Method for carrying out laser additive manufacturing in cells

Abstract

The invention discloses a method for carrying out laser additive manufacturing in cells, and belongs to the technical field of optics. The method comprises the steps that a substrate comprising a cavity is constructed, the substrate is pretreated, and an opening is formed in the top of the cavity; the method comprises the following steps: injecting a polymer solution into a cavity of a pretreated substrate, centrifuging cells to be subjected to laser additive manufacturing, dispersing a suspension containing the cells into the polymer solution after centrifuging, enabling the polymer solution to enter the cells, and sealing the cavity, the polymerizable solution comprises a light-induced polymerizable material, a cross-linking agent and a photoinitiator; carrying out polymerization processing on the polymer solution permeating into the cells through a two-photon polymerization femtosecond laser direct writing system; and putting the polymerized and processed cell into a complete medium for soaking and fixing to obtain the cell containing the laser additive manufacturing microstructure. The method can be used for manufacturing subcellular scale units and devices in cells, and is convenient to operate and small in damage to the cells.

Description

Technical Field

[0001] The present invention relates to the field of optical technologies, and particularly to a method for laser additive manufacturing inside cells. Background Art

[0002] Near-infrared femtosecond lasers have strong penetration power, and the two-photon polymerization direct writing technology derived therefrom has high forming accuracy and flexible configuration. It has broad application prospects in the fields of micro-nano optical devices, precision medicine, micro-nano robots, and micro-nano sensors. Cells are the basis of the life system, and modifying cells or constructing structural unit devices inside cells to change and regulate cell functions is a very important topic in synthetic biology.

[0003] Traditional methods for constructing micro-nano units inside cells rely on complex means such as gene editing. The structures of the products produced through the gene expression products of cells themselves are regulated by genes, and the manufacture of arbitrarily complex three-dimensional structures cannot be achieved. At the same time, the materials for producing the products are synthesized by the cells themselves, and the structures of foreign materials cannot be constructed. The poor extensibility of materials and structures makes it difficult for current manufacturing methods to achieve the construction of arbitrary three-dimensional units inside cells. Summary of the Invention

[0004] To solve the problems in the prior art, the present invention proposes a method for laser additive manufacturing inside cells.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention proposes a method for laser additive manufacturing inside cells, including the following steps:

[0007] 1) Construct a substrate containing a chamber, and preprocess the substrate to obtain a preprocessed substrate; wherein, the top of the chamber has an opening;

[0008] 2) Inject a polymerizable solution into the chamber of the preprocessed substrate, then centrifuge the cells to be subjected to laser additive manufacturing, and after centrifugation, disperse the cell-containing suspension in the polymerizable solution, so that the polymerizable solution penetrates into the cells and seals the chamber;

[0009] Wherein, the polymerizable solution includes a photo-induced polymerizable material, an initiator, and a cross-linking agent, and the photo-induced polymerizable material is one or more of a polymerizable biomedical material, a polymerizable DNA subjected to end modification, or a polymerizable polypeptide with a specially designed sequence;

[0010] 3) Perform polymerization processing on the polymerizable solution that has penetrated into the cells through a two-photon polymerization femtosecond laser direct writing system;

[0011] 4) Immerse the polymerized cells in a complete medium for soaking and fixing to obtain cells containing microstructures fabricated by laser additive manufacturing.

[0012] Further, in step 2), the mass ratio of the photo-polymerizable material: initiator: cross-linking agent is (100-1000): 1: (50-500);

[0013] The polymerizable biomedical material is polyethylene glycol diacrylate, poly(lactic-co-glycolic acid), gelatin methacrylate, polycaprolactone, poly(N-isopropylacrylamide), or methoxypolyethylene glycol methacrylate;

[0014] The end-modification method of the polymerizable DNA with a special sequence includes: turning the polymerizable DNA into two Y-motif structures using the base complementary pairing principle, then obtaining a DNA hairpin structure containing a photocleavage site, and then using femtosecond laser to cleave the photocleavage site on the DNA hairpin structure, causing the DNA hairpin structure to break and form short DNA single strands capable of connecting the two Y-motif structures. Furthermore, the short DNA single strands form a planar or three-dimensional network structure of DNA with the two Y-motif structures through self-assembly;

[0015] The special sequence design method of the polymerizable polypeptide with a special sequence includes: replacing non-polar amino acids in the polymerizable polypeptide with tyrosine or lysine to increase the content of tyrosine and lysine in the polypeptide chain.

