WO2023193483A1 - 用于质谱成像样本制备的自动电喷雾喷涂装置及方法 - Google Patents
用于质谱成像样本制备的自动电喷雾喷涂装置及方法 Download PDFInfo
- Publication number
- WO2023193483A1 WO2023193483A1 PCT/CN2022/142308 CN2022142308W WO2023193483A1 WO 2023193483 A1 WO2023193483 A1 WO 2023193483A1 CN 2022142308 W CN2022142308 W CN 2022142308W WO 2023193483 A1 WO2023193483 A1 WO 2023193483A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- spray capillary
- automatic
- sample preparation
- mass spectrometry
- matrix
- Prior art date
Links
- 238000005507 spraying Methods 0.000 title claims abstract description 77
- 238000003384 imaging method Methods 0.000 title claims abstract description 69
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000001819 mass spectrum Methods 0.000 title abstract description 5
- 239000007921 spray Substances 0.000 claims abstract description 120
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 92
- 238000001212 derivatisation Methods 0.000 claims abstract description 80
- 239000011159 matrix material Substances 0.000 claims abstract description 75
- 238000002360 preparation method Methods 0.000 claims abstract description 55
- 238000002347 injection Methods 0.000 claims abstract description 30
- 239000007924 injection Substances 0.000 claims abstract description 30
- 230000007246 mechanism Effects 0.000 claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 230000008569 process Effects 0.000 claims abstract description 9
- 238000004949 mass spectrometry Methods 0.000 claims description 69
- 238000010438 heat treatment Methods 0.000 claims description 22
- 239000011521 glass Substances 0.000 claims description 11
- 238000001035 drying Methods 0.000 claims description 7
- 238000009434 installation Methods 0.000 claims description 5
- 238000003032 molecular docking Methods 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 4
- 239000004020 conductor Substances 0.000 claims description 4
- 239000012212 insulator Substances 0.000 claims description 4
- 239000002699 waste material Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 230000013011 mating Effects 0.000 claims 2
- 239000000243 solution Substances 0.000 abstract description 25
- 238000011065 in-situ storage Methods 0.000 abstract description 11
- 238000002425 crystallisation Methods 0.000 abstract description 7
- 230000008025 crystallization Effects 0.000 abstract description 7
- 239000013078 crystal Substances 0.000 abstract description 6
- 238000009827 uniform distribution Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 42
- 238000005516 engineering process Methods 0.000 description 30
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 15
- 239000000126 substance Substances 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 238000000576 coating method Methods 0.000 description 5
- 230000005684 electric field Effects 0.000 description 5
- 239000011248 coating agent Substances 0.000 description 4
- 238000001704 evaporation Methods 0.000 description 4
- 230000008020 evaporation Effects 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000003795 desorption Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000007781 pre-processing Methods 0.000 description 3
- 206010018338 Glioma Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010146 3D printing Methods 0.000 description 1
- 239000004696 Poly ether ether ketone Substances 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- JUPQTSLXMOCDHR-UHFFFAOYSA-N benzene-1,4-diol;bis(4-fluorophenyl)methanone Chemical compound OC1=CC=C(O)C=C1.C1=CC(F)=CC=C1C(=O)C1=CC=C(F)C=C1 JUPQTSLXMOCDHR-UHFFFAOYSA-N 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000000752 ionisation method Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920002530 polyetherether ketone Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Classifications
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/10—Ion sources; Ion guns
- H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
- H01J49/165—Electrospray ionisation
- H01J49/167—Capillaries and nozzles specially adapted therefor
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/26—Mass spectrometers or separator tubes
Definitions
- the present invention relates to the field of mass spectrometry imaging technology, and in particular to an automatic electrospray spraying device and method for mass spectrometry imaging sample preparation.
- Histopathological examination is an important tool for medical diagnosis.
- important diseases such as cancer need to be judged by histopathological examination.
- traditional histopathological examination is based on optical observation after staining of sections, and the information is relatively small.
- mass spectrometry imaging technology further provides chemical information such as metabolite changes, which can further help differentiate tissues.
- Stoeckli et al. first confirmed the huge potential of mass spectrometry imaging technology in tumor research. This article discussed how to use mass spectrometry imaging technology to reveal the chemical spatial structure of glioblastoma tissue sections. Subsequently, mass spectrometry imaging technology was confirmed to be able to directly perform tissue analysis. Analyze and locate gliomas, and grade the malignancy of gliomas.
- MALDI mass spectrometry imaging technology is currently a relatively advanced technology in the field of mass spectrometry imaging.
- MALDI mass spectrometry molecular imaging is completed under the control of specialized mass spectrometry imaging software using a mass spectrometer that analyzes the standard molecular weight of chemical and biological molecules by measuring the mass-to-charge ratio.
- the tissue to be studied is frozen and sectioned to obtain extremely thin tissue slices.
- derivatization is needed based on the analyte. If necessary, the derivatization reagent is applied to the surface of the tissue slice in the form of tiny droplets and controlled.
- the temperature allows the derivatization reaction to occur, omitting the modification step if not required, then coating the tissue section evenly with the matrix and placing the section on the target of the mass spectrometer.
- Observe the sample through the computer screen use the mass spectrometry imaging software of the MALDI system, select the part to be imaged, first define the size of the image, and divide the image into a two-dimensional lattice composed of several points according to the size to determine the spacing between laser point bombardments.
- the laser beam irradiates the tissue slices on the target disk through this grating pattern, and the software controls to start collecting mass spectrometry data.
- the laser beam continuously scans the tissue slices, and the molecules released by the tissue sample under the excitation of the laser beam are The mass spectrometer identifies and obtains the mass-to-charge ratio (m/z) information of each point on the sample, and then converts the molecular weight information of each point into pixels on the photo. At each point, all mass spectral data are averaged to obtain a complete mass spectrum that represents the distribution of compounds in that region.
- the instrument gradually collects mass spectrometry data of tissue sections, and finally obtains mass spectrometry data of the entire set of tissue sections with spatial information. In this way, "molecular imaging" of tissue samples can be completed.
- the types of biomolecules contained in the tissue region can be determined, and the peak height or peak area can be selected to represent the relative abundance of biomolecules.
- the colored spots in the image represent the localization of the compounds, and the shade of color of each spot is related to the size of the signal detected by the laser at each point or pixel.
- the method of spraying derivatization reagents and matrix causes large droplets of derivatization reagents to destroy in-situ information, the inability to control the derivatization reaction temperature, uneven distribution of matrix on the tissue surface, and the crystallization of large particles. problems, seriously affecting the quality of mass spectrometry imaging, and the preparation process takes too long.
- the present invention provides an automatic electrospray spraying device for mass spectrometry imaging sample preparation.
- the micron-level droplets generated by the electrospray principle can realize the spraying of derivatization reagents and substrates.
- the liquid The droplet fineness has been improved by orders of magnitude, ensuring that the derivatization reaction does not destroy the in-situ information, and ensures that the matrix crystallization grain size is small and evenly distributed. It is also equipped with a heating plate to control the temperature to meet the derivatization reaction conditions and matrix drying. requirements to ensure mass spectrometry imaging quality.
