A single cell in situ culture chip and a method for isolating in situ pure culture thereof

Abstract

The present invention discloses a single cell in situ culture chip and a method for separating in situ pure culture, comprising: covering a layer of microporous membrane on a metal-coated glass sheet to form a microporous sorting chip, and covering a layer of nano-scale polymer membrane on the microporous membrane. The three-layer structure together constitutes an in situ culture chip with multiple groups of micro-diffusion chambers, which can provide naturally existing growth factors and nutrients for cells in the micro-diffusion chambers, so that those microbial species that cannot be cultured in the experiment can grow in situ in the natural environment. The randomness of species culture is reduced, the culture rate of microorganisms in a sample in a single environment is increased, and the single microorganism can be effectively separated with high throughput, so as to realize parallel culture and separation of previously uncultured microbial species from various environments.

Description

Technical Field

[0001] The patent of this invention involves emerging cross-disciplinary technology fields such as materials, optics and biomedical engineering, especially a single-cell in situ culture chip and a method for separating its in situ pure culture, which realizes the high-throughput in situ culture of single cells of "uncultivable" microbial species in experiments. Background Art

[0002] More than 99% of microbial species in nature will not grow on synthetic media in vitro, and rRNA and metagenomic approaches have demonstrated the amazing diversity of these uncultivated species. At the same time, almost every therapeutic area - autoimmune diseases, infectious diseases, central nervous system, metabolic diseases, and even cancer - is associated with alterations in the microbiome in some way. Therefore, obtaining this "missing" microbial diversity is of great significance to both basic and applied sciences.

[0003] However, people's analysis of microorganisms is based on the average level of group cells, which blurs our understanding of microbial diversity, obscures the correspondence between cell functional phenotypes and genes, and loses the information of single-cell heterogeneity.

[0004] To address this challenge, a method has been developed to culture microorganisms in an in situ diffusion chamber. The basic principle of this method is that diffusion will provide naturally occurring growth factors to the cells in the chamber and allow those microbial species that are "unculturable" in the experiment to grow in the diffusion chamber in their natural environment. This method produces microbial recoveries many times higher than standard laboratory techniques. Even so, this method is laborious, has a high degree of randomness, a small number of diffusion chambers, cannot guarantee that all microorganisms in the sample are cultured in a single diffusion chamber, and cannot effectively and high-throughput isolate a large number of individual microorganisms, which limits the applicability of this method.

[0005] The research on single cell sorting technology is a hot topic in biological research at home and abroad. Single cell sorting technology based on the principle of Laser Induced Forward Transfer (LIFT) can accurately sort designated single cells for genetic analysis under microscopic conditions and is widely used in research in the fields of biology, medicine, food, etc.

[0006] However, LIFT technology has certain limitations. It is impossible to capture single cells in liquid during LIFT sorting. Multiple cells may be sorted at one time. The laser generates high-temperature steam on the sacrificial layer. The heat generated is absorbed by the cells near the sacrificial layer, causing cell damage and death. This greatly reduces the success rate of single-cell live sorting and loses its great scientific research value. At present, there is no visual and precise single-cell live sorting method that can effectively capture single cells in liquid and reduce sorting damage. Summary of the invention

[0007] In order to overcome the shortcomings of the prior art, the present invention provides a single-cell in situ culture chip and a method for separating in situ pure cultures thereof. Through a special three-layer structure chip design, the randomness of species culture is reduced, the culture rate of microorganisms in the sample in a single environment is increased, and effective high-throughput separation of single microorganisms is achieved, thereby realizing parallel culture and separation of previously uncultured microbial species from various environments.

[0008] In order to solve the above technical problems, the present invention provides a single cell in situ culture chip and a method for isolating an in situ pure culture thereof, wherein:

[0009] A single cell in situ culture chip, comprising:

[0010] A layer of microporous membrane is covered on the metal-coated glass sheet to form a microporous sorting chip, and a layer of nano-scale polymer membrane is covered on the microporous membrane. The above three-layer structure together constitutes an in-situ culture chip with multiple sets of micro-diffusion chambers, which can provide naturally occurring growth factors and nutrients for cells in the micro-diffusion chambers, allowing those microbial species that cannot be cultured in the experiment to grow in situ in the natural environment.

