analyzed using cryoEF. 14 The data were plotted on equal-area Mollweide projections. The shaded scales represent the local Gaussian kernel density (i.e., probability distribution function, PDF) of the distribution for all sampled orientations (see Figures 13(a)-(b), Figure 6). The kernel density bandwidth was set to 5°. These plots also show the normalized probability that particles interact with the surface in specific directions, quantifying the strength of this interaction. Some variability in the assigned orientation may be due to local bending of the foil, wrinkles in graphene, bulging of ice, or bending of the movable parts of ribosome subunits, all of which are less than 5°. High-resolution crystal structure of 30S ribosomes (1J5E 12 The image is converted to a surface, and shading is applied using the surface Coulomb potential calculated in UCSF Chimera. 23The dominant orientation of particles on graphene was illustrated, and the interaction interfaces were analyzed (see Figures 13(c)-(f)). Efficiency metrics were used to evaluate whether each orientation distribution could provide a uniform Fourier space sample enabling high-resolution reconstruction. 14 The efficiency was calculated for each separate orientation distribution from approximately 10,000 particles. These efficiencies can be used to make reasonable decisions regarding the optimal surface or combination of surfaces for further data collection.

[0161] In this example, the selected surface is graphene functionalized with amines and phenols, providing an overall efficiency of 0.8, thereby enabling near-atomic structure determination with approximately 100 times fewer particles compared to, for example, an efficiency of 0.3 for a dataset of unsupported 30S ribosomes in ice. 14 (See Figure 6).

[0162] Figure 6 shows the improvement in efficiency and resolution by optimizing the orientation distribution of 30S ribosome subunits on functionalized graphene. Gold standard FSC plots are shown for 30S in unsupported ice (dashed line) with a resolution of 6.9 Å from 12,600 particles, and 30S on amine / phenol-functionalized graphene (solid line) with a resolution of 3.94 Å from 26,483 particles.

[0163] These functionalization combinations were predicted to maximize the efficiency of the orientation distribution. The difference in resolution is not due to a doubling of the number of particles, as shown by reconstruction from a subset of 12,600 particles in the high-efficiency dataset (dotted line) that reached 4.68 Å. The inset shows the point spreading function (PSF) corresponding to each orientation distribution, with the PSF extending in the weakest resolution direction.

[0164] (d) Tomography Using a Titan Krios operating at 300 keV, a Falcon 3 direct electron detector was used to collect a uniaxial bidirectional tilt series of the same sample in 5° increments from -50° to +50° in integral mode. Each image was exposed for 1 second (40 frames) at a nominal magnification of 37,000x for 2e - / Å 2 The video was motion-corrected using MotionCorr. 20 The tomographic images were aligned without a reference point and reconstructed using SIRT in Etomo (IMOD). 24 .

[0165] (e) Discussion These experiments revealed a functional interaction map of the surface of 30S ribosome subunits. This allows for the creation of a physical model of particle-surface interactions that explains the observed orientation distribution. On carboxylated supports, the dominant view is that the protein-rich outer side of 30S faces the surface (Figure 13(d)). This side has Arg S2 226 Lys S10 80 Lys S4 166,169Positively charged amino acid residues such as are exposed, which appears to stabilize contact with the surface. The same orientation is more preferred on thiol-functionalized surfaces (Figure 4(b)), consistent with the lower pKa of the thiol group. The next most common orientation on carboxylated graphene is with the 50S side of the 30S subunit facing the surface (Figure 13(c)). The interaction causing this orientation is likely similar to that occurring at the 30S-50S interface in vivo. The putative anchor points include the Arg / Lys-rich S13 chain near the 30S head and the 16S rRNA 5′ domain on the body surface. This orientation is very favorable on partially hydrogenated graphene (Figure 4(e)). On the other hand, this orientation is rarely seen on partially hydrogenated graphene treated with amylamine, which is consistent with the higher pKa of the amine group. Of all the surfaces tested, amylamine / hydrogen-functionalized graphene minimized the orientation bias of the tested samples. However, some acidic amino acid residues (Glu S6 24,31 , Glu S13 58 ) can be identified as the putative interaction point of the 30S subunit in contact with the amine-graphene surface (Figure 13(e)-(f)). Since the Debye screening length in the buffer used is approximately 10 Å, only electrostatic interactions with amino acid side chains in closer contact with the graphene surface are considered. The breadth of the field of view around each preferred direction is determined by the particle shape and how the particle restricts rotation around the fixed interaction point.

