CN112067534B - Single cell mass spectrometry system and method - Google Patents
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- 238000004949 mass spectrometry Methods 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 20
- 238000000605 extraction Methods 0.000 claims abstract description 86
- 239000007788 liquid Substances 0.000 claims abstract description 40
- 238000002955 isolation Methods 0.000 claims abstract description 25
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 239000000126 substance Substances 0.000 claims abstract description 10
- 239000007921 spray Substances 0.000 claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims description 125
- 238000004458 analytical method Methods 0.000 claims description 15
- 210000000130 stem cell Anatomy 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims 2
- 238000001819 mass spectrum Methods 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000016359 Fibronectins Human genes 0.000 description 2
- 108010067306 Fibronectins Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
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- 238000006073 displacement reaction Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1404—Handling flow, e.g. hydrodynamic focusing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a single-cell mass spectrometry system and a method, wherein the single-cell mass spectrometry system comprises a bearing piece, and a plurality of cells are suitable for being borne by the bearing piece and are mutually separated; the extraction unit comprises an extraction liquid pipeline and a discharge electrode, wherein the extraction liquid in the pipeline forms spray emitted from an opening at the bottom end of the pipeline under the action of the discharge electrode; the pipeline or the bearing piece is driven by the moving unit, so that the bottom end of the extraction liquid pipeline selectively moves to the upper side of any cell; the isolation unit is used for forming a space for isolating any cell from other cells; the bottom end of the extract pipe is suitable for being positioned in the space; the collecting unit is used for collecting substances after the cells in the space are extracted; the detection unit is used for detecting whether the cells in the space are sufficiently extracted. The invention has the advantages of high precision, high efficiency and the like.
Description
Technical Field
The present invention relates to cell analysis, and more particularly to single cell mass spectrometry systems and methods.
Background
Cells are the basic units that make up life bodies, and methods for single cell analysis are needed to understand the changes and behavior of individual cells at various stages in a complex and diverse environment. The mass spectrum is a multi-component simultaneous analysis method, according to the molecular weight difference of various components in the cells, the mass spectrum can form spectrum peaks arranged according to mass numbers in the mass spectrum, and the molecular information of the various components in the cells can be obtained through multi-stage mass spectrum analysis. Because the mass spectrometry does not need to be marked and the information of the molecules to be detected does not need to be known in advance, various unknown components in the cells can be rapidly identified, and the histology information of proteins and even small molecule metabolites in the cells can be obtained. In addition, the mass spectrum can easily obtain the isotope information of each component molecule, and the accurate quantification of various molecules to be detected in cells can be realized by adopting an isotope internal standard and dilution technology. Mass spectrometry single cell analysis has therefore recently received high attention and is believed to play an important role in the study of single cell histology.
One problem with the current single cell detection field is: cell extraction is not uniform and fully considered, is obtained simply by a limited experiment of researchers, does not consider the specificity of cells, and has inaccurate obtained results and complicated steps.
There are many implementation manners of single-cell mass spectrometry, one of the more common and effective ways is to use a U-shaped spray needle to realize in-situ extraction and introduce high-pressure ionization into a mass spectrometry port, and the method has two problems:
1.U type probe does not move, and the operation platform moves: resulting in micro-displacement of cells, inaccurate positioning and cross contamination.
The u-shaped groove partially blocks the microscope field of view: the operator can't utilize the microscope camera to fix a position the cell position, only can remove U type probe after extracting a cell, observe and then operate after the next position, and is comparatively loaded down with trivial details, and the location is inaccurate.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a single-cell mass spectrometry system with high efficiency and high precision.
