CN209858475U - In-situ extraction and online mass spectrum detection device for living unicells - Google Patents
In-situ extraction and online mass spectrum detection device for living unicells Download PDFInfo
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- CN209858475U CN209858475U CN201821971989.0U CN201821971989U CN209858475U CN 209858475 U CN209858475 U CN 209858475U CN 201821971989 U CN201821971989 U CN 201821971989U CN 209858475 U CN209858475 U CN 209858475U
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Abstract
The utility model provides a living body unicellular normal position extraction and online mass spectrometry detection device, it includes: the microfluidic platform is used for transmitting the extraction fluid to the single cell probe; a cell manipulation platform on which cells to be detected are cultured; the single cell probe is positioned on the cell manipulation platform and used for isolating a target single cell to be detected and realizing single cell extraction to obtain a single cell extract; an in-line ionization device for receiving the single-cell extract and forming an ionized spray from the single-cell extract in-line; and the detection device is used for receiving the ionized spray formed by the online ionization device and detecting the ionized spray. When the device is used for cell detection, protein and nucleic acid information in cells can be maximally reserved for subsequent analysis after the extracted cells detect the markers of the cells.
Description
Technical Field
The utility model belongs to the technical field of unicellular mass spectrometry research, a living body unicellular normal position extraction and online mass spectrometry detection device is related to.
Background
The single cell analysis technology is used for analyzing cell markers, metabolites, genomes, proteomes and the like on a single cell level by combining a microfluidic technology with electrochemical analysis, fluorescence analysis, mass spectrometry and other technologies. With the progress of analytical methods, more and more single cell detection techniques are in force. Single cell analysis has attracted much attention because it can provide important information for disease diagnosis and gene sequencing with low cost.
The mass spectrum is used as an important means for analyzing the chemical components of the single cell, and has the characteristics of high sensitivity, high throughput and simultaneous analysis of various important components. In the existing and widely used single-cell mass spectrum technology, because the single-cell entrapment rate is low, the experimental error is difficult to avoid in the single-cell droplet technology; the single cell probe technology can realize the analysis of cell contents, but the puncturing position at each time cannot be determined, and the whole information of the whole single cell cannot be embodied. Therefore, the development of a single-cell mass spectrometry technology capable of realizing original detection has a great promoting effect on the field.
SUMMERY OF THE UTILITY MODEL
In order to solve the problem that exists among the current unicellular mass spectrometry, the utility model provides a living body unicellular normal position extraction and online mass spectrometry detection device uses the device to carry out cell detection time measuring, detects its marker after the cell through the extraction, can maximize remain intracellular protein and nucleic acid information and be used for subsequent analysis.
The utility model provides an aspect provides a living body unicellular normal position extraction and online mass spectrometry detection device, the device includes:
the microfluidic platform is used for transmitting the extraction fluid to the single cell probe;
a cell manipulation platform on which cells to be detected are cultured;
the single cell probe is positioned on the cell manipulation platform and used for isolating a target single cell to be detected and realizing single cell extraction to obtain a single cell extract;
an in-line ionization device for receiving the single-cell extract and forming an ionized spray from the single-cell extract in-line;
and the detection device is used for receiving the ionized spray formed by the online ionization device and detecting the ionized spray.
According to a preferred embodiment of the present invention, the single cell probe comprises a fluid inlet, a single cell extraction chamber and a fluid outlet, which are sequentially arranged along the flow direction of the extraction fluid, and the fluid inlet and the fluid outlet are communicated with the single cell extraction chamber. The extraction fluid enters the single-cell extraction chamber from the fluid inlet and then exits from the fluid outlet.
According to a preferred embodiment of the present invention, the single-cell extraction chamber is configured to accommodate a single cell, preferably, the single-cell extraction chamber is configured to accommodate only a single cell.
According to a preferred embodiment of the present invention, the cell manipulation platform comprises:
an object stage;
the cell culture device is positioned on the objective table, and cell culture solution is contained in the cell culture device and is used for culturing cells to be detected;
and the aligning device is arranged below the objective table and is used for observing and positioning the target single cell to be extracted.
According to a preferred embodiment of the present invention, the cells are planted in the cell culture apparatus at a density of 100-.
In some embodiments of the invention, the cell culture device is a petri dish, which is placed on a stage.
