Method for preparing gold rule microsieve supported membranes

Abstract

The application discloses a preparation method of a gold regular microsieve supporting membrane, which comprises the following steps: covering a carbon regular microsieve supporting membrane on a carrier net; preparing a gold membrane with a regular microsieve structure on the carbon regular microsieve supporting membrane covered on the carrier net; and removing the carbon regular microsieve supporting membrane covered on the carrier net, so that the gold membrane is covered on the carrier net, and the gold regular microsieve supporting membrane is formed. The carbon regular microsieve supporting membrane which is easy to remove is introduced to play a bridging role between the carrier net and the gold regular microsieve supporting membrane, the gold membrane with the regular microsieve structure is prepared on the carbon regular microsieve supporting membrane, and then the carbon regular microsieve supporting membrane is removed, so that the gold regular microsieve supporting membrane is obtained, and a technical blank of preparing the gold regular microsieve supporting membrane is broken.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cryo-TEM, in particular to a preparation method of gold regular micro-sieve support film for cryo-TEM. BACKGROUND

[0002] In the field of cryo-TEM, carbon regular micro-sieve support film is widely used.

[0003] Amorphous carbon is a commonly used material due to its simple preparation, no harm to biological samples and good hydrophilic performance. However, carbon material is a semiconductor, and its conductive performance decreases rapidly at low temperature. For example, the resistivity of 22nm-thick amorphous carbon film is 5×10 -4 Ωm at room temperature, and its resistivity increases by a geometric progression at minus 196 degrees Celsius (liquid nitrogen temperature), which is too large to be measured.

[0004] In addition, other conductive materials such as silicon carbide and silicon-titanium alloy have good conductive performance, but poor toughness and are easy to break. Moreover, it is difficult to process them into metal wires for thermal evaporation and uniform particles, which makes it difficult to control the amount of thermal evaporation and leads to damage to the tungsten wire basket during the thermal evaporation process, and the preparation is difficult.

[0005] In contrast, gold has excellent conductive and mechanical properties and remains stable at low temperature, so it is considered to be a suitable support film material for cryo-TEM. However, it is difficult to prepare gold regular micro-sieve support film using the preparation method of commonly used carbon support film and alloy regular micro-sieve film (for example, the preparation method of nickel-titanium amorphous alloy micro-array support film disclosed in CN108660426B, and the preparation method of carbon support film disclosed in CN103528866B). Therefore, it is of great significance to develop a gold regular micro-sieve support film for the development of cryo-TEM technology. SUMMARY

[0006] The prior art generally prepares a carbon support film or an alloy support film by coating or spraying a hydrophilic agent on a regular micro-protrusion template; then, depositing a carbon support film or an alloy support film on the template with the hydrophilic agent on the surface; and then, separating the regular micro-protrusion template from the carbon support film or the alloy support film in water. However, it is difficult to prepare a gold regular micro-sieve support film by using the above method (a method of directly depositing a gold film on the template with the hydrophilic agent on the surface). The reason is that the gold regular micro-sieve support film is difficult to separate from the template with the hydrophilic agent on the surface, and the gold regular micro-sieve support film is often broken when the gold regular micro-sieve support film is separated from the template. The inventor repeatedly adjusted the preparation method of the gold regular micro-sieve support film on the basis of the foregoing method, but failed to successfully prepare the gold regular micro-sieve support film.

[0007] One day, the inventor broke out of the inherent thinking of the foregoing conventional preparation method, abandoned the conventional method of separating the gold regular micro-sieve support film deposited on the template with the hydrophilic agent on the surface, and obtained the gold regular micro-sieve support film by removing the gold regular micro-sieve support film. However, the inventor found that the conventional regular micro-protrusion template was also difficult to remove from the gold regular micro-sieve support film when the gold regular micro-sieve support film was prepared by using this preparation method. By chance, the inventor prepared a gold regular micro-sieve support film on a carbon regular micro-sieve support film, found that the carbon regular micro-sieve support film placed on the support net could be removed from the gold regular micro-sieve support film, and the gold regular micro-sieve support film remained intact when the carbon regular micro-sieve support film between the support net and the gold regular micro-sieve support film was removed. Therefore, a preparation method of a gold regular micro-sieve support film is provided.

