CN116297594A - Carbon nano tube carrier net, preparation method and application thereof - Google Patents

Abstract

The invention provides a carbon nanotube carrier network, its preparation method and application. The invention discloses a carbon nanotube carrier network, which includes a carrier network, one side surface of the carrier network is at least partially covered with two or more layers of stacked carbon nanotube fiber films, and the carbon nanotube fiber film is composed of a shaft The fibers in each carbon nanotube fiber film basically have the same orientation, and the fibers in adjacent carbon nanotube fiber films have different orientations. The carbon nanotube grid provided by the present invention can be applied to cryo-electron microscope analysis of biological samples, for example, can be used for high-resolution cryo-electron microscope structure reconstruction of biological samples.

Description

Carbon nanotube carrier network, its preparation method and use

technical field

The invention belongs to the technical field of biological cryo-electron microscopy, and relates to a carbon nanotube carrier network, its preparation method and application, more specifically, to a carrier network covered with carbon nanotubes applied to the cryo-electron microscope analysis of biological samples, which can be used for high High-resolution cryo-EM structural reconstruction of biological samples.

Background technique

With the development of cryo-electron microscopy technology, especially the breakthroughs in data processing and camera hardware in recent years, this method has broken through the limitations of previous resolutions, making structural analysis at near-atomic resolution more common. The cryo-electron microscopy technique performs rapid freezing of samples in solution under physiological conditions, and stores the samples in ice in an amorphous glass state. Then observe with a transmission electron microscope and collect pictures. Finally, the three-dimensional reconstruction of the sample is carried out by combining relevant algorithms and image processing technology. In cryo-electron microscopy, in order to resolve high-resolution protein structures, a commonly used method is single-particle cryo-electron microscopy, which collects different orientation information of frozen samples, and finally reconstructs the three-dimensional structure. After several years of application and development, the process of image collection and data processing of this technology has become relatively mature. However, the development of technology for the production of frozen samples in the early stage is still progressing slowly.

The support grid used in cryo-TEM sample preparation is generally covered with a layer of porous carbon film. The surface properties of this layer of carbon film are uneven, the conductivity is poor, the mechanical rigidity is weak, and the biological macromolecules will drift when taking pictures, thus Affects the resolution of the final structure. In addition, due to the weak adsorption capacity of porous carbon membranes for biomacromolecules, most of the samples in the pores will exist at the gas-liquid interface, causing problems such as dominant orientation or sample denaturation. Therefore, researchers usually cover additional support films on the porous carbon film, such as amorphous ultra-thin carbon film and other materials, but these support films will bring large background noise, and the conductivity is weak, and they are limited Due to poor rigidity at liquid nitrogen temperature and other reasons, the application value of these support films is low.

Carbon nanotubes, as one-dimensional carbon nanomaterials, have many excellent electrical, mechanical and chemical properties, so they have attracted increasing attention. With the continuous deepening of research on nanomaterials, broad application prospects of carbon nanotubes are also emerging, for example, for field emission electron sources, nano field effect transistors, hydrogen storage materials, and high-strength fibers. Carbon nanotubes can be classified into single-wall carbon nanotubes and multi-wall carbon nanotubes according to the number of layers of carbon atoms forming a tube wall. Few-walled carbon nanotubes are three-dimensional tubular nanomaterials composed of a single layer or a few layers (for example, 2 to 7 layers) of carbon atoms, and have excellent optical, electrical conductivity, mechanical strength, and thermal conductivity.

Citation 1 discloses a transmission electron microscope grid with a special structure and its preparation method. However, the hollow holes of the metal grid framework are partially covered with a carbon nanotube self-interweaving thin film layer, which is suitable for observation and characterization of metal deposition. And because it still covers the hollow holes of the grid in the form of a thin film, rather than distributed on the hollow holes in the form of bundles, it is not much different from the support film of the cryo-TEM grid used for biological samples, and cannot reduce the impact caused by the film. background noise. At the same time, the coverage of the hollow holes of the film is not high, which is not as good as the above-mentioned existing products, and the adsorption effect of the film on biomolecules has not been characterized. Therefore, the self-interweaving film layer of carbon nanotubes is not conducive to the detection and analysis of biological macromolecules by cryo-electron microscopy.

Citation 2 discloses an electron microscope grid, and discloses that its side surface is covered with one or more of amorphous carbon, graphene, and carbon nanotubes as a supporting film. However, Citation 2 does not describe in detail what kind of carbon nanotubes are used and the preparation method of the grid with carbon nanotubes, nor is it used to prepare cryo-electron microscopy samples for the detection of biomacromolecules.

It can be seen that the grid applied to cryo-electron microscopy analysis of biological samples still needs to be further developed in this field, especially the feasibility and effect of applying carbon nanotubes to the grid are unknown. In particular, the mode of action and the effect of action between carbon nanotubes and biological samples have not been explored in any way. Based on this, the present invention provides a new type of grid applied to cryo-electron microscopy analysis of biological samples, that is, a carbon nanotube electron microscope grid.

Citation

Citation 1: CN114689414A

Citation 2: CN113192816A

Contents of the invention

The problem to be solved by the invention

Based on the above-mentioned problems in the prior art, the present invention provides a carbon nanotube carrier network, its preparation method and application.

solutions to problems

[1]. A carbon nanotube carrier network, which includes a carrier network, one side surface of the carrier network is at least partially covered with more than two layers of carbon nanotube fiber films stacked, and the carbon nanotube fiber film is formed by a shaft The fibers in each carbon nanotube fiber film basically have the same orientation, and the fibers in adjacent carbon nanotube fiber films have different orientations.

