Micro-fluidic chip for normal Raman microscope in-situ unlabeled single cell imaging and application of micro-fluidic chip

Abstract

The invention belongs to the technical field of biology, and discloses a micro-fluidic chip for in-situ single-cell non-label imaging of an upright Raman microscope. Comprising a quartz substrate subjected to surface treatment, a detachable cell chamber, a glass sheet provided with a through hole and a gravity flow channel control system, the cell chamber is used for cell culture before in-situ cell imaging, after cell culture is completed, the cell chamber is taken off, the glass sheet is bonded with the quartz substrate, the glass sheet is used for packaging a chip, and the gravity flow channel control system is used for controlling the gravity flow channel. The gravity flow channel control system is connected with the through hole and is used for guiding fluid to flow in the chip; the micro-fluidic chip can effectively package living cells and is compatible with a forward Raman microscope, so that in-situ unlabeled Raman imaging of single cells under the microscope is realized, experimental operation is simplified, more efficient cell treatment and real-time monitoring capabilities are provided, imaging stability and accuracy are improved, and the micro-fluidic chip is suitable for large-scale popularization and application. The method is of great significance to application of drug screening, biochemical research and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically designs a microfluidic chip for in-situ single-cell non-labeling imaging with an upright Raman microscope and application thereof. Background Art

[0002] As a spectral analysis and imaging method based on the Raman scattering effect, Raman imaging technology has been widely used in the biomedical field in recent years. Unlike traditional fluorescence microscopy technology that requires sample labeling, the biggest advantage of Raman imaging technology is its non-invasive and label-free characteristics, which enables it to perform high-resolution imaging and analysis of the chemical components inside cells without destroying the sample or affecting the normal physiological activities of the cells. Raman imaging can achieve qualitative and quantitative analysis of the intrinsic components of the sample by detecting the scattering spectrum of the sample molecules under the excitation of light of a specific wavelength. Specifically, Raman imaging can provide spatial distribution information of molecules such as lipids, proteins, and nucleic acids in cells, thus showing unique advantages in cell metabolism, pathological research, and drug screening.

[0003] However, despite the many advantages of Raman imaging technology, it still faces some challenges in practical applications. Traditional biological microscopes usually use an inverted microscope structure, which is more suitable for processing and observing biological samples such as cells. However, in order to take into account more usage scenarios (such as materials, environment, etc.), Raman microscopes in the prior art often use an upright imaging optical path. Although the design of the upright optical path optimizes the imaging efficiency and optical path structure, there are still compatibility issues in the application of living cell monitoring imaging.

[0004] However, there is currently a lack of microfluidic chips on the market that are compatible with upright Raman microscopes, which limits the application potential of Raman imaging technology in cell research and drug screening. Summary of the invention

[0005] In view of the technical problems existing in the above-mentioned prior art, the present invention provides a microfluidic chip for in situ single-cell label-free imaging using an upright Raman microscope.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] In the first aspect, the present invention provides a microfluidic chip for in situ single-cell non-labeling imaging under an upright Raman microscope, comprising a surface-treated quartz substrate, a cell chamber, a glass sheet and a gravity flow channel control system, wherein the glass sheet is provided with a through hole, and the cell chamber is detachably mounted on the surface of the quartz substrate for cell culture before in situ single-cell imaging. After the cell culture is completed, the cell chamber is removed, and the glass sheet is bonded to the quartz substrate. The glass sheet is used to encapsulate the chip, and the gravity flow channel control system is connected to the through hole of the glass sheet for guiding the flow of fluid inside the chip. The gravity flow channel control system comprises a syringe, a microfluidic switch, a hose, and an L-shaped steel needle. The bottom end of the syringe is sequentially connected to the hose and the L-shaped steel needle, and the microfluidic switch is used to control the outflow of fluid in the syringe.

