A method and device for sampling and in-situ detection of extracellular substances

Abstract

The present invention provides a method and device for sampling and in-situ detection of extracellular secretions, including injecting an organic solvent into the microfluidic probe, using the organic solvent to extract the exogenous substances of the tested part of the tested cell to obtain the tested sample and send it to the detection device. The microfluidic probe includes a capillary for receiving and circulating the organic solvent and sampling, the capillary having a U-shaped structure, the top of the U-shaped structure having a sampling port, the top of the U-shaped structure is moved to the tested part of the tested cell, the organic solvent circulating in the capillary extracts the exogenous substances of the tested part of the tested cell through the sampling port, and then obtains the tested sample. The method and device of the present invention can sample and detect extracellular secretions in situ while keeping the cells in their original state, thereby realizing analysis of extracellular secretions at the subcellular level.

Description

Technical Field

[0001] The present invention belongs to the field of cell research technology, and relates to a method and device for sampling and in-situ detection of extracellular secretions, and in particular to a method and device for sampling and in-situ detection of extracellular secretions based on microfluidics technology. Background Art

[0002] As the most basic structural and functional unit of most living organisms, the behavior of cells will directly affect the properties of living organisms, so cells have become an enduring research hotspot. Cells can exchange substances with the outside world through endocytosis and exocytosis, and this behavior of exchanging substances can reflect the state of cells. For example, people can detect the presence of cancer cells through exosomes, which is of great reference value for the early diagnosis of cancer. In recent years, people's research on cells has progressed to the single-cell level, but even for a single cell, its different parts will have certain differences, so research at the subcellular level has gradually attracted the attention of scientific researchers.

[0003] There are many ways to study cells, such as optical imaging, fluorescence analysis, electrophoresis analysis, chromatography and mass spectrometry. Among them, mass spectrometry detection technology is widely used in cell analysis due to its advantages such as fast analysis speed, strong universality and high resolution. At present, cell mass spectrometry analysis has also developed to the single-cell level, which can analyze the components in a single cell. The patch clamp technology has realized the analysis of part of the cell contents. However, once this mass spectrometry analysis is performed, it will inevitably have a serious impact on the cell, and the analysis of cell status is not necessarily achieved only by analyzing its contents. Therefore, it is very important to develop a subcellular level analysis technology for exosomes under normal cell survival conditions. Summary of the invention

[0004] The purpose of the present invention is to address the technical problems existing in the prior art and to provide a method and device for sampling and in-situ detection of extracellular secretions using microfluidic technology. The method and device of the present invention can sample and detect extracellular secretions in situ while keeping the cells in their original state, thereby realizing analysis of extracellular secretions at the subcellular level.

[0005] To achieve the purpose of the present invention, the present invention provides a method for sampling and in-situ detection of extracellular secretions, comprising:

[0006] Cultivating cells to be tested in a cell culture device;

[0007] Moving the microfluidic probe to a desired location of a cell to be tested;

[0008] An organic solvent is injected into the microfluidic probe, and the exogenous substances of the tested part of the tested cell are extracted by the organic solvent to obtain the tested sample, which is then sent to the detection device.

[0009] According to some embodiments of the present invention, the microfluidic probe comprises:

[0010] A capillary tube for receiving and circulating the organic solvent and sampling;

[0011] A tapered end connected to one end of the capillary tube, used to deliver the sample to be tested into a detection device;

[0012] A retaining ring is used to secure the capillary and the tapered end.

[0013] According to a preferred embodiment of the present invention, the capillary tube has a U-shaped structure, and a sampling port is formed at the top of the U-shaped structure.

[0014] According to some embodiments of the present invention, one end of the capillary is connected to an organic solvent source, and the other end is connected to the tapered end.

[0015] In some specific embodiments, the top of the U-shaped structure of the capillary is moved to the test site of the cell to be tested, and an organic solvent is injected into the capillary through an organic solvent source and allowed to flow. Since the organic solvent will not leak to the outside through the sampling port due to surface tension limitations at the sampling port, the organic solvent extracts the exogenous substances of the test site of the cell to be tested through the sampling port opened at the top of the U-shaped structure, thereby obtaining a test sample containing the exogenous substances of the test site of the cell to be tested.

[0016] According to a preferred embodiment of the present invention, the organic solvent includes one or more of hydrocarbon compounds, hydroxyl compounds, nitrogen-containing compounds and oxygen-containing compounds, preferably includes one or more of methanol, ethanol, propanol, butanol, acetonitrile, acetone, methane, ethane, propane, butane, pentane and hexane.