[2]. According to the carbon nanotube grid described in [1], the fibers in the carbon nanotube fiber film are formed by carbon nanotubes connected head to tail.

[3]. According to the carbon nanotube carrier network described in [1] or [2], the average length of the carbon nanotubes forming the fibers of the carbon nanotube fiber film is 100-800 μm, preferably 200-500 μm, more preferably 200 μm ~300μm.

[4]. The carbon nanotube grid according to any one of [1] to [3], wherein the fibers in the carbon nanotube fiber film are obtained by drawing a super-aligned carbon nanotube array.

[5]. According to the carbon nanotube grid described in any one of [1] to [4], the orientation angle of the fibers in the adjacent carbon nanotube fiber films is 45-90°, preferably 60 to 90°, more preferably 75 to 90°.

[6]. According to the carbon nanotube carrier network described in any one of [1] to [5], the coverage of the carbon nanotube fiber film on the surface of the carrier network is more than 10%, preferably more than 30% , more preferably 50% or more.

[7]. According to the carbon nanotube carrier network described in any one of [1] to [6], the thickness of each layer of the carbon nanotube fiber film is 5 to 30 nanometers, preferably 5 to 20 nanometers, more preferably 5 to 10 nanometers.

[8]. The carbon nanotube support network according to any one of [1] to [7], wherein the carbon nanotube support network has been hydrophilized.

[9]. The method for preparing a carbon nanotube carrier network according to any one of [1] to [8], which includes:

The step of forming a carbon nanotube fiber film: preparing a carbon nanotube array, drawing the carbon nanotube array to form a carbon nanotube fiber film;

The step of stacking carbon nanotube fiber membranes: taking at least two layers of carbon nanotube fiber membranes obtained in the step of forming carbon nanotube fiber membranes to form a stacked carbon nanotube fiber membrane, wherein the adjacent carbon nanotube fiber membranes The orientation of the fibers in the nanotube fiber membrane is different;

A step of transferring the stacked carbon nanotube fiber membrane: transferring the stacked carbon nanotube fiber membrane obtained in the step of stacking the carbon nanotube fiber membrane to a grid, so that the stacked carbon nanotube fiber membrane is at least partially ground cover the carrier network;

And, optionally, a step of hydrophilizing the grid covering the laminated carbon nanotube fiber membrane.

[10]. The method for preparing a carbon nanotube grid according to [9], wherein, in the step of forming a carbon nanotube fiber film, a super-parallel carbon nanotube array is grown by chemical vapor deposition, Drawing the super-aligned carbon nanotube array to form a carbon nanotube fiber film.

[11]. According to [9] or [10], the preparation method of the carbon nanotube grid, wherein, in the step of laminating the carbon nanotube fiber membrane, laying at least two layers of carbon nanotube fiber membrane On the substrate, make the carbon nanotube fiber membrane float on the liquid, press the frame with the adhesive on the surface layer to float on the liquid surface, place at least two layers of carbon nanotube fiber membranes, and place at least two layers of carbon nanotube fibers The tube fiber membrane is lifted to form a laminated carbon nanotube fiber membrane.

[12]. The method for preparing a carbon nanotube grid according to any one of [9] to [11], wherein the step of transferring the laminated carbon nanotube fiber membrane includes:

the step of coating the surface of the grid with an adhesive; and,

The step of laminating the laminated carbon nanotube fiber membrane on the grid;

Preferably, the binder is terpineol.

[13]. The carbon nanotube grid as described in any one of [1] to [8] or the carbon nanotube grid prepared by the preparation method as described in any one of [9] to [12] Use in preparing samples for cryo-electron microscopy;

Preferably, said sample comprises a biological sample.

The effect of the invention

Compared with the traditional two-dimensional supporting film, the specific surface area of the three-dimensional material carbon nanotubes in the carbon nanotube carrier network provided by the present invention is increased, which introduces more contact areas for the combined samples, and has a good sample Adsorption capacity. At the same time, the carbon nanotubes can maintain the liquid through the surface tension and play the role of a supporting film, but it does not introduce the background noise carried by the additional supporting film. Therefore, using carbon nanotubes as a grid to prepare biological cryo-electron microscopy samples can Better resolution of high-resolution structures. Moreover, the method for carrying carbon nanotube electron microscope grid prepared by the present invention has simple operation process and can be prepared on a large scale. In the present invention, the coverage and density of the carbon nanotubes are detected by a transmission electron microscope, and more than 95% of the openings of the grid can be covered with carbon nanotubes with a suitable density, and a single bundle of carbon nanotubes distributed in a network shape can be obtained. , reducing carbon nanotube thickness and potential imaging noise. The test on the biological sample of 20S proteasome shows that the grid can be applied to the three-dimensional reconstruction of cryo-electron microscopy, and can obtain better resolution.

Description of drawings

Figure 1 is the flow chart of carbon nanotube grids for freezing samples and the design of carbon nanotube grids; a in Figure 1 is plasma glow discharge (plasma) treatment of grids under _O2 atmosphere to make them hydrophilic , drop the solution onto the surface of the grid, use filter paper to absorb excess water on the surface of the grid so that there is only a thin layer of water film, insert the grid into the cold trap to prepare frozen samples; b in Figure 1 is the design of the grid, the grid The square holes on the surface are covered with mesh-shaped carbon nanotubes. After the solution is added to the surface of the mesh, biological samples are adsorbed around the carbon nanotubes.

