Lipid in-situ mass spectrometry method and device based on online selective enrichment-optical desorption

Abstract

The invention relates to the technical field of chemical substance detection, in particular to a lipid in-situ mass spectrometry method and device based on online selective enrichment-photoanalysis, and the method comprises the following steps: (1) extracting lipid in a sample into an alkaline protic solvent by adopting an in-situ liquid extraction technology to obtain an in-situ extraction system solution; (2) sucking the in-situ extraction system solution through a transmission pipeline under vacuum or negative pressure, and leading the in-situ extraction system solution to a mass spectrum for mass spectrum detection, wherein a titanium dioxide nano-particle coating is arranged in the transmission pipeline; and (3) after the lipid mass spectrum band in the in-situ extraction system solution is completely subjected to mass spectrum detection, irradiating the titanium dioxide nano-particle coating by adopting an ultraviolet lamp, continuously introducing the alkaline protic solvent, sucking the alkaline protic solvent through a transmission pipeline, and introducing the alkaline protic solvent to a mass spectrum for mass spectrum detection. According to the method disclosed by the invention, online selective enrichment of glycolipid can be realized while in-situ liquid extraction is carried out, meanwhile, the glycosphingolipid is directly analyzed by using ultraviolet light under the condition that solvent switching is not needed, the analysis rate of the glycosphingolipid on the surface of titanium dioxide and the operation convenience are greatly improved, and finally, the detection sensitivity of the glycosphingolipid is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical substance detection, and in particular to a lipid in situ mass spectrometry analysis method and device based on online selective enrichment-photoanalysis. Background Art

[0002] Spatially resolved lipidomics is a popular branch of lipidomics. It uses spatially resolved mass spectrometry technology to obtain the spatial distribution and changes of various lipids in biological samples, such as tissues and cells, in a single test, thereby obtaining multi-channel chemical change information in the disease and physiological process of the living body. It is an important pillar of research such as disease diagnosis, pathological research and disease treatment. The structure of lipids is complex and there are many classifications. Among them, glycosphingolipids are a class of glycolipid substances widely distributed on the surface of animal cell membranes. They play an important biological role in regulating cell recognition, adhesion, proliferation and apoptosis. However, the high coverage detection and complete structural characterization of glycosphingolipids in spatial lipidomics have always been a difficult problem. In terms of detection, due to the large matrix effect of direct mass spectrometry detection of biological sample micro-areas, highly ionized and highly abundant lipids will inhibit the ionization of glycosphingolipids with low ionization efficiency and low signal response. At the same time, since glycosphingolipids and phospholipids are in the same mass spectrum segment, a large number of phospholipid peaks will also interfere with the coverage of sphingolipid peaks. In terms of structural characterization, the sample volume in the sample micro-area is small, and glycolipids, which have very low signals, will not have enough lipid content to achieve secondary mass spectrometry characterization. In addition, glycolipids themselves have complex structures, making it even more difficult to obtain abundant secondary fragments. Currently, the secondary mass spectra of glycosphingolipids are mainly composed of fragments related to the drop of sugar head, and there is little information on fragments related to fatty acid chain structure and hydroxyl and double bond modifications.