[0008] Furthermore, the quartz substrate is hydrophilized and modified with cell adhesion polymers. The quartz substrate is preferably made of high-purity, non-fluorescent, ultraviolet-near infrared fully transparent quartz.

[0009] The quartz substrate is treated with hydrophilization and modified with cell adhesion polymers in the following steps: treating the quartz substrate with concentrated sulfuric acid-hydrogen peroxide washing solution to remove dirt on the surface of the quartz substrate and hydroxylate it in large quantities to obtain a hydrophilic surface, wherein the volume ratio of concentrated sulfuric acid to hydrogen peroxide is 3:1, and after cleaning with deionized water, the quartz substrate with the hydrophilic surface is immersed in a cell adhesion polymer solution for incubation to functionalize its surface, and the incubation concentration is 0.1-1wt.%, the incubation temperature is 25-37°C, and the incubation time is 0.1-2h. After the incubation is completed, it is rinsed with a phosphate buffer solution and dried naturally.

[0010] Furthermore, the cell adhesion polymer solution is selected from one or more of collagen, gelatin, poly-L-lysine, and hyaluronic acid.

[0011] The preferred material of the cell chamber is one of silica gel and polydimethylsiloxane (PDMS), which has adhesion to the quartz substrate. The cell chamber is attached to the surface of the quartz substrate and can be used for cell culture. The cells to be cultured can be selected from human breast cancer MDA-MB-231 cells, rat breast cancer 4T1 cells, and human cervical cancer Hela cells. The cells are cultured in a standard culture bottle, and the quartz substrate is inoculated when the growth density reaches 80%. The specific method is to digest the cells with trypsin, add a certain proportion of culture medium and then centrifuge, redisperse the obtained cell precipitate in fresh culture medium, and directly add it to the quartz substrate loaded with a detachable chamber, and the inoculation density is 5000-50000 cells / chamber.

[0012] Furthermore, the through hole diameter of the glass sheet is 100-1000 μm.

[0013] Furthermore, a PDMS block is arranged on the through hole of the glass sheet, a through hole is arranged in the middle of the PDMS block, and the through hole of the glass sheet is connected with the through hole of the PDMS block.

[0014] Furthermore, the glass sheet is bonded to the quartz substrate by double-sided adhesive tape, and the distance between the glass sheet and the quartz substrate is 50-500 μm. The portion without double-sided adhesive tape between the glass sheet and the quartz substrate forms a microfluidic fluid channel, and the width and height through which the microfluidic fluid flows can be controlled by adjusting the size and thickness of the double-sided adhesive tape to control various parameters of the channel.

[0015] Furthermore, the microfluid flow rate of the gravity flow channel control system is 0.1-1 mL / min.

[0016] The quartz substrate is hydrophilized and modified with cell adhesion polymers to promote cell adhesion and adhesion. The quartz substrate provides a stable and suitable cell culture environment to ensure the stability and activity of cells under the microscope; the cell chamber is designed as a detachable structure, which is convenient for removing and reinstalling the cell chamber without destroying the cell culture environment, thereby achieving multiple imaging and analysis. The glass slide is used to package the chip, and the glass slide and the quartz substrate are bonded by waterproof double-sided tape to ensure the sealing of the chip, while allowing the connection of fluid channels and fluid exchange; the gravity flow channel control system can achieve precise control of cell culture fluid and reagents by utilizing the gravity flow channel system to ensure experimental repeatability and reliability.

[0017] In a second aspect, the present invention provides an application of a microfluidic chip for in situ label-free imaging of single cells using an upright Raman microscope, wherein the application is that the microfluidic chip performs label-free in situ imaging of multiple biological components in a single living cell, wherein the multiple biological components in the single living cell include lipids, proteins, and water molecules.