Figure 2 is the image of the 300-mesh gold grid without carbon nanotubes and the carbon nanotube grid under the transmission electron microscope; a in Figure 2 and b in Figure 2 are the 300-mesh gold grid covered with carbon The images of the nanotubes before and after under the transmission electron microscope, c and d in Figure 2 are the transmission electron microscope images of the carbon nanotube grid (no protein sample) at different magnifications, visible in the center of d in Figure 2 A single bundle of carbon nanotubes.

Figure 3 is the frozen transmission electron microscope image of 20S proteasome on the carbon nanotube grid; a, b, and c in Figure 3 are the frozen transmission electron microscope images under the magnifications of 155 times, 1200 times and 69000 times, respectively, Figure 3 White arrows in c identify some 20S proteasomes.

Fig. 4 is a schematic diagram of the results of preparing 20S proteasome samples using carbon nanotube grids. Among them, a in Figure 4 is the two-dimensional protein classification result obtained after processing the cryo-transmission electron microscope image collected by the 20S proteasome sample on the carbon nanotube support network; b in Figure 4 is the 20S proteasome sample on the carbon nanotube The FSC curve of the three-dimensional reconstruction results on the grid shows that the carbon nanotube grid can obtain a high-resolution structure; c and d in Figure 4 are the three-dimensional reconstruction results of the 20S proteasome on the carbon nanotube grid, respectively. View (sideview) and top view (topview), the scale bar (scalebar) is 50 Angstrom (Angstrom).

Detailed ways

Hereinafter, the content of the present invention will be described in detail. The description of the technical features described below is based on representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. It should be noted:

In this specification, the numerical range represented by "numerical value A - numerical value B" means the range which includes numerical value A and B of an end point.

In this specification, the use of "substantially" means that there is an error of 5° or less from the standard or ideal state.

In this specification, the meaning expressed by "may" includes the meaning of performing certain processing and not performing certain processing.

In this specification, "optional" or "optionally" means that the next described event or situation may or may not occur, and that the description includes situations where the event occurs and situations where the event does not occur.

In this specification, references to "some specific/preferred embodiments", "other specific/preferred embodiments", "embodiments" and the like refer to specific elements described in relation to the embodiments (for example, A feature, structure, property, and/or characteristic) is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

In this specification, the terms "comprising" and "having", as well as any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, device, product or equipment that includes a series of steps is not limited to the listed steps or modules, but optionally also includes steps that are not listed, or optionally also includes for these processes, Other steps inherent in a method, product, or apparatus.

In this specification, reference to "plurality" means two or more. "And/or" describes the association relationship of associated objects, indicating that there may be three types of relationships, for example, A and/or B may indicate: A exists alone, A and B exist simultaneously, and B exists independently. The character "/" generally indicates that the contextual objects are an "or" relationship.

The technical solution of the present invention is specifically described below.

<Carbon nanotube grid>

In some embodiments of the present invention, a carbon nanotube carrier network is provided, which includes a carrier network, one side surface of the carrier network is at least partially covered with two or more layers of carbon nanotube fiber membranes stacked, the The carbon nanotube fiber membrane is composed of fibers formed by axially aligned carbon nanotubes, the fibers in each of the carbon nanotube fiber membranes basically have the same orientation, and the adjacent carbon nanotube fiber membranes The orientation of the fibers is different. The carrier net covered with the carbon nanotube fiber membrane provided by the invention is used to make biological frozen samples, and is applied to the sample preparation process of biological cryo-electron microscopes. Due to the large specific surface area of carbon nanotubes, the contact area of the solid-liquid interface can be increased, which is helpful for the adsorption of biological sample particles. At the same time, there is surface tension between carbon nanotubes, which has a supporting effect on the solution and can act as a supporting film instead of The commonly used amorphous ultra-thin carbon film can reduce the introduction of background noise and can be used as a grid for preparing biological frozen samples.

In the present invention, there is no particular limitation on the material of the carrier grid, for example, the material of the carrier grid is selected from copper, nickel, molybdenum, titanium, and gold. In some preferred embodiments, the grid can be made of gold. In some embodiments, the grid is provided with an array of wells. In the present invention, there is no particular limitation on the shape of the holes, for example, square holes, round holes, triangular holes, hexagonal holes, oval holes, but not limited thereto. In some optional embodiments, the mesh of the carrying net is 100-1000 mesh, preferably 200-800 mesh, more preferably 300-500 mesh. Selecting a gold carrier grid and matching the above-mentioned mesh size, the layered carbon nanotube fiber film covered on the surface of the carbon nanotube carrier network provided by the present invention can effectively improve the effect of preparing cryo-electron microscope samples.

In some embodiments of the present invention, the fibers in the carbon nanotube fiber film are formed of carbon nanotubes connected end to end. In some embodiments of the present invention, the average length of the carbon nanotubes of the fibers forming the carbon nanotube fiber film is 100-800 μm, preferably 200-500 μm, more preferably 200-300 μm.

In some embodiments of the present invention, the fibers in the carbon nanotube fiber film are obtained by drawing a carbon nanotube array. Carbon nanotube arrays refer to a type of carbon nanotubes grown on the surface of a certain substrate. The tubes grow cooperatively in a certain direction and are arranged in parallel to form a bunch of neatly grown carbon tubes. Compared with agglomerated carbon nanotubes, it has longer length and higher growth density, and also has the advantages of better orientation and easy dispersion.