According to the utility model discloses a preferred embodiment, aligning device is microscope objective, and the cell in the culture dish can be observed through microscope objective, can confirm accurately whether the unicellular probe aims at the single cell.
According to a preferred embodiment of the present invention, the microfluidic platform comprises:
a fluid line comprising a first fluid line connected to the fluid inlet and a second fluid line connected to the fluid outlet;
a peristaltic syringe pump for driving the extraction fluid in the fluid line.
The peristaltic injection pump drives the extraction fluid to enter the first fluid pipeline, the extraction fluid enters the single cell probe through the first fluid pipeline through the fluid inlet of the single cell probe, then the single cell is extracted by flowing through the single cell extraction chamber, and the single cell extract flows into the second fluid pipeline through the fluid outlet and enters the online ionization device.
According to a preferred embodiment of the present invention, the peristaltic injection pump drives the extraction fluid in the fluid line at a rate of 0.2-1 μ L/min, preferably at a rate of 0.5 μ L/min, enabling continuous flow extraction.
According to a preferred embodiment of the present invention, the first fluid line and the second fluid line have the same inner diameter, preferably an inner diameter of 20-100 μm, preferably 50 μm.
According to the utility model discloses a preferred embodiment, online ionization device includes power, needle end and metal two-way valve, the metal two-way valve is used for receiving unicellular extract and is linked together with the needle end, and the needle end provides the ionization spraying, and the metal two-way valve is connected to the power.
According to the preferred embodiment of the present invention, the power supply is a commercial high voltage power supply with a voltage of 2000 and 4500V, which is used to provide the voltage required by the needle-shaped tip to form the spray.
Preferably, the detection device comprises a detection device sample inlet and a detection device main body, and the needle-shaped tail end is over against the detection device sample inlet, so that the needle-shaped tail end directly enters the detection device for detection after forming ionized spray.
According to a preferred embodiment of the present invention, the single cell probe is prepared by a soft lithography technique. Preferably, the single cell probe is made of a plastic material with elasticity and good light transmittance, and is preferably made of polydimethylsiloxane.
The device provided by the utility model can be at unicellular mass spectrum detection and unicellular drug screening.
A method for in-situ extraction and online mass spectrometry detection of living unicells comprises the following steps:
step S1, providing the living single cell in-situ extraction and online mass spectrum detection device;
step S2, culturing cells to be detected on a cell manipulation platform;
s3, vertically approaching the single cell probe to a cell manipulation platform, adjusting the cell manipulation platform to enable the target cell to be detected to be placed below the single cell probe, and adjusting the cell manipulation platform to enable the single cell probe to be attached to the cell manipulation platform, so that the target single cell to be detected is isolated;
and step S4, transmitting the extraction fluid to the single cell probe through the microfluidic platform, flowing into the online ionization device, forming ionized spray through the online ionization device, and entering the detection device for detection.
According to a preferred embodiment of the present invention, the extraction fluid is methanol. The methanol has a cell fixing function, and the cells are fixed in situ after extraction, so that a large amount of undetected molecular information can be reserved for subsequent analysis.
When the device is applied to screening of single-cell drugs, the extraction fluid is replaced by a culture medium solution containing drugs, so that the metabolism level of the cells to the drugs can be monitored in real time, and the screening of the single-cell drugs can be carried out.
The utility model has the advantages that:
1. the utility model discloses collect microfluid continuous flow control, unicellular normal position extraction, online real-time ionization and detect in an organic whole, can carry out normal position target extraction to the adherent cell of cultivateing in the culture dish to realize normal position mass spectrum and detect, realize the little damage ground to the normal position marker analysis of unicellular.
2. The utility model discloses a single cell probe can sample one by one the unicellular, can be the at utmost after sample analysis remain residual material composition and content under the cell adherence growth state moreover, can be used for other analysis.
3. The cell analysis of the utility model is fast, and can complete the sampling analysis of single cells in a short time.