- An automatic electrospray spraying device for mass spectrometry imaging sample preparation including:
- An injection mechanism whose outlet end is connected to the inlet end of the spray capillary tube;
- a high-voltage power supply is electrically connected to the spray capillary.
- it also includes a first connecting piece
- the first connecting piece is a conductor, one end of which is connected to the outlet end of the injection mechanism, the other end of which is connected to the inlet end of the spray capillary tube, and is electrically connected to the high-voltage power supply.
- the first connecting piece includes a two-way connection.
- the outlet end of the spray capillary tube has a closed-end structure.
- the injection mechanism includes a syringe pump
- the outlet end of the syringe capillary tube of the syringe pump is connected with the inlet end of the spray capillary tube.
- it also includes:
- Heating plate for heating the slide.
- it also includes a robotic arm
- the spray capillary is provided at the end of the robotic arm.
- it also includes a connecting piece
- the connecting piece is an insulator, and one end thereof is provided with a docking structure for cooperating with the end of the robotic arm, and the other end is provided with an installation structure for cooperating with the spray capillary tube.
- An automatic electrospray spraying method for mass spectrometry imaging sample preparation using the above-mentioned automatic electrospray spraying device for mass spectrometry imaging sample preparation for automatic spraying, the method includes the following steps:
- the robot arm drives the spray capillary down into the derivatization reagent or matrix reagent bottle, and drives the injection mechanism to make the spray capillary absorb the derivatization reagent or matrix reagent;
- step S3 it also includes:
- the automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by the present invention generates micron-level micro-droplets through the electrospray principle to realize spraying of derivatization reagents and substrates.
- the technology has improved the fineness of the droplets by orders of magnitude, ensuring that the derivatization reaction does not destroy the in-situ information, and ensures that the matrix crystallization grain size is small and evenly distributed.
- the temperature control can ensure the smooth derivatization reaction and matrix drying. to ensure the quality of mass spectrometry imaging.
- Figure 1 is a schematic structural diagram of an automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by an embodiment of the present invention
- Figure 2 is a schematic structural diagram of an automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by another embodiment of the present invention
- Figure 3 is a structural diagram of an automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by an embodiment of the present invention
- Figure 4 is a flow chart of an automatic electrospray spraying method for mass spectrometry imaging sample preparation provided by an embodiment of the present invention.
- 1 is a high-voltage power supply
- 2 is the first connecting piece
- 3 is a spray capillary
- 4 is a syringe pump
- 5 is a robotic arm
- 6 is a glass slide
- 7 is a heating plate
- 8 is an electrospray
- 9 is a tissue section.
- 10 is the second connecting piece.
- the automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by embodiments of the present invention, as shown in Figure 1, includes:
- An injection mechanism whose outlet end is connected to the inlet end of the spray capillary tube 3;
- the high-voltage power supply 1 is electrically connected to the spray capillary 3.
- the injection mechanism can not only spray material from the spray capillary 3, but also absorb material from the spray capillary 3. That is, the injection mechanism can also drive the spray capillary 3 to absorb derivatization reagents or matrix reagents. Details are described below.
- the high-voltage power supply 1 is used to provide a high-voltage electric field for the spray capillary tube 3 , wherein the high-voltage power supply 1 is electrically connected to a portion of the spray capillary tube 3 near its outlet end.
- this solution is based on the electrospray principle.
- the injection mechanism drives the derivatization reagent or matrix reagent to fill the spray capillary 3.
- this solution can enable the derivatization reagent and matrix reagent to be dispersed into micron-sized droplets through electrospray. That is, this solution can realize the electrospray of the derivatization reagent and matrix, ensuring that the derivatization reaction does not destroy the original site. information, and ensures that the grain size of the matrix crystal is small and evenly distributed.
- the automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by the embodiment of the present invention can generate micron-sized droplets through the electrospray principle to realize spraying of derivatization reagents and substrates.
- the original technology has improved the fineness of droplets by orders of magnitude, ensuring that the derivatization reaction does not destroy the in-situ information, and helps ensure that the grain size of the matrix crystal is small and evenly distributed, ensuring the quality of mass spectrometry imaging.
- the automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by the embodiment of the present invention also includes a first connector 2;
- the first connecting piece 2 is a conductor, and one end thereof is connected to the outlet end of the injection mechanism (ie, connected and conducted), and the other end is connected to the inlet end of the spray capillary tube 3 and is electrically connected to the high-voltage power supply 1 .
- This solution is designed in this way, which not only improves the connection effect between the outlet end of the injection mechanism and the inlet end of the spray capillary tube 3, but also facilitates the electrical conduction between the high-voltage power supply 1 and the spray capillary tube 3.
- the first connecting piece 2 includes a two-way connection.
- the first connecting piece 2 of this solution is designed in this way and has the characteristics of simple structure and convenient connection.
- both ends of the two-way connection are connected to the casing and corresponding components through PEEK connectors.
- the outlet end of the spray capillary tube 3 has a closed structure.
- the injection mechanism includes a syringe pump 4;
- the outlet end of the syringe capillary tube of the syringe pump 4 is connected with the inlet end of the spray capillary tube 3 . That is, the outlet end of the output capillary tube of the syringe pump 4 is connected with the inlet end of the spray capillary tube 3 .
- this solution uses syringe pump 4 as the injection mechanism, which has the characteristics of uniform and stable injection and accurate flow control.
- the automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by the embodiment of the present invention also includes:
- the heating piece 7 is used to heat the glass slide 6; wherein, the heating piece 7 is arranged below the glass slide 6.
- a heating plate 7 it is convenient to increase the temperature of the tissue slice 9, meet the derivatization reaction conditions, ensure the smooth progress of the derivatization reaction, and accelerate the evaporation of the matrix to avoid the mass spectrometry imaging sample preparation process from taking too long.
- the automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by the embodiment of the present invention also includes a robotic arm 5;
- the spray capillary 3 is provided at the end of the robotic arm 5 .
- This solution is designed so that the robotic arm 5 can drive the spray capillary 3 to spray on a designated area to complete the preparation of derivatization reagents and matrix coatings for tissue sections, thereby helping to achieve rapid and flexible spraying of derivatization reagents and matrix.
- this solution uses the robotic arm 5 to drive the spray capillary 3 to operate, and can also flexibly control the spray area and the number of spray layers, thereby achieving parameterized control and programming of the derivatization reagent and matrix spray.
- the automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by the embodiment of the present invention also includes a second connector 10;
- the second connector 10 is an insulator, and one end thereof is provided with a docking structure for cooperating with the end of the robotic arm 5 , and the other end is provided with an installation structure for cooperating with the spray capillary tube 3 . That is to say, the spray capillary 3 is fixed to the end of the robotic arm 5 through the second connector 10 .