In some preferred embodiments, the carbon nanotube array is a super-aligned carbon nanotube array (Super-aligned Carbon Nanotube, referred to as SACNT), that is, the fibers in the carbon nanotube fiber film are Tube arrays are obtained. The super-aligned carbon nanotube array is a vertically arranged super-aligned carbon nanotube array perpendicular to the surface of the substrate. This carbon nanotube has the characteristics of clean surface, strong intertube force and highly ordered arrangement. Ordinary carbon nanotube arrays are not completely parallel, but bend to a certain extent, and the overall appearance is more disordered. In the super-parallel carbon nanotube array, the carbon nanotubes are arranged more orderly, and the degree of alignment is extremely high, so it is called a super-parallel carbon nanotube array. The reason why the carbon nanotubes in the superparallel carbon nanotube array can achieve a highly ordered arrangement among the carbon nanotubes is that the particle size of the catalyst varies in a narrow range and the nucleation density is high. Compared with ordinary carbon nanotube arrays, the surface density of super-aligned carbon nanotube arrays is higher and the diameter distribution of carbon nanotubes is more concentrated.

Generally, a carbon nanotube fiber film can be obtained from a super-aligned carbon nanotube array. The carbon nanotube fiber film is composed of fibers formed by axially aligned carbon nanotubes. The fibers in each carbon nanotube fiber film basically have same orientation. For example, use tweezers to pull out a bundle of carbon nanotubes from the superparallel array, and then a continuous ribbon (fiber) of carbon nanotubes can be obtained, the width of which is determined by the width of the initial carbon nanotube bundle. And from this initial narrow ribbon (fiber) can rapidly expand into the carbon nanotube fiber membrane with the same width as the silicon wafer as the substrate, which is composed of fibers formed by axially aligned carbon nanotubes, carbon nanotube fibers The fibers in the membrane are formed from carbon nanotubes connected end to end. For another example, it is also possible to use a blade to directly scrape out the carbon nanotubes located in a straight line simultaneously from the silicon wafer, and directly obtain a carbon nanotube fiber film with the same width as the length of the blade. The carbon nanotube fiber film drawn from the superparallel carbon nanoarray is very thin. Therefore, this ultra-thin carbon nanotube fiber film has very low density, high transparency, good electrical conductivity, and extremely small heat capacity per unit area. In addition, because the carbon nanotubes are arranged in parallel along the pulling direction, it can will show polarization.

In some embodiments of the present invention, the array of superparallel carbon nanotubes includes or consists of few-walled carbon nanotubes.

In some embodiments, the surface of one side of the carbon nanotube grid provided by the present invention is at least partially covered with more than two layers of stacked carbon nanotube fiber membranes. In some optional embodiments, at least 10% or more, 30% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more of the surface of one side of the carrying net The area is covered with more than two layers of laminated carbon nanotube fiber membranes. In some preferred embodiments, at least 90%, 95%, and 99% of the surface of one side of the grid is covered with more than two layers of carbon nanotube fiber membranes.

In some embodiments, the surface of one side of the carbon nanotube grid is at least partially covered with two, three, four or five or more layers of carbon nanotube fiber membranes. In some preferred embodiments, the surface of one side of the carbon nanotube grid is at least partially covered with two layers of laminated carbon nanotube fiber membranes.

In some specific embodiments, the orientation angle of the fibers in the adjacent carbon nanotube fiber films is 45-90°, preferably 60-90°, more preferably 75-90°, even more preferably 80-90°.

In some embodiments, the average height (ie, the average growth height) of the carbon nanotubes in the super-aligned carbon nanotube array is 100-800 μm, preferably 200-500 μm, more preferably 200-300 μm. At this height, a carbon nanotube fiber film with a thickness of 5-30 nm can be obtained by drawing, preferably a carbon nanotube fiber film with a thickness of 5-20 nm, more preferably a carbon nanotube fiber film with a thickness of 5-10 nm. Under this thickness, it has the advantages of suitable density, high transparency, good electrical conductivity, and extremely small heat capacity per unit area.

In some embodiments of the present invention, the super-aligned carbon nanotube array has higher nucleation density, narrower carbon tube diameter distribution, cleaner carbon tube surface and better alignment arrangement than ordinary carbon nanotube arrays , these advantages lead to strong van der Waals force between the carbon tubes in the super-aligned carbon nanotube array, and the super-aligned carbon nanotube film can be extracted from it. The carbon nanotubes in the film are connected end to end, and are connected by van der Waals force , and aligned in the same direction. The super-aligned carbon nanotube film can be laid out on any substrate according to the design, and can also be self-supported and suspended on the frame. Compared with disordered carbon nanotube films prepared by other methods, super-parallel carbon nanotube films have fewer defects and better mutual contact. The thickness of the film depends on the height of the array, usually on the order of tens of nanometers .

In some embodiments of the present invention, since the arrays of super-aligned carbon nanotubes on the silicon wafer substrate are all composed of multi-walled carbon nanotubes, the resulting super-aligned carbon nanotube films are mostly multi-walled. Tubes, the diameter of which is mostly between 10-20 nanometers. From the perspective of mechanical properties, the super-aligned carbon nanotube film has good self-supporting properties, and can be prepared by pumping without being attached to any substrate. The carbon nanotubes in it have good directionality. If the multiple preparations are all along one direction, a higher-density super-aligned carbon nanotube film can be obtained; if the carbon nanotubes are stretched in parallel or perpendicular directions, a lower-density super-aligned carbon nanotube can be obtained film; if there is a certain angle overlap between different layers during the preparation process, the preparation of a super-parallel carbon nanotube film network can be realized. From the point of view of electrical properties, since multi-walled carbon nanotubes are usually metallic, the superparallel carbon nanotube film presents the conductive properties of metal. The carbon nanotube film exhibits anisotropic electrical conductivity, and the conductivity along the direction of the carbon nanotube is higher than that perpendicular to the direction of the carbon nanotube.

