WO2020116990A1 - Graphene composite for electron microscopic observation and method for manufacturing specimen substrate - Google Patents

Abstract

When used in electron microscopic observation, a graphene composite of the present invention can allow an accurate and clear observation of a specimen to be observed. Particularly, in the case of a graphene composite supported by a support layer consisting of crystalline material, a specimen to be observed can be formed to be crystallized, and, in the case of a graphene composite supported by a support layer consisting of a non-crystalline material, an insulating bio specimen or the like can be accurately and clearly observed, and even a specimen that cannot be mounted on a sole graphene layer can be stably mounted on the graphene composite due to the chemical transparency of graphene.

Description

Method for manufacturing graphene composite and sample substrate for electron microscope observation

The present invention relates to a graphene complex for electron microscopy that can observe a sample to be observed clearly and accurately.

The crystalline phase of a specific material allows the characteristics of the material to be grasped in more detail, and the material made of the crystalline phase has superior physical and chemical properties compared to the amorphous material. In general, a very high temperature is required to form a crystalline phase of a material, or it is performed in a solution phase, because it undergoes a process of reassembling the constituent molecules of the material. In the case of a solution phase, it is relatively easy to produce a single crystal of a soluble material, because crystal growth is made by homogeneous nucleation, and in the case of heterogeneous nucleation that forms crystals on a solid substrate, there is a problem that it is difficult to greatly increase the crystal size. do. In particular, in the case of biomolecules such as proteins, cells, and DNA, it is impossible to increase the temperature or make a saturated solution, so single crystal growth is very difficult. Single crystals such as protein and DNA are very important technologies for determining the structure of biomolecules, and have been having difficulty in observing these biomaterials precisely and revealing their characteristics.

On the other hand, graphene having a large sheet shape composed of hybridized SP ² bonds of carbon atoms is a perfectly flat and defect-free material. Therefore, since the discovery of graphene, it has attracted attention as the best template material for epitaxial growth of various 2D materials.

An object of the present invention is to provide a graphene complex for electron microscope observation that can observe a sample to be observed clearly and accurately.

1. Graphene complex for electron microscopic observation, including a graphene layer and a support layer located on one surface of the graphene layer.

2. In the above 1, the graphene complex is a graphene complex in which the sample to be observed is located on the other surface of the graphene layer.

3. In the above 2, wherein the sample to be observed is at least one graphene complex selected from the group consisting of oligonucleotides, proteins, polypeptides, cells, organelles, protozoa, viruses, metals, polymers and semiconductors.

4. In the above 1, the support layer is a graphene composite made of an amorphous material.

5. In the above 1, the support layer is a graphene composite made of a crystalline material.

6. In the above 5, wherein the crystalline material is at least one selected from the group consisting of metal, silica, quartz, ceramic, paper, plastic, polymer, cloth and composites thereof Graphene composite.

7. In the above 1, wherein the graphene layer is a single-layer graphene layer graphene complex.

8. In the above 1, wherein the graphene complex is 10 to 50 Å thick graphene complex.

9. In the above 1, the microscope is a graphene complex selected from the group consisting of transmission electron microscope, scanning electron microscope, scanning probe microscope, scanning tunneling microscope, confocal microscope, X-ray microscope, optical microscope and tomography microscope. .

10. A method of manufacturing an electron microscope sample substrate comprising forming a crystalline sample on the other surface of the graphene layer of the composite of 5 above.

11. The method of 10 above, wherein the crystalline sample is at least one selected from the group consisting of oligonucleotides, proteins, polypeptides, cells, organelles in the cell, protozoa, viruses, metals, polymers, and semiconductors.

When the graphene composite of the present invention is used for electron microscopic observation, a sample to be observed can be accurately and clearly observed. In particular, in the case of a graphene composite supported by a support layer made of a crystalline material, a sample to be observed can be crystallinely formed, and in the case of a graphene composite supported by a support layer made of an amorphous material, an insulating bio sample The back can be accurately and clearly observed, and even in the case of a sample that could not be placed on a single graphene layer, it has the advantage of being stably placed due to the chemical transparency of graphene.

