CN116403883A - Mass spectrum detection device and method for single cells - Google Patents
Mass spectrum detection device and method for single cells Download PDFInfo
- Publication number
- CN116403883A CN116403883A CN202111615141.0A CN202111615141A CN116403883A CN 116403883 A CN116403883 A CN 116403883A CN 202111615141 A CN202111615141 A CN 202111615141A CN 116403883 A CN116403883 A CN 116403883A
- Authority
- CN
- China
- Prior art keywords
- fluorescence
- laser
- sample
- pulse laser
- electrode
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 40
- 238000001819 mass spectrum Methods 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 19
- 150000002500 ions Chemical class 0.000 claims abstract description 34
- 239000002207 metabolite Substances 0.000 claims abstract description 27
- 230000007246 mechanism Effects 0.000 claims abstract description 22
- 239000012634 fragment Substances 0.000 claims abstract description 21
- 238000010494 dissociation reaction Methods 0.000 claims abstract description 7
- 230000005593 dissociations Effects 0.000 claims abstract description 7
- 239000002245 particle Substances 0.000 claims abstract description 5
- 238000004949 mass spectrometry Methods 0.000 claims description 32
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 14
- 230000003287 optical effect Effects 0.000 claims description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- 239000007789 gas Substances 0.000 claims description 10
- 239000013307 optical fiber Substances 0.000 claims description 9
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 235000019253 formic acid Nutrition 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 230000033001 locomotion Effects 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 230000005684 electric field Effects 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 230000029918 bioluminescence Effects 0.000 claims description 2
- 238000005415 bioluminescence Methods 0.000 claims description 2
- 238000007599 discharging Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 82
- 238000004458 analytical method Methods 0.000 abstract description 21
- 238000000132 electrospray ionisation Methods 0.000 abstract description 14
- 238000005516 engineering process Methods 0.000 abstract description 13
- 210000000805 cytoplasm Anatomy 0.000 abstract description 6
- 238000001727 in vivo Methods 0.000 abstract description 4
- 230000004907 flux Effects 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 56
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 239000003550 marker Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000000053 physical method Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 210000004895 subcellular structure Anatomy 0.000 description 2
- 108010026552 Proteome Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000026058 directional locomotion Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000010249 in-situ analysis Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002705 metabolomic analysis Methods 0.000 description 1
- 230000001431 metabolomic effect Effects 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 239000012855 volatile organic compound Substances 0.000 description 1
Images
Classifications
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/0027—Methods for using particle spectrometers
-
- 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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
-
- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
-
- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
-
- 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
- G01N27/64—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 using wave or particle radiation to ionise a gas, e.g. in an ionisation chamber
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/0027—Methods for using particle spectrometers
- H01J49/0031—Step by step routines describing the use of the apparatus
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
- H01J49/0459—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for solid samples
- H01J49/0463—Desorption by laser or particle beam, followed by ionisation as a separate step
-
- 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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N2021/0106—General arrangement of respective parts
- G01N2021/0112—Apparatus in one mechanical, optical or electronic block
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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
Abstract
The invention provides a single-cell mass spectrum detection device and method, and relates to the technical field of single-cell mass spectrum detection. The device comprises a sealed cavity, a sample injector, a pulse laser, a nano-liter electrospray mechanism, an electrode assembly and a mass spectrum sample inlet; two opposite sides of the sealed cavity are respectively provided with a sample inlet and a discharge outlet; the sample injector is inserted into the sealed cavity through the sample inlet and used for focusing single-cell particles into a beam; the pulse laser is used for carrying out laser dissociation on single cells to form cell fragments and metabolites; the nano-liter electrospray mechanism is used for ionizing cell fragments and metabolites to form sample ions; the electrode assembly is arranged in the sealed cavity and is used for dragging sample ions to enter the mass spectrum sample inlet; the mass spectrum sample inlet is communicated with the sealed cavity. The method can effectively realize analysis of single cell cytoplasm or in-vivo metabolites by treating through preionization and secondary electrospray ionization technologies, and has the advantages of rapidness, high flux, multi-component analysis and the like.
Description
Technical Field
The invention relates to the technical field of single-cell mass spectrometry detection, in particular to a single-cell mass spectrometry detection device and method.
Background
The improvement of the research capability of single cells can greatly promote the development of basic research of clinical medicine and life science. However, for the research of single-cell proteome and metabolome, the conventional analysis and detection means have the defects of insufficient sensitivity, difficult multi-component analysis and the like when being applied to single-cell analysis due to extremely low content of substances to be detected.