<Preparation method of carbon nanotube support network>

In some embodiments of the present invention, a method for preparing a carbon nanotube-supported network is provided, which includes:

The step of forming a carbon nanotube fiber film: preparing a carbon nanotube array, drawing the carbon nanotube array to form a carbon nanotube fiber film;

The step of stacking carbon nanotube fiber membranes: taking at least two layers of carbon nanotube fiber membranes obtained in the step of forming carbon nanotube fiber membranes to form stacked carbon nanotube fiber membranes, and adjacent carbon nanotube fiber membranes The orientation of the fibers in the fibrous film is different;

A step of transferring the stacked carbon nanotube fiber membrane: transferring the stacked carbon nanotube fiber membrane obtained in the step of stacking the carbon nanotube fiber membrane to a grid, so that the stacked carbon nanotube fiber membrane is at least partially ground cover the carrier network;

And, optionally, a step of hydrophilizing the grid covering the laminated carbon nanotube fiber membrane.

(Step of forming carbon nanotube fiber film)

In the present invention, a carbon nanotube fiber film is formed by preparing a carbon nanotube array and drawing the carbon nanotube array. In the present invention, there is no particular limitation on the method of preparing the carbon nanotube array, for example, but not limited to: arc discharge method, chemical vapor deposition (CVD) and laser evaporation method can be used. In some preferred embodiments, the carbon nanotube arrays are prepared by chemical vapor deposition (CVD).

In some preferred embodiments, chemical vapor deposition (CVD) is used to grow a super-aligned carbon nanotube array, and the super-aligned carbon nanotube array is drawn to form a carbon nanotube fiber film. The chemical vapor deposition method has the advantages of low synthesis temperature, good controllability, compatibility with the existing semiconductor manufacturing process, and easy mass production. It can prepare super-aligned carbon nanotube arrays on large-scale substrates, and can be extracted from them. Silk, spinning or film formation greatly expands the application of carbon nanotubes at the macro scale.

In some specific embodiments, adopting CVD method to prepare super-parallel carbon nanotube array comprises the following steps:

Adopt nanoscale metal catalyst (for example: the nano particle of transition group metal iron) on silicon substrate (for example the silicon substrate of eight-inch size, or other any suitable size, those skilled in the art can according to the area of the prepared net selection), the growth temperature is 550-1200°C (preferably 600-800°C, more preferably 700-800°C), the carbon source gas is one or more of acetylene, ethylene, and methane, and the growth time is 10-20 minutes (preferably 10-18 minutes, more preferably 10-15 minutes), the average height of the obtained carbon nanotubes is 100-800 microns.

In some preferred embodiments, for drawing the super-aligned carbon nanotube array to form a carbon nanotube fiber film, the fiber density in the carbon nanotube fiber film can be reduced. Exemplarily, the carbon nanotube fiber membrane can be spread on two parallel silicone collars supported by a guide rail, and by stretching the length of the silicone collar, the direction is perpendicular to the axial arrangement of the carbon nanotubes (for example, the tensile strain is 100% ), a carbon nanotube fiber membrane with a single layer of 50% of the original density can be obtained. Correspondingly, a two-layer carbon nanotube fiber membrane with an original density of 25% can be obtained.

(Procedure for stacking carbon nanotube fiber membranes)

In some embodiments, at least two layers of carbon nanotube fiber membranes obtained in the step of forming carbon nanotube fiber membranes are taken, and at least two layers of carbon nanotube fiber membranes are composited to form a laminated carbon nanotube fiber membrane, Wherein, the orientations of fibers in adjacent carbon nanotube fiber films are different.

In some specific embodiments, at least two layers of carbon nanotube fiber membranes are composited to form a laminated carbon nanotube fiber membrane. Specifically, laying at least two layers of carbon nanotube fiber films on the substrate, making at least two layers of carbon nanotube fiber films float on the liquid, and making the orientation of fibers in adjacent carbon nanotube fiber films different , the substrate with at least two layers of carbon nanotube fiber membranes floating on the liquid surface is pressed by a frame coated with an adhesive, and at least two layers of carbon nanotube fiber membranes are lifted to form stacked carbon nanotubes For fibrous membranes, the above method is referred to herein as the liquid transfer method.

In some preferred embodiments, the two layers of carbon nanotube fiber membranes can be composited using a liquid transfer method to form a laminated carbon nanotube fiber membrane. Exemplarily, the liquid transfer method includes the following steps:

Get the carbon nanotube fiber film obtained in the step of forming the carbon nanotube fiber film (for example, preferably the carbon nanotube fiber film formed by the fibers obtained by drawing the super-arranged carbon nanotube array), two layers of carbon The orientation of the fibers in the nanotube fiber membrane is crossed at 90 degrees, and spread evenly on a flat substrate (for example, a glass slide with hydrophilic properties), and the substrate with the carbon nanotube fiber membrane is placed in the In a container filled with liquid (eg, deionized water), two layers of carbon nanotube fiber membranes with fibers oriented at 90 degrees crossing float above the surface of the liquid, and the substrate then falls to the bottom of the container. Then use a metal copper round or square frame coated with an adhesive (optional adhesive: silicone or hot melt adhesive) to press the carbon nanotube fiber film floating on the liquid surface from top to bottom and slow down. Slowly pick it up, use absorbent paper to gently absorb the remaining water droplets on the surface, and then make a complete two-layer carbon nanotube fiber membrane in which the orientation of the fibers intersects each other.

