CN115799038A

CN115799038A - In-situ liquid extraction sampling probe with high spatial resolution and high stability for in-situ mass spectrum and preparation method and application thereof

Info

Publication number: CN115799038A
Application number: CN202211286572.1A
Authority: CN
Inventors: 吴倩; 赵志皓
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2022-10-20
Filing date: 2022-10-20
Publication date: 2023-03-14

Abstract

The invention discloses an in-situ liquid extraction sampling probe with high spatial resolution and high stability for in-situ mass spectrometry.A quartz inner capillary tube is coaxially sleeved in a quartz outer capillary tube; the quartz outer capillary tube and the quartz inner capillary tube extend from the tip end with the smaller outer diameter to the end B with the larger outer diameter; the external diameter of the tip of the quartz outer capillary tube is 10-200 μm, and the external diameter of the tip of the quartz inner capillary tube is 2-30 μm; the end B of the quartz outer capillary tube is connected with one outlet of the tee joint; the end B of the quartz inner capillary tube penetrates through the inner cavity of the tee joint and is exposed out of the other outlet of the tee joint, and the tip of the quartz inner capillary tube is positioned in the quartz outer capillary tube and close to one side of the tip. The probe integrates high single cell level spatial resolution, low intra-probe band diffusion and high liquid node stability. Also discloses a preparation method of the in-situ liquid extraction sampling probe and application of the in-situ liquid extraction sampling probe in-situ mass spectrometry detection.

Description

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

Technical Field

The invention belongs to the field of pretreatment of mass spectrometry detection, 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

In-situ Mass Spectrometry (ms) or Mass Spectrometry Imaging (MSI) with spatial resolution is a novel Mass spectrometric detection technique. The method combines the multichannel detection characteristic of mass spectrum and the real-time sampling and space resolution characteristics of various in-situ sampling ionization probes. The characteristic of multi-channel simultaneous detection meets the high-throughput non-target analysis required by proteomics and metabonomics in system biology. Meanwhile, the spatial resolution capability of the molecular probe can obtain the spatial distribution information of various molecules, and the requirement of molecular mechanism exploration of biology in a tissue functional area and a cell layer on the spatial resolution is met. For example, the study of the mechanism of neural signal transduction in brain science relies on the tracking of the distribution of the content of various signal transduction substances in the brain in different brain functional areas; the identification of cancer cell metastasis cannot be separated from metabolomic analysis on a single cell scale within tissues.

In order to realize the spatial resolution detection of mass spectra, various sampling or ionization techniques applied to mass spectrometry imaging are continuously developed. One of the spatially resolved sampling techniques is the in situ liquid extraction technique (spatial liquid extraction techniques). The method realizes the extraction effect on the analyte in the sample in a certain micro area by generating a stable liquid junction point with the diameter from micron to sub-millimeter between a probe and the surface of the sample, and then sends the extraction solution into a mass spectrum for detection in real time through the control of a liquid flow path. Compared with other ionization sampling methods, the method has higher sampling efficiency and higher sensitivity theoretically; meanwhile, the device is simple and flexible, and the manufacturing cost is low; the system can be used together with different mass spectrum ion sources to realize online mass spectrum imaging. The probe structure can be classified into flow-probe technology (flow-probe), single-probe technology (single-probe), liquid Extraction Surface Analysis (LESA), nano-flow desorption electrospray (nanodesii), swan probe technology (swanprobe), and the like. The single-probe technology can achieve the highest spatial resolution (8.5 mu m) at present, has the advantage that a needle tip can be inserted into a cell, and can realize high-spatial-resolution mass spectrum imaging and even single cell analysis. Other probes are difficult to achieve at present. However, due to the limitations and disadvantages of the probe flow path structure, the diffusion of the analyte after extraction is very severe, which reduces the detected analyte signal and the analytical sensitivity. Meanwhile, the liquid junction of the probe is difficult to stabilize. The earliest micro-liquid junction surface sampling technology has higher liquid junction stability, but the spatial resolution is very low, generally about 300-1000 μm, due to the restriction of the diameter of the capillary at present. At present, with the requirements of the biological field on the mass spectrum imaging spatial resolution and imaging quality and the front-edge exploration of single cell analysis, probes with high stability, small molecular diffusion in pipelines and high spatial resolution are in urgent need to be developed.

