Micro-nano processing method for living organism surface

Abstract

The invention discloses a micro-nano processing method for a living organism surface, which comprises the following steps: inducing a biological sample to be processed to enter a strong stress resistance state according to the characteristics of the biological sample to be processed, and processing a micro-nano pattern on the living organism surface by utilizing an ice etching method; and after the processing is completed, the biological sample in a strong stress resistance state is subjected to reactivation operation, so that micro-nano processing of the living body biological surface is realized. The process is based on the ice etching technology, overcomes the defect that the micro-nano pattern cannot be directly processed on the living organism by the existing micro-nano processing process, has the advantages of simple process steps, in-situ processing, higher precision and good biocompatibility, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of micro-nano processing and preparation, and specifically relates to a living biological material preparation technology, and in particular to a processing technology based on ice engraving technology to achieve micro-nano structure on the surface of living biological materials. Background Art

[0002] Micro-nano processing technology can be used to process fine structures on various planar substrates, and it has a wide range of applications in integrated circuit manufacturing, sensor technology, micro-electromechanical systems, and nanomaterial preparation.

[0003] The current mainstream micro-nano processing technologies include: photon lithography, two-photon polymerization, nanoimprinting, electron beam etching, scanning probe direct writing and electron beam induced deposition. However, there is currently no micro-nano processing method that can directly process and manufacture micro-nano structures on the surface of living organisms. Because the surface of living organisms is uneven, most of the current processing methods cannot process on uneven surfaces, and the current processing technologies all include operations that living organisms cannot withstand, such as etching, high-temperature baking, toxic organic solvents, strong electron radiation and vacuum.

[0004] Ice lithography is a new type of micro-nano processing method. It uses ice as an electron beam resist and has been proven to be a micro-nano manufacturing method with broad prospects. The US patent document with publication number US 8,790,863, B2 (publication date: 2014.07.29) discloses an ice lithography method using water ice as a precursor. The freezing process and the presence of ice film in this technology will reduce the damage of electron radiation to the substrate. This method has outstanding advantages in the processing of biological samples. However, so far, there has been no application of ice lithography technology for direct processing on biological samples. Direct processing of micro-nano structures on biological samples may promote the use of multiple physical means such as light, electricity, heat, and magnetic fields to accurately and efficiently affect, detect, and manipulate organisms at the micro-nano scale. Summary of the invention

[0005] The present invention provides a processing method for realizing micro-nanostructure on the surface of living biological materials based on ice engraving technology. The method has simple process steps, can be processed in situ, has high processing accuracy, has good biocompatibility, and can process fragile and uneven living biological samples.

[0006] The present invention provides a micro-nano processing method for the surface of living organisms, comprising: according to the characteristics of a biological sample to be processed, inducing it to enter a strong stress resistance state, and using an ice engraving method to process micro-nano patterns on the surface of the living organism; after the processing is completed, a revival operation is performed on the biological sample in the strong stress resistance state to achieve micro-nano processing on the surface of the living organism.

[0007] Preferably, the substrate material, precursor material, vacuuming rate, vacuum degree, cooling and heating speed, ice layer thickness, and electron beam exposure parameters in the micro-nano processing process are determined according to the tolerance of the biological sample in its strong stress resistance state.

[0008] The present invention provides a micro-nano processing method for the surface of living organisms, comprising: inducing a biological sample to be processed to enter a state of strong stress resistance according to its characteristics, determining the substrate material, vacuum pumping rate, vacuum degree, cooling and heating speed, minimum temperature, ice thickness, and electron beam exposure parameters in the processing process according to the tolerance of the organism in this state, and using electron beams to expose the precursor material attached to the sample surface, thereby achieving the purpose of processing micro-nano patterns on the surface of living organisms, and finally providing suitable conditions according to the characteristics of the biological sample, so that the biological sample will move with a customized structure.