- the docking structure of the second connector 10 can be designed as: a slot that matches the size of the two-finger gripper to facilitate the grasping of the two-finger gripper;
- the installation structure of the second connector 10 can be designed as a vertical hole to facilitate the insertion and fixation of the spray capillary 3 .
- the docking structure and installation structure of the second connector 10 are not limited to this, and can also be other forms of structures, which will not be described again here.
- the second connecting member 10 can be a connecting block.
- Embodiments of the present invention also provide an automatic electrospray spraying method for mass spectrometry imaging sample preparation.
- the automatic electrospray spraying device for mass spectrometry imaging sample preparation as described above is used for automatic spraying. As shown in Figure 4, the The method includes the following steps:
- the robot arm drives the spray capillary down into the derivatization reagent or matrix reagent bottle, and drives the injection mechanism to make the spray capillary absorb the derivatization reagent or matrix reagent, that is, the derivatization reagent or matrix reagent is filled in the Spray the capillary; of course, close the injection mechanism after absorbing the derivatization reagent or matrix reagent;
- step S3 it also includes:
- Mass spectrometry imaging is an important detection and visualization technology in biochemical analysis and research, and matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging technology is one of the representatives of advanced technologies.
- Sample preparation is a necessary step in MALDI mass spectrometry imaging analysis that has a decisive impact on the analysis quality and is time-consuming.
- the ideal sample preparation mainly includes: choosing whether to perform derivatization according to needs, and if necessary, spraying the derivatization reagent through tiny droplets on the surface of the tissue section, and control the reaction temperature. After the reaction is completed, wait for the tissue section to dry. If not necessary, omit this step.
- the present invention is to realize automated, high-quality spraying of derivatization reagents and matrix, thereby providing uniform, fast, flexible and fully automatic sample preparation technology, methods and devices for MALDI mass spectrometry imaging.
- the derivatization reagent and matrix are dispersed into micron-sized droplets through electrospray, ensuring that the derivatization reaction does not destroy the in-situ information and ensuring that the matrix crystallization grain size is small and uniform.
- the heating plate controls the temperature to ensure the smooth progress of the derivatization reaction and the evaporation of the matrix.
- the spray area and the number of spray layers can be flexibly controlled, thereby achieving parameterized control and programming of the derivatization reagent and matrix spray.
- high-voltage power supply, heating plate and syringe pump switching of multiple derivatization reagents and multiple matrices as well as automatic spraying can be achieved, further expanding the use of this technology.
- mass spectrometry analysis a method that uses electric and magnetic fields to separate moving ions according to their mass-to-charge ratio and then detect them. Substances can be identified by their mass-to-charge ratio.
- Mass spectrometry imaging is an imaging method based on mass spectrometry technology. This method directly scans biological samples for imaging through mass spectrometry, and can simultaneously analyze the spatial distribution characteristics of hundreds of molecules on the same tissue slice or tissue chip. Simply put, mass spectrometry imaging technology relies on mass spectrometry, and is controlled by specialized mass spectrometry imaging software, using a mass spectrometer that analyzes the standard molecular weight of chemical and biological molecules by measuring the mass-to-charge ratio.
- Tissue sectioning The biological sample tissue is frozen and cut into thin slices and adhered to a glass slide for observation of tissue morphology, cell morphology, imaging analysis, etc.
- MALDI Matrix-assisted laser desorption/ionization (Matrix-assisted laser desorption/ionization) is a new mass spectrometry ionization technology.
- Derivatization is a method that uses chemical transformation to convert compounds into substances with similar chemical structures. It is widely used in instrumental analysis. The main function is to convert substances that are difficult to analyze into substances that are similar to their chemical structures but easy to analyze, so as to facilitate quantification and separation.
- the reagents used for derivatization are liquid reagents and will be prepared in advance before use.
- the matrix is a substance that co-crystallizes with the substance to be measured, absorbs the incident laser energy and transfers it to the molecules to be measured, while preventing its direct irradiation from causing damage to the sample to be measured. It has an important influence on the desorption ionization process.
- the matrix is a liquid reagent that is pre-mixed before use.
- MALDI imaging technology Scan prepared sample slices in a laser array to obtain mass spectrometry analysis results with spatial information, which can be visualized as imaging results.
- Electrospray Add a high-voltage electric field to the exit of the capillary tube. Under the action of the electric field force and Coulomb force, the liquid reagent will generate Taylor cone spray at the exit of the capillary tube and atomize into small charged droplets. The diameter is in the micron range.
- the present invention is a technology, system and device for pre-processing tissue slice derivatization and matrix coating for MALDI mass spectrometry imaging technology. Its function is to drive the derivatization reagent or matrix to fill the spray capillary through a syringe pump, and then drive it with high voltage electricity. Next, the derivatization reagent or matrix in the spray capillary is sprayed out through the electrospray principle, and the spray capillary is driven by the robotic arm to spray on the designated area, and the temperature is controlled by the heating plate to complete the derivatization and matrix coating preparation of tissue sections. The heating plate can increase the temperature of tissue sections to meet the derivatization reaction conditions, accelerate the evaporation of the matrix, and control the crystal quality.
- the overall structure of the present invention is shown in Figure 1.
- the spray capillary described in this solution is a pipe structure with a small inner diameter, as shown in Figure 2.
- the inner diameter is usually 0.3 to 1 mm, and the length is 10 cm. Its front end opening narrows to 0.01 mm, which is the spray outlet, and its rear end is Connect the syringe pump connector.
- the connecting piece of the syringe pump is a metal conductor, which is connected to the high-voltage power supply, so that the liquid in the spray capillary tube is connected to the high-voltage power supply.
- the spray capillary is fixed at the end of the robotic arm and connected to the connecting piece of the robotic arm.
- the connecting piece of the robotic arm is an insulator to prevent short circuit.
- the spray capillary and its connectors are installed at the end of the robotic arm.
- the spray capillary is connected to the high-voltage power supply through flexible wires, and to the syringe pump through a flexible capillary tube. connected.
- the work flow is: turning on the heating plate and preheating.
- the tissue sections are placed on the glass slide. After drying, they are placed in the designated position, and the temperature is controlled by the heating plate below.
- Derivatization reagents and matrix are pre-prepared liquid reagents, stored in reagent bottles and placed in designated locations.
- the spray capillary extends into the reagent bottle driven by the robotic arm, and is driven by the syringe pump to draw derivatization reagents or matrix reagents into the capillary tube. Thereafter, the spray capillary moves to the initial position outside the scope of the slide, turns on the high-voltage power supply, and waits for a few seconds until the spray stabilizes.
- the spray capillary moves to a set height above the glass slide and moves horizontally according to the set program to complete the spraying of derivatization reagent or matrix.
- the spray capillary is moved out of the slide area and the high-voltage power supply is turned off.
- the robotic arm drives the spray capillary to the waste liquid area to discharge the remaining reagents, and then to the pure water area to repeatedly pump and empty the spray capillary through the syringe pump to clean the spray capillary. If more than one reagent is required, repeat the above process. The process is shown in Figure 4.