(Step of transferring laminated carbon nanotube fiber membrane)

In some embodiments, the layered carbon nanotube fiber membrane obtained in the step of layering the carbon nanotube fiber membrane is transferred to a carrier such that the layered carbon nanotube fiber film at least partially covers the carrier net, to obtain a carrier net covered with a carbon nanotube fiber film.

In the present invention, there is no particular limitation on the material of the carrier grid, for example, the material of the carrier grid is selected from copper, nickel, molybdenum, titanium, and gold. In some preferred embodiments, the grid can be made of gold. In some embodiments, the grid is provided with an array of wells. In the present invention, there is no particular limitation on the shape of the holes, for example, square holes, round holes, triangular holes, hexagonal holes, oval holes, but not limited thereto. In some preferred embodiments, the grid is a gold grid. In some optional embodiments, the mesh of the carrying net is 100-1000 mesh, preferably 200-800 mesh, more preferably 300-500 mesh.

In some specific embodiments, the transfer laminated carbon nanotube fiber membrane comprises:

1) A step of coating the surface of the carrier net with an adhesive.

In the step of coating the surface of the grid with an adhesive, exemplary, first coat a layer of terpineol or other organic solvents on a flat substrate (for example: a glass slide with hydrophilic properties), Then use the carrier net to gently press it on the substrate, and a layer of terpineol will stick to the surface of the carrier net.

In some specific embodiments, the transfer laminated carbon nanotube fiber membrane also includes:

2) A step of attaching the stacked carbon nanotube fiber membranes to the grid.

Specifically, the laminated carbon nanotube fiber membrane obtained in the step of laminating the carbon nanotube fiber membrane is pasted on the grid coated with a binder, and the binder is removed.

In some specific embodiments, as an example, a plurality of grids coated with a binder (such as terpineol) can be placed on a new substrate with the surface facing up, and then the two prepared by liquid transfer method The carbon nanotube fiber film in which the orientation of the fibers intersects each other is attached to the upper net, and placed on a heating plate at 130-170 degrees Celsius (°C), preferably 150°C, heated for 1-5 minutes, and evaporated to remove loose adhesive. oleyl alcohol.

In some optional embodiments, the transfer of the laminated carbon nanotube fiber membrane further includes: a step of cutting the laminated carbon nanotube fiber membrane.

Specifically, after attaching the stacked carbon nanotube fiber membranes to the grid, the stacked carbon nanotube fiber membranes extending out of the grid are removed. There is no particular limitation on the cutting method in the present invention, for example, preferably, a laser can be used for cutting (for example, a YAG marking laser is used). Those skilled in the art can also choose any other suitable method for cleavage.

<Procedure of Hydrophilicizing Treatment of the Covered and Laminated Carbon Nanotube Fiber Membrane>

In some optional embodiments of the present invention, the preparation method of the carbon nanotube grid further includes hydrophilizing the grid covered with carbon nanotubes obtained in the step of transferring the laminated carbon nanotube fiber membrane A treatment step to increase the adsorptive properties of the carrier grid.

In the present invention, there is no particular limitation on the method of hydrophilization treatment. For example, preferably, oxygen glow discharge can be used to perform hydrophilization treatment on the grid covered with carbon nanotubes. In some specific embodiments, the grid covered with carbon nanotubes is placed in an airtight container, and after being vacuumed to 10 ⁻³ to 10 ⁻⁴ Torr, oxygen glow discharge treatment is performed for 10 to 30 s (preferably 20 s).

<Applications of carbon nanotube grids>

Use of the carbon nanotube grid provided by the invention and the carbon nanotube grid prepared by the method for preparing the carbon nanotube grid provided by the invention in the preparation of cryo-electron microscope samples.

In some specific embodiments, the sample comprises a biological sample. In the present invention, there are no special restrictions on biological samples, which can be any samples suitable for analysis by cryo-electron microscopy. Biological samples include biological macromolecules, such as proteins; biological macromolecular complexes, such as protein-protein complexes, protein-nucleic acid Complexes, drug target proteins and small molecule compounds, etc.; and other large biological entities, such as bacteria and viruses; but not limited thereto. For different types of biological samples, those skilled in the art can select appropriate liquids to form corresponding biological sample solutions, so as to facilitate dropping onto the carbon nanotube grid provided by the present invention.

The following examples further illustrate the present disclosure, but not as a limitation to the present disclosure. Specific materials and their sources used in embodiments of the present disclosure are provided below. However, it should be understood that these are only exemplary and not intended to limit the present disclosure, and materials with the same or similar type, model, quality, property or function as the following reagents and instruments can be used to implement the present disclosure. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment: the preparation of carbon nanotube carrier network

1) Formation of carbon nanotube fiber membrane

In this embodiment, a CVD method is used to prepare a super-aligned carbon nanotube array, and the super-aligned carbon nanotube array is pulled to obtain a carbon nanotube fiber film.

The preparation of super-parallel carbon nanotube arrays by CVD method comprises the following steps:

Growth of carbon nanotubes: adopt CVD method to grow super-parallel carbon nanotube arrays, nanoscale metal catalysts (nanoparticles of transition metal iron used in this embodiment) are born on eight-inch silicon wafer substrates, and the growth temperature is 600 to 800 ℃, the carbon source gas is acetylene, and the growth time is 10-20 minutes. The average height of the obtained carbon nanotubes is 200-300 microns. The carbon nanotubes located in a straight line can be directly scraped from the silicon wafer with a blade and drawn. A carbon nanotube fiber film with the same width as the length of the blade is obtained, and the carbon nanotube fiber film is composed of fibers formed by axially arranged carbon nanotubes.

2) Stretching the carbon nanotube fiber membrane.