The invention aims at realizing the following technical scheme:
a single cell mass spectrometry system comprising a carrier, a plurality of cells adapted to be carried by the carrier and separated from one another; the single cell mass spectrometry system further comprises:
the extraction unit comprises an extraction liquid pipeline and a discharge electrode, wherein the extraction liquid in the pipeline forms spray emitted from an opening at the bottom end of the pipeline under the action of the discharge electrode;
a moving unit, the pipeline or the bearing piece is driven by the moving unit, so that the bottom end of the extraction liquid pipeline selectively moves to the upper side of any cell;
a separation unit for forming a space separating any one cell from other cells; the bottom end of the extract pipe is suitable for being positioned in the space;
the collecting unit is used for collecting substances after the cells in the space are extracted;
and the detection unit is used for detecting whether the cells in the space are sufficiently extracted.
Another object of the present invention is to provide a single cell mass spectrometry method using the single cell mass spectrometry system, which is achieved by the following technical scheme:
a single cell mass spectrometry method, the single cell mass spectrometry method comprising:
detecting a position of a cell on the carrier;
the carrier and the extraction liquid pipeline relatively move, so that the bottom end of the extraction liquid pipeline moves to the upper side of the single cell at the designated position;
the isolation unit forms a space for isolating single cells from other cells at the bottom end of the extraction liquid pipeline and at a designated position;
the extraction liquid in the extraction liquid pipeline forms spray emitted from the bottom end opening of the pipeline under the action of a discharge electrode;
single cells at the designated location are extracted until extraction is sufficient;
the extracted substances in the space are collected by a collecting unit and sent to downstream analysis.
Compared with the prior art, the invention has the following beneficial effects:
1. the analysis precision is high;
whether the cells are extracted sufficiently is scientifically, accurately and efficiently judged by using a detection unit such as a laser sensor, and experience judgment is not relied on any more, so that the efficiency and the precision of subsequent analysis are obviously improved;
the scientific setting of the cell quality range is beneficial to improving the judgment accuracy of whether the extraction is sufficient or not, and further improving the accuracy of cell analysis;
2. the efficiency is high;
the extraction, the judgment of whether the extraction is sufficient or not and the collection of the extracted substances are carried out simultaneously, so that the efficiency of the whole analysis is obviously improved;
the detection unit efficiently judges the position of the cells, replaces the low-efficiency microscopic detection, and further improves the efficiency of the whole analysis;
3. automatic analysis is realized;
the detection unit is used for accurately and efficiently judging whether cells exist at each position of the bearing piece and whether extraction is carried out or not, and the cell positioning is not carried out by a microscope, so that a foundation is laid for the automatic detection of single cells.
Drawings
The present disclosure will become more readily understood with reference to the accompanying drawings. As will be readily appreciated by those skilled in the art: the drawings are only for illustrating the technical scheme of the present invention and are not intended to limit the scope of the present invention. In the figure:
FIG. 1 is a schematic diagram of a single cell mass spectrometry system according to an embodiment of the present invention.
Fig. 2 is a schematic structural view of a detection unit according to an embodiment of the present invention.
Detailed Description
Figures 1-2 and the following description depict alternative embodiments of the invention to teach those skilled in the art how to make and reproduce the invention. In order to teach the technical solution of the present invention, some conventional aspects have been simplified or omitted. Those skilled in the art will appreciate variations or alternatives derived from these embodiments that fall within the scope of the invention. Those skilled in the art will appreciate that the features described below can be combined in various ways to form multiple variations of the invention. Thus, the invention is not limited to the following alternative embodiments, but only by the claims and their equivalents.
Example 1:
fig. 1 schematically shows a structural diagram of a single cell mass spectrometry system of embodiment 1 of the present invention, as shown in fig. 1, comprising:
a carrier 11, by which a plurality of cells are adapted to be carried and separated from each other;
the extraction unit 21 comprises an extraction liquid pipeline and a discharge electrode, wherein the extraction liquid in the pipeline forms spray emitted from an opening at the bottom end of the pipeline under the action of the discharge electrode;
a moving unit, such as a three-dimensional moving platform, driven by the moving unit, the duct or carrier selectively moves the bottom end of the extraction liquid duct to the upper side of any cell;
a spacer unit 41, such as a spacer cover, for forming a space for separating any one cell from other cells; the bottom end of the extract pipe is suitable for being positioned in the space;
a collection unit 31 for collecting the extracted substances of the cells in the space;
and the detection unit is used for detecting whether the cells in the space are sufficiently extracted.