In recent years, mass spectrometry has been widely used as a detection technique with high sensitivity in single-cell metabolomics and proteomics analysis. The most widely used of these are typically flow cytometry in combination with inductively coupled plasma mass spectrometry (ICP-MS), whereby detection of single cell body samples is achieved by means of a metal probe label. However, the difficulty of multi-component labeling of metal probes makes it disadvantageous for high-throughput, multi-channel rapid detection. In order to achieve simultaneous detection of multiple components in single cells, open ion sources based on electrospray ionization (ESI) have been developed. The electrospray ionization technology is a soft ionization technology, can directly obtain molecular ion peaks of a sample, and is beneficial to spectrogram analysis. Currently, single cell electrospray mass spectrometry-based methods are largely divided into two forms. The first method is to extract the cytoplasmic substances by using the extraction substances and then to perform electrospray analysis by pretreatment; the second is to extract cytoplasm directly by physical method (such as probe) without pretreatment and then to conduct electrospray analysis. These methods all limit high throughput analysis of single cell electrospray mass spectrometry.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a mass spectrum detection device and a mass spectrum detection method for single cells.
The invention is realized in the following way:
in a first aspect, the invention provides a mass spectrometry detection device for single cells, comprising a sealed cavity, a sample injector, a pulse laser, a nano-liter electrospray mechanism, an electrode assembly and a mass spectrometry sample inlet;
two opposite sides of the sealed cavity are respectively provided with a sample inlet and a discharge outlet for entering and discharging single cell airflow;
the sample injector is used for focusing single-cell particles into a beam, and is inserted into the sealed cavity through the sample inlet;
the pulse laser is used for generating pulse laser to carry out laser dissociation on single cells to form cell fragments and metabolites;
the nano-liter electrospray mechanism is used for ionizing the cell fragments and the metabolites to form sample ions;
the electrode assembly is arranged in the sealed cavity and is used for generating a pulse electric field to draw the sample ions into the mass spectrum sample inlet;
and the mass spectrum sample inlet is communicated with the sealed cavity.
In an alternative embodiment, the nano-liter electrospray mechanism comprises a reagent bottle, a high-pressure gas pipe, a high-pressure electrode and a capillary, wherein the high-pressure gas pipe, the high-pressure electrode and the capillary are all inserted into the reagent bottle, the reagent bottle is filled with a formic acid aqueous solution, and the capillary is inserted into the sealed cavity.
In an alternative embodiment, the volume percentage concentration of the formic acid aqueous solution is 0.1-0.2%, nitrogen with the pressure of 0.11-0.15MPa is introduced into the high-pressure gas pipe, and a voltage of 3-5KV is applied to the high-pressure electrode.
In an alternative embodiment, the mass spectrometry detection device of single cells further comprises a continuous laser and a fluorescence detector, the continuous laser and the fluorescence detector being located between the sample injector and the pulsed laser;
the continuous laser is used for exciting single cells marked by fluorescence so that the single cells generate specific biological fluorescence;
the fluorescence detector is used for detecting the biological fluorescence and sending a signal to the pulse laser after detecting a fluorescence pulse signal.
In an alternative embodiment, the spacing between the continuous laser and the pulsed laser is 0.5-1.5mm;
in an alternative embodiment, the fluorescence detector comprises a focusing lens, an optical fiber, an optical filter and a photomultiplier, wherein the focusing lens is vertically arranged in the direction of continuous laser emitted by the continuous laser, the focusing lens collects fluorescence and scattered light, the optical fiber guides the collected fluorescence and scattered light into the optical filter, the optical filter removes the scattered light, and a fluorescence signal enters the photomultiplier and is converted into a fluorescence pulse signal to be sent to the pulse laser.
In alternative embodiments, the sample injector is a nozzle or an aerodynamic lens.
In a second aspect, the present invention provides a method for mass spectrometry detection of single cells, comprising performing with a mass spectrometry detection device of single cells as described in the previous embodiments; the method comprises the following steps:
focusing and introducing gaseous single suspension cells into the sealed cavity through the injector; the focused single cells move downwards along the central axis direction of the sample injector;
the single cells in motion are dissociated by the pulsed laser generated by the pulsed laser into cell fragments and metabolites;
the formed cell fragments and metabolites are ionized by electrospray sprayed by the nano-liter electrospray mechanism to form sample ions;
the formed sample ions are drawn into the mass spectrometry sample inlet via the electrode assembly.