1 is a SEM sample obtained by placing a section sample on a Kapton film without carbon coating (A), a SEM observation by coating the sample surface with carbon (B), and a graphene layer positioned on top using a Kapton film as a support layer. It shows the SEM observation result (C) by placing a target section sample on the graphene composite of the invention.

2 and 3 are the results of imaging each of the cases in which the present invention graphene complex was used (FIG. 2) and the kapton film alone was not used (FIG. 3).

4 is a result of observing under a microscope by growing a lysozyme protein crystal using the graphene complex of the present invention.

5 to 7 shows the growth pattern of gold crystals over time after PECVD as a support layer (copper foil, SiO ₂ /Si), as a result of observation under a microscope by growing gold crystals using the graphene composite of the present invention. . The thickness of the gold thin film was grown to 1 nm, 3 nm, 3.4 nm, 4.5 nm, 5 nm, and 7 nm, and the current resistance exhibited by the thin film was measured as 145 Ω for 3 nm and 75 Ω for 5 nm.

8 shows an atomic force microscope (AFM) picture of the gold crystal film formed on the graphene of FIG. 6.

FIG. 9 shows that a crystal film having a thickness of about 3 nm was formed as a result of showing the thickness according to the position (horizontal) of the gold crystal film of FIG.

10 shows the out-of-plane XRD (X-ray Diffraction) spectrum of the crystal film of FIG. 9, and the Au (111) crystal plane mainly appears and the thickness of the crystal layer is 2-3 layers of gold atoms, as seen in the Laue Peak on the right. You can see that it is done.

11 shows the UV/VIS spectrum of the gold single crystal layer formed on the graphene layer supported on the quartz support layer to evaluate the optical properties of the gold crystal film. 11, the transmittance decreases as the gold crystal layer deposition thickness increases.

12 and 13 are electron microscope images showing the growth of an indium single crystal formed on a graphene layer supported on a copper foil support layer, which is formed at 277K and 100K, respectively.

FIG. 14 shows an AFM image of crystals after transferring the indium single crystal layer of FIG. 12 to a silicon substrate with PMMA.

15 is a graph showing the thickness according to the position (horizontal) of the indium single crystal layer of FIG. 14, and it can be seen that 10-30 nm thick crystals were grown.

16 is an electron microscope image showing single crystal growth of tin formed on a graphene layer supported on a copper foil support layer.

17 to 19 are electron microscopic images showing the growth of Pd, Pt, and Co single crystals formed on the graphene layers supported on the copper foil support layers, respectively.

Hereinafter, the present invention will be described in detail.

The present invention provides a graphene complex for electron microscopic observation, including a graphene layer and a support layer located on one surface of the graphene layer.

The graphene composite is a sample supporting substrate for electron microscopy observation, and the sample to be observed can be clearly and precisely observed due to the excellent conductivity and chemical transparency of the graphene layer made of carbon, and the bio sample having insulating properties is also clearly observed. It has the advantage that it is possible.

When observing the electron microscope using the graphene complex, the sample to be observed may be located on the upper surface, the lower surface, and both sides of the graphene complex in a range capable of observing the electron microscope, but specifically, the other surface of the graphene layer That is, it may be located on a surface that is not supported as a support layer among both surfaces of the graphene layer.

As a kind of a sample that can be observed under an electron microscope using the graphene complex, there is no particular limitation. Specifically, oligonucleotides, proteins, polypeptides, cells, organelles, protozoa, viruses, metals, polymers, organic molecules and semiconductors It may be at least one selected from the group consisting of.

The support layer can be used without limitation as long as it is used as a sample support layer for an electron microscope in the prior art, and can be varied depending on the type of electron microscope or the nature of the sample to be observed. It may be a crystalline support layer made of a material or an amorphous support layer made of a material that is not crystalline.

