CN111908455A - Reduced graphene oxide film and preparation method thereof - Google Patents
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Abstract
The invention discloses a reduced graphene oxide film and a preparation method thereof. The preparation method of the reduced graphene oxide film comprises the following steps: transferring the graphene oxide onto an electron microscope carrier net, and reducing to obtain the graphene oxide; the reduction steps are as follows: and heating to 300-600 ℃ in an argon hydrogen environment, and keeping the temperature constant to realize the reduction of the graphene oxide. The reduced graphene oxide film has stronger conductivity, so that drift caused by electrons during photographing exposure can be reduced; compared with graphene oxide, the reduced graphene oxide has higher conductivity, and the problem of sample drift caused by light beam irradiation is reduced. In addition, the reduced graphene oxide is thinner, and thus the background noise of the transmission electron micrograph is also lower. The reduced graphene oxide film provided by the invention can bring great convenience to sample preparation and later three-dimensional reconstruction of a cryoelectron microscope.
Description
Technical Field
The invention relates to a reduced graphene oxide film and a preparation method thereof, belonging to the technical field of biological cryoelectron microscopes.
Background
The long-term development of the cryoelectron microscope technology, especially the breakthrough development in data processing and camera hardware in recent years, makes the method break through the limitation of the previous resolution, and the structure analysis of the near atomic resolution becomes more common. This technique rapidly freezes a sample dissolved in water under physiological conditions, so that the sample is preserved in amorphous glassy ice. And observed again with a transmission electron microscope and photographs were collected. And finally, carrying out three-dimensional reconstruction on the molecules by combining a related algorithm and an image processing technology. Previously, this method has not been widely used because of the inability to achieve very high resolution. The invention of direct electron detection cameras and optimization of related algorithms have substantially solved this problem over the last two decades, enabling cryoelectron microscopy to achieve near atomic resolution. The revolutionary breakthrough of the cryoelectron microscope enables the cryoelectron microscope to be developed vigorously. China also catches the opportunity, obtains a series of achievements by applying the technology, and occupies a place in the field of cryo-electron microscopes.
Through application and development in recent years, image collection and processing in the later period of the cryoelectron microscope technology are mature. However, the technical development of frozen sample preparation has been relatively ineffective. Up to now, the repeatability of samples is still not high, and the conditions for making samples still need to be continuously searched according to different samples. Good frozen samples require that the amorphous glassy ice be of suitable thickness, have a uniform distribution of biological macromolecules and be in a non-denatured state. Due to the maturity of the post-image processing technology, the problem of the early-stage sample preparation is gradually highlighted. A good sample often becomes the key to obtaining high resolution structures.
A porous carbon film is generally covered on a sample supporting net applied to the preparation of a cryoelectron microscope sample, if no supporting film is additionally covered, most of samples in holes exist in a gas-liquid interface, and biological macromolecules existing in the gas-liquid interface are often denatured to influence the structure. The porous carbon film has non-uniform surface properties, poor electrical conductivity, and weak mechanical rigidity. Poor conductivity can cause the biomacromolecules to drift during photographing, thereby affecting the resolution of the final structure. Typically, to address these problems, researchers will lay an amorphous, ultra-thin carbon film on top of a porous carbon film. However, the amorphous carbon film brings about a large background noise, which causes a great trouble in post-image processing. In recent years, there have been problems in attempting to use a material such as silicon nitride or gold as a support film. However, these grids have not been widely used due to problems of poor conductivity, poor rigidity at liquid nitrogen temperature, immature manufacturing process, etc. Later, graphene, a two-dimensional material with little background, was considered as a support film. Graphene is thin and electrons can easily pass through it. In addition, the ultrahigh conductivity and mechanical strength of graphene make it an ideal mesh-carrying material. However, due to the hydrophobic nature of graphene, biological macromolecules do not readily attach. One method is to oxidize graphene, which has hydrophilicity and can be combined with biological macromolecules. However, this reduces the conductivity of the graphene, which can cause a series of difficulties. In addition, the surface of the graphene oxide has a plurality of functional groups, so that the thickness of the graphene film is increased, and the background noise is stronger. Therefore, it is necessary to provide an ideal novel material for supporting the grid of the cryoelectron microscope.
