CN107389627B - Single-drop single-cell culture and cell metabolite real-time detection device - Google Patents
Single-drop single-cell culture and cell metabolite real-time detection device Download PDFInfo
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- CN107389627B CN107389627B CN201710383315.2A CN201710383315A CN107389627B CN 107389627 B CN107389627 B CN 107389627B CN 201710383315 A CN201710383315 A CN 201710383315A CN 107389627 B CN107389627 B CN 107389627B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
Abstract
The invention provides a single-drop single cell culture and cell metabolite real-time detection device, which comprises: a capillary electroosmotic flow assembly to form a single droplet containing a single cell at a capillary tip; observation components arranged at two opposite sides of the capillary tip and used for observing the state of the cells in the liquid drop; and the detection components are arranged on the other two sides of the capillary tip and are used for detecting cell metabolites in the liquid drop in real time. The device can realize the simultaneous on-line continuous monitoring of the cell metabolism on the level of fluorescence imaging and molecules, and can better understand the whole process of the cell growth and metabolism.
Description
Technical Field
The invention relates to the technical field of cell research, in particular to a single-drop single-cell culture and cell metabolite real-time detection device.
Background
In recent years, the cost of life, food, clothing, medical care, and the like has been increasing due to the increase in the world population. The resource limitation makes us to save cost in all aspects, thereby creating a new cell research platform, namely microfluidic technology. Capillary electrophoresis, as a microfluidic technology, has the advantages of high throughput, low reagent consumption, easy integration, easy operation and the like, is widely concerned, and has wide application prospect compared with the traditional cell research platform. At present, the droplet analysis method is a very popular single cell analysis method. This method has unique advantages in analyzing the lysate in single cells and is of great significance in single cell analysis because the liquid drop can be used as a separate chemical reaction vessel in the analysis system. Capillary electrophoresis is an analytical means for separating trace substances by electrophoresis under microfluid, and a single-cell sample in a capillary can be accurately and controllably separated by utilizing an electroosmotic flow phenomenon in the analytical means, so that the analytical means becomes an important tool means in single-cell analysis. In the research field of single cell analysis, how to establish an analysis device and method which can be used for the metabolic analysis of cells on line, quickly, efficiently and simply becomes very important and becomes a great challenge at present.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides a single-droplet single-cell culture and cell metabolite real-time detection device based on capillary electroosmotic flow driving.
In order to achieve the purpose of the invention, the invention provides a single-drop single-cell culture and cell metabolite real-time detection device, which comprises:
a capillary electroosmotic flow module forming a single droplet containing a single cell at a capillary tip;
observation components for observing the state of the cells in the liquid drop are arranged on two opposite sides of the capillary tip;
and the detection assembly for detecting cell metabolites in the liquid drop in real time is arranged on the other two sides of the capillary tip.
According to some embodiments of the invention, the capillary electroosmotic flow assembly comprises:
a sample cell containing a cell sample;
the inlet of the capillary is communicated with the sample cell, and the outlet end of the capillary is a capillary tip;
an electrode located above the sample cell;
and a power source connected to the electrodes and supplying power thereto.
In some embodiments, the outlet end of the capillary is polished and silanized to obtain a smooth surface with hydrophobic properties, thereby stabilizing the generation of droplets at the tip of the capillary.
According to a preferred embodiment of the invention, the electrode is a high voltage dc electrode connectable to a high voltage dc power supply, and the size of the droplets formed at the tip of the capillary can be controlled by adjusting the range of voltage output.
According to a preferred embodiment of the invention, the capillary is filled with a solution, and the solution in the capillary is driven by the electrode to form a single droplet at the tip of the capillary.
In some specific embodiments, the capillary has an inner diameter of 50-200 μm. In this range, different kinds of cells can smoothly flow in the capillary, and the detection sensitivity can be improved to some extent.
According to some embodiments of the invention, the cells within the sample cell enter the microdroplet by electroosmotic flow, forming a single droplet containing a single cell.
According to a preferred embodiment of the present invention, the viewing assembly is located around the capillary tip, and comprises:
a laser emitting device located at one side of the capillary tip;
and the observation device is positioned on the opposite side of the laser emission device.
