A lipid in situ mass spectrometry analysis method and apparatus based on online selective enrichment-optical desorption

Abstract

This invention relates to the field of chemical substance detection technology, specifically to a lipid in-situ mass spectrometry analysis method and apparatus based on online selective enrichment-photometric desorption. The method includes the following steps: (1) extracting lipids from the sample into an alkaline proton solvent using in-situ liquid extraction technology to obtain an in-situ extraction system solution; (2) drawing the in-situ extraction system solution through a transfer pipeline under vacuum or negative pressure for mass spectrometry detection, wherein a titanium dioxide nanoparticle coating is provided in the transfer pipeline; (3) after the lipid bands in the in-situ extraction system solution have been completely detected by mass spectrometry, irradiating the titanium dioxide nanoparticle coating with an ultraviolet lamp, continuing to pass an alkaline proton solvent, drawing it through the transfer pipeline for mass spectrometry detection. This invention enables online selective enrichment of glycolipids during in-situ liquid extraction, and direct desorption of glycosphingolipids using ultraviolet light without solvent switching, greatly improving the rate of glycosphingolipid desorption on the titanium dioxide surface and the ease of operation, ultimately improving the detection sensitivity of glycosphingolipids.

Description

Technical Field

[0001] This invention relates to the field of chemical substance detection technology, specifically to a lipid in situ mass spectrometry analysis method and apparatus based on online selective enrichment-optical desorption. Background Technology

[0002] Spatial-resolved lipidomics is a popular branch of lipidomics. It uses spatially resolved mass spectrometry to obtain the spatial distribution and changes of various lipids in biological samples, such as tissues and cells, in a single detection. This provides multi-channel chemical information on disease and physiological processes, making it a crucial pillar for research in disease diagnosis, pathology, and treatment. Lipids have complex structures and numerous classifications. Glycosphingolipids are a class of glycolipids widely distributed on the surface of animal cell membranes, playing important biological roles in regulating cell recognition, adhesion, proliferation, and apoptosis. However, high-coverage detection and complete structural characterization of glycosphingolipids in spatial lipidomics remain challenging. In terms of detection, direct mass spectrometry detection of micro-regions in biological samples suffers from significant matrix effects. Highly ionized and abundant lipids inhibit the ionization of glycosphingolipids, which have low ionization efficiency and low signal response. Furthermore, since glycosphingolipids and phospholipids reside in the same mass spectrometric region, numerous phospholipid peaks also interfere with and cover sphingolipid peaks. In terms of structural characterization, the small sample volume in micro-regions means that lipids with inherently low signal strength, such as glycolipids, will not have sufficient lipid content for secondary mass spectrometry characterization. Furthermore, the complex structure of glycolipids makes obtaining abundant secondary fragments even more difficult. Currently, secondary mass spectrometry of glycosphingolipids mainly focuses on fragments related to glycosyl head loss, with very little information on fragments related to fatty acid chain structure, hydroxyl groups, and double bond modifications.

[0003] To address the issues of ion suppression and peak interference in spatial lipidomics analyses, several novel spatially resolved mass spectrometry (SMS) methods have been developed. Firstly, in pretreatment methods for SMS analysis, some techniques are designed for the detection of low-abundance, low-ionization-capacity lipids. For example, in-situ tissue derivatization and in-situ tissue decomposition utilize in-situ derivatization and decomposition reactions on the tissue surface that do not disrupt the tissue's spatial distribution to enhance the ionization capacity of low-ionization substances or decompose and remove high-abundance substances. These methods are more suitable for target SMS analysis of specific lipid classes and may lose information about other lipids during detection. Secondly, some new high-ionization-capacity SMS techniques have been developed, such as matrix-assisted laser desorption / ionization (MALDI-2) after ionization. However, while these methods can comprehensively and effectively enhance the ionization capacity of all lipids, they cannot eliminate peak overlap and interference from high-abundance lipids on low-abundance lipids. In addition, some solid-phase extraction (SPE)-based separation methods have been used to improve the detection coverage of low-abundance lipids in in-situ mass spectrometry, such as surface-assisted laser desorption / ionization, tissue imprinting mass spectrometry, and in-situ liquid extraction techniques based on online adsorption desorption. These separation-based techniques have the potential to improve the coverage of target lipids without losing other lipids, while also eliminating peak interference due to separation. However, these SPE-based in-situ mass spectrometry methods typically require complex processes such as sample loading, adsorption, washing, and elution to achieve the separation of high-abundance lipids and enrichment of low-abundance lipids. Furthermore, they require replacing the solvent in the entire system, which is a cumbersome and lengthy process for the micro / nanofluidic systems commonly used in in-situ mass spectrometry, and can increase band diffusion and reduce sensitivity.