[0003] In order to solve the problems of ion suppression and spectral peak interference in these spatial lipidomics analyses, many new spatially resolved mass spectrometry methods have been developed. First, in the pre-treatment methods of spatially resolved mass spectrometry analysis, some methods are aimed at the detection of low-abundance and low-ionization-ability lipids. For example, in situ tissue derivatization and in situ tissue decomposition methods use in situ derivatization reactions and decomposition reactions that occur on the tissue surface without destroying the spatial distribution of the tissue to improve the ionization ability of low-ionized substances or decompose and remove high-abundance substances. These methods are more suitable for targeted spatially resolved mass spectrometry analysis for the detection of a certain type of lipids, and the information of other lipids will be lost in the detection. Secondly, some new spatially resolved ionization technologies with high ionization ability have been developed, such as post-ionization matrix-assisted laser desorption ionization (MALDI2), but these methods can comprehensively and effectively improve the ionization ability of all lipids but cannot eliminate the spectral peak overlap and interference of high-abundance lipids on low-abundance lipids. In addition, some methods based on solid phase extraction separation have also been used to improve the detection coverage of in situ mass spectrometry for low-abundance lipids, such as surface-assisted laser desorption ionization, tissue imprint mass spectrometry imaging, and in situ liquid extraction technology based on online adsorption analysis. This separation-based technology has the potential to increase the coverage of target lipids without losing other lipids, and at the same time eliminate spectral peak interference due to separation. However, these in situ mass spectrometry methods based on solid phase extraction separation usually have to go through complex processes such as sample adsorption, washing, and elution to achieve the purpose of separating high-abundance lipids and enriching low-abundance lipids, and the solvent of the entire system needs to be replaced. For the micro-nanoflow system commonly used in in situ mass spectrometry, it is a cumbersome and lengthy operation process, and it will increase the diffusion of the spectral band and reduce the sensitivity.

[0004] Currently, there are few reports on in situ mass spectrometry methods that can quickly enrich and separate glycosphingolipids without losing other lipids and obtain accurate qualitative information in in situ spatially resolved mass spectrometry such as mass spectrometry imaging or single-cell mass spectrometry. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the pretreatment and separation steps of lipid detection are lengthy, which will increase the diffusion of the spectral band and reduce the sensitivity. The shortcomings and defects mentioned in the above background technology are overcome, and a lipid in situ mass spectrometry analysis method and device based on online selective enrichment-photoanalysis is provided.

[0006] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0007] A lipid in situ mass spectrometry analysis method based on online selective enrichment-photoanalysis comprises the following steps:

[0008] (1) extracting lipids in the sample into an alkaline proton solvent using an in situ liquid extraction technique to obtain an in situ extraction system solution;

[0009] (2) sucking the in-situ extraction system solution through a transmission pipeline by vacuum to a mass spectrometer for mass spectrometry detection, wherein a titanium dioxide nanoparticle coating is arranged in the transmission pipeline;

[0010] (3) After the lipid spectrum bands in the in-situ extraction system solution have been completely detected by mass spectrometry, the titanium dioxide nanoparticle coating is irradiated with an ultraviolet lamp, and the alkaline proton solvent is continuously introduced and inhaled through a transmission pipeline to the mass spectrometer for mass spectrometry detection.

[0011] Preferably, the alkaline protic solvent comprises aqueous ammonia, methanol and acetonitrile, wherein the concentration of aqueous ammonia is 1%-10% by volume of the alkaline protic solvent, more preferably 5%; and the volume ratio of methanol to acetonitrile is 1:1.

[0012] Preferably, the titanium dioxide nanoparticles include calcined and / or uncalcined pure TiO 2 Nanoparticles or doped TiO 2 At least one of the nanoparticles has a particle size of 1-10 nm, and is more preferably calcined anatase TiO 2 Nanoparticles.

[0013] Preferably, the titanium dioxide nanoparticle coating is in-situ polymerized by a sol-gel method and then calcined on the inner wall of the transmission pipeline, or the titanium dioxide nanoparticles are dispersed in an organic solvent, injected into a tube and vacuum-coated on the inner wall of the transmission pipeline; the transmission pipeline includes a capillary or the inner wall of a quantitative loop connected to the sampling probe outlet.

[0014] Preferably, the inner diameter of the capillary is 50-100 μm, and the length of the titanium dioxide nanoparticle coating is not greater than the length of the ultraviolet light irradiation area.

[0015] Preferably, the flow rate of the in-situ extraction system solution is 0.5-9 μL / min, more preferably 1-2 μL / min.

[0016] Preferably, the wavelength of ultraviolet light irradiated by the ultraviolet lamp is 200-400nm, and the ultraviolet light irradiation time is 1-10min.

[0017] Preferably, the in-situ liquid extraction technology continuously pumps the in-situ extraction system solution into the sampling probe, and simultaneously continuously draws the in-situ extraction system solution into the mass spectrometer by vacuum or negative pressure, forming a liquid node at the tip of the sampling probe, and extraction is achieved when the liquid node contacts the sample surface.