In an alternative embodiment, the gaseous single suspension cells have a fluorescent label;
the moving single cell, before being dissociated by the pulse laser, further comprises exciting the fluorescent-labeled single cell with a continuous laser to cause the single cell to generate bioluminescence;
detecting the biological fluorescence by adopting a fluorescence detector;
and after the fluorescence detector detects the biological fluorescence, a signal is sent to the pulse laser, and the pulse laser generates pulse laser to dissociate the single cells.
In an alternative embodiment, the electrode assembly comprises a first electrode and a second electrode which are oppositely arranged, and the second electrode is arranged at the mass spectrum sample inlet; the electrode assembly drawing the sample ions into the mass spectrometry sample inlet comprises: before the pulse laser generates pulse laser, the first electrode is adjusted to be low level, and the second electrode is adjusted to be high level; and when the pulse laser generates pulse laser, the first electrode is regulated to be at a high level, and the second electrode is regulated to be at a low level.
The invention has the following beneficial effects: the mass spectrum detection device and the mass spectrum detection method for the single cells are characterized in that high-flux sample injection is carried out by adopting an aerodynamic technology, and then a pulse laser and a nano-liter electrospray mechanism are combined, wherein the pulse laser preionizes the single cells to form cell fragments and metabolites; then, the nano liter electrospray ionization technology is used for processing, so that cell fragments and metabolites can form sample ions, analysis of single cell cytoplasm or in-vivo metabolites can be effectively realized, and the method has the advantages of rapidness, high flux, multi-component analysis and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic structural diagram of a mass spectrum detection device for single cells according to a first embodiment of the present application;
FIG. 2 is a schematic structural diagram of a single cell mass spectrum detection device according to the first embodiment of the present application when a continuous laser and a fluorescence detector are combined;
FIG. 3 is a schematic structural diagram of a nano-liter electrospray mechanism of a single-cell mass spectrometry detection device according to a first embodiment of the present application;
FIG. 4 is a timing chart of operation of the fluorescence detector, the pulse laser, the first electrode and the second electrode in the single-cell mass spectrometry detection device according to the first embodiment of the present application in a linkage mode.
Icon: 100-mass spectrometry detection device of single cells; 110-sealing the cavity; 111-sample inlet; 120-sample injector; 130-a continuous laser; 140-fluorescence detector; 141-a focusing lens; 142-optical fiber; 143-an optical filter; 144-photomultiplier tubes; 150-pulse laser; 160-nanoliter electrospray mechanism; 161-reagent bottles; 162-high pressure gas pipe; 163-high voltage electrode; 164-capillary; 170-an electrode assembly; 171-a first electrode; 172-a second electrode; 180-mass spectrum sample inlet.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.
In the description of the present invention, it should be noted that, if the terms "upper", "lower", "inner", "outer", and the like indicate an azimuth or a positional relationship based on the azimuth or the positional relationship shown in the drawings, or the azimuth or the positional relationship in which the inventive product is conventionally put in use, it is merely for convenience of describing the present invention and simplifying the description, and it is not indicated or implied that the apparatus or element referred to must have a specific azimuth, be configured and operated in a specific azimuth, and thus it should not be construed as limiting the present invention.
Furthermore, the terms "first," "second," and the like, if any, are used merely for distinguishing between descriptions and not for indicating or implying a relative importance.
It should be noted that the features of the embodiments of the present invention may be combined with each other without conflict.
First embodiment
Referring to fig. 1, the present embodiment provides a single cell mass spectrum detection apparatus 100, which includes a sealed cavity 110, a sample injector 120, a continuous laser 130, a fluorescence detector 140, a pulse laser 150, a nano-liter electrospray mechanism 160, an electrode assembly 170, and a mass spectrum sample inlet 180.
The sealed cavity 110 is used for guiding the single-cell airflow to enter and discharge, and two opposite sides of the sealed cavity 110 are respectively provided with a sample inlet 111 and a discharge outlet (not shown) for the single-cell airflow to enter and discharge.
The injector 120 is inserted into the sealed cavity 110 through the injection port 111, thereby injecting single cell particles into the sealed cavity 110. The injector 120 in this application may be a nozzle or an aerodynamic lens that functions to aerodynamically focus single cell particles into bundles for later fluorescence analysis and laser dissociation.