[0016] In step 3), if the cells to be fabricated by laser additive manufacturing contain polymerizable autologous proteins, the two-photon polymerization femtosecond laser direct writing system will also polymerize the material composed of the autologous proteins, initiator, and cross-linking agent that penetrate into the cells.

[0017] In a second aspect, the present invention provides cells containing micro-nano structures obtained by the method of performing laser additive manufacturing inside the cells as described above.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] The present invention adopts the technical means of two-photon polymerization direct writing, overcomes the technical problem in the prior art that three-dimensional complex subcellular structures cannot be constructed inside cells, and thus enables laser additive manufacturing inside cells, that is, the construction of three-dimensional complex subcellular structures. The present invention uses a photoinducible polymerizable material, a photoinitiator, and a crosslinking agent together as the basic material for constructing the structure, and the photoinducible polymerizable material is one or more of a polymerizable biomedical material, a polymerizable DNA with terminal modification, or a polymerizable polypeptide with a specially designed sequence, overcoming the problems of apoptosis and a large amount of rejection of cells during and after the printing process inside cells, and thus enabling the construction of three-dimensional complex subcellular structures inside living cells with good biocompatibility. The present invention adopts the technical means of a printing platform jointly constructed by a micro-structure, an aptamer, and a sealed chamber, and thus overcomes the technical problems of positioning and clamping during cell printing, and thus achieves the technical effect of stably constructing three-dimensional complex high-fidelity subcellular structures inside cells.

[0020] The method for laser additive manufacturing inside cells provided by the present invention solves the problem that it is difficult to construct arbitrary three-dimensional units in cells. Its preparation process does not require complex gene editing, is efficient and intuitive, and demonstrates the possibility of constructing micro-nano devices inside cells. The method for laser additive manufacturing inside cells is based on a two-photon polymerization femtosecond laser direct writing system, and the intracellular polymer micro-nano structures prepared by it have the characteristics of high integration, high precision, and high freedom, and can be applied to cutting-edge fields such as cell sensing, cell tracing, and precise drug delivery, and have broad application prospects. Brief Description of the Drawings

[0021] Figure 1 is a flowchart of the steps of the method for laser additive manufacturing inside cells of the present invention;

[0022] Figure 2 is a schematic diagram of laser additive manufacturing inside cells provided by the present invention;

[0023] Figure 3 is a physical picture of inside cells before and after laser additive manufacturing under a 60× optical microscope;

[0024] Figure 4 is a model diagram of a PDMS sealed chamber;

[0025] Figure 5 is an example diagram of the process of modifying and designing DNA. Detailed Embodiments

[0026] The following further elaborates and explains the present invention in conjunction with specific embodiments. The described embodiments are only examples of the present disclosure content and do not delimit the scope of limitation. Without conflict, the technical features of each embodiment of the present invention can be combined accordingly. Refer to Figure 1, Figure 1 is a flowchart of the steps of the method for intracellular laser additive manufacturing provided by the present invention. The present invention provides a method for intracellular laser additive manufacturing, which includes the following steps:

[0027] 1) Construct a substrate containing a chamber, preprocess the substrate to obtain a preprocessed substrate; wherein, the top of the chamber has an opening;

[0028] 2) Inject a polymerizable solution into the chamber of the preprocessed substrate, then centrifuge the cells to be subjected to laser additive manufacturing, and after centrifugation, disperse the cell-containing suspension in the polymerizable solution, so that the polymerizable solution penetrates into the cells and seals the chamber;

[0029] Wherein, the polymerizable solution includes a photoinduced polymerizable material, an initiator and a crosslinking agent, and the photoinduced polymerizable material is one or more of a polymerizable biomedical material, a polymerizable DNA subjected to end modification, or a polymerizable polypeptide with a specially designed sequence;

[0030] 3) Perform polymerization processing on the polymerizable solution that has penetrated into the cells through a two-photon polymerization femtosecond laser direct writing system;

[0031] 4) Immerse and fix the polymerized cells in a complete culture medium to obtain cells containing laser additive manufacturing microstructures.