Disclosure of Invention
The invention aims to provide a reduced graphene oxide film (loaded on an electron microscope carrier) and application thereof in a biological cryoelectron microscope.
Due to the ultra-strong conductivity, mechanical property, uniformity and ultrathin characteristic of the graphene, the problems of strong background noise, poor conductivity, macromolecule distribution on a gas-liquid interface and the like existing in the conventional electron microscope carrier net can be partially or even completely eliminated. The graphene oxide solves the hydrophobic characteristic of graphene, but reduces the conductivity, and the reduced conductivity can be compensated after reduction. In addition, a plurality of functional groups exist between graphene oxide layers, the interlayer spacing is large, and the interlayer spacing is reduced after reduction, so that the noise is relatively small.
The preparation method of the reduced graphene oxide film provided by the invention comprises the following steps:
and transferring the graphene oxide onto an electron microscope carrier, and reducing to obtain the reduced graphene oxide film.
The graphene oxide can be prepared by adopting an improved Hummers method, and specifically comprises the following steps:
adding graphene (such as 3g) into a mixture with the volume ratio of 9: 1 (e.g. 360ml and 40ml respectively) of H2SO4/H3PO4The solution and potassium permanganate (e.g., 18g) are mixed and heated to 50 ℃ with constant stirring (e.g., 12 h). After the reaction was complete, the temperature was reduced to room temperature and 30% H was added to the ice-water bath2O2The solution (e.g., 3ml) is filtered through a filter (300um) to remove larger fragments. Centrifuging at room temperature at 4000rpm for 4H, removing supernatant, and precipitating with 200ml H2O, 30% HCl and absolute ethyl alcohol for heavy suspension, and absolute ethyl alcohol for heavy suspension.
After the resuspension steps described above, centrifugation was carried out for 4h at 4000rpm and the supernatant was removed.
And after the second time of resuspension and centrifugation by absolute ethyl alcohol, drying in a vacuum drying oven for later use.
Transferring the graphene oxide according to the following method:
diluting graphene oxide with a methanol-water mixed solution, ultrasonically dispersing, and transferring to a (gold) electron microscope carrier, wherein the method specifically comprises the following steps:
taking 1ml of high-concentration graphene oxide dissolved in water, and adding 7ml of dispersion buffer (the dispersion buffer is water and methanol (CH)3OH) in a volume ratio of 1: 5) mixing, and ultrasonic treating for 10 min. The solution after ultrasonic treatment was equally distributed to 6 EP tubes of 1.5ml, and centrifuged at 4000g at room temperature for 10min (to remove fragmented graphene oxide). The supernatant was removed and resuspended with 500ul of dispersion buffer per tube, followed by sonication for 2 min. 500g was centrifuged at room temperature and low speed for 1min (to remove undispersed graphene oxide), the supernatant was transferred to a new EP tube, coated with tinfoil and stored at 4 ℃.
The transfer method comprises the following specific steps:
carrying out thin air glow discharge hydrophilization treatment on a (gold) electron microscope carrier net: placing the net in a closed container, and vacuumizing to 10%-3～10-4After holding, the low gear voltage is processed for 10 s. Container washingAfter a net charge of distilled water to just over four short bars, the metal sheet at the bottom of the container was placed on the short bars. A piece of filter paper of the same size as the metal piece was cut and laid on the metal piece, and then the treated carrier net was placed on the filter paper with the right side up (taking care that the carrier nets were not overlapped). Slowly, gently and uniformly dropwise adding 200ul of the treated graphene oxide solution to the liquid level, then opening a valve of a water outlet pipe, and allowing water in the container to slowly flow out (1-2 drops per second). The filter paper is taken out and placed in a culture dish to naturally dry the net (the net can be placed in an oven at 65 ℃ to accelerate drying).
