In-situ liquid extraction sampling probe with high spatial resolution and high stability for in-situ mass spectrometry, and preparation method and application thereof

Abstract

The application discloses an in-situ liquid extraction sampling probe with high spatial resolution and high stability for in-situ mass spectrometry, wherein a quartz inner capillary is coaxially sleeved in the inside of a quartz outer capillary; the quartz outer capillary and the quartz inner capillary are both extended to a B end with a larger outer diameter from a tip with a smaller outer diameter; the tip outer diameter of the quartz outer capillary is 10-200 μm, and the tip outer diameter of the quartz inner capillary is 2-30 μm; the B end of the quartz outer capillary is connected with one of the outlets of a three-way joint; the B end of the quartz inner capillary passes through the inner cavity of the three-way joint and is exposed from the other outlet of the three-way joint, and the tip of the quartz inner capillary is located in the inside of the quartz outer capillary and close to the tip side. The probe integrates high spatial resolution at a single cell level, low spectrum band diffusion in the probe and high liquid node stability. The application also discloses a preparation method of the in-situ liquid extraction sampling probe and application of the in-situ liquid extraction sampling probe in in-situ mass spectrometry detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of mass spectrometry pretreatment, and particularly relates to an in-situ liquid extraction sampling probe with high spatial resolution and high stability for in-situ mass spectrometry, and a preparation method and application thereof. BACKGROUND

[0002] Ambient mass spectrometry or mass spectrometry imaging (MSI) with spatial resolution is a new type of mass spectrometry detection technology. It combines the multi-channel detection characteristics of mass spectrometry, the real-time sampling of various in-situ sampling ionization probes, and the spatial resolution characteristics. The multi-channel simultaneous detection characteristics meet the high-throughput non-target analysis requirements of proteomics and metabolomics in systems biology. At the same time, it has the spatial resolution capability to obtain the spatial distribution information of various molecules, and also meets the spatial resolution requirements of biological exploration of molecular mechanisms at the tissue functional region and cell level. For example, the mechanism research of neural signal conduction in brain science relies on the tracking of the content distribution changes of various signal substances in different brain functional blocks; the recognition of cancer cell diffusion and metastasis cannot be separated from the analysis of metabolomics at the single cell scale in the tissue.

[0003] To realize spatially resolved detection of mass spectrometry, various sampling or ionization techniques applied to mass spectrometry imaging are continuously developed. Ambient liquid extraction techniques are one of the spatially resolved sampling techniques. It realizes extraction of analytes in a sample in a certain small area by generating a stable micron to sub-millimeter diameter liquid node between the probe and the sample surface, and then controls the extraction solution to be sent into mass spectrometry in real time through a liquid flow path for detection. Compared with other ionization sampling methods, it has higher sampling efficiency and higher sensitivity in theory; at the same time, the device is simple and flexible, and the cost is low; it can be used with different mass spectrometry ion sources to realize online mass spectrometry imaging. At present, the structure of the probe can be divided into flow-probe, single-probe, liquid extraction surface analysis (LESA), nanospray desorption electrospray ionization (nanoDESI), swan probe and the like. Among them, the single-probe technique can currently achieve the highest spatial resolution (8.5 μm), and it has the advantage that the needle tip can be inserted into cells, so that mass spectrometry imaging with high spatial resolution or even single-cell analysis can be realized. At present, other probes are difficult to achieve. However, due to the limitations and drawbacks of the structure of the probe flow path, the diffusion of the extracted analytes is very serious, which reduces the signal of the detected analytes and decreases the analysis sensitivity. At the same time, the liquid node of this probe is difficult to stabilize. Although the earliest micro-liquid node surface sampling technique has higher liquid node stability, it is currently limited by the diameter of the capillary, and the spatial resolution is very low, usually about 300-1000 μm. At present, with the requirements of the biological field for the spatial resolution and imaging quality of mass spectrometry imaging, as well as the frontier exploration of single-cell analysis, a probe with high stability, small intravascular molecular diffusion and high spatial resolution is urgently needed to be developed. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the deficiencies and defects mentioned in the above background art, and to provide an in-situ liquid sampling probe with high stability, small intravascular molecular diffusion and high spatial resolution.

[0005] To solve the above technical problems, the technical solution provided by the present application is:

[0006] A high spatial resolution and high stability in-situ liquid extraction sampling probe for in-situ mass spectrometry, comprising a quartz outer capillary and a quartz inner capillary, the quartz inner capillary is coaxially sleeved in the inside of the quartz outer capillary; the outer diameter of the quartz inner capillary is smaller than the inner diameter of the quartz outer capillary;

[0007] The quartz outer capillary and the quartz inner capillary both extend from a tip with a small outer diameter to a B end with a large outer diameter; the outer diameter of the tip of the quartz outer capillary is 10-200 μm, and the outer diameter of the tip of the quartz inner capillary is 2-30 μm;

[0008] The B end of the quartz outer capillary is connected to one of the outlets of the tee joint.