- the spray nozzle of the spray capillary is drawn by a capillary needle puller.
- the high-voltage power connector (the second connector) is a commodity
- the heating plate is a commodity
- the mechanical arm connector is processed by 3D printing.
- the present invention uses the principle of electrospray to generate micron-level droplets to achieve spraying of derivatization reagents and substrates. Compared with the original technology, the fineness of the droplets is improved by an order of magnitude. Spraying is achieved by driving the spray with a mechanical arm, which is more flexible than the original technology. , more efficient and smarter; through the joint control of the robotic arm, electrospray, and drive pump, the derivatization reagent and matrix spraying tasks can be parameterized and customized, and can be easily extended to mixed spraying of multiple reagents or multiple matrices, fully automatically Achieve complex sample pre-processing tasks; achieve temperature control through the heating plate to meet derivatization reaction conditions and accelerate matrix evaporation.
- the mechanical arm drives the electrospray to realize the parameterized setting of the spraying task
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Plasma & Fusion (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
本发明公开了用于质谱成像样本制备的自动电喷雾喷涂装置及方法，其中，该用于质谱成像样本制备的自动电喷雾喷涂装置包括：喷雾毛细管；其出口端与所述喷雾毛细管的进口端连通的注射机构；与喷雾毛细管为电连接的高压电源；用于带动喷雾毛细管运动的机械臂。本方案通过电喷雾原理生成微米级微小雾滴可实现衍生化试剂与基质喷涂，其相较原有技术在液滴精细程度上有数量级的提升，保证了衍生化反应不破坏原位信息，而且保证了基质结晶的晶粒尺度小且分布均匀，确保质谱成像质量。此外，本方案还使用机械臂操作喷涂过程，使得喷涂过程更灵活、效率更高，且可自动吸取、更换试剂，实现全自动多试剂喷涂的样本制备，如衍生化试剂与基质叠加喷涂等。
Description
本申请要求于2022年4月8日提交中国专利局、申请号为CN202210366595.7、发明名称为“用于质谱成像样本制备的自动电喷雾喷涂装置及方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
本发明涉及质谱成像技术领域，特别涉及用于质谱成像样本制备的自动电喷雾喷涂装置及方法。
质谱成像技术在医学中的应用：组织病理检查是医疗诊断的重要工具，特别是癌症等重要疾病要通过组织病理检查判断类型，但传统的组织病理检查是通过切片染色后进行光学观察，信息较为单一，而质谱成像技术则进一步提供了代谢物变化等化学信息，可以进一步帮助区分组织。如2001年Stoeckli等首次证实质谱成像技术在肿瘤研究中的巨大潜能，该文论述了如何应用质谱成像技术揭示胶质母细胞瘤组织切片的化学空间结构，随后质谱成像技术被证实可直接进行组织分析定位神经胶质瘤，并进行神经胶质瘤的恶性程度分级。
MALDI质谱成像技术：MALDI质谱成像技术是目前质谱成像领域较先进的一种技术。MALDI质谱分子成像是在专门的质谱成像软件控制下，使用一台通过测定质荷比来分析化学、生物分子的标准分子量的质谱仪来完成的。被用来研究的组织经过冰冻切片来获得极薄的组织片，首先，根据分析物质不同判断是否需要进行衍生化，如果需要，将衍生化试剂通过微小液滴形式施加于组织切片表面，并控制温度使衍生化反应发生，如果不需要则省略改步，接着用基质均匀涂覆组织切片并将切片置入质谱仪的靶上。通过计算机屏幕观察样品，利用MALDI系统的质谱成像软件，选择拟成像部分，首先定义图像的尺寸，根据尺寸大小将图像均分为若干点组成的二维点阵，来确定激光点轰击的间距。激光束通过这个光栅图案照射到靶盘上的组织切片，软件控制开始采集质谱数据，在质谱仪中，激光束对组织切片进行连续的扫描，组织样品在激光束的激发下释放出的分子被质谱仪所鉴定从而获得样品上每个点的质荷比（m/ z）信息，然后将各个点的分子量信息转化为照片上的像素点。在每个点上，所有质谱数据经平均化处理获得一幅代表该区域内化合物分布情况的完整质谱图。仪器逐步采集组织切片的质谱数据，最后得到具有空间信息的整套组织切片的质谱数据。这样就可以完成对组织样品的“分子成像”。设定m/ z 的范围，即可确定该组织区域所含生物分子的种类，并选定峰高或者峰面积来代表生物分子的相对丰度。图像中的彩色斑点代表化合物的定位，每个斑点颜色的深浅与激光在每一个点或像素上检测到的信号大小相关。
传统的MALDI成像样本制备技术：做MALDI成像必须使用冷冻切片，且不能用OCT等包埋试剂包埋，因为OCT中的多聚物会严重干扰质谱，组织切片转移到导电载玻片上后，真空干燥，根据实际需要进行衍生化（喷涂衍生化试剂并控制反应温度），然后喷涂基质，送入仪器进行分析。
传统样本制备技术存在的问题：对组织切片进行衍生化前处理，需要保持其原位信息，因此需要通过微小液滴的方式向组织表面施加衍生化试剂，现在市面上没有专门用于组织切片原位衍生化的商用仪器，制备操作都是基于基质喷涂商用仪器，其喷涂液滴较大，破坏原位信息，同时仪器无法满足温度等衍生化反应条件，影响或破坏衍生化效果。基质在组织切片表面干燥结晶过程中，会形成较大的晶体颗粒，破坏基质包被均匀性，影响质谱成像结果，降低信号质量与空间分辨率。商用仪器使用超声雾化基质沉降在组织表面，结晶质量提升，但仍不满足50微米的分析精度，且只适用于全片喷涂单一基质，耗时长，同时仪器不稳定，常发生堵塞等意外。