In the above preparation method, the reduction step is as follows:
and gradually heating to 300-600 ℃ in an argon hydrogen environment, and keeping the temperature constant to realize reduction of the graphene oxide.
In the preparation method, the flow rate of the argon gas can be 80-120 sccm/min, and specifically can be 100 sccm/min;
the volume content of argon in the argon-hydrogen gas can be 90-98%, such as 95%.
The heating rate in the heating step can be 2-5 ℃/min, and particularly the heating can be carried out at the rate of 2 ℃/min.
In the preparation method, the constant temperature time can be 60-100 min, for example, the constant temperature is 60min at 300 ℃;
and naturally cooling to room temperature after the reduction is finished.
The method for preparing the reduced graphene oxide electron microscope grid supporting film is simple, the required raw materials are convenient to obtain, the large-scale preparation can be realized, and hundreds of reduced graphene oxide electron microscope grid supporting films can be prepared at one time. According to the invention, the integrity and the coverage rate of the reduced graphene oxide are detected by adopting a transmission electron microscope, the coverage rate can reach 80-90%, the number of layers can reach a thin layer or even a single layer, and the background noise of a sample in electron microscope imaging is greatly reduced. Through the test of three biological samples (20S proteasome, apoferritin and ribosome), the grid is applied to the three-dimensional reconstruction of a cryoelectron microscope, and the resolution ratio of the grid is better than that of graphene oxide.
Compared with an ultrathin carbon film and graphene oxide as supporting films, the reduced graphene oxide film provided by the invention has stronger conductivity, so that drift caused by electrons during photographing exposure can be reduced. The ultra-thin carbon film, although thin, is amorphous carbon, the surface properties are not uniform, and reduced graphene oxide, which is more uniform in properties and thinner, can greatly reduce background noise. Compared with a porous carbon film without an additional supporting film, the biomacromolecules are more attached to the surface of the reduced graphene oxide rather than a gas-liquid interface, so that the denaturation of the biomacromolecules and the formation of dominant orientation are reduced. The uneven surface of the porous carbon film can also cause uneven distribution of biological macromolecules, so that the molecules are difficult to enter pores frequently, and the problem of reducing graphene oxide does not exist. Compared with graphene oxide, the reduced graphene oxide has higher conductivity, and the problem of sample drift caused by light beam irradiation is reduced. In addition, the reduced graphene oxide is thinner, and thus the background noise of the transmission electron micrograph is also lower.
The fabrication of frozen samples has been a major challenge due to the shortcomings of current support membranes. The search for a more stable, more conductive, and less background noise support film is the focus of current sample preparation. The reduced graphene oxide is the support film which is most suitable for practical application at present, and has other characteristics such as ultrahigh mechanical strength and the like while overcoming the defects of the conventional support film. The method can be used for preparing samples of a cryoelectron microscope and performing later three-dimensional reconstruction, and is expected to bring great convenience to large-scale production of the single-layer reduced graphene oxide grid.
Drawings
Fig. 1(a) is a schematic diagram of reduced graphene oxide electron microscope carrier mesh preparation, and fig. 1(b) and fig. 1(c) are a transmission electron microscope image and a diffraction image of a support film covered with a single layer of reduced graphene oxide electron microscope carrier mesh, respectively.
Fig. 2 is a schematic diagram of the interlayer spacing between graphene oxide and reduced graphene oxide and the sample preparation effect, wherein fig. 2(a) and fig. 2(b) are schematic diagrams of the interlayer spacing between graphene oxide and reduced graphene oxide and the frozen sample irradiated by an electron beam, respectively.
FIG. 3 is an oxidationThe characteristics of the graphene and reduced graphene oxide electron microscope carrier net supporting film are represented, wherein fig. 3(a) is a transmission electron microscope image of the graphene oxide electron microscope carrier net supporting film, fig. 3(b) is an image of the graphene oxide electron microscope carrier net supporting film under high power (50000 x), and the lower image is an image obtained by passing the upper image through 80e/A for four times2Fig. 3(c) and 3(d) show the results of the reduced graphene oxide electron microscope supporting film corresponding to fig. 2(a) and 2(b), respectively.