[0018] Based on a general inventive concept, the present invention also provides an in situ mass spectrometry analysis device for lipids based on online selective enrichment-photoanalysis, wherein the in situ mass spectrometry analysis device is operated using the described method, and the device comprises an in situ extraction sampling probe and a mass spectrometer, wherein the sampling probe is connected to the mass spectrometer.

[0019] Preferably, when the in-situ liquid extract is drawn into the mass spectrometer in the probe by negative pressure aspiration of a mass spectrometer ion source spray, the probe outlet is connected to the mass spectrometer inlet through a capillary, and titanium dioxide is coated in the capillary between the probe outlet and the mass spectrometer inlet;

[0020] When the in-situ liquid extract is drawn into the mass spectrometer in the probe by using a vacuum pump to draw the solution into a quantitative loop, the probe outlet is connected to a port of a one-position six-way valve, the adjacent port of this port is connected to a waste pipe, and the adjacent ports of the waste pipe port and the probe outlet port are respectively connected by a quantitative loop, one of the remaining two ports of the six-way valve is connected to a microfluidic pump, and the other is connected to the mass spectrometer inlet through a capillary, and a titanium dioxide coating is applied in the connecting capillary between the six-way valve port and the mass spectrometer inlet.

[0021] Preferably, the sampling probe comprises at least one of a capillary coaxial sleeve, a double-hole quartz tube, a goose-shaped tube or a folded tube, and the mass spectrometry ion source comprises an electrospray ionization source or an atmospheric pressure chemical ionization source.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The method of the present invention can achieve online selective enrichment of glycolipids during in-situ liquid extraction, and directly analyze glycosphingolipids using ultraviolet light without solvent switching, which greatly improves the rate of analysis of glycosphingolipids on the titanium dioxide surface and the simplicity of operation, and ultimately improves the detection sensitivity of glycosphingolipids;

[0024] (2) In addition to enriching glycosphingolipids, the method of the present invention can also separate other lipids from glycosphingolipids during the entire process, and detect them by mass spectrometry before glycosphingolipids. This method solves the interference between the spectral peaks of phospholipids and glycolipids, and does not lose the information of other lipids;

[0025] (3) The method of the present invention not only rapidly analyzes glycosphingolipids online, but also deglycosylates glycosphingolipids into ceramides through a photocatalytic reaction. Ceramides have more abundant secondary mass spectrometry fragments than glycosphingolipids, which can provide information for the structural characterization of the fatty acid chains of glycosphingolipids.

[0026] (4) The present invention provides a lipid in situ mass spectrometry analysis device based on online selective enrichment-photoanalysis, which can achieve rapid enrichment and separation of glycosphingolipids without losing other lipids and obtain accurate mass spectrometry analysis information. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 Schematic diagram of the in-situ liquid extraction-online selective enrichment-photoanalysis mass spectrometry system of the present invention (A) and the principle schematic diagram of glycolipid deglycosylation photoanalysis (B);

[0029] Figure 2 Characterization diagram of the titanium dioxide porous coating tube, including microscope image (A) and electron microscope image (B);

[0030] Figure 3 These are the adsorption breakthrough curves of various lipids in the coated tube at various flow rates during the online adsorption process in Example 1; wherein PC represents acetylcholine phospholipids, CB represents cerebroside (hexaglycoceramide), SM represents acetylcholine sphingomyelin, and Cer represents ceramide.

[0031] Figure 4 The comparison of the analysis kinetic curves of glycosphingolipids at different flow rates during the online optical analysis in Example 1 and the injection curves of the glycolipid injection mass spectrometry without adsorption analysis;

[0032] Figure 5 The mass spectra of in situ mass spectrometry detection of brain tissue using the new online enrichment-optical decomposition in situ mass spectrometry method and the direct in situ mass spectrometry method without online enrichment-optical decomposition in Example 2 are compared; wherein PC represents acetylcholine phospholipids, CB represents cerebroside (hexaglycoceramide), SM represents acetylcholine sphingomyelin, Cer represents ceramide, ST represents sulfatide, and PE represents phosphatidylethanolamine.