[0032] The method for intracellular laser additive manufacturing provided by the present invention, through multiple steps, including providing a substrate, preprocessing the substrate, preparing a polymerizable solution, two-photon polymerization laser direct writing processing, and fixing, etc., finally obtains cells containing laser additive manufacturing microstructures. Specifically, in step 1), constructing a substrate containing a chamber specifically includes the following steps:

[0033] Use the casting method to form a PDMS chamber with a square bottom surface on a glass substrate. The thickness of the fence surrounding the PDMS chamber is 1-3 mm, the height of the fence is 50-100 μm, and the inner side length of the PDMS chamber is 3-5 mm.

[0034] The fence of the PDMS chamber determines the relevant dimensions according to the general cell size and the working distance in the z direction of the two-photon laser direct writing device. The upper bottom surface of the PDMS chamber structure is in the shape of a square fence. Functionally, it is mainly used to place the polymerizable solution containing a certain cell culture medium and seal the solution through the top glass slide to ensure reducing the drift phenomenon and large positioning error during the printing process due to the flow of the sealed solution.

[0035] Specifically, also in step 1), preprocessing the substrate includes:

[0036] 1.1) Ultrasonically clean the substrate for 1 - 2 h, then dry the surface of the substrate with nitrogen and bake it. Put the baked substrate into an ultraviolet ozone cleaner to activate the surface of the substrate and increase the surface adhesion of the substrate;

[0037] 1.2) Add photoresist IP-dip into the chamber of the substrate, then seal the substrate and fix it on the sample holder of the two-photon polymerization femtosecond laser direct writing system;

[0038] Import the stent model file of the fixed cell array into the two-photon polymerization laser direct writing system to obtain the optical path running trajectory of the two-photon polymerization femtosecond laser direct writing system; Run the two-photon polymerization femtosecond laser direct writing system to print and form microstructures in the chamber of the substrate;

[0039] 1.3) Add RCA solution into the chamber with microstructures to physically clean the microstructures to remove organic pollutants; Then perform UV treatment on the substrate to improve the surface activity of the microstructures; Then activate the surface of the microstructures by plasma treatment;

[0040] 1.4) Obtain aptamers and construct an aptamer solution. Add it into a 2.5% glutaraldehyde solution in the chamber and incubate for 1 - 2 h, then add the aptamer solution and incubate for 12 - 24 h to obtain the microstructures modified with aptamers;

[0041] Then treat the microstructures modified with aptamers with 1% BSA or 1 mM MCH for 30 min to block the non-specific adsorption sites of the aptamers, and complete the pretreatment of the substrate.

[0042] Further, in step 1.1), the ultraviolet light wavelength of the ultraviolet ozone cleaner is 185 nm, the ozone concentration is 10 - 100 ppm, the treatment time is 10 - 30 min, the temperature is 30 - 50 °C, the air flow rate is 1 - 5 L / min, and the ultraviolet lamp power is 10 - 100 W.

[0043] Further, the functions of steps 1.2) - 1.4) are to further avoid positioning errors or other interferences during the printing process. Therefore, import the stent model file of the fixed cell array into the two-photon polymerization laser direct writing system to obtain the optical path running trajectory of the two-photon polymerization femtosecond laser direct writing system. Then drop the commercial photoresist IP-dip into the chamber of the substrate, then seal the substrate and fix it on the sample holder and insert it into the sample clamp of the two-photon polymerization femtosecond laser direct writing system. Focus the laser on the upper plane of the substrate to print and form microstructures. Modify the aptamer onto the microstructures. The following are the detailed steps:

[0044] Step 1: Cleaning the surface of the microstructures

[0045] First, perform physical cleaning: clean with RCA solution (H2O2 + NH4OH + H2O) to remove organic contaminants.

[0046] Secondly, perform UV treatment: use UV ozone treatment for 5 - 10 minutes to improve surface activity.

[0047] Step 2: Chemical modification of the interface

[0048] Surface activate the microstructure by plasma treatment to make it have aptamer immobilization sites.

[0049] Step 3: Aptamer immobilization

[0050] Obtain the aptamer and construct an aptamer solution. Among them, the aptamer is a short single - stranded deoxyribonucleic acid or ribonucleic acid molecule with a length of 20 - 100 nucleotides, usually obtained by screening through the systematic evolution of ligands by exponential enrichment (SELEX) technology;

[0051] Add 2.5% glutaraldehyde solution to the chamber and incubate for 1 - 2 h, then add the aptamer solution and incubate for 12 - 24 h to obtain the microstructure modified with the aptamer.