In order to achieve accurate positioning of cells and isolation between cells, preventing other cells from being affected when extracting individual cells at a designated location, further, the carrier has grooves distributed in an array, each groove being adapted to receive an individual cell.
In order to extract single cells at a designated location accurately, further, the tube includes a first portion disposed obliquely and a second portion disposed vertically, the bottom end of the second portion being pointed;
the discharge electrode is a conductive coating provided at the tip or an electrode sheet inserted into the tube.
In order to achieve mutual isolation between cells, to prevent other cells from being affected when extracting individual cells at a designated location, further, the isolation unit includes:
the isolation cover is matched with the bearing piece from top to bottom to form the space; .
In order to facilitate the extraction of individual cells at a given location, the screen is further provided with a through-hole adapted to be passed through by the extraction liquid conduit or is fixedly connected to the extraction liquid conduit.
In order to collect information of the extracted cells, further, the collecting unit includes a collecting pipe passing through the isolating unit.
In order to accurately locate the cell position and accurately judge whether the extraction is sufficient, further, the detection unit includes a sensor provided in each groove for detecting the mass of the individual cells in the space.
The single-cell mass spectrometry method of the embodiment of the invention, namely the working method using the single-cell mass spectrometry system, comprises the following steps:
detecting a position of a cell on the carrier;
the carrier and the extraction liquid pipeline relatively move, so that the bottom end of the extraction liquid pipeline moves to the upper side of the single cell at the designated position;
the isolation unit forms a space for isolating single cells from other cells at the bottom end of the extraction liquid pipeline and at a designated position; forming the space is performed simultaneously with or in time with the movement of the extraction liquid pipeline, such as forming the space before the movement of the extraction liquid pipeline is performed, or forming the space after the movement of the extraction liquid pipeline is performed, and the movement of the extraction liquid pipeline is performed before the formation of the space;
the extraction liquid in the extraction liquid pipeline forms spray emitted from the bottom end opening of the pipeline under the action of a discharge electrode;
single cells at the designated location are extracted until extraction is sufficient;
the extracted substances in the space are collected by a collecting unit and sent to downstream analysis.
In order to accurately and efficiently judge whether the extraction of the cells is sufficient, further, the judgment mode of the sufficient extraction of the single cells is as follows:
detecting the mass of the cells at the designated location, and judging whether the mass is within a mass range;
if the extraction is in the mass range, the extraction is sufficient;
if the extraction is out of the mass range, the extraction is not satisfied.
In order to accurately and efficiently judge whether the extraction of the cells is sufficient, further, the mass range is as follows:
for stem cells, the mass range is [0.3m 1 ，0.4m 1 ]，m 1 Is the mass of stem cells before extraction;
for living cells, the mass range is [0.03m 2 ，0.05m 2 ]，m 2 Is the quality of the living cells before extraction.
Example 2:
application example of the single cell mass spectrometry system and method according to embodiment 1 of the present invention.