In one embodiment of the present invention, as a material constituting the crystalline support layer, specifically, may be at least one selected from the group consisting of metal, silica, quartz, ceramic, paper, plastic, polymer, cloth, and composites thereof. , It is not particularly limited.

When the graphene is supported by the crystalline support layer, a sample that has already been prepared may be placed on the graphene layer, but it may be observed by forming a crystalline sample on the graphene layer. In this case, the crystallinity of the crystalline sample can be further improved to more clearly observe the internal structure such as the crystal structure.

A sample substrate in the case of using a crystalline support layer as an embodiment of the present invention may be prepared by including a step of forming a crystalline sample on the graphene layer of the present invention graphene composite.

The crystalline sample may be at least one selected from the group consisting of oligonucleotides, proteins, polypeptides, cells, intracellular organelles, protozoa, viruses, metals, polymers, organic molecules and semiconductors, but is not limited thereto, and crystallinity Any sample to be observed can be freely selected and formed on the graphene layer.

The crystalline sample can be applied without any particular limitation on the method of forming the sample as an observation subject of an electron microscope. For example, a chemical vapor deposition method, sputtering, etc. may be mentioned, but an appropriate method may be applied depending on the material.

In one embodiment of the present invention, as a material constituting the amorphous support layer, specifically, it may be at least one selected from the group consisting of amorphous polymers, amorphous inorganic materials and composites thereof, but is not particularly limited thereto.

It has been difficult to load insulating samples such as conventional biomolecules on a single graphene layer, but when using a graphene layer supported on a support layer made of a previously loaded amorphous material, it has been found to be stably loaded. It is believed that this is achieved by allowing graphene to penetrate the chemical and physical properties of the support layer.

In addition, in this case, since graphene has excellent electrical conductivity, there is no need for a separate sample carbon coating for electrical conductivity, and thus the contamination of the sample can be prevented. The advantage is that you can proceed with the experiment.

The graphene layer included in the graphene composite may be a single graphene layer, a double graphene layer, or multiple graphene layers, but is preferably a single graphene layer or a thin graphene layer in terms of forming a thin graphene composite for microscopic observation.

The graphene layer is located on one surface of the support layer, and is not limited to a specific surface such as upper, lower, or both sides of the support layer, but considering the aspect of placing the target material on the graphene layer during electron microscope observation , It is preferably located on the upper portion of the support layer.

The graphene layer is located on one surface of the support layer by at least one method from the group consisting of chemical vapor deposition, vapor deposition, molecular beam deposition, vacuum deposition, activation reaction deposition, mechanical separation, fluid collision separation, and chemical separation. It may be, but is not limited thereto, and any method known in the art can be freely selected as long as it can effectively position the graphene layer on the support layer.

The graphene composite is a graphene composite for electron microscope observation, and has no particular limitation on its thickness, and may be set in various ranges depending on the type of electron microscope and the nature of the support layer, but has a thinner thickness to improve electron permeability, etc. To obtain the effect of, for example, it may be 5 to 70, 6 to 65, 7 to 60, 8 to 55, 9 to 55, 10 to 50 mm 2, preferably 10 to 50 mm thick.

The graphene composite is a graphene composite for electron microscopic observation, and is based on chemical transparency of graphene and conductivity of a carbon-based material. Therefore, as a kind of microscope, TEM (transmission electron microscope), SEM (scanning electron microscope), AFM (scanning probe microscope), STM (scanning tunneling microscope), confocal microscope (confocal microscope), X-ray microscope, optical microscope and It may be one selected from the group consisting of a tomographic microscope (tomography microscope), but is not limited thereto.

Hereinafter, examples will be described in detail to specifically describe the present invention.