[0052] Step 4: Surface blocking

[0053] Treat with 1% concentration of BSA (bovine serum albumin) or 1 mM MCH (Mean Corpuscular Hemoglobin) for 30 min to block the non - specific adsorption sites of the aptamer and reduce background noise.

[0054] During the experiment, stability tests and surface characterization and verification are also carried out on the microstructure modified with the aptamer. Among them, the stability test is: optimize the pH, ionic strength, and temperature stability to ensure that the aptamer remains functional under experimental conditions. The surface characterization and verification are:

[0055] First, perform optical or electrochemical characterization, and use XPS (X - ray photoelectron spectroscopy) or AFM (atomic force microscopy) to analyze the surface modification.

[0056] Or use SPR (surface plasmon resonance) or EIS (electrochemical impedance spectroscopy) to detect the aptamer binding effect.

[0057] Secondly, perform functional tests, and confirm the activity of the aptamer through target - binding experiments (such as fluorescence labeling, colorimetric analysis, electrochemical signal detection).

[0058] After the above treatment, the printing of microstructures in a sealed chamber is completed. Then, a suitable chemical fixation method is selected according to the microstructure material to enable the aptamer to specifically recognize the target molecule. Finally, the aptamer is modified on the microstructure to bind it to the cell membrane, thereby playing a role in fixing the cell.

[0059] Specifically, in step 2), the photoinducible polymerizable material refers to a material that can change from a monomer or short-chain polymer to a long-chain polymer under the initiation of an initiator under femtosecond or continuous laser conditions, that is, the photoinducible polymerizable material is based on the photoinducible polymerizable characteristics of the material to ensure that it can be polymerized under the irradiation of a femtosecond laser with a certain power, so as to enable laser micro-nano three-dimensional manufacturing. In the present invention, the photoinducible polymerizable material is one or more of a polymerizable biomedical material, a polymerizable DNA with end modification, or a polymerizable polypeptide with a specially designed sequence. This makes it not produce serious rejection reactions when used in biological experiments, ensures that the cells still have activity and basically normal metabolic capacity after laser micro-nano three-dimensional manufacturing, and avoids damage to the normal physiological environment and function of cells caused by other non-natural materials or non-biomedical materials.

[0060] A biomedical material refers to a material that, except for natural materials, has been evaluated and confirmed for its safety and effectiveness by the US Food and Drug Administration (FDA) alone or as part of a medical device or drug delivery system, and exhibits good biocompatibility, mechanical properties, and long-term stability in clinical trials and laboratory tests and is suitable for medical use. These materials can be used for diagnosis, treatment, repair, or replacement of human tissues, organs, or functions. Therefore, the polymerizable biomedical materials of the present invention include: polyethylene glycol diacrylate (PEGDA), poly(lactic-co-glycolic acid) (PLGA), gelatin methacrylate (GelMA), polycaprolactone (PCL), poly(N-isopropylacrylamide) (PNIPAAm), polyethylene glycol methyl ether methacrylate (PEGMA), etc.

[0061] Completely natural DNA single strands will form hydrogen bonds between the single strands based on the principle of base complementary pairing and self-assemble into a DNA double helix structure. However, this basic structure cannot be used to manufacture large-molecular-weight DNA micro-nano structures, such as hydrogel micro-nano structures with DNA as the main raw material, etc. Therefore, it is necessary to design the basic structure of DNA so that it can achieve a reaction similar to the monomer polymerization process. The structural design means of DNA mainly include forming two Y-motif structures by using the principle of base complementary pairing. The Y-motif structure is a three-way cross structure self-assembled by three complementary single-stranded DNAs through base pairing, and its shape is similar to the letter "Y". It usually consists of a core intersection point and three double-stranded DNA branches. In a specific embodiment of the present invention, the two Y-motif structures can be referred to Figure 5Two Y-shaped DNAs, YA and YB, are then obtained, and a DNA hairpin structure containing a photocleavage site is acquired. Subsequently, femtosecond laser is used to cleave the photocleavage site on the DNA hairpin structure containing the photocleavage site (refer to Figure 5 the inactive linker INACITVE LINKER in it), causing it to break and form short DNA single strands capable of connecting the two Y-motif structures. Then, the short DNA single strands self-assemble with the two Y-motif structures to form a planar or three-dimensional network-structured DNA. Finally, under the condition of femtosecond laser, the construction of three-dimensional micro-nano structures using DNA as raw material can be achieved.