Disclosure of Invention

The technical problem to be solved by the present invention is to overcome the above mentioned disadvantages and drawbacks of the background art, and to provide an in situ liquid sampling probe having high stability, small in-line molecular diffusion, and high spatial resolution.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

an in-situ liquid extraction sampling probe aiming at high spatial resolution and high stability of an in-situ mass spectrum comprises a quartz outer capillary tube and a quartz inner capillary tube, wherein the quartz inner capillary tube is coaxially sleeved inside the quartz outer capillary tube; the outer diameter of the quartz inner capillary is smaller than the inner diameter of the quartz outer capillary;

the quartz outer capillary tube and the quartz inner capillary tube both extend from the tip with the smaller outer diameter to the end B with the larger outer diameter; the external diameter of the tip of the quartz outer capillary tube is 10-200 μm, and the external diameter of the tip of the quartz inner capillary tube is 2-30 μm;

the end B of the quartz outer capillary tube is connected with one outlet of the tee joint;

the end B of the capillary tube in the quartz penetrates through the inner cavity of the tee joint and is exposed out of the other outlet of the tee joint, and the tip of the capillary tube in the quartz is located in the capillary tube outside the quartz and close to one side of the tip.

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

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.

Preferably, the tip of the quartz inner capillary has a spacing of 0 to 50 μm with respect to the tip of the quartz outer capillary.

Preferably, the end B of the quartz outer capillary is connected with one outlet of the tee joint through a switching sleeve and a joint; and the quartz inner capillary tube and the other outlet of the tee joint are fixed by using a switching sleeve and a joint.

More preferably, the tee is a PEEK tee, the adapter sleeve is an FEP adapter sleeve, and the fitting is a standard 1/16' PEEK fitting.

More preferably, the third outlet of the tee is connected to a syringe pump through a transfer tube.

More preferably, the exposed B-end of the capillary tube in quartz (using an FEP adapter sleeve and a standard 1/16"PEEK fitting) is connected to the mass spectrometer ion source inlet or to a vacuum chamber (using a silicone gasket), which in turn is connected to a vacuum pump.

More preferably, a syringe pump (with a certain flow rate) connected with the third outlet of the tee is used for pumping an extraction solution into the quartz outer capillary, the extraction solution flows into the tip and is sucked into the quartz inner capillary by vacuum formed by spraying of the mass spectrometry ion source or vacuum generated by a vacuum pump connected with the vacuum cavity.

The preparation method of the in-situ liquid extraction sampling probe comprises the following steps:

s1, drawing two quartz capillary tubes with different inner and outer diameters on a butane lighter, and then clamping the quartz capillary tubes with forceps while the quartz capillary tubes are hot to generate tips with two different opening diameters, wherein the other ends of the quartz capillary tubes, which are not drawn to be thin, are B ends, so that a quartz outer capillary tube with a larger diameter and a quartz inner capillary tube with a smaller diameter are obtained;

s2, the tip of the inner quartz capillary tube is sleeved into one end of the tip of the outer quartz capillary tube to form a coaxial sleeve, the B end of the outer quartz capillary tube (an FEP switching sleeve and a standard 1/16 'PEEK connector) is connected with one outlet of the tee joint, the B end of the inner quartz capillary tube penetrates through the inner cavity of the tee joint and is exposed out of the other outlet of the tee joint, and the inner quartz capillary tube is fixed at the outlet of the tee joint (the FEP switching sleeve and the standard 1/16' PEEK connector); a third outlet of the tee joint is connected with the injection pump through a transmission pipe; the B end of the exposed quartz inner capillary tube (by utilizing an FEP adapter sleeve and a standard 1/16' PEEK joint) is connected with the mass spectrometry ion source inlet or (by utilizing a silica gel sealing gasket) is connected with a vacuum cavity, and the vacuum cavity is connected with a vacuum pump;

and S3, adjusting the distance between the tip of the quartz inner capillary and the tip of the quartz outer capillary under a microscope and fixing.

Based on a general inventive concept, the invention also provides an application of the in-situ liquid extraction sampling probe in-situ mass spectrometry detection.

The operation method of the application comprises the following steps:

(1) An injection pump connected with a third outlet of the tee joint pumps an extraction solution into the quartz outer capillary of the probe at a certain flow rate, and the solution flows into the tip and is sucked into the quartz inner capillary by vacuum formed by spraying of a mass spectrometry ionization source or vacuum generated by a vacuum pump connected with a vacuum cavity;

(2) The probe tip is suspended above the surface of a sample to be detected, and the distance between the probe and the sample is controlled to be 5-50 mu m; the extraction solution is dropped to make contact between the probe tip and the sample surface and form a liquid junction between the probe tip and the sample.