Example 1 Observation of a bio sample using a graphene complex supported on a flexible support layer

1. Experimental method

(1) Preparation of graphene complex

The graphene material is grown for about 1 hour in a methane and hydrogen atmosphere at 1000° C. using a copper foil cleaned with a nickel etchant in a hot wall furnace as a catalyst. More specifically, graphene was manufactured on a 4.8 μm thick copper thin film (Alfa Aesar) by low pressure chemical vapor deposition (CVD). Prior to synthesis, the copper film was immersed in a nickel etchant (transene, TFB) and washed. The specific growth conditions are as follows.

First, a copper thin film was put in a reactor, and the pressure was reduced, followed by heating to 1000° C. and annealing for 20 minutes under 100 sccm H ₂ conditions. 30 sccm CH ₄ and 30 sccm H ₂ were introduced for 40 minutes to promote graphene growth. The furnace was cooled to room temperature.

The graphene material grown on the copper foil as described above can be used as an electron microscope substrate capable of growing crystals without additional treatment, but when optical transparency is required, the graphene grown on the copper foil is transferred to a transparent substrate. use. The image of the biosample of the present invention was used by transferring a graphene sample grown on a copper foil onto a kapton foil via PMMA or by transferring a graphene sample onto a kapton foil using an electrostatic film.

(2) Preparation of mouse brain cell sample substrate

Cell sections are made by staining cell membranes with osmium, uranium, lead, etc., and forming thin sections of tens of nanometers in flexible membranes (Kapton films, etc.) with clean surfaces using ultra-thin sectioning machines using biothin tissue embedded in resin. It was used in experiments to observe the microstructure of.

(3) Preparation of lysozyme protein sample substrate

The graphene material grown on the copper foil on the glass substrate was transferred and used as a substrate for growing the lysozyme protein.

2. Experimental results

(1) Observation of mouse brain cell sample

These experiments are mainly used to obtain microstructures of successive sections of biological samples using a scanning electron microscope to analyze the tertiary structure of cells. In general, the flexible membrane used in this experiment lacks conductivity and is observed by coating carbon (C) or the like while attaching the sample section (section) to the flexible membrane for proper structural analysis of the biological sample (see the schematic diagram of FIG. 1B). ).

Referring to Figures 1A, B, when a section sample is placed on a Kapton film without carbon (C) coating, it can be confirmed that the section of the section sample is darkly observed due to the charging phenomenon, so that the proper structure cannot be confirmed (Fig. 1A, yellow). arrow). This is a phenomenon in which extra electrons remaining on the surface of the sample are observed dark as the sample signal. When the sample surface is coated with carbon using the carbon coating method, which has been studied to remove the charging phenomenon (FIG. 1B), the structure of the target sample can be confirmed by reducing the charging phenomenon, but carbon on the sample As a limitation of the type of covering, the result of reducing the resolution of the observed image is somewhat reduced, resulting in a difference in the image resolution of each sample according to the degree of sample coating, and additional experiments such as immunostaining after imaging cannot be additionally performed. This exists.

However, referring to FIG. 1C, a sample of a target fragment is placed on the graphene complex of the present invention in which a graphene layer is positioned on a Kapton film as a support layer, and the results observed by SEM can be confirmed. In comparison, the charging phenomenon has been eliminated, and the resolution of the image is very high enough to clearly identify the structure. In addition, since the surface of the sample is also exposed, it is possible to proceed with additional experimental processes such as immunostaining after imaging.

2 and 3, more specific effects and examples can be confirmed. 2 shows that when using the graphene composite of the present invention, the microstructure of biological tissues, such as cell membranes, is well shown in various sample thicknesses and various acceleration voltages, it can be utilized regardless of sample thickness and acceleration voltage. . Figure 3 shows the effect of improving the conductivity of the graphene coating at the boundary coated with the graphene composite of the present invention on the surface. In the case of the Kapton film using the graphene composite of the present invention, the conductivity is increased compared to the case where it is not, so the charging phenomenon does not appear, and the effect of improving the conductivity is to remove the excess electrons accumulated in the biological tissue sample having insulating characteristics, thereby removing the sample. By reducing the charging phenomenon, clear observation of the microstructure is possible.