[0033] Figure 6 The figure is a Venn diagram showing the types and numbers of lipids detected by in situ mass spectrometry of brain tissue using the new method and the traditional method in Example 2; wherein PC represents acetylcholine phospholipids, LPC represents lysoacetylcholine phospholipids, PE represents phosphatidylethanolamine, LPE represents lysophosphatidylethanolamine, HexCer represents hexaglycoceramide, LacCer represents lactic acid ceramide, PLs represents phospholipids, SLs represents sphingolipids, and GLs represents glycerides.

[0034] Figure 7 The adsorption and analysis mass spectra obtained by detecting a single nerve cell HT22 using the new method in Example 3, DETAILED DESCRIPTION

[0035] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0036] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0037] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0038] A high coverage lipid in situ mass spectrometry analysis method based on online selective enrichment-photoanalysis according to a specific embodiment of the present invention comprises the following steps:

[0039] S1. Build an in-situ liquid extraction-online enrichment-optical desorption mass spectrometry device. Figure 1 As shown. The microfluidic sampling probe is fixed on the z-axis platform of the three-axis platform, and the sample is fixed on the xy-axis platform. The positional relationship between the positioning probe and the sample is controlled by three-axis movement. When the microfluidic sampling probe is directly connected to the mass spectrometer, the in-situ micro-liquid node sampling-mass spectrometry technology is to continuously pump the extraction liquid into the sampling probe, and at the same time, the extraction liquid is continuously sucked into the mass spectrometer through the vacuum degree of the mass spectrometer ion source nozzle, forming a liquid node at the tip of the probe, and the extraction is achieved when the liquid node contacts the sample surface. The liquid node formed between the probe and the sample surface is observed in real time by a microscopic camera. The capillary connected between the probe outlet and the mass spectrometer is a capillary coated with titanium dioxide. When used in conjunction with the mass spectrometer through a six-way valve, the probe outlet is connected to a six-way valve connected to a quantitative loop, and the six-way valve is connected to a vacuum pump. The extraction liquid is continuously sucked into the quantitative loop by the vacuum pump to form a liquid node at the tip of the probe. The six-way valve switching enables the quantitative loop to switch between the sampling system and the liquid chromatography-mass spectrometry injection system. The titanium dioxide coating is on the capillary wall connecting the six-way valve and the mass spectrometer inlet. The sampling probe can be at least one of a capillary coaxial sleeve, a double-hole quartz tube, a goose-shaped tube or a folded tube. The mass spectrometer ion source can be an electrospray ionization source or an atmospheric pressure chemical ionization source.

[0040] The titanium dioxide particles may be calcined and / or uncalcined pure TiO 2 Nanoparticles or doped TiO 2 Nanoparticles: In a specific embodiment, the titanium dioxide nanoparticles have a particle size of 1-10 nm.

[0041] The inner diameter of the titanium dioxide open capillary tube should be less than 300 μm, preferably 50-100 μm, and more preferably 100 μm.

[0042] The open capillary tube with titanium dioxide nanoparticles coated on the inner wall can be prepared by in-situ polymerizing the whole material by the sol-gel method and then pushing out the main body of the material by a high-pressure pump to leave a porous coating on the tube wall. Alternatively, the nanoparticles can be directly dispersed in titanium dioxide sol, injected into the tube and vacuum coated.