[0008] The preparation method of the gold regular micro-sieve support film includes the following steps:

[0009] Covering a carbon regular micro-sieve support film on a support net;

[0010] Preparing a gold film with a regular micro-sieve structure on the carbon regular micro-sieve support film covered on the support net;

[0011] Removing the carbon regular micro-sieve support film covered on the support net, so that the gold film is covered on the support net to form a gold regular micro-sieve support film.

[0012] This invention introduces a readily removable carbon-based regular microsieve support membrane as a bridging agent, allowing the fabrication of a gold film with a regular microsieve structure on the carbon-based support membrane. Removing the carbon-based support membrane yields the gold-based regular microsieve support membrane, filling a technological gap in its preparation. The resulting gold-based regular microsieve support membrane effectively suppresses sample drift caused by electron beam irradiation, resulting in higher density and better dispersion of biological samples within the pores. Furthermore, since gold is a heavy metal, it significantly enhances the contrast in cryo-electron microscopy, greatly improving the efficiency of automated cryo-electron microscopy procedures in identifying meshes and thus increasing the efficiency of automated data collection. This solves the problem of indistinguishability between the carbon-based support membrane and the mesh caused by the low contrast of the carbon-based support membrane. These advantages make this invention promising for broad applications in cryo-transmission electron microscopy.

[0013] In some embodiments, the thickness of the carbon-regular microsieve support film ranges from 20 nm ± 5 nm. Preferably, the thickness of the carbon-regular microsieve support film is 20 nm. This is to avoid the carbon-regular microsieve support film being too thin and prone to breakage, making it impossible to prepare a gold film with a regular microsieve structure on the carbon-regular microsieve support film; at the same time, it avoids the carbon-regular microsieve support film being too thick and difficult to remove.

[0014] In some embodiments, the carbon-regular microsieve support membrane is an amorphous carbon-regular microsieve support membrane.

[0015] In some embodiments, the carbon-regular micro-sieve support film is formed by depositing carbon powder sputtered from the carbon rod onto a regular micro-convex template by thermal evaporation of the carbon rod, thereby forming the carbon-regular micro-sieve support film.

[0016] In some embodiments, a gold film with a regular microsieve structure is prepared on a carbon regular microsieve support film covering a carrier mesh by means of thermal evaporation of gold wires onto the carbon regular microsieve support film to form a gold film.

[0017] In some embodiments, the gold wire is placed in a tungsten wire basket during thermal vapor deposition. Since the melting point of tungsten is much higher than that of gold, the gold wire in the tungsten wire basket can be heated by the tungsten wire in the basket during thermal vapor deposition, and the tungsten wire in the basket can be kept intact when the gold wire begins to melt and evaporate gold atoms.

[0018] In some embodiments, the vacuum value is controlled at 1×10⁻⁶ during the thermal evaporation of the gold wire. -4 Pa~2×10 -4Pa (Pa). Generally speaking, the lower the vacuum value, the easier it is to complete the thermal evaporation of gold wire. However, considering that the lower the vacuum value, the higher the requirements for equipment, and taking into account both the preparation cost and the thermal evaporation efficiency of gold wire, the thermal evaporation vacuum value of gold wire is controlled at 1×10⁻⁶. -4 Pa~2×10 -4 Pa.

[0019] In some embodiments, during the thermal evaporation deposition of gold wire, the distance between the tungsten wire basket and the carbon regular microsieve support film is 60 mm to 80 mm to obtain a better evaporation deposition effect.

[0020] In the process of developing the method for preparing the gold-dot microsieve support membrane of this invention, the inventors tried many methods, but failed to remove the carbon-dot microsieve support membrane from the gold film. It was only by chance that the inventors successfully removed the carbon-dot microsieve support membrane from the gold film using plasma cleaning. Therefore, in some embodiments, the removal of the carbon-dot microsieve support membrane covering the carrier mesh is carried out by plasma cleaning.

