CN111965093B - Single cell mass spectrometry device and method - Google Patents
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Abstract
The invention provides a single-cell mass spectrometry device and a method, wherein the single-cell mass spectrometry device comprises a bearing part and a mass spectrometer, the bearing part is used for bearing a plurality of cells which are separated from each other, and the mass spectrometer is provided with a sample inlet; the transmission unit comprises a capillary needle, a moving part, a negative pressure generation module, a driving module and a bracket; the two ends of the capillary needle are open and are arranged on the bracket; the moving part is selectively in a first state and a second state; the negative pressure generating module is communicated with a space between the moving piece in the capillary needle and the first opening end, so that the pressure in the space is lower than that outside the capillary needle; the driving module is used for rotating the capillary needle on the vertical surface, so that the second opening end of the capillary needle corresponds to the sample inlet; the electrode is arranged at the second opening end and is suitable for being connected with a power supply; the first moving unit is used for driving the carrier or the conveying unit, so that the distance between the first opening end of the capillary needle driven by the driving module and the cell on the carrier is 0-1 mm. The invention has the advantages of high analysis efficiency and the like.
Description
Technical Field
The invention relates to cell analysis, in particular to a single-cell mass spectrometry device and a single-cell mass spectrometry method.
Background
Cells are the basic units constituting life bodies, and the change and behavior of each stage of a single cell in a complex and changeable environment are known, so that a method for analyzing the single cell is needed. The mass spectrum is a method for simultaneously analyzing multiple components, and can form spectral peaks arranged according to mass numbers in the mass spectrum according to different molecular weights of various components in cells, and further obtain molecular information of various components in the cells through multi-stage mass spectrum analysis. The mass spectrometry does not need to be marked and does not need to know the information of the molecules to be detected in advance, so that various unknown components in the cells can be rapidly identified, and the omics information of the proteins and even the micromolecular metabolites in the cells can be obtained. In addition, the mass spectrum can easily obtain isotope information of each component molecule, and accurate quantification of various molecules to be detected in cells can be realized by adopting isotope internal standards and dilution technology. Therefore, mass spectrometry single cell analysis has recently received high attention and is considered to play an important role in omics analysis and study of single cells.
The existing detection methods of single cell mass spectrum mainly comprise three types:
1. fluorescence flow cytometry: when multiple indices are detected simultaneously, up to 18 indices can be detected simultaneously due to the overlap of fluorescence spectra and dye limitations.
2. Mass flow cytometry: the multi-parameter detection is carried out on the single cells by utilizing the mass spectrum principle, the characteristic of high-speed analysis of the traditional flow cytometer is inherited, the high resolution capability of mass spectrum detection is realized, and a metal label antibody is required.
3. Mass spectrometry micromanipulation cell method: the method of adopting single-drop extraction and utilizing the ionization of the electric spray ion source solves the problems, but still has the following technical problems:
A. the formation of picoliter single liquid drops is unstable, so that ionized spray cannot be formed, and single operation is ineffective;
B. the capillary tube is turned to the mass spectrum sample inlet by rotating about 225 degrees after the extraction liquid is sucked, the technical means enables the liquid drops to move towards the other end of the capillary tube under the influence of gravity when the needle head faces upwards, the liquid drops are greatly reduced after the liquid drops rotate to the mass spectrum sample inlet, the mass spectrum detection efficiency is low, and even signals cannot be detected.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides the single-cell mass spectrometry device which has high analysis efficiency, stable work, high sensitivity and automation.