The continuous laser 130 is used for exciting the single cells marked by fluorescence, so that the single cells generate specific biological fluorescence; the continuous laser 130 in this application may be selected from a variety of sources including, but not limited to, lasers with wavelengths 355nm, 405nm, 633 nm. Specifically, the wavelength of the continuous laser light emitted by the continuous laser 130 may be selected according to the fluorescent marker, and after the continuous laser light is emitted by the continuous laser 130, the continuous laser light may be focused into a spot of about 300 μm by the focusing lens 141, and the focused spot is on the moving track of the single cell after passing through the injector 120.
The fluorescence detector 140 is used to detect bio-fluorescence and send a signal to the pulse laser 150 after detecting a fluorescence pulse signal. In this application, fluorescence detector 140 may collect bio-fluorescence and scattered light, and then remove scattered light from the continuous laser light through an optical filter (e.g., filter 143) to detect the excited fluorescence.
Specifically, referring to fig. 2, the fluorescence detector 140 in the present application includes a focusing lens 141, an optical fiber 142, an optical filter 143 and a photomultiplier 144, the focusing lens 141 is vertically disposed in a direction of continuous laser light emitted by the continuous laser 130, the focusing lens 141 collects fluorescence and scattered light, a probe of the optical fiber 142 introduces the collected fluorescence and scattered light into the optical filter 143, the optical filter 143 removes the scattered light, and a fluorescence signal enters the photomultiplier 144 and is converted into a fluorescence pulse signal to be sent to the pulse laser 150. The fluorescent detector 140 is provided to clearly know when a single cell arrives at the pulsed laser, so that the single cell can be hit when the pulsed laser 150 is emitted. After the fluorescence detector 140 detects that a single cell arrives, a fluorescence pulse signal is sent to the pulse laser to trigger, and the pulse laser emits laser after receiving the fluorescence pulse signal.
The pulse laser 150 is used for generating pulse laser after receiving the fluorescent pulse signal to laser-dissociate single cells to form cell fragments and metabolites; the spacing between the continuous laser 130 and the pulsed laser 150 is 0.5-1.5mm; the pulse laser 150 in the present application is a high-energy pulse laser 150, which has an extremely high response speed, after the fluorescence detector 140 detects the bio-fluorescence, a signal is sent to the pulse laser 150, the pulse laser 150 receives the signal and emits laser light within a few microseconds, and single cells are subjected to laser dissociation to form cell fragments or other subcellular structures, so that rapid mass spectrum structural analysis can be performed. Because the sealed cavity 110 is in the atmospheric pressure condition in this application, the pulsed laser 150 in this application generates a smaller amount of ions at the atmospheric pressure, and is mainly used to ablate dissociated cells, resulting in the formation of cell debris and metabolites or other subcellular structures. Because the amount of ions excited by the pulse laser 150 is small, the pulse laser 150 can be prevented from directly dissociating single cells to form too small ions, which is not easy for subsequent mass spectrometry.
It should be noted that, since the pulsed laser light generated by the pulsed laser 150 may also excite the fluorescent marker, but the pulsed laser 150 is different from the fluorescent light generated by the continuous laser 130, by selecting the appropriate aperture of the optical fiber 142, only the light spot of the continuous laser 130 exciting the fluorescent marker may be detected by the fluorescent detector 140, and the light spot of the pulsed laser 150 exciting the fluorescent marker may not be detected by the fluorescent detector 140, so that the fluorescent detector 140 may be effectively prevented from being triggered secondarily. The specific aperture of the optical fiber 142 is not limited in this application, and may be determined according to the selection of the fluorescent marker, the pulsed laser 150, and the link laser in practical applications.
The nanoliter electrospray mechanism 160 is used to ionize cell debris and metabolites to form sample ions. The nano-liter electrospray mechanism 160 provided herein is a primary ion generation module that ionizes cell debris and metabolites to form sample ions for subsequent mass spectrometry.
In this application, referring to fig. 3, the nano-liter electrospray mechanism 160 includes a reagent bottle 161, a high-pressure gas tube 162, a high-pressure electrode 163 and a capillary tube 164, the high-pressure gas tube 162, the high-pressure electrode 163 and the capillary tube 164 are all inserted into the reagent bottle 161, the reagent bottle 161 is filled with a formic acid aqueous solution, and the capillary tube 164 is inserted into the sealed cavity 110. Wherein, the volume percentage concentration of the formic acid aqueous solution is 0.1-0.2%, nitrogen with the pressure of 0.11-0.15MPa is introduced into the high-pressure gas tube 162, 3-5KV voltage is applied to the high-pressure electrode 163, the formic acid aqueous solution can form electrospray under the action of high voltage and high pressure to be discharged from the capillary 164, and the electrospray can ionize cell fragments and metabolites in the sealed cavity 110 to form sample ions.