[0009] The B end of the quartz inner capillary is exposed from the other outlet of the tee joint through the inner cavity of the tee joint, and the tip of the quartz inner capillary is located inside the quartz outer capillary and close to the tip side.

[0010] Preferably, the length of the quartz outer capillary is 3-5 cm, and the length of the quartz inner capillary is 10-20 cm.

[0011] Preferably, the difference between the outer diameter of the B end of the quartz inner capillary and the inner diameter of the B end of the quartz outer capillary is 50-100 μm.

[0012] Preferably, the distance between the tip of the quartz inner capillary and the tip of the quartz outer capillary is 0-50 μm.

[0013] Preferably, the B end of the quartz outer capillary is connected to one of the outlets of the tee joint through an adapter sleeve and a joint, and the quartz inner capillary is fixed at the other outlet of the tee joint using an adapter sleeve and a joint.

[0014] More preferably, the tee joint is a PEEK tee joint, the adapter sleeve is an FEP adapter sleeve, and the joint is a standard 1 / 16" PEEK joint.

[0015] More preferably, the third outlet of the tee joint is connected to a syringe pump through a transmission tube.

[0016] More preferably, the exposed B end of the quartz inner capillary (using an FEP adapter sleeve and a standard 1 / 16" PEEK joint) is connected to the inlet of a mass spectrometry ion source or (using a silica gel sealing gasket) to a vacuum chamber, which is connected to a vacuum pump.

[0017] More preferably, the syringe pump connected to the third outlet of the tee joint pumps the extraction solution into the quartz outer capillary at a certain flow rate, the extraction solution flows into the tip and is then sucked into the quartz inner capillary by the vacuum generated by the vacuum pump connected to the vacuum chamber or the vacuum generated by the ion source spray.

[0018] The preparation method of the in-situ liquid extraction sampling probe described above comprises the following steps:

[0019] S1. Two quartz capillary tubes with different inner and outer diameters are pulled to different opening diameters by a butane lighter, and then are clamped by a pair of tweezers while hot to produce two tips with different opening diameters. The other end of the quartz capillary tube which is not pulled is the B end. A quartz outer capillary tube with a larger diameter and a quartz inner capillary tube with a smaller diameter are obtained.

[0020] S2. The tip of the quartz inner capillary tube is sleeved into the inside of the tip of the quartz outer capillary tube to form a coaxial sleeve. The B end of the quartz outer capillary tube is connected to one of the outlets of a three-way joint by using a FEP adapter sleeve and a standard 1 / 16" PEEK joint. The B end of the quartz inner capillary tube is exposed from the other outlet of the three-way joint by passing through the inner cavity of the three-way joint, and is fixed at the outlet by using a FEP adapter sleeve and a standard 1 / 16" PEEK joint. The third outlet of the three-way joint is connected to a syringe pump through a transmission tube. The exposed B end of the quartz inner capillary tube is connected to the inlet of a mass spectrometry ion source or is connected to a vacuum chamber by using a silica gel sealing gasket. The vacuum chamber is connected to a vacuum pump.

[0021] S3. The distance between the tips of the quartz inner capillary tube and the quartz outer capillary tube is adjusted under a microscope and is fixed.

[0022] Based on the overall inventive concept, the application further provides an application of the in-situ liquid extraction sampling probe in in-situ mass spectrometry detection.

[0023] The operation method of the application comprises the following steps:

[0024] (1) A syringe pump connected to the third outlet of the three-way joint pumps an extraction solution into the quartz outer capillary tube of the probe at a certain flow rate. The solution flows into the tips and is then sucked into the quartz inner capillary tube by a vacuum generated by a mass spectrometry ion source spray or a vacuum pump connected to a vacuum chamber.

[0025] (2) The probe tips are suspended above the surface of a sample to be detected. The distance between the probe and the sample is controlled to be 5-50 μm. The extraction solution is dropped to contact the probe tips and the sample surface, and a liquid junction is formed between the probe tips and the sample.

[0026] Preferably, in step (1), the syringe pump is set to a liquid pushing flow rate of 0-50 μL / min, preferably 0-2 μL / min.

[0027] Preferably, the mass spectrometry ion source is an electrospray ionization source or an atmospheric pressure chemical ionization source.