The purpose of the invention is realized by the following technical scheme:
the single-cell mass spectrometry device comprises a bearing piece and a mass spectrometer, wherein the bearing piece is used for bearing a plurality of cells which are separated from each other, and the mass spectrometer is provided with a sample inlet; the single-cell mass spectrometry apparatus further comprises:
the device comprises a conveying unit, a control unit and a control unit, wherein the conveying unit comprises a capillary needle, a moving piece, a negative pressure generating module, a driving module and a bracket; the two ends of the capillary needle are open and are arranged on the bracket; when the moving piece is in a first state, the inside of the capillary needle is separated into two parts by the moving piece, and when the moving piece is in a second state, the two parts are communicated; the negative pressure generating module is communicated with a space between the moving member in the capillary needle in the first state and the first opening end of the capillary needle, so that the pressure in the space is lower than that outside the capillary needle; the driving module is used for rotating the capillary needle on a vertical surface, so that the second opening end of the capillary needle corresponds to the sample inlet of the mass spectrometer;
an electrode disposed at the second open end and adapted for connection to a power source;
a first moving unit, configured to drive the carrier or the transport unit, so that a distance between the first open end of the capillary needle and the cell on the carrier driven by the driving module is 0-1 mm.
The invention also aims to provide a single-cell analysis method using the single-cell mass spectrometry device, and the invention aims to be realized by the following technical scheme:
according to the single cell analysis method of the single cell mass spectrometry device, the single cell analysis method comprises the following steps:
the first moving unit drives the bearing piece or the bracket, and the bearing piece and the bracket move relatively;
the driving module drives the capillary needle to rotate in the forward direction, so that the first opening end is adjacent to the selected cells, and the selected cells are extracted by the extraction liquid;
the moving part is in a first state, and the negative pressure generating module works;
the extraction material of the selected cells passes through the first open end and into the space;
the driving module drives the capillary needle to rotate reversely, and the second opening end of the capillary needle corresponds to the sample inlet of the mass spectrometer;
the moving member is in a second state, and the extraction substances in the space flow downwards to the second opening end;
and discharging the electrode, wherein the extraction substance is ionized and sprayed out from the second opening end to enter the sample inlet.
Compared with the prior art, the invention has the beneficial effects that:
1. the analysis efficiency is high;
the conveying unit is specially designed, the capillary needles with openings at two ends are adopted, the extraction substances enter and temporarily store from one end and exit from the other end after rotation, complex lifting and rotation are not needed, the conveying efficiency of the extraction substances is improved, and the overall analysis efficiency is improved;
when the technical means of moving the bearing piece is adopted, the capillary needles in the conveying unit can realize the conveying function only by rotating in the vertical plane, so that the conveying efficiency and the analysis efficiency are further improved;
2. the work is stable;
the extract liquid in the capillary is heated by the emergent light of the laser, so that single liquid drops with stable output are generated;
by controlling the laser energy, the inner diameter of the capillary and the irradiation position of emergent light of the laser, the stable generation of 1-600pL single liquid drops is realized;
3. the sensitivity is high;
utilizing a negative pressure generating module to generate negative pressure in a space (formed by the moving part in the first state and the capillary needle), so as to suck the extraction substance into the space; in the rotation process of the capillary needle and the like, due to the action of the negative pressure generation module and the inclined direction (the position of the space is higher than the position of the second opening end) of the capillary needle after rotation, the extraction substance in the space cannot leak, enough extraction liquid is obtained, and the sensitivity and the detection efficiency of subsequent mass spectrometry are improved;
4. automatic analysis is realized;
the automatic moving unit, the automatic driving module and the automatic moving piece driving device realize automatic generation of single liquid drops, automatic absorption of extraction substances, automatic movement of capillary needles, ionization and mass spectrometry of the extraction substances, realize full automation of single cell analysis, do not need manual intervention, and are more accurate in operation.
Drawings
The disclosure of the present invention will become more readily understood with reference to the accompanying drawings. As is readily understood by those skilled in the art: these drawings are only for illustrating the technical solutions of the present invention and are not intended to limit the scope of the present invention. In the figure:
FIG. 1 is a flow diagram of a method of single cell analysis according to an embodiment of the invention;
FIG. 2 is an experimental picture of a single drop of a selected cell encapsulated by an embodiment of the invention;
FIG. 3 is a primary mass spectrum of a single cell according to an embodiment of the present invention.
Detailed Description
Fig. 1-3 and the following description depict alternative embodiments of the invention to teach those skilled in the art how to make and use the invention. Some conventional aspects have been simplified or omitted for the purpose of explaining the technical solution of the present invention. Those skilled in the art will appreciate that variations or substitutions from these embodiments will be 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 present invention is not limited to the following alternative embodiments, but is only limited by the claims and their equivalents.