[0166] Figures 13(a)-(b) plot the orientation distribution of 30S ribosome subunits on two types of graphene functionalizations ((a) graphene + hydrogen + carboxyl, i.e., partially hydrogenated graphene with carboxyl functionalization derived from hexanoic acid, and (b) graphene + hydrogen + amine, i.e., partially hydrogenated graphene with amine functionalization derived from amylamine) from a single patterned grid, plotted using Mollweide projection. Each point represents an observed particle, and the scale represents the calculated normalized probability density for observing particles in a particular orientation. E represents the efficiency of each orientation distribution. 14 The two most likely view directions, along with their surrounding variations, are labeled.

[0167] Figure 4 shows, more completely, the Mollweide projection orientation distribution plots of 30S ribosomes on graphene functionalized with (a) hexanoic acid, (b) 1-pentanethiol, (c) amylamine, and (d) 4-pentylphenol, all of which were transported in helium plasma and preceded by partial hydrogenation; (e) the Mollweide projection orientation distribution plot of 30S ribosomes on partially hydrogenated graphene; and (f) the Mollweide projection orientation distribution plot of 30S ribosomes not supported by ice. Each point represents the orientation of the particle observed on the graphene surface, and the scale represents the calculated normalized probability distribution function (PDF) when the particle is observed in a field of view given by Euler angles (φ, θ). All plots are shown on the same scale as the peaks corresponding to the saturated primary field of view in (e) to (f). The efficiency E of each orientation distribution is shown.

[0168] (Example 4) Cryo-electron microscopy of human 20S proteasome (a) Preparing the grid Human 20S proteasome (Enzo) samples were buffered with pH 8.3 TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA) to adjust the concentration to 0.8 mg / mL. A four-quadrant grid was prepared by exposing half of the grid to hydrogen plasma for 180 seconds, rotating the mask 90°, exposing half of the grid to amylamine under helium plasma for 30 seconds, rotating the mask 180°, and exposing the remaining half of the grid to hexanoic acid under helium plasma for 30 seconds. Vitrification was performed using the same method as described above.

[0169] (b) Imaging and data acquisition Microscope images were taken using a Titan Krios microscope operating at 300kV with a Falcon 3 detector in integration mode. The flux was 17e. - / Å 2 The exposure time was 2 seconds, and a 70 μm objective aperture was used. The nominal magnification was 59,000x, corresponding to 1.34 Å / pix, and calibration was performed using reflections from anatase (TiO2) nanoparticles dispersed on a separate calibration grid. The grid quadrant was determined based on the orientation of the asymmetric grid center mark as described above (see Figure 2).

[0170] Figure 2 shows experimental efforts to control the wettability of functionalized graphene and the orientation distribution of 20S proteasomes. Figures 2(a)–(d) are representative micrographs of 20S proteasomes on graphene in ice, obtained from four quadrants of a single patterned polyfunctionalized graphene grid. The quadrant of the grid from which the micrograph was taken is highlighted in each inset. Hexanoic acid treatment (Figure 2(b)) makes the grid hydrophobic, reducing the wettability of the graphene surface and causing partial drying of the proteins. This can be resolved by pre-treating the grid with hydrogen plasma (Figure 2(b)), which makes the surface hydrophilic, allowing for the formation of a uniform thin layer of ice independently of subsequent functionalization. Thus, it has been demonstrated that initial hydrogen plasma treatment can maintain the macroscopic surface wettability necessary for the formation of a thin layer of ice, even after subsequent functionalization with compounds that may make the graphene surface hydrophobic.

[0171] The axes dividing the grid into four distinct functionalized quadrants were defined by orienting easily visible grid rim marks (Figure 2(e), inset) parallel or perpendicular to the edges of the mask. Quadrants were also identified by finding the position and orientation of asymmetric grid center marks (Figure 2(e), inset) in low-magnification mode of the TEM. The inset in Figure 2(e) is an optical micrograph of the true relative orientation of the rim and center marks. The scale bars are 500 Å for Figures 2(a)-(d) and 50 μm for the inset in Figure 2(e). As shown in Figure 2(f), contact angle measurements of the functionalized graphene can be used to monitor the degree of surface modification and evaluate the hydrophilicity of the resulting surface.