也就是说，在传统样本制备技术中，衍生化试剂与基质喷涂的方式存在衍生化试剂液滴大破坏原位信息、无法控制衍生化反应温度、基质在组织表面分布不均匀、大颗粒的结晶的问题，严重影响质谱成像质量，且制备过程耗时过长的问题。
有鉴于此，本发明提供了一种用于质谱成像样本制备的自动电喷雾喷涂装置，通过电喷雾原理生成微米级微小雾滴可实现衍生化试剂与基质喷涂，其相较原有技术在液滴精细程度上有数量级的提升，保证了衍生化反应不破坏原位信息，而且保证了基质结晶的晶粒尺度小且分布均匀，同时配备有加热片控制温度，满足衍生化反应条件与基质干燥要求，确保质谱成像质量。
为实现上述目的，本发明提供如下技术方案：
一种用于质谱成像样本制备的自动电喷雾喷涂装置，包括：
喷雾毛细管；
其出口端与所述喷雾毛细管的进口端连接的注射机构；
与所述喷雾毛细管为电连接的高压电源。
优选地，还包括第一连接件；
所述第一连接件为导体，且其一端与所述注射机构的出口端连通，另一端与所述喷雾毛细管的进口端连通，并与所述高压电源为电连接。
优选地，所述第一连接件包括二通。
优选地，所述喷雾毛细管的出口端为收口结构。
优选地，所述注射机构包括注射泵；
所述注射泵的注射器毛细管的出口端与所述喷雾毛细管的进口端连通。
优选地，还包括：
用于加热所述载玻片的加热片。
优选地，还包括机械臂；
所述喷雾毛细管设置于所述机械臂的末端。
优选地，还包括连接件；
所述连接件为绝缘体，且其一端设有用于同所述机械臂的末端配合的对接结构，另一端设有用于同所述喷雾毛细管配合的安装结构。
一种用于质谱成像样本制备的自动电喷雾喷涂方法，采用如上所述的用于质谱成像样本制备的自动电喷雾喷涂装置进行自动喷涂，该方法包括如下步骤：
S1、根据样本制备流程，通过机械臂带动喷雾毛细管下伸入衍生化试剂或基质试剂瓶中，驱动注射机构以使得喷雾毛细管吸取衍生化试剂或基质试剂；
S2、通过机械臂带动喷雾毛细管移至载玻片范围外的初始位置，开启高压电源，驱动注射机构使得喷雾毛细管缓慢推出衍生化试剂或基质试剂，等待直到喷雾毛细管喷雾稳定；同时，根据衍生化试剂反应温度要求或基质试剂干燥温度要求，调整加热片温度；
S3、通过机械臂带动喷雾毛细管移至载玻片上方设定高度，并按设定程序水平移动，完成衍生化试剂或基质喷涂。
优选地，在所述步骤S3之后，还包括：
S4、通过机械臂带动喷雾毛细管移出载玻片区域，关闭高压电源及停止注射机构；
S5、通过机械臂带动喷雾毛细管到废液区域排出剩余基质，并到纯水区域通过注射机构反复抽吸、排空清洗喷雾毛细管；
S6、洗净喷雾毛细管后，可根据样本制备要求，返回S1，吸取新的试剂进行下一轮喷涂，即重复S1到S6，当所有样本制备喷涂任务完成后，结束流程。
从上述的技术方案可以看出，本发明提供的用于质谱成像样本制备的自动电喷雾喷涂装置，通过电喷雾原理生成微米级微小雾滴可实现衍生化试剂与基质喷涂，其相较原有技术在液滴精细程度上有数量级的提升，保证了衍生化反应不破坏原位信息，而且保证了基质结晶的晶粒尺度小且分布均匀，同时通过控温可以保证衍生化反应与基质干燥顺利进行，确保质谱成像质量。
图1为本发明实施例提供的用于质谱成像样本制备的自动电喷雾喷涂装置的结构简图；
图2为本发明另一实施例提供的用于质谱成像样本制备的自动电喷雾喷涂装置的结构简图；
图3为本发明实施例提供的用于质谱成像样本制备的自动电喷雾喷涂装置的结构组成图；
图4为本发明实施例提供的用于质谱成像样本制备的自动电喷雾喷涂方法的流程图。
其中，1为高压电源，2为第一连接件，3为喷雾毛细管，4为注射泵，5为机械臂，6为载玻片，7为加热片，8为电喷雾，9为组织切片，10为第二连接件。
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明实施例提供的用于质谱成像样本制备的自动电喷雾喷涂装置，如图1所示，包括：
喷雾毛细管3；
其出口端与喷雾毛细管3的进口端连接的注射机构；
与喷雾毛细管3为电连接的高压电源1。
需要说明的是，注射机构不仅可实现喷雾毛细管3的喷料，还可实现喷雾毛细管3的吸料，即注射机构还可驱使喷雾毛细管3吸取衍生化试剂或基质试剂，详情见下文描述。此外，高压电源1用于为喷雾毛细管3提供高压电场，其中，高压电源1与喷雾毛细管3靠近其出口端的部位为电连接。另外，本方案基于电喷雾原理，首先通过注射机构驱动衍生化试剂或基质试剂填充喷雾毛细管3，然后在高压电源1和注射机构的驱动下，将喷雾毛细管3内的衍生化试剂或基质试剂喷射出去形成电喷雾8（如图2所示）。也就是说，本方案可使得衍生化试剂和基质试剂通过电喷雾的方式被分散为微米级小液滴，即本方案可实现衍生化试剂和基质的电喷雾，保证衍生化反应不破坏原位信息，并保证了基质结晶的晶粒尺度小且分布均匀。
从上述的技术方案可以看出，本发明实施例提供的用于质谱成像样本制备的自动电喷雾喷涂装置，通过电喷雾原理生成微米级微小雾滴可实现衍生化试剂和基质喷涂，其相较原有技术在液滴精细程度上有数量级的提升，保证衍生化反应不破坏原位信息，而且有助于保证基质结晶的晶粒尺度小且分布均匀，确保质谱成像质量。
在本方案中，如图1所示，本发明实施例提供的用于质谱成像样本制备的自动电喷雾喷涂装置还包括第一连接件2；
第一连接件2为导体，且其一端与注射机构的出口端连通（即连接和导通），另一端与喷雾毛细管3的进口端连通，并与高压电源1为电连接。本方案如此设计，不仅提升了注射机构出口端与喷雾毛细管3进口端的连接效果，而且还便于实现高压电源1与喷雾毛细管3的电导通。
具体地，第一连接件2包括二通。本方案的第一连接件2如此设计，具有结构简单、连接便捷等特点。此外，该二通的两端均通过PEEK接头与套管和对应的部件实现连接。
进一步地，为了提升喷雾毛细管3的电喷雾效果；相应地，如图2所示，
喷雾毛细管3的出口端为收口结构。
再进一步地，如图1所示，注射机构包括注射泵4；
注射泵4的注射器毛细管的出口端与喷雾毛细管3的进口端连通。即为注射泵4的输出毛细管的出口端与喷雾毛细管3的进口端连通。其中，本方案选用注射泵4作为注射机构，具有注射均匀平稳、流量控制精准等特点。
为了进一步优化上述技术方案，如图1所示，本发明实施例提供的用于质谱成像样本制备的自动电喷雾喷涂装置还包括：
用于加热载玻片6的加热片7；其中，加热片7设置于载玻片6的下方。本方案通过增设加热片7，便于提高组织切片9的温度，满足衍生化反应条件，确保衍生化反应顺利进行，并加速基质的挥干，避免质谱成像样本制备过程耗时过长。
在本方案中，如图1所示，本发明实施例提供的用于质谱成像样本制备的自动电喷雾喷涂装置还包括机械臂5；
喷雾毛细管3设置于机械臂5的末端。本方案如此设计，以便于通过机械臂5带动喷雾毛细管3喷涂于指定区域，完成组织切片的衍生化试剂和基质包被制备，从而有助于实现衍生化试剂和基质的快速、灵活的喷涂。当然，本方案通过机械臂5带动喷雾毛细管3作业，还可灵活控制喷涂区域与喷涂层数，从而实现对衍生化试剂和基质喷涂的参数化控制与编程。
具体地，如图1所示，本发明实施例提供的用于质谱成像样本制备的自动电喷雾喷涂装置还包括第二连接件10；
第二连接件10为绝缘体，且其一端设有用于同机械臂5的末端配合的对接结构，另一端设有用于同喷雾毛细管3配合的安装结构。也就是说，喷雾毛细管3通过第二连接件10固定于机械臂5的末端。其中，以机械臂5的末端为二指夹爪为例，该第二连接件10的对接结构可设计为：与二指夹爪尺寸匹配的卡槽，便于二指夹爪的抓持；该第二连接件10的安装结构可设计为垂直孔，便于喷雾毛细管3的插入固定。当然，该第二连接件10的对接结构和安装结构不局限于此，还可为其它形式的结构，此处不再赘述。此外，该第二连接件10可为连接块。