Preferably, in step (1), the syringe pump is set to a push flow rate of 0 to 50. Mu.L/min, preferably 0 to 2. Mu.L/min.

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

Preferably, the diameter of the liquid junction is adjusted to substantially coincide with the diameter of the probe tip.

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

Preferably, the sample is a solid or semi-solid, preferably a commercial product such as animal and plant tissue, microbial colonies, cell crawlers, wrapping paper or murals.

Preferably, the liquid junction point is adjusted as follows: under the condition of fixed liquid pushing flow rate, the vacuum degree of the vacuum pump is regulated to generate a stable liquid junction point with the diameter basically consistent with that of the probe. When a mass spectrometer ion source is used for providing vacuum degree, a stable liquid junction point with the diameter basically consistent with that of the probe is generated by adjusting the flow rate of the liquid pushing of the injection pump.

Compared with the prior art, the invention has the following beneficial effects:

1. aiming at an in-situ liquid extraction sampling probe with high spatial resolution and high stability of an in-situ mass spectrum, the invention draws the tip of an inner quartz outer capillary tube 1 of a liquid flow probe to form a smaller (10-200 mu m) probe tip with adjustable size; simultaneously, the relation between the distance between the inner quartz outer capillary tube 1 and an extraction outflow curve is explored through experiments, and the optimal distance (0-50 mu m) between the inner quartz outer capillary tube 1 and the probe with minimum inner diffusion and stable liquid nodes is obtained through optimization; the novel probe integrates high single cell level spatial resolution, low probe internal band diffusion and high liquid node stability.

2. Compared with the traditional liquid flow probe, the probe of the invention solves the most problems of all the reported probes, and the novel in-situ liquid extraction probe technology comprises the following steps:

1) The spatial resolution of the probe is improved by more than 10 times, the minimum sampling diameter can reach 10 mu m, and the traditional liquid flow probe is more than 300 mu m;

2) Compared with the reported single probe, the band diffusion of the analyte after the probe is extracted is reduced by about 5 times, and the liquid node can be formed in a suspension state, and the single probe can only form the liquid node under the condition of accurately controlling the distance between the probe samples, so the condition required by the stability of the liquid node is lower;

3) Compared with the reported novel probes, namely the nanodESI probe and the swan probe, the needle tip type probe is very favorable for being inserted into cells to analyze single cells, and the first two probes cannot be inserted into the cells; and the spatial resolution of the probe of the present invention is also higher than the first two probes.

3. The preparation method of the invention has simple operation and low cost.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a schematic diagram of a microcannula probe construction;

illustration of the drawings: 1. a quartz outer capillary tube; 2. a quartz inner capillary tube; 3. adapting a sleeve; 4. a joint; 5. a tee joint; 6. a conveying pipe; 7. the distance between the inner and outer capillaries of the probe.

FIG. 2 is a photomicrograph of a microcannula probe.

FIG. 3 is a graph of the extraction time of the dye dripped into the tissue by using a single probe and a micro-cannula probe with the same size in the example 1, which is a real-time sampling-visible absorption spectrum detection; wherein: the flow rate of the extract was 3. Mu. In the absorption spectrum (A), 1. Mu. In the absorption spectrum (B) and 0.5 absorbance spectrum (C).

FIG. 4 is a block diagram of an apparatus for performing in situ liquid extraction-mass spectrometry imaging experiments in example 2.

FIG. 5 is an optical image (A) of mouse brain tissue imaged with the novel probe (probe size 200 μm) and a mass spectrum image (B) of each lipid in example 2.

FIG. 6 is a microscope picture of the liquid node formed on the cell slide for the microcannula probe.

FIG. 7 is a mass spectrum of single cell sampling mass spectrometry detection of HT22 neuronal cells using 30 μm-sized microcannula probes as described in example 3.