[0017] According to some embodiments of the present invention, one end of the tapered end is connected to the capillary, and the other end is a tip, which is opposite to the sampling port of the detection device. The sample solution to be tested in the capillary forms a spray at the outlet of the tip and enters the sampling port of the detection device.

[0018] In some specific embodiments, the distance between the tip and the injection port of the detection device is 1±0.1 cm.

[0019] It should be noted that the tip being opposite to the injection port of the detection device means that the center line of the tip and the injection port of the detection device are opposite to each other on a straight line.

[0020] According to some embodiments of the present invention, the organic solvent is loaded with voltage (voltage range is 500V-5000V, preferably 2000V), the voltage-loaded organic solvent is injected from one end of the capillary, and the voltage-loaded liquid forms an ionized spray at the tip of the conical end due to the action of the voltage and enters the inlet of the mass spectrometer detector.

[0021] According to a preferred embodiment of the present invention, the detection device is a mass spectrometer detector.

[0022] Another aspect of the present invention provides a device for sampling and in-situ detection of extracellular secretion substances, comprising:

[0023] A cell manipulation platform on which cells to be tested are cultured;

[0024] The microfluidic probe located above the cell manipulation platform is used to sample and obtain a test sample containing exogenous substances from a test site of a test cell, and send the sample to a detection device;

[0025] The detection device connected to the microfluidic probe is used to receive the sample to be detected and detect it.

[0026] According to some embodiments of the present invention, the cell manipulation platform comprises:

[0027] Stage;

[0028] A cell culture device located on the upper surface of the stage, wherein the cell culture device contains a cell culture medium solution for culturing cells to be tested;

[0029] The alignment device disposed below the stage is used for observing and positioning the cells to be tested.

[0030] According to a preferred embodiment of the present invention, the cell culture device is preferably a culture dish, which is arranged on the upper surface of the stage and contains a cell culture medium solution for culturing the cells to be tested.

[0031] In some specific embodiments, the cells to be tested are 10 2 ~10 4 The density of each square centimeter was planted in the culture device.

[0032] According to a preferred embodiment of the present invention, the alignment device is disposed below the stage and comprises a microscope objective lens, which can be used to observe and locate the cells to be tested.

[0033] According to some embodiments of the present invention, the microfluidic probe comprises:

[0034] A capillary tube for receiving and circulating the organic solvent and sampling;

[0035] A tapered end connected to one end of the capillary tube, used to deliver the sample to be tested into a detection device;

[0036] A retaining ring is used to secure the capillary and the tapered end.

[0037] According to a preferred embodiment of the present invention, the capillary tube has a U-shaped structure, and a sampling port is formed at the top of the U-shaped structure.

[0038] According to some embodiments of the present invention, one end of the capillary is connected to an organic solvent source, and the other end is connected to the tapered end.

[0039] According to some embodiments of the present invention, the inner diameter of the capillary is 40-250 microns, preferably 40 microns.

[0040] According to a preferred embodiment of the present invention, the diameter of the sampling port is 5-30 microns, preferably 5 microns.

[0041] According to some embodiments of the present invention, one end of the tapered end is connected to the capillary, and the other end is a tip; the tip is opposite to the sampling port of the detection device, and the sample solution to be tested in the capillary forms a spray at the tip outlet and enters the sampling port of the detection device.

[0042] In some specific embodiments, the distance between the tip and the injection port of the detection device is 1±0.1 cm.

[0043] It should be noted that the tip being opposite to the injection port of the detection device means that the center line of the tip and the injection port of the detection device are opposite to each other on a straight line.

[0044] According to some embodiments of the present invention, the organic solvent is loaded with voltage (voltage range is 500V-5000V, preferably 2000V), the voltage-loaded organic solvent is injected from one end of the capillary, and the voltage-loaded liquid forms an ionized spray at the tip of the conical end due to the action of the voltage and enters the inlet of the mass spectrometer detector.

[0045] In some specific embodiments, the inner diameter of the tip is 10 microns.

[0046] According to some embodiments of the present invention, the microfluidic probe further comprises:

[0047] A supporting device for fixing the capillary tube;

[0048] The supporting device is connected to the moving device of the stage to move the supporting device on the stage, thereby moving the capillary to move the top of the U-shaped structure to the testing site of the cell to be tested.

[0049] According to some embodiments of the present invention, there is no particular limitation on the supporting device, as long as it can fix the capillary tube; preferably, the supporting device is a transparent hard PVC plate, which is bonded to the capillary tube by an adhesive and fixedly connected to the moving device.