In this application example, the carrier is of a matrix structure, with rectangular grooves distributed in an array, each groove being adapted to receive a single cell;
the isolation unit adopts an isolation cover, the (equivalent) inner diameter of the isolation cover from top to bottom is increased, and the bottom end of the isolation cover is rectangular matched with the groove of the bearing piece; the isolation cover is buckled on the grooves of the bearing piece from top to bottom to form a space isolated from other grooves, and only one rectangular groove is arranged in the space, namely only one cell is arranged in the space;
a moving unit which adopts a three-dimensional moving platform and is used for driving the bearing piece to move in three dimensions, such as movement in a horizontal plane and movement in a vertical direction, so that the isolation cover moves (relatively) to any rectangular groove, thereby forming the space;
the extraction unit comprises an extraction liquid pipeline and a discharge electrode, wherein the extraction liquid comprises an inclined part and a vertical part, the part of the vertical part connected with the inclined part is in a bent shape, and the bottom end of the vertical part is in a tip shape; the discharge electrode is a conductive coating outside the bottom end or an electrode plate inserted into the pipeline and is suitable for being connected with external high voltage; the vertical portion passes through the top of the cage, thereby entering the cage;
the collecting unit adopts a pipeline, and the pipeline passes through the isolation cover, so that substances after cells in the space are extracted are transmitted to the downstream through the pipeline under negative pressure for analysis;
the detection unit is used for detecting whether the cells in the space are sufficiently extracted or not; as shown in fig. 2, the laser sensor specifically includes:
cantilever 112 of transparent silica gel coated with collagen or fibronectin may take the form of an ultrathin metal sheet coated with collagen or fibronectin film. The cantilever is suspended at a corner above the recess of the carrier 11 and the cells are stuck to the lower end of the cantilever. The blue pulse laser 111 then emits light at its fixed end, causing the entire arm to oscillate. Another infrared laser 113 measures these oscillations at the free end of the cell suspension. By comparing the degree of oscillation and comparing the difference between the cell placed on the measuring arm and not placed on the measuring arm, the weight thereof can be calculated, the data is displayed on the screen of the computer as a curve showing the change of weight with time, when the weight is not changed any more, the high-voltage discharge is stopped, and the weight of the cell at this time is acquired, the weight at this time being used for comparison with the mass range.
In the device, the extraction unit, the isolation cover and the collection unit are fixedly arranged, and the bearing piece is driven to move in three dimensions.
The single-cell mass spectrometry method of the embodiment of the invention, namely the working method using the single-cell mass spectrometry system, comprises the following steps:
the position of the rectangular groove with cells is determined by using the output value of the detection unit in each groove, specifically: the output value is within the cell mass range, i.e. the presence of cells is confirmed;
the moving unit drives the carrier so that the single cells at the designated positions are on the lower side of the bottom end;
the isolation cover is buckled on the upper side of the single cell at the appointed position, namely the upper side of the rectangular groove at the appointed position, and the isolation cover and the rectangular groove form a space isolated from other cells;
switching on high-voltage electricity, wherein the extraction liquid in the extraction liquid pipeline forms spray emergent from the bottom end opening of the pipeline under the action of a discharge electrode;
single cells at the designated location are extracted until extraction is sufficient; the judgment mode of the full extraction is as follows:
detecting the mass of the single cells at the designated position, and judging whether the mass is in a mass range;
if the extraction is in the mass range, the extraction is sufficient; for stem cells, the mass range is [0.3m 1 ，0.4m 1 ]，m 1 Is the mass of stem cells before extraction; for living cells, the mass range is [0.03m 2 ，0.05m 2 ]，m 2 Is the mass of living cells before extraction;
if the extraction is out of the mass range, the extraction is not satisfied;
the extracted substances in the space are collected by a collecting unit and sent to a downstream mass spectrometer for analysis.
Example 3:
an application example of the single cell mass spectrometry system and method according to embodiment 1 of the present invention is different from embodiment 2 in that:
the carrier is fixed, the mass spectrometer is fixed, the isolation cover, the extraction unit and the collection unit are relatively fixed, and the isolation cover is driven by the same moving unit to move in three dimensions, so that the isolation cover is buckled on a rectangular groove with cells at a designated position; the bottom end of the extraction liquid pipeline is positioned on the upper side of the single cell in the space while the isolation cover is buckled on the rectangular groove to form an isolated space.
Example 4:
an application example of the single cell mass spectrometry system and method according to embodiment 1 of the present invention is different from embodiment 2 in that:
the carrier is fixed, and the mass spectrometer is fixed; the isolation cover and the collecting unit are relatively fixed and driven by the first moving unit to move in three dimensions; the extraction unit is driven by the second moving unit to move in three dimensions; the shield has a through hole adapted to be passed through by the vertical portion of the extraction liquid conduit.