[0011] Furthermore, the microporous membrane is a biocompatible thin film, which is covered on a metal-coated glass sheet using a micro-nano processing technology;

[0012] As an example, the metal-coated glass sheet refers to: a nano-scale metal film is coated on the glass sheet by magnetron sputtering.

[0013] As an example, the nanoscale polymer membrane may be a polycarbonate membrane or a cellulose acetate filter membrane with a pore size of less than 0.1 μm.

[0014] As an example, the non-culturable refers to: nonculture, which are microorganisms that have been isolated but cannot be cultured and multiplied in large quantities in the laboratory; these microorganisms cannot find suitable culture conditions through existing laboratory technology and can only be propagated and cultured in situ.

[0015] As an example, the micro-nano processing technology includes three methods:

[0016] Method 1:

[0017] ① Use a Parylene deposition instrument to deposit a Parylene C layer of the required thickness onto the metal-coated glass sheet;

[0018] ② Spin-coat photoresist on the polyparaxylene C layer, and prepare the required micropore diameter, shape, and density on the metal-coated glass by photolithography patterning to form a densely packed micropore array;

[0019] ③ Perform reactive ion etching (RIE) on the polyparaxylene C layer that leaks out after development until the metal layer is exposed;

[0020] ④ The prepared micropore sorting chip is immersed in acetone to wash away the photoresist from the sorting chip, thereby obtaining a micropore sorting chip.

[0021] As an example, the photoresist is used as an etching template for Parylene and will be completely washed off in the end;

[0022] As an example, each micropore in the close-packed micropore array is: equal in size, shape, and spacing;

[0023] As an example, the shape of the micropores is: isohexagonal, circular or other shapes;

[0024] Method 2:

[0025] ① Make Si negative master using photolithography and deep reactive ion etching (DRIE);

[0026] ② Use PDMS to mold a microfluidic chip mold with a densely packed microcolumn structure, bond it to a glass sheet, and then inject a film-forming material (PDMS, UV curing material, etc.) to cure it. After curing, remove the mold to obtain a microwell array chip;

[0027] ③ Combine it with the metal-coated glass sheet through plasma action to obtain a micro diffusion chamber.

[0028] Method 3:

[0029] Spin-coat photoresist on a metal-coated glass sheet and develop the micro-diffusion chamber by photolithography patterning;

[0030] When the pore size of the micro diffusion chamber is 20um-80um, it is convenient to load a single cell into each diffusion micro chamber, and the resulting culture is monospecific; when the thickness of the micro diffusion chamber is 3um-15um, it is convenient to separate the microorganisms into pure cultures after growth.

[0031] Based on a method for separating pure in situ cultures of a single-cell in situ culture chip, combined with laser-induced forward transfer technology, a single cell that has completed in situ culture is visually separated to obtain pure in situ cultures, and the growth and sorting of microbial species that cannot be cultured in the laboratory in a diffusion chamber in a natural environment are realized, including:

[0032] Step 1: Place the microporous sorting chip in ethanol for disinfection, and rinse it with particle-free DNA-grade water after drying; perform hydrophilic treatment on the rinsed microporous sorting chip under the action of plasma, and then perform microbial spotting treatment; then use a nanoscale polymer film to cover the microporous sorting chip for sealing to form an in-situ culture chip;

[0033] Step 2: immersing the in situ culture chip into a cell suspension of target culture; at this time, multiple groups of micro-diffusion chambers jointly capture a cell suspension containing a certain number of cells and a certain volume; each micro-diffusion chamber captures one cell; due to fluid dynamics and surface wettability, the cells are individually "trapped" in their respective micro-diffusion chambers and separated from each other;

[0034] As an illustration, the number of cells depends on the dilution level;

[0035] As an example, the use of nanoscale polymer membranes can effectively prevent cells from migrating out of a single diffusion chamber.