The nano-liter electrospray ionization technology is developed based on ESI, and has the advantages of low sample amount, high ionization efficiency, capability of simultaneously analyzing various samples and the like. The secondary electrospray ionization technology (SESI) formed by the nanoliter electrospray ionization technology can realize in-situ analysis of samples, and is already applied to the aspects of VOCs detection, aerosol extraction analysis and the like. However, few analyses directed to single cells have been studied directly using SESI sources, and it is difficult to directly extract the cytoplasmic material by secondary electrospray due mainly to the influence of cell structure. In this application, the single cells are laser dissociated by the pulse laser 150 to form cell fragments and metabolites, and then the cell fragments and the metabolites are ionized by the nano-liter electrospray mechanism 160 to form sample ions, so that the nano-liter electrospray mechanism 160 can effectively obtain substances in cytoplasm, and the size of the sample ions is better controlled, so that the subsequent multi-component mass spectrometry is facilitated.
The electrode assembly 170 is disposed in the sealed cavity 110, and the electrode assembly 170 is configured to generate a pulsed electric field to draw sample ions into the mass spectrometry sample inlet 180.
In the present application, the electrode assembly 170 includes a first electrode 171 and a second electrode 172 disposed opposite to each other, and the second electrode 172 is disposed at the mass spectrum sample inlet 180; the directional movement of ions can be realized by adjusting the output levels of the first electrode 171 and the second electrode 172, specifically, please refer to fig. 4, the ions move along the direction from the high level to the low level, when the ions do not need to enter the mass spectrum sample inlet 180, the first electrode 171 is set to be the low level, the second electrode 172 is the high level, at this time, the ions enter the mass spectrum sample inlet 180 to be blocked, when the ions need to enter the mass spectrum sample inlet 180, the first electrode 171 is set to be the high level, the second electrode 172 is the low level, and thus the ions are pulled to enter the mass spectrum sample inlet 180 by adjusting the levels of the first electrode 171 and the second electrode 172, so as to perform mass spectrum detection.
In this application, the mass spectrum sample inlet 180 is communicated with the sealed cavity 110, and the other end of the mass spectrum sample inlet 180 is communicated with a mass spectrometer, and the mass spectrum sample inlet 180 in this application is a universal mass spectrometer interface for introducing atmospheric pressure ions into the mass spectrometer in the vacuum system for analysis. The inlet flow of the common atmospheric pressure interface is 1-2L/min.
The method combines aerodynamic sample injection technology, fluorescence detection, pulsed laser pre-dissociation and secondary electrospray ionization, and realizes single-cell high-flux, multi-component and high-sensitivity mass spectrum detection. The single-cell high-flux sample injection is performed by an aerodynamic lens sample injection technology, the single-cell movement track is further detected by fluorescence detection, the cell dissociation is further realized by pulse laser, and finally the multi-component and high-sensitivity detection of the single cell is realized by a high-ionization-efficiency secondary electrospray ionization technology.
The fluorescence detector 140 is adopted to track single cells, the pulse laser 150 is adopted to conduct pre-ionization, then the nano-liter electrospray mechanism 160 is utilized to conduct secondary electrospray ionization, single cell cytoplasm or in-vivo metabolite analysis can be achieved, and analysis speed is high. Compared with a pretreatment method or a physical method for analysis, the method has the advantages of rapidness, high throughput, multi-component analysis and the like.