In the single-cell mass spectrometry method, under the drive of a first moving unit, a shielding cover is buckled on a rectangular groove with cells at a designated position to form a space isolated from other cells; then, the vertical portion of the extraction liquid pipeline is driven by the second moving unit to move in three dimensions and finally move downwards through the through hole, so that the bottom end of the vertical portion enters the space and is positioned on the upper side of the single cells in the space.
Claims (6)
1. A single cell mass spectrometry system comprising a carrier, a plurality of cells adapted to be carried by the carrier and separated from one another; wherein the single cell mass spectrometry system further comprises:
the extraction unit comprises an extraction liquid pipeline and a discharge electrode, wherein the extraction liquid in the pipeline forms spray emitted from an opening at the bottom end of the pipeline under the action of the discharge electrode;
a moving unit, the pipeline or the bearing piece is driven by the moving unit, so that the bottom end of the extraction liquid pipeline selectively moves to the upper side of any cell;
a separation unit for forming a space separating any one cell from other cells; the bottom end of the extract pipe is suitable for being positioned in the space;
the collecting unit is used for collecting substances after the cells in the space are extracted;
a detection unit for detecting whether or not the cells in the space are sufficiently extracted; the carrier has grooves distributed in an array, each groove being adapted to receive a single cell; the detection unit comprises a sensor, wherein the sensor is arranged in each groove and is used for detecting the quality of single cells in the space and judging whether the extraction is sufficient or not according to the quality;
the isolation cover is matched with the bearing piece from top to bottom to form the space.
2. The single cell mass spectrometry system of claim 1, wherein the conduit comprises a first portion disposed obliquely and a second portion disposed vertically, the second portion having a bottom end that is pointed;
the discharge electrode is a conductive coating provided at the tip or an electrode sheet inserted into the tube.
3. The single cell mass spectrometry system of claim 1, wherein the shield has a through hole adapted for the passage of the extraction fluid conduit or the shield is fixedly connected to the extraction fluid conduit.
4. The single cell mass spectrometry system of claim 1, wherein the collection unit comprises a collection conduit that passes through the isolation unit.
5. The single cell mass spectrometry method of the single cell mass spectrometry system according to any one of claims 1 to 4, which is:
detecting a position of a cell on the carrier;
the carrier and the extraction liquid pipeline relatively move, so that the bottom end of the extraction liquid pipeline moves to the upper side of the single cell at the designated position;
the isolation unit forms a space for isolating single cells from other cells at the bottom end of the extraction liquid pipeline and at a designated position;
the extraction liquid in the extraction liquid pipeline forms spray emitted from the bottom end opening of the pipeline under the action of a discharge electrode;
single cells at the designated location are extracted until extraction is sufficient; the method for judging the full extraction of the single cells comprises the following steps:
detecting the mass of the cells at the designated location, and judging whether the mass is within a mass range;
if the extraction is in the mass range, the extraction is sufficient;
if the extraction is out of the mass range, the extraction is not satisfied;
the extracted substances in the space are collected by a collecting unit and sent to downstream analysis.
6. The method of single cell mass spectrometry of claim 5, wherein the mass range is:
for stem cells, the mass range is [0.3m 1 ，0.4m 1 ]，m 1 Is the mass of stem cells before extraction;
for living cells, the mass range is [0.03m 2 ，0.05m 2 ]，m 2 Is the quality of the living cells before extraction.
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| CN112444583B (en) * | 2021-02-01 | 2021-05-07 | 宁波大学 | Cell detection device and method based on paper-based discharge technology |
| CN114910546B (en) * | 2022-07-13 | 2022-11-11 | 宁波华仪宁创智能科技有限公司 | Single cell mass spectrometry device and method based on extraction technology |
| CN115266896B (en) * | 2022-09-26 | 2023-01-13 | 宁波华仪宁创智能科技有限公司 | Biological sample analysis system and method |
| CN117405803B (en) * | 2023-10-25 | 2024-03-22 | 苏州市药品检验检测研究中心（苏州市药品不良反应监测中心） | Cell detection device and method based on paper-based discharge technology |
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