[0036] Step 3: Then fix the in situ culture chip in the original environment of the cells, which can ensure that the hatched fixed cells are provided with their naturally existing nutrients and growth factors;

[0037] Step 4: After the incubation is completed, the in situ culture chip is disassembled, the nanoscale polymer membrane is removed, and the microporous sorting chip is retained; then, the identification and sorting of pure cultures are performed automatically on a specially designed LIFT sorting device, and combined with sequencing for subsequent further analysis;

[0038] Step 5: Structural design of LIFT sorting device:

[0039] The LIFT sorting device includes: a single cell sorting module, a white light (or fluorescence) imaging module, a chip fixing device and a receiving device;

[0040] As an example, the single cell sorting module includes: a 532nm pulsed laser, a half-wave plate, a polarization beam splitter, a first beam expander, a second beam expander, a first reflector, a second reflector, and a first microscope objective lens;

[0041] As an example, the duration of the 532nm pulse laser is 5ns;

[0042] As an example, the white light imaging module includes: a second microscope objective, a third reflector, a convex lens, a beam splitter, a multi-channel filter, an LED and a camera;

[0043] As an example, the chip fixing device is a 3D motion platform;

[0044] As an example, the receiving device is a receiving dish, which is placed in a machined groove below the micropore sorting chip and moved by electrical control;

[0045] Step 6: Single cell live sorting is performed using a LIFT sorting device combined with a micropore sorting chip:

[0046] First, the LIFT sorting device was exposed to ultraviolet light for at least 30 minutes to eliminate other bacterial contamination;

[0047] Then, place the microwell sorting chip on the 3D motion platform;

[0048] Finally, all sorting and collection processes are observed and recorded by a CCD camera connected to a computer; the above sorting operation is controlled by one button to sort the target cells into a receiving device.

[0049] Compared with the existing metal-plated chip spot sorting, the microporous sorting chip overcomes the influence of liquid surface tension, disperses cells into single-cell micro-droplets, achieves single-cell fixation, and has a stable and efficient single-cell capture efficiency;

[0050] Furthermore, compared with spot sorting on a conventional metal-plated chip, the through-hole membrane has no liquid layer on the surface to hinder sorting, which greatly overcomes the constraint of liquid surface tension on single-cell sorting;

[0051] As an example, the effect of sorting temperature on single-cell activity was investigated by simulating the photothermal conversion process of the chip sacrificial layer. The results confirmed that compared with the traditional LIFT sorting method, the new sorting method greatly reduced the thermal damage of sorting; therefore, the through-hole membrane combined with the LIFT single-cell sorting technology can achieve accurate liquid living single-cell sorting.

[0052] Beneficial effects of the present invention:

[0053] The present invention reduces the randomness of species cultivation through a special three-layer chip design, improves the cultivation rate of microorganisms in a sample in a single environment, satisfies the requirement of effective high-throughput separation of single microorganisms, and realizes parallel cultivation and separation of previously uncultured microbial species from various environments.

[0054] On traditional Raman signal enhancement chips, cells in liquid cannot be fixed, making it difficult to obtain stable single-cell Raman spectra with high signal-to-noise ratio. By combining a through-hole membrane with a metal-plated chip, single cells can be fixed, greatly increasing the stability and signal-to-noise ratio of liquid single-cell Raman spectra, laying the foundation for single-cell liquid Raman identification and sorting.

[0055] The principle of single-cell sorting captured by microporous structure combines fluid dynamics and surface wettability, so it is different from the precise and stable single-cell sorting method using LIFT technology on traditional metal-coated chips. On traditional sorting chips, cells have lateral migration, which makes it impossible to sort accurately, or there are jets in the sorting process that cause multiple cells to be sorted together. Microporous sorting chips are different from traditional sorting chips. Due to the hydrophobic properties of the chip material, single cells form single-cell microdroplets, which are fixed in the micropores to achieve single-cell capture, facilitating subsequent single-cell sorting and cultivation. Therefore, this is an important factor in using a microporous array combined with the LIFT sorting method for single-cell capture and single-cell sorting.