本发明实施例还提供了一种用于质谱成像样本制备的自动电喷雾喷涂方法，采用如上所述的用于质谱成像样本制备的自动电喷雾喷涂装置进行自动喷涂，如图4所示，该方法包括如下步骤：
S1、根据样本制备流程，通过机械臂带动喷雾毛细管下伸入衍生化试剂或基质试剂瓶中，驱动注射机构以使得喷雾毛细管吸取衍生化试剂或基质试剂，即使得衍生化试剂或基质试剂填充于喷雾毛细管内；当然，在吸取衍生化试剂或基质试剂后关闭注射机构；
S2、通过机械臂带动喷雾毛细管移至载玻片范围外的初始位置，开启高压电源，驱动注射机构使得喷雾毛细管缓慢推出衍生化试剂或基质试剂，等待直到喷雾毛细管喷雾稳定；同时，根据衍生化试剂反应温度要求或基质试剂干燥温度要求，调整加热片温度；
S3、通过机械臂带动喷雾毛细管移至载玻片上方设定高度，并按设定程序水平移动，完成衍生化试剂或基质喷涂。由于本方案采用了上述的用于质谱成像样本制备的自动电喷雾喷涂装置进行喷涂，因此其也就具有相应的有益效果，具体可以参照前面说明，在此不再赘述。
在本方案中，在所述步骤S3之后，还包括：
S4、通过机械臂带动喷雾毛细管移出载玻片区域，关闭高压电源和停止注射机构；
S5、通过机械臂带动喷雾毛细管到废液区域排出剩余基质，并到纯水区域通过注射机构反复抽吸、排空清洗喷雾毛细管；本方案如此设计，以便于喷雾毛细管的清洗。
S6、洗净喷雾毛细管后，可根据样本制备要求，返回S1，吸取新的试剂进行下一轮喷涂，即重复S1到S6，当所有样本制备喷涂任务完成后，结束流程。当然，可根据样本制备要求，需要先后完成衍生化试剂和基质的喷涂，实现衍生化试剂与基质的叠加喷涂。也就是说，在前一轮喷涂中吸取衍生化试剂进行喷涂，在后一轮喷涂中接着吸取基质试剂进行喷涂。
下面结合具体实施例对本方案作进一步介绍：
质谱成像是生化分析与研究中重要的检测与可视化技术，基质辅助激光解吸电离（MALDI）质谱成像技术是其中一种先进技术代表。样本制备是MALDI质谱成像分析中对分析质量有决定性影响同时耗时较多的必要环节，理想的样本制备主要是：根据需要选择是否进行衍生化，如需要，将衍生化试剂通过微小液滴喷涂于组织切片表面，并控制反应温度，反应结束等待组织切片干燥，如不需要则省略这一步。当前缺乏现成的商业设备完成组织切片的原位衍生化反应，替代方案是使用商业基质喷涂设备代替，但存在液滴大破坏原位信息，以及无法控制反应温度，影响反应顺利进行的问题。其后，需要将基质均匀地附着在组织切片表面，但当前基质喷涂的方式，都存在基质在组织表面分布不均匀、大颗粒的结晶，严重影响质谱成像质量，且制备过程耗时过长的问题。
本发明是为了实现自动化、高质量的衍生化试剂和基质喷涂，从而为MALDI质谱成像提供喷涂均匀、快速、灵活、全自动的样本制备技术、方法与装置。衍生化试剂和基质通过电喷雾的方式被分散为微米级小液滴，保证衍生化反应不破坏原位信息，并保证了基质结晶的晶粒尺度小且均匀。加热片控制温度，保证衍生化反应顺利进行及基质的挥干。通过机械臂带动电喷雾喷头，可以灵活控制喷涂区域与喷涂层数，从而实现对衍生化试剂和基质喷涂的参数化控制与编程。通过机械臂、高压电源、加热片与注射泵的集成与联控，可以实现多衍生化试剂与多基质的切换以及自动喷涂，进一步扩展了该技术的用途。
此外，质谱分析：用电场和磁场将运动的离子按它们的质荷比分离后进行检测的方法，可以通过质荷比辨识物质。
质谱成像技术：质谱成像是以质谱技术为基础的成像方法，该方法通过质谱直接扫描生物样品成像，可以在同一张组织切片或组织芯片上同时分析数百种分子的空间分布特征。简单而言，质谱成像技术就是借助于质谱的方法，再配套上专门的质谱成像软件控制下，使用一台通过测定质荷比来分析化学、生物分子的标准分子量的质谱仪来成像的方法。
组织切片：将生物样本组织冰冻后切为薄片并黏附于载玻片上，用于观察组织形态、细胞形态、成像分析等。
MALDI：基质辅助激光解吸离子化技术（Matrix-assisted laser desorption/ionization)是一种新的质谱离子化技术。
衍生化：衍生化是一种利用化学变换把化合物转化成类似化学结构的物质，在仪器分析中被广泛应用。主要作用是把难于分析的物质转化为与其化学结构相似但易于分析的物质，便于量化和分离。衍生化所用试剂是液态试剂，使用时会预先调配好。
基质：在MALDI技术中，基质是和待测物质共结晶、吸收入射激光能量并转移给待测分子，同时防止其直接照射致使待测样品被破坏的物质，对解吸离子化过程有重要影响。基质是液态试剂，使用时会预先调配好。
MALDI成像技术：通过激光阵列化地扫描制备好的样本切片，得到带有空间信息的质谱分析结果，从而可视化为成像结果。
电喷雾：在毛细管的出口处加一高压电场，在电场力和库仑力的作用下，液态试剂会在毛细管的出口处会发生泰勒锥喷雾，雾化成细小的带有电荷的雾滴，雾滴直径在微米级。
本发明是为了MALDI质谱成像技术进行组织切片衍生化与基质包被前处理的技术、系统与装置，其功能是通过注射泵驱动衍生化试剂或基质填充喷雾毛细管，其后在高压电的驱动下，将喷雾毛细管内的衍生化试剂或基质通过电喷雾原理喷出，通过机械臂带动喷雾毛细管，喷涂于指定区域，并通过加热片控制温度，完成组织切片的衍生化与基质包被制备。加热片可以提高组织切片温度，满足衍生化反应条件，并加速基质挥干，控制结晶质量。本发明整体结构如图1所示。
通过机械臂、注射泵、加热片以及高压电源的联合控制，可以实现指定区域喷涂指定量衍生化试剂与基质的参数化控制，并可以自由切换不同衍生化试剂与基质，自动实现复杂的前处理过程。该系统可进一步扩展到其他试剂的喷涂，如酶解试剂等，实现全面的样本前处理功能。
本方案所述喷雾毛细管是内径细小的管道结构，如图2所示，内径通常为0.3到1毫米，长度10厘米，其前端开口收细为0.01毫米，是喷雾的喷出口，其后端与注射泵的连接件相连。注射泵的连接件为金属导体，与高压电源相连，使喷雾毛细管内的液体与高压电源导通。喷雾毛细管被固定于机械臂末端，与机械臂的连接件相连，机械臂的连接件为绝缘体，防止短路。此外，注射泵、机械臂和高压电源等大体积设备均置于台面上，喷雾毛细管及其连接件安装于机械臂末端，喷雾毛细管与高压电源间通过柔性导线相连，与注射泵间通过柔性毛细管相连。
工作原理：当在毛细管的出口处加一高压电场，在库仑力的作用下，试剂在毛细管的出口处会发生喷雾，雾化成细小的带有电荷的雾滴，就是所谓的电喷雾，如图2所示。喷雾的形成原理使静电库仑力克服液滴表面张力，导致液滴破裂成为细小雾滴，这个现象叫做泰勒锥。通过调节电场强度、毛细管开口内径以及试剂黏度等参数可以调节雾滴粒径与泰勒锥宽度。当机械臂带动喷雾毛细管水平运动时，就会在下方形成泰勒锥覆盖的喷雾区域，通过调节机械臂运动方向、运动次数、往返此时以及喷雾毛细管开口距离组织切片的距离，可以调节喷雾区域大小、厚度以及任务完成速度等。
如图3和图4所示，工作流程：加热片开启、预热。组织切片承载于载玻片上，经干燥后，置于指定位置，由下方加热片控制温度。衍生化试剂与基质为预先调配好的液态试剂，存储于试剂瓶中，置于指定位置。喷雾毛细管在机械臂的带动下伸入试剂瓶中，经注射泵驱动，吸取衍生化试剂或基质试剂到毛细管中。其后，喷雾毛细管移动到载玻片范围外的初始位置，开启高压电源，等待几秒，直到喷雾稳定。其后，喷雾毛细管移动到载玻片上方设定高度，并按设定程序水平移动，完成衍生化试剂或基质喷涂。喷涂完成后，喷雾毛细管移出载玻片区域，关闭高压电源。机械臂带动喷雾毛细管到废液区域排出剩余试剂，并到纯水区域通过注射泵反复抽吸、排空清洗喷雾毛细管。如有多于一种试剂的喷涂需求，则重复上述过程。流程如图4所示。