[0172] Based on previous experiments involving partial hydrogenation, a typical exposure result would result in graphene reaching 1 nm 2 It can be estimated that approximately one functional group is added per sample. Functionalization with carboxyl and thiol groups tends to make the surface hydrophobic, as can be seen from the increased contact angle, which can be problematic when freezing samples for cryo-electron microscopy. This problem can be avoided by pre-treating the sample with hydrogen plasma for 180 seconds before a typical 30-second exposure to the functionalizing chemical (data points shown in black).

[0173] This method, which involves partially hydrogenating the graphene before functionalization, reliably produced a surface with sufficient wettability to prepare the cryo-sample.

[0174] (Example 5) Cryo-electron microscopy of apoferritin In this example, to demonstrate that this support is suitable for high-resolution structural determination, the structure of horse spleen apoferritin is determined using amylamine-functionalized graphene on a gold support.

[0175] (a) Preparing the grid Apoferritin (Sigma) from the spleen of horses (Equus cabarus) was buffered with 100 nM phosphate-buffered saline at pH 7.4 to a concentration of 11.7 mg / mL. Graphene-coated grids treated with He plasma carrying amylamine for 30 seconds were used. Vitrification was performed using the same method as described above.

[0176] (b) Imaging and data acquisition The video was acquired in count mode using a Titan Krios microscope operating at 300kV with a Falcon 3 detector. The nominal magnification was 120,000x, the pixel size was 0.6495 Å / pix, and calibration was performed using the 2.347 Å gold reflection in the Fourier transform of a micrograph of a gold substrate foil taken under the same conditions as data acquisition. A 100 μm objective aperture was used for data acquisition. The video was acquired with an exposure time of 30.01 seconds, and the frames were combined into 38 fractions (31 frames per fraction). The flux was 0.53 e - Set to / pix / second, and the total fluence of each video is 37e - / Å 2 That was the case.

[0177] Figure 7(a) is a micrograph of apoferritin particles in glassy ice on amylamine-functionalized graphene. When such electron microscope images are taken at appropriate high magnification, the graphene lattice can be directly seen.

[0178] (c) Analysis and modeling of single-particle data Motion correction of the microscope images was performed with Relion 3, CTF fitting was done with Gctf, and particle pickup was done manually with EMAN2. 2D and 3D classification, 3D refinement, CTF refinement, particle polishing, and post-processing were all performed with Relion 3. As a result, the EM density map reached the gold standard FSC resolution of 2.14 Å, and the corresponding high-resolution crystal structure (2W0O) was obtained. 25 ) matched.

[0179] JPEG0007874967000005.jpg105147

[0180] A 3D map using the fitted model was rendered in Chimera. Analysis of the particle orientation distribution contributing to the final reconstruction using cryoEF showed an efficiency of 0.7. For motion analysis, the displacement of each particle throughout the entire video was calculated by subtracting the motion of the entire microscope image from the particle motion calculated by Bayesian polishing using Relion. 26 This was necessary because the dominant component of the motion was the microscope stage drifting at a speed of 0.6 Å / second during a 30-second exposure.

[0181] (d) Discussion Figure 14 details high-resolution structural determination using a polyfunctional graphene-on-gold support. Figure 14(a) shows a contour density map of apoferritin, indicating the amino acid side chains within the structure. Figure 14(b) shows a Fourier shell correlation plot (FSC) of the apoferritin structure. Figure 14(c) shows the mean square displacement of unsupported gold nanoparticles on an all-gold support (thin circles) or unsupported gold nanoparticles on graphene (dark cross). Adding the graphene film doubles both the motion at the start of irradiation and the diffusive motion in subsequent frames. The dashed line is a linear fit of the data excluding the first frame. Adding graphene to the all-gold sample support results in a slope of 0.13 ± 0.1 Å. 2 / ( e - / Å 2 ) from 0.048±0.005Å 2 / ( e - / Å 2 ) has decreased. Figure 14(d) shows the B-factor calculated as a function of electron fluence for the apoferritin dataset. - / Å 2 The B-factor in this context corresponds to the initial non-diffusion transport stage. The B-factor is 10-20 e - / Å 2 With a fluence in the range of 7.7±0.8 Å 2 / ( e - It decreases at an almost constant rate (the dashed line is a linear fit).