[0009] A micro-nano processing method for the surface of a living organism comprises the following steps:

[0010] (1) Inducing biological samples to enter a state of strong stress resistance, allowing them to adhere to the substrate material;

[0011] (2) placing the prepared biological sample and substrate together in a chamber of a scanning electron microscope, and cooling the substrate and the biological sample together and maintaining the low temperature;

[0012] (3) introducing a precursor material into the cavity in a gas phase, and the precursor material forms an ice film on the surface of the biological sample;

[0013] (4) exposing the ice film in step (3) with an electron beam;

[0014] (5) raising the temperature to remove the unexposed ice film and obtain a biological sample with a structure on the surface;

[0015] (6) The sample is taken out of the cavity of the scanning electron microscope to create suitable conditions for the biological sample, that is, the resurrection conditions corresponding to the biological sample, and the biological sample enters an active state with the customized micro-nanostructure.

[0016] In the present invention, the living biological samples are tardigrades, extreme psychrophilic bacteria and other organisms that can resist dryness, low temperature and vacuum environment in a dormant, suspended animation or cryptobiotic state.

[0017] In the present invention, the selection of the biological samples requires literature research and preliminary experiments to determine whether they can withstand the experimental operating environment.

[0018] In order to achieve the purpose of making micro-nano patterns on the surface of living organisms, the present invention needs to determine suitable processing parameters after preliminary experiments. In the present invention, the preliminary experiment on biological samples needs to be determined according to the working environment of the scanning electron microscope actually selected. For example: if an environmental scanning electron microscope is selected, it is necessary to determine in the preliminary experiment whether the organism in a state of strong stress resistance can withstand at least a vacuum environment of 1KPa, whether the organism can survive for more than 5 hours when the water content in the organism is less than 30% of the active state, whether the organism in a state of strong stress resistance can withstand electron radiation of 1keV voltage for about 5s, and whether the organism can withstand the vacuum pumping rate of the scanning electron microscope. Furthermore, for example, for tardigrades, if a field emission scanning electron microscope is selected, it should be determined in the preliminary experiment whether it can withstand a vacuum environment higher than 6*10 -7 The vacuum degree of mba and the water content below 10% of the active state can survive for more than 5 hours. The slow-moving in the strong stress resistance state can withstand the electron radiation of 1keV voltage for about 5s and the vacuum pumping rate can withstand about 3mbar / s.

[0019] In the present invention, the biological sample is preferably a species within the phylum Tardigrades.

[0020] In the present invention, the length of the biological sample in an active state is 50um-1000um, preferably 200um-500um.

[0021] In step (1) of the present invention, the strong stress resistance state refers to the state in which the biological sample enters a state with stronger resistance to drying, vacuum and freezing. For example, tardigrades have extremely strong resistance to vacuum, drying, freezing and electron radiation in a state of water-deficient cryptobiosis, so the strong stress resistance state of tardigrades can be selected as a water-deficient cryptobiosis state.

[0022] In step (1) of the present invention, the base material is conducive to the attachment of biological samples, and can assist the biological samples to smoothly enter and maintain a strong stress resistance state. For example, for tardigrades, a loose, porous and highly hydrophilic material can be selected. The loose, porous structure can assist active tardigrades to crawl, and the highly hydrophilic and loose, porous structure can ensure that the tardigrades discharge water from the body at a slower speed during the process of entering the water-deficient cryptobiotic state, thereby increasing the success rate of entering the water-deficient cryptobiotic state.

[0023] In step (1) of the present invention, the base material should have good thermal conductivity so that it can assist in rapidly and evenly cooling the biological sample in step (2).

[0024] In step (1) of the present invention, the substrate material should have good electrical conductivity so that it can assist in positioning the biological sample and reduce charge accumulation in step (3).

[0025] That is, preferably, the base material is a material that can assist the biological sample to smoothly enter and maintain a strong stress resistance state, has a certain conductivity to ensure that it does not generate charge accumulation under the observation of a scanning electron microscope, and has a certain thermal conductivity to ensure that the biological sample can be quickly and evenly cooled to a low temperature.

