Ultrasonic substrate spraying device and MALDI mass spectrum imaging substrate spraying method

Abstract

The invention relates to the technical field of matrix-assisted laser desorption ionization mass spectrometry imaging, in particular to an ultrasonic matrix spraying device and an MALDI mass spectrometry imaging matrix spraying method. The device comprises a vacuum atomization chamber, a sample bearing assembly, a matrix spraying assembly, a spraying assembly timing control unit, a spraying assembly driving unit, a vacuum assembly and an air flow assembly unit, wherein the matrix spraying assembly is used for spraying micron-sized matrix liquid drops into the vacuum atomization chamber; the spraying assembly timing control unit is used for controlling starting and stopping of the matrix spraying assembly. The spraying assembly driving unit drives the substrate spraying assembly to move, and the vacuum assembly and air flow assembly unit is used for providing a vacuum environment and air flow for the vacuum atomization cavity. According to the method, micron-sized matrix liquid drops are uniformly deposited on the surface of a biological sample to be sprayed by utilizing air pressure in a vacuum environment, so that a fine and uniform matrix and analyte co-crystallization layer is formed, and tissue in-situ analyte high-spatial-resolution imaging is realized.

Description

Technical Field

[0001] The invention relates to the technical field of matrix-assisted laser desorption ionization mass spectrometry imaging, and in particular to an ultrasonic matrix spraying device and a MALDI mass spectrometry imaging matrix spraying method. Background Art

[0002] It is well known that the synthesis and accumulation of proteins and metabolites in cells often have precise spatial distribution characteristics, and their physiological functions are often closely related to their spatial distribution in tissues and even cells. Even for cells of the same type, the protein, lipid and metabolite characteristics of each cell are different. This complexity, coupled with the diversity of different cell types in tissues, has led to widespread attention to spatial omics technologies at high-resolution scales and even single-cell scales.

[0003] Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry imaging technology can be directly used for the characterization of the molecular structure, relative abundance and spatial distribution of proteins, lipids and metabolites in situ of tissue sections, and is an important tool for the analysis of spatial proteome, spatial lipidome and spatial metabolome. The characteristic of MALDI is that the co-crystallization of the sample and the matrix is ​​irradiated with laser, and the matrix absorbs energy during the irradiation of the laser and transmits it to the molecule to be measured. The molecule to be measured absorbs energy and ionizes to form ions, which are then detected by the mass spectrometer. Therefore, the co-crystallization formation of the analyte and the matrix, as well as factors such as the uniformity of crystallization and the size of crystal particles are crucial for the sensitivity, spatial resolution and repeatability of MALDI mass spectrometry imaging. At present, the matrix deposition methods commonly used in MALDI mass spectrometry imaging research are mainly matrix sublimation method and matrix spraying method. The matrix sublimation method uses a heated vacuum device to sublimate the matrix and form a uniform crystal layer on the surface of the tissue section, which is suitable for high spatial resolution analysis mass spectrometry imaging analysis, but this method is not suitable for matrices that require high sublimation temperatures, and it is also difficult to control the thickness of the matrix crystal layer. The matrix spraying method, by spraying the matrix solution on the surface of the tissue slice, extracts the analyte to the sample surface on the one hand, and forms a co-crystallization layer of the matrix and the analyte on the surface of the tissue slice after the solvent evaporates, which assists the ionization of the analyte and provides higher sensitivity and spatial resolution for MALDI mass spectrometry imaging of lipids and proteins. At present, commercial matrix spraying devices mainly use mesh atomization (Bruker ImagePrep), ultrasonic atomization (SoniCoat), electrospray and compressed air atomization (HTX-TM-Sprayer) technology to produce micron-sized matrix solution droplets, which use gravity sedimentation and high-speed sheath gas to deposit the matrix solution droplets on the surface of the tissue slice, which is easy to produce a "sweet spot effect" that is not conducive to the acquisition of high spatial resolution mass spectrometry imaging data. Summary of the invention

