Double-sheath liquid micro-nano liquid cone electrospray ionization mass spectrometry ion source system, use method and application

Abstract

The invention provides a double-sheath liquid micro-nano liquid cone electrospray ionization mass spectrometry ion source system, a use method and application, and relates to the field of mass spectrometry analysis, the double-sheath liquid micro-nano liquid cone electrospray ionization mass spectrometry ion source system comprises a double-sheath liquid pipeline module, an integrated micro-flow pump control module and a high-voltage potential loading module, the double-sheath fluid pipeline module comprises a sample quartz capillary tube, an inner sheath fluid quartz capillary tube and an outer sheath fluid stainless steel tube which are coaxially arranged, and the outlet ends of the pipelines are sequentially arranged to form a three-stage layered embedded coaxial outlet with a concave-convex structure; the integrated micro-flow pump control module is connected with the double-sheath fluid pipeline module and is used for conveying sample fluid, inner sheath fluid and outer sheath fluid; and the output end of the high-voltage potential loading module is connected with the inner sheath liquid quartz capillary tube. The inner sheath fluid provides an ionization environment, the outer sheath fluid reduces the surface tension, assists in rapid desolvation and forms a protective barrier, the ionization efficiency and the spray stability are remarkably improved under dual cooperation, matrix effect interference is reduced, the efficiency and sensitivity of mass spectrometry are greatly improved, and the application range of mass spectrometry is greatly widened.

Description

Technical Field

[0001] The present invention relates to the technical field of mass spectrometry analysis, and particularly relates to a dual-sheath liquid micro-nano liquid cone electrospray mass spectrometry ion source system, a usage method and an application thereof. Background Art

[0002] As a highly sensitive and selective analytical method, mass spectrometry technology plays a crucial role in the fields of modern analytical chemistry, biochemistry, environmental science, etc. Among them, electrospray ionization (ESI) is widely used in various mass spectrometry analyses because of its wide applicability to liquid-phase samples. Traditional ESI converts a liquid into charged droplets through a high-voltage electric field, and forms gas-phase ions through the evaporation of the solvent to enter the mass spectrometer for analysis. However, traditional ESI still has many limitations when facing complex samples.

[0003] In the analysis of high-salt matrices and complex biological samples by traditional ESI ion sources, coexisting substances in the samples will significantly reduce the ionization efficiency of target molecules, resulting in a decrease in signal intensity and poor repeatability. Existing research shows that matrix effect is one of the main factors affecting electrospray ionization efficiency. In addition, the adaptability of traditional ESI to liquid flow rate is limited, and it usually performs best in the range of 1 μL / min to 10 μL / min. When the flow rate exceeds or is lower than this range, the spray stability decreases, resulting in a significant reduction in ionization efficiency.

[0004] Another significant problem is the design of the spray needle. During long-term use, traditional ESI spray needles are prone to contamination by high-salt matrices or impurities in complex samples, resulting in signal drift, reduced ionization efficiency, increased equipment maintenance costs, and downtime. Such contamination not only affects the reliability of analysis results but also significantly shortens the service life of the equipment. Traditional ion source designs are difficult to avoid matrix effects, especially in the analysis of complex samples, where insufficient sensitivity is exhibited. Although existing technologies can form a spray through a liquid cone, there are still limitations. For example, Chinese Patent CN114109756 A discloses a high-conductivity electrolyte aqueous solution electrospray system and method. The device includes two layers of coaxially arranged capillaries. The inner capillary extends a length from the outer capillary. The inner capillary injection pump delivers the electrolyte aqueous solution to the inner capillary, and the outer capillary injection pump delivers the ionic liquid to the outer capillary. A high electric potential is applied to the inner and outer capillaries through a high-voltage power supply to directly form a liquid cone spray. Although this device can achieve a relatively stable liquid cone spray, the ionic liquid transported in the outer capillary of this device only serves as a coating layer to prevent the evaporation of the electrolyte and form a thin film to stabilize the liquid cone, and it is not clearly promoted in improving the mass spectrometry ionization efficiency. In addition, the high electric potential of this device is directly applied to the double-layer capillary, and the electric field distribution is uneven. The application scenario of this device is mainly for electrospray thruster applications, focusing on solving the problem of liquid volatilization in a vacuum environment.