[0182] As a result of performing standard data collection and processing, a reconstruction was carried out from 41,202 particles at a resolution of 2.1 Å (0.143 FSC 16 ); see Table 2, Figures 7 and 14). The resolution of this reconstructed map is equivalent to the highest resolution reported so far for this sample, but 17 the amount of data required is approximately half, as indicated by the clear density of the side chains in the map (Figure 14(a)). This includes complete aspartic acid and glutamic acid residues, and the density beyond Cβ is often absent in EM maps 18 . The decay rate of high-resolution information as a function of fluence after irradiation with the first 5 - 10 e - / Å 2 in this dataset (Figure 14(d)) is similar to the measurement results of radiation damage in two-dimensional protein crystals 19 . The movement of particles on multifunctional graphene on a gold support was compared with past measurement results on several other supports 4 (Figures 8 and 9). Figure 8 shows the root mean square (RMS) displacement during radiation exposure of apoferritin particles in unsupported ice on a carbon grid (top) and particles on graphene-on-gold (bottom). The particles in unsupported ice are from a dataset corresponding to the highest resolution structure published so far of commercially available horse spleen apoferritin 17 . The error bars are the standard error of the mean. Functionalized graphene on a gold support reduced the initial movement, displacement velocity, and dispersion of movement of particles during irradiation compared to a porous amorphous carbon support. Figure 9 plots the root mean square (RMS) displacement of ribosomes with MW 2 MDa against electron fluence. Figures 9(a)-(d) and (f) reproduce previously published studies 4 . The movement on graphene on a gold support in Figure 9(e) was measured independently from the apoferritin dataset obtained here. For this comparison, the effective diffusion constant was scaled according to the molecular weight (MW 1 / 3 ) using the Stokes-Einstein equation 9The motion of a sample on a graphene-on-gold support is diffusive (mean square displacement is directly proportional to fluence), except in the very early stages of irradiation.

[0183] Adding a graphene layer to an all-gold support reduced particle motion during electron beam irradiation by half. Compared to graphene on a carbon support, particle motion was reduced to one-third on graphene on a gold support. The reduction in particle motion was also confirmed by tracking the motion of individual gold nanoparticles on an all-gold support with and without the graphene layer (Figure 14(c), Figure 10). Figure 10 shows the reduction in nanoparticle motion on an all-gold grid coated with graphene. Gold nanoparticles (10 nm in diameter, 6 MDa in mass) were imaged in unsupported glassy ice on an all-gold grid (Figure 10(a)) and in glassy ice on graphene on an all-gold grid (Figure 10(d)) at a magnification that included the edge of the pores in the field of view for drift tracking. The trajectories of the particles on the motion-corrected whole microscope images were plotted at 200x magnification (Figure 10(b) and Figure 10(e)). The dashed rectangles correspond to the regions in the micrograph, the empty circles are the initial particle positions, and the filled circles are 20e - / Å 2 This is the final particle position after the total fluence. In Figures 10(c) and 10(f), the root mean square (RMS) displacement of gold nanoparticles is plotted against electron fluence for the shown micrographs (upper panel), and for the average of n=19 micrographs of the same type (gold) and n=8 micrographs (graphene on gold) (lower panel).

[0184] Importantly, the addition of the graphene layer not only suppresses random particle movement, but also reduces movement during the first few electron beam irradiations, improving the quality of the initial frame (B-factor) with less molecular damage from the electron beam (Figure 14(c)-(d)). This twofold reduction may be because the graphene layer restricts the bulk movement of thin ice films within each pore, which occurs when stress is released at the start of electron beam irradiation.