[0004] In view of the above problems, the present invention aims to provide an ultrasonic matrix spraying device and a MALDI mass spectrometry imaging matrix spraying method, which utilizes the gas pressure deposition principle to ultrasonically atomize the matrix solution under vacuum conditions, so that ultrafine droplets evenly fill the vacuum atomization chamber, and then utilizes gas pressure to evenly deposit the ultrafine droplets on the surface of the tissue slice, and assists in analyte extraction and matrix crystallization by contact heating and non-contact methods. This method can ensure the formation of a fine and uniform matrix and analyte co-crystallization layer on the surface of the tissue slice, and realize high spatial resolution imaging of analytes in situ in the tissue.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] On the one hand, the present invention provides an ultrasonic matrix spraying device, comprising a vacuum atomization chamber, a sample carrying component, a matrix spraying component, a spraying component timing control unit, and a vacuum component and an air flow component unit, wherein the sample carrying component is arranged in the vacuum atomization chamber, and the sample carrying component is used to carry the biological sample to be sprayed; the matrix spraying component is arranged in the vacuum atomization chamber, and the matrix spraying component is used to spray micron-sized matrix droplets into the vacuum atomization chamber; the spraying component timing control unit is used to control the start and end of the matrix spraying component; the spraying component driving unit is arranged at the top of the vacuum atomization chamber and connected to the matrix spraying component, and the spraying component driving unit is used to drive the matrix spraying component to move; the vacuum component and the air flow component unit are connected to the vacuum atomization chamber, and the vacuum component and the air flow component unit are used to provide a vacuum environment and airflow for the vacuum atomization chamber, so that the micron-sized matrix droplets are uniformly deposited on the surface of the biological sample to be sprayed under the vacuum environment by using air pressure.

[0007] The matrix spraying assembly comprises a liquid spraying head, which utilizes ultrasonic waves to break the matrix solution into micron-sized droplets.

[0008] The liquid spray head has an annular gas nozzle, which is used for spraying micron-sized liquid droplets at multiple angles.

[0009] The timing control unit of the spraying assembly controls the opening and closing of the liquid spray head and has a second-level countdown function of 0 to 999 minutes.

[0010] The spraying assembly driving unit can drive the matrix spraying assembly to move horizontally in the left and right directions, and the moving speed is adjustable according to the sample spraying requirements.

[0011] The sample carrying assembly comprises a glass slide holder and a glass slide, wherein the glass slide holder is arranged at the bottom of the vacuum atomization chamber, and the glass slide is arranged on the glass slide holder for carrying the biological sample to be sprayed.

[0012] The ultrasonic matrix spraying device also includes a load-bearing component temperature control unit, which includes a heating plate and a temperature probe fixed below the slide holder. The heating plate is used to heat the biological sample to be sprayed, and the temperature probe is used to control the temperature of the biological sample to be sprayed.

[0013] The ultrasonic matrix spraying device also includes an illumination component and an illumination component heat dissipation unit arranged on the vacuum atomization chamber, wherein the illumination component provides visible light, 365nm and 460nm multi-wavelength illumination, and the illumination component heat dissipation unit is used for heat dissipation of the illumination component.

[0014] Another aspect of the present invention provides a MALDI mass spectrometry imaging matrix spraying method, which uses the ultrasonic matrix spraying device as described above to perform MALDI mass spectrometry imaging matrix spraying.

[0015] The biological samples to be sprayed include animal tissues, plant tissues, and tumor tissues. The matrix spraying component ultrasonically sprays the matrix solution under vacuum conditions, so that micron-sized matrix droplets evenly fill the vacuum atomization chamber, and then uses air pressure to evenly deposit the micron-sized matrix droplets on the surface of the biological sample to be sprayed, and assists in analyte extraction and matrix crystallization through contact and non-contact heating methods.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0017] 1. The present invention adopts a liquid spray head, uses ultrasound to promote liquid oscillation to break the matrix solution into small droplets, and uses gravity sedimentation or air pressure sedimentation to deposit and spray the sample surface. Compared with the commercial electrospray MALDI matrix sprayer that produces charged droplets, resulting in poor imaging of some analytes with the same charge, the droplets produced by the liquid spray head of the present invention are not charged and are more suitable for the full spectrum characterization of non-targeted tissue surface proteins, lipids, metabolites or drugs.