[0026] In the present invention, the base material is composed of a loose and porous hydrophilic support and a conductive component loaded on the support; the conductive component includes any one or more of silicon, platinum, gold, germanium, titanium, chromium, vanadium, tungsten, silver, palladium, aluminum, manganese, zinc, copper, cadmium, tin, iron, lead, antimony, nickel, cobalt, bismuth, titanium dioxide, zinc selenide, gallium nitride, gallium arsenide, indium arsenide, zinc sulfide, cadmium sulfide, cadmium selenide, zinc oxide, gallium phosphide, titanium nitride, magnesium oxide, cadmium oxide, aluminum oxide, silicon dioxide, silicon nitride, indium phosphide, aluminum-doped zinc oxide, indium tin oxide, carbon nanotubes, and graphene; the hydrophilic support is a combination of any one or more of nylon microporous filter membrane, resin, polytetrafluoroethylene, and polyester membrane.

[0027] In step (1) of the present invention, the substrate material is preferably a combination of graphene, carbon nanotubes and resin materials, for example, commercially available or self-made carbon paper can be selected.

[0028] In the present invention, the thickness of the base material is preferably 100um-300um.

[0029] In the present invention, the scanning electron microscope is one of a field emission scanning electron microscope, an environmental scanning electron microscope, a low temperature scanning electron microscope, a transmission electron microscope, and a spherical aberration corrected transmission electron microscope. A suitable scanning electron microscope is selected according to the vacuum degree and dehydration degree that the organism can withstand. For example, for tardigrade samples, a field emission scanning electron microscope and an environmental scanning electron microscope can be selected.

[0030] In step (2) of the present invention, the temperature range is 80-140 K. For example, 90 K, 100 K, 110 K, 120 K, 130 K, 140 K, and specific values ​​between the above values. For the sake of simplicity and space limitation, the present invention will no longer exhaustively list the specific values ​​included in the range.

[0031] In step (2) of the present invention, the low temperature range is a temperature range in which the biological sample can survive and the precursor material can form ice; that is, the low temperature range is a temperature range in which the biological sample can survive and the precursor material can form ice under the corresponding air pressure. On the premise that the biological sample can survive, the frozen state of the precursor material is preferably amorphous ice.

[0032] In step (2) of the present invention, preferably, the low temperature range is 120K-130K.

[0033] In the present invention, the cooling in step (2) only needs to cool the biological sample. In order to protect the biological sample and improve the overall conductivity and thermal conductivity of the sample, in actual operation, the biological sample attached to the substrate material and the substrate are placed together on a movable sample stage in the chamber of the scanning electron microscope. In order to further reduce charge accumulation and enhance the cooling efficiency, carbon conductive glue or copper conductive glue is preferably used to stick the substrate material to the surface of the sample stage. The purpose of cooling the substrate material and the biological sample is achieved by cooling the sample stage.

[0034] The cooling rate in step (2) and the heating rate in step (5) of the present invention need to be set according to the characteristics of the selected biological sample to ensure that the biological sample is frozen and restored at an appropriate rate. At the same time, it is necessary to consider whether the sum of the time required for cooling and heating and the time required for exposure meets the survival time of the organism in the corresponding scanning electron microscope environment. These can all be determined through preliminary experiments. For example, for tardigrades, the cooling rate can be set to 1.3°C / min for the first 1.5 hours, followed by cooling at a rate of 0.5°C / min for 1 hour, and finally cooling to the target temperature at a rate of 0.05°C / min, such as 130K; for tardigrades, the heating rate can be set to 1.5°C / min.

[0035] In the step (2) of the present invention, the cooling method may be to use liquid nitrogen for temperature transfer or to use a refrigeration chip for cooling.

[0036] In step (2) of the present invention, after the temperature is lowered to the set minimum temperature, it should be maintained for at least ten minutes before proceeding to the next step to ensure that the surface temperature of the biological sample is uniform.

[0037] In the present invention, the precursor materials used in the ice engraving method can be directly purchased or prepared by themselves, and the precursor materials include but are not limited to any one of ether compounds, alcohol compounds, hydrocarbon compounds, aldehyde compounds, ketone compounds, and carboxyl compounds, or a combination of two or more thereof.

[0038] Further, the precursor material includes, but is not limited to, any one or a combination of two or more organic substances such as anisole, phenethyl ether, phenylethyl alcohol, n-pentanol, benzoic acid, gallic acid, octane, nonane, undecane, tetradecane, benzylmethane, phenylethylethane, styrene, benzylamine, benzaldehyde, phenol, aniline, toluene, ethylbenzene, butylbenzene, xylene, trimethylbenzene, acetaminophen, chlorobenzene, chlorotoluene, dichlorobenzene, iodobenzene, bromobenzene, bromostyrene, bromobenzoic acid, etc. The ice film is a solid film formed after the precursor material is condensed.