Fig. 4 is a comparison of application of graphene oxide and reduced graphene oxide to a cryo-electron microscope, where fig. 3(a), fig. 4(b), and fig. 4(c) are transmission electron microscope images of 20S proteasome, apoferritin, and ribosome on a graphene oxide electron microscope mesh support film, respectively, and fig. 4(d), fig. 4(e), and fig. 4(f) are results of fig. 4(a), fig. 4(b), and fig. 4(c) of the reduced graphene oxide electron microscope mesh support film, respectively.
Fig. 5 shows the distribution of particles on a reduced graphene oxide electron microscope mesh support film and the three-dimensional reconstruction results of three samples using the reduced graphene oxide electron microscope mesh support film and the reduced graphene oxide electron microscope mesh support film. FIG. 5(a) is plotted from the particle coordinates in the tomogram reconstruction; fig. 5(b) is an FSC curve (R in abscissa is resolution) of three-dimensional reconstruction results of 20S proteasomes on graphene oxide and reduced graphene oxide cargo nets using nearly equal numbers of particles; fig. 5(c) is the three-dimensional reconstruction results of the three samples on the graphene oxide electron microscope supporting film (gray) and the reduced graphene oxide electron microscope supporting film (yellow).
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of reduced graphene oxide Electron microscopy Supported Membrane
Fig. 1(a) is a schematic diagram of reduced graphene oxide electron microscope mesh preparation, and the principle is that a solution containing graphene oxide is dripped into a container filled with water. The liquid level of the solution is higher than the carrier net arranged in the solution, and the graphene oxide floats above the liquid level. And opening the water outlet valve to enable the liquid level to slowly fall, and then attaching the graphene oxide to the carrying net. Naturally drying the graphene oxide electron microscope carrying net, and then carrying out high-temperature reduction at 300 ℃ to obtain the reduced graphene oxide electron microscope carrying net, wherein the specific process is as follows:
1. preparation of graphene oxide
Adding a graphene powder into 3g of graphene, wherein the volume ratio is 9: 1H2SO4/H3PO4The solution (400ml) and 18g of potassium permanganate were mixed and heated to 50 ℃ with constant stirring for 12 h. After the reaction was complete, the temperature was reduced to room temperature and 3ml of 30% H were added to the ice-water bath2O2The solution was filtered through a filter (300um) to remove larger fragments. Centrifuging at room temperature at 4000rpm for 4H, removing supernatant, and precipitating with 200ml H2And (3) resuspending O, 30% HCl and absolute ethyl alcohol, centrifuging for 4 hours at 4000rpm after each resuspension, removing supernatant, then resuspending the mixture by using absolute ethyl alcohol, and drying the mixture in a vacuum drying oven for later use after centrifugation.
2. Transfer of graphene oxide
Taking 1ml of high-concentration graphene oxide dissolved in water, and adding 7ml of dispersion buffer (the dispersion buffer is water and methanol (CH)3OH) in a volume ratio of 1: 5) mixing, and ultrasonic treating for 10 min. The solution after ultrasonic treatment was equally distributed to 6 EP tubes of 1.5ml, and centrifuged at 4000g at room temperature for 10min (to remove fragmented graphene oxide). The supernatant was removed and resuspended with 500ul of dispersion buffer per tube, followed by sonication for 2 min. 500g was centrifuged at room temperature and low speed for 1min (to remove undispersed graphene oxide), the supernatant was transferred to a new EP tube, coated with tinfoil and stored at 4 ℃. Then transferred onto a metal mesh: carrying out rarefied air glow discharge hydrophilization treatment on a gold electron microscope carrier net: placing the net in a closed container, and vacuumizing to 10%-3～10-4After the second step, the low-level voltage is processed for 10 s. In the figure, the vessel is cleaned and filled with distilled water until just over four short bars, and the metal sheet at the bottom of the vessel is placed on the short bars. Cutting a piece of filter paper with the size similar to that of the metal sheet, spreading the filter paper on the metal sheet, and then placing the treated carrier net on the filter paper with the right side facing upwards (multiple carriers)The nets need not overlap). Slowly, gently and uniformly dropwise adding 200ul of the treated graphene oxide solution to the liquid level, then opening a valve of a water outlet pipe, and allowing water in the container to slowly flow out (1-2 drops per second). The filter paper is taken out and placed in a culture dish to naturally dry the net (the net can be placed in an oven at 65 ℃ to accelerate drying).