[0028] Preferably, the diameter of the liquid junction point needs to be adjusted to be substantially the same as the diameter of the probe tips.

[0029] Preferably, the extraction solvent is an organic solvent or a mixed solvent of organic solvent and water which is compatible with the ion source of mass spectrometer and has extraction effect on the analyte in the sample, preferably methanol, acetonitrile, water and a mixed solvent thereof.

[0030] Preferably, the sample is a solid or semi-solid, preferably animal plant tissue, microbial colony, cell slide, packaging paper or mural, etc.

[0031] Preferably, the liquid node is adjusted in the following manner: under a fixed liquid flow rate, a stable liquid node with a diameter substantially consistent with the diameter of the probe is generated by adjusting the vacuum degree of the vacuum pump. When the vacuum degree is provided by the ion source of mass spectrometer, a stable liquid node with a diameter substantially consistent with the diameter of the probe is generated by adjusting the liquid flow rate of the injection pump.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] 1. The present application is a high spatial resolution and high stability in-situ liquid extraction sampling probe for in-situ mass spectrometry. The tip of the inner quartz outer capillary 1 of the liquid flow probe is drawn to form a smaller (10-200 μm) probe tip with adjustable size. The optimal inner quartz outer capillary 1 spacing (0-50 μm) that minimizes the probe internal diffusion and stabilizes the liquid node is obtained by experimental exploration of the relationship between the inner quartz outer capillary 1 spacing and the extraction flow curve. This new probe integrates high spatial resolution at single cell level, low probe internal band diffusion and high liquid node stability.

[0034] 2. Compared with the traditional liquid flow probe, the present application is a new type of in-situ liquid extraction probe technology that solves most of the problems of all reported probes, as follows:

[0035] 1) The spatial resolution of the present application is improved by more than 10 times, and the minimum sampling diameter can be 10 μm, while the traditional liquid flow probe is above 300 μm.

[0036] 2) Compared with the reported single probe, the spectrum band diffusion of the analyte after extraction by the present application is reduced by about 5 times, and the liquid node can be formed in a suspended state, while the single probe can only form a liquid node under the condition of precise control of the probe sample spacing, so the conditions required for the stability of the liquid node are lower.

[0037] 3) Compared with the two new types of probes, nanoDESI and swan probe, the needle type probe of the present application is very suitable for insertion into the cell for single cell analysis, while the former two probes cannot be inserted into the cell; and the spatial resolution of the present application is also higher than that of the former two probes.

[0038] 3. The preparation method of the present application is simple to operate and low in cost. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0040] Figure 1 is a structural diagram of a micro-sleeved probe;

[0041] Legend: 1, quartz outer capillary; 2, quartz inner capillary; 3, adapter sleeve; 4, joint; 5, tee; 6, transmission tube; 7, distance between probe inner and outer capillaries.

[0042] Figure 2 is a microscope photo of a micro-sleeved probe.

[0043] Figure 3 is an extraction time curve diagram of real-time sampling-detecting of visible absorption spectrum of a dye added dropwise in a tissue by using a single probe and a micro-sleeved probe of the same size in Example 1; wherein: the flow rate of the extraction liquid is 3 μm, absorption spectrum (A), 1 μm, absorption spectrum (B) and 0.5 μm, absorption spectrum (C).

[0044] Figure 4 is a structural diagram of a device for in-situ liquid extraction-mass spectrometry imaging experiment in Example 2.

[0045] Figure 5 is an optical imaging diagram (A) and a mass spectrometry imaging diagram (B) of each lipid of a mouse brain tissue imaged by using a new probe (the size of the probe is 200 μm) in Example 2.

[0046] Figure 6 is a microscope photo of a liquid node formed on a cell slide by a micro-sleeved probe.

[0047] Figure 7 is a mass spectrometry diagram of single-cell sampling mass spectrometry detection of HT22 neuron cells by using a 30 μm-sized micro-sleeved probe in Example 3. DETAILED DESCRIPTION

[0048] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present application is not limited to the following specific embodiments.

[0049] Unless otherwise defined, all terms used in the description herein, including technical terms, terms of trade, instruments and equipment, etc., have the same meaning as commonly understood by one of ordinary skill in the art. The technical 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 application.

[0050] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or prepared by existing methods.