[0185] Reference To more fully describe and disclose the present invention and the state of the art to which it relates, numerous publications are cited. A complete citation of these documents is given below. The entirety of each of these documents is incorporated herein. 1. J. Dubochet, et al., Q. Rev. Biophys. 21, 129 (1988). 2. RA Crowther, The Resolution Revolution: Recent Advances in Cryo-EM , vol. 579 of Methods in Enzymology (Elsevier, 2016). 3. RM Glaeser, Current Opinion in Colloid and Interface Science 34, 1 (2018). 4. C. Russo, LA Passmore, Current Opinion in Structural Biology 37, 81 (2016). 5. AK Geim, Science 324, 1530 (2009). 6. RS Pantelic, JC Meyer, U. Kaiser, W. Baumeister, JM Plitzko, J. Struct. Biol. 170, 152 (2010). 7. RS Pantelic, et al., J. Struct. Biol. 174, 234 (2011). 8. CJ Russo, LA Passmore, Nat. Methods 11, 649 (2014). 9. A. Einstein, Annalen der Physik 322, 549 (1905). 10. C. Russo, LA Passmore, Science 346, 1377 (2014). 11. T. E. Daubert, R. P. Danner, Physical and thermodynamic properties of pure chemicals: data compilation (Taylor & Francis, Washington DC, 1989). 12. B. Wimberly, et al., Nature 407, 327 (2000). 13. T. Hussain, J. L. Llacer, B. T. Wimberly, J. S. Kieft, V. Ramakrishnan, Cell 167, 133 (2016). 14. K. Naydenova, C. J. Russo, Nature Communications 8, 1 (2017). 15. A. J. Noble, et al., eLife 7, e34257 (2018). 16. P. B. Rosenthal, R. Henderson, J. Mol. Biol, 333, 721 (2003). 17. J. Zivanov, et al., eLife 7, e42166 (2018). 18. C. F. Hryc, et al., Proc Natl Acad Sci USA 114, 3103 (2017). 19. J. Hattne, et al., Structure 26, 759 (2018). 20. X. Li, et al., Nat. Methods 10, 584 (2013). 21. G. Tang, et al., Journal of Structural Biology 157, 38 (2007). 22. K. Zhang, Journal of Structural Biology 193, 1 (2016). 23. E. F. Pettersen, et al., J. Comput. Chem. 25, 1605 (2004). 24. D. N. Mastronarde, S. R. Held, Journal of Structural Biology 197, 102 (2017). 25. N. de Val, J.-P. Declercq, C. K. Lim, R. R. Crichton, J. Inorg. Biochem. 112, 77 (2012). 26. J. Zivanov, T. Nakane, S. H. W. Scheres, IUCrJ 6, 5 (2019). 27. National Institute of Standards and Technology, NIST X-ray Photoelectron Spectroscopy Database, vol. 20899 of NIST Standard Reference Database (Gaithersburg MD, 2000). 28. K. Naydenova, C. J. Russo, Microscopy and Microanalysis 24, 880 (2018). 29. C. A. Schneider, W. S. Rasband, K. W. Eliceiri, Nat. Methods 9, 671 (2012). 30. C. Russo, S. Scotcher, M. Kyte, Review of Scientific Instruments 87, 114302 (2016). 31. C. J. Russo, L. A. Passmore, Journal of Structural Biology 193, 33 (2016). Some embodiments of the present invention are described in the following sections (1)-(24). (1) An apparatus for functionalizing graphene or graphene oxide, Plasma generator; A processing chamber connected to the plasma generator; A substrate holder for electron microscopy, preferably cryo-electron microscopy, arranged to hold a substrate having a graphene layer or graphene oxide layer thereon within a processing chamber, and having a mounting area configured for mounting the substrate therein; A shutter that is movable between an open position in which the mounting area of ​​the substrate holder is exposed to the processing chamber and a closed position in which the mounting area of ​​the substrate holder is hidden; An injector for injecting the functionalized compound into the processing chamber; and A helium source that supplies helium to the plasma generator. A device that includes this. (2) The apparatus according to (1) above, further comprising a mask for masking the region of the substrate. (3) The apparatus according to (2) above, wherein the mask is movable. (4) The apparatus according to (2) or (3) above, wherein the mask is removable from the rest of the apparatus. (5) The apparatus according to any one of (2) to (4) above, wherein the apparatus is configured such that the mask does not come into contact with the graphene layer or graphene oxide layer of the substrate when the substrate is held in the substrate holder. (6) The apparatus according to (5) above, wherein the apparatus is configured such that when the substrate is held in the substrate holder, the mask is located less than 200 μm from the surface of the substrate. (7) The apparatus according to any one of (1) to (6) above, further comprising a hydrogen source for supplying hydrogen to the plasma generator. (8) The apparatus according to any one of (1) to (7) above, further comprising a viewport opening into the processing chamber and a UV-vis spectrometer configured to analyze radiation emitted from the processing chamber through the viewport. (9) The apparatus according to any one of (1) to (8) above, further comprising one or more functional compound sources, each connected to the injector, for supplying the functional compound or a plurality of functional compounds to the injector. (10) The apparatus according to any one of (1) to (9) above, wherein the apparatus comprises a plurality of injectors, and each injector is connected to a respective functional compound source for supplying the functional compound to the respective injector. (11) The apparatus according to any one of (1) to (10) above, wherein the injector or a plurality of