[0018] 2. The present invention uses ultrasound to achieve the emission of micron-sized droplets. At the same time, the co-crystallization temperature of the sample to be sprayed is controllable, which is conducive to the formation of finer matrix and analyte co-crystallization, and realizes MALDI mass spectrometry imaging with high sensitivity and high spatial resolution.

[0019] 3. The present invention introduces a vacuum component and an airflow component unit to make the micron-sized matrix droplets evenly deposited on the surface of the sample to be sprayed. The vacuum component makes the matrix droplets evenly fill the vacuum atomization chamber. The air pressure formed by the nitrogen flow evenly deposits the matrix droplets on the surface of the sample to be sprayed. The reported ultrasonic matrix sprayer relies on gravity sedimentation, and the matrix crystallization is more uniform.

[0020] 4. The present invention adopts a temperature-controlled sample-carrying component, which has the functions of heating and temperature control, and can realize functions such as in-situ enzymatic hydrolysis, derivatization, and matrix recrystallization.

[0021] 5. The present invention adopts a multi-wavelength illumination component, which has the functions of visible light, 395nm and 460nm multi-band illumination and temperature control, and can realize in-situ photocatalysis, derivatization and other functions.

[0022] In the present invention, the above-mentioned technical solutions can also be combined with each other to achieve more preferred combination solutions. Other features and advantages of the present invention will be described in the subsequent description, and some of the advantages can be clearly seen from the description. The purpose and other advantages of the present invention can be obtained through the contents indicated in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings are only for the purpose of illustrating specific embodiments and are not to be considered as limiting the present invention. Like symbols denote like components throughout the drawings.

[0024] Figure 1 It is a structural schematic diagram of an ultrasonic matrix spraying device of the present invention;

[0025] In the figure: 1-stepping motor moving platform, 2-liquid nozzle, 3-heating plate, 4-vacuum atomization chamber, 5-vacuum air valve, 6-multi-wavelength light source;

[0026] Figure 2 This is an in situ mass spectrometry image of mouse brain tissue of the intact protein in Example 1 of the present invention;

[0027] Figure 3 This is an in situ mass spectrometry image of lipids in mouse brain tissue in Example 2 of the present invention;

[0028] Figure 4 This is an in situ mass spectrometry image of the mouse brain tissue of the neurotransmitter in Example 3 of the present invention. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0030] like Figure 1As shown, on the one hand, the present invention provides an ultrasonic matrix spraying device, including a vacuum atomization chamber 4, a sample carrying component, a matrix spraying component, a spraying component timing control unit, a spraying component driving unit, and a vacuum component and an air flow component unit, wherein the sample carrying component is arranged in the vacuum atomization chamber 4, and the sample carrying component is used to carry the biological sample to be sprayed; the matrix spraying component is arranged in the vacuum atomization chamber, and the matrix spraying component is used to spray micron-sized matrix droplets into the vacuum atomization chamber 4; the spraying component timing control unit is used to control the start and end of the matrix spraying component; the spraying component driving unit is arranged at the top of the vacuum atomization chamber 4 and is connected to the matrix spraying component, and the spraying component driving unit is used to drive the matrix spraying component to move; the vacuum component and the air flow component unit are connected to the vacuum atomization chamber, and the vacuum component and the air flow component unit are used to provide a vacuum environment and airflow for the vacuum atomization chamber 4, so that the micron-sized matrix droplets are evenly deposited on the surface of the biological sample to be sprayed under the vacuum environment by using air pressure.

[0031] In an embodiment of the present invention, the sample carrying assembly includes a slide holder and a slide, wherein the slide holder is arranged at the bottom of the vacuum atomization chamber 4, and the slide is arranged on the slide holder for carrying the biological sample to be sprayed.

