Method and chip for capturing single cells and application of chip

Abstract

The invention provides a method for capturing single cells, a chip and application thereof, the method comprises the following steps: (1) fixing metal for capturing on a substrate in a manner of photoetching the substrate coated with photoresist by using a photoetching machine, then spraying metal or vacuum evaporation, and washing off the photoresist to obtain metal points in dot matrix arrangement; or, a single layer of small balls are tightly arranged on the substrate, metal spraying is conducted on the surfaces of the small balls, then the small balls are imprinted on another glass sheet, and metal points arranged in a dot matrix mode are obtained; and (2) capturing a single cell by adopting the fixed substrate, wherein the surface of the single cell contains recognition molecules with specific metal adsorption capacity. According to the method, single cell separation and regular arrangement can be realized, so that accurate positioning of cells is realized, and subsequent quantitative detection and analysis are carried out.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell separation, and particularly relates to a method for capturing single cells, a chip and application thereof. BACKGROUND

[0002] Single cell technology opens up a new direction for biological omics research-single cell omics. Single cell technology can analyze the cell types present in a tissue, identify the state of the cells, study the heterogeneity between cells, and reveal the molecular mechanisms of differentiation and development, disease occurrence and development, and tumor drug resistance through gene expression changes at the single cell level by sequencing single cells.

[0003] There are no two identical leaves in the world, and there are no two identical cells in the world. Single cells are all unique, and they can differ greatly in size, protein level and expressed RNA transcripts. Single cell analysis (SCA) is the study of single cells. Studying single cells can not only better understand cell heterogeneity, but also better interpret diseases.

[0004] The first step of single cell analysis is cell capture and separation. Single cells are initially obtained by gradient dilution, which is tedious and prone to contamination. Subsequently, single cells are separated by a device similar to a syringe using a micromanipulation method, or a laser capture microdissection technique (LCM), but the operability is not strong. Later, the sorting of a flow cytometer becomes the most commonly used method for separating single cells, but the environmental requirements are too high. Therefore, it is of great significance to provide a new simple, easy, convenient and fast single cell separation method for single cell omics research. SUMMARY

[0005] In view of the deficiencies in the prior art, the purpose of the present application is to provide a method for capturing single cells, a chip and application thereof. The method can realize single cell separation and can quantitatively lyse single cells in situ, control the number of lysed cells, and perform subsequent quantitative detection and analysis.

[0006] To achieve the purpose of the present application, the following technical solutions are adopted:

[0007] In a first aspect, the present application provides a method for capturing single cells, which comprises:

[0008] (1) fixing a metal for capturing on a substrate, the fixing method comprising: using a photoetching machine to photoetch a substrate coated with photoresist, then performing gold spraying or vacuum evaporation, and washing away the photoresist to obtain metal dots arranged in a dot matrix; or, closely arranging a single layer of small balls on a substrate, spraying gold on the surface of the small balls, and then pressing on another glass sheet to obtain metal dots arranged in a dot matrix;

[0009] (2) using the fixed substrate to capture single cells, the surface of the single cells containing recognition molecules having metal adsorption ability.

[0010] In the present application, the single cells contain recognition molecules, which can make molecules such as proteins, peptides, polysaccharides, etc. have metal adsorption ability. When the cells added with the recognition molecules approach the substrate, the cells will be adsorbed on the corresponding metal points, and since the spacing of the metal point array is small, only one cell can be present on one site, thereby realizing the separation of single cells. When subsequent cell analysis is performed, the cells can be broken by laser or other means as needed, so as to analyze the substances in the cells. In addition, the number of broken cells can be set, so as to control the test concentration.

[0011] Preferably, in step (1), the metal used for capturing is selected from any one or a combination of at least two of gold, calcium, manganese, iron, copper, zinc, molybdenum, cobalt, magnesium, mercury, chromium or cadmium.

[0012] Preferably, in step (1), the substrate is selected from any one or a combination of at least two of a glass slide, a silicon wafer, a copper sheet or a steel sheet.

[0013] Preferably, in step (1), the spacing between the metal points in the array is 1-50 μm, for example, it can be 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm, etc.

[0014] Preferably, in step (1), the diameter of the small balls in the array is 1-50 μm, for example, it can be 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm, etc.