In this embodiment, the fiber density in the carbon nanotube fiber film obtained in step 1) is reduced by a stretching device. It specifically includes: laying the carbon nanotube fiber film obtained in step 1) on two parallel silicone collars supported by a guide rail, and stretching the length of the silicone collar to align the direction perpendicular to the axial direction of the carbon nanotubes (in this paper In the embodiment, the tensile strain is 100%), and a single-layer carbon nanotube fiber membrane with an original density of 50% can be obtained. Correspondingly, a two-layer carbon nanotube fiber membrane with an original density of 25% can be obtained.

The thickness of the carbon nanotube fiber film obtained in this embodiment is about 10 nanometers.

3) Laminated carbon nanotube fiber membrane

In this example, a double-layer laminated carbon nanotube fiber membrane was prepared by a liquid transfer method.

In this embodiment, the stacking of carbon nanotube fiber membranes specifically includes: the orientation of the fibers in the stretched carbon nanotube fiber membranes obtained in the two layers of step 2) are 90-degree crossed, and evenly laid on a flat surface On the glass slide substrate with hydrophilic property (Shitai CIOTEST pathological grade microscope slide, REF.101271101P), dip a little organic solvent (for example, ethanol, isopropanol) along both sides of the substrate with both sides of the razor blade Cut off the rest of the carbon nanotube fiber film, and wipe off the excess carbon nanotube fiber along the four sides of the substrate with a cotton swab. The substrate with two layers of carbon nanotube fiber film is placed in a container filled with deionized water. 1. The carbon nanotube fiber membrane whose fiber orientation is 90-degree cross will float on the water surface. Then use a metal copper round or square frame coated with silicone adhesive on the surface to press the carbon nanotube fiber membrane from top to bottom, then slowly pick it up, and gently absorb the surface residue of the carbon nanotube fiber membrane with absorbent paper. A complete two-layer carbon nanotube fiber membrane with intersecting fiber orientations, that is, a laminated carbon nanotube fiber membrane.

4) transfer laminated carbon nanotube fiber membrane

The stacked carbon nanotube fiber membrane obtained in step 3) is transferred to a gold carrier grid; specifically includes:

First, coat a layer of terpineol (Tianjin Damao Reagent, Jin Q/HG3042-99) on a flat and hydrophilic glass slide substrate (Shitai CIOTEST pathological grade microscope slide, REF.101271101P) , and then gently press the gold grid (Gilder Grids, G300-G3) on the substrate, and the surface of the gold grid will be glued with a layer of terpineol as a binder.

Afterwards, put a plurality of carrier nets with terpineol on the new substrate with the surface of the side with terpineol facing up, and then stick the laminated carbon nanotube fiber membrane obtained in step 3) on the gold substrate. Put the net on the heating plate and heat it at 150° C. for 3 minutes to remove terpineol by evaporation.

Afterwards, the laminated carbon nanotube fiber film was cut using a 12-watt YAG marking laser with a wavelength of 1.064 μm, a laser speed of 100 mm/s, and a laser power of 100%. Draw a circle with the same size as the carrier grid on the laser control software, mark it with a laser on the substrate, then move the laser translation stage under the 10x microscope, and use a marker pen to make a circle mark on the visible screen Mark outline.

Put the substrate with the carrier grid under the microscope, align the circle of the gold carrier grid with the marked outline on the visible screen, and then move the laser translation stage back to the original position and mark it with the laser, and it will be removed Remove the carbon nanotubes around the outer ring of the gold grid. Repeat the whole marking process to cut the carbon tubes on the rest of the gold grid, and then carefully remove the grid covered with carbon nanotubes with tweezers to obtain the gold grid covered with the carbon nanotube fiber film.

5) Hydrophilic treatment of the grid on the covered and laminated carbon nanotube fiber membrane

The carrier net covered with the carbon nanotube fiber membrane obtained in step 4) is subjected to hydrophilization treatment to increase the adsorption performance.

The specific steps of hydrophilic treatment are as follows:

The gold carrier grid covered with the carbon nanotube fiber membrane is subjected to plasma glow discharge hydrophilic treatment in an oxygen atmosphere: the carrier grid is placed in a closed container, and after vacuuming to 10 ^-4 Torr, oxygen glow discharge treatment In 20 seconds, the carbon nanotube grid is obtained, which can be used for the subsequent preparation of biological frozen samples suitable for cryo-electron microscopy.

test case

The coverage and distribution of the carbon nanotube grids prepared in the examples were detected under a transmission electron microscope, and the experimental results are shown in FIG. 2 . A in Figure 2 and b in Figure 2 are images of 300-mesh gold grids (Gilder Grids, G300-G3) before and after covering the intersecting carbon nanotube fiber membrane under the transmission electron microscope, and the cross-arranged carbon nanotubes can be seen The tube fiber membrane covers the fully loaded web surface area well. Figure 2 c and Figure 2 d are transmission electron microscope images of carbon nanotube grids (no protein samples) at different magnifications. It can be seen that carbon nanotubes are more uniformly distributed in single bundles or at least bundles. A single bundle of carbon nanotubes can be seen in the center of d in Figure 2 . The above data show that a carbon nanotube carrier network with high coverage (in this embodiment, 95% of the holes are covered with carbon nanotube film), uniform distribution, and the orientation of the fibers in the two-layer carbon nanotube fiber film can be prepared. The angle is substantially about 90°.

Application example

The carbon nanotube grid prepared in the example was used to prepare biological frozen samples, and the flow chart is shown in Figure 1.