[0004] Currently, there are few reported in-situ mass spectrometry methods that can rapidly 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 this invention is that the pretreatment and separation steps for lipid detection are lengthy, which increases the diffusion of spectral bands and reduces sensitivity. This invention overcomes the shortcomings and defects mentioned in the background art and provides a lipid in situ mass spectrometry analysis method and device based on online selective enrichment-optical desorption.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

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

[0008] (1) The lipids in the sample were extracted into an alkaline protic solvent by in-situ liquid extraction technology to obtain an in-situ extraction system solution;

[0009] (2) The in-situ extraction system solution is drawn into the mass spectrometer through a transmission pipeline by vacuum and then detected by mass spectrometry. A titanium dioxide nanoparticle coating is provided in the transmission pipeline.

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

[0011] Preferably, the alkaline protic solvent includes ammonia, methanol, and acetonitrile, wherein the concentration of ammonia is 1%-10% of the 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 at least one of calcined and / or uncalcined pure TiO2 nanoparticles or doped TiO2 nanoparticles, with a particle size of 1-10 nm, more preferably calcined anatase TiO2 nanoparticles.

[0013] Preferably, the titanium dioxide nanoparticle coating is applied to the inner wall of the transmission pipeline by in-situ polymerization using a sol-gel method, followed by calcination; or the titanium dioxide nanoparticles are dispersed in an organic solvent, injected into the pipeline, and then vacuum-coated onto the inner wall of the transmission pipeline. The transmission pipeline includes the inner wall of a capillary tube or 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 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 the ultraviolet light irradiated by the ultraviolet lamp is 200-400nm, and the irradiation time is 1-10min.

[0017] Preferably, the in-situ liquid extraction technology involves continuously pumping the in-situ extraction system solution into the sampling probe, while simultaneously drawing the in-situ extraction system solution into the mass spectrometer through vacuum or negative pressure, forming a liquid node at the tip of the sampling probe, and achieving extraction when the liquid node contacts the sample surface.

[0018] Based on a general inventive concept, the present invention also provides a lipid in situ mass spectrometry analysis device based on online selective enrichment-optical desorption, wherein the in situ mass spectrometry analysis device is operated using the method described above, and the device includes 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 aspirated into the mass spectrometer via negative pressure aspiration through a mass spectrometer ion source spray, the probe outlet is connected to the mass spectrometer inlet via a capillary tube, and a titanium dioxide coating is applied to the capillary tube between the probe outlet and the mass spectrometer inlet.

[0020] When the in-situ liquid extract is drawn into the mass spectrometer via a vacuum pump, the probe outlet is connected to one port of a two-position six-way valve. The adjacent port of this port is connected to a waste liquid tube. The adjacent ports of the waste liquid tube port and the probe outlet port are 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 via a capillary tube. A titanium dioxide coating is applied to the capillary tube connecting the six-way valve port and the mass spectrometer inlet.

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

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) The method of the present invention can achieve online selective enrichment of glycolipids while performing in-situ liquid extraction, and can directly decompose glycolipids with ultraviolet light without solvent switching, which greatly improves the rate of glycolipid decomposition on titanium dioxide surface and the ease of operation, and ultimately improves the detection sensitivity of glycolipids.

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

[0025] (3) The method of the present invention not only performs online rapid optical desorption of glycosphingolipids, but also deglycosylates glycosphingolipids into ceramides through photocatalytic reaction. Ceramides have more secondary mass spectrometry fragments than glycosphingolipids, which will provide information for the structural characterization of glycosphingolipid fatty acid chains.