[0005] In the field of single-cell analysis, existing technologies also have limitations. For example, Chinese Patent CN109950126 A discloses a high-throughput single-cell electrospray mass spectrometry device. The device includes three layers of coaxially arranged capillary needles. The inner capillary transports the cell suspension, the middle capillary transports the sheath liquid extractant, and the outer capillary forms an electrospray. Although this device can achieve the sorting of cells into the mass spectrometer for analysis, it still retains the traditional needle structure (the tip diameter is 10μm), and is prone to contamination and blockage. In addition, the high voltage of this device is directly applied to the outer wall of the outermost capillary needle, and the electric field distribution is uneven, which easily causes unstable spraying. This device mainly focuses on the sorting and fragmentation of cells, rather than optimizing the ionization efficiency and spraying stability. Therefore, it still faces significant challenges when dealing with complex matrix samples.

[0006] In addition, existing ion source technologies also have deficiencies in the ability of on-line chemical reactions. The application of traditional ESI technology in real-time analysis is limited and it is difficult to support the realization of on-line chemical reactions, which are increasingly emphasized in dynamic sample analysis and metabolic research.

[0007] In summary, existing electrospray mass spectrometry ion source technologies still have obvious deficiencies in ionization efficiency, spraying stability, anti-contamination ability, flow rate adaptability, and on-line chemical reaction ability. There is an urgent need for a new type of ion source technology to overcome these limitations and improve the application efficiency and reliability of mass spectrometry analysis in complex samples. Summary of the Invention

[0008] The object of the present invention is to provide a dual-sheath liquid micro / nano liquid cone electrospray mass spectrometry ion source system, its usage method and application. By combining innovative technologies such as needleless design, dual-sheath liquid system, optimized electric field loading, and integrated micro-flow pump control, the core problems faced by traditional electrospray ion sources in the analysis of high-salt and complex samples are solved, and the efficiency, sensitivity, stability, and applicable range of mass spectrometry analysis are significantly improved.

[0009] To achieve the above object, the present invention is realized through the following technical solutions: A dual-sheath liquid micro / nano liquid cone electrospray mass spectrometry ion source system, comprising: A dual-sheath liquid pipeline module, including a sample quartz capillary, an inner-sheath liquid quartz capillary, and an outer-sheath liquid stainless steel tube arranged coaxially. The sample quartz capillary is located at the central position, the inner-sheath liquid quartz capillary wraps the sample quartz capillary, and the outer-sheath liquid stainless steel tube wraps the inner-sheath liquid quartz capillary; the outlet ends of the sample quartz capillary, the inner-sheath liquid quartz capillary, and the outer-sheath liquid stainless steel tube are arranged in sequence to form a three-level hierarchical embedded coaxial outlet with a concave-convex structure; An integrated micro-flow pump control module, which is connected to the dual-sheath liquid pipeline module and is used to respectively transport sample liquid to the sample quartz capillary, inner-sheath liquid to the inner-sheath liquid quartz capillary, and outer-sheath liquid to the outer-sheath liquid stainless steel tube; The flow rate of the sample liquid, inner-sheath liquid, or outer-sheath liquid transported by the integrated micro-flow pump control module is less than or equal to 20 μL / min; A high-voltage potential loading module, the output end of which is connected to the inner-sheath liquid quartz capillary, and is used to transfer the high-voltage electric field to the three-level hierarchical embedded coaxial outlet through the inner-sheath liquid, so that the high-voltage electric field is evenly distributed at the three-level hierarchical embedded coaxial outlet, avoiding interference with sample analysis caused by local heating of the wire directly applying the electric field at the three-level hierarchical embedded coaxial outlet.

[0010] Among them, a liquid cone is formed at the three-level hierarchical embedded coaxial outlet under the action of the high-voltage electric field to generate charged ions.