[0050] According to a preferred embodiment of the present invention, the detection device is a mass spectrometer detector.

[0051] The working process and working principle of the device of the present invention are as follows:

[0052] The cells to be tested are cultured in a culture dish on a stage, and the stage and the moving device are adjusted to make the U-shaped top of the capillary of the microfluidic probe close to the tested part of the cells to be tested by observation through an alignment device; an organic solvent loaded with voltage is injected from one end of the capillary, the organic solvent flows in the capillary, and the organic solvent extracts the exogenous substances of the tested part of the cells to be tested through a sampling port opened at the top of the U-shaped structure, thereby obtaining a test sample containing the exogenous substances of the tested part of the cells to be tested; the test sample enters the conical end from the other end of the capillary, and due to the action of the voltage, an ionized spray is formed at the tip of the conical end and enters the sampling port of the mass spectrometer detector for detection.

[0053] The advantages and beneficial technical effects of the present invention are as follows:

[0054] (1) The present invention can sample and detect extracellular secretions in situ while keeping the cells in their original state.

[0055] (2) The present invention uses extraction technology to replace the solvent of the cell from the culture medium to an organic solvent, which greatly improves the ionization efficiency and weakens the influence of water-soluble substances (such as salts in the culture medium), thereby greatly improving the sensitivity.

[0056] (3) The present invention can detect in real time the types and levels of secretions from different external parts of a single cell in culture, thereby obtaining metabolic information at the subcellular level when damage is caused to the cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The present invention will be described in more detail below based on embodiments and with reference to the accompanying drawings, wherein:

[0058] Figure 1 This is a schematic structural diagram of a device for sampling and in-situ detection of extracellular secretions according to an embodiment of the present invention;

[0059] Figure 2 A schematic diagram of the structure of a microfluidic probe according to an embodiment of the present invention;

[0060] Figure 3 This is a schematic diagram of the structure of a cell manipulation platform according to an embodiment of the present invention;

[0061] Explanation of the reference numerals: 1. microfluidic probe; 2. cell manipulation platform; 3. first capillary port; 4. capillary; 5. fixing device; 6. fixing ring; 7. tapered end; 8. sample to be tested; 9. injection port; 10. cell culture device; 11. cells to be tested; 12. stage; 13. alignment device; 14. cell culture medium solution; 15. sampling port.

[0062] In the drawings, the same reference numerals are used for the same components and the drawings are not drawn to scale. DETAILED DESCRIPTION

[0063] The present invention will be further described below in conjunction with the accompanying drawings.

[0064] like Figure 1 As shown, the extracellular secretion substance sampling and in-situ detection device of the present invention comprises a microfluidic probe 1, a cell manipulation platform 2 and a detection device.

[0065] The microfluidic probe 1 comprises: a capillary 4, a tapered end 7, a fixing ring 6 and a fixing device 5; wherein the capillary 4 has a U-shaped structure, and a sampling port 15 is provided at the top of the U-shaped structure; the capillary 4 is preferably made of glass, with an inner diameter of 40-250 microns, and the diameter of the sampling port 15 is preferably 5-30 microns; the first port 3 of the capillary 4 is connected to an organic solvent source, and the second port is connected to the tapered end 7 through the fixing ring 6; the end of the tapered end 7 away from the capillary is a tip, with an inner diameter of 10 microns, and the tip is opposite to the sampling port of the detection device; the capillary 4 is fixed by the fixing device 5, and the fixing device 5 is connected to the stage 12 through the moving device, so as to move the supporting device on the stage, thereby moving the capillary 4.

[0066] The cell manipulation platform 2 includes a stage 12, a cell culture device 10 and an alignment device 13; wherein the cell culture device 10 is located on the upper surface of the stage 12, which contains a cell culture medium solution 14 and cultured cells 11 to be tested; the alignment device 13 is arranged below the stage 12, and is preferably a microscope objective lens, for observing and positioning the cells to be tested.

[0067] The detection device is preferably a mass spectrometer detector, which includes an injection port 9 connected to / opposite to the tip of the tapered end 7 .