[0062] The polarity and type of amino acids are directly related to their suitability for three-dimensional micro-nano manufacturing. Generally speaking, amino acids that show polarity in the photoinducible polymerizable solution can be used for printing, such as tyrosine and lysine. However, for completely natural polypeptides, due to the uneven content of various amino acids, there may be a situation where there is no tyrosine but a large amount of non-polar amino acids, and thus they cannot be applied to three-dimensional micro-nano manufacturing. Therefore, the polypeptide sequence needs to be specially designed, specifically by increasing the content of tyrosine and lysine in the polypeptide chain.

[0063] The specially designed polymerizable polypeptide specifically refers to a polypeptide sequence using tyrosine or amino acids that are polar in the solvent as the main raw material, with a molecular weight less than 1000 kDa to ensure that the material can smoothly enter the cell from outside the cell through passive transport along the concentration gradient across the cell membrane. The polymerizable DNA with terminal modification specifically refers to a DNA sequence containing a specially designed photocleavage site that can achieve photoinduced cleavage and self-assembly, with a molecular weight less than 1000 kDa to ensure that the material can smoothly enter the cell from outside the cell through passive transport along the concentration gradient across the cell membrane. Natural polypeptides and DNA cannot be printed and need to be specially designed to solve the original printability problems of the materials.

[0064] A photoinitiator refers to a material that can generate reactive centers and initiate the polymerization of polymerizable materials under femtosecond or continuous laser conditions. A crosslinking agent refers to a material that can be used to strengthen the network structure of polymerizable materials during the polymerization process. Therefore, generally, three materials are required to participate in the reaction to achieve laser additive manufacturing in cells. The proportional relationship of the three materials depends on the specific types of the materials and there is a certain proportional range. Generally speaking, the mass ratio of photoinducible polymerizable material: initiator: crosslinking agent is approximately (100 - 1000):1:(50 - 500).

[0065] Specifically, also in step 2), the centrifugation speed is 800 - 1000 rpm and the time is 4 - 5 min. After sealing the chamber, wait for 10 - 15 min before proceeding to step 3) to allow the polymerizable solution to penetrate into the cells.

[0066] See Figure 4 , Figure 4 which is a schematic diagram of the PDMS sealed chamber model required in the method for intracellular laser additive manufacturing provided by the present invention. The sealed chamber consists of a top cover glass, a PDMS cavity fence, and a glass substrate from top to bottom.

[0067] Specifically, in step 3), the two-photon polymerization laser direct writing system is a two-photon three-dimensional lithography machine produced by Nanoscribe GmbH, Germany.

[0068] And if the cells to be subjected to laser additive manufacturing contain polymerizable autologous proteins, the two-photon polymerization femtosecond laser direct writing system will also perform polymerization processing on the substance composed of the autologous proteins, the initiator and the crosslinking agent that penetrate into the cells.

[0069] See Figure 2 and Figure 3 , Figure 2 which is a schematic diagram of laser additive manufacturing in cells based on two-photon polymerization technology provided by the present invention. Figure 2 On the left side of is the equipment optical path diagram of the two-photon polymerization femtosecond laser direct writing system, and on the right side is a partially enlarged schematic diagram of the cell sample on the sample stage. Figure 3 which is a bright-field view of laser additive manufacturing in cells based on two-photon polymerization technology provided by the present invention. Figure 3 On the left side of is the cell image before laser additive manufacturing of the second square structure (a square structure has been printed inside the cell in the left figure for identifying the structure during printing), Figure 3 and on the right side is the cell image after laser additive manufacturing.

[0070] Polymerization processing of the polymerizable solution that penetrates into the cells is performed by the two-photon polymerization femtosecond laser direct writing system, including the following steps:

[0071] Drop 10 - 20 μL of immersion oil in the chamber projection area on the side of the substrate without the chamber to match the refractive index of the objective lens; then fix the substrate on the sample stage and insert it into the sample holder of the two-photon polymerization femtosecond laser direct writing system; then adopt the oil immersion mode, adjust the position of the objective lens of the two-photon polymerization femtosecond laser direct writing system to focus the laser in the sealed chamber on the substrate; and import the file of the structure to be processed into the operating software of the two-photon polymerization femtosecond laser direct writing system;

[0072] Then adjust the laser processing parameters, further focus the laser inside the cells, and finally control the two-photon polymerization femtosecond laser direct writing system to perform polymerization laser direct writing.