[0032] Furthermore, an ultrasonic matrix spraying device provided by the present invention also includes a load-bearing component temperature control unit, which includes a heating plate 3 and a temperature probe fixed under the slide holder, the heating plate 3 is used to heat the biological sample to be sprayed, and the temperature probe is used to control the temperature of the biological sample to be sprayed.

[0033] In an embodiment of the present invention, the matrix spraying assembly includes a liquid spray head 2, which uses ultrasound to break the matrix solution into micron-sized droplets. Specifically, the liquid spray head 2 has an annular gas nozzle, which is used to spray micron-sized droplets at multiple angles.

[0034] In the embodiment of the present invention, the timing control unit of the spray assembly controls the opening and closing of the liquid spray head 2 to have a second-level countdown function of 0 to 999 minutes.

[0035] In an embodiment of the present invention, the spray assembly driving unit can drive the substrate spray assembly to move horizontally in the left and right directions, and the moving speed is adjustable according to the sample spraying requirements.

[0036] Furthermore, the spray assembly driving unit includes a slide rail, a slider, a nozzle clamp, a connector and a slider driving device, wherein one end of the nozzle clamp is connected to the slider through the connector, and the other end clamps the liquid nozzle 2, the slider is sleeved on the slide rail and can move left and right along the slide rail, and the slider driving device drives the slider to realize the horizontal movement of the liquid nozzle 2 in the left and right direction. Specifically, the spray assembly driving unit adopts a stepper motor moving platform 1.

[0037] In an embodiment of the present invention, the vacuum component and air flow component unit includes a vacuum component and an air flow component, the vacuum component includes a vacuum pipeline, a vacuum pump and a vacuum air valve 5, the vacuum pipeline is connected to the exhaust port provided in the vacuum atomization chamber 4 and the vacuum pump, and the vacuum air valve 5 is arranged at the exhaust port. The vacuum component is used to remove the gas in the vacuum atomization chamber 4. The air flow component includes an air supply pipeline, a pressure gauge and an air filter, wherein the air supply pipeline is connected to the air inlet of the vacuum atomization chamber 4, and the pressure gauge and the air filter are arranged on the air supply pipeline. The vacuum component is used to inject filtered air into the vacuum atomization chamber 4.

[0038] Furthermore, the ultrasonic matrix spraying device provided by the present invention further includes an illumination component and an illumination component heat dissipation unit arranged on the vacuum atomization chamber 4, wherein the illumination component provides visible light, 365nm and 460nm multi-wavelength illumination, and the illumination component heat dissipation unit is used to dissipate heat from the illumination component. Specifically, the illumination component heat dissipation unit includes a heat sink and a temperature probe.

[0039] Furthermore, a temperature alarm unit is provided in the vacuum atomization chamber 4 for monitoring the temperature in the vacuum atomization chamber 4. When the temperature of the vacuum atomization chamber 4 exceeds a set value, the temperature alarm unit alarms.

[0040] Another aspect of the present invention provides a MALDI mass spectrometry imaging matrix spraying method, which utilizes an ultrasonic matrix spraying device as described in any of the above embodiments to perform MALDI mass spectrometry imaging matrix spraying.

[0041] In this embodiment, the biological samples to be sprayed include animal tissues, plant tissues, and tumor tissues. The matrix spraying component ultrasonically sprays the matrix solution under vacuum conditions, so that the micron-sized matrix droplets evenly fill the vacuum atomization chamber 4, and then uses air pressure to evenly deposit the micron-sized matrix droplets on the surface of the biological sample to be sprayed, and assists in analyte extraction and matrix crystallization through contact and non-contact heating methods.