(2) Observation of lysozyme protein samples

In the observation of protein crystals, not only crystallinity but also crystal size is very important. In order to form crystal nuclei from a protein solution to continuously grow crystals, not only does the concentration of the protein decrease but continuously flows into the crystals, but also a supporting layer capable of easily growing crystals is required. When the graphene is placed on the support layer, transparency can be secured while growing the protein crystal, so that the crystal can be easily observed and the hydrophilicity of the surface can be controlled through plasma treatment and chemical treatment.

Referring to FIG. 4, the lysozyme protein crystal is grown using a graphene complex and the results observed under a microscope can be confirmed. As a result of growing the crystal using the graphene complex of the present invention, nucleation is easier than in the case where it is not. It can be seen that the crystal growth is fast and large.

Example 2. Growth and observation of a metal single crystal using a graphene composite supported on a crystalline support layer

1. Experimental method

(1) Preparation of graphene complex

1) Use of copper foil support layer

Graphene was prepared on a 4.8 μm thick copper thin film (Alfa Aesar) by low pressure chemical vapor deposition (CVD). Prior to synthesis, the copper film was immersed in a nickel etchant (transene, TFB) and washed. The specific growth conditions are as follows.

First, a copper thin film was put in a reactor, and the pressure was reduced, followed by heating to 1000° C. and annealing for 20 minutes under 100 sccm H ₂ conditions. 30 sccm CH ₄ and 30 sccm H ₂ were introduced for 40 minutes to promote graphene growth. The furnace was cooled to room temperature.

2) SiO ₂ /Si or quartz support layer used

To transfer the graphene layer grown on the copper foil onto another support layer, PMMA is coated on the graphene surface and transferred to a desired substrate by etching copper, or after attaching an electrostatic film to the graphene surface and etching the copper to the desired substrate. The method of transcription was used. At this time, transfer the graphene coated with PMMA onto the SiO ₂ /Si or quartz support layer, dry it sufficiently at room temperature to remove water, and then dry it completely at a temperature of 100℃ or less to improve adhesion. Then, the PMMA was soaked and removed in acetone to complete the graphene complex. In the case of the electrostatic film, a graphene layer attached to the electrostatic film was attached to the target substrate, and then the electrostatic film was compressed to transfer and remove the graphene.

(2) Sample substrate manufacturing

In the case of graphene grown on the surface of the copper foil as described above, since the copper foil serving as a support layer also shows excellent crystal phase due to high growth temperature, the graphene composite supported by the copper foil support layer and the SiO ₂ /Si or quartz support layer All supported graphene composites correspond to graphene composites supported by a crystalline support layer. Metal crystals were grown through vacuum thermal vapor deposition on the graphene composite surface thus manufactured.

Initially the vacuum thermal evaporation chamber was depressurized to 2.0 X 10 ^-6 mbar, and pure metal pellets (99.999%, iTASCO T) were placed in a molybdenum boat (iTASCO) and then evaporated. The deposition was performed at room temperature (300K) or low temperature (277K~100K). The thickness of the thin film was monitored with a quartz crystal microbalance (QCM).

2. Experimental results

(1) Growth and observation of Au single crystal

5 shows an electron microscope image of a gold single crystal formed on a graphene layer supported on a copper foil support layer. When gold is deposited on the graphene layer supported on the crystalline copper foil support layer, it can be seen that fractal crystals grow. In addition, it was observed that the connectivity of crystals was significantly reduced on the graphene surface left in the atmosphere for a long time (Fig. 5, 18h, 1-2 weeks exposed).

6 and 7 show electron microscope images of gold single crystals formed on a graphene layer supported on a SiO ₂ /Si support layer. Again, it can be seen that the crystals in the form of fractals grow as if supported on the copper foil support layer. However, the size of the crystal is smaller than that of the graphene supported on the crystalline support layer.