According to some embodiments of the invention, the laser emitting device comprises a point light source laser and an optical fiber connected in sequence.
According to a preferred embodiment of the present invention, the observation device comprises an optical filter, a camera and a computer connected in sequence; the optical filter is positioned on the opposite side of the optical fiber and is positioned on the same straight line with the optical fiber.
The point light source laser generates laser which is emitted to the periphery of the single liquid drop through the optical fiber, the single liquid drop containing single cells is irradiated, redundant optical fibers are filtered through the optical filter, fluorescence imaging of cells and culture media is recorded through the camera and transmitted to the computer, and therefore real-time online observation of the cells wrapped in the liquid drop is achieved.
According to some preferred embodiments of the present invention, the detection assembly is located at the other two ends around the capillary tip, and includes:
an ionizing assembly located to one side of the capillary tip;
a detector located on an opposite side of the ionizing assembly.
According to the real-time detection device of the invention, the other two ends are the other two ends opposite to the observation assembly.
According to a preferred embodiment of the present invention, the ionizing assembly comprises:
the capillary tube is positioned at one side of the tip of the capillary tube, and the air flow controller and the air source are sequentially connected with the needle tube.
In some embodiments, the needle tube is made of plastic, and the diameter of the gas outlet is 50-200 μm.
According to a preferred embodiment of the present invention, the detector includes, but is not limited to, a quadrupole-time-of-flight tandem mass spectrometer, an ion cyclotron resonance mass spectrometer, and an orbital ion trap mass spectrometer.
According to some embodiments of the present invention, the gas flows out from the gas source, and is blown out from the needle tube after passing through the gas flow controller, so as to obtain a constant gas flow, and the volatile substance on the surface of the single droplet is blown to the vicinity of the detector on the opposite side of the needle tube, so that the cell metabolite is detected on line through the detector.
According to a preferred embodiment of the present invention, the gas includes, but is not limited to, high purity nitrogen, oxygen, and synthetic air.
According to a preferred embodiment of the present invention, the flow rate of the gas flow can be adjusted by adjusting the gas flow controller and changing the gas type, so as to select the appropriate gas and ionization state for different cell samples, thereby achieving a more optimal ionization effect.
According to some preferred embodiments of the present invention, after the cells are cultured for a short time at the tip and the signal is detected, the voltage of the dc power supply can be adjusted to recover the droplets and the cells in the droplets, and the recovered cells can be added into the culture dish and then the culture medium can be added to continue culturing, so as to achieve the purpose of circulating culture detection and achieve zero consumption for the scarce sample.
According to the preferred embodiment of the invention, the cell drug is added into the liquid drop through the sample cell, so that the metabolism condition of the cell in drug environment stimulation can be researched, and the whole process of the cell on drug metabolism under the microfluidic system can be comprehensively understood.
The working process and the working principle of the device are as follows:
high-voltage direct current is transmitted to the electrode through a power supply, the electrode is electrified to drive liquid in the capillary, and a single liquid drop is formed at the tip of the capillary; the cells in the sample pool enter the single liquid drop under the action of electroosmotic flow to form a culture state that the single liquid drop wraps the single cells. The point light source laser generates laser which is emitted to the periphery of the single liquid drop through the optical fiber to irradiate the single liquid drop containing the single cell, redundant optical fibers are filtered through the optical filter positioned at the opposite side of the single liquid drop, and the fluorescence imaging of the cell and the culture medium is recorded through the camera and transmitted to the computer, so that the real-time online observation of the cell wrapped in the liquid drop is realized. Meanwhile, gas flows out of the gas cylinder, is blown out of the needle-shaped pipe through the gas flow controller to form constant-speed gas flow, volatile substances on the surface of the single liquid drop are blown to the position close to a detector on the opposite side of the needle-shaped pipe, and the cell metabolites are detected on line through the detector.
The invention has the advantages and beneficial technical effects as follows:
(1) the real-time detection device can directly carry out real-time online detection on the cell culture solution.
(2) According to the real-time detection device, the solution in the sample to be detected cannot enter the detector, the desalting step is omitted, and the detection method is simplified.