Detailed Description

In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

The method of the invention firstly carries out the manufacture of the probe, and the specific structure of the probe is shown in figure 1. In one embodiment of the present invention, the probe uses a polyimide film-protected fused silica capillary tube having an outer diameter of 360 μm and an inner diameter of 250 μm as an outer tube (i.e., quartz outer capillary tube 1), and a polyimide film-protected fused silica capillary tube having an outer diameter of 150 μm and an inner diameter of 100 μm as an inner tube (i.e., quartz inner capillary tube 2). The outer polyimide film with the length of 2cm at the lower end of each of the two quartz capillary tubes is burnt by candle flame, then the area is heated by a butane lighter, the capillary tubes are rapidly stretched to 2 times the length by tweezers, and the thinnest part is rapidly cut off by surgical scissors after the fire is cut off, so that a probe tip with a flat tip and the diameter of 10-30 mu m can be obtained, as shown in figure 2; the tip can be further adjusted to a larger tip diameter by cutting with scissors or sanding with ground glass. The end of all capillaries opposite the tip is referred to as the B-end. Connecting the B end of quartz outer capillary tube 1 to one of the outlets of PEEK tee 5 using FEP (fluorinated ethylene propylene copolymer) adapter sleeve 3 and standard 1/16' PEEK joint 4; then the tip of the quartz inner capillary 2 is inserted into the quartz outer capillary 1 from the other outlet of the tee joint 5, and the FEP adapter sleeve 3 and a standard 1/16' PEEK joint 4 are used for fixing the quartz inner capillary 2 at the outlet; the retraction distance of the needle point 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 7 between the inner capillary and the outer capillary of the probe is adjusted to 0-50 mu m, the joint 4 can be screwed to fix the inner capillary 2 of the quartz. The length of the quartz inner capillary 2 is about 10-20cm; the quartz outer capillary 1 is about 3-5cm in length. Specific values of the above parameters are shown in the following examples.

The invention fixes the probe on the z-axis of the three-axis stepping electric platform, and can move up and down to control the distance between the probe and the sample. And the sample is fixed on the xy axis of the three-axis platform and can move back and forth and left and right to control the relative position of the probe on the sample.

The third outlet of the tee joint 5 is connected with an injector of a syringe pump through a transmission pipe 6 (liquid supply capillary) and is used for pumping extraction liquid into the quartz outer capillary 1. The quartz inner capillary 2 is connected with a mass spectrometer ion source or a vacuum pump to provide vacuum degree to suck the extraction liquid into the quartz inner capillary 2. As shown in FIG. 4, the specific connection mode is shown in the specific embodiment.

The present invention utilizes a z-axis moving probe to control the distance between the sample and the probe. And observing the real-time distance between the sample and the probe and the stability of the liquid node through a micro camera.

The three-axis stepping electric platform is a platform capable of linearly moving in the xyz three directions, and the precision and the moving range of the three-axis stepping motor are determined according to the size of an imaging sample and the imaging spatial resolution. Three-axis stepper motor platforms are well known to those skilled in the art.

The vacuum pump is mainly a diaphragm pump with a vacuum degree controller.

The mass spectrometry ion source can be an electrospray ionization source, an atmospheric pressure chemical ionization source, and the like.

Example 1:

an in-situ liquid extraction sampling probe with high spatial resolution and high stability for in-situ mass spectrometry is shown in figure 1, and comprises a quartz outer capillary tube 1 and a quartz inner capillary tube 2, wherein the quartz inner capillary tube 2 is coaxially sleeved in the quartz outer capillary tube 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 tube 1 and the quartz inner capillary tube 2 both extend from the tip end with the smaller outer diameter to the B end with the larger outer diameter; the external diameter of the tip of the quartz outer capillary 1 is 50 micrometers, and the external diameter of the tip of the quartz inner capillary 2 is 10 micrometers; the length of the quartz outer capillary tube 1 is 3cm, and the length of the quartz inner capillary tube 2 is 15cm; the difference between the outer diameter of the end B of the quartz inner capillary 2 and the inner diameter of the end B of the quartz outer capillary 1 is 100 mu m; the tip of the quartz inner capillary 2 was spaced 20 μm from the tip of the quartz outer capillary 1.

The B end of the quartz outer capillary 1 is connected with one outlet 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 passes through the inner cavity of the PEEK tee joint 5 and is exposed out of the other outlet of the PEEK tee joint 5, the FEP adapter sleeve 3 and a standard 1/16' PEEK joint 4 are used for fixing the quartz inner capillary 2 at the outlet, and the tip end of the quartz inner capillary 2 is located inside the quartz outer capillary 1 and close to one side of the tip end.