[0021] Plasma cleaning allows for precise control of the removal rate and extent of carbon-doped microsieve support membranes, ensuring the integrity and uniformity of the gold-doped microsieve support membranes. Compared to traditional chemical methods, plasma cleaning offers higher precision and controllability, effectively avoiding potential damage to the gold-doped microsieve support membranes and the substrate by chemical reagents. Furthermore, the plasma cleaning process is conducted at lower temperatures, further reducing the impact of high temperatures on the substrate and the gold-doped microsieve support membranes, thereby improving the mechanical stability and electrical conductivity of the gold-doped microsieve support membranes. Gold-doped microsieve support membranes prepared using this method exhibit better performance under cryo-transmission electron microscopy, effectively suppressing electron beam-induced sample drift and improving the distribution density and dispersion of biological samples.

[0022] In some embodiments, the carrier mesh is hydrophilically treated before the carbon-regular microsieve support membrane is covered onto it. This facilitates the coating of the carbon-regular microsieve support membrane onto the carrier mesh.

[0023] In some implementations, during the plasma cleaning process, the gas in the plasma cleaner is first evacuated as much as possible to ensure a vacuum value of 1×10⁻⁶. -2 Pa~2×10 -2 To achieve good cleaning results while considering equipment costs, a mixture of argon and oxygen is then introduced, with the argon-oxygen ratio controlled at 8:1 to 10:1, and the pressure maintained within the range of 2.5 × 10⁻⁶. 3 Pa ~ 3.0 × 10 3 Preferably, the ratio of argon to oxygen is controlled at 9:1, and the pressure range is 2.8 × 10⁻⁶.3 Pa; and / or the power is set to ≤8W for forward power and ≤2W for reverse power to ensure cleaning efficiency while avoiding damage to the gold film; and / or the cleaning time is 6 minutes or more.

[0024] This invention uses argon as a catalyst. Argon can dilute and cool the ion source, making it more stable and efficient. It assists the ion source in achieving efficient energy transfer at lower temperatures, thus achieving a higher degree of ionization. Oxygen is added during the plasma cleaning process to generate radio frequency plasma. This plasma reacts with the carbon in the carbon-regular microsieve support membrane to form carbon dioxide or carbon monoxide. The formed carbon dioxide or carbon monoxide can be discharged from the plasma cleaner using a vacuum pump, thereby removing the carbon-regular microsieve support membrane. Cleaning for at least 6 minutes based on the above parameters ensures complete removal of the carbon-regular microsieve support membrane. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the process flow for preparing the gold rule microsieve support membrane according to an embodiment of the present invention. Detailed Implementation

[0026] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0027] It should also be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising" or "including" include not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. The terminology used herein is generally that commonly used by those skilled in the art; in case of any discrepancy with commonly used terminology, the terminology used herein shall prevail.

[0028] In this paper, the term "mesh" refers to a mesh structure. Its cross-sectional outer contour can be circular or square; generally, its cross-sectional outer contour diameter is 3 mm; the mesh openings of the mesh can be circular or square. A typical mesh has 300 meshes, with each mesh having a side length of 60 μm.

[0029] In this paper, the term "regular micro-convex template" refers to regularly arranged protrusions on the surface of the template; the cross-section of these protrusions can be circular or square; the diameter of the cross-section of these protrusions is generally 1μm to 2μm (micrometers), and the spacing between the protrusions is also 1μm to 2μm.

[0030] In this paper, the term "regular microsieve" refers to a microporous structure arranged regularly on a support membrane. Since the microporous structure on the support membrane is defined by a regular micro-convex template, the diameter of these micropores is generally 1 μm to 2 μm, and the spacing between the micropores is generally 1 μm to 2 μm. The array of micropores forms a sieve-like structure, hence the name "regular microsieve".