Example 1:
the single-cell mass spectrometer of embodiment 1 of the present invention comprises:
the device comprises a bearing piece and a mass spectrometer, wherein the bearing piece is used for bearing a plurality of cells which are separated from each other, and the mass spectrometer is provided with a sample inlet;
the device comprises a conveying unit, a control unit and a control unit, wherein the conveying unit comprises a capillary needle, a moving piece, a negative pressure generating module, a driving module and a bracket; the two ends of the capillary needle are open and are arranged on the bracket; when the moving piece is in a first state, the inside of the capillary needle is separated into two parts by the moving piece, and when the moving piece is in a second state, the two parts are communicated; the negative pressure generating module is communicated with a space between the moving member in the capillary needle in the first state and the first opening end of the capillary needle, so that the pressure in the space is lower than that outside the capillary needle; the driving module is used for rotating the capillary needle on a vertical surface, so that the second opening end of the capillary needle corresponds to the sample inlet of the mass spectrometer;
an electrode disposed at the second open end and adapted for connection to a power source;
the first moving unit is used for driving the bearing member or the conveying unit, so that the first open end of the capillary needle driven by the driving module faces any cell on the bearing member, and the distance between the first open end and the cell on the bearing member is 0-1 mm.
In order to reduce the influence of movement on the cells, the bearing piece and the sample inlet are further static, and the first moving unit drives the support to translate and rotate in a horizontal plane; alternatively, the first and second electrodes may be,
the sample inlet and the support are static, and the first moving unit drives the bearing piece to translate in the horizontal plane.
In order to realize the conversion between the first state and the second state, further, the shape of the moving part is matched with the inner wall of the capillary needle and is rotatably arranged in the capillary needle; the driving module drives the moving element to rotate forwards and backwards: the moving part is in a first state after rotating in the forward direction, and the moving part is in a second state after rotating in the reverse direction; alternatively, the first and second electrodes may be,
the moving member is selectively positioned in the capillary needle and moved out of the capillary needle under the driving of the driving module: the moving part is in the capillary needle and is in the first state, and the moving part is moved out of the capillary needle and is in the second state.
To form a stable single droplet, further, the single-cell mass spectrometry apparatus further comprises a droplet generation unit comprising:
a capillary tube adapted to contain an extraction fluid therein;
the emergent light of the laser irradiates the end part of the open end of the capillary;
a second moving unit for driving the carrier or the capillary so as to be on an upper side of any one of the cells on the carrier at the open end.
To drop a single droplet on a selected cell, further, the first moving unit moves the holder, the second moving unit moves the capillary, and the carrier is stationary; alternatively, the first and second electrodes may be,
the capillary and the sample inlet are static, and the first moving unit and the second moving unit are shared and move the bearing piece.
In order to form stable picoliter single liquid drops, the inner diameter of the capillary is 3-6 microns, the inner diameter of the opening end is 1-3 microns, the emergent light energy is 40-200 muJ, and the distance between the position of the emergent light irradiating the capillary and the opening end is 30-70 microns.
In order to realize automatic analysis, further, the driving module adopts a motor, and the moving member is driven by the motor.
Fig. 1 is a flow chart of a single cell analysis method according to an embodiment of the present invention, and as shown in fig. 1, the single cell analysis method includes:
the first moving unit drives the bearing piece or the bracket, and the bearing piece and the bracket move relatively;
the driving module drives the capillary needle to rotate in the forward direction, so that the first opening end is adjacent to the selected cells, and the selected cells are extracted by the extraction liquid;
the moving part is in a first state, and the negative pressure generating module works;
the extraction material of the selected cells passes through the first open end and into the space;
the driving module drives the capillary needle to rotate reversely, and the second opening end of the capillary needle corresponds to the sample inlet of the mass spectrometer;
the moving member is in a second state, and the extraction substances in the space flow downwards to the second opening end;
and discharging the electrode, wherein the extraction substance is ionized and sprayed out from the second opening end to enter the sample inlet.