A third outlet of the PEEK tee joint 5 is connected with the injection pump through a transmission pipe 6; the B end of the exposed quartz inner capillary 2 is connected with the inlet of a flow cell of a diode array detector (the flow cell is a 200nL micro flow cell), the outlet of the flow cell is connected with a vacuum cavity, and the vacuum cavity is connected with a numerical control diaphragm pump; the injection pump pumps the extraction solution into the quartz outer capillary 1, the extraction solution flows into the tip and is sucked into the quartz inner capillary 2 by the vacuum generated by the numerical control diaphragm pump connected with the vacuum cavity.

In this embodiment, the difference between the in-situ liquid extraction sampling time curves of the microcannula probe and the single probe is compared, and the specific steps are as follows:

(1) Preparing a probe: the specific implementation process of the fabrication of the micro-cannula probe is described in the fabrication section of the probe in the specific embodiment, here, the tip diameter of the capillary 2 in the drawn quartz is 10 μm, and the tip diameter of the capillary 1 outside the drawn quartz is 50 μm. The quartz inner capillary 2 is set back at a distance of 20 μm from the quartz outer capillary 1. The length of the quartz inner capillary tube 2 is 15cm, and the length of the quartz outer capillary tube 1 is 3cm.

For comparison with the new probe, a single probe was also prepared. The double-hole silicon boric acid glass tube is quickly thinned on flame and then is cut off to the diameter of 50 mu m at the tip by using tweezers. The two holes at the upper end of the double-hole glass tube are respectively inserted into a fused quartz capillary tube with the outer diameter of 360 mu m and the inner diameter of 250 mu m (inlet tube) and the outer diameter of 150 mu m and the inner diameter of 100 mu m (outlet tube) to be used as a liquid transmission tube, and the interface is sealed by ultraviolet glue.

(2) The construction of the in-situ liquid extraction-ultraviolet detection device: the probe is attached to the tri-axial platform in the manner described in the detailed description. For the micro-cannula probe, the third outlet of the tee is connected with the injection pump, the B end of the capillary 2 in the quartz is connected with the inlet of the flow cell of the diode array detector (the flow cell is a 200nL micro flow cell), the outlet of the flow cell is connected with the vacuum cavity, and the vacuum cavity is connected with the numerical control diaphragm pump.

For a single probe, the inlet tube is connected to a syringe pump and the outlet tube is connected to the flow cell inlet.

(3) Preparing an azo dye spot plate: the mouse brain tissue was cut into slices with a thickness of 10 μm using a cryomicrotome and then hot mounted onto a glass plate. 2 μm of 1mg/mL disperse Red 1 methanol solution was repeatedly dropped on the brain tissue to form a circular spot of the azo dye having a diameter of 3 mm. 3 duplicate spots were formed for use.

(4) Detecting an in-situ liquid sampling time curve: and fixing the manufactured spot plate on an xy-axis sample table, and performing single-point extraction on the central position of the spot by using methanol as an extraction solvent. The probe sample spacing was 10 μm, and the flow rate of the syringe pump was 0.5. Mu.L/min, 1. Mu.L/min, and 3. Mu.L/min. Each flow rate was repeated once in three replicate samples, and different probes were extracted adjacent to each spot to make comparisons.

(5) And (4) analyzing results: as can be seen from fig. 3, at any flow rate, the width of the band produced by microcannula probe sampling is smaller than that of a single probe. Particularly, at a small flow rate, when the flow rate is 1, the half-peak width of a time curve generated by sampling of the micro-cannula probe is about 0.5min, and a single probe reaches about 2min at any flow. When the flow rate is reduced to 0.5 mu L/min, the half-peak width generated by the micro-cannula probe is about 1min, and the single probe can reach 5min. From this, it was found that the sample solution diffused in the flow path of the single probe about 5 times as much as the micro cannula probe. Such diffusion will produce dilution of the sample ultimately leading to a decrease in signal.