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Figure 1 An exemplary method for preparing a gold-rule microsieve support membrane according to an embodiment of the present invention is shown. The method for preparing the gold-rule microsieve support membrane includes the following steps:

[0033] S11: Cover the carrier mesh with a carbon-regular microsieve support membrane;

[0034] S12: Prepare a gold film with a regular microsieve structure on a carbon regular microsieve support membrane covered on a carrier mesh;

[0035] S13: Remove the carbon-regular micro-sieve support membrane covering the carrier mesh so that the gold film covers the carrier mesh, forming the gold-regular micro-sieve support membrane.

[0036] In step S11, the mesh is hydrophilically treated using glow discharge before being covered with the carbon regular microsieve support membrane. The mesh can be made of metal mesh such as copper, nickel, or gold, or an alloy mesh.

[0037] In step S11, the carbon-regular microsieve support membrane is coated onto the carrier mesh through the following steps:

[0038] First, a carbon thin film (carbon regular microsieve support film) with a regular micro-convex structure is prepared on a template with regular micro-convexity. Specifically, the process includes: first, preparing regular micro-sieves with a pore size range of 1μm to 2μm and a pore spacing range of 1μm to 20μm on a template using photolithography; then, immersing the regular micro-convex template in a mixture of sulfuric acid and hydrogen peroxide for cleaning; then repeatedly immersing the regular micro-convex template in distilled water to wash away the sulfuric acid and hydrogen peroxide mixture; next, drying the regular micro-convex template after washing off the sulfuric acid and hydrogen peroxide mixture; then, spraying a layer of hydrophilic agent onto the dried regular micro-convex template using vacuum spraying; finally, depositing an amorphous carbon film with regular micro-sieves on the surfaces of the hydrophilic agent and the regular micro-convex template using spraying. The specific operation of the spraying is as follows: using a DV-502B thermal evaporation equipment, placing a carbon rod on a carbon rod sputtering device, maintaining a distance of 60mm to 80mm between the carbon rod and the regular micro-convex template to be coated, and maintaining a vacuum value of 1×10 -4 Pa~2×10 -4 At a pressure of Pa, the carbon rod is heated until carbon powder is ejected from it, depositing onto a regularly convex template to form a thin carbon film with regularly shaped micro-pores. During the carbon powder ejection process, the vacuum value is controlled to be less than 5 × 10⁻⁶. -4 Pa, the thickness of the carbon film is controlled at 20nm±5nm to ensure that the prepared carbon film has regular micro-sieve pores corresponding to the regular micro-convexities on the template; the template can be, for example, mica sheet, glass slide, etc.

[0039] Then, the carbon film with a regular micro-sieve structure is separated from the regular micro-convex template. The process includes the following steps: injecting double-distilled water (or ultrapure water) into a transfer tank, placing the template with the carbon regular micro-sieve support membrane deposited on it into the water of the transfer tank, so that the template is separated from the carbon regular micro-sieve support membrane. After the carbon regular micro-sieve support membrane is separated from the template, it floats on the water surface, while the template sinks to the bottom of the transfer tank.

[0040] Next, a carbon-regular microsieve support membrane is coated onto the carrier mesh. Specifically, this involves: first, placing the carrier mesh frame, which has undergone hydrophilic treatment via glow discharge from an ion sputtering apparatus, into a transfer cell; placing the hydrophilicated carrier mesh on the carrier mesh frame, ensuring the water level in the transfer cell submerges the mesh; then, drifting the carbon-regular microsieve support membrane above the carrier mesh; next, slowly opening the drain valve of the transfer cell, controlling the flow rate to allow water to drain slowly until the water level in the transfer cell drops below the carrier mesh, thus covering it with the carbon-regular microsieve support membrane; then removing the carrier mesh frame and the carrier mesh covered with the carbon-regular microsieve support membrane from the transfer cell and drying them; finally, detaching the carrier mesh from the carrier mesh frame to obtain the carrier mesh covered with the carbon-regular microsieve support membrane. The hydrophilic treatment via glow discharge from an ion sputtering apparatus can be performed using commonly used processes and equipment in the prior art, and this invention is not limited to this method.