[0026] (4) This invention provides a lipid in situ mass spectrometry analysis device based on online selective enrichment-optical desorption. Using this device, it is possible to rapidly enrich and separate glycosphingolipids without losing other lipids and obtain accurate qualitative mass spectrometry analysis information. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram (A) of the in-situ liquid extraction-online selective enrichment-optical desorption mass spectrometry system of the present invention and a schematic diagram (B) of the principle of glycolipid deglycosylation optical desorption.

[0029] Figure 2 Characterization images of the porous titanium dioxide coated tube, including microscopic image (A) and electron microscopic image (B);

[0030] Figure 3 The images show the adsorption breakthrough curves of various lipids in the coated tube at different flow rates during the online adsorption process in Example 1; where PC represents acetylcholine phospholipids, CB represents cerebrosides (hexaglycosylceramides), SM represents acetylcholine sphingomyelins, and Cer represents ceramides.

[0031] Figure 4 This is a comparison of the desorption kinetics curves of glycosphingolipids at different flow rates during online photodesorption in Example 1 with the injection curves of glycosphingolipids without adsorption desorption by mass spectrometry.

[0032] Figure 5 This is a comparison of mass spectra of brain tissue in situ mass spectrometry detection using the novel online enrichment-optical desorption in situ mass spectrometry method in Example 2 and the direct in situ mass spectrometry method without online enrichment-optical desorption. In this comparison, PC represents acetylcholine phospholipids, CB represents cerebrosides (hexaglycosylceramide), SM represents acetylcholine sphingomyelin, Cer represents ceramide, ST represents cerebroside sulfate, and PE represents phosphatidylethanolamine.

[0033] Figure 6 This 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; where PC represents acetylcholine phospholipids, LPC represents lysoacetylcholine phospholipids, PE represents phosphatidylethanolamine, LPE represents lysophosphatidylethanolamine, HexCer represents hexaglycosylceramide, LacCer represents lactic acid ceramide, PLs represent phospholipids, SLs represent sphingolipids, and GLs represent glycerides.

[0034] Figure 7 The adsorption and desorption mass spectra obtained by the new method in Example 3 for detecting HT22 in a single nerve cell are shown. Detailed Implementation

[0035] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0036] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

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

[0038] A specific embodiment of the present invention provides a high-coverage lipid in-situ mass spectrometry analysis method based on online selective enrichment-optical desorption, comprising the following steps:

[0039] S1. Construct an in-situ liquid extraction-online enrichment-optical desorption mass spectrometry (ODMS) device. For example... Figure 1 As shown, the microfluidic sampling probe is fixed on the z-axis platform of a triaxial platform, while the sample is fixed on the x and y-axis platforms. The positional relationship between the probe and the sample is controlled by triaxial movement. When the microfluidic sampling probe is directly connected to a mass spectrometer, in-situ microliquid node sampling-mass spectrometry (MLMS) involves continuously pumping extractant into the sampling probe while simultaneously drawing the extractant into the mass spectrometer through the vacuum of the mass spectrometer ion source nozzle, forming a liquid node at the probe tip. Extraction is achieved when the liquid node contacts the sample surface. A microscope camera is used to observe the liquid node formed between the probe and the sample surface in real time. The capillary connecting the probe outlet and the mass spectrometer is coated with titanium dioxide. When coupled with the mass spectrometer via a six-way valve, the probe outlet is connected to a six-way valve with a quantitative loop, which is then connected to a vacuum pump. The vacuum pump continuously draws the extractant into the quantitative loop, forming a liquid node at the probe tip. Switching the six-way valve allows the quantitative loop to switch between the sampling system and the liquid chromatography-mass spectrometry injection system. A titanium dioxide coating is applied to the capillary wall connecting the six-way valve and the mass spectrometer inlet. The sampling probe can be at least one of the following: a capillary coaxial sleeve, a two-hole quartz tube, a gooseneck 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 can be calcined and / or uncalcined pure TiO2 nanoparticles or doped TiO2 nanoparticles. In one specific embodiment, the titanium dioxide nanoparticles have a particle size of 1-10 nm.

[0041] The inner diameter of the titanium dioxide open capillary needs to be <300μm, preferably 50-100μm, and more preferably 100μm.