[0043] S2. Using ammonia-methanol / acetonitrile (1:1, v / v) solution as the extracting liquid, the various lipids in the micro-area of ​​the biological sample are extracted into the solvent by in-situ liquid extraction micro-sampling technology, and then the extracting liquid is pumped into the mass spectrometer or quantitative loop by the negative pressure of the mass spectrometer or vacuum pump. When the extracting liquid is pumped into the mass spectrometer by the negative pressure formed by the spray of the mass spectrometer ion source, the extracting liquid will pass through the titanium dioxide coating on the way into the mass spectrometer ion source, so that the glycosphingolipid lipids are captured by the titanium dioxide coating, while other lipids enter the mass spectrometer and are detected. The flow rate of the extracting liquid is balanced and controlled by adjusting the size of the injection pump that delivers the extracting liquid and the spray sheath gas of the mass spectrometer ion source; when the extracting liquid is pumped into the mass spectrometer by the negative pressure generated by the vacuum pump, the extracting liquid enters the quantitative loop and is collected. Once the quantitative loop is full of extracting liquid, the six-way valve will rotate to connect the quantitative loop with the injection pump and mass spectrometer of another flow path. At this time, the syringe pump of the other flow path will push the extract in the quantitative loop into the mass spectrometer. On the way into the mass spectrometer, the extract will pass through the titanium dioxide coating, so that the glycosphingolipids are also captured by the coating, while other lipids flow into the mass spectrometer for detection. In this mode, the flow rate of the extract flowing through the coating is completely controlled by the syringe pump.

[0044] The biological samples mainly include various animal and plant tissue sections, cell slides, cell culture dishes, etc.

[0045] The concentration of aqueous ammonia in the aqueous ammonia-methanol / acetonitrile (1:1, v / v) solution is usually 1%-10%.

[0046] S3. After the lipid bands in the sample extract have completely passed through the coated tube, ultraviolet light is used to irradiate the coated tube to achieve deglycosylation of the adsorbed glycolipids and resolve them from the coated tube. During the irradiation process, the clean ammonia-methanol / acetonitrile (1:1, v / v) solution still passes through the coated tube at the same flow rate as mentioned above and flows into the mass spectrometer for detection.

[0047] The wavelength of ultraviolet light can be 200-400nm, preferably 200-300nm, and more preferably 250nm. Since the maximum absorption wavelength of titanium dioxide to ultraviolet light is about 250nm, ultraviolet light with a wavelength of 200-300nm can be better absorbed by titanium dioxide, and the energy generated is higher than the energy band gap of titanium dioxide electronic transition 3.2eV. The ultraviolet light generated by the ultraviolet lamp needs to completely cover the capillary containing the titanium dioxide coating, and maintain a certain irradiation distance. The irradiation distance mainly affects the unit area power of the light, and the optimal distance is related to the unit area light intensity of the ultraviolet lamp. The ultraviolet lamp used is not limited, for example, it can be any mercury lamp, xenon lamp, etc. that produces 200-400nm passband or single band light. Among them, the mercury lamp with stronger ultraviolet band intensity is preferred. Usually, if the capillary surface is protected by a coating that is opaque to ultraviolet light (such as a polyimide coating), the outer coating needs to be burned off at high temperature to remove it so that the coating can be exposed to ultraviolet light.

[0048] The photolysis reaction time can be 1-10 minutes, preferably 2 minutes. This is related to the rate of photocatalytic deglycosylation of glycolipids adsorbed on the surface of the material. From the experimental results, the adsorbed glycolipids can be completely eluted in about 2 minutes.

[0049] The principle of glycolipid analysis is as follows Figure 1 As shown, glycolipids are selectively adsorbed on the titanium dioxide surface due to bidentate chelation between the sugar head and Lewis acid sites in titanium dioxide in an alkaline environment. When ultraviolet light is irradiated to the titanium dioxide surface, titanium dioxide absorbs ultraviolet light to generate photogenerated electrons and holes. The holes oxidize the water adsorbed on the surface into hydroxyl radicals, so that the glycolipid adsorbed on the titanium dioxide surface is selectively oxidized and decomposed into ceramide by hydroxyl radicals. The sugar head is further oxidized and decomposed into small molecules on the titanium dioxide surface, and the dropped ceramide is resolved into the solution as a product because it cannot be adsorbed on the titanium dioxide surface.

[0050] In situ detection can be performed by using the above-mentioned in situ mass spectrometry technology to perform in situ mass spectrometry detection at a single spatial point of the biological sample, such as a certain position of a functional area of ​​a tissue or the position of a single cell in a cell culture dish; in situ mass spectrometry technology can also be used to scan a certain area on the surface of the sample for detection; a certain area can also be scanned point by point to obtain imaging results.