[0041] In step S12, the gold film with a regular microsieve structure is prepared on the carbon regular microsieve support film covering the mesh using the following steps: First, the mesh covered with the carbon regular microsieve support film is laid flat on a glass slide, with the side covered by the carbon regular microsieve support film facing upwards. Then, the mesh covered with the carbon regular microsieve support film is placed in a DV-502B thermal evaporation plating instrument for gold plating. During the gold plating process, the gold wire is placed in a tungsten wire basket, and the distance between the tungsten wire basket and the carbon regular microsieve support film to be plated is 60mm to 80mm, and the vacuum value is 1×10⁻⁶. -4 Pa~2×10 -4 At a pressure of Pa, the tungsten wire basket is heated until the gold wire melts and completely evaporates, causing gold to deposit on the carbon-based microsieve support film to form a gold film. The gold film has regular microsieve pores that correspond to the regular microsieve pores on the carbon-based microsieve support film. The thickness of the gold film... (angstrom).

[0042] In step S13, the carbon-regular microsieve support film covering the carrier mesh is removed so that the gold film covers the carrier mesh, forming the gold-regular microsieve support film. This is prepared by the following steps: the carbon-regular microsieve support film is removed using radio frequency plasma in a plasma cleaner. During the plasma cleaning process, the gas in the plasma cleaner is first evacuated as much as possible to ensure a vacuum value of 1×10⁻⁶. -2 Pa~2×10 -2 Pa, then a mixture of argon and oxygen is introduced, while controlling the ratio of argon to oxygen to be 8:1 to 10:1, and ensuring the gas pressure range is 2.5 × 10 Pa. 3 Pa ~ 3.0 × 10 3 Preferably, the ratio of argon to oxygen is controlled at 9:1, and the pressure range is 2.8 × 10⁻⁶. 3Pa; and / or power settings of ≤8W forward power and ≤2W reverse power; and / or cleaning time of 6 minutes or more. Separating the gold film from the carrier mesh yields the desired gold-regular microsieve support membrane.

[0043] This invention introduces a readily removable carbon-based regular microsieve support membrane as a bridging agent, allowing the fabrication of a gold film with a regular microsieve structure on the carbon-based support membrane. Removing the carbon-based support membrane yields the gold-based regular microsieve support membrane, thus filling a technological gap in its preparation. The resulting gold-based regular microsieve support membrane effectively suppresses sample drift caused by electron beam irradiation, resulting in a higher density and better dispersion of biological samples within the pores. Furthermore, since gold is a heavy metal, it significantly improves the contrast in cryo-electron microscopy, greatly enhancing the efficiency of automated cryo-electron microscopy programs in identifying meshes and consequently improving automated data collection efficiency. This solves the problem of indistinguishability between the carbon-based regular microsieve support membrane and the mesh due to the low contrast of the carbon-based support membrane.

[0044] In another embodiment, an amorphous carbon film can be deposited on the surface of a template with regularly spaced micropores using chemical vapor deposition (CVD). Specifically, a regularly convex template is placed in a CVD reaction chamber, and a carbon-containing gas, such as methane or acetylene, is introduced. Under high temperature and low pressure conditions, the carbon-containing gas decomposes on the surface of the regularly convex template, generating carbon atoms which are deposited on the template to form an amorphous carbon regularly spaced micropore support film. This method can be carried out at relatively low temperatures, avoiding potential damage to the template from high temperatures, while also ensuring the uniformity and adhesion of the carbon film. Although the process of preparing an amorphous carbon regularly spaced micropore support film by CVD is more complex than that by thermal evaporation, it can still produce an amorphous carbon regularly spaced micropore support film capable of supporting a gold film.