[0011] Furthermore: The inner diameter of the sample quartz capillary is 30 μm - 70 μm, and the outer diameter is 100 μm - 200 μm; the inner diameter of the inner-sheath liquid quartz capillary is 200 μm - 250 μm, and the outer diameter is 300 μm - 400 μm; the inner diameter of the outer-sheath liquid stainless steel tube is 500 μm - 600 μm, and the outer diameter is 1000 μm - 1500 μm; The nozzle of the sample quartz capillary at the outlet of the three-stage hierarchical embedded coaxial structure is indented by 2 mm - 5 mm compared to the nozzle of the inner sheath liquid quartz capillary, and the nozzle of the inner sheath liquid quartz capillary protrudes 0.5 mm - 2 mm from the nozzle of the outer sheath liquid stainless steel tube, enabling the formation of a functional gradient field partition including a sample premixing zone, a charge pre-distribution zone, a surface tension attenuation zone, and a rapid desolvation zone.

[0012] Furthermore: The double sheath liquid pipeline module further includes: PEEK three-way joints, which are used to connect and fix the sample quartz capillary, the inner sheath liquid quartz capillary, and the outer sheath liquid stainless steel tube; among them, the PEEK three-way joint includes two T-shaped PEEK three-way joints, which are respectively used to achieve the independent and co-directional transportation of the inner sheath liquid and the outer sheath liquid; The T-shaped PEEK three-way joint is divided into a first T-shaped three-way joint and a second T-shaped three-way joint. The sample quartz capillary is inserted into the main pipeline input port of the first T-shaped three-way joint and sequentially passes through the main pipeline output port of the first T-shaped three-way joint, the main pipeline input port of the second T-shaped three-way joint, and the main pipeline output port of the second T-shaped three-way joint; One end of the inner sheath liquid quartz capillary wraps the sample quartz capillary and is connected to the main pipeline output port of the first T-shaped three-way joint, and the other end sequentially passes through the main pipeline input port of the second T-shaped three-way joint and the main pipeline output port of the second T-shaped three-way joint; One end of the outer sheath liquid stainless steel tube wraps the inner sheath liquid quartz capillary extending from the main pipeline output port of the second T-shaped three-way joint and is connected to the main pipeline output port of the second T-shaped three-way joint; The sample quartz capillary, the inner sheath liquid quartz capillary, and the outer sheath liquid stainless steel tube are all connected to the T-shaped PEEK three-way joint through joints.

[0013] Furthermore: The integrated micro-flow pump control module includes integrated micro-flow pump control module one, integrated micro-flow pump control module two, and integrated micro-flow pump control module three with the same structure; Integrated micro-flow pump control module one transports the outer sheath liquid to the outer sheath liquid stainless steel tube, integrated micro-flow pump control module two transports the inner sheath liquid to the inner sheath liquid quartz capillary, and integrated micro-flow pump control module three transports the sample liquid to the sample quartz capillary; Integrated micro-flow pump control module one, integrated micro-flow pump control module two, and integrated micro-flow pump control module three all include: A stepper motor, which is used to independently control the flow rate of the sample liquid, the inner sheath liquid, or the outer sheath liquid; A lead screw and a lead screw nut. The lead screw is connected to the stepper motor, and the lead screw nut is arranged on the lead screw and is used to convert the rotational motion of the stepper motor into a linear motion; A Luer connector glass syringe, which is used to store the sample liquid, the inner sheath liquid, or the outer sheath liquid; A micro linear slide rail and a slider are used to ensure the stable pushing movement of the push rod of the luer connector glass syringe; one end of the slider clamps the outer shell of the luer connector glass syringe and is arranged on the micro linear slide rail, and the other end of the slider is connected to the lead screw nut; A fixed block is also arranged on the micro linear slide rail, and the push rod of the luer connector glass syringe abuts against the fixed block.

[0014] After the output end of the luer connector glass syringe of the integrated micro flow pump control module I is connected to the branch input port of the second T-shaped tee through a connector, it is connected to the outer sheath liquid stainless steel tube; After the output end of the luer connector glass syringe of the integrated micro flow pump control module II is connected to the branch input port of the first T-shaped tee through a connector, it is connected to the inner sheath liquid quartz capillary; The output end of the luer connector glass syringe of the integrated micro flow pump control module III is connected to the sample quartz capillary through a connector.

[0015] Furthermore: It also includes: A multi-axis linkage microscopic positioning module, which includes a spatial position fine-tuning component and a digital microscope component. The spatial position fine-tuning component is used to realize the fine-tuning of the spatial position between the liquid cone and the mass spectrometry inlet, and the digital microscope component is used to display the position of the liquid cone and the mass spectrometry inlet and the spraying state of the liquid cone in real time; A mass spectrometry interface connection module, which is used to fix the double-sheath liquid micro-nano liquid cone electrospray mass spectrometry ion source system on the mass spectrometer.

[0016] Furthermore: The inner sheath liquid is an aqueous solution containing volatile organic acid / base, which is used to provide an ionization environment for sample molecules and load a high-voltage potential over a long distance; the outer sheath liquid is an organic solvent, which is used to reduce the liquid surface tension and accelerate the evaporation of liquid droplets.

[0017] The present invention also provides a method for using the above double-sheath liquid micro-nano liquid cone electrospray mass spectrometry ion source system, including the following steps: S1: Through the integrated micro flow pump control module, sample liquid is transported to the sample quartz capillary, inner sheath liquid is transported to the inner sheath liquid quartz capillary, and outer sheath liquid is transported to the outer sheath liquid stainless steel tube based on a preset flow rate ratio; S2: Through the high-voltage potential loading module, a high-voltage electric field is applied to the inner sheath liquid, so that the high-voltage electric field is conducted to the three-stage stratified embedded coaxial outlet through the inner sheath liquid; S3: Under the action of the high-voltage electric field, a liquid cone spray is formed at the three-stage stratified embedded coaxial outlet, and through droplet cleavage and rapid evaporation of the solvent, the ionization of sample molecules is realized; S4: Adjust the liquid cone spray through the multi-axis linkage microscopic positioning module, and guide the ions into the mass spectrometer for analysis to achieve the best mass spectrometry signal response.

[0018] Further: in step S1, the flow rate ratio of the sample solution, the inner sheath liquid, and the outer sheath liquid is 0.5 - 1:0.5 - 2:1 - 10; preferably 0.5:1:2.

[0019] The present invention also provides the application of the double - sheath liquid micro - nano liquid cone electrospray mass spectrometry ion source system described above, or the method using the above double - sheath liquid micro - nano liquid cone electrospray mass spectrometry ion source system in the mass spectrometry analysis of high - salt matrix samples, biological samples or single cells.

[0020] The high - salt matrix sample refers to a sample with 10 mM concentration of PBS (phosphate - buffered saline) or physiological saline as the matrix.

[0021] In summary, the present invention features "three - stage hierarchical embedded coaxial outlet + multi - functional gradient sheath liquid synergistic effect + modular compact configuration design". Through staged sheath liquid wrapping, sheath liquid delayed loading mechanism and optimized design of electric field zoning, a functional gradient field of "sample premixing - charge pre - distribution - surface tension attenuation - rapid desolvation" is formed. The inner sheath liquid provides an ionization environment, and the outer sheath liquid reduces the surface tension, assists in rapid desolvation and forms a protective barrier. Through the dual cooperation of the functionalized inner and outer sheath liquids, the ionization efficiency and spray stability are significantly improved, and the matrix effect interference is reduced. The present invention effectively solves the problems faced by traditional electrospray ion sources in the analysis of high - salt and complex biological samples, such as pollution blockage, poor salt tolerance and low ionization efficiency, greatly improves the efficiency, sensitivity and application range of mass spectrometry analysis, and is particularly suitable for the analysis of complex high - salt biological samples and single - cell research.

[0022] Compared with the prior art, the present invention has the following beneficial effects: First, the present invention adopts a spray - needle - free design, cancels the metal spray needle or capillary needle tip structure formed by laser - burning and pulling of capillary tubes in traditional electrospray systems that are vulnerable to contamination, and replaces it with a three - stage hierarchical embedded coaxial outlet to directly form a liquid cone. This fundamentally solves the problems of signal drift and reduced ionization efficiency caused by spray - needle contamination or blockage. This design significantly improves the anti - contamination ability of the device in the analysis of high - salt matrix and complex biological samples, extends the service life of the equipment, and reduces the maintenance frequency and downtime.

[0023] II. The innovative dual-sheath liquid system in the present invention significantly improves the ionization efficiency and stability through the synergistic effect of the inner and outer sheath liquids. Among them, the inner sheath liquid (such as 1% formic acid solution) provides a stable protonation environment for the sample, while the outer sheath liquid (such as acetonitrile) greatly enhances the spray stability by reducing the surface tension and forming a stable Taylor cone. This dual-sheath liquid design can significantly reduce the inhibitory effect of matrix effects on ionization efficiency, especially for the analysis of high-salt samples, which has important advantages, enabling the sensitivity and accuracy of complex sample analysis to be significantly improved and realizing single-cell analysis.

[0024] III. The present invention optimizes the electric field distribution by loading high voltage at a long distance in the inner sheath liquid, avoiding problems such as unstable flow rate, wire heat release, and uneven electric field distribution caused by the built-in wire at the traditional liquid outlet. The uniformly distributed electric field makes the formation of the liquid cone more stable, and the stretching, evaporation, and cracking processes of the liquid droplets in the electric field are more efficient, thereby further improving the ionization efficiency and signal stability.

[0025] IV. The three-layer liquid coaxial flow design of the present invention, combined with precisely controlled flow rate ratios (such as 1:2:4), realizes the hydrodynamic balance of the sample liquid, inner sheath liquid, and outer sheath liquid at the outlet, ensuring the stability of the spray cone shape and the spray efficiency. This design significantly expands the flow rate adaptation range of the device, enabling it to adapt to various flow rate requirements from nanoliters to microliters.

[0026] V. The present invention can be applied to mass spectrometry. Through reasonable spatial layout design of each module, the modular compact configuration reduces the volume of the core components of the system to 300mm×150mm×200 mm, meeting the demand for small and portable use. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a schematic structural diagram of a dual-sheath liquid micro-nano liquid cone electrospray mass spectrometry ion source system; Figure 2 is a schematic semi-sectional structural diagram of a dual-sheath liquid pipeline module; Figure 3 is Figure 2 a simplified diagram after adjusting the ratio; Figure 4 is a schematic structural diagram of an integrated micro-flow pump control module I; Figure 5 is a schematic structural diagram of a spatial position fine-tuning component; Figure 6 is a schematic diagram of the relationship between voltage and spray stability; Figure 7 is a schematic diagram of the relationship between different sheath liquids and spray stability and signal intensity; Figure 8 is a flow rate ratio optimization diagram; Figure 9 It is a comparison diagram of the presence or absence of sheath fluid; Figure 10 It is a linear detection diagram of reference substances; Figure 11 It is a detection limit test diagram; Figure 12 It is an intra-day precision test diagram; Figure 13 It is an inter-day precision test diagram; Figure 14 It is a salt tolerance test diagram; Figure 15 It is a detection diagram of metabolites of human multiple myeloma peripheral blood B lymphocytes (8226); Figure 16 It is a detection diagram of metabolites of human acute myeloid leukemia cells - 3; Figure 17 It is a detection diagram of metabolites of human myeloma cells - H929; Figure 18 It is a detection diagram of metabolites of human monocytic leukemia cells - 1; Figure 19 It is a classification distribution diagram of metabolites of human multiple myeloma peripheral blood B lymphocytes (8226); Figure 20 It is a classification distribution diagram of metabolites of human acute myeloid leukemia cells - 3; Figure 21 It is a classification distribution diagram of metabolites of human myeloma cells - H929; Figure 22 It is a classification distribution diagram of metabolites of human monocytic leukemia cells - 1; Figure 23 It is a PCA analysis diagram of the metabolomics of four types of cells.

[0028] In the figure: 1. Dual-sheath fluid pipeline module; 1.1. Stainless steel tube for outer sheath fluid; 1.2. Quartz capillary for inner sheath fluid; 1.3. Quartz capillary for sample; 1.4. Three-way joint; 1.41. First T-shaped three-way joint; 1.42. Second T-shaped three-way joint; 1.5. Outer sheath fluid; 1.6. Inner sheath fluid; 1.7. Sample fluid; 1.8. Liquid cone spray; 2. Integrated micro-flow pump control module; 2.1. Integrated micro-flow pump control module one; 2.11. Stepper motor; 2.12. Lead screw; 2.13. Coupling; 2.14. Luer connector glass syringe; 2.15. Round joint; 2.2. Integrated micro-flow pump control module two; 2.3. Integrated micro-flow pump control module three; 3. High-voltage potential loading module; 3.1. High-voltage output wire; 4. Mass spectrometry interface connection module; 5. Digital microscope assembly; 6. Spatial position fine-tuning component; 6.1. Three-axis stage; 6.2. M3 screw; 6.3. T-shaped nut. Detailed implementation mode

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the accompanying drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation of the present invention. In addition, the terms "first", "second", and "third" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0031] Embodiment 1 As Figure 1 shown, the present invention provides a dual-sheath liquid micro-nano liquid cone electrospray mass spectrometry ion source system, which mainly includes five main parts: a dual-sheath liquid pipeline module 1, an integrated micro-flow pump control module 2, a multi-axis linkage microscopic positioning module, a high-voltage potential loading module 3, and a mass spectrometry interface connection module 4.

[0032] As Figures 2 - 3 shown, among them Figure 2 is the actual half-sectional structure diagram of the dual-sheath liquid pipeline module 1. However, the structure involved is too small, so on the basis of Figure 2 add Figure 3 , Figure 3 is the simplified structure diagram after magnifying the structural ratio of the sample quartz capillary 1.3, the inner-sheath liquid quartz capillary 1.2, and the outer-sheath liquid stainless steel tube 1.1 in Figure 2 to clearly show the installation and cooperation relationship between the components of the dual-sheath liquid pipeline module 1. The dual-sheath liquid pipeline module 1 includes a sample quartz capillary 1.3, an inner-sheath liquid quartz capillary 1.2, and an outer-sheath liquid stainless steel tube 1.1 arranged coaxially. Among them, in some embodiments, the sample quartz capillary 1.3 is used to accurately transport the sample solution. The outer diameter of the sample quartz capillary 1.3 is 200 μm, and the inner diameter is 50 μm; the outer diameter of the inner-sheath liquid quartz capillary 1.2 is 365 μm, and the inner diameter is 250 μm; the outer diameter of the outer-sheath liquid stainless steel tube 1.1 is 1000 μm, and the inner diameter is 500 μm. These three capillaries are connected and fixed through two T-shaped PEEK tees 1.4 and joints to realize the independent and co-directional transportation of the inner-sheath liquid 1.6 and the outer-sheath liquid 1.5.

[0033] The T-shaped PEEK tee is divided into a first T-shaped tee 1.41 and a second T-shaped tee 1.42. The sample quartz capillary 1.3 is inserted from the main line input port of the first T-shaped tee 1.41, and passes through the main line output port of the first T-shaped tee 1.41, the main line input port of the second T-shaped tee 1.42, and the main line output port of the second T-shaped tee 1.42 in sequence; One end of the inner sheath liquid quartz capillary 1.2 wraps around the sample quartz capillary 1.3 and is connected to the main line output port of the first T-type tee 1.41, and the other end passes through the main line input port of the second T-type tee 1.42 and the main line output port of the second T-type tee 1.42 in sequence; One end of the outer sheath liquid stainless steel tube 1.1 wraps around the inner sheath liquid quartz capillary 1.2 extending out of the main pipe outlet of the second T-shaped tee 1.42, and is connected to the main pipe outlet of the second T-shaped tee 1.42; The sample quartz capillary 1.3, the inner sheath liquid quartz capillary 1.2 and the outer sheath liquid stainless steel tube 1.1 are all connected to a T-shaped PEEK tee 1.4 via a joint.

[0034] The orifice of the sample quartz capillary 1.3 at the three-level layered embedded coaxial outlet is retracted by 2mm-5mm compared to the orifice of the inner sheath liquid quartz capillary 1.2, and the orifice of the inner sheath liquid quartz capillary 1.2 protrudes from the orifice of the outer sheath liquid stainless steel tube 1.1 by 0.5mm-2mm, so that functional gradient field partitions of sample premixing zone, charge predistribution zone, surface tension attenuation zone and rapid desolvation zone can be formed.

[0035] In this embodiment, European standard 120mm×20mm×200mm aluminum profiles, M3 screws 6.2 and T-nuts 6.3 are used to fix two T-shaped tees 1.4 to the grooves of the aluminum profiles, and the ports of the sheath liquid tees 1.4 are adjusted to be flush with the ports of the aluminum profiles.

[0036] like Figure 1 As shown, the integrated micro-flow pump control module 2 includes an integrated micro-flow pump control module 1 2.1, an integrated micro-flow pump control module 2 2.2, and an integrated micro-flow pump control module 3 2.3 with the same structure, which are respectively used to transport outer sheath liquid 1.5 to the outer sheath liquid stainless steel tube 1.1, transport inner sheath liquid 1.6 to the inner sheath liquid quartz capillary 1.2, and transport sample liquid 1.7 to the sample quartz capillary 1.3.

[0037] Combination Figure 4 As shown, taking the integrated micro-flow pump control module 2.1 as an example, the integrated micro-flow pump control module 2.1 includes: A stepper motor 2.11 is used to independently control the flow rate of the sample liquid 1.7, the inner sheath liquid 1.6 or the outer sheath liquid 1.5; The lead screw 2.12 and the lead screw nut, the lead screw 2.12 is connected to the stepping motor 2.11 through a coupling 2.13, and the lead screw nut is arranged on the lead screw 2.12 for converting the rotational motion of the stepping motor 2.11 into a linear motion; The Luer connector glass syringe 2.14 is used for storing the sample liquid 1.7, the inner sheath liquid 1.6 or the outer sheath liquid 1.5; The micro linear slide rail and the slider are used to ensure the stable pushing motion of the push rod of the Luer connector glass syringe 2.14; one end of the slider clamps the outer shell of the Luer connector glass syringe 2.14 and is arranged on the micro linear slide rail, and the other end of the slider is connected to the lead screw nut; A fixed block is also arranged on the micro linear slide rail, and the push rod of the Luer connector glass syringe 2.14 abuts against the fixed block.

[0038] The output end of the Luer connector glass syringe of the integrated micro flow pump control module 2.1 is connected to one end of the input pipeline through a round connector 2.15. After the other end of the input pipeline is connected to the branch input port of the second T-shaped tee, it is connected to the outer sheath liquid stainless steel pipe 1.1; The output end of the Luer connector glass syringe of the integrated micro flow pump control module 2.2 is connected to one end of the input pipeline through a round connector 2.15. After the other end of the input pipeline is connected to the branch input port of the first T-shaped tee, it is connected to the inner sheath liquid quartz capillary 1.2; The output end of the Luer connector glass syringe of the integrated micro flow pump control module 2.3 is connected to one end of the input pipeline through a round connector 2.15, and the other end of the input pipeline is connected to the sample quartz capillary 1.3.