[0042] Specifically, the present invention provides a MALDI mass spectrometry imaging matrix spraying method, which comprises the following steps:

[0043] 1) Connect the host power supply and vacuum pump power supply;

[0044] 2) Adjust the vacuum pump valve and air valve to the closed state;

[0045] 3) Turn on the switch of the heating plate 3 and adjust the heating parameters of the slide holder;

[0046] 4) Setting the spraying time parameter, incubation time parameter, and drying time parameter of the liquid nozzle 2;

[0047] 5) Set the left and right moving speed parameters of the stepper motor mobile platform 1;

[0048] 6) Add the matrix solution into the liquid bottle of the liquid spray head 2, and install the liquid spray head 2 on the liquid spray head bracket with the nozzle facing upward;

[0049] 7) Place the slide glass on the slide glass holder and close the door of the vacuum atomization chamber 4;

[0050] 8) Turn on the moving switch of the stepper motor moving platform 1, and wait until the liquid spray head 2 moves to the waiting position;

[0051] 9) Turn on the vacuum pump switch, open the valve of the vacuum air valve 5, and when the vacuum in the vacuum atomization chamber 4 reaches 0.06MPa, close the vacuum pump valve;

[0052] 10) Turn on the switch of the liquid spray head 2 to start the spraying and incubation process. After the incubation is completed, open the vacuum pump valve and the air valve to start drying. After the drying is completed, close the vacuum pump valve and the air valve, and repeat step 10) until the spraying is completed;

[0053] 11) After spraying is completed, turn off the liquid nozzle 2 switch, the movement switch of the stepper motor moving platform 1, the base heating switch and the vacuum pump switch;

[0054] 12) Open the door of the vacuum atomization chamber 4 and take out the glass slide;

[0055] 13) Maintain and clean the liquid ejecting head 2.

[0056] The following is further described with specific examples.

[0057] Example 1: In situ MALDI mass spectrometry imaging of intact proteins

[0058] (1) Preparation of matrix solution: α-Cyano-4-hydroxycinnamic acid (CHCA) at a concentration of 7 mg / mL was dissolved in 60% acetonitrile containing 0.2% TFA aqueous solution.

[0059] (2) Use Figure 1 The device shown in the figure has the following operating steps:

[0060] 1) Connect the host power supply and vacuum pump power supply;

[0061] 2) Adjust the vacuum pump valve and air valve to the closed state;

[0062] 3) Turn on the heating switch of the heating plate 3 and adjust the heating temperature of the heating plate 3 to 37° C.;

[0063] 4) Set the spraying time of liquid nozzle 2 to 15 s, the incubation time to 30 s, and the drying time to 60 s;

[0064] 5) Set the left and right moving speed of the stepper motor moving platform 1 to 5 mm / s;

[0065] 6) Add 2 mL of CHCA solution into the liquid bottle of the liquid spray head 2, and install the liquid spray head 2 on the liquid spray head bracket with the nozzle facing upward;

[0066] 7) placing the ITO glass slide with the mouse brain tissue slice on the glass slide holder, and closing the door of the vacuum atomization chamber 4;

[0067] 8) Turn on the moving switch of the stepper motor moving platform 1, and wait until the liquid spray head 2 moves to the waiting position;

[0068] 9) Turn on the vacuum pump switch, open the vacuum pump valve, and when the vacuum in the vacuum atomization chamber 4 reaches 0.06MPa, close the vacuum pump valve;

[0069] 10) Turn on the switch of the liquid spray head 2 to start the spraying and incubation process. After the incubation is completed, open the vacuum pump valve and the air valve to start drying. After the drying is completed, close the vacuum pump valve and the air valve, and repeat step 10) until the spraying is completed;

[0070] 11) After spraying is completed, turn off the switch of the liquid spray head 2, the moving switch of the stepping motor moving platform 1, the heating switch of the heating plate 3 and the vacuum pump switch;

[0071] 12) Open the door of the vacuum atomization chamber 4 and take out the glass slide;

[0072] 13) Maintain and clean the liquid ejecting head 2.

[0073] (3) A commercial matrix sprayer was used as the control group: The commercial matrix sprayer used was ImagePrep TM (Bruker), the ITO slide with mouse brain tissue sections was placed on the base, 2 mL of CHCA solution was added to the solvent bottle, and the matrix was sprayed using the CHCA default program.