[0015] Preferably, in step (2), the recognition molecules having metal adsorption ability are selected from any one or a combination of at least two of HIS tag, Flag-tag, SNAP-Tag, glutathione S-transferase tag, c-Myc tag, luciferase tag, metallothionein tag, hemoglobin tag, myoglobin tag, cytochrome C tag, azurin tag or iron-sulfur protein tag.

[0016] In the present application, there are many ways to combine the recognition molecules having metal adsorption ability and the metal, the principle of which is that after the recognition molecules having metal adsorption ability are expressed by the cells, part of them is on the outside of the cell membrane and part of them is inside the cell membrane, and the part on the outside is combined with the metal. Among them, sulfhydryl recognizes gold and lead; His, histidine recognizes nickel.

[0017] In a second aspect, the present application provides a chip for capturing single cells, which comprises a substrate and a metal dot array arranged on the substrate.

[0018] The chip for capturing single cells is prepared by a method comprising the following steps:

[0019] (A) arranging a monolayer of densely packed small balls with a diameter of 1-50 μm on the substrate;

[0020] (B) spraying metal particles on the small balls to adhere the metal particles to the surface of the small balls;

[0021] (C) pressing another substrate on the substrate with the small balls adhered thereto to make the metal particles sprayed on the top of the small balls adhere to the other substrate, thereby obtaining a metal dot array substrate with a distance interval.

[0022] Preferably, the chip for capturing single cells is prepared by a method comprising the following steps:

[0023] (a) performing photolithography on the substrate coated with photoresist by using a photolithography machine;

[0024] (b) covering the part of the substrate not protected by the photoresist with a metal coating by using gold spraying or evaporation;

[0025] (c) washing off the photoresist to obtain a metal dot array substrate with a distance interval.

[0026] In a third aspect, the present application provides a method for single cell quantitative analysis, which comprises:

[0027] The single cell is physically separated by using the chip for capturing single cells according to the second aspect, and the single cell is treated in situ to obtain intracellular substances for quantitative analysis.

[0028] Preferably, the method for treating the single cell in situ comprises ionizing the cell by using laser bombardment.

[0029] In the present application, after the single cell is captured by using the chip for capturing single cells, the molecular fingerprint of the substances contained in the cell can be determined by laser Raman analysis to perform quantitative and qualitative analysis of the substances contained in the cell. Alternatively, the cell is ionized by laser bombardment and then mass spectrometry analysis is performed. When mass spectrometry analysis is performed, the substrate needs to be made of conductive material, such as conductive silicon wafer, indium tin oxide glass, etc. In this way, an electric field can be formed between the substrate and the mass spectrometer by applying voltage, and the ionized substances can enter the mass spectrometer under the action of the electric field, and then the composition of the substances can be determined by mass spectrometry analysis.

[0030] In a fourth aspect, the present application provides the use of the chip for capturing single cells according to the second aspect in the preparation of a product for single cell omics analysis.

[0031] The numerical range described in the present application includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed, and the present application does not exhaustively list the specific point values included in the range for the sake of brevity and simplicity.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] The present application does not need to perform gradient dilution on cells, nor does it need a complex cell separation environment, but only needs the cells to carry metal recognition molecules, so that the chip provided by the present application can be used to separate and distribute single cells. Moreover, the single cells obtained by the method are arranged in a two-dimensional plane in an orderly manner, and the spacing between the cells is controllable, which brings great convenience for subsequent analysis and provides a premise for rapid analysis. In-situ lysis analysis, non-destructive Raman spectroscopy analysis and analysis based on laser ionization analysis can all be performed using the present technology. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is a schematic diagram of the chip in Example 1.

[0035] Figure 2 is a schematic diagram of the process of adhering a layer of metal particles to the surface of the small ball in the preparation of the chip in Example 2.

[0036] Figure 3 is a schematic diagram of the preparation of the glass slide with a metal matrix in the preparation of the chip in Example 2. DETAILED DESCRIPTION

[0037] The technical solutions of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations on the present application.

[0038] If a specific technology or condition is not specified in the embodiments, it is performed according to the technology or condition described in the literature in the art or according to the product instruction. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be commercially available through a regular channel.