[0051] The method of the present application first produces a probe, the specific structure of which is shown in Figure 1 In one embodiment of the present application, the probe uses a fused quartz capillary with an outer diameter of 360 μm and an inner diameter of 250 μm as the outer tube (i.e. quartz outer capillary 1), and a fused quartz capillary with an outer diameter of 150 μm and an inner diameter of 100 μm as the inner tube (i.e. quartz inner capillary 2). The outer layer of polyimide film of both types of quartz capillary is burned off for a length of 2 cm at the lower end by candle flame, and then the region is heated by a butane lighter while the capillary is quickly stretched to twice the length by using tweezers. After the flame is turned off, the thinnest part is quickly cut off by surgical scissors, and a probe needle tip with a flat tip and a diameter of 10-30 μm is obtained, as shown in Figure 2 The tip can be further adjusted to a larger diameter by cutting off with scissors or polishing with a ground glass. The end of all capillaries opposite to the tip is called the B end. The B end of the quartz outer capillary 1 is connected to one of the outlets of a PEEK tee joint 5 by using a FEP adapter sleeve 3 and a standard 1 / 16" PEEK joint 4; then the tip of the quartz inner capillary 2 is inserted into the other outlet of the tee joint 5 until it extends into the interior of the quartz outer capillary 1, and at the same time the quartz inner capillary 2 is fixed at the outlet by using a FEP adapter sleeve 3 and a standard 1 / 16" PEEK joint 4. The retracted distance of the tip of the quartz inner capillary 2 relative to the quartz outer capillary 1 can be adjusted by moving the quartz inner capillary 2; after the distance between the inner and outer capillaries 7 is adjusted to 0-50 μm, the quartz inner capillary 2 is fixed by tightening the joint 4. The length of the quartz inner capillary 2 is about 10-20 cm; the length of the quartz outer capillary 1 is about 3-5 cm. The specific values of the above parameters are shown in the following examples.

[0052] The probe is fixed on the z-axis of a three-axis stepping motor platform, which can move up and down to control the distance between the probe and the sample. The sample is fixed on the xy-axis of the three-axis platform, which can move forward and backward and left and right to control the relative position of the probe on the sample.

[0053] The third outlet of the tee 5 is connected to the syringe of the injection pump through a transmission pipe 6 (a liquid capillary) for pumping the extraction liquid into the quartz outer capillary 1. Figure 4 The specific connection mode can be seen from the specific embodiments.

[0054] The present application controls the distance between the sample and the probe by moving the probe along the z-axis. The real-time distance between the sample and the probe and the stability of the liquid node are observed by a microscope camera.

[0055] The three-axis stepping motor platform is a platform that can be linearly moved in the xyz three directions. The precision and moving range of the three-axis stepping motor are determined according to the size of the imaging sample and the imaging spatial resolution. The three-axis stepping motor platform is a technology familiar to those skilled in the art.

[0056] The vacuum pump is mainly a diaphragm pump with a vacuum degree controller.

[0057] The mass spectrometry ion source can be an electrospray ionization source and an atmospheric pressure chemical ionization source.

[0058] Embodiment 1:

[0059] A high spatial resolution and high stability in-situ liquid extraction sampling probe for in-situ mass spectrometry, as shown in the structural diagram Figure 1 The quartz outer capillary 1 and the quartz inner capillary 2 are coaxially sleeved inside the quartz outer capillary 1; the outer diameter of the quartz inner capillary 2 is smaller than the inner diameter of the quartz outer capillary 1.

[0060] The quartz outer capillary 1 and the quartz inner capillary 2 are both extended by a small-diameter tip to a large-diameter B end; the tip outer diameter of the quartz outer capillary 1 is 50 μm, and the tip outer diameter of the quartz inner capillary 2 is 10 μm; the length of the quartz outer capillary 1 is 3 cm, and the length of the quartz inner capillary 2 is 15 cm; the difference between the B end outer diameter of the quartz inner capillary 2 and the B end inner diameter of the quartz outer capillary 1 is 100 μm; the tip of the quartz inner capillary 2 is spaced apart from the tip of the quartz outer capillary 1 by 20 μm.

[0061] The B end of the quartz outer capillary 1 is connected to one of the outlets of the PEEK tee 5 through the FEP adapter sleeve 3 and the standard 1 / 16" PEEK joint 4; the B end of the quartz inner capillary 2 is exposed from the other outlet of the PEEK tee 5 by passing through the inner cavity of the PEEK tee 5, and the quartz inner capillary 2 is fixed at the outlet by using the FEP adapter sleeve 3 and the standard 1 / 16" PEEK joint 4, and the tip of the quartz inner capillary 2 is located inside the quartz outer capillary 1 and close to the tip side.

[0062] The third outlet of the PEEK tee 5 is connected to a syringe pump through a transfer tube 6. The B end of the exposed quartz inner capillary 2 is connected to the inlet of a flow cell of a diode array detector (the flow cell is a 200 nL micro flow cell), and the outlet of the flow cell is connected to a vacuum chamber, which is connected to a digital membrane pump. The syringe pump pumps the extraction solution into the quartz outer capillary 1, and the extraction solution flows into the tip and is then sucked into the quartz inner capillary 2 by the vacuum generated by the digital membrane pump connected to the vacuum chamber.