加工：喷雾毛细管的喷雾口通过毛细拉针仪拉制实现，高压电源连接件（即为第二连接件）为商品，加热片为商品，机械臂连接件由3D打印方式加工。
本发明的优点：
本发明通过电喷雾原理生成微米级微小雾滴实现衍生化试剂与基质喷涂，相较原有技术在液滴精细程度上有数量级的提升；通过机械臂带动喷雾实现喷涂，较原有技术更灵活、更有效率且更智能；通过机械臂、电喷雾、驱动泵的联合控制，可对衍生化试剂与基质喷涂任务实施参数化定制，并轻易扩展到多试剂或多基质的混合喷涂，全自动实现复杂的样品前处理任务；通过加热片，实现温度控制，满足衍生化反应条件及加速基质挥干。
本发明的关键点和欲保护点：
1、通过电喷雾方式实现衍生化、基质喷涂以及酶解等综合前处理任务；
2、通过机械臂带动电喷雾实现喷涂任务的参数化设定；
3、通过机械臂、高压电源、注射泵、喷雾毛细管与加热片的集成联控，实现全自动的MALDI成像组织切片样本制备装置。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (10)
- 一种用于质谱成像样本制备的自动电喷雾喷涂装置，其特征在于，包括：喷雾毛细管（3）；其出口端与所述喷雾毛细管（3）的进口端连通的注射机构；与所述喷雾毛细管（3）为电连接的高压电源（1）。
- 根据权利要求1所述的用于质谱成像样本制备的自动电喷雾喷涂装置，其特征在于，还包括第一连接件（2）；所述第一连接件（2）为导体，且其一端与所述注射机构的出口端连通，另一端与所述喷雾毛细管（3）的进口端连通，并与所述高压电源（1）为电连接。
- 根据权利要求2所述的用于质谱成像样本制备的自动电喷雾喷涂装置，其特征在于，所述第一连接件（2）包括二通。
- 根据权利要求1所述的用于质谱成像样本制备的自动电喷雾喷涂装置，其特征在于，所述喷雾毛细管（3）的出口端为收口结构。
- 根据权利要求1所述的用于质谱成像样本制备的自动电喷雾喷涂装置，其特征在于，所述注射机构包括注射泵（4）；所述注射泵（4）的注射器毛细管的出口端与所述喷雾毛细管（3）的进口端连通。
- 根据权利要求1所述的用于质谱成像样本制备的自动电喷雾喷涂装置，其特征在于，还包括：用于加热所述载玻片（6）的加热片（7）。
- 根据权利要求6所述的用于质谱成像样本制备的自动电喷雾喷涂装置，其特征在于，还包括机械臂（5）；所述喷雾毛细管（3）设置于所述机械臂（5）的末端。
- 根据权利要求7所述的用于质谱成像样本制备的自动电喷雾喷涂装置，其特征在于，还包括第二连接件（10）；所述第二连接件（10）为绝缘体，且其一端设有用于同所述机械臂（5）的末端配合的对接结构，另一端设有用于同所述喷雾毛细管（3）配合的安装结构。
- 一种用于质谱成像样本制备的自动电喷雾喷涂方法，其特征在于，采用如权利要求7所述的用于质谱成像样本制备的自动电喷雾喷涂装置进行自动喷涂，该方法包括如下步骤：S1、根据样本制备流程，通过机械臂带动喷雾毛细管下伸入衍生化试剂或基质试剂瓶中，驱动注射机构以使得喷雾毛细管吸取衍生化试剂或基质试剂；S2、通过机械臂带动喷雾毛细管移至载玻片范围外的初始位置，开启高压电源，驱动注射机构使得喷雾毛细管缓慢推出衍生化试剂或基质试剂，等待直到喷雾毛细管喷雾稳定；同时，根据衍生化试剂反应温度要求或基质试剂干燥温度要求，调整加热片温度；S3、通过机械臂带动喷雾毛细管移至载玻片上方设定高度，并按设定程序水平移动，完成衍生化试剂或基质喷涂。
- 根据权利要求9所述的用于质谱成像样本制备的自动电喷雾喷涂方法，其特征在于，在所述步骤S3之后，还包括：S4、通过机械臂带动喷雾毛细管移出载玻片区域，关闭高压电源及停止注射机构；S5、通过机械臂带动喷雾毛细管到废液区域排出剩余基质，并到纯水区域通过注射机构反复抽吸、排空清洗喷雾毛细管；S6、洗净喷雾毛细管后，可根据样本制备要求，返回S1，吸取新的试剂进行下一轮喷涂，即重复S1到S6，当所有样本制备喷涂任务完成后，结束流程。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210366595.7 | 2022-04-08 | ||
| CN202210366595.7A CN114724921A (zh) | 2022-04-08 | 2022-04-08 | 用于质谱成像样本制备的自动电喷雾喷涂装置及方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2023193483A1 true WO2023193483A1 (zh) | 2023-10-12 |
Family
ID=82241400
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2022/142308 WO2023193483A1 (zh) | 2022-04-08 | 2022-12-27 | 用于质谱成像样本制备的自动电喷雾喷涂装置及方法 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN114724921A (zh) |
| WO (1) | WO2023193483A1 (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114724921A (zh) * | 2022-04-08 | 2022-07-08 | 中国科学院深圳先进技术研究院 | 用于质谱成像样本制备的自动电喷雾喷涂装置及方法 |
| CN116046877B (zh) * | 2023-04-03 | 2024-03-15 | 深圳先进技术研究院 | 疾病模型代谢分析方法、包埋模具及喷涂装置 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN204842003U (zh) * | 2014-12-08 | 2015-12-09 | 中国医学科学院基础医学研究所 | 一种用于质谱成像的基质喷涂装置 |
| CN106404885A (zh) * | 2015-07-28 | 2017-02-15 | 中国科学院化学研究所 | 用于基质辅助激光解析电离质谱成像的喷雾系统和分析生物样本的方法 |
| CN106683973A (zh) * | 2017-01-17 | 2017-05-17 | 中国科学技术大学 | 一种用于质谱仪的电喷雾电离源装置及系统 |
| CN111257406A (zh) * | 2020-03-27 | 2020-06-09 | 中国科学院成都生物研究所 | 一种与质谱联用的除盐装置及在线除盐、质谱检测方法 |
| CN114724921A (zh) * | 2022-04-08 | 2022-07-08 | 中国科学院深圳先进技术研究院 | 用于质谱成像样本制备的自动电喷雾喷涂装置及方法 |
-
2022
- 2022-04-08 CN CN202210366595.7A patent/CN114724921A/zh active Pending
- 2022-12-27 WO PCT/CN2022/142308 patent/WO2023193483A1/zh unknown