3. High temperature reduction of graphene oxide
And gradually heating to 300 ℃ at a heating rate of 2 ℃/min under an argon-hydrogen environment (the flow rate is 100sccm/min), maintaining the temperature to perform high-temperature reduction on the graphene oxide for 60min, and naturally cooling to room temperature after reduction to obtain the reduced graphene oxide supporting film.
The transmission electron microscope image and the diffraction image of the support film covered with the single-layer reduced graphene oxide electron microscope grid prepared in this embodiment are respectively shown in fig. 1(b) and fig. 1(c), and it can be seen that the graphene is uniformly covered on the electron microscope grid, the background noise of the support film is low, and the diffraction image also shows an obvious single-layer graphene diffraction mode (regular hexagon, one point on the right side is shielded).
Fig. 2 is a schematic view of the interlayer spacing and the sample preparation effect of the graphene oxide and reduced graphene oxide electron microscope carrier supporting film prepared in this embodiment; fig. 2(a) is a schematic diagram of the graphene oxide layer spacing and the frozen sample irradiated by the electron beam, and fig. 2(b) is a schematic diagram of the frozen sample irradiated by the reduced graphene oxide layer spacing and the light beam, which shows that the layer spacing of the reduced graphene oxide is smaller and the background noise of the sample is less.
Fig. 3 is a characteristic representation of the graphene oxide and reduced graphene oxide electron microscope carrier supporting film prepared in this example; wherein, FIG. 3(a) is a transmission electron microscope image of graphene oxide, FIG. 3(b) is an image of graphene oxide with a mesh at high magnification (50000X), and the lower image is an image of the upper image taken four times2The result after irradiation; fig. 3(c) and 3(d) show the results of reducing graphene oxide corresponding to fig. 3(a) and 3(b), respectively, and it can be seen that some bubbles exist on the graphene oxide, and the bubbles become more and more obvious after irradiation; this phenomenon was not observed in the case of reduced graphene oxide. The presence of these bubbles suggests oxidationThe graphene layers have a large pitch.
Example 2 application of reduced graphene oxide supporting film for electron microscopy
Frozen samples were prepared, using 20S proteasome, apoferritin and ribosomes as examples.
The grid covering the reduced graphene oxide prepared in example 1 was subjected to discharge hydrophilization treatment: the vacuum pumping time in the treatment process is 15s, and the low-grade discharge time is 15 s. Subsequently, 4ul of 20S proteasome solution was added dropwise to a reduced graphene oxide electron microscopy grid support membrane and then transferred to a FEI Vitrobot (frozen sampling apparatus). The humidity of the Vitrobot chamber was adjusted to 100% and the temperature was adjusted to 12 ℃. The filter paper nip time was set to 2s, and force was set to-2. After the filter paper clip absorbs the redundant liquid, the carrying net is rapidly inserted into the liquid ethane, so that the water on the carrying net is rapidly frozen to form glass state ice, and then the glass state ice is transferred to an electron microscope for data acquisition.
The ferritin frozen sample and the ribosome frozen sample were obtained separately as described above.