[0073] During the laser focusing process, there are technical problems with focusing, which is different from the general case of supporting three-dimensional construction with the substrate as the base surface. In this method, it is required to perform suspended three-dimensional component manufacturing inside the cell. It is necessary to slightly adjust the distance of the piezoelectric platform in the z direction to make the laser focus inside the cell, so as to avoid the result that three-dimensional micro-nano manufacturing cannot be carried out inside the cell due to focusing on the base surface or other planes. At the same time, it should be noted that the focusing time in this step should not be too long. Long-term operation will cause the cells to sink to the bottom or even adhere to the wall, making it impossible to carry out three-dimensional micro-nano manufacturing inside the cell.

[0074] Specifically, step 4) includes the following steps:

[0075] Sterilize the outside of the substrate after polymerization processing, and then place it in a secondary biosafety cabinet in a sterile environment. Then, remove the upper glass slide used for sealing the chamber, transfer the polymerized cells into the culture medium, and then centrifuge the mixed solution to obtain a cell suspension containing cells, that is, obtain the cells after laser additive manufacturing; among them, the centrifugation speed is 800-1000 rpm, and the time is 4-5 min.

[0076] On the other hand, the present invention also provides a cell containing micro-nano structures obtained by using the method of laser additive manufacturing inside the cell.

[0077] Generally speaking, different from the traditional method of producing products inside cells through gene editing of the endogenous source, the method of laser additive manufacturing inside the cell provided by the present invention solves the problem that it is difficult for cells to achieve arbitrary three-dimensional unit construction. Its preparation process does not require complex gene editing, is efficient and intuitive, and demonstrates the possibility of constructing micro-nano devices inside cells. The method of laser additive manufacturing inside cells is based on a two-photon polymerization femtosecond laser direct writing system, and the polymer micro-nano structures prepared inside the cells have the characteristics of high integration, high precision, and high freedom, and can be applied to frontier fields such as cell sensing, cell tracing, and precise drug delivery, and have broad application prospects.

[0078] The above embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. For those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A method for performing laser additive manufacturing inside a cell, characterized in that, It includes the following steps: 1) Construct a substrate including a chamber, and pre-treat the substrate to obtain a pre-treated substrate; wherein, the top of the chamber has an opening; 2) Inject a polymerizable solution into the chamber of the pre-treated substrate, then centrifuge the cells to be subjected to laser additive manufacturing, and after centrifugation, disperse the cell-containing suspension in the polymerizable solution, so that the polymerizable solution penetrates into the cells and seals the chamber; Wherein, the polymerizable solution includes a photo-induced polymerizable material, an initiator and a cross-linking agent, and the photo-induced polymerizable material is one or more of a polymerizable biomedical material, a polymerizable DNA subjected to end modification, or a polymerizable polypeptide with a specially designed sequence; 3) Perform polymerization processing on the polymerizable solution that has penetrated into the cells through a two-photon polymerization femtosecond laser direct writing system; 4) Immerse and fix the polymerized cells in a complete culture medium to obtain cells containing microstructures manufactured by laser additive manufacturing.

2. The method for laser additive manufacturing inside a cell according to claim 1, wherein In step 1), the construction of the substrate including a chamber includes: Using a casting method to form a PDMS chamber with a square bottom surface on a glass substrate, the thickness of the fence surrounding the PDMS chamber is 1-3 mm, the height of the fence is 50-100 μm, and the inner side length of the PDMS chamber is 3-5 mm.

3. The method for laser additive manufacturing inside a cell according to claim 1, wherein In step 1), the pre-treatment of the substrate includes: 1.1) Ultrasonically clean the substrate for 1-2 h, then dry the surface of the substrate with nitrogen and bake it, put the baked substrate into an ultraviolet ozone cleaner to perform surface activation on the substrate and increase the surface adhesion of the substrate; 1.2) Add a photoresist IP-dip to the chamber of the substrate, then seal the substrate and fix it on a sample holder and insert it into the sample clamp of a two-photon polymerization femtosecond laser direct writing system; Import the stent model file of the fixed cell array into the two-photon polymerization laser direct writing system to obtain the optical path running trajectory of the two-photon polymerization femtosecond laser direct writing system; operate the two-photon polymerization femtosecond laser direct writing system to print and form microstructures in the chamber of the substrate; 1.3) Add an RCA solution to the chamber with microstructures to physically clean and remove organic pollutants from the microstructures; then perform UV treatment on the substrate to improve the surface activity of the microstructures; then perform surface activation on the microstructures by means of plasma treatment; 1.4) Obtain aptamers and construct an aptamer solution, add it to a 2.5% concentration of glutaraldehyde solution in the chamber and incubate for 1-2 h, then add the aptamer solution and incubate for 12-24 h to obtain microstructures modified with aptamers; Then treat the aptamer-modified microstructures with 1% concentration of BSA or 1 mM of MCH for 30 min to block the non-specific adsorption sites of the aptamers, and complete the pre-treatment of the substrate.