[0042] Open capillary tubes with titanium dioxide nanoparticles coated on the inner wall can be polymerized in situ using the sol-gel method, and then the main body of the material can be pushed out by a high-pressure pump, leaving a porous coating on the tube wall. Alternatively, the nanoparticles can be directly dispersed in titanium dioxide sol, injected into the tube, and coated under vacuum.

[0043] S2. Using an ammonia-methanol / acetonitrile (1:1, v / v) solution as the extractant, in-situ liquid extraction microsampling technology is employed to extract various lipids from micro-regions of biological samples into the solvent. The extractant is then drawn into the mass spectrometer or quantitative loop using a mass spectrometer or vacuum pump under negative pressure. When the extractant is drawn in under negative pressure generated by the mass spectrometer ion source spray, it passes through a titanium dioxide coating on its way to the ion source. This allows glycosphingolipids to be captured by the titanium dioxide coating, while other lipids are detected by the mass spectrometer. The flow rate of the extractant is balanced and controlled by adjusting the injection pump supplying the extractant and the gas flow rate of the mass spectrometer ion source spray sheath. When the extractant is drawn in under negative pressure generated by the vacuum pump, it enters the quantitative loop for collection. Once the quantitative loop is full of extractant, a six-way valve rotates, connecting the quantitative loop to the injection pump and mass spectrometer in another flow path. Meanwhile, the syringe pump in the other flow path propels the extract in the quantitative loop into the mass spectrometer. During its journey, the extract passes through a titanium dioxide coating, where glycosphingolipids are captured, while other lipids flow into the mass spectrometer for detection. In this mode, the flow rate of the extract through the coating is entirely controlled by the syringe pump.

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

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

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

[0047] The ultraviolet light wavelength can be 200-400 nm, preferably 200-300 nm, and more preferably 250 nm. Since titanium dioxide's maximum absorption wavelength for ultraviolet light is approximately 250 nm, ultraviolet light with a wavelength of 200-300 nm is better absorbed by titanium dioxide, and the resulting energy is higher than the band gap of titanium dioxide's electronic transitions (3.2 eV). 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 power per unit area of ​​light, and the optimal distance is related to the light intensity per unit area of ​​the ultraviolet lamp. There are no restrictions on the type of ultraviolet lamp used; for example, it can be any mercury lamp, xenon lamp, etc., that produces light in the 200-400 nm passband or a single wavelength band. Among these, mercury lamps with stronger ultraviolet intensity are preferred. If the capillary surface is protected with an opaque coating (such as a polyimide coating), the outer coating needs to be burned off at high temperature to allow the coating to receive ultraviolet light.

[0048] The photodesorption reaction time can be 1-10 min, preferably 2 min. This is related to the rate of photocatalytic deglycosylation of glycolipids adsorbed on the material surface. Experimental results show that glycolipids adsorbed in about 2 min can generally be completely eluted.

[0049] The principle of glycolipid desorption is as follows: Figure 1 As shown, glycolipids are selectively adsorbed onto the surface of titanium dioxide due to the bidentate chelation between the glycosidic heads and the Lewis acidic sites in titanium dioxide under alkaline conditions. When ultraviolet light irradiates the surface of titanium dioxide, the titanium dioxide absorbs the ultraviolet light and generates photogenerated electrons and holes. The holes oxidize the water adsorbed on the surface into hydroxyl radicals, thereby selectively oxidizing and decomposing the glycolipids adsorbed on the titanium dioxide surface into ceramides. The glycosidic heads are further oxidized and decomposed into smaller molecules on the titanium dioxide surface, while the detached ceramides, unable to adsorb onto the titanium dioxide surface, are released into the solution as products.

[0050] In-situ detection can be performed using the aforementioned in-situ mass spectrometry technique at a single spatial point in a biological sample, such as a location in a functional region of a tissue or the location of a single cell in a cell culture dish; it can also be performed by scanning a certain area on the sample surface; or it can be performed by scanning a certain area point by point to obtain imaging results.

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

[0052] The sampling probe scans at a speed of 0-1000 μm / s. Within this speed range, the micro-liquid nodes formed by the probe are stable, and the micro-liquid nodes always maintain a constant liquid node coverage area without the generation of bubbles.

[0053] The present invention will be further described below through examples.