Second embodiment
The present embodiment provides a mass spectrometry detection method of a single cell, which is performed using the mass spectrometry detection apparatus 100 of a single cell provided in the first embodiment. Specifically, the method for mass spectrometry detection of single cells provided in this embodiment includes the following steps:
s1, forming gaseous single suspension cells by atomizing single cell suspension with fluorescent markers, focusing the gaseous single suspension cells by a sample injector 120 and introducing the gaseous single suspension cells into a sealed cavity 110; the focused single cells move downward along the central axis direction of the injector 120;
s2, exciting the single cells marked by fluorescence by adopting a continuous laser 130 to enable the single cells to generate biological fluorescence;
s3, detecting biological fluorescence by adopting a fluorescence detector 140;
s4, after the fluorescence detector 140 detects biological fluorescence, a signal is sent to the pulse laser 150, and the pulse laser 150 generates pulse laser to dissociate single cells to form cell fragments and metabolites;
s5, ionizing formed cell fragments and metabolites by electrospray sprayed by the nano-liter electrospray mechanism 160 to form sample ions;
s6, before the pulse laser 150 generates the pulse laser, the first electrode 171 is adjusted to be at a low level, and the second electrode 172 is adjusted to be at a high level; after the pulse laser 150 generates the pulse laser light, the first electrode 171 is adjusted to a high level, and the second electrode 172 is adjusted to a low level. The formed sample ions are drawn through electrode assembly 170 into mass spectrometry sample inlet 180.
According to the mass spectrum detection method of the single cells, the aerodynamic technology is adopted for sample injection, then the continuous laser 130 and the fluorescence detector 140 are combined to judge the motion trail of the single cells, after the fluorescence detector 140 detects biological fluorescence, signals are sent to the pulse laser 150, and the pulse laser 150 generates pulse laser to dissociate the single cells so as to form cell fragments and metabolites; in the application, the pulse laser 150 preionizes single cells, and then the single cells are treated by using the nano-liter electrospray ionization mechanism 160 to perform secondary electrospray ionization technology, so that cell fragments and metabolites can form sample ions, analysis of single cell cytoplasm or in-vivo metabolites can be effectively realized, and the method has the advantages of rapidness, high flux, multi-component analysis and the like.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention, are intended to be included within the scope of the present invention.
Claims (10)
1. The mass spectrum detection device for the single cells is characterized by comprising a sealed cavity, a sample injector, a pulse laser, a nano-liter electrospray mechanism, an electrode assembly and a mass spectrum sample inlet;
two opposite sides of the sealed cavity are respectively provided with a sample inlet and a discharge outlet for entering and discharging single cell airflow; the sample injector is used for focusing single-cell particles into a beam, and is inserted into the sealed cavity through the sample inlet; the pulse laser is used for generating pulse laser to carry out laser dissociation on single cells to form cell fragments and metabolites; the nano-liter electrospray mechanism is used for ionizing the cell fragments and the metabolites to form sample ions; the electrode assembly is arranged in the sealed cavity and is used for generating a pulse electric field to draw the sample ions into the mass spectrum sample inlet; and the mass spectrum sample inlet is communicated with the sealed cavity.
2. The mass spectrometry detection device for single cells according to claim 1, wherein the nano-liter electrospray mechanism comprises a reagent bottle, a high-pressure gas tube, a high-pressure electrode and a capillary tube, wherein the high-pressure gas tube, the high-pressure electrode and the capillary tube are all inserted into the reagent bottle, the reagent bottle is filled with a formic acid aqueous solution, and the capillary tube is inserted into the sealed cavity.
3. The mass spectrometry detection device for single cells according to claim 2, wherein the volume percentage concentration of the formic acid aqueous solution is 0.1-0.2%, nitrogen with the pressure of 0.11-0.15MPa is introduced into the high-pressure gas pipe, and a voltage of 3-5KV is applied to the high-pressure electrode.
4. The single cell mass spectrometry detection apparatus of claim 1, further comprising a continuous laser and a fluorescence detector, the continuous laser and the fluorescence detector being located between the sample injector and the pulsed laser;
the continuous laser is used for exciting single cells marked by fluorescence so that the single cells generate specific biological fluorescence;
the fluorescence detector is used for detecting the biological fluorescence and sending a signal to the pulse laser after detecting a fluorescence pulse signal.
5. The single cell mass spectrometry detection apparatus according to claim 4, wherein a distance between the continuous laser and the pulsed laser is 0.5 to 1.5mm.
6. The apparatus according to claim 4, wherein the fluorescence detector comprises a focusing lens, an optical fiber, an optical filter and a photomultiplier, wherein the focusing lens is vertically arranged in a direction of continuous laser light emitted by the continuous laser, the focusing lens collects fluorescence and scattered light, the optical fiber introduces the collected fluorescence and scattered light into the optical filter, the optical filter removes the scattered light, and a fluorescence signal enters the photomultiplier and is converted into a fluorescence pulse signal to be sent to the pulse laser.