4. The method for laser additive manufacturing in cells according to claim 3, characterized in that, In step 1.1), the ultraviolet light wavelength of the ultraviolet ozone cleaner is 185 nm, the ozone concentration is 10-100 ppm, the treatment time is 10-30 min, the temperature is 30-50 °C, the air flow rate is 1-5 L / min, and the ultraviolet lamp power is 10-100 W.

5. The method for laser additive manufacturing inside a cell according to claim 1, characterized in that, In step 2), the centrifugation speed is 800 - 1000 rpm and the time is 4 - 5 min; After sealing the chamber, wait for 10 - 15 min before performing step 3) to allow the polymerizable solution to penetrate into the cells.

6. The method for laser additive manufacturing in cells according to claim 1, wherein In step 2), the mass ratio of the photo - polymerizable material: initiator: cross - linker is (100 - 1000):1:(50 - 500); The polymerizable biomedical material is polyethylene glycol diacrylate, poly(lactic - co - glycolic acid), gelatin methacrylate, polycaprolactone, poly(N - isopropylacrylamide), or methoxy polyethylene glycol methacrylate; The end - modification method of the polymerizable DNA for end - modification includes: using the base complementary pairing principle to turn the polymerizable DNA into two Y - motif structures, then obtaining a DNA hairpin structure containing a photocleavage site, and then using femtosecond laser to cut the photocleavage site on the DNA hairpin structure, so that the DNA hairpin structure breaks to form short DNA single strands capable of connecting the two Y - motif structures. Furthermore, the short DNA single strands form a planar or three - dimensional network - structured DNA with the two Y - motif structures through self - assembly; The sequence special - design method of the polymerizable polypeptide with a specially designed sequence includes: replacing non - polar amino acids in the polymerizable polypeptide with tyrosine or lysine to increase the content of tyrosine and lysine in the polypeptide chain.

7. The method for laser additive manufacturing inside cells according to claim 1, characterized in that, In step 3), if the cells to be subjected to laser additive manufacturing contain polymerizable autologous proteins, the two - photon polymerization femtosecond laser direct - writing system will also perform polymerization processing on the substance composed of the autologous proteins, the initiator, and the cross - linker that have penetrated into the cells.

8. The method for performing laser additive manufacturing inside a cell according to claim 1, characterized in that, Step 3) includes: Drop 10 - 20 μL of immersion oil in the chamber projection area on the side of the substrate without the chamber to match the refractive index of the objective lens; then fix the substrate on the sample holder and insert it into the sample holder of the two - photon polymerization femtosecond laser direct - writing system; then adopt the oil - immersion mode, adjust the position of the objective lens of the two - photon polymerization femtosecond laser direct - writing system so that the laser is focused in the sealed chamber on the substrate; and import the file of the structure to be processed into the operating software of the two - photon polymerization femtosecond laser direct - writing system; Then adjust the laser processing parameters, further focus the laser inside the cells, and finally control the two - photon polymerization femtosecond laser direct - writing system to perform polymerization laser direct - writing.

9. The method for laser additive manufacturing inside a cell according to claim 4, characterized in that, Step 4) includes: Sterilize the outside of the substrate after polymerization processing, and then place it in a secondary biosafety cabinet in a sterile environment. Then remove the upper glass slide used to seal the chamber, transfer the polymerized cells into the culture medium, and then centrifuge the mixed solution. Take the cell - containing suspension to obtain cells containing micro - and nano - structures, that is, obtain the cells after laser additive manufacturing; among them, the centrifugation speed is 800 - 1000 rpm and the time is 4 - 5 min.

10. A cell containing micro - and nano - structures obtained by the method of performing laser additive manufacturing inside the cell according to any one of claims 1 - 9.

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