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN204842003U (zh) * | 2014-12-08 | 2015-12-09 | 中国医学科学院基础医学研究所 | 一种用于质谱成像的基质喷涂装置 |
| CN106404885A (zh) * | 2015-07-28 | 2017-02-15 | 中国科学院化学研究所 | 用于基质辅助激光解析电离质谱成像的喷雾系统和分析生物样本的方法 |
| CN106683973A (zh) * | 2017-01-17 | 2017-05-17 | 中国科学技术大学 | 一种用于质谱仪的电喷雾电离源装置及系统 |
| CN111257406A (zh) * | 2020-03-27 | 2020-06-09 | 中国科学院成都生物研究所 | 一种与质谱联用的除盐装置及在线除盐、质谱检测方法 |
| CN114724921A (zh) * | 2022-04-08 | 2022-07-08 | 中国科学院深圳先进技术研究院 | 用于质谱成像样本制备的自动电喷雾喷涂装置及方法 |
Non-Patent Citations (1)
| Title |
|---|
| YANFENG CHEN, JEREMY ALLEGOOD, YING LIU, ELAINE WANG, BEGOÑA CACHÓN-GONZÁLEZ, TIMOTHY M. COX, ALFRED H. MERRILL,, M. CAMERON SULLA: "Imaging MALDI Mass Spectrometry Using an Oscillating Capillary Nebulizer Matrix Coating System and Its Application to Analysis of Lipids in Brain from a Mouse Model of Tay−Sachs/Sandhoff Disease", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 80, no. 8, 1 April 2008 (2008-04-01), US , pages 2780 - 2788, XP055284276, ISSN: 0003-2700, DOI: 10.1021/ac702350g * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114724921A (zh) | 2022-07-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2023193483A1 (zh) | 用于质谱成像样本制备的自动电喷雾喷涂装置及方法 | |
| US11391651B2 (en) | Capture probe | |
| CA2434305C (en) | Sample deposition method and system | |
| CA2440833C (en) | Piezoelectric charged droplet source | |
| DE60306355T2 (de) | Mikromatrixprobenausleser mittels elektrospraymassenspektrometrie | |
| WO2010047399A1 (ja) | 探針を用いたイオン化方法および装置、ならびに分析方法および装置 | |
| WO2007126141A1 (ja) | エレクトロスプレーによるイオン化方法および装置 | |
| US8816274B2 (en) | Mass spectrometer | |
| AU2918399A (en) | Electrospray device for mass spectrometer | |
| US6444980B1 (en) | Apparatus for production and extraction of charged particles | |
| US7095018B2 (en) | Deposition of samples and sample matrix for enhancing the sensitivity of matrix assisted laser desorption/ionization mass spectrometry | |
| CA2518706A1 (en) | Method and apparatus for coupling an analyte supply to an electrodynamic droplet processor | |
| US20120132799A1 (en) | Mass Spectrometer | |
| CN1933092A (zh) | 用于离子产生增强的设备和方法 | |
| Kossakovski et al. | Spatially resolved chemical analysis with an NSOM-based laser desorption microprobe | |
| Zhao et al. | Automated and miniaturized pico-liter metabolite extraction system for single-cell mass spectrometry | |
| JP2002116184A (ja) | 半導体デバイス異物分析装置およびシステム | |
| CN108344793A (zh) | 一种基质及其制备方法、代谢分子的质谱分析检测方法 | |
| Zhang et al. | On-line single droplet deposition for MALDI mass spectrometry | |
| Meng et al. | Microlensed fiber allows subcellular imaging by laser-based mass spectrometry | |
| KR20190006334A (ko) | 살아있는 생체 시료의 분석을 위한 고해상도 대기압 질량분석 이미징 시스템 및 이의 용도 | |
| Ozdemir et al. | Advancing mass spectrometry-based chemical imaging: A noncontact continuous flow surface probe in mass spectrometry for enhanced signal detection and spatial resolution | |
| EP3502654A1 (en) | Method for applying liquids to a substrate | |
| CN216978930U (zh) | 基于激光解吸辅助碳纤维离子化的敞开式质谱成像装置 | |
| CN116046877B (zh) | 疾病模型代谢分析方法、包埋模具及喷涂装置 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22936408 Country of ref document: EP Kind code of ref document: A1 |