[0074] (4) In situ mass spectrometry imaging of mouse brain tissue slices: MALDI-TOF mass spectrometry was used to perform mass spectrometry imaging analysis on mouse brain tissue slices. The positive ion linear mode was used, the mass-to-charge ratio acquisition range was set to 4000-20000, the laser energy was 50%, the acquisition rate was 200 Hz, and the spatial resolution was set to 20, 50 and 100 μm respectively. The results are shown in Figure 2. Figure 2 As shown, the matrix sprayer described in the present application makes the matrix crystals of CHCA smaller and more uniform, and can achieve protein spatial distribution imaging at a spatial resolution of 20, 50 and 100 μm.

[0075] Example 2: In situ MALDI mass spectrometry imaging of lipids in tissues

[0076] (1) Prepare the matrix solution: 2,5-dihydroxybenzoic acid (DHB) at a concentration of 15 mg / mL is dissolved in 90% acetonitrile containing 0.1% TFA aqueous solution.

[0077] (2) Use Figure 1 The device shown in the figure has the following operating steps:

[0078] 1) Connect the host power supply and vacuum pump power supply;

[0079] 2) Adjust the vacuum pump valve and air valve to the closed state;

[0080] 3) Turn on the heating switch of the heating plate 3 and adjust the heating temperature of the heating plate 3 to 37° C.;

[0081] 4) Set the spraying time of liquid nozzle 2 to 15 s, the incubation time to 30 s, and the drying time to 30 s;

[0082] 5) Set the left and right moving speed of the stepper motor moving platform 1 to 5 mm / s;

[0083] 6) Add 2 mL of DHB solution into the liquid bottle of the liquid spray head 2, and install the liquid spray head 2 on the liquid spray head bracket with the nozzle facing upward;

[0084] 7) placing the ITO glass slide with the mouse brain tissue slice on the glass slide holder, and closing the door of the vacuum atomization chamber 4;

[0085] 8) Turn on the moving switch of the stepper motor moving platform 1, and wait until the liquid spray head 2 moves to the waiting position;

[0086] 9) Turn on the vacuum pump switch, open the vacuum pump valve, and when the vacuum in the vacuum atomization chamber 4 reaches 0.06MPa, close the vacuum pump valve;

[0087] 10) Turn on the switch of the liquid spray head 2 to start the spraying and incubation process. After the incubation is completed, open the vacuum pump valve and the air valve to start drying. After the drying is completed, close the vacuum pump valve and the air valve, and repeat step 10) until the spraying is completed;

[0088] 11) After spraying is completed, turn off the switch of the liquid spray head 2, the moving switch of the stepping motor moving platform 1, the heating switch of the heating plate 3 and the vacuum pump switch;

[0089] 12) Open the door of the vacuum atomization chamber 4 and take out the glass slide;

[0090] 13) Maintain and clean the liquid ejecting head 2.

[0091] (3) In situ mass spectrometry imaging of mouse brain tissue slices: MALDI-TOF mass spectrometry was used to perform mass spectrometry imaging analysis on mouse brain tissue slices. The positive ion reflectron mode was used, the mass-to-charge ratio acquisition range was set to 500-1000, the laser energy was 90%, the acquisition rate was 200 Hz, and the spatial resolution was set to 20, 50 and 100 μm respectively. The results are shown in Figure 2. Figure 3 As shown, the matrix sprayer described in the present application makes the matrix crystals of DHB small and uniform, and can achieve spatial distribution imaging of lipids such as PA (38:4), sulfatide (18:1 / 18:0 (OH)) and hemoglobin at a spatial resolution of 100 μm.

[0092] Example 3: In situ derivatization of neurotransmitters and MALDI mass spectrometry imaging

[0093] (1) Preparation of matrix and derivatization reagent solutions: α-cyano-4-hydroxycinnamic acid (CHCA) at a concentration of 7 mg / mL was dissolved in 60% acetonitrile containing 0.2% TFA aqueous solution; Preparation of derivatization reagent solution: 2,4,6-trimethylpyridinium borate (Py1) at a concentration of 2.5 mmol / L was dissolved in 50% methanol containing 0.2% TEA aqueous solution.