[0018] The present invention has the following beneficial effects: the microfluidic chip and gravity flow channel control system of the present invention ensure the efficient packaging and fluid control of the microfluidic chip through precise design and processing, and provide a stable and reliable platform for in situ cell imaging under an upright Raman microscope. At the same time, various parameters of the microfluidic fluid are controllable and easy to operate. This design not only improves the flexibility and accuracy of the experiment, but also provides a new technical means for cell biology research and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : A schematic diagram of the structure of a microfluidic chip for in situ single-cell label-free imaging using an upright Raman microscope in the present invention;

[0020] Figure 2 : A schematic diagram of the structure of the quartz substrate carrying the cell chamber in the present invention;

[0021] Figure 3 : A schematic diagram of the structure of the glass sheet in the present invention;

[0022] Figure 4 : A schematic diagram of the structure of a glass sheet bonded to a PDMS block in the present invention;

[0023] Figure 5 : A physical picture of the microfluidic chip after packaging in the present invention;

[0024] Figure 6 : A physical diagram of the gravity flow channel control system of the present invention;

[0025] Figure 7 : Bright field photograph of MDA-MB-231 cells encapsulated in the chip;

[0026] Figure 8 : Raman scattering spectrum of a single cell;

[0027] Fig. 9 :In situ Raman imaging of MDA-MB-231 cells, imaging band 2800-3000cm -1 .

[0028] In the figure: 1. quartz substrate; 2. cell chamber; 3. glass slide; 4. gravity flow channel control system; 5. PDMS block; 6. microscope objective lens; 7. waste liquid collection device. DETAILED DESCRIPTION

[0029] The present invention is further described and defined by the following examples but is not limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified. The present invention is further described below in conjunction with the accompanying drawings.

[0030] Example 1: Construction of microfluidic chip

[0031] Hydrophilization of quartz substrate: Schematic diagram of the structure of the microfluidic chip used for in situ single-cell label-free imaging under upright Raman microscopy. Figure 1 As shown, a high-purity quartz sheet with a size of 50×25×0.5 mm is used as the substrate, and its material is JGS1, with a light transmittance range of 185-2500nm, and its surface is polished to ensure flatness. The quartz substrate 1 is immersed in a newly configured piranha washing solution (concentrated sulfuric acid: hydrogen peroxide = 3:1, providing a basis for modifying the cell adhesion polymer on the surface of the quartz substrate), and is taken out after 10 minutes to remove the dirt on the surface of the quartz substrate and hydroxylate a large amount of it to obtain a hydrophilic surface, and then washed three times with deionized water and dried with nitrogen for use.

[0032] Modification of cell adhesion polymer on its surface: Use poly-L-lysine as cell adhesion polymer. Prepare poly-L-lysine into 0.1wt.% aqueous solution, immerse the hydrophilized quartz substrate 1 in the poly-L-lysine aqueous solution for incubation at 37°C for 2h, rinse with phosphate buffer after incubation, and use it after natural drying.

[0033] Installation of detachable cell chamber: The cell chamber 2 is made of silicone, with a size of 10×10×5 mm and a capacity of 200 μL. Clean the cell chamber 2 with disinfectant alcohol and dry it, then stick it to the center of the quartz substrate 1 and press it gently with your fingers for 1 minute (such as Figure 2 As a test of the degree of adhesion, the cell chamber 2 can be picked up with tweezers. If the quartz substrate 1 can be lifted up at the same time, it means that the adhesion is firm.