[0039] In the present invention, the precursor material can be condensed at low temperature to form a uniform ice film on the uneven surface of the biological sample with uniform temperature, wherein the ice is preferably amorphous. The ice film will form a cross-linked structure during the subsequent exposure process, and the presence of the ice film weakens the damage caused by electron beam radiation to the biological sample, thus playing a certain protective role.

[0040] In the present invention, the thickness of the ice film and the electron beam exposure parameters are mutually constrained. The electron beam exposure parameters can be determined by the required pattern thickness, and the minimum thickness can also be determined by the known electron beam exposure parameters.

[0041] In the present invention, the thickness of the ice film is greater than or equal to 90% of the maximum penetration depth of electrons of the corresponding voltage in the ice film to protect the sample from electron radiation damage to the greatest extent. For example: if a voltage of 1keV is selected for pattern exposure, the ice layer thickness should be set to be greater than or equal to 38nm; if a voltage of 2keV is selected for pattern exposure, the ice layer thickness should be set to be greater than or equal to 115nm; if a voltage of 3keV is selected for pattern exposure, the ice layer thickness should be set to be greater than or equal to 230nm. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the ice layer thickness ranges corresponding to the remaining voltage values. The specific thickness setting can be adjusted according to the electron radiation tolerance of the biological sample.

[0042] In the present invention, the thickness of the ice film is 50nm-500nm, and is preferably 200nm-300nm in consideration of the accuracy of in-situ patterning and the degree of protection of the sample.

[0043] In the present invention, the ice film and the electron beam interact with each other, and chemical bonds in the ice film material are broken and cross-linked, wherein the degree of cross-linking is greater than the degree of breaking, and the exposed areas are solid at room temperature, and the unexposed areas are gaseous at room temperature. After the ice film and the electron beam react, the composition of the ice film changes, and the carbon content increases, which weakens its toxicity and enhances its compatibility with carbon-based biomaterials.

[0044] In the present invention, the step (4) exposes the ice film in step (3) according to the customized pattern, and selects different voltages and doses for exposure according to the thickness of the ice layer and the grayscale of the pattern to obtain a biological sample with a customized pattern structure on the surface. The specific steps include: converting the customized pattern into a grayscale image, then converting the information of the grayscale image into a computer-readable code, customizing exposure parameters according to a curve diagram of the relationship between the exposure dose and the thickness of the corresponding precursor ice film at the corresponding voltage, and performing exposure according to the pattern information.

[0045] In the present invention, in the step (4), the electron beam energy ranges from 1keV to 30keV, such as 1keV, 2keV, 3keV, 4keV, 5keV, 10keV, 15keV, 20keV, 25keV, 30keV, and specific values ​​between the above values. Due to space limitations and for the sake of simplicity, the present invention will no longer exhaustively list the specific values ​​included in the range.

[0046] In the present invention, in step (4), according to the electron radiation tolerance of the biological sample, the visibility of the ice film to electrons, and the thickness of the ice layer, preferably, the electron beam energy is less than 5 keV, and specifically, the electron beam energy is preferably 1 keV-5 keV.

[0047] In the present invention, in step (4), the surface dose required for the electron beam exposure is 0.1 mC / cm 2 -50mC / cm 2 For example, 0.1mC / cm 2 , 0.2mC / cm 2 、0.3mC / cm 2 , 0.4mC / cm 2 , 0.5mC / cm 2 , 1mC / cm 2 , 2mC / cm 2 、3mC / cm 2 , 4mC / cm 2 , 5mC / cm 2 、10mC / cm 2 、20mC / cm 2 、30mC / cm 2 、40mC / cm 2 、50mC / cm 2 , and specific point values ​​between the above numerical values. Due to space limitations and for the sake of brevity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0048] In the present invention, in step (4), the dose range is preferably 1 mC / cm 2 -10mC / cm 2 .