[0094] (2) Use Figure 1 The device shown in the figure has the following operating steps:

[0095] 1) Connect the host power supply and vacuum pump power supply;

[0096] 2) Adjust the vacuum pump valve and air valve to the closed state;

[0097] 3) Turn on the heating switch of the heating plate 3 and adjust the heating temperature of the heating plate 3 to 37° C.;

[0098] 4) Set the spraying time of liquid nozzle 2 to 15 s, the incubation time to 30 s, and the drying time to 60 s;

[0099] 5) Set the left and right moving speed of the stepper motor moving platform 1 to 5 mm / s;

[0100] 6) Add 0.5 mL of Py1 solution into the liquid bottle of the liquid spray head 2, and install the liquid spray head 2 on the liquid spray head bracket with the nozzle facing upward;

[0101] 7) placing the ITO glass slide with the mouse brain tissue slice on the glass slide holder, and closing the door of the vacuum atomization chamber 4;

[0102] 8) Turn on the moving switch of the stepper motor moving platform 1, and wait until the liquid spray head 2 moves to the waiting position;

[0103] 9) Turn on the vacuum pump switch, open the vacuum pump valve, and when the vacuum in the vacuum atomization chamber 4 reaches 0.06MPa, close the vacuum pump valve;

[0104] 10) Turn on the switch of the liquid spray head 2 to start the spraying and incubation process. After the incubation is completed, open the vacuum pump valve and the air valve to start drying. After the drying is completed, close the vacuum pump valve and the air valve, and repeat step 10) until the spraying is completed;

[0105] 11) Open the door of the vacuum atomization chamber 4, take out the glass slide, and maintain and clean the liquid nozzle 2.

[0106] 12) Add 2 mL of CHCA solution into the liquid bottle of the liquid spray head 2, and install the liquid spray head 2 on the liquid spray head bracket with the nozzle facing upward;

[0107] 13) placing the ITO glass slide with the mouse brain tissue slice on the glass slide holder, and closing the door of the vacuum atomization chamber 4;

[0108] 14) Turn on the moving switch of the stepper motor moving platform 1, and wait until the liquid spray head 2 moves to the waiting position;

[0109] 15) Turn on the vacuum pump switch, open the vacuum pump valve, and when the vacuum in the vacuum atomization chamber 4 reaches 0.06MPa, close the vacuum pump valve;

[0110] 16) Turn on the switch of the liquid spray head 2 to start the spraying and incubation process. After the incubation is completed, open the vacuum pump valve and the air valve to start drying. After the drying is completed, close the vacuum pump valve and the air valve, and repeat step 16) until the spraying is completed;

[0111] 17) After spraying is completed, turn off the switch of the liquid spray head 2, the moving switch of the stepping motor moving platform 1, the heating switch of the heating plate 3 and the vacuum pump switch;

[0112] 18) Open the door of the vacuum atomization chamber 4 and take out the glass slide;

[0113] 19) Maintain and clean the liquid ejecting head 2.

[0114] (3) In situ mass spectrometry imaging of mouse brain tissue slices: MALDI-TOF mass spectrometry was used to perform mass spectrometry imaging analysis on mouse brain tissue slices. The positive ion reflectron mode was used, the mass-to-charge ratio acquisition range was set to 100-1000, the laser energy was 90%, the acquisition frequency was 200 Hz, and the spatial resolution was set to 100 μm. The results are shown in Figure 2. Figure 4 As shown, the matrix sprayer described in the present application can realize in situ tissue derivatization and mass spectrometry imaging, and can realize spatial distribution imaging of neurotransmitters such as γ-aminobutyric acid at a spatial resolution of 100 μm.

[0115] The substrates and derivatization reagents in the above examples were purchased from commercial sources, wherein α-cyano-4-hydroxycinnamic acid (CHCA) was purchased from Sigma-Aldrich (CAS 28166-41-8), 2,5-dihydroxybenzoic acid (DHB) was purchased from Sigma-Aldrich (CAS 207-718-5), and 2,4,6-trimethylpyrylium tetrafluoroborate (Py1) was purchased from TCI (CAS 773-01-3).