[0063] The embodiment compares the differences in in-situ liquid extraction sampling time curves of the micro-sleeved capillary probe and the single probe, and the specific steps are as follows:

[0064] (1) Preparation of the probe: The specific implementation process of the micro-sleeved capillary probe is described in the probe preparation part of the specific embodiment. Here, the diameter of the tip of the drawn quartz inner capillary 2 is 10 μm, and the diameter of the tip of the quartz outer capillary 1 is 50 μm. The retracted distance between the quartz inner capillary 2 and the quartz outer capillary 1 is 20 μm. The length of the quartz inner capillary 2 is 15 cm, and the length of the quartz outer capillary 1 is 3 cm.

[0065] In order to compare with the new probe, a single probe is also made. The double-hole borosilicate glass tube is quickly thinned on the flame and then cut to a tip diameter of 50 μm with tweezers. The upper end of the double-hole glass tube is inserted into a fused quartz capillary with an outer diameter of 360 μm and an inner diameter of 250 μm (inlet tube) and an outer diameter of 150 μm and an inner diameter of 100 μm (outlet tube) as a liquid transfer tube, and is sealed at the interface with ultraviolet glue.

[0066] (2) Construction of in-situ liquid extraction-ultraviolet detection device: The connection mode of the probe on the three-axis platform is as described in the specific embodiment. For the micro-sleeved capillary probe, the third outlet of the tee is connected to a syringe pump, the B end of the quartz inner capillary 2 is connected to the inlet of a flow cell of a diode array detector (the flow cell is a 200 nL micro flow cell), and the outlet of the flow cell is connected to a vacuum chamber, which is connected to a digital membrane pump.

[0067] For the single probe, the inlet tube is connected to the syringe pump, and the outlet tube is connected to the inlet of the flow cell.

[0068] (3) Preparation of azo dye spot plate: The mouse brain tissue is cut into 10 μm thick slices with a freezing microtome, and then is hot-mounted on a glass plate. 2 μm 1 mg / mL disperse red 1 methanol solution is repeatedly dropped on the brain tissue to form azo dye circular spots with a diameter of 3 mm. Three repeated spots are formed for use.

[0069] (4) Detection of in-situ liquid sampling time curve: The prepared spot plate was fixed on the xy-axis sample stage, and methanol was used as the extraction solvent to extract the center of the spot at a single point. The probe-sample spacing was 10 μm, and the injection pump flow rate was 0.5 μL / min, 1 μL / min, and 3 μL / min. Each flow rate was repeated once in three replicate samples, and different probes were used to extract at adjacent positions of each spot for comparison.

[0070] (5) Results Analysis: Figure 3 It can be seen that, at any flow rate, the width of the spectral band produced by the microcannula probe sampling is smaller than that of the single probe. Especially at low flow rates, when the flow rate is 1, the half-width at half-maximum (WHM) of the time curve produced by the microcannula probe sampling is approximately 0.5 min, while that of the single probe reaches approximately 2 min. When the flow rate is further reduced to 0.5 μL / min, the WHM of the microcannula probe is approximately 1 min, while that of the single probe reaches 5 min. This indicates that the diffusion of the sampling solution in the flow path of the single probe is approximately five times that of the microcannula probe. Such diffusion will dilute the sample, ultimately leading to a decrease in signal strength.

[0071] Example 2:

[0072] A high spatial resolution and high stability in-situ liquid extraction sampling probe for in-situ mass spectrometry is shown in the schematic diagram below. Figure 1 As shown, it includes an outer quartz capillary tube 1 and an inner quartz capillary tube 2, with the inner quartz capillary tube 2 coaxially fitted inside the outer quartz capillary tube 1; the outer diameter of the inner quartz capillary tube 2 is smaller than the inner diameter of the outer quartz capillary tube 1.

[0073] Both the outer quartz capillary 1 and the inner quartz capillary 2 extend from a tip with a smaller outer diameter to a B-end with a larger outer diameter. The outer diameter of the tip of the outer quartz capillary 1 is 200 μm, and the outer diameter of the tip of the inner quartz capillary 2 is 30 μm. The length of the outer quartz capillary 1 is 3 cm, and the length of the inner quartz capillary 2 is 15 cm. The difference between the outer diameter of the B-end of the inner quartz capillary 2 and the inner diameter of the B-end of the outer quartz capillary 1 is 100 μm. The distance between the tip of the inner quartz capillary 2 and the tip of the outer quartz capillary 1 is 20 μm.