[0056] The present invention not only realizes the sorting and cultivation of single-cell living liquid, but also helps to obtain stable single-cell Raman spectroscopy signals in liquid. On traditional chips, cells in liquid cannot be fixed, making it difficult to obtain stable single-cell Raman spectra with high signal-to-noise ratio. The single-cell fixation and single-cell capture methods provided by the microporous sorting chip will greatly increase the stability of the single-cell Raman spectroscopy signals collected in liquid, reduce the signal interference of liquid disturbance, and lay the foundation for single-cell liquid Raman identification and sorting. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The present invention discloses a single cell in situ culture chip and a method for separating pure in situ culture products thereof, and an optical path diagram for ejection sorting of single cells in living liquid.

[0058] Figure 2 It is a schematic diagram of the structure of a micropore sorting chip for a single cell in situ culture chip and a method for separating in situ pure cultures thereof according to the present invention.

[0059] Figure 3 This is a simulation analysis diagram of the effect of sorting temperature on single cell activity in a single cell in situ culture chip and a method for separating in situ pure culture of the present invention.

[0060] Figure 4 It is a schematic diagram of cell loading before sorting in a single-cell in situ culture chip and a method for separating in situ pure cultures thereof according to the present invention.

[0061] Figure 5 Schematic diagram of cell capture of a single cell in situ culture chip and a method for separating in situ pure culture of the present invention

[0062] Figure 6 This is a diagram of the single cell identification and sorting process of a single cell in situ culture chip and a method for separating pure in situ cultures of the present invention.

[0063] Figure 7 This is a single cell sorting receiving diagram of a single cell in situ culture chip and a method for separating in situ pure cultures thereof according to the present invention.

[0064] Figure 8 It is a schematic diagram of the separation process operation of a single cell in situ culture chip and a method for separating in situ pure culture thereof of the present invention. DETAILED DESCRIPTION

[0065] The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0066] Reference Figures 1 to 8 As shown, the present invention provides a single cell in situ culture chip and a method for isolating an in situ pure culture thereof, wherein:

[0067] A single cell in situ culture chip, comprising:

[0068] A layer of microporous membrane is covered on a metal-coated glass sheet to form a microporous sorting chip, and a layer of nanoscale polymer membrane is covered on the microporous membrane. The above three-layer structure together constitutes an in-situ culture chip with multiple sets of micro-diffusion chambers, which can provide naturally occurring growth factors and nutrients for the cells in the micro-diffusion chambers, allowing those microbial species that cannot be cultured in the experiment to grow in situ in the natural environment.

[0069] Furthermore, the microporous membrane is a biocompatible thin film, which is covered on a metal-coated glass sheet using a micro-nano processing technology;

[0070] As an example, the metal-coated glass sheet refers to: a nano-scale metal film is coated on the glass sheet by magnetron sputtering.

[0071] As an example, the nanoscale polymer membrane may be a polycarbonate membrane or a cellulose acetate filter membrane with a pore size of less than 0.1 μm.

[0072] As an example, the non-culturable refers to: nonculture, which are microorganisms that have been isolated but cannot be cultured and multiplied in large quantities in the laboratory; these microorganisms cannot find suitable culture conditions through existing laboratory technology and can only be propagated and cultured in situ.

[0073] As an example, the micro-nano processing technology includes three methods:

[0074] Method 1:

[0075] ① Use a Parylene deposition instrument to deposit a Parylene C layer of the required thickness onto the metal-coated glass sheet;

[0076] ② Spin-coat photoresist on the polyparaxylene C layer, and prepare the required micropore diameter, shape, and density on the metal-coated glass by photolithography patterning to form a densely packed micropore array;

[0077] ③ Perform reactive ion etching (RIE) on the polyparaxylene C layer that leaks out after development until the metal layer is exposed;

[0078] ④ The prepared micropore sorting chip is immersed in acetone to wash away the photoresist from the sorting chip, thereby obtaining a micropore sorting chip.

[0079] As an example, the photoresist is used as an etching template for Parylene and will be completely washed off in the end;

[0080] As an example, each micropore in the close-packed micropore array is: equal in size, shape, and spacing;

[0081] As an example, the shape of the micropores is: isohexagonal, circular or other shapes;

[0082] Method 2:

[0083] ① Make Si negative master using photolithography and deep reactive ion etching (DRIE);

[0084] ② Use PDMS to mold a microfluidic chip mold with a densely packed microcolumn structure, bond it to a glass sheet, and then inject a film-forming material (PDMS, UV curing material, etc.) to cure it. After curing, remove the mold to obtain a microwell array chip;

[0085] ③ Combine it with the metal-coated glass sheet through plasma action to obtain a micro diffusion chamber.