[0116] The present invention provides an ultrasonic matrix spraying device, which can spray a variety of MALDI matrices and derivatization reagents on the surface of tissues under vacuum assistance. The device uses the principle of air pressure deposition to ultrasonically spray the matrix solution under vacuum conditions, so that ultrafine droplets evenly fill the vacuum atomization chamber, and then uses air pressure to evenly deposit the ultrafine droplets on the surface of tissue sections, and assists in analyte extraction and matrix crystallization through contact and non-contact heating. The method provided by the present invention can ensure the formation of a fine and uniform matrix and analyte co-crystallization layer on the surface of tissue sections, and realize high spatial resolution imaging of analytes in situ in tissues.

[0117] The above description is only an embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, expansion, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. An ultrasonic matrix spraying device, characterized in that: include: Vacuum atomization chamber; A sample carrying assembly is disposed in the vacuum atomization chamber, and is used to carry the biological sample to be sprayed; The matrix spraying assembly is arranged in the vacuum atomization chamber, and the matrix spraying assembly is used to spray micron-sized matrix droplets into the vacuum atomization chamber; A spray assembly timing control unit, used to control the start and end of the substrate spray assembly; The spray assembly driving unit is arranged at the top of the vacuum atomization chamber and connected to the substrate spray assembly. The spray assembly driving unit is used to drive the substrate spray assembly to move. The vacuum component and air flow component unit are connected to the vacuum atomization chamber. The vacuum component and air flow component unit are used to provide a vacuum environment and airflow for the vacuum atomization chamber, so that micron-sized matrix droplets are evenly deposited on the surface of the biological sample to be sprayed using air pressure under a vacuum environment.

2. The ultrasonic matrix spraying device according to claim 1, characterized in that: The matrix spraying assembly comprises a liquid spraying head, which utilizes ultrasonic waves to break the matrix solution into micron-sized droplets.

3. The ultrasonic matrix spraying device according to claim 2, characterized in that: The liquid spray head has an annular gas nozzle, which is used for spraying micron-sized liquid droplets at multiple angles.

4. The ultrasonic matrix spraying device according to claim 2, characterized in that: The timing control unit of the spraying assembly controls the opening and closing of the liquid spray head and has a second-level countdown function of 0 to 999 minutes.

5. The ultrasonic matrix spraying device according to claim 1, characterized in that: The spraying assembly driving unit can drive the matrix spraying assembly to move horizontally in the left and right directions, and the moving speed is adjustable according to the sample spraying requirements.

6. The ultrasonic matrix spraying device according to claim 1, characterized in that: The sample carrying assembly comprises a glass slide holder and a glass slide, wherein the glass slide holder is arranged at the bottom of the vacuum atomization chamber, and the glass slide is arranged on the glass slide holder for carrying the biological sample to be sprayed.

7. The ultrasonic matrix spraying device according to claim 6, characterized in that: It also includes a bearing component temperature control unit, which includes a heating plate and a temperature probe fixed below the slide holder, the heating plate is used to heat the biological sample to be sprayed, and the temperature probe is used to control the temperature of the biological sample to be sprayed.

8. The ultrasonic matrix spraying device according to claim 1, characterized in that: It also includes an illumination component and an illumination component heat dissipation unit arranged on the vacuum atomization chamber, wherein the illumination component provides visible light, 365nm and 460nm multi-wavelength illumination, and the illumination component heat dissipation unit is used for heat dissipation of the illumination component.

9. A MALDI mass spectrometry imaging matrix spraying method, characterized in that: The ultrasonic matrix spraying device according to any one of claims 1 to 8 is used to perform MALDI mass spectrometry imaging matrix spraying.

10. The MALDI mass spectrometry imaging matrix spraying method according to claim 9, characterized in that: The biological samples to be sprayed include animal tissues, plant tissues, and tumor tissues. The matrix spraying component ultrasonically sprays the matrix solution under vacuum conditions, so that micron-sized matrix droplets evenly fill the vacuum atomization chamber, and then uses air pressure to evenly deposit the micron-sized matrix droplets on the surface of the biological sample to be sprayed, and assists in analyte extraction and matrix crystallization through contact and non-contact heating methods.

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