[0039] Example 1

[0040] The present embodiment provides a chip for capturing single cells, which comprises a substrate and metal dots arranged in a dot matrix on the substrate; the chip for capturing single cells is prepared by a method comprising the following steps:

[0041] First, a mask for the photolithography is prepared, then the photoresist is coated on the substrate, and then the photolithography is performed using a photolithography machine (H94-30 type). After the photolithography, the substrate of the dot matrix part loses the protection of the photoresist and is exposed. Then, the parts of the substrate without the protection of the photoresist can be covered with a metal coating by spraying or evaporation. Finally, the photoresist is washed away to obtain a neat metal dot matrix on the substrate. The interval of the dot matrix obtained by using this method can be adjusted at will in the range of 1-50 μm, the size of the dot can also be adjusted at will, the pattern and size can also be adjusted according to the needs, and the flexibility is relatively large. The processing precision is determined by the precision of the photolithography machine, and the processing precision of the above machine can reach 1 μm. Figure 1 is a schematic diagram of a chip, Figure 1 A is a glass slide coated with a matrix of metal copper, and free cysteine Cys; B is a substrate adsorbed with cysteine Cys.

[0042] Example 2

[0043] The present embodiment provides a chip for capturing single cells, which comprises a substrate and metal dots arranged in a dot matrix on the substrate; and the chip for capturing single cells is prepared by a method comprising the following steps:

[0044] (A) A small ball with a diameter of 1-50 μm is laid on a glass slide (other planar materials can also be selected), and the balls are arranged closely with each other. The small balls are distributed in a single layer due to the surface adsorption force and gravity, and then an adhesive is added to firmly adhere the small balls to the glass slide;

[0045] (B) A metal particle is sprayed to the small balls using a spraying device, so that a layer of metal particles is adhered to the surface of the small balls;

[0046] (C) Another glass slide is pressed on the glass slide with the small balls adhered thereto, so that the metal particles sprayed on the small balls are adhered to the glass slide, and a metal dot matrix glass slide with a distance interval is obtained. The preparation schematic diagram of the chip is shown in Figure 2 and Figure 3 Figure 2 is a schematic diagram of the small balls with a layer of metal particles adhered to the surface during the preparation of the chip. Figure 3 is a preparation schematic diagram of the glass slide with a metal matrix during the preparation of the chip.

[0047] Example 3

[0048] The present embodiment provides a method for capturing single cells, which comprises: capturing single cells by using the chip for capturing single cells in Example 1, wherein the surface of the single cell contains a recognition molecule with a metal adsorption capacity, and the recognition molecule is a cysteine-rich polypeptide tag. The method comprises the following steps:

[0049] ​(1) Take the above-mentioned prepared chip with dilute acid solution to clean, remove the surface dirt, organic matter and oxide layer, and expose metal atoms.

[0050] (2) The treated chip is immersed in a cell solution (the cell surface expresses a cysteine-rich polypeptide tag) for 30 minutes.

[0051] (3) Take out the chip and clean the chip with normal saline to remove cells and impurities that do not have adsorption effect with metal points.

[0052] (4) Stand for 10 minutes and dry the excess water on the chip surface.

[0053] Example 4

[0054] The present embodiment provides a single cell quantitative analysis method, which comprises: performing site-specific laser Raman detection on the chip capturing single cells in Example 3, specifically as follows:

[0055] (1) Take the prepared single cell chip and place it on the stage of the Raman laser analyzer.

[0056] (2) Set the parameters of the analyzer, the size of each displacement (0-50 μm).

[0057] (3) Scan and collect data one by one.

[0058] (4) According to the data of the molecular fingerprint library, the data of each single cell can be analyzed to analyze the metabolite content of the cell, such as the content of amino acids, fat content, etc.

[0059] Example 5

[0060] The present embodiment provides a method for capturing single cells, which comprises: capturing single cells by using the chip for capturing single cells in Example 2, wherein the surface of the single cell contains recognition molecules with metal adsorption capacity, and the recognition molecules are cysteine tags. Specifically, it comprises the following steps:

[0061] (1) Take the above-mentioned prepared chip with dilute acid solution to clean, remove the surface dirt, organic matter and oxide layer, and expose metal atoms.

[0062] (2) The treated chip is immersed in a cell solution (the cell surface expresses a cysteine-rich polypeptide tag) for 30 minutes.

[0063] (3) Take out the chip and clean the chip with normal saline to remove cells and impurities that do not have adsorption effect with metal points.

[0064] (4) Stand for 10 minutes and dry the excess water on the chip surface.

[0065] Example 6

[0066] The present embodiment provides a single cell quantitative analysis method, which comprises: carrying out site-specific laser bombardment on the chip capturing single cells in Example 5, after bombardment, the cell material is ionized and enters the mass spectrometer under the action of an electric field, and then the composition of the material is determined by mass spectrometric analysis, specifically as follows:

[0067] (1) Take the chip fixed with cells to the mass spectrometry imaging instrument (MALDI-MSI), this method requires that the substrate material used is a conductive material, such as an indium tin oxide glass sheet.