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

[0055] 1) Capillary Coating: A capillary (356 μm O.D. / 100 μm I.D., UV-transparent coating) was activated with 1 mol / L NaOH, then washed with pure water, 0.01 mol / L HCl aqueous solution, methanol, and pure water, respectively. A sol-gel method was used with butyl phthalate as a precursor and acetic acid as a protective agent. Hydrolysis was performed in an ice bath to obtain a sol. P25 titanium dioxide nanoparticles were ultrasonically dispersed in the sol. The dispersed nanoparticle sol was pushed into the cleaned capillary and reacted at room temperature for half an hour, then pushed out. The resulting coated capillary was dried at 60°C. This process was repeated 6 times. The titanium dioxide nanoparticle coating is shown below. Figure 2 As shown, the length is 5cm.

[0056] 2) Online adsorption process: 10 μg / mL lipid standard mixtures (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 then... Figure 3 The flow rate shown is pumped into the coated capillary, and the solution is then fed into a mass spectrometer for real-time detection.

[0057] 3) Online optical desorption / resorption 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. The solution was then fed into the mass spectrometer for real-time detection. After the standard solution was completely pumped in, the coated capillary was irradiated for 5 min with an unfiltered mercury lamp (200-400 nm passband), maintaining a mercury lamp current of 8 mA and an irradiation distance of 10 cm. The 6% ammonia-methanol / acetonitrile (1:1, v / v) solution flowing out during irradiation was also fed into the mass spectrometer for detection.

[0058] 4) Mass spectrometry analysis: The mass spectrometry was performed using an electrospray ionization 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, with the voltage applied to the spray needle at +4500V and -3000V, the ion source temperature at 200℃, and the nebulized gas (N2) flow rate at 1.5L / min. The mass analyzer was used in a single-stage mass spectrometry full scan mode, with a scan range of 500-1000 m / z.

[0059] 5) Results Analysis: From the results ( Figure 3 It can be seen that this coated tube does not adsorb lipids other than glycolipids, but it has a near 100% adsorption efficiency for glycolipids at a certain flow rate and volume. The lower the flow rate, the larger the volume at which the coated tube can achieve 100% adsorption of glycolipids, indicating that a low flow rate is beneficial for increasing the breakthrough volume of the coated tube. However, when performing online analysis (… Figure 4 The flow rate of the solution also has a significant impact on the resolved spectrum; the lower the flow rate, the narrower the resolved spectrum and the higher the resolved signal peak. Therefore, online adsorption and photodesorption only achieve maximum efficiency at low flow rates. Compared with the lipid spectrum of direct injection without adsorption and desorption, it can be found that adsorption-desorption greatly enhances the glycolipid signal, indicating that the online process has a certain enrichment capacity.

[0060] Example 2: Analysis of lipid composition at single tissue sites using in-situ liquid extraction online adsorption-optical desorption mass spectrometry and comparison with conventional in-situ liquid extraction mass spectrometry.

[0061] 1) Setup of the in-situ liquid extraction apparatus: The in-situ liquid extraction probe is a stretched coaxial sleeve probe (outer capillary dimensions 251μm ID / 356μm OD, inner capillary dimensions 100μm ID / 163μm OD). One end of both the inner and outer tubes is stretched to a narrow opening, with the outer tube having an outer diameter of 200µm and the inner tube having an inner diameter of 50µm. The probe inlet is connected to the syringe 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, as described in Example 1. The titanium dioxide coating is 5cm long, and the outer membrane of this section of the capillary is burned off with an open flame. A mercury lamp is placed in this area.

[0062] 2) In-situ liquid extraction and online adsorption of tissue: In-situ liquid extraction was performed on a specific location of a brain tissue slice using the probe described above. The probe was injected into the syringe pump at a flow rate of 2 μL / min. The mass spectrometer maintained a stable liquid node by adjusting the negative pressure of the electrospray sheath to balance the positive pressure of the syringe pump. The extract was coated with titanium dioxide and then entered the mass spectrometer for detection.

[0063] 3) Online optical analysis: After 2 minutes of single-point extraction, the probe is lifted, and the coated tube is irradiated with a mercury lamp. At this time, the extract continues to flow into the mass spectrometer at a flow rate of 2 μL / min for detection. Irradiation is stopped after 2 minutes of mercury lamp irradiation.