Drawings
FIG. 1 is a diagram of an in-situ extraction and online mass spectrometry device for single cells in vivo according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a single cell probe according to an embodiment of the present invention;
wherein, 1 represents a peristaltic injection pump, 2-1 represents a first fluid pipeline, 2-2 represents a second fluid pipeline, 3 represents a culture dish, 4 represents a microscope objective, 5 represents a single-cell probe, 6 represents a cell, 7 represents a metal two-way valve, 8 represents a power supply, 9 represents a needle-shaped end, 10 represents a sample inlet of a detection device, 11 represents a fluid inlet, 12 represents a single-cell extraction chamber, and 13 represents a fluid outlet.
Detailed Description
The present invention will be described in detail with reference to the following examples, but the present invention is not limited to the following examples.
Example 1
As shown in fig. 1 and fig. 2, an in-situ extraction and online mass spectrometry apparatus for single cell in vivo comprises:
the micro-fluidic platform comprises a peristaltic injection pump 1 and fluid pipelines, wherein the fluid pipelines comprise a first fluid pipeline 2-1 and a second fluid pipeline 2-2, and the peristaltic injection pump is used for driving extraction fluid in the fluid pipelines; ,
the cell manipulation platform comprises an object stage (not shown in figure 1), a culture dish 3 arranged on the object stage, a cell culture solution contained in the culture dish 3, cells to be detected cultured in the culture dish, and a microscope objective 4 arranged below the object stage and used for observing and positioning the cells to be detected;
the single cell probe is positioned above the cell manipulation platform and comprises a fluid inlet 11, a single cell extraction chamber 12 and a fluid outlet 13 which are sequentially arranged along the fluid flow direction, and the fluid inlet and the fluid outlet are communicated with the single cell extraction chamber. A first fluid pipeline 2-1 of the micro-fluidic platform is connected with a fluid inlet 11, and a fluid outlet 13 is connected with a second fluid pipeline 2-2;
the online ionization device comprises a power supply 8, a needle-shaped tail end 9 and a metal two-way valve 7, wherein the metal two-way valve 7 is connected with the needle-shaped tail end 9, the metal two-way valve 7 receives the single-cell extract from the second fluid pipeline 2-2 and transmits the single-cell extract to the needle-shaped tail end 9, the needle-shaped tail end 9 forms spray, and the power supply 8 is connected with the metal two-way valve 7 and used for providing voltage required by the needle-shaped tail end to form the spray;
and the detection device comprises a detection device sample inlet 10 and a detection device body (not shown in figure 1), wherein the detection device sample inlet 10 is opposite to the needle-shaped tail end 9, so that the spray formed by the needle-shaped tail end enters the detection device for detection.
A method for in-situ extraction and online mass spectrometry detection of living unicells comprises the following steps:
providing the living single cell in-situ extraction and online mass spectrum detection device,
the cells are planted in the culture dish 3 at the density of 100-10000 cells/square centimeter, the single cell probe 5 is vertically close to the culture dish 3, the target cell 6 to be detected is adjusted to be right below the single cell probe through the objective table, and the cells 6 in the culture dish 3 can be observed through the microscope objective 4, so that whether the single cell probe is aligned with the single cell is determined. Make single cell probe 5 hug closely the bottom surface of culture dish 3 through adjusting the objective table, after single cell probe and the bottom surface laminating of culture dish, can seal cell 6 in single cell probe to cell 6 and the culture medium solution and other cells isolation around will the target is detected. Injecting an extraction fluid into a fluid pipeline 2-1 through a peristaltic injection pump 1, enabling the extraction fluid to enter a single cell extraction chamber 12 through a fluid inlet 11, realizing single cell extraction in the single cell extraction chamber 12, enabling a single cell extraction liquid to flow out of a single cell probe 5 through a fluid outlet 13, transmitting the single cell extraction liquid to a needle-shaped tail end 9 through a second fluid pipeline 2-2 and a metal two-way valve 7, conducting a high-voltage potential to the needle-shaped tail end 9 through a solution, continuously forming spray at the needle-shaped tail end 9, and enabling the spray to enter a detection device through a sample inlet of the detection device for online detection.
After a certain time, the marker on the cell 6 is extracted into the needle-shaped end 9 to form a continuous ionized spray, and the extracted cell 6 is fixed on the culture dish 3 for subsequent other analysis.
It should be noted that the above-mentioned embodiments are only used for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as specified within the scope of the claims and modifications may be made without departing from the scope and spirit of the invention. Although the present invention has been described herein with reference to particular means, materials and embodiments, it is not intended to be limited to the particulars disclosed herein, but rather the present invention extends to all other methods and applications having the same functionality.