[0049] In the present invention, the electron beam exposure time and residence time can be determined according to actual pattern requirements, and can be calculated by a computer according to the shape of the processed pattern.

[0050] In step (4), it is necessary to select a voltage for in-situ observation based on the electron radiation tolerance of the biological sample and the thickness of the ice layer, and to select a suitable electron beam voltage and dose for exposure based on the electron radiation tolerance of the biological sample and the thickness of the ice layer.

[0051] The method of removing the ice film by heating in step (5) is sublimation removal. This process does not involve the transition from solid phase to liquid phase, and the precursor in the gas phase is pumped out of the cavity by a pump.

[0052] The suitable conditions in step (6) are the temperature, humidity, osmotic pressure, and air pressure environment that allow the biological sample to move autonomously. For example, for a tardigrade in a water-deficient cryptobiotic state, providing it with a temperature of 25°C, a double-distilled water liquid environment, and a normal pressure environment can assist it in converting from a cryptobiotic state to an active state.

[0053] Preferably, the biological sample is a species in the phylum Tardigrade; the strong stress resistance state is the state of Tardigrades in water-deficient cryptozoology; the substrate material is a combination of graphene, carbon nanotubes and resin materials; the low temperature range is 120K-130K; the surface dose required for the electron beam exposure used in the ice engraving method is 0.1mC / cm 2 -10mC / cm 2 ; The electron beam energy is the energy corresponding to the voltage of 1keV-5keV.

[0054] The present invention provides a micro-nano processing method for the surface of a living organism, which is a novel micro-nano processing method with simple process steps, in-situ processing capability, high resolution and good biocompatibility.

[0055] The micro-nano processing method provided by the present invention solves the problem that the existing micro-nano processing methods cannot directly process micro-nano patterns in situ on biological samples. It has the advantages of simple process steps, in-situ processing, high precision, and good biocompatibility. It has good application prospects and may promote the use of various physical means such as light, electricity, heat, and magnetic fields at the micro-nano scale to accurately and efficiently affect, detect, and manipulate organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The present invention is a process roadmap for a micro-nano processing method for the surface of a living organism.

[0057] Figure 2 This is a schematic diagram of a preparation method of a micro-nano processing method for a living biological surface of the present invention. Taking tardigrades as an example, firstly, the tardigrades that have entered a stress resistance state are placed on a substrate, and then the sample is cooled, the precursor is deposited, the electron beam is exposed, and the temperature is increased. Finally, suitable conditions are created for the tardigrades, and they will move with customized patterns.

[0058] Figure 3The pattern prepared on the tardigrade sample using the process of the present invention is shown: the logo of West Lake University. DETAILED DESCRIPTION

[0059] For ease of understanding, the present invention is listed in the following embodiments. Those skilled in the art will understand that the following examples are only preferred embodiments of the present invention and are only used to help understand the present invention and should not be considered to limit the scope of the present invention.

[0060] like Figure 1 As shown, a method for processing micro-nano structures on the surface of living biological samples based on ice engraving technology, the technical route of which includes inducing the organism to enter a stress-resistant state, cooling, precursor deposition, electron beam exposure, rewarming, and the organism recovering from the stress-resistant state to an active state.

[0061] Embodiment 1:

[0062] Taking tardigrades as an example, before processing, a processing condition experiment was conducted on tardigrades. The experiment showed that when they entered the water-deficient cryptobiotic state, the substrate (carbon paper) was reasonably selected. After entering the water-deficient cryptobiotic state, the vacuum rate (less than 3.38 mbar / s) and vacuum degree (higher than 6*10 -7 mbar), cooling rate (cooling rate of 1.3℃ / min in the first 1.5 hours, then cooling at a rate of 0.5℃ / min for 1h, and finally cooling to 130K at a rate of 0.05℃ / min), heating rate (greater than 1.5℃ / min), and electron beam exposure parameters (voltage and corresponding dose less than 5keV), the tardigrades can remain intact in a living state and can enter an active state through conventional recovery conditions. The thickness of the ice layer can be determined according to actual needs and electron beam exposure parameters to ensure that the thickness of the ice layer is greater than or equal to 90% of the penetration depth of the electron beam in the ice layer.