8 and 9 show AFM images and thicknesses of gold crystals grown on the graphene of FIG. 5, respectively.

10 shows XRD analysis results of the gold single crystal film of FIG. 6. It can be seen that the Au <111> plane occupies the majority, and it can be seen from the Laue pattern of the Au <111> peak of the right enlarged image that the crystal film is composed of 2-3 layers of gold atoms.

11 shows the UV/VIS spectrum of the grown gold crystal layer. Referring to this, as the deposition thickness increases, the transparency of the film decreases, and it can be seen that the location of the resonance peak shifts slightly to a long wavelength. This means that the crystal size increases with increasing deposition thickness.

(2) Growth and observation of In single crystal

12 and 13 are electron microscope images showing the growth of an indium single crystal formed on a graphene layer supported on a copper foil support layer, which is formed at 277K and 100K, respectively. Referring to this, it can be seen that the crystal of FIG. 13 is formed in a very small size compared to the crystal of FIG. 12 because the metal atoms lose kinetic energy due to the low temperature of the substrate and crystallize as soon as it hits the substrate. On a substrate close to room temperature, metal atoms move relatively freely to form a larger crystal phase.

14 and 15 show the thickness of the AFM images and crystals obtained after transferring indium crystals grown on copper foil to a silicon substrate using a PMMA film, respectively.

(3) Growth and observation of Sn single crystal

16 is an electron microscope image showing single crystal growth of tin formed on a graphene layer supported on a copper foil support layer. Referring to this, increasing the deposition thickness increases the crystal size, but there was no significant difference in crystals formed at 277K and 307K. Thus, it can be seen that the temperature of the substrate is preferably 0 degrees Celsius or more in order to maximize the crystal size.

(4) Growth and observation of Pd, Pt, Co single crystal

17 to 19 are electron microscopic images showing the growth of Pd, Pt, and Co single crystals formed on the graphene layers supported on the copper foil support layers, respectively. Likewise, in the case of Au, In, and Sn, small crystals in a fractal form are grown, but in the case of Pd, Pt, and Co, the binding energy with graphene is higher than in the case of Au, In, Sn, and the crystal size is high. Very small

Claims (11)

1. Graphene complex for electron microscopic observation, including a graphene layer and a support layer located on one surface of the graphene layer.
2. The method according to claim 1, wherein the graphene complex is a graphene complex in which the sample to be observed is located on the other surface of the graphene layer.
3. The method according to claim 2, The sample to be observed is at least one graphene complex selected from the group consisting of oligonucleotides, proteins, polypeptides, cells, organelles, protozoa, viruses, metals, polymers and semiconductors.
4. The method according to claim 1, The support layer is a graphene composite made of an amorphous material.
5. The method according to claim 1, The support layer is a graphene composite made of a crystalline material.
6. The method according to claim 5, wherein the crystalline material is a metal, silica, quartz, ceramics, paper, plastics, polymers, fabrics and at least one selected from the group consisting of composites of graphene composite.
7. The graphene composite according to claim 1, wherein the graphene layer is a single-layer graphene layer.
8. The method according to claim 1, The graphene complex is 10 to 50 mm thick graphene complex.
9. The method according to claim 1, The microscope is a graphene complex selected from the group consisting of a transmission electron microscope, a scanning electron microscope, a scanning probe microscope, a scanning tunneling microscope, a confocal microscope, an X-ray microscope, an optical microscope and a tomography microscope.
10. Method of manufacturing an electron microscope sample substrate comprising the step of forming a crystalline sample on the other surface of the graphene layer of the composite of claim 5.
11. The method according to claim 10, wherein the crystalline sample is at least one selected from the group consisting of oligonucleotides, proteins, polypeptides, cells, organelles, protozoa, viruses, metals, polymers and semiconductors.

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