[0074] The B end of the quartz outer capillary tube 1 is connected to one of the outlets of the PEEK tee 5 via the FEP adapter sleeve 3 and the standard 1 / 16” PEEK connector 4; the B end of the quartz inner capillary tube 2 passes through the inner cavity of the PEEK tee 5 and protrudes from the other outlet of the PEEK tee 5. The outlet is fixed by the FEP adapter sleeve 3 and the standard 1 / 16” PEEK connector 4. The tip of the quartz inner capillary tube 2 is located inside the quartz outer capillary tube 1 and close to the tip side.

[0075] The third outlet of the PEEK tee 5 is connected to a syringe pump through a transfer tube 6. The B end of the exposed quartz inner capillary 2 is directly connected to the ion source inlet of an electrospray ionization source-ion trap-time of flight mass spectrometer. The syringe pump pumps the extraction solution into the quartz outer capillary 1, and the extraction solution flows into the tip and is then sucked into the quartz inner capillary 2 by the negative pressure generated by the ion source spraying of the mass spectrometer.

[0076] The present embodiment uses a micro cannula probe to image lipids in the mouse brain, and the specific steps are as follows:

[0077] (1) Preparation of micro cannula probe: The micro cannula probe is made according to the description in the probe making part of the specific embodiment. Here, the diameter of the tip of the drawn quartz inner capillary 2 is 30 μm, and the diameter of the tip of the quartz outer capillary 1 is 200 μm. The retracted distance between the quartz inner capillary 2 and the quartz outer capillary 1 is 20 μm. The length of the quartz inner capillary 2 is 15 cm, and the length of the quartz outer capillary 1 is 3 cm.

[0078] (2) Construction of in situ liquid extraction-mass spectrometry imaging device: As shown in Figure 4 , the connection mode of the probe on the three-axis platform is as described in the specific embodiment. For the micro cannula probe, the third outlet of the tee is connected to a syringe pump, and the B end of the quartz inner capillary 2 is directly connected to the ion source inlet of an electrospray ionization source-ion trap-time of flight mass spectrometer.

[0079] (3) Tissue sample pretreatment: The mouse brain tissue is cut into 10 μm thick slices using a freezing microtome, then hot mounted on a glass plate, and the slices are placed in a desiccator for use. The dried slices are scanned by a scanner for comparison with the mass spectrometry imaging graph.

[0080] (4) Mass spectrometry imaging of mouse brain tissue: The tissue slices are fixed on the xy-axis sample stage, and methanol-acetonitrile is used as the extraction solvent and pumped into the tip of the probe. The liquid is then sucked into the quartz inner capillary 2 by the negative pressure generated by the ion source spraying of the mass spectrometer. At a flow rate of 5 μL / min, the syringe pump can balance the negative pressure to form a stable liquid junction under a sheath flow gas flow rate of 1.5 L / min. The probe is used to scan the sample surface point by point with a fixed sample spacing of 10 μm. The residence time of each point is 17 s, and the moving speed of the probe between points is 200 μm / s, with a step of 200 μm between points. The probe scans the sample surface in a zigzag path to form a 20*25 point matrix. During the scanning process, the mass spectrometer collects signals, and the final mass spectrometry signals are restored to the spatial position corresponding to the signal height of the substance, and finally recombined into an imaging graph.

[0081] (5) Result analysis: As shown in Figure 5It can be seen that the mass spectrometry imaging using the micro jacketed probe can image multiple lipids in the mouse brain tissue. The signal distribution of these lipids in each region of the tissue has a great correlation with the functional region of the tissue itself. By comparing the mass spectrometry imaging map with the optical imaging map, it can be found that the signal distribution of multiple lipids is very consistent with the optical appearance of the tissue. Meanwhile, the content of different lipids in different positions also has a great difference.

[0082] Example 3

[0083] A high spatial resolution and high stability in-situ liquid extraction sampling probe for in-situ mass spectrometry has a structure as shown in the figure, which comprises a quartz outer capillary 1 and a quartz inner capillary 2, and the quartz inner capillary 2 is coaxially sleeved in the inside of the quartz outer capillary 1; the outer diameter of the quartz inner capillary 2 is smaller than the inner diameter of the quartz outer capillary 1. Figure 1

[0084] The quartz outer capillary 1 and the quartz inner capillary 2 are both extended by a small-diameter tip to a large-diameter B end; the tip outer diameter of the quartz outer capillary 1 is 30 μm, and the tip outer diameter of the quartz inner capillary 2 is 10 μm; the length of the quartz outer capillary 1 is 3 cm, and the length of the quartz inner capillary 2 is 15 cm; the difference between the B end outer diameter of the quartz inner capillary 2 and the B end inner diameter of the quartz outer capillary 1 is 100 μm; the distance between the tip of the quartz inner capillary 2 and the tip of the quartz outer capillary 1 is 5 μm.