[0051] Scanning imaging refers to the use of in-situ mass spectrometry technology with spatial resolution to analyze and ionize each pixel on the sample surface one by one, and finally recombining the signal of each pixel into the signal of lipids at different spatial positions of the sample, thereby obtaining signal imaging images of different lipids.

[0052] The scanning speed of the sampling probe is 0-1000 μm / s. The micro-liquid node formed by the probe is stable within this speed range. The micro-liquid node always maintains a constant liquid node coverage area and no bubbles are generated.

[0053] The present invention is further described below by way of examples.

[0054] Example 1: Investigation of adsorption capacity and desorption kinetics in online adsorption-photodesorption process

[0055] 1) Capillary coating: Activate the capillary (356μmO.D. / 100μmI.D., UV-transparent coating) with 1mol / LNaOH, and then clean the capillary with pure water, 0.01mol / LHCl aqueous solution, methanol, and pure water, respectively. Use the sol-gel method with butyl phthalate as a precursor and acetic acid as a protective agent, and hydrolyze in an ice bath to obtain a sol. Ultrasonic disperse P25 titanium dioxide nanoparticles in the sol, push the sol with dispersed nanoparticles into the cleaned capillary, react at room temperature for half an hour, and then push it out. The obtained coated capillary is dried at 60°C. Then repeat the above process 6 times. Titanium dioxide nanoparticle coating such as Figure 2 As shown, the length is 5cm.

[0056] 2) Online adsorption process: 10 μg / mL of lipid standard mixture (PC (16:0 / 18:1), Cer (d18:1 / 18:0), SM (d18:1 / 16:0), GlcCer (d18:2 / 16:0) and PI (18:2 / 16:0)) were dissolved in 6% ammonia-methanol / acetonitrile (1:1, v / v) solution and the adsorption was carried out as follows. Figure 3 The indicated flow rates pump the coated capillaries and allow the solution to enter the mass spectrometer for real-time detection.

[0057] 3) Online photoanalysis process: 25 μL of 10 μg / mL GlcCer (d18:2 / 16:0) was dissolved in a 6% ammonia-methanol / acetonitrile (1:1, v / v) solution, and loaded onto the coated capillary at a flow rate of 2 μL / min using a six-way valve, and the solution was allowed to enter the mass spectrometer for real-time detection. After the standard solution was pumped in, an unfiltered mercury lamp (200-400nm passband) was used to irradiate the coated capillary for 5 minutes, keeping the mercury lamp current at 8 mA and the irradiation distance at 10 cm. The 6% ammonia-methanol / acetonitrile (1:1, v / v) solution that flowed out during the irradiation process also entered the mass spectrometer for detection.

[0058] 4) Mass spectrometry analysis: The mass spectrometer was an electrospray ion source-ion trap tandem time-of-flight mass spectrometer (IT-TOF, Shimadzu Corporation, Japan). The ion source operated in positive and negative ion switching mode, the voltage applied to the spray needle was +4500V and -3000V, the ion source temperature was 200°C, and the nebulizing gas (N2 ) flow rate 1.5 L / min. The mass analyzer performed a first-stage mass spectrometry full scan mode with a scan range of 500-1000 m / z.

[0059] 5) Result analysis: From the results ( Figure 3 ) It can be seen that this coated tube has no adsorption on lipids other than glycolipids, but has an adsorption efficiency of nearly 100% for glycolipids at a certain flow rate and volume. When the flow rate is lower, the volume of the coated tube that can achieve 100% adsorption of glycolipids is larger, indicating that low flow rate is conducive to the increase of the breakthrough volume of the coated tube. When online analysis is performed ( Figure 4 ), the flow rate of the solution also has a great influence on the analytical band. The smaller the flow rate, the narrower the analytical band and the higher the analytical signal peak. Therefore, online adsorption and optical analysis have the highest efficiency only at low flow rates. Compared with the lipid bands of direct injection without adsorption analysis, it can be found that adsorption-analysis greatly increases the signal of glycolipids, indicating that the online process has a certain enrichment ability.