In order to improve the extraction effect of the selected cells, further, the selected cells are extracted in a manner that:
the second moving unit drives the capillary or the bearing piece to be driven, and the capillary and the bearing piece move relatively, so that the outlet end of the capillary is positioned at the upper side of the selected cell;
the emergent light of the laser irradiates the end part of the outlet end of the capillary;
an expanded bubble is formed in the extraction fluid within the end portion, and the extraction fluid is pushed to drop from the outlet end of the capillary tube, and the formed drop is dropped on the selected cell, so that the selected cell is extracted.
In order to prevent the extraction material sucked into the capillary needle from leaking, further, the negative pressure generating module works continuously from the time of sucking the extraction material to the time of converting the moving member from the first state to the second state.
Example 2:
an application example of the single-cell mass spectrometry apparatus and method according to embodiment 1 of the present invention.
In the application example, the bearing member is of a matrix structure and is provided with rectangular grooves distributed in an array manner, and each groove is suitable for accommodating a single cell; the bearing piece is placed still;
the capillary tube is arranged on a second moving unit, such as a two-dimensional moving platform, and the outlet end of the capillary tube is positioned on the upper side of any selected cell under the driving of the second moving unit; the inner diameter of the capillary is 3-6 microns, such as 3 microns, 5 microns or 6 microns, the inner diameter of the opening end is 1-3 microns, such as 1 microns, 2 microns or 3 microns, the emergent light energy is 40-200 muJ, the position of the capillary irradiated by the emergent light is 30-70 microns away from the opening end, so that a skin-upgraded (1-600 pL) single liquid drop is formed from the outlet end of the capillary, accurately drops on the selected cell, wraps the selected cell and extracts the selected cell; the laser outputs pulse laser, and the pulse laser irradiates the capillary after beam expanding, collimation and reflection in sequence;
the capillary needle is rotatably arranged on the bracket and is driven by the first motor; the moving piece is rotatably arranged in the capillary needle, and is driven by a second motor outside the capillary needle to rotate in a forward direction, the edge of the moving piece is contacted and sealed with the inner wall of the capillary needle, namely the moving piece is in a first state, and the inside of the capillary needle is isolated into two parts; when the moving piece is driven by the second motor to rotate reversely, the edge of the moving piece is separated from contact with the inner wall of the capillary needle, namely the moving piece is in a second state, and the two parts in the capillary needle are communicated;
the negative pressure generating module adopts a suction pump and is communicated with the moving part in the first state and the space between the first opening ends of the capillary tubes through a hose;
an electrode disposed at the second open end and adapted to be connected to a high voltage power supply;
the first moving unit drives the support and is matched with the driving module in a driving mode, so that the first open end of the capillary needle faces any cell on the bearing piece, and the second open end of the capillary needle corresponds to a sample inlet of the mass spectrometer.
The single cell analysis method of the embodiment of the present invention, that is, the working method of the single cell mass spectrometry apparatus of the embodiment of the present invention, includes:
the second moving unit drives the capillary tube, and the capillary tube and the bearing piece move relatively, so that the outlet end of the capillary tube is positioned at the upper side of the selected cell, and the distance between the outlet end and the selected cell is larger than zero and smaller than or equal to 1mm, such as 0.1mm, 0.3mm, 0.6mm, 0.8mm and 1 mm;
the emergent light of the laser irradiates the end part of the outlet end of the capillary;
forming expanded bubbles in the extraction liquid in the end part, pushing the extraction liquid to drop from the outlet end of the capillary tube, and enabling the formed drops to drop on the selected cells and wrap the selected cells so as to extract the selected cells;
the first moving unit drives the bracket, and the bearing piece and the bracket move relatively;
the driving module drives the capillary needle to rotate in the positive direction, so that the first open end is adjacent to the extracted selected cells, and the height of the first open end is lower than that of the second open end;
the first motor drives the moving part to rotate in the positive direction, so that the moving part is in a first state, and the negative pressure generating module works;
the extraction material of the selected cells passes through the first open end and into the space;
the first moving unit drives the bracket, and the driving module drives the capillary needle to rotate reversely, so that the second opening end of the capillary needle corresponds to the sample inlet of the mass spectrometer, and the height of the second opening end is lower than that of the first opening end;
the first motor drives the moving piece to rotate reversely, so that the moving piece is in a second state, and the extraction substances in the space flow downwards to the second opening end;
discharging the electrode, wherein the extraction substance is ionized and sprayed out from the second opening end to enter the sample inlet;
when the moving member is switched from the first state to the second state from the extraction of the extraction material, the negative pressure generation module continuously works.