[0045] In another embodiment, a gold film can be deposited on the side of the mesh with the carbon-regular microsieve support film by electroplating, instead of using thermal evaporation. The specific steps are as follows: First, the mesh and the carbon-regular microsieve support film are immersed in an electroplating solution containing gold ions. An electric current is used to reduce and deposit the gold ions on the surface of the carbon-regular microsieve support film, forming a gold film with a regular microsieve structure. Then, the mesh and the gold-plated carbon-regular microsieve support film are removed from the electroplating solution and cleaned to remove any residual electroplating solution. Finally, a plasma cleaner is used to remove the carbon-regular microsieve support film, leaving the gold-regular microsieve support film covering the mesh. This method can be performed at a lower temperature, avoiding potential damage to the mesh and carbon-regular microsieve support film caused by high temperatures, while also ensuring the uniformity and adhesion of the gold-regular microsieve support film. Compared to thermal evaporation, electroplating is more difficult to use for preparing gold films on carbon-based microsieve support films with a diameter of 3 mm. Moreover, unlike thermal evaporation which only deposits gold on one side of the carbon-based microsieve support film, electroplating deposits gold on all sides of the film simultaneously, resulting in higher raw material consumption. Furthermore, compared to the pollution-free operation of thermal evaporation, electroplating generates chemical waste liquid, which can easily cause environmental pollution.

[0046] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a gold-standard microsieve support membrane, characterized in that, Includes the following steps: A carbon-regular micro-sieve support membrane is coated on the carrier mesh; A gold film with a regular microsieve structure was prepared on the carbon regular microsieve support membrane; Remove the carbon-regular micro-sieve support membrane to allow the gold film to cover the carrier mesh, forming a gold-regular micro-sieve support membrane; The carbon-regular micro-sieve support membrane covering the carrier mesh is removed by plasma cleaning.

2. The method for preparing the gold rule microsieve support membrane according to claim 1, characterized in that, The thickness of the carbon-regular microsieve support membrane ranges from 20 nm to 5 nm.

3. The method for preparing the gold rule microsieve support membrane according to claim 1, characterized in that, The carbon-regular micro-sieve support film is formed by thermally vaporizing carbon rods to deposit carbon powder sprayed from the carbon rods onto a regular micro-convex template, thereby forming the carbon-regular micro-sieve support film.

4. The method for preparing the gold rule microsieve support membrane according to any one of claims 1 to 3, characterized in that, A gold film with a regular microsieve structure is prepared on a carbon regular microsieve support film covering a carrier mesh by means of thermal evaporation of gold wires to deposit gold evaporated on the carbon regular microsieve support film to form the gold film.

5. The method for preparing the gold rule microsieve support membrane according to claim 4, characterized in that, When hot-dip evaporation is performed on the gold wire, the gold wire is placed in a tungsten wire basket.

6. The method for preparing the gold rule microsieve support membrane according to claim 4, characterized in that, When performing thermal evaporation coating on gold wire, the vacuum value is controlled at 1×10⁻⁶. -4 Pa ~2×10 -4 Pa.

7. The method for preparing the gold rule microsieve support membrane according to claim 5, characterized in that, When hot-dip evaporation is performed on the gold wire, the distance between the tungsten wire basket and the carbon regular micro-sieve support membrane is 60mm~80mm.

8. The method for preparing the gold rule microsieve support membrane according to any one of claims 1 to 3, characterized in that, Before covering the carrier mesh with a carbon-regular microsieve support membrane, the carrier mesh is subjected to a hydrophilic treatment.

9. The method for preparing the gold rule microsieve support membrane according to claim 8, characterized in that, During the plasma cleaning process, the vacuum value is first controlled to 1×10⁻⁶. -2 Pa ~2×10 -2 Pa, then a mixture of argon and oxygen is introduced, while controlling the ratio of argon to oxygen to be 8:1 to 10:1, and ensuring the gas pressure ranges from 2.5 × 10 Pa. 3 Pa ~ 3.0 × 10 3 Pa; and / or The power is set to ≤8W for forward power and ≤2W for reverse power.

10. The method for preparing the gold rule microsieve support membrane according to claim 8, characterized in that, The cleaning time is more than 6 minutes.

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