[0034] Perforated glass sheet and PDMS block production: The glass sheet 3 is made of high-transmittance optical glass, with specific dimensions of 40×22×0.15 mm. The through hole on the glass sheet 3 is a circular hole with an aperture of 1 mm, and the center of the circular hole is 2 mm away from the short side to ensure that the flow channel is unobstructed and that the objective lens 6 of the subsequent microscope has enough space to approach the quartz substrate when imaging. Figure 3 As shown. In order to make the PDMS block 5, the silicone base material and the curing agent are mixed in a ratio of 10:1, stirred thoroughly, and then the mixed PDMS is poured into a pre-prepared square mold (2×2mm), and the mold is placed in a vacuum degassing machine for degassing, and then cured in an oven at 80°C for 2 hours. After curing, the PDMS is removed from the mold, and a hole with a diameter of 0.5mm is punched on the PDMS block using precision laser drilling technology. The gravity flow channel control system 4 is connected to the through hole on the glass sheet 3 through the PDMS block 5 to ensure smooth fluid flow. The through hole on the other side of the glass sheet 3 is connected to the waste liquid collection device 7. Use a plasma cleaner to clean the surface of the PDMS block 5 and the punched glass sheet 3, and then align the position of the hole in the PDMS block 5 with the through hole on the glass sheet 3, and press it tightly. You can press with your fingers for 2 minutes to ensure a tight fit (such as Figure 4 shown).

[0035] MDA-MB-231 cell culture and inoculation: Human breast cancer MDA-MB-231 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) in a cell culture incubator at 37°C and 5% CO2. When the cell growth density reached 80%, the cells were digested with trypsin, centrifuged after adding culture medium at a ratio of 1:1, and the resulting cell pellet was redispersed in fresh culture medium. The treated cell suspension was directly added to the quartz substrate 1 loaded with cell chamber 2, with an inoculation density of 5000 cells / chamber. The quartz substrate 1 was placed in a cell culture incubator at 37°C and 5% CO2 for 24 hours to ensure cell adhesion and proliferation.

[0036] Sealing and flow channel: Take out the quartz substrate 1 that has been inoculated and cultured with cells, and carefully remove the cell chamber 2. Align the glass sheet 3 with the quartz substrate 1, and glue the two together with waterproof double-sided tape to ensure the sealing of the chip and the smooth connection of the fluid channel. See the actual photo for details. Figure 5 . An L-shaped steel needle (outer diameter 0.6mm, inner diameter 0.5mm) with a microfluidic hose (inner diameter 0.5mm) at one end is inserted into the hole of the PDMS block with holes, and the other end of the hose is connected to the syringe. By adjusting the height of the syringe, gravity is used to achieve precise control of the fluid. See the actual photo for details. Figure 6 When the syringe is 30 cm above the chip, the fluid flow rate is 0.3 mL / min, and the extracellular fluid flows through the flow channel to maintain the stability of the osmotic pressure inside and outside the cells to ensure the vitality of the cells.

[0037] Example 2: In situ Raman imaging of living cells

[0038] This example demonstrates the process of in situ Raman imaging of living cells using the constructed microfluidic chip. By combining an upright Raman microscope with microfluidic technology, high-resolution imaging and analysis of living cells were successfully achieved.

[0039] After completing the encapsulation of living cells in Example 1, the assembled microfluidic chip was placed on the stage of an upright Raman microscope, and the microscope parameters were adjusted to obtain the best imaging effect. Figure 7 As shown in Figure 1, Raman imaging of living cells in the chip is then started. The laser beam (638 nm) of the microscope excites the molecules in the cells, and the Raman scattering spectrum ( Figure 8 ), can detect intracellular water (3300cm -1 ), lipids (2880cm -1 )、protein(1649cm -1 )、nucleic acid (788、1044cm -1 ) and other components. Taking lipids as an example, for 2800-3000cm -1By imaging the Raman signals inside the cell, the spatial distribution information of lipid components such as lipid droplets and biomembranes in single cells can be obtained. Fig. 9 ). During the entire experiment, the gravity flow system 4 accurately controls the extracellular fluid, ensuring the vitality and normal physiological state of the cells in the microfluidic chip.