[0068] Example 1

[0069] In this embodiment, a glass capillary tube with an inner diameter of 100 μm is selected, and a sampling port is opened at the top of the U-shaped structure, and the diameter of the sampling port is 5 μm;

[0070] In the culture dish 10 on the stage 12, 2 ~10 4Plant and culture the cells 11 to be tested at a density of 100 cells / cm2; observe through the alignment device 13, adjust the stage 12 and the moving device so that the sampling port 15 at the U-shaped top of the capillary 4 is aligned with the tested part of the cells 11 to be tested;

[0071] n-hexane with a high voltage of 2000V is injected from the first port 3 of the capillary to flow through the capillary 4, contacting with the culture medium solution 14 through the sampling port 15 and extracting the exogenous substances of the test site of the test cell to be tested, thereby obtaining a test sample 8 containing the exogenous substances of the test site of the test cell to be tested; the test sample 8 enters the conical end 7 from the other end of the capillary, and forms an ionized spray due to the high voltage at the conical end 7, which enters the sampling port 9 of the mass spectrometer detector, and then is detected to generate a mass spectrometer signal.

[0072] Although the present invention has been described with reference to the preferred embodiments, the structure, size, location and shape of each component may be changed without departing from the scope of the present invention. On the basis of the technical solution of the present invention, any improvements and equivalent transformations of individual components based on the principles of the present invention should not be excluded from the scope of protection of the present invention.

[0073] In the description of the present invention, it should be understood that the terms "upper", "lower", "front", "rear", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance. The present invention is described in detail below in conjunction with the drawings and embodiments.

Claims

1. A method for sampling and in-situ detection of extracellular secretions, comprising: Cultivating cells to be tested in a cell culture device; Moving the microfluidic probe to a desired location of a cell to be tested; Injecting an organic solvent into the microfluidic probe, extracting the exogenous substances of the tested part of the tested cell with the organic solvent to obtain a test sample, and sending the sample to a detection device; The microfluidic probe comprises: a capillary tube for receiving and circulating the organic solvent; A tapered end connected to one end of the capillary tube, used to deliver the sample to be tested into a detection device; a fixing ring for fixing the capillary and the tapered end; The capillary tube has a U-shaped structure, and a sampling port is opened at the top of the U-shaped structure; The top of the U-shaped structure of the capillary is moved to the test site of the test cell, and the organic solvent flowing in the capillary extracts the exogenous substances of the test site of the test cell through the sampling port opened at the top of the U-shaped structure, thereby obtaining a test sample containing the exogenous substances of the test site of the test cell; The organic solvent carries voltage, and the organic solvent carrying voltage is injected from one end of the capillary. The liquid carrying voltage forms ionized spray at the tip of the conical end due to the action of voltage and enters the detection device.

2. The method according to claim 1, characterized in that: The organic solvent includes one or more of hydrocarbon compounds, nitrogen-containing compounds and oxygen-containing compounds.

3. The method according to claim 2, characterized in that The organic solvent includes one or more of methanol, ethanol, propanol, butanol, acetonitrile, acetone, methane, ethane, propane, butane, pentane and hexane.

4. A device for sampling and in-situ detection of extracellular secretion substances, using the in-situ detection method according to any one of claims 1 to 3, characterized in that: include: A cell manipulation platform on which cells to be tested are cultured; The microfluidic probe located above the cell manipulation platform is used to sample and obtain a test sample containing exogenous substances from a test site of a test cell, and send the sample to a detection device; The detection device connected to the microfluidic probe is used to receive the sample to be detected and detect it.

5. The device according to claim 4, characterized in that The cell manipulation platform comprises: Stage; A cell culture device located on the upper surface of the stage, wherein the cell culture device contains a cell culture medium solution for culturing cells to be tested; The alignment device disposed below the stage is used for observing and positioning the cells to be tested.

6. The device according to claim 5, characterized in that The microfluidic probe comprises: a capillary tube for receiving and circulating the organic solvent; A tapered end connected to one end of the capillary tube, used to deliver the sample to be tested into a detection device; A retaining ring is used to secure the capillary and the tapered end.

7. The device according to claim 6, characterized in that The capillary has a U-shaped structure, and a sampling port is opened at the top of the U-shaped structure; and / or the inner diameter of the capillary is 40-250 microns; and / or the diameter of the sampling port is 5-30 microns.

8. The device according to claim 7, characterized in that The inner diameter of the capillary is 40 microns.

9. The device according to claim 7, characterized in that The diameter of the sampling port is 5 microns.

10. The device according to claim 6, characterized in that One end of the tapered end is connected to the capillary, and the other end is a tip; The tip is opposite to the sampling port of the detection device, and the sample solution to be detected in the capillary forms a spray at the outlet of the tip and enters the sampling port of the detection device.

11. The device according to claim 6, characterized in that The microfluidic probe further comprises: A supporting device for fixing the capillary tube; The supporting device is connected to the moving device of the stage to move the supporting device on the stage, thereby moving the capillary to move the top of the U-shaped structure to the testing site of the cell to be tested.

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