[0085] The B end of the quartz outer capillary 1 is connected with one of the outlets of a PEEK tee joint 5 through an FEP adapter sleeve 3 and a standard 1 / 16" PEEK joint 4; the B end of the quartz inner capillary 2 is exposed from the other outlet of the PEEK tee joint 5 through the inner cavity of the PEEK tee joint 5, and the quartz inner capillary 2 is fixed at the outlet by using the FEP adapter sleeve 3 and the standard 1 / 16" PEEK joint 4, and the tip of the quartz inner capillary 2 is located in the inside of the quartz outer capillary 1 and close to the tip side.

[0086] The third outlet of the PEEK tee joint 5 is connected with a syringe pump through a transmission tube 6; the exposed B end of the quartz inner capillary 2 is directly connected with the ion source inlet of an electrospray ionization source-ion trap-time-of-flight mass spectrometer; the syringe pump pumps the extraction solution into the quartz outer capillary 1, the extraction solution flows into the tip and is then inhaled into the quartz inner capillary 2 by the vacuum formed by the ion source spraying of the mass spectrometer.

[0087] In this embodiment, the micro jacketed probe is used for single-cell detection of lipids in HT22 neuron cells, and the specific steps are as follows:

[0088] ​(1) Preparation of microcapillary probe: The microcapillary probe was prepared as described in the probe preparation section of the detailed embodiment. The inner capillary 2 was drawn to have a tip diameter of 10 μm and the outer capillary 1 was drawn to have a tip diameter of 30 μm. The inner capillary 2 was drawn to have a length of 15 cm and the outer capillary 1 was drawn to have a length of 3 cm.

[0089] (2) Construction of in situ liquid extraction single cell-mass spectrometry platform: The probe was connected to the three-axis platform as described in the detailed embodiment. For the microcapillary probe, the third outlet of the tee was connected to a syringe pump and the B end of the inner capillary 2 was directly connected to the ion source inlet of the electrospray ionization source-ion trap-time of flight mass spectrometer. The three-axis platform was constructed on a fluorescence inverted microscope.

[0090] (3) Pretreatment of cell sample: After the HT22 cell line was purchased, it was subcultured in a culture dish. The culture medium was Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin solution, and the temperature was 37°C and the environment was 5% CO2. After the cell density reached 90%, the cells in the culture dish were digested with trypsin, and the digested suspension cells were distributed into a 6-well plate containing a crawling sheet for further culture. The crawling sheet finally grown to a certain density was used for the following single cell analysis experiment.

[0091] (4) Single cell analysis: The cultured crawling sheet was attached to a glass plate, and the glass plate was placed on the three-axis platform. The probe was controlled by the z-axis to modulate the distance between the glass plate and the probe to form a stable liquid junction point with a distance of about 10 μm, and then a cell was found on the crawling sheet for extraction by the microscope. During the extraction process, the extraction solution was 1% formic acid-methanol, the ion source sheath gas flow rate was 0.5 L / min, and the balanced sampling flow rate was about 0.5 μL / min. During the sampling process, the mass spectrometer collected signals to obtain real-time mass spectra.

[0092] (5) Result analysis: First, it can be seen from Figure 6 that the size of the probe liquid node is basically consistent with the size of the HT22 neuron cell body, indicating that the probe can distinguish single cells. At the same time, it can be seen from Figure 7 that the common phospholipid substances on the biological cell membrane were indeed collected on the single cell, and a few glycosphingolipids were also detected, indicating the rationality of the detection results.

[0093] Overall, the present application is aimed at the problem of serious intra-probe diffusion and liquid node instability of the current single-probe with the highest spatial resolution, and improves and innovates the traditional liquid flow probe with better stability. A high spatial resolution and high stability in-situ liquid extraction sampling probe for in-situ mass spectrometry is provided, which includes drawing one end of two fused silica capillaries with different diameters to a tip with a diameter of 10-200 μm, then combining the two capillaries into a coaxial sleeve by a tee joint, and adjusting the tip distance between the inner silica capillary 2 and the outer silica capillary 1 to be between 0-50 μm, thereby obtaining a micro coaxial sleeve probe with a tip diameter of 10-200 μm. The outer capillary is pumped from one outlet of the tee joint, and the probe tip forms a stable liquid node by negative pressure generated by a vacuum pump or a mass spectrometry ion source connected to the inner silica capillary 2, so as to perform spatially resolved in-situ extraction sampling and online mass spectrometry detection on the sample surface. Compared with the traditional in-situ liquid extraction sleeve probe, the new probe can increase the spatial resolution by more than ten times, and compared with the single probe with high spatial resolution that has appeared in recent years, the new probe has higher stability and lower diffusion of extracted analytes. The probe can be applied to single cell analysis and tissue mass spectrometry imaging analysis with high spatial resolution.