(3) The real-time detection device can analyze trace cell metabolites under the condition of consuming a very small amount of biological cell samples by connecting the microfluidic capillary electroosmosis system with the high-resolution mass spectrometer in series, and has the characteristics of high sensitivity and low detection limit.
(4) The real-time detection device has small limitation on the size and the type of the detected cells, so that the device can be used for researching the metabolic process in various cell culture states and has wide applicability.
Drawings
The invention will be described in more detail hereinafter on the basis of embodiments and with reference to the accompanying drawings. Wherein:
FIG. 1 is a schematic structural diagram of a single-drop single-cell culture and real-time cell metabolite detection device according to an embodiment of the present invention;
description of reference numerals: 1. a power source; 2. an electrode; 3. a sample cell; 4. a capillary tube; 5. a capillary tip; 6. a single droplet; 7. a laser; 8. an optical fiber; 9. an optical filter; 10. a camera; 11. a computer; 12. a gas cylinder; 13. an airflow controller; 14. a needle-like tube; 15. a detector.
In the drawings, like parts are provided with like reference numerals. The drawings are not to scale.
Detailed Description
The invention will be further explained with reference to the drawings.
As shown in FIG. 1, the single-drop single-cell culture and cell metabolite real-time detection device of the present invention comprises: a capillary electroosmotic flow module, a viewing module, and a detection module.
Capillary electroosmotic flow assembly comprising: a power supply 1, an electrode 2, a sample cell 3 and a capillary tube 4; the electrode 2 is a high-voltage direct-current electrode, a power supply 1 bit high-voltage direct-current electrode connected with the electrode, and the output voltage is 0-10 KV; the sample cell 3 and the electrode 2 are positioned at the inlet of the capillary tube 4, the outlet end of the capillary tube 4 is subjected to silanization treatment after being polished to obtain a conical surface with a smooth surface and hydrophobic property, a capillary tip 5 is formed, high-voltage direct-current voltage is output through the power supply 1, the solution in the capillary tube 4 and the cells in the sample cell 3 are treated through the electrode, and a single liquid drop 6 wrapping a single cell can be formed at the capillary tip 5.
The observation assembly includes: a laser 7, an optical fiber 8, an optical filter 9, a camera 10 and a computer 11; a laser 7-site light source laser which can generate laser with a certain wavelength; the laser 7 is connected with the optical fiber 8, the optical fiber 8 is positioned on one side around the capillary tip 5, the optical filter 9 is positioned on one side around the capillary tip 5 opposite to the optical fiber 8, the optical filter is sequentially connected with the camera 10 and the computer 11 through pipelines, and the camera 10 is a high-magnification camera and can record fluorescence imaging of cells and culture media.
The detection assembly comprises an ionization assembly and a detector 15, wherein the ionization assembly comprises a gas steel cylinder 12, a gas flow controller 13 and a needle-shaped tube 14; the needle-shaped tube 14 is positioned at one side around the tip 5 of the capillary tube and is sequentially communicated with the gas flow controller 13 and the gas steel cylinder 12 through pipelines, gas (nitrogen, oxygen, synthetic air and the like) is filled in the gas steel cylinder 12, the gas flow controller 13 can adjust the gas flow and further adjust the gas flow, and the needle-shaped tube 14 is a needle-shaped plastic tube; the detector 15 is a high resolution mass spectrometer detector.
Example 1
In the embodiment, a capillary tube with the inner diameter of 100 mu m is selected, and the tip of the capillary tube is polished and subjected to silanization hydrophobic modification to form an inverted cone capillary tip 5; the capillary 4 contains PBS buffer solution, Caco-2 cell suspension containing culture medium is added into the sample cell 3, 2000V high-voltage direct current is transmitted to the electrode 2 through the power supply 1, the solution in the capillary 4 is driven by high voltage electricity, and a single liquid drop is formed at the tip of the capillary; the cells in the sample pool 3 slowly enter the single liquid drop under the action of electroosmotic flow to obtain a single liquid drop 6 wrapping the single cell, and a culture state that the single cell is wrapped by the liquid drop is formed.