Fig. 2 is an actual experimental diagram of the picoliter-level single droplet encapsulated cells obtained in this embodiment, in which the cells are completely covered by the single droplet, so that the cells can be completely extracted, and meanwhile, the cells do not contact nearby cells, so that cross contamination is avoided, and the droplets have small volumes, so that the obtained extract has high actual detection substance concentration (more information in unit conditions), and is more convenient for mass spectrometry detection and analysis.
FIG. 3 is a primary mass spectrum of a single cell of the present example, from which a mass spectrum signal of spermine was obtained, and this signal further confirmed the spermine-based component in the single cell, which is an important substance for promoting cell proliferation. In addition, other substances which are not specifically concluded by scientists for a while, such as 278.69, 175.09 and the like, are detected, and can be further researched by using the single-cell mass spectrometry device, so that a data basis is provided for further single-cell metabonomics and molecular biology research, and a new chapter of single-cell metabonomics is opened.
Example 3:
the application example of the single-cell mass spectrometry device and method in embodiment 1 of the present invention is different from embodiment 2 in that:
1. the capillary tube is static; the second motor drives the capillary needle to rotate only in the vertical plane, so that the second opening end corresponds to the sample inlet of the mass spectrometer;
the first moving unit and the second moving unit are used in common, the carrier is driven to move in two dimensions in a horizontal plane so that the outlet end of the capillary tube is positioned on the upper side of any selected cell, and the first open end of the capillary needle is positioned adjacent to the selected cell under the cooperation of the second motor.
2. The moving member is selectively positioned in the capillary needle and moved out of the capillary needle under the drive of the first motor: the moving part is in the capillary needle and is in a first state: the inside of the capillary needle is separated into two parts, and the capillary needle is moved out to be in a second state: the two parts in the capillary needle are communicated; the first motor is connected with the moving piece in a gear transmission mode, and the moving piece moves perpendicular to the central axis of the capillary needle in a lead screw or slide rail transmission mode.
Example 4:
the application example of the single-cell mass spectrometry device and method in embodiment 1 of the present invention is different from embodiment 2 in that:
1. the moving part is selectively positioned in the capillary needle and moved out of the capillary needle under the driving of the screw rod: the moving part is in the capillary needle and is in a first state: the capillary needle is internally separated into two parts; moving out the capillary needle to be in a second state: the two parts in the capillary needle are communicated; the moving direction of the moving piece is vertical to the central axis of the capillary needle;
2. first motor and second motor sharing, the one end drive capillary needle of the axis of rotation of motor is rotatory, and simultaneously, the other end of axis of rotation is connected with the lead screw through flexible drive mechanism, and that is, only one motor has not only driven the rotation of capillary needle, has still driven the removal of moving member.
Claims (9)
1. The single-cell mass spectrometry device comprises a bearing piece and a mass spectrometer, wherein the bearing piece is used for bearing a plurality of cells which are separated from each other, and the mass spectrometer is provided with a sample inlet; characterized in that the single cell mass spectrometry device further comprises:
the device comprises a conveying unit, a control unit and a control unit, wherein the conveying unit comprises a capillary needle, a moving piece, a negative pressure generating module, a driving module and a bracket; the two ends of the capillary needle are open and are arranged on the bracket; when the moving piece is in a first state, the inside of the capillary needle is separated into two parts by the moving piece, and when the moving piece is in a second state, the two parts are communicated; the negative pressure generating module is communicated with a space between the moving member in the capillary needle in the first state and the first opening end of the capillary needle, so that the pressure in the space is lower than that outside the capillary needle; the driving module is used for rotating the capillary needle on a vertical surface, so that the second opening end of the capillary needle corresponds to the sample inlet of the mass spectrometer;
an electrode disposed at the second open end and adapted for connection to a power source;
a first moving unit, configured to drive the carrier or the transport unit, so that a distance between the first open end of the capillary needle driven by the driving module and the cell on the carrier is 0-1 mm;
a droplet generation unit comprising:
a capillary tube adapted to contain an extraction fluid therein;
the emergent light of the laser irradiates the end part of the open end of the capillary;
a second moving unit for driving the carrier or the capillary so as to be on an upper side of any one of the cells on the carrier at the open end.