[0060] Example 2: Analysis of lipid composition at a single point in tissue using in situ liquid extraction online adsorption-photodesorption mass spectrometry and comparison with conventional in situ liquid extraction mass spectrometry

[0061] 1) Construction of in-situ liquid extraction device: The in-situ liquid extraction probe uses a stretched coaxial sleeve probe (external capillary size 251μm ID / 356μm OD, internal capillary size 100μm ID / 163μm OD), a section of the inner and outer tubes are pulled to a narrow mouth, the outer tube port is 200um outer diameter, and the inner tube port is 50um inner diameter. The probe inlet is connected to the injection pump, and the probe outlet is directly connected to the mass spectrometer. The inner wall of the capillary connecting the probe and the mass spectrometer is coated with titanium dioxide nanoparticles, and the method is the same as in Example 1. The length of the titanium dioxide coating is 5cm, and the outer membrane of this section of the capillary is burned with an open flame. A mercury lamp is placed in this area.

[0062] 2) In-situ liquid extraction and online adsorption of tissue: The above probe is used to perform in-situ liquid extraction at a certain position of brain tissue slices. The syringe pump pumps into the probe at a flow rate of 2μL / min. The mass spectrometer adjusts the electrospray sheath gas flow to balance its negative pressure with the positive pressure of the syringe pump to keep the liquid node stable. The extract passes through the titanium dioxide coating and enters the mass spectrometer for detection.

[0063] 3) Online optical analysis: After 2 minutes of single-point extraction, the probe is lifted and the mercury lamp illuminates the coating tube position. At this time, the extract still flows into the mass spectrometer at a flow rate of 2 μL / min for detection. The mercury lamp stops irradiation after 2 minutes.

[0064] 4) Mass spectrometry analysis: Same as Example 1.

[0065] 5) Result analysis: Figure 5It can be seen that after online adsorption by the coated tube, a large amount of phospholipids, glycerides and sugar-free sphingolipids were detected by mass spectrometry, and when the light was photoresolved, many peaks were detected in the mass range of 500-700 by mass spectrometry. Through the primary mass spectrometry and its secondary mass spectrometry search, it can be qualitatively determined that they are ceramides after deglycosylation of glycolipids. If the in-situ liquid extraction device without titanium dioxide coating is used to directly detect brain tissue slices, very few glycolipid peaks are obtained, and they are also interfered by a large number of phospholipid peaks around them. Figure 6 The number of different lipids detected in the two methods was counted, and it can be seen that the new method detected 9 times more glycosphingolipids than the traditional method, including 20 HexCer and 13 LacCer, while the traditional method could only detect 4 HexCer.

[0066] Example 3: Analysis of lipid composition of single neurons using in situ liquid extraction and online adsorption-photodesorption mass spectrometry

[0067] 1) Construction of in-situ liquid extraction device: same as in Example 2.

[0068] 2) In-situ liquid extraction and online adsorption of single neurons: An inverted microscope is used to find single neurons in the field of view, and the probe is moved above the neuron through a three-axis platform for in-situ liquid extraction. The syringe pump pumps into the probe at a flow rate of 2μL / min. The mass spectrometer adjusts the electrospray sheath gas flow to balance the negative pressure with the positive pressure of the syringe pump to keep the liquid node stable. The extract passes through the titanium dioxide coating and enters the mass spectrometer for detection.

[0069] 3) Online optical analysis: Same as Example 2

[0070] 4) Mass spectrometry analysis: Same as Example 1.

[0071] 5) Result analysis: Figure 7 It can be seen that before optical analysis, mass spectrometry detected more phospholipids in neurons, while after optical analysis, a large number of ceramide peaks appeared. By adding sugar groups to these ceramides, their original lipid structure can be restored.

[0072] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Therefore, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the scope of protection of the technical solution of the present invention.