[0063] Based on the above experimental conclusions, the "West Lake University logo" was created on the live tardigrade sample using ice engraving. The specific process is shown in Figure 2 (a)- Figure 2 (f).

[0064] (1) Figure 2In (a), the tardigrade in an active state is placed on a base material, and at room temperature, the tardigrade is gradually put into a water-deficient cryptobiotic state by natural drying. After 2 hours of natural drying, it is placed in a drying cabinet for at least 24 hours to ensure that the tardigrade is completely in a water-deficient cryptobiotic state. The carbon paper formed by combining graphene, carbon nanotubes and resin materials is selected as the base material, which can be purchased (the base material used in this embodiment is purchased from the general-purpose carbon nanocomposite paper of Kunming Natai Technology Co., Ltd.). The base material is cut into squares with a side length of 0.5 cm for subsequent processing operations.

[0065] (2) Figure 2 In (b), the prepared biological sample and substrate were placed in the chamber of the field emission scanning electron microscope. The substrate was attached to the movable sample stage using conductive copper tape. The system vacuum value was reduced to 5*10 for 5 min. - 5 The substrate and biological sample were cooled to 130 K together: the cooling rate was 1.3 °C / min for the first 1.5 h, then 0.5 °C / min for 1 h, and finally 0.05 °C / min to 130 K.

[0066] (3) Figure 2 In (c), the temperature is maintained low, gaseous anisole is introduced as a precursor directly above the sample, and the valve switch of the anisole gas chamber is opened so that anisole is dispersed on the sample at a rate of about 1:1.

[0067] The deposition rate is 1 nm per second, and finally a uniform ice film with a thickness of about 150 nm is formed.

[0068] (4) Figure 2 (d), keep the temperature low, use scanning electron microscope to observe the tardigrade quickly in situ, select 1keV voltage here to reduce the damage caused by electron radiation, find its back and focus quickly. Through Monte Carlo simulation, it is concluded that 2keV voltage will not penetrate 150nm thick ice layer and has a deeper penetration depth than lower acceleration voltage, so 2keV acceleration voltage is selected for exposure operation. At the same time, the grayscale image of the customized pattern "West Lake University Logo" is imported into the computer, and the maximum exposure dose of 5mC / cm is selected according to the exposure dose and thickness relationship curve of anisole at 2keV acceleration voltage. 2 , set the pattern size to 6μm*6μm. After setting the parameters, use a voltage of 2keV to expose the ice film on the back of the tardigrade. The precise exposure position is controlled by moving the sample stage and deflecting the electron beam.

[0069] (5) Figure 2 (e) The substrate and biological sample were heated to room temperature at a rate of 1.5°C.

[0070] Every minute, the anisole ice film irradiated by the electron beam remains in solid form after heating, and the remaining ice film sublimates and leaves the sample during the heating process.

[0071] (6) After taking the biological sample and substrate out of the chamber of the scanning electron microscope, place them in a culture dish, add double distilled water until the biological sample is completely submerged, and place the culture dish containing the biological sample in a constant temperature box at 25°C. After about 1 hour, the biological sample will enter an active state with the customized micro-nanostructure. If the biological sample remains in a water-deficient cryptobiotic state for too long, the time it takes to recover to an active state will be extended.

[0072] Figure 3 (a) is a scanning electron microscope image of a tardigrade covered with an anisole ice film after cooling. Figure 3 (b) is a scanning electron microscope image of a tardigrade with the Westlake University logo pattern on its back. Figure 3 It can be seen that micro-nanoscale patterns can be produced at any position on the surface of biological samples by ice engraving.

[0073] For other organisms that can resist dryness, low temperature and vacuum environment in dormant, suspended animation or cryptogenetic state, similar methods can be used to first determine the substrate material, vacuum pumping rate, vacuum degree, temperature drop and temperature rise rate, electron beam exposure parameters, etc. that can ensure that they will not die during the processing process through preliminary experiments. Then, the above method of the present invention can be used to realize the processing of micro-nano patterns on their surfaces.