2. The single-cell mass spectrometry apparatus of claim 1, wherein the carrier and the sample inlet are stationary, and the first movement unit drives the support to translate and rotate in a horizontal plane; alternatively, the first and second electrodes may be,
the sample inlet and the support are static, and the first moving unit drives the bearing piece to translate in the horizontal plane.
3. The single-cell mass spectrometry apparatus of claim 1, wherein the moving member is shaped to match an inner wall of the capillary needle and is rotatably disposed within the capillary needle; the driving module drives the moving element to rotate forwards and backwards: the moving part is in a first state after rotating in the forward direction, and the moving part is in a second state after rotating in the reverse direction; alternatively, the first and second electrodes may be,
the moving member is selectively positioned in the capillary needle and moved out of the capillary needle under the driving of the driving module: the moving part is in the capillary needle and is in the first state, and the moving part is moved out of the capillary needle and is in the second state.
4. The single-cell mass spectrometry apparatus of claim 1, wherein the first movement unit moves the carriage, the second movement unit moves the capillary, and the carrier is stationary; alternatively, the first and second electrodes may be,
the capillary and the sample inlet are static, and the first moving unit and the second moving unit are shared and move the bearing piece.
5. The single-cell mass spectrometry apparatus according to claim 1, wherein the inner diameter of the capillary is 3 to 6 μm, the inner diameter of the open end is 1 to 3 μm, the energy of the emitted light is 4 to 200 μ J, and the position of the capillary irradiated with the emitted light is 3 to 10 μm from the open end.
6. The single-cell mass spectrometry apparatus of claim 1, wherein the drive module employs a motor, and the moving member is driven by the motor.
7. The single-cell mass spectrometry apparatus of any one of claims 1 to 6, wherein the single-cell analysis method comprises:
the first moving unit drives the bearing piece or the bracket, and the bearing piece and the bracket move relatively;
the driving module drives the capillary needle to rotate in the forward direction, so that the first opening end is adjacent to the selected cells, and the selected cells are extracted by the extraction liquid;
the moving part is in a first state, and the negative pressure generating module works;
the extraction material of the selected cells passes through the first open end and into the space;
the driving module drives the capillary needle to rotate reversely, and the second opening end of the capillary needle corresponds to the sample inlet of the mass spectrometer;
the moving member is in a second state, and the extraction substances in the space flow downwards to the second opening end;
and discharging the electrode, wherein the extraction substance is ionized and sprayed out from the second opening end to enter the sample inlet.
8. The single cell analysis method of claim 7, wherein the selected cells are extracted by:
the second moving unit drives the capillary or the bearing piece to be driven, and the capillary and the bearing piece move relatively, so that the outlet end of the capillary is positioned at the upper side of the selected cell;
the emergent light of the laser irradiates the end part of the outlet end of the capillary;
an expanded bubble is formed in the extraction fluid within the end portion, and the extraction fluid is pushed to drop from the outlet end of the capillary tube, and the formed drop is dropped on the selected cell, so that the selected cell is extracted.
9. The single-cell analysis method according to claim 7, wherein the negative pressure generation module is continuously operated from the time of sucking the extraction material to the time of switching the moving member from the first state to the second state.
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Denomination of invention: Single cell mass spectrometry analysis device and method Effective date of registration: 20220119 Granted publication date: 20210205 Pledgee: China Construction Bank Co.,Ltd. Ningbo housing and urban construction sub branch Pledgor: CHINA INNOVATION INSTRUMENT Co.,Ltd. Registration number: Y2022330000127 |
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