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

[0065] 5) Results Analysis: From Figure 5 It can be seen that after online adsorption through the coated tube, a large number of phospholipids, glycerides, and glycosyl-free sphingolipids were detected by mass spectrometry. When light was applied and photolysis occurred, mass spectrometry detected many peaks in the mass range of 500-700. Through primary mass spectrometry and secondary mass spectrometry, it can be identified that they are ceramides after deglycosylation of glycolipids. However, if brain tissue slices are directly detected without the in-situ liquid extraction device with titanium dioxide coating, very few glycolipid peaks are obtained, and they are also interfered with by a large number of surrounding phospholipid peaks. Figure 6 The number of different lipids detected by the two methods was statistically analyzed. 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 a single neuron using in-situ liquid extraction, online adsorption-optical desorption mass spectrometry.

[0067] 1) Construction of the 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 was used to locate individual neurons in the field of view. The probe was moved above the neuron via a triaxial platform for in-situ liquid extraction. The probe was pumped in at a flow rate of 2 μL / min using a syringe pump. The mass spectrometer maintained a stable liquid node by adjusting the negative pressure of the electrospray sheath to balance the positive pressure of the syringe pump. The extract was coated with titanium dioxide and then entered the mass spectrometer for detection.

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

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

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

[0072] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention should fall within the protection scope of the present invention.

Claims

1. A lipid in situ mass spectrometry analysis method based on online selective enrichment-optical desorption, characterized in that, Includes the following steps: (1) The lipids in the sample were extracted into an alkaline protic solvent using in-situ liquid extraction technology to obtain an in-situ extraction system solution; (2) The in-situ extraction system solution is drawn into the mass spectrometer through the transmission pipeline by negative pressure and detected by mass spectrometry. A titanium dioxide nanoparticle coating is provided in the transmission pipeline. (3) After the lipid spectrum in the in-situ extraction system solution has been completely detected by mass spectrometry, the titanium dioxide nanoparticle coating is irradiated with ultraviolet light, and an alkaline proton solvent is continued to be introduced and drawn into the mass spectrometer through the transmission pipeline for mass spectrometry detection.

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

1.

3. The in-situ mass spectrometry analysis method as described in 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, with a particle size of 1-10 nm.

4. The in-situ mass spectrometry analysis method as described in claim 1, characterized in that, The titanium dioxide nanoparticle coating is applied to the inner wall of the transmission pipeline by in-situ polymerization of the sol-gel method and calcination, or by dispersing titanium dioxide nanoparticles in an organic solvent, injecting them into the pipeline, and then applying them to the inner wall of the transmission pipeline under vacuum. The transmission pipeline includes the inner wall of a capillary tube or a quantitative loop connected to the sampling probe outlet.

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

6. The in-situ mass spectrometry analysis method as described in 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 as described in claim 1, characterized in that, The ultraviolet light irradiated by the ultraviolet lamp has a wavelength of 200-400nm and an irradiation time of 1-10min.

8. The in-situ mass spectrometry analysis method according to any one of claims 1-7, characterized in that, The in-situ liquid extraction technology involves continuously pumping the in-situ extraction system solution into the sampling probe, while simultaneously drawing the in-situ extraction system solution into the mass spectrometer using negative pressure, forming a liquid node at the tip of the sampling probe. Extraction is achieved when the liquid node comes into contact with the sample surface.

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

10. The in-situ mass spectrometry analysis apparatus as described in claim 9, characterized in that, When the in-situ liquid extract is drawn into the probe and aspirated into the mass spectrometer by negative pressure aspiration through a mass spectrometer ion source spray, the probe outlet is connected to the mass spectrometer inlet via a capillary tube, and a titanium dioxide coating is applied to the capillary tube between the probe outlet and the mass spectrometer inlet. When the in-situ liquid extract is drawn into the mass spectrometer via a vacuum pump, the probe outlet is connected to one port of a two-position six-way valve. The adjacent port of this port is connected to a waste liquid tube. The adjacent ports of the waste liquid tube port and the probe outlet port are 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 via a capillary tube. A titanium dioxide coating is applied to the capillary tube connecting the six-way valve port and the mass spectrometer inlet.

11. The in-situ mass spectrometry analysis apparatus as described in claim 10, 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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