injectors is a gas injector or a plurality of gas injectors. (12) A sample support for cryo-electron microscopy, Metal foil substrate; and A functionalized graphene layer or graphene oxide layer on the substrate having multiple different functionalizations. A sample support for cryo-electron microscopy, including the following: (13) The sample support for cryo-electron microscopy according to (12) above, wherein the graphene or graphene oxide is also partially hydrogenated. (14) The substrate is a sample support for cryo-electron microscopy according to (12) or (13) above, having pores on which the functionalized graphene layer or graphene oxide layer is suspended. (15) A method for functionalizing graphene or graphene oxide on a substrate for use in electron microscopy, preferably cryo-electron microscopy, comprising the following steps: (a) Exposing a substrate having a graphene layer or graphene oxide layer thereon to a plasma containing helium and at least one functionalized compound. A method that includes this. (16) The method according to (15), wherein, prior to step (a), a mask is applied to the substrate having the graphene layer or graphene oxide layer to selectively expose only a portion of the substrate. (17) The method according to (16) above, wherein the mask is moved during step (a). (18) A method for polyfunctionalizing graphene or graphene oxide on a substrate for use in electron microscopy, preferably cryo-electron microscopy, comprising the following steps: (a) Masking the region of the substrate having a graphene layer or graphene oxide layer; (b) Exposing the unmasked portion to a plasma containing helium and at least one functionalized compound to form a functionalized zone; (c) To remove the masking from the substrate; (d) Repeat steps (a) to (c) using different functionalized compounds to form a substrate having a graphene layer with multiple zones having different functionalizations. A method that includes this. (19) The method according to any one of (15) to (18) above, wherein the exposure time of the substrate to the plasma is controlled by using a shutter that is fixed on the substrate and movable between an open position that exposes the substrate and a closed position that hides the substrate. (20) The aforementioned functionalized compound or each functionalized compound is defined by the following general formula (I): Equation (I) One or more compounds selected from the following: In the formula, R1 and R2 are independently selected from the group consisting of H, halos (e.g., -F, -Cl, -Br), thiols (-SH), aminos (-NH2), carboxyls (-COOH), and hydroxyphenyls (-C6H4OH). One of R1 and R2 is not H; and n is an integer between 1 and 10. The method described in any of the above (15) to (19). (21) The method according to any one of (15) to (20) above, wherein the functionalized compound or each functionalized compound is one or more selected from the group consisting of amylamine, hexanoic acid, 1-pentanethiol, and 4-pentylphenol. (22) The method according to any one of (15) to (21) above, further comprising the step of exposing a substrate having a graphene layer or a graphene oxide layer thereon to a hydrogen-containing plasma to hydrogenate the graphene layer or the graphene oxide layer, or a portion of the graphene layer or graphene oxide layer. (23) The method according to any one of (15) to (22) above, wherein the substrate is a gold cryo-electron microscope grid. (24) An apparatus for performing any of the methods described in (15) to (23) above.

Claims

1. An apparatus for functionalizing graphene or graphene oxide, Plasma generator; A processing chamber connected to the plasma generator; A substrate holder for electron microscopy, arranged to hold a substrate having a graphene layer or graphene oxide layer thereon within the processing chamber, and having a mounting area configured for mounting the substrate therein; A shutter that is movable between an open position in which the mounting area of ​​the substrate holder is exposed to the processing chamber and a closed position in which the mounting area of ​​the substrate holder is hidden; An injector for injecting the functionalized compound into the processing chamber; One or more functional compound sources, each of which is connected to the injector for supplying the functional compound or a plurality of functional compounds to the injector; and A helium source that supplies helium to the plasma generator. A device that includes this.

2. The system further comprises a mask for masking the region of the substrate. The apparatus according to claim 1.

3. The system further comprises a hydrogen source for supplying hydrogen to the plasma generator, and / or The apparatus according to claim 1, further comprising a viewport opening into the processing chamber and a UV-vis spectrometer configured to analyze radiation emitted from the processing chamber through the viewport.

4. The apparatus further comprises a plurality of injectors, and each injector is connected to its respective functional compound source in order to supply the functional compound to each injector. The apparatus according to claim 1.

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