Example 2:

an in-situ liquid extraction sampling probe aiming at high spatial resolution and high stability of in-situ mass spectrometry has a structure schematic as shown in figure 1, and comprises a quartz outer capillary tube 1 and a quartz inner capillary tube 2, wherein the quartz inner capillary tube 2 is coaxially sleeved in the quartz outer capillary tube 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 tube 1 and the quartz inner capillary tube 2 both extend from the tip end with the smaller outer diameter to the B end with the larger outer diameter; the external diameter of the tip of the quartz outer capillary 1 is 200 μm, and the external diameter of the tip of the quartz inner capillary 2 is 30 μm; the length of the quartz outer capillary tube 1 is 3cm, and the length of the quartz inner capillary tube 2 is 15cm; the difference between the outer diameter of the end B of the quartz inner capillary 2 and the inner diameter of the end B of the quartz outer capillary 1 is 100 mu m; the tip of the quartz inner capillary 2 is spaced 20 μm apart from the tip of the quartz outer capillary 1.

The B end of the quartz outer capillary 1 is connected with one outlet 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 passes through the inner cavity of the PEEK tee joint 5 and is exposed out of the other outlet of the PEEK tee joint 5, the FEP adapter sleeve 3 and a standard 1/16' PEEK joint 4 are used for fixing the quartz inner capillary 2 at the outlet, and the tip end of the quartz inner capillary 2 is positioned inside the quartz outer capillary 1 and close to one side of the tip end.

A third outlet of the PEEK tee joint 5 is connected with the injection pump through a transmission pipe 6; the B end of the exposed quartz inner capillary 2 is directly connected with an ion source inlet of an electrospray ionization source-ion trap-time-of-flight mass spectrum; the injection pump pumps the extraction solution into the quartz outer capillary 1, the extraction solution flows into the tip and is sucked into the quartz inner capillary 2 by the vacuum formed by the mass spectrometry ion source spray.

This example uses a microcannula probe to image lipids in the mouse brain, and the specific steps are as follows:

(1) Preparing a micro-sleeve probe: fabrication of the microcannula probe the fabrication was carried out as described in the probe fabrication section of the embodiment where the tip diameter of the particular drawn quartz inner capillary 2 was 30 μm and the tip diameter of the quartz outer capillary 1 was 200 μm. The quartz inner capillary 2 is set back at a distance of 20 μm with respect to the quartz outer capillary 1. The length of the quartz inner capillary tube 2 is 15cm, and the length of the quartz outer capillary tube 1 is 3cm.

(2) Building an in-situ liquid extraction-mass spectrum imaging device: as shown in fig. 4, the probe is attached to the tri-axial platform in the manner described in the detailed description. For the micro-sleeve probe, the third outlet of the tee is connected with an injection pump, and the B end of the quartz inner capillary 2 is directly connected with an ion source inlet of an electrospray ionization source-ion trap-time-of-flight mass spectrum.

(3) Pretreatment of a tissue sample: mouse brain tissue was cut into slices having a thickness of 10 μm using a cryomicrotome, then hot-mounted on a glass plate, and the slices were put into a desiccator for use. The dried sections were picture scanned with a scanner for comparison with a mass spectrometric image.

(4) Mass spectrometric imaging of mouse brain tissue: fixing the tissue slice on an xy-axis sample table, pumping methanol-acetonitrile serving as an extraction solvent into the probe tip, and sucking the liquid by using negative pressure generated by mass spectrometry ion source spraying. A steady liquid junction can be formed by balancing the negative pressure with a syringe pump flow rate of 5. Mu.L/min at a 1.5L/min nebulizer sheath flow rate. The sample surface was scanned point by point with the probe with the sample pitch of the probe kept constant at 10 μm, the dwell time of each point was 17s, the probe moving speed between points was 200 μm/s, and the step between points was 200 μm. The probe is scanned point by point in a zigzag path to form a matrix of 20 x 25 points. And performing signal acquisition on the mass spectrum in the scanning process, reducing the finally obtained mass spectrum signal into a substance signal height corresponding to the spatial position according to the probe driving route, and finally recombining into an imaging graph.

(5) And (4) analyzing results: as can be seen in fig. 5, mass spectrometry imaging using a microcannula probe can image multiple lipids in mouse brain tissue. The signal distribution of these lipids in various regions of the tissue is greatly related to the functional regions of the tissue itself. Comparing the mass spectrum imaging graph with the optical imaging graph can find that the distribution of various lipid signals is very fit with the optical appearance of the tissue. Meanwhile, the content of different lipids at different positions is greatly different.

Example 3:

the quartz outer capillary tube 1 and the quartz inner capillary tube 2 both extend from the tip end with the smaller outer diameter to the B end with the larger outer diameter; the external diameter of the tip of the quartz outer capillary tube 1 is 30 micrometers, and the external diameter of the tip of the quartz inner capillary tube 2 is 10 micrometers; the length of the quartz outer capillary tube 1 is 3cm, and the length of the quartz inner capillary tube 2 is 15cm; the difference between the outer diameter of the end B of the quartz inner capillary tube 2 and the inner diameter of the end B of the quartz outer capillary tube 1 is 100 mu m; the tip of the quartz inner capillary 2 was spaced 5 μm from the tip of the quartz outer capillary 1.

The end B of the quartz outer capillary tube 1 is connected with one outlet 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 passes through the inner cavity of the PEEK tee joint 5 and is exposed out of the other outlet of the PEEK tee joint 5, the FEP adapter sleeve 3 and a standard 1/16' PEEK joint 4 are used for fixing the quartz inner capillary 2 at the outlet, and the tip end of the quartz inner capillary 2 is positioned inside the quartz outer capillary 1 and close to one side of the tip end.

A third outlet of the PEEK tee joint 5 is connected with the injection pump through a transmission pipe 6; the B end of the exposed quartz inner capillary 2 is directly connected with an ion source inlet of an electrospray ionization source-ion trap-time-of-flight mass spectrum; the injection pump pumps the extraction solution into the quartz outer capillary 1, the extraction solution flows into the tip and is sucked into the quartz inner capillary 2 by the vacuum formed by the mass spectrometry ion source.

In this example, the microcannula probe is used to perform single cell detection on lipid in HT22 neuronal cells, and the specific steps are as follows:

(1) Preparing a micro-sleeve probe: fabrication of the microcannula probe the fabrication was carried out as described in the probe fabrication section of the embodiment, where the tip diameter of the particular drawn quartz inner capillary 2 was 10 μm and the tip diameter of the quartz outer capillary 1 was 30 μm. The quartz inner capillary 2 is set back at a distance of 5 μm from the quartz outer capillary 1. The length of the quartz inner capillary tube 2 is 15cm, and the length of the quartz outer capillary tube 1 is 3cm.

(2) Building an in-situ liquid extraction single cell-mass spectrum platform: the probe is attached to the tri-axial platform in the manner described in the detailed description. For the micro-sleeve probe, the third outlet of the tee is connected with an injection pump, and the B end of the quartz inner capillary 2 is directly connected with an ion source inlet of an electrospray ionization source-ion trap-time-of-flight mass spectrum. The three-axis platform is built on a fluorescence inverted microscope.

(3) Pretreatment of a cell sample: HT22 cell line purchased and subcultured in Petri dishes in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% streptomycin double antibody solution at 37 ℃ in an environment of 5% CO ₂ . After the cell density reached 90%, the cells in the culture dish were digested with trypsin, and the digested suspension cells were distributed to 6-well plates with slides for further culture. The final slide grown to a certain density was used for the single cell analysis experiments described below.

(4) Single cell analysis: and attaching the cultured climbing piece to a glass plate, and then placing the glass plate on a three-axis platform. The probe forms a stable liquid node with the space between the glass plates regulated by about 10 mu m through z-axis control, and then a cell is found on the slide by a microscope for extraction. During extraction, the extraction solution was 1% formic acid-methanol, the ion source sheath flow rate was 0.5L/min, and the equilibrated sampling flow rate was about 0.5. Mu.L/min. And acquiring a signal by the mass spectrum in the sampling process to obtain a real-time mass spectrum.

(5) And (4) analyzing results: first, as can be seen from FIG. 6, the size of the probe liquid node is substantially consistent with the soma size of HT22 neuronal cells, indicating that the probe can resolve single cells. Meanwhile, as is clear from FIG. 7, phospholipids which are generally found in biological cell membranes were collected from individual cells, and very individual glycosphingolipids were detected. And the rationality of the detection result is explained.

In general, the invention aims at the problems of serious probe internal diffusion and unstable liquid node existing in the single probe with the highest spatial resolution at present, and improves and innovates the traditional liquid flow probe with better stability. The method comprises the steps of pulling one end of two fused quartz capillaries with different diameters to form a tip with the diameter of 10-200 mu m, combining the two capillaries into a coaxial sleeve by using a tee joint, and adjusting the distance between the tips of a quartz inner capillary 2 and a quartz outer capillary 1 to be 0-50 mu m so as to obtain a micro coaxial sleeve probe with the tip diameter of 10-200 mu m. Pumping liquid from an outlet of the tee joint to an external capillary, and enabling a probe tip to form a stable liquid node through negative pressure generated by a vacuum pump or a mass spectrum ion source connected with the quartz inner capillary 2 to perform space-resolved in-situ extraction sampling and online mass spectrum detection on the surface of a sample. The new probe can improve the spatial resolution by more than ten times compared with the traditional in-situ liquid extraction casing probe, and has higher stability and lower diffusion of the extracted analyte compared with the single probe with high spatial resolution which is newly appeared in recent years. The probe can be applied to single cell analysis with high spatial resolution, tissue mass spectrometry imaging analysis and the like.

Claims

1. The in-situ liquid extraction sampling probe aiming at high spatial resolution and high stability of in-situ mass spectrometry is characterized by comprising a quartz outer capillary tube (1) and a quartz inner capillary tube (2), wherein the quartz inner capillary tube (2) is coaxially sleeved in the quartz outer capillary tube (1); the outer diameter of the quartz inner capillary tube (2) is smaller than the inner diameter of the quartz outer capillary tube (1);

the quartz outer capillary tube (1) and the quartz inner capillary tube (2) both extend from the tip with the smaller outer diameter to the B end with the larger outer diameter; the external diameter of the tip of the quartz outer capillary (1) is 10-200 μm, and the external diameter of the tip of the quartz inner capillary (2) is 2-30 μm;

the end B of the quartz outer capillary tube (1) is connected with one outlet of the tee joint (5);

the end B of the capillary tube (2) in the quartz penetrates through the inner cavity of the tee joint (5) and is exposed from the other outlet of the tee joint (5), and the tip of the capillary tube (2) in the quartz is located in the capillary tube (1) outside the quartz and close to one side of the tip.

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

3. The in situ liquid extraction sampling probe of claim 1, wherein 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 (2) is 50-100 μ ι η.

4. The in situ liquid extraction sampling probe of claim 1, wherein the spacing of the tip of the quartz inner capillary (2) relative to the tip of the quartz outer capillary (1) is 0-50 μ ι η.

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

6. The in situ liquid extraction sampling probe of claim 5, wherein the tee (5) is a PEEK tee, the adapter sleeve (3) is an FEP adapter sleeve, and the fitting (4) is a standard 1/16"PEEK fitting.

7. The in situ liquid extraction sampling probe according to any of claims 1-6 wherein the third outlet of the tee (5) is connected to a syringe pump via a transfer tube (6); the B end of the exposed quartz inner capillary tube (2) is connected with a mass spectrum ion source inlet or a vacuum cavity, and the vacuum cavity is connected with a vacuum pump.

8. The in situ liquid extraction sampling probe of claim 7, wherein a syringe pump connected to the third outlet of the tee (5) pumps an extraction solution into the outer quartz capillary (1), and the extraction solution flows into the tip and is drawn into the inner quartz capillary (2) by a vacuum created by the mass spectrometer ion source spray or by a vacuum pump connected to the vacuum chamber.

9. A method of making an in situ liquid extraction sampling probe according to any one of claims 1 to 8, comprising the steps of:

s1, drawing two quartz capillary tubes with different inner and outer diameters on a butane lighter, and then clamping the quartz capillary tubes with tweezers while the quartz capillary tubes are hot to generate tips with two different opening diameters, wherein the other non-drawn end of the quartz capillary tube is an end B, so that a quartz outer capillary tube with a larger diameter and a quartz inner capillary tube with a smaller diameter are obtained;

s2, sleeving the tip of the quartz inner capillary tube into the end of the tip of the quartz outer capillary tube to form a coaxial sleeve, connecting the end B of the quartz outer capillary tube with one outlet of the tee joint, penetrating the end B of the quartz inner capillary tube through the inner cavity of the tee joint and exposing the end B of the quartz inner capillary tube from the other outlet of the tee joint, and fixing the quartz inner capillary tube at the outlet of the tee joint; a third outlet of the tee joint is connected with the injection pump through a transmission pipe; the exposed B end of the capillary tube in the quartz is connected with an ion source inlet of the mass spectrum or a vacuum cavity, and the vacuum cavity is connected with a vacuum pump;

10. Use of an in situ liquid extraction sampling probe according to any one of claims 1 to 8 in situ mass spectrometry.