Claims

1. A lipid in situ mass spectrometry analysis method based on online selective enrichment-photoanalysis, characterized in that: The steps include: (1) extracting lipids in the sample into an alkaline proton solvent using an in situ liquid extraction technique to obtain an in situ extraction system solution; (2) sucking the in-situ extraction system solution through a transmission pipeline to a mass spectrometer for mass spectrometry detection by negative pressure, wherein a titanium dioxide nanoparticle coating is provided in the transmission pipeline; (3) After the lipid spectrum bands in the in-situ extraction system solution have been completely detected by mass spectrometry, the titanium dioxide nanoparticle coating is irradiated with an ultraviolet lamp, and the alkaline proton solvent is continuously introduced and inhaled through a transmission pipeline to the mass spectrometer for mass spectrometry detection.

2. The in-situ mass spectrometry analysis method according to claim 1, characterized in that: The alkaline proton solvent comprises ammonia water, methanol and acetonitrile, wherein the concentration of ammonia water is 1%-10% of the volume of the alkaline proton solvent, and the volume ratio of methanol to acetonitrile is 1:

1.

3. The in-situ mass spectrometry method according to claim 1, characterized in that: The titanium dioxide nanoparticles include at least one of calcined and / or uncalcined pure TiO2 nanoparticles or doped TiO2 nanoparticles, and the particle size thereof is 1-10 nm.

4. The in-situ mass spectrometry analysis method according to claim 1, characterized in that: The titanium dioxide nanoparticle coating is in-situ polymerized by a sol-gel method and then calcined on the inner wall of the transmission pipeline, or the titanium dioxide nanoparticles are dispersed in an organic solvent, injected into a tube and vacuum-coated on the inner wall of the transmission pipeline; the transmission pipeline includes a capillary tube or the inner wall of a quantitative loop connected to the sampling probe outlet.

5. The in-situ mass spectrometry method according to claim 4, characterized in that: The inner diameter of the capillary is 50-100 μm, and the length of the titanium dioxide nanoparticle coating is not greater than the length of the ultraviolet light irradiation area.

6. The in-situ mass spectrometry analysis method according to claim 1, characterized in that: The flow rate of the in-situ extraction system solution is 0.5-9 μL / min.

7. The in-situ mass spectrometry analysis method according to claim 1, characterized in that: The wavelength of ultraviolet light irradiated by the ultraviolet lamp is 200-400nm, and the ultraviolet light irradiation time is 1-10min.

8. The in-situ mass spectrometry analysis method according to any one of claims 1 to 7, characterized in that: The in-situ liquid extraction technology continuously pumps the in-situ extraction system solution into the sampling probe, and simultaneously continuously draws the in-situ extraction system solution into the mass spectrometer through negative pressure, forming a liquid node at the tip of the sampling probe, and the extraction is achieved when the liquid node contacts the sample surface.

9. A lipid in situ mass spectrometry analysis device based on online selective enrichment-photoanalysis, characterized in that: The in-situ mass spectrometry analysis device is operated using the method according to any one of claims 1 to 8. The device comprises an in-situ extraction sampling probe and a mass spectrometer, and the sampling probe is connected to the mass spectrometer.

10. The in-situ mass spectrometry device according to claim 9, characterized in that: When the in-situ liquid extract is sucked into the mass spectrometer in the probe by negative pressure suction of the mass spectrometer ion source spray, the probe outlet is connected to the mass spectrometer inlet through a capillary, and titanium dioxide is coated in the capillary between the probe outlet and the mass spectrometer inlet; When the in-situ liquid extract is drawn into the mass spectrometer in the probe by using a vacuum pump to draw the solution into a quantitative loop, the probe outlet is connected to a port of a one-position six-way valve, the adjacent port of this port is connected to a waste pipe, and the adjacent ports of the waste pipe port and the probe outlet port are respectively connected by a quantitative loop, one of the remaining two ports of the six-way valve is connected to a microfluidic pump, and the other is connected to the mass spectrometer inlet through a capillary, and a titanium dioxide coating is applied in the connecting capillary between the six-way valve port and the mass spectrometer inlet.

11. The in-situ mass spectrometry device according to claim 9, characterized in that: The sampling probe includes at least one of a capillary coaxial sleeve, a double-hole quartz tube, a goose-shaped tube or a folded tube, and the mass spectrometry ion source includes an electrospray ionization source or an atmospheric pressure chemical ionization source.

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