Claims (10)
1. The utility model provides a living body unicell in situ extraction and online mass spectrometry detection device which characterized in that includes:
the microfluidic platform is used for transmitting the extraction fluid to the single cell probe;
a cell manipulation platform on which cells to be detected are cultured;
the single cell probe is positioned on the cell manipulation platform and used for isolating a target single cell to be detected and realizing single cell extraction to obtain a single cell extract;
an in-line ionization device for receiving the single-cell extract and forming an ionized spray from the single-cell extract in-line;
and the detection device is used for receiving the ionized spray formed by the online ionization device and detecting the ionized spray.
2. The device of claim 1, wherein the single cell probe comprises a fluid inlet, a single cell extraction chamber and a fluid outlet, which are arranged in sequence along the flow direction of the extraction fluid, and the fluid inlet and the fluid outlet are communicated with the single cell extraction chamber.
3. The device of claim 2, wherein the single-cell extraction chamber is configured to accommodate a single cell.
4. The device of claim 2, wherein the single cell extraction chamber is configured to accommodate only a single cell.
5. The device of claim 1, wherein the cell manipulation platform comprises:
an object stage;
the cell culture device is positioned on the objective table, and cell culture solution is contained in the cell culture device and is used for culturing cells to be detected;
and the alignment device is arranged below the objective table and is used for observing and positioning the target single cell to be detected.
6. The apparatus of claim 5, wherein the alignment device is a microscope objective.
7. The device of claim 2, wherein the microfluidic platform comprises:
a fluid line comprising a first fluid line connected to the fluid inlet and a second fluid line connected to the fluid outlet;
a peristaltic syringe pump for driving the extraction fluid in the fluid line.
8. The device of claim 7, wherein the first and second fluid conduits have an inner diameter of 20-100 μm.
9. The apparatus of claim 1, wherein the in-line ionization device comprises a power source, a needle-shaped tip, and a metal two-way valve for receiving the single-cell extract and communicating with the needle-shaped tip, the power source being connected to the metal two-way valve.
10. The device of claim 9, wherein the detection device comprises a detection device inlet and a detection device body, and the needle-shaped end faces the detection device inlet.
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| CN201821971989.0U CN209858475U (en) | 2018-11-27 | 2018-11-27 | In-situ extraction and online mass spectrum detection device for living unicells |
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| CN201821971989.0U CN209858475U (en) | 2018-11-27 | 2018-11-27 | In-situ extraction and online mass spectrum detection device for living unicells |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109557163A (en) * | 2018-11-27 | 2019-04-02 | 清华大学 | A kind of Living single cell situ extracting and on-line mass spectroscopy detection device and application |
| CN111397985A (en) * | 2020-04-14 | 2020-07-10 | 清华大学 | Single cell mass spectrometry |
| CN111624250A (en) * | 2020-06-03 | 2020-09-04 | 东华理工大学 | Online mass spectrometry system for arsenic with different forms on iron mineral surface |
| CN111808750A (en) * | 2020-07-15 | 2020-10-23 | 清华大学 | Micro-fluidic chip device, in-situ detection method for living unicells and application |
-
2018
- 2018-11-27 CN CN201821971989.0U patent/CN209858475U/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109557163A (en) * | 2018-11-27 | 2019-04-02 | 清华大学 | A kind of Living single cell situ extracting and on-line mass spectroscopy detection device and application |
| CN109557163B (en) * | 2018-11-27 | 2023-09-15 | 清华大学 | Living single-cell in-situ extraction and online mass spectrum detection device and application |
| CN111397985A (en) * | 2020-04-14 | 2020-07-10 | 清华大学 | Single cell mass spectrometry |
| CN111624250A (en) * | 2020-06-03 | 2020-09-04 | 东华理工大学 | Online mass spectrometry system for arsenic with different forms on iron mineral surface |
| CN111624250B (en) * | 2020-06-03 | 2022-06-10 | 东华理工大学 | Online mass spectrometry system for arsenic with different forms on iron mineral surface |
| CN111808750A (en) * | 2020-07-15 | 2020-10-23 | 清华大学 | Micro-fluidic chip device, in-situ detection method for living unicells and application |
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