[0086] Method 3:

[0087] Spin-coat photoresist on a metal-coated glass sheet and develop the micro-diffusion chamber by photolithography patterning;

[0088] When the pore size of the micro diffusion chamber is 20um-80um, it is convenient to load a single cell into each diffusion micro chamber, and the resulting culture is monospecific; when the thickness of the micro diffusion chamber is 3um-15um, it is convenient to separate the microorganisms into pure cultures after growth.

[0089] Based on a method for separating pure in situ cultures of a single-cell in situ culture chip, combined with laser-induced forward transfer technology, a single cell that has completed in situ culture is visually separated to obtain pure in situ cultures, and the growth and sorting of microbial species that cannot be cultured in the laboratory in a diffusion chamber in a natural environment are realized, including:

[0090] Step 1: Place the microporous sorting chip in ethanol for disinfection, and rinse it with particle-free DNA-grade water after drying; perform hydrophilic treatment on the rinsed microporous sorting chip under the action of plasma, and then perform microbial spotting treatment; then use a nanoscale polymer film to cover the microporous sorting chip for sealing to form an in-situ culture chip;

[0091] Step 2: immersing the in situ culture chip into a cell suspension of target culture; at this time, multiple groups of micro-diffusion chambers jointly capture a cell suspension containing a certain number of cells and a certain volume; each micro-diffusion chamber captures one cell; due to fluid dynamics and surface wettability, the cells are individually "trapped" in their respective micro-diffusion chambers and separated from each other;

[0092] As an illustration, the number of cells depends on the dilution level;

[0093] As an example, the use of nanoscale polymer membranes can effectively prevent cells from migrating out of a single diffusion chamber.

[0094] Step 3: Then fix the in situ culture chip in the original environment of the cells, which can ensure that the hatched fixed cells are provided with their naturally existing nutrients and growth factors;

[0095] Step 4: After the incubation is completed, the in situ culture chip is disassembled, the nanoscale polymer membrane is removed, and the microporous sorting chip is retained; then, the identification and sorting of pure cultures are performed automatically on a specially designed LIFT sorting device, and combined with sequencing for subsequent further analysis;

[0096] Step 5: Structural design of LIFT sorting device:

[0097] The LIFT sorting device includes: a single cell sorting module, a white light (or fluorescence) imaging module, a chip fixing device 116 and a receiving device 117;

[0098] As an example, the single cell sorting module includes: a 532nm pulse laser (duration 5ns) 101, a half-wave plate 102, a polarization beam splitter 103, a first beam expander 104, a second beam expander 105, a first reflector 106, a second reflector 107 and a first microscope objective 108;

[0099] As an example, the white light imaging module includes: a second microscope objective lens 109, a third reflector 110, a convex lens 111, a beam splitter 112, a multi-channel filter 113, an LED 114 and a camera 115;

[0100] As an example, the micro-hole sorting chip fixing device 116 is a 3D motion platform;

[0101] As an example, the receiving device 117 is a receiving dish, which is placed in a machined groove below the micropore sorting chip and moved by electrical control;

[0102] Step 6: Single cell live sorting is performed using the LIFT sorting device combined with a micropore sorting chip:

[0103] First, the LIFT sorting device was exposed to ultraviolet light for at least 30 minutes to eliminate other bacterial contamination;

[0104] Then, place the microwell sorting chip on the 3D motion platform;

[0105] Finally, all sorting and collection processes are observed and recorded by a CCD camera connected to a computer; the above sorting operation is controlled by one button to sort the target cells into a receiving device.

[0106] Compared with the existing metal-plated chip spot sorting, the microporous sorting chip overcomes the influence of liquid surface tension, disperses cells into single-cell micro-droplets, achieves single-cell fixation, and has a stable and efficient single-cell capture efficiency;

[0107] Furthermore, compared with spot sorting on a conventional metal-plated chip, the through-hole membrane has no liquid layer on the surface to hinder sorting, which greatly overcomes the constraint of liquid surface tension on single-cell sorting;

[0108] Reference Figure 3 As shown, by simulating the photothermal conversion process of the chip sacrificial layer, the effect of sorting temperature on single-cell activity was explored. The results confirmed that compared with the traditional LIFT sorting method, the new sorting method greatly reduced the thermal damage of sorting; therefore, the through-hole membrane combined with the LIFT single-cell sorting technology can achieve accurate liquid living single-cell sorting.

[0109] As an example, the Figure 6 and Figure 7 The culture dish can be replaced with a receiving substrate, which can be a cover glass placed at 500um or other distances from the microporous membrane, to facilitate the observation of single cells being accurately sorted from the micropores;

[0110] On traditional Raman signal enhancement chips, cells in liquid cannot be fixed, making it difficult to obtain stable single-cell Raman spectra with high signal-to-noise ratio. By combining a through-hole membrane with a metal-plated chip, single cells can be fixed, greatly increasing the stability and signal-to-noise ratio of liquid single-cell Raman spectra, laying the foundation for single-cell liquid Raman identification and sorting.

[0111] The principle of single-cell sorting captured by microporous structure combines fluid dynamics and surface wettability, so it is different from the precise and stable single-cell sorting method using LIFT technology on traditional metal-coated chips. On traditional sorting chips, cells have lateral migration, which makes it impossible to sort accurately, or there are jets in the sorting process that cause multiple cells to be sorted together. Microporous sorting chips are different from traditional sorting chips. Due to the hydrophobic properties of the chip material, single cells form single-cell microdroplets, which are fixed in the micropores to achieve single-cell capture, facilitating subsequent single-cell sorting and cultivation. Therefore, this is an important factor in using a microporous array combined with the LIFT sorting method for single-cell capture and single-cell sorting.

[0112] The present invention not only realizes the sorting and cultivation of single-cell living liquid, but also helps to obtain stable single-cell Raman spectroscopy signals in liquid. On traditional chips, cells in liquid cannot be fixed, making it difficult to obtain stable single-cell Raman spectra with high signal-to-noise ratio. The single-cell fixation and single-cell capture methods provided by the microporous sorting chip will greatly increase the stability of the single-cell Raman spectroscopy signals collected in liquid, reduce the signal interference of liquid disturbance, and lay the foundation for single-cell liquid Raman identification and sorting.

[0113] The above are only preferred embodiments of the present invention. It should be understood that the description of the above embodiments is only used to help understand the method and core ideas of the present invention, and is not used to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, etc. made within the ideas and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A single cell in situ culture chip, characterized in that: include: A layer of microporous membrane is covered on a metal-coated glass sheet to form a microporous sorting chip, and a layer of nanoscale polymer membrane is covered on the microporous membrane. The nanoscale polymer membrane is covered on the microporous sorting chip for sealing to form an in-situ culture chip; the above three-layer structure together constitutes an in-situ culture chip with multiple groups of micro-diffusion chambers, which can provide naturally occurring growth factors and nutrients for cells in the micro-diffusion chambers, so that those microbial species that cannot be cultured in the experiment can grow in-situ in the natural environment; The microporous membrane is a biocompatible thin film, which is covered on a metal-coated glass sheet by micro-nano processing technology; The metal-coated glass sheet refers to: a glass sheet on which a nano-scale metal film is coated by magnetron sputtering; The nano-scale polymer membrane is a polycarbonate membrane or a cellulose acetate filter membrane with a pore size of less than 0.1 μm. The use of the nano-scale polymer membrane can effectively prevent cells from migrating out of a single micro-diffusion chamber.

2. A single cell in situ culture chip according to claim 1, characterized in that: The micro-nano processing technology comprises: ① Use a polyparaxylene deposition instrument to deposit a polyparaxylene C layer of the required thickness onto the metal-coated glass sheet; ② Spin-coat photoresist on the polyparaxylene C layer, and prepare the required micropore diameter, shape, and density on the metal-coated glass by photolithography patterning to form a densely packed micropore array; ③ Perform reactive ion etching on the polyparaxylene C layer that leaks out after development until the metal layer is exposed; ④ The prepared micropore sorting chip is immersed in acetone to wash away the photoresist from the sorting chip, thereby obtaining a micropore sorting chip.

3. A single cell in situ culture chip according to claim 2, characterized in that: The photoresist is used as an etching template for Parylene and will be completely washed off in the end.

4. The single cell in situ culture chip according to claim 2, characterized in that: Each micropore in the densely packed micropore array has the same size, shape and spacing.

5. The single cell in situ culture chip according to claim 4, characterized in that: The micropores are in the shape of an equihexagon, a circle or other shapes.

6. The single cell in situ culture chip according to claim 1, characterized in that: The micro-nano processing technology comprises: ① Use photolithography and deep reactive ion etching to make Si negative master; ② Use PDMS to mold a microfluidic chip mold with a densely packed microcolumn structure, bond it to a glass sheet, inject a film-forming material, and solidify it. After solidification, remove the mold to obtain a microwell array chip; ③ Combine it with the metal-coated glass sheet through plasma action to obtain a micro diffusion chamber.

7. The single cell in situ culture chip according to claim 1, characterized in that: The micro-nano processing technology comprises: Spin-coat photoresist on a metal-coated glass sheet and develop the micro-diffusion chamber by photolithography patterning; When the pore size of the micro diffusion chamber is 20um-80um, it is convenient to load a single cell into each diffusion micro chamber, and the resulting culture is monospecific; when the thickness of the micro diffusion chamber is 3um-15um, it is convenient to separate the microorganisms into pure cultures after growth.

8. A method for separating pure in situ cultures of a single cell in situ culture chip according to any one of claims 1 to 7, combined with laser-induced forward transfer technology, to visually separate single cells that have completed in situ culture to obtain pure in situ cultures, and to achieve the growth and sorting of laboratory-uncultivable microbial species in a diffusion chamber in a natural environment, characterized in that: include: Step 1: Place the micropore sorting chip in ethanol for disinfection, and rinse with particle-free DNA-grade water after drying; The washed microporous sorting chip is subjected to a hydrophilic treatment under the action of plasma, and then subjected to a microbial spotting treatment; a nanoscale polymer film is then used to cover the microporous sorting chip for sealing, thereby forming an in-situ culture chip; Step 2: immersing the in situ culture chip into a cell suspension of target culture; at this time, multiple groups of micro-diffusion chambers jointly capture a cell suspension containing a certain number of cells and a certain volume; each micro-diffusion chamber captures one cell; due to fluid dynamics and surface wettability, the cells are individually fixed in their respective micro-diffusion chambers and separated from each other; Step 3: Then fix the in situ culture chip in the original environment of the cells, which can ensure that the hatched fixed cells are provided with their naturally existing nutrients and growth factors; Step 4: After the incubation is completed, the in situ culture chip is disassembled, the nanoscale polymer membrane is removed, and the microporous sorting chip is retained; then, the identification and sorting of pure cultures are performed automatically on a specially designed LIFT sorting device, and combined with sequencing for subsequent further analysis; Step 5: Structural design of LIFT sorting device: The LIFT sorting device includes: a single cell sorting module, a white light imaging module, a chip fixing device and a receiving device; The single cell sorting module comprises: a 532nm pulse laser, a half-wave plate, a polarization beam splitter, a first beam expander, a second beam expander, a first reflector, a second reflector and a first microscope objective lens; the duration of the 532nm pulse laser is 5ns; The white light imaging module comprises: a second microscope objective lens, a third reflector, a convex lens, a beam splitter, a multi-channel filter, an LED and a camera; Step 6: Single cell live sorting is performed using a LIFT sorting device combined with a micropore sorting chip: First, the LIFT sorting device was exposed to ultraviolet light for at least 30 minutes to eliminate other bacterial contamination; Then, place the microwell sorting chip on the 3D motion platform; Finally, all sorting and collection processes are observed and recorded by a CCD camera connected to a computer; the above sorting operation is controlled by one button to sort the target cells into a receiving device.

9. The separation method according to claim 8, characterized in that The chip fixing device is a 3D motion platform.

10. The separation method according to claim 8, characterized in that The receiving device is a receiving dish, which is placed in a machined groove below the micropore sorting chip and moved by electrical control.

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