Claims

1. A high spatial resolution, high stability in-situ liquid extraction sampling probe for in-situ mass spectrometry, characterized in that, The quartz outer capillary (1) and the quartz inner capillary (2) are coaxially sleeved in the quartz outer capillary (1); the outer diameter of the quartz inner capillary (2) is smaller than the inner diameter of the quartz outer capillary (1); The quartz outer capillary (1) and the quartz inner capillary (2) are both extended from a small-diameter tip to a large-diameter B end; the tip outer diameter of the quartz outer capillary (1) is 10-200 µm, and the tip outer diameter of the quartz inner capillary (2) is 2-30 µm; the difference between the outer diameter of the B end of the quartz inner capillary (2) and the inner diameter of the B end of the quartz outer capillary (1) is 50-100 µm; the distance between the tip of the quartz inner capillary (2) and the tip of the quartz outer capillary (1) is 0-50 µm; The B end of the quartz outer capillary (1) is connected with one of the outlets of the three-way joint (5); The B end of the quartz inner capillary (2) is exposed from the other outlet of the three-way joint (5) through the inner cavity of the three-way joint (5), and the tip of the quartz inner capillary (2) is located in the interior of the quartz outer capillary (1) and close to the tip side.

2. The liquid extraction sampling probe in situ as claimed in claim 1, wherein, The length of the quartz outer capillary (1) is 3-5 cm, and the length of the quartz inner capillary (2) is 10-20 cm.

3. The liquid extraction sampling probe in situ according to claim 1, wherein, The B end of the quartz outer capillary (1) is connected with one of the outlets of the three-way joint (5) through the adapter sleeve (3) and the joint (4); the quartz inner capillary (2) is fixed at the other outlet of the three-way joint (5) by using the adapter sleeve (3) and the joint (4).

4. The liquid extraction sampling probe in situ as claimed in claim 3, wherein, The three-way joint (5) is a PEEK three-way joint, the adapter sleeve (3) is an FEP adapter sleeve, and the joint (4) is a standard 1 / 16'' PEEK joint.

5. The liquid extraction sampling probe in situ according to any one of claims 1-4, wherein, The third outlet of the three-way joint (5) is connected with an injection pump through a transmission pipe (6); the exposed B end of the quartz inner capillary (2) is connected with a mass spectrometry ion source inlet or a vacuum cavity, and the vacuum cavity is further connected with a vacuum pump.

6. The liquid extraction sampling probe in situ as claimed in claim 5, wherein, The injection pump connected with the third outlet of the three-way joint (5) pumps the extraction solution into the quartz outer capillary (1), and the extraction solution flows into the tip and is then inhaled into the quartz inner capillary (2) by the vacuum generated by the mass spectrometry ion source spray or the vacuum pump connected with the vacuum cavity.

7. A method of making an in-situ liquid extraction sampling probe according to any one of claims 1 to 6, wherein, The method comprises the following steps: S1. Two quartz capillaries with different inner and outer diameters are thinned on a butane lighter, and then clamped and cut by forceps while hot to generate two tips with different opening diameters, and the other end of the quartz capillary which is not thinned is a B end, thereby obtaining a quartz outer capillary with a larger diameter and a quartz inner capillary with a smaller diameter; S2. The tip of the quartz inner capillary is sleeved into the interior of the tip of the quartz outer capillary to form a coaxial sleeve, the B end of the quartz outer capillary is connected with one of the outlets of the three-way joint, the B end of the quartz inner capillary is exposed from the other outlet of the three-way joint through the inner cavity of the three-way joint, and the quartz inner capillary is fixed at the outlet; the third outlet of the three-way joint is connected with an injection pump through a transmission pipe; the exposed B end of the quartz inner capillary is connected with a mass spectrometry ion source inlet or a vacuum cavity, and the vacuum cavity is further connected with a vacuum pump. S3. Adjust the distance between the inner and outer quartz capillary tips under the microscope and secure.

8. Use of the in-situ liquid extraction sampling probe according to any one of claims 1 to 6 in in-situ mass spectrometry detection.

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