[0040] The above description is only a preferred embodiment of the present invention, but is not intended to limit the present invention. Those skilled in the art may make possible changes and modifications to the present invention using the technical contents disclosed above without departing from the scope of the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. A microfluidic chip for in situ single-cell label-free imaging using an upright Raman microscope, characterized in that: The invention comprises a quartz substrate (1) after surface treatment, a cell chamber (2), a glass sheet (3) and a gravity flow channel control system (4), wherein the glass sheet (3) is provided with a through hole, and the cell chamber (4) can be detachably mounted on the surface of the quartz substrate (1) for cell culture before in-situ single-cell imaging. After the cell culture is completed, the cell chamber (2) is removed, and the glass sheet (3) is bonded to the quartz substrate (1). The glass sheet (3) is used to package a chip, and the gravity flow channel control system (4) is connected to the through hole of the glass sheet (3) for guiding the flow of fluid inside the chip. The gravity flow channel control system (4) comprises a syringe, a microfluidic switch, a hose, and an L-shaped steel needle. The bottom end of the syringe is connected to the hose and the L-shaped steel needle in sequence, and the microfluidic switch is used to control the outflow of fluid in the syringe.

2. The microfluidic chip for in situ single-cell label-free imaging using an upright Raman microscope according to claim 1, characterized in that: The quartz substrate (1) is hydrophilized and modified with cell adhesion polymers.

3. The microfluidic chip for in situ single-cell label-free imaging using an upright Raman microscope according to claim 2, characterized in that: The quartz substrate (1) is subjected to a hydrophilic treatment and modified with a cell adhesion polymer, and the steps are as follows: treating the quartz substrate (1) with a concentrated sulfuric acid-hydrogen peroxide washing solution to remove dirt on the surface of the quartz substrate (1) and simultaneously perform a large amount of hydroxylation to obtain a hydrophilic surface, wherein the volume ratio of concentrated sulfuric acid to hydrogen peroxide is 3:1; after cleaning with deionized water, the quartz substrate (1) with a hydrophilic surface is immersed in a cell adhesion polymer solution for incubation to functionalize the surface; the incubation concentration is 0.1-1wt.%, the incubation temperature is 25-37°C, and the incubation time is 0.1-2h; after the incubation is completed, the substrate is rinsed with a phosphate buffer solution and dried naturally.

4. The microfluidic chip for in situ single-cell label-free imaging using an upright Raman microscope according to claim 3, characterized in that: The cell adhesion polymer solution is selected from one or more of collagen, gelatin, poly-L-lysine, and hyaluronic acid.

5. The microfluidic chip for in situ single-cell label-free imaging using an upright Raman microscope according to claim 1, characterized in that: The cell chamber (2) is made of silica gel or polydimethylsiloxane, which is sticky to the quartz substrate (1). The cell chamber (2) can be used for cell culture by attaching it to the surface of the quartz substrate (1).

6. The microfluidic chip for in situ single-cell label-free imaging using an upright Raman microscope according to claim 1, characterized in that: The through hole diameter of the glass sheet (2) is 100-1000 μm.

7. The microfluidic chip for in situ single-cell label-free imaging using an upright Raman microscope according to claim 1, characterized in that: A PDMS block (5) is arranged on the through hole of the glass sheet (2), a through hole is arranged in the middle of the PDMS block (5), and the through hole of the glass sheet (2) is connected with the through hole of the PDMS block (5).

8. The microfluidic chip for in situ single-cell label-free imaging using an upright Raman microscope according to claim 1, characterized in that: The glass sheet (2) is bonded to the quartz substrate (1) by means of a double-sided adhesive tape, and the distance between the glass sheet (2) and the quartz substrate (1) is 50-500 μm.

9. The microfluidic chip for in situ single-cell label-free imaging using an upright Raman microscope according to claim 1, characterized in that: The microfluid flow rate of the gravity flow channel control system (4) is 0.1-1 mL / min.

10. An application of a microfluidic chip for in situ single-cell label-free imaging using an upright Raman microscope, characterized in that: The application is to use the microfluidic chip according to any one of claims 1 to 9 to perform label-free in situ imaging of multiple biological components in a single living cell, wherein the multiple biological components in the single living cell include lipids, proteins, and water molecules.

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