Claims

1. A micro-nano processing method for the surface of a living organism, characterized in that: include: According to the characteristics of the biological sample to be processed, it is induced to enter a state of strong stress resistance, and micro-nano patterns are processed on the surface of living organisms using ice engraving method; After processing is completed, the biological samples in a state of strong stress resistance are revived to achieve micro-nano processing of the surface of living organisms.

2. The micro-nano processing method for the surface of a living organism according to claim 1, characterized in that: The steps include: (1) Induce biological samples into a state of strong stress resistance and attach them to the substrate material; (2) placing the prepared biological sample and substrate together in a chamber of a scanning electron microscope, and cooling the substrate and the biological sample together and maintaining a low temperature; (3) introducing a precursor material into the cavity in a gas phase, and the precursor material forms an ice film on the surface of the biological sample; (4) exposing the ice film in step (3) with an electron beam; (5) raising the temperature to remove the unexposed ice film and obtain a biological sample with a structure on the surface; (6) The sample is taken out of the cavity of the scanning electron microscope to create corresponding revival conditions for the biological sample, and the biological sample enters an active state with the customized micro-nanostructure.

3. The micro-nano processing method for the surface of a living organism according to claim 1, characterized in that: According to the tolerance of biological samples in their strong stress resistance state, the substrate material, precursor material, vacuum pumping rate, vacuum degree, cooling and heating rates, ice layer thickness, and electron beam exposure parameters in the micro-nano processing process are determined.

4. The micro-nano processing method for the surface of a living organism according to claim 1, characterized in that: The biological samples are tardigrades, extreme psychrophilic bacteria and other organisms that can resist dryness, low temperature and vacuum environment in a dormant, suspended animation or cryptobiotic state.

5. The micro-nano processing method for the surface of a living organism according to claim 1, characterized in that: The base material can assist biological samples to smoothly enter and maintain a strong stress resistance state, has a certain conductivity to ensure that no charge accumulation occurs under the observation of a scanning electron microscope, and has a certain thermal conductivity to ensure that the biological samples can be quickly and evenly cooled to a low temperature.

6. The micro-nano processing method for the surface of a living organism according to claim 5, characterized in that: The base material is composed of a loose and porous hydrophilic support and a conductive component loaded on the support; the conductive component includes any one or more of silicon, platinum, gold, germanium, titanium, chromium, vanadium, tungsten, silver, palladium, aluminum, manganese, zinc, copper, cadmium, tin, iron, lead, antimony, nickel, cobalt, bismuth, titanium dioxide, zinc selenide, gallium nitride, gallium arsenide, indium arsenide, zinc sulfide, cadmium sulfide, cadmium selenide, zinc oxide, gallium phosphide, titanium nitride, magnesium oxide, cadmium oxide, aluminum oxide, silicon dioxide, silicon nitride, indium phosphide, aluminum-doped zinc oxide, indium tin oxide, carbon nanotubes, and graphene; the hydrophilic support is a combination of any one or more of nylon microporous filter membrane, resin, polytetrafluoroethylene, and polyester membrane.

7. The micro-nano processing method for the surface of a living organism according to claim 1, characterized in that: The precursor material used in the ice engraving method is any one or a combination of two or more of ether compounds, alcohol compounds, hydrocarbon compounds, aldehyde compounds, ketone compounds, and carboxyl compounds, and the ice film is a solid film formed after the precursor material is condensed.

8. The micro-nano processing method for the surface of a living organism according to claim 1, characterized in that: The thickness of the ice film is greater than or equal to 90% of the maximum penetration depth of electrons in the ice film corresponding to the voltage.

9. The micro-nano processing method for the surface of a living organism according to claim 1, characterized in that: The low temperature during ice engraving is a temperature range in which biological samples can survive and a temperature at which the precursor material can form ice under the corresponding gas pressure.

10. The micro-nano processing method for living biological surface according to claim 1, characterized in that: The biological sample is a species in the phylum Tardigrade; the strong stress resistance state is the state of Tardigrades in water-deficient cryptozoology; the substrate material is a combination of graphene, carbon nanotubes and resin materials; the low temperature range is 120K-130K; the surface dose required for the electron beam exposure used in the ice engraving method is 0.1mC / cm 2 -10mC / cm 2 ;Electron beam energy is 1keV-5keV.

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