Laser generated by a point light source laser 7 is emitted to the periphery of the single liquid drop 6 through an optical fiber 8, the single liquid drop 6 containing single cells is irradiated, redundant optical fibers are filtered through an optical filter 9 positioned on the opposite side of the single liquid drop, fluorescence imaging of cells and culture media is recorded through a camera 10 and is transmitted to a computer 11, and the states of the liquid drops and the cells are observed in real time on line through a computer display.
After the liquid drops and the cells are cultured for 5 minutes, a valve of a nitrogen steel cylinder 12 is opened, nitrogen gas flows out of the steel cylinder, and is blown out by a needle tube 14 after passing through an air flow controller 13 to form constant-speed air flow which is not lower than 5L/min and not higher than 15L/min, volatile substances on the surface of the liquid drops 6 are blown to the position close to a detector 15 on the opposite side of the needle tube 14, so that the detector 15 obtains stable signals, and the small molecule volatile substances and the metabolites of the cells in the liquid drops are analyzed through mass spectrum peaks, and the real-time metabolic state of the cells can be obtained.
After the data recording is finished once, the output voltage of the power supply 1 is adjusted to 2500-.
While the invention has been described with reference to a preferred embodiment, various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In particular, the technical features mentioned in the embodiments can be combined in any way as long as there is no structural conflict. It is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (6)
1. A single drop single cell culture and cell metabolite real-time detection device comprises:
a capillary electroosmotic flow module forming a single droplet containing a single cell at a capillary tip; the inner diameter of the capillary tube is 50-200 μm;
observation components for observing the state of the cells in the liquid drop are arranged on two opposite sides of the capillary tip;
a detection assembly for real-time detection of cell metabolites in the droplet, disposed on the other two sides of the capillary tip, wherein the detection assembly comprises: an ionizing assembly located to one side of the capillary tip; a detector located on an opposite side of the ionizing assembly; the ionizing assembly includes: the needle-shaped tube is positioned on one side of the tip end of the capillary tube, and the air flow controller and the air source are sequentially connected with the needle-shaped tube; the gas flowing out of the gas source is blown out of the needle-shaped tube after passing through the gas flow controller to obtain a constant-speed gas flow, the volatile substances on the surface of the single liquid drop are blown to the position close to a detector on the opposite side of the needle-shaped tube, and the cell metabolites are detected on line through the detector;
the capillary electroosmotic flow assembly comprising:
a sample cell containing a cell sample;
the inlet of the capillary is communicated with the sample cell, and the outlet end of the capillary is a capillary tip;
an electrode located above the sample cell;
and a power source connected to the electrodes and supplying power thereto.
2. The real-time detection device of claim 1, wherein the capillary contains a solution, and the solution in the capillary is driven by the electrodes to form a single droplet at the tip of the capillary.
3. The real-time detection device according to claim 1 or 2, wherein the cells in the sample cell enter the micro-droplets through electroosmotic flow to form single droplets containing single cells.
4. The real-time detection device of claim 1, wherein the observation assembly comprises:
a laser emitting device located at one side of the capillary tip;
and the observation device is positioned on the opposite side of the laser emission device.
5. The real-time detection device according to claim 4, wherein the laser emitting device comprises a point light source laser and an optical fiber which are connected in sequence; the point light source laser generates laser light which is emitted to the periphery of the single liquid drop through the optical fiber.
6. The real-time detection device according to claim 4 or 5, wherein the observation device comprises an optical filter, a camera and a computer which are connected in sequence; the optical filter is positioned on the opposite side of the optical fiber; the filter filters the unwanted light beam, the camera records the fluorescence image of the cells and the culture medium and transmits the fluorescence image to the computer.
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| CN110426445B (en) * | 2019-08-01 | 2021-07-27 | 东北大学 | Three-dimensional ordered spiral focusing nano particle/single cell analysis system and use method |
| CN113008637B (en) * | 2019-12-20 | 2022-08-02 | 苏州昊通仪器科技有限公司 | Single cell sample preparation and processing device and single cell sample microdroplet processing method |
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| NL1016887C2 (en) * | 2000-12-15 | 2002-06-18 | Tno | Method and device for detecting and identifying bio-aerosol particles in the air. |
| CA2448387C (en) * | 2001-05-24 | 2008-02-05 | New Objective, Inc. | Method and apparatus for feedback controlled electrospray |
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