7. The single cell mass spectrometry detection apparatus of claim 1, wherein the sample injector is a nozzle or an aerodynamic lens.
8. A method for mass spectrometry of single cells, comprising using the single cell mass spectrometry device of claim 1; the method comprises the following steps:
focusing and introducing gaseous single suspension cells into the sealed cavity through the injector; the focused single cells move downwards along the central axis direction of the sample injector;
the single cells in motion are dissociated by the pulsed laser generated by the pulsed laser into cell fragments and metabolites;
the formed cell fragments and metabolites are ionized by electrospray sprayed by the nano-liter electrospray mechanism to form sample ions;
the formed sample ions are drawn into the mass spectrometry sample inlet via the electrode assembly.
9. The method for mass spectrometry detection of single cells according to claim 8, wherein the single suspension cells in a gaseous state have fluorescent markers;
the moving single cell, before being dissociated by the pulse laser, further comprises exciting the fluorescent-labeled single cell with a continuous laser to cause the single cell to generate bioluminescence;
detecting the biological fluorescence by adopting a fluorescence detector;
and after the fluorescence detector detects the biological fluorescence, a signal is sent to the pulse laser, and the pulse laser generates pulse laser to dissociate the single cells.
10. The method of claim 8, wherein the electrode assembly comprises a first electrode and a second electrode disposed opposite each other, the second electrode disposed at the mass spectrometry inlet; the electrode assembly drawing the sample ions into the mass spectrometry sample inlet comprises: before the pulse laser generates pulse laser, the first electrode is adjusted to be low level, and the second electrode is adjusted to be high level; and when the pulse laser generates pulse laser, the first electrode is regulated to be at a high level, and the second electrode is regulated to be at a low level.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111615141.0A CN116403883A (en) | 2021-12-27 | 2021-12-27 | Mass spectrum detection device and method for single cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111615141.0A CN116403883A (en) | 2021-12-27 | 2021-12-27 | Mass spectrum detection device and method for single cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116403883A true CN116403883A (en) | 2023-07-07 |
Family
ID=87018454
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111615141.0A Pending CN116403883A (en) | 2021-12-27 | 2021-12-27 | Mass spectrum detection device and method for single cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116403883A (en) |
-
2021
- 2021-12-27 CN CN202111615141.0A patent/CN116403883A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9146180B2 (en) | Systems and methods for laser assisted sample transfer to solution for chemical analysis | |
| JP4982635B2 (en) | Flow cytometry method and apparatus linked to elemental analysis | |
| US6906322B2 (en) | Charged particle source with droplet control for mass spectrometry | |
| US6797947B2 (en) | Internal introduction of lock masses in mass spectrometer systems | |
| US7078679B2 (en) | Inductive detection for mass spectrometry | |
| US10622199B2 (en) | Laser ablation system | |
| US20140283628A1 (en) | Apparatus and method for liquid sample introduction | |
| JP4159252B2 (en) | Atmospheric particle analyzer | |
| US20130306855A1 (en) | Efficient detection of ion species utilizing fluorescence and optics | |
| Allen et al. | Mass spectrometric analyzer for individual aerosol particles | |
| CN108695135B (en) | Ion source and method for generating elemental ions from aerosol particles | |
| CN110333282B (en) | Flow type single-cell spectrum analyzer and using method thereof | |
| US20230215714A1 (en) | Plasma and sampling geometries for imaging mass cytometry | |
| CN102854240A (en) | Ion trap mass spectrometer for organic aerosol ionized by vacuum ultraviolet light | |
| CN108630517B (en) | Method and device for plasma ionization of atmospheric particulates | |
| CN101290304A (en) | Substance assistant laser desorpted ionized biological aerosol mass spectrometer | |
| JP2015511704A (en) | Improved interface for mass spectrometer equipment | |
| CN208187913U (en) | Atmospheric particulates heavy metal and its isotope on-line water flushing device | |
| CN116403883A (en) | Mass spectrum detection device and method for single cells | |
| CN114018883B (en) | Flow cytometry multi-spectrum analyzer and application method thereof | |
| JPH1137974A (en) | Particle composition analyzer | |
| US11651945B2 (en) | Rapid droplet introduction interface (RDII) for mass spectrometry | |
| GB2554430A (en) | Apparatus and method for detecting molecular chirality | |
| CN117686481A (en) | Aerosol isotope single particle detection device based on laser multispectral | |
| JP2005283391A (en) | Liquid chromatograph mass spectrometer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |