Microwell array chip and single cell sorting method based on laser-induced forward transfer

Abstract

The present invention provides a microporous array chip and a single cell sorting method for laser-induced forward transfer. A layer of microporous membrane is covered on a metal-coated glass sheet. The microporous membrane is a biocompatible thin film and is covered on the metal-coated glass sheet using a micro-nano processing technology. Due to the hydrophobic properties of the microporous array chip material, a single cell forms a single cell micro-droplet, which is fixed in the micropores to achieve single cell capture, facilitating subsequent single cell sorting. The stability and signal-to-noise ratio of the liquid single cell Raman spectrum are greatly increased, laying a foundation for single cell liquid Raman identification and sorting.

Description

Technical Field

[0001] The patent of this invention involves emerging cross-technical fields such as materials, optics and biomedical engineering, especially microwell array chips and their laser-induced forward transfer single cell sorting method. Background Art

[0002] Cells are the basic units of biological organisms. With the in-depth study of life sciences, scientists have discovered that scientific research based on single cells can fully interpret cell heterogeneity, analyze the essence and laws of life from a deeper level, and provide a scientific basis for studying the origin of life, the evolution and treatment of diseases. Therefore, single cell sorting technology is a prerequisite for single cell research. Single cell sorting technologies include fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), laser capture microdissection (LCM), laser induced forward transfer (LIFT), optical tweezers, artificial microscopy and microfluidics.

[0003] In FACS or MACS, cells are labeled with fluorescently activated antigens or proteins. Then, based on the scattering and fluorescence signals obtained from the cells, they are sorted by fluid force or magnetic force, respectively. However, these methods cannot separate single cells from a small population of cells and cannot be used to obtain single cells or adherent cells that lack known antigens.

[0004] In LCM, a high-power laser is used to cut a thin layer of medium to obtain the target cells, and the damage of high-power laser to cells is fatal. Optical tweezers use optical trapping force to capture single cells in suspension. The efficiency of single-cell capture is low, and the throughput of sorting is low. Artificial microscopy is a technology in which experimenters use high-precision instruments under a microscope to select cells from a cell population. It has low throughput, high difficulty in operation, and low degree of automation. Microfluidic technology is based on the size, shape, dielectric constant, protein phenotype and other characteristics of cells of a specific population, and designs precise micro-nano structures to perform batch sorting of cells of the same population; it has a small sample dosage, a high degree of automation, low reagent cost, and a wide range of applications, but it requires that cells must be suspended in microfluidics, and the concentration of cells is difficult to control, making it difficult to accurately screen single cells, and excessive cells often lead to microchannel blockage.

[0005] Laser Induced Forward Transfer (LIFT) uses the interaction between laser and matter to accurately locate the target directly through microscopic imaging, and ejects and sorts it instantly by irradiating the sacrificial layer with pulsed laser. When using LIFT for single cell sorting, the laser acts on the traditional metal-coated chip through the objective lens to generate a driving force, so that the sample loaded on the metal-coated surface is ejected onto the receiving substrate. This sorting technology meets the requirements of lower sample loading, which is conducive to the collection of target cells in rare samples; while meeting the requirements of conventional size cell sorting, it has significant advantages in accurately sorting smaller microbial cells (1-5um) or irregularly shaped cells.

[0006] 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.

[0007] However, LIFT technology has certain limitations. During the LIFT sorting process, single cells cannot be captured in liquid, and multiple cells may be sorted at one time. In addition, the laser acts on the sacrificial layer to produce high-temperature steam. The generated heat 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.

[0008] Currently, there is no visual and precise single-cell live sorting method that can effectively capture single cells and reduce sorting damage in liquids. Summary of the invention

[0009] In order to overcome the shortcomings of the prior art, the present invention provides a microwell array chip and a single cell sorting method of laser-induced forward transfer thereof, so as to achieve single cell capture, solve the damage to cell activity during the sorting process, realize effective single cell sorting in living liquids, and provide an opportunity for measuring single cell Raman in liquids.

[0010] In order to solve the above technical problems, the present invention provides a microwell array chip and a single cell sorting method by laser-induced forward transfer, wherein:

[0011] Microwell array chip, including:

[0012] On the metal-coated glass sheet, a layer of microporous film is covered;

[0013] Furthermore, the microporous membrane is a biocompatible thin film, which is covered on a metal-coated glass sheet using a micro-nano processing technology;

[0014] Due to the hydrophobic properties of the microwell array chip material, single cells form single-cell microdroplets and are fixed in the microwells to achieve single-cell capture, facilitating subsequent single-cell sorting.

[0015] As an example, the metal-coated glass sheet refers to: a nano-scale metal film is coated on the glass sheet by magnetron sputtering.

[0016] As an example, the micro-nano processing technology includes three methods:

[0017] Method 1:

[0018] ① Use a Parylene deposition instrument to deposit a Parylene C layer of the required thickness onto the metal-coated glass sheet;

[0019] 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;

[0020] ③ Perform reactive ion etching (RIE) on the polyparaxylene C layer that leaks out after development until the metal layer is exposed;

[0021] ④ The prepared microwell array chip is immersed in acetone to wash away the photoresist from the sorting chip to obtain a microwell array chip.

[0022] As an example, the photoresist is used as an etching template for Parylene and will be completely washed off in the end;

[0023] As an example, each micropore in the close-packed micropore array is: equal in size, shape, and spacing;

[0024] As an example, the shape of the micropores is: isohexagonal, circular or other shapes;

[0025] Method 2:

[0026] ① Make Si negative master using photolithography and deep reactive ion etching (DRIE);

[0027] 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;

[0028] ③ Combine it with the metal-coated glass sheet through plasma action to obtain a micro diffusion chamber.

[0029] Method 3:

[0030] Spin-coat photoresist on a metal-coated glass sheet and develop the micro-diffusion chamber by photolithography patterning;

[0031] 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.

[0032] The single cell sorting method based on laser-induced forward transfer of microwell array chip includes:

[0033] Step 1: Structural design of LIFT sorting device:

[0034] 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;

[0035] 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;

[0036] As an example, the duration of the 532nm pulse laser is 5ns;

[0037] 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;

[0038] As an example, the chip fixing device is a 3D motion platform;

[0039] As an example, the receiving device is a receiving dish, which is placed in a machined groove below the microwell array chip and moved by electrical control;

[0040] Step 2: Single cell live sorting using the LIFT sorting device combined with a microwell array chip:

[0041] First, the LIFT sorting device and microwell array chip were exposed to ultraviolet light for at least 30 minutes to eliminate other bacterial contamination;

[0042] Secondly, the microwell array chip was treated with hydrophilicity under the action of O2 plasma; then 2-3ul of cell suspension was dripped onto the microwell array chip for single cell capture for about 10s;

[0043] Then, the microwell array chip is placed on the 3D motion platform;

[0044] 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.

[0045] Compared with the existing metal-plated chip spot sorting, the microwell array chip overcomes the influence of liquid surface tension, disperses cells into single-cell microdroplets, achieves single-cell fixation, and has a stable and efficient single-cell capture efficiency;

[0046] 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;

[0047] 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.

[0048] Beneficial effects of the present invention:

[0049] 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.

[0050] 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 array 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 the microporous array combined with the LIFT sorting method for single-cell capture and single-cell sorting.

[0051] 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 microwell array 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

[0052] Figure 1 It is a light path diagram of single cell ejection sorting of living liquid of the microwell array chip and single cell sorting method of laser induced forward transfer of the present invention.

[0053] Figure 2 It is a schematic diagram of the structure of the microwell array chip of the present invention and the single cell sorting method of the microwell array chip by laser induced forward transfer.

[0054] Figure 3 This is a simulation analysis diagram of the effect of sorting temperature on single cell activity in the microwell array chip and the single cell sorting method using laser induced forward transfer of the present invention.

[0055] Figure 4 This is a schematic diagram of cell loading before sorting in the microwell array chip and the single cell sorting method using laser-induced forward transfer of the present invention.

[0056] Figure 5 It is a schematic diagram of cell capture of the microwell array chip and the single cell sorting method of laser-induced forward transfer thereof of the present invention.

[0057] Figure 6 This is a diagram of the single cell identification and sorting process of the microwell array chip and the single cell sorting method of laser-induced forward transfer of the present invention.

[0058] Figure 7 It is a single cell sorting receiving diagram of the microwell array chip of the present invention and the single cell sorting method of laser induced forward transfer thereof, which is directly used for culture. DETAILED DESCRIPTION

[0059] The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0060] Reference Figures 1 to 7 As shown, a microwell array chip and a single cell sorting method using laser-induced forward transfer, wherein:

[0061] Microwell array chip, including:

[0062] On the metal-coated glass sheet, a layer of microporous film is covered;

[0063] Furthermore, the microporous membrane is a biocompatible film, which is covered on a metal-coated glass sheet using a micro-nano processing technology; due to the hydrophobic properties of the microporous array chip material, a single cell forms a single-cell micro-droplet, which is fixed in the micropores to achieve single-cell capture, facilitating subsequent single-cell sorting.

[0064] As an example, the metal-coated glass sheet refers to: a nano-scale metal film is coated on the glass sheet by magnetron sputtering.

[0065] As an example, the micro-nano processing technology includes three methods:

[0066] Method 1:

[0067] ① Use a Parylene deposition instrument to deposit a Parylene C layer of the required thickness onto the metal-coated glass sheet;

[0068] 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;

[0069] ③ Perform reactive ion etching (RIE) on the polyparaxylene C layer that leaks out after development until the metal layer is exposed;

[0070] ④ The prepared microwell array chip is immersed in acetone to wash away the photoresist from the sorting chip to obtain a microwell array chip.

[0071] As an example, the photoresist is used as an etching template for Parylene and will be completely washed off in the end;

[0072] As an example, each micropore in the close-packed micropore array is: equal in size, shape, and spacing;

[0073] As an example, the shape of the micropores is: equihexagonal or circular;

[0074] Method 2:

[0075] ① Make Si negative master using photolithography and deep reactive ion etching (DRIE);

[0076] 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;

[0077] ③ Combine it with a metal-coated glass sheet through plasma action to obtain a micro diffusion chamber and a microporous array chip.

[0078] Method 3:

[0079] Spin-coat photoresist on a metal-coated glass sheet, and obtain a micro-diffusion chamber by photolithography patterning and development to obtain a microwell array chip;

[0080] 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.

[0081] The single cell sorting method based on laser-induced forward transfer of microwell array chip includes:

[0082] Step 1: Structural design of LIFT sorting device:

[0083] 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;

[0084] 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;

[0085] 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;

[0086] As an example, the microwell array chip fixing device 116 is a 3D motion platform;

[0087] As an example, the receiving device 117 is a receiving dish, which is placed in a machined groove below the microwell array chip and moved by electrical control;

[0088] Step 2: Single cell live sorting using the LIFT sorting device combined with a microwell array chip:

[0089] First, the LIFT sorting device and microwell array chip were exposed to ultraviolet light for at least 30 minutes to eliminate other bacterial contamination;

[0090] Secondly, the microwell array chip was treated with hydrophilicity under the action of O2 plasma; then 2-3ul of cell suspension was dripped onto the microwell array chip for single cell capture for about 10s;

[0091] Then, the microwell array chip is placed on the 3D motion platform;

[0092] 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.

[0093] Compared with the existing metal-plated chip spot sorting, the microwell array chip overcomes the influence of liquid surface tension, disperses cells into single-cell microdroplets, achieves single-cell fixation, and has a stable and efficient single-cell capture efficiency;

[0094] 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;

[0095] 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.

[0096] 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;

[0097] 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.

[0098] 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 array 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 the microporous array combined with the LIFT sorting method for single-cell capture and single-cell sorting.

[0099] 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 microwell array 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.

[0100] 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 microwell array chip, characterized in that: include: On the metal-coated glass sheet, a layer of microporous film is covered; The microporous membrane is a biocompatible thin film, which is covered on a metal-coated glass sheet using a micro-nano processing technology; due to the hydrophobic properties of the microporous array chip, a single cell forms a single-cell micro-droplet, which is fixed in the micropores to achieve single-cell capture, facilitating subsequent single-cell sorting; The metal-coated glass sheet refers to: coating a nano-scale metal film on a glass sheet by magnetron sputtering; and performing a hydrophilic treatment on a microporous array chip under the action of O2 plasma.

2. The microwell array chip according to claim 1, characterized in that: The micro-nano processing technology includes: method 1, 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 microwell array chip is immersed in acetone to wash away the photoresist from the sorting chip to obtain a microwell array chip.

3. The microwell array chip according to claim 2, characterized in that: The photoresist is used as an etch template for the polyparaxylene and will eventually be completely washed away.

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

5. The microwell array chip according to claim 1, characterized in that: The micro-nano processing technology also includes: ① Use photolithography and deep counter-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 a metal-coated glass sheet through plasma action to obtain a micro diffusion chamber and a microporous array chip.

6. The microwell array chip according to claim 1, characterized in that: The micro-nano processing technology also includes: Photoresist is spin-coated on a metal-coated glass sheet, and a micro-diffusion chamber is obtained by photolithographic patterning and development to obtain a micropore array chip; when the pore size of the micro-diffusion chamber is 20um-80um, it is convenient to load a single cell in each diffusion micro-chamber, and the obtained 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.

7. A single cell sorting method based on the laser-induced forward transfer of the microwell array chip according to any one of claims 1 to 6, characterized in that: include: Step 1: 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 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 2: Single cell live sorting using the LIFT sorting device combined with a microwell array chip: First, the LIFT sorting device and microwell array chip were exposed to ultraviolet light for at least 30 minutes to eliminate other bacterial contamination; Secondly, the microwell array chip was treated with hydrophilicity under the action of O2 plasma; then 2-3ul of cell suspension was dripped onto the microwell array chip for single cell capture for 10s; Then, the microwell array chip is placed 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.

8. The single cell sorting method according to claim 7, characterized in that: The chip fixing device is a 3D motion platform, and the receiving device is a receiving dish, which is placed in a machined groove below the microwell array chip and moved by electrical control.

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