[0068] (2) Set the parameters of mass spectrometry imaging, including the number of bombardment times, the time and laser energy of each time, the position of each point, voltage, and data acquisition parameters.

[0069] (3) Data acquisition is carried out according to the set program and parameters.

[0070] (4) Use the data in the mass spectrometry library as a reference to analyze the data, which can analyze the types of proteins, lipids and metabolites, and can be quantified.

[0071] In summary, the present application provides a chip for capturing single cells and a method for capturing single cells, which can realize single cell separation and regular arrangement, thereby realizing precise positioning of cells and subsequent quantitative detection and analysis. After capturing the cells, when analyzing the separated single cells, site-specific analysis of the cells can be realized, and the number of cells to be crushed can be set, so as to control the test concentration. The chip for capturing single cells provided by the present application has important application value in single cell omics analysis.

[0072] The applicant declares that the above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by those skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. A method of capturing single cells, characterized in that, The method comprises: (1) fixing a metal for capturing on a substrate, the fixing method comprising: using a photoetching machine to photoetch a substrate coated with photoresist, then performing gold spraying or vacuum evaporation, and washing away the photoresist to obtain metal dots arranged in a dot matrix; or, closely arranging single-layer small balls on a substrate, spraying gold on the surface of the small balls, and then pressing on another glass sheet to obtain metal dots arranged in a dot matrix; (2) using the fixed substrate to capture a single cell, the surface of the single cell containing a recognition molecule having a specific metal adsorption capacity.

2. The method of capturing a single cell according to claim 1, wherein, In step (1), the metal for capturing is selected from any one or a combination of at least two of gold, calcium, manganese, iron, copper, zinc, molybdenum, cobalt, magnesium, mercury, chromium, or cadmium.

3. The method of capturing a single cell according to claim 1 or 2, wherein, In step (1), the substrate is selected from any one or a combination of at least two of a glass sheet, a silicon sheet, a copper sheet, or a steel sheet.

4. The method of capturing single cells according to any one of claims 1-3, wherein, In step (1), the spacing between the metal dots arranged in a dot matrix is 1-50 μm.

5. The method of capturing single cells according to any one of claims 1-4, wherein, In step (1), the diameter of the small balls arranged in a dot matrix is 1-50 μm.

6. The method of capturing single cells according to any one of claims 1-5, wherein, In step (2), the recognition molecule having a metal adsorption capacity is selected from any one or a combination of at least two of a HIS tag, a Flag-tag tag, a SNAP-Tag tag, a glutathione sulphydryl transferase tag, a c-Myc tag, a luciferase tag, a metallothionein tag, a hemoglobin tag, a myoglobin tag, a cytochrome C tag, a blue copper protein tag, or an iron-sulfur protein tag.

7. A chip for capturing single cells, characterized by The chip comprises a substrate and metal dots arranged in a dot matrix on the substrate; The chip for capturing a single cell is prepared by a method comprising the following steps: (A) closely arranging a single layer of small balls with a diameter of 1-50 μm on a substrate; (B) spraying metal particles on the small balls to make the surface of the small balls stick to the metal particles; (C) using another substrate to press on the small balls on the carrier substrate, so that the metal particles sprayed on the top of the small balls are adhered to the other substrate to obtain a metal dot matrix substrate with a distance interval; Preferably, the chip for capturing a single cell is prepared by a method comprising the following steps: (a) using a photoetching machine to photoetch a substrate coated with photoresist; (b) using gold spraying or evaporation to cover the substrate part without photoresist protection with a metal coating; (c) after washing away the photoresist, a metal dot matrix substrate with a distance interval is obtained.

8. A method of single cell quantitative analysis, comprising: The analysis method comprises: The single cell is physically separated from the chip for capturing a single cell according to claim 7, and the single cell is treated in situ to obtain intracellular substances for quantitative analysis.

9. The method of claim 8, wherein the single-cell protein is quantified by measuring the amount of the one or more of the biomarkers in the single-cell protein. The method for treating a single cell in situ comprises using laser bombardment to ionize the cell.

10. Use of the chip for capturing a single cell according to claim 7 in the preparation of a single cell omics analysis product.

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