Specifically, the self-locking tweezers were used to take out the grid covered with carbon nanotubes prepared in the embodiment, that is, the grid of carbon nanotubes, and loaded it on the Vitrobot freezing sample preparation instrument. Transfer 2μl of 20S proteasome solution expressed and purified by E.coli in the laboratory to the grid at 100% humidity and 8°C. Set the Blot force to -2, the Blot time to 1s, and the wait time to 10s. Use Vitrobot Special filter paper absorbs excess liquid on the grid, then quickly placed in a liquid ethane environment and transferred to liquid nitrogen for storage.

The experimental results are shown in Figures 3 to 4. In the absence of a support membrane, the solution between carbon nanotubes can be frozen to form a thin ice layer by virtue of surface tension, which reduces the background noise caused by the support membrane. It can be seen that the frozen transmission electron microscope images under different magnifications (155 times, 1200 times, 69000 times) all have clear imaging effects. In c of Fig. 3, it can be seen that the 20S proteasome is mainly distributed near the carbon nanotubes in the ice layer, which shows that the carbon nanotubes have an adsorption effect on biological samples. The cryo-TEM images of the above protein samples were collected and subjected to two-dimensional classification, and the two-dimensional classification results of proteins with different orientation distributions could be obtained, which indicated that the protein samples showed different orientations on the carbon nanotube support network. The resolution of the above data can reach 3.04 angstroms after three-dimensional reconstruction, which shows that the carbon nanotube grid can be used for high-resolution structure analysis of protein samples.

Claims (13)

1. A carbon nanotube carrier network, characterized in that it includes a carrier network, one side surface of the carrier network is at least partially covered with more than two layers of carbon nanotube fiber membranes stacked, the carbon nanotube fiber membrane It is composed of fibers formed by axially aligned carbon nanotubes, the fibers in each carbon nanotube fiber film basically have the same orientation, and the fibers in adjacent carbon nanotube fiber films have different orientations. 2. The carbon nanotube carrier network according to claim 1, characterized in that, the fibers in the carbon nanotube fiber film are formed by carbon nanotubes connected head to tail. 3. The carbon nanotube carrier network according to claim 1 or 2, characterized in that the average length of the carbon nanotubes forming the fibers of the carbon nanotube fiber film is 100 to 800 μm, preferably 200 to 500 μm, more preferably 200 μm. ~300μm. 4. The carbon nanotube carrier network according to any one of claims 1-3, characterized in that, the fibers in the carbon nanotube fiber film are obtained by drawing a super-aligned carbon nanotube array. 5. The carbon nanotube carrier network according to any one of claims 1 to 4, wherein the orientation angle of fibers in the adjacent carbon nanotube fiber films is 45 to 90°, preferably 60 to 90°, more preferably 75 to 90°. 6. The carbon nanotube carrier network according to any one of claims 1 to 5, characterized in that the coverage of the carbon nanotube fiber film on the surface of the carrier network is more than 10%, preferably more than 30% , more preferably 50% or more. 7. The carbon nanotube carrier network according to any one of claims 1 to 6, characterized in that, the thickness of each layer of the carbon nanotube fiber film is 5 to 30 nanometers, preferably 5 to 20 nanometers, more preferably 5 to 10 nanometers. 8. The carbon nanotube carrier network according to any one of claims 1-7, characterized in that the carbon nanotube carrier network has been hydrophilized. 9. The preparation method of the carbon nanotube carrier network according to any one of claims 1 to 8, comprising: The step of forming a carbon nanotube fiber film: preparing a carbon nanotube array, drawing the carbon nanotube array to form a carbon nanotube fiber film; The step of stacking carbon nanotube fiber membranes: taking at least two layers of carbon nanotube fiber membranes obtained in the step of forming carbon nanotube fiber membranes to form a stacked carbon nanotube fiber membrane, wherein the adjacent carbon nanotube fiber membranes The orientation of the fibers in the nanotube fiber membrane is different; A step of transferring the stacked carbon nanotube fiber membrane: transferring the stacked carbon nanotube fiber membrane obtained in the step of stacking the carbon nanotube fiber membrane to a grid, so that the stacked carbon nanotube fiber membrane is at least partially ground cover the carrier network; And, optionally, a step of hydrophilizing the grid covering the laminated carbon nanotube fiber membrane. 10. the preparation method of carbon nanotube carrier net according to claim 9, wherein, in the step of described formation carbon nanotube fiber membrane, adopt the chemical vapor deposition method method to grow super along row carbon nanotube array, draw The super-aligned carbon nanotube array forms a carbon nanotube fiber film. 11. The preparation method of the carbon nanotube carrier network according to claim 9 or 10, wherein, in the step of stacking carbon nanotube fiber membranes, at least two layers of carbon nanotube fiber membranes are laid on the substrate , make the carbon nanotube fiber membrane float on the liquid, press the frame with adhesive on the surface layer and place at least two layers of carbon nanotube fiber membrane floating on the liquid surface, and lift the at least two layers of carbon nanotube fiber membrane to form a laminated carbon nanotube fiber film. 12. The preparation method of the carbon nanotube carrier network according to any one of claims 9 to 11, wherein the step of transferring the laminated carbon nanotube fiber membrane comprises: the step of coating the surface of the carrier with an adhesive; and, The step of laminating the laminated carbon nanotube fiber membrane on the grid; Preferably, the binder is terpineol. 13. The carbon nanotube carrier network according to any one of claims 1 to 8 or the carbon nanotube carrier network prepared by the preparation method according to any one of claims 9 to 12 in the preparation of cryo-electron microscope samples the use of; Preferably, said sample comprises a biological sample.

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