The cryoelectron microscopy data were collected using a Tecnai Arctica cryoelectron microscopy equipped with a Falcon II camera at an acceleration voltage of 200 kV. The under-focus value range of the collected photos is-2.0 to-3.2 um, and the pixel size is
The exposure time was 1.2 s. Each picture was made by stacking the results of 19 low dose exposures, for a total dose of about
The collected photographic data were first corrected for electron beam induced sample drift using the MotionCor2 algorithm and then the CTF and under focus values were estimated using the CTFFIND4 algorithm. Particles were then picked using the relion2.1 software and subjected to iteratively repeated two-dimensional classification. And removing the particles with poor two-dimensional classification results, and performing three-dimensional reconstruction by using the residual particles.
The present embodiment also employs an electron tomography technique. Collecting electron tomography photos, using Titan Krios cryo-electron microscope equipped with Gatan K2 camera, 300kV acceleratingA voltage. The electron irradiation dose of each photo is about
The total dosage is about
Magnification of 64000 x. For each deflection series, photographs were collected from +51 to-51 in 3 steps at an under-focus value of approximately-5.0 um. Each photograph was calibrated using MotionCor2, and all deflection series were calibrated and reconstructed using IMOD with a binning factor of 4, corresponding to a pixel size of
And (4) analyzing results:
fig. 4 is a comparison of graphene oxide and reduced graphene oxide applied to a cryo-electron microscope, fig. 4(a) and fig. 4(d) are transmission electron microscope images of 20S proteasome on a graphene oxide and reduced graphene oxide electron microscope mesh supporting film, respectively, fig. 4(b) and fig. 4(e) are transmission electron microscope images of apoferritin on a graphene oxide and reduced graphene oxide electron microscope mesh supporting film, respectively, and fig. 4(e) and fig. 4(f) are transmission electron microscope images of ribosomes on a graphene oxide and reduced graphene oxide electron microscope mesh supporting film, respectively.
Fig. 5 shows the distribution of particles on a reduced graphene oxide electron microscope mesh support film and the three-dimensional reconstruction results of three samples using the reduced graphene oxide electron microscope mesh support film and the reduced graphene oxide electron microscope mesh support film. Fig. 5(a) shows that the particles are mostly attached to the reduced graphene oxide electron-microscope supporting film. And selecting a representative tomogry three-dimensional reconstruction result, and establishing a rectangular coordinate system by taking the direction perpendicular to the direction of the reduced graphene oxide electron microscope carrier net supporting film as a Z axis. Neglecting the Y-axis direction coordinates of the particles, and drawing the X-axis direction coordinates and the Z-axis direction coordinates to obtain a graph shown in FIG. 5 (a); fig. 5(b) is an FSC curve of three-dimensional reconstruction results of 20S proteasomes on a graphene oxide electron microscope mesh support film and a reduced graphene oxide electron microscope mesh support film using almost equal numbers of particles, the curve indicating that the reduced graphene oxide electron microscope mesh support film can obtain a structure with higher resolution (R in abscissa is resolution); fig. 5(c) shows three-dimensional reconstruction results of three samples on a graphene oxide electron microscope mesh supporting film (GO, left side) and a reduced graphene oxide electron microscope mesh supporting film (RGO, right side), and the structures obtained by reducing the graphene oxide electron microscope mesh supporting film can show more details.
Claims (6)
1. A preparation method of a reduced graphene oxide film comprises the following steps:
and transferring the graphene oxide onto an electron microscope carrier, and reducing to obtain the reduced graphene oxide film.
2. The method of claim 1, wherein: the reduction steps are as follows:
and heating to 300-600 ℃ in an argon hydrogen environment, and keeping the temperature constant to realize the reduction of the graphene oxide.
3. The method of claim 2, wherein: the flow rate of the argon and hydrogen is 80-120 sccm/min;
the heating rate in the heating step is 2-5 ℃/min.
4. The production method according to claim 2 or 3, characterized in that: the volume content of argon in the argon gas is 90-98%.
5. The production method according to any one of claims 2 to 4, characterized in that: the constant temperature time is 60-100 min.
6. The reduced graphene oxide membrane prepared by the method of any one of claims 1-5.
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