[0039] Each liquid path (sample liquid 1.7, inner sheath liquid 1.6, outer sheath liquid 1.5) of the integrated micro flow pump control module is respectively connected to an independent syringe and a stepping motor 2.11. The micro linear slide rail and the slider ensure the stable pushing motion of the push rod of the syringe, avoiding flow rate fluctuations. The stepping motor 2.11 drives the syringe to push the liquid to the sample quartz capillary 1.3, the inner sheath liquid quartz capillary 1.2 and the outer sheath liquid stainless steel pipe 1.1, and controls the flow rate with a micro-liter precision. The driving of the stepping motor 2.11 is controlled by the ESP32 chip through the A4988 driver, accurately controlling the liquid flow rate and supporting independent adjustment of multiple flow paths.

[0040] To realize the fine adjustment of the spatial position between the liquid cone spray 1.8 and the mass spectrometry inlet, the present invention further includes a multi-axis linkage micro-positioning module to obtain the best mass spectrometry signal response. The multi-axis linkage micro-positioning module includes a spatial position fine adjustment component 6 and a digital microscope component 5, as Figure 1 and Figure 5As shown in the figure, in the present invention, the aluminum profile groove in the above-assembled double sheath liquid pipeline is assembled with a manual fine-tuning three-axis stage 6.1 of model LD60-CM-2 (XYZ-axis three-dimensional) through M3 screws 6.2 and T-shaped nuts 6.3 to realize the adjustment of the three-dimensional spatial position of the liquid cone spray 1.8 and the mass spectrometry inlet. The side groove of the aluminum profile is assembled with a commercially available digital electron microscope through M3 screws 6.2 and T-shaped nuts 6.3 to realize the real-time display and recording of the position of the liquid cone spray 1.8 and the mass spectrometry inlet and the liquid cone spray.

[0041] The high-voltage potential loading module 3 uses an external high-voltage power supply to provide a high-voltage electric field of 3 kV to 6 kV. The electric field directly acts on the liquid interface of the three-stage layered embedded coaxial outlet through the inner sheath liquid 1.6. The high-voltage potential loading module 3 is connected to the inner sheath liquid 1.6 flow path through a T-shaped PEEK tee 1.4 joint with a high-voltage output wire 3.1, and the output high voltage is loaded into the inner sheath liquid 1.6, so that the high-voltage electric field is applied to the liquid outlet end through the inner sheath liquid 1.6 at a long distance, making the electric field more evenly and stably distributed at the three-stage layered embedded coaxial outlet. The high-voltage ground wire is grounded together with the mass spectrometer. The high-voltage electric field makes the liquid cone spray 1.8 spray towards the mass spectrometry inlet direction to generate charged ions. This design avoids problems such as unstable flow rate, wire heat dissipation, and uneven electric field distribution caused by arranging wires inside the liquid outlet. In addition, the use of T-shaped PEEK and PEEK joints with good insulation properties for full-enclosure covering connection with the inner sheath liquid PEEK tube ensures the safety of the mass spectrometer and the operator.

[0042] The device of the present invention belongs to a portable and compact ion source, and can be fixed through 4 channels in a 120 mm × 80 mm × 200 mm aluminum profile by M5 screws to adapt to the ion source connection module including but not limited to the ThermoFisher Q-Exactive mass spectrometer. The three-stage layered embedded multi-functional double sheath liquid electrospray mass spectrometry ion source system is directly fixed on the mass spectrometer through the mass spectrometry interface connection module 4.

[0043] Embodiment 2 The present invention also provides a method for using the double sheath liquid micro-nano liquid cone electrospray mass spectrometry ion source system, including the following steps: S1: Through the integrated micro-flow pump control module 2, sample liquid 1.7 is delivered to the sample quartz capillary 1.3, inner sheath liquid 1.6 is delivered to the inner sheath liquid quartz capillary 1.2, and outer sheath liquid 1.5 is delivered to the outer sheath liquid stainless steel tube 1.1 based on a preset flow rate ratio. In a preferred embodiment, the flow rate ratio of the sample liquid 1.7, the inner sheath liquid 1.6, and the outer sheath liquid 1.5 is 1:2:4.

[0044] S2: Through the high-voltage potential loading module 3, a high-voltage electric field is applied to the inner sheath liquid 1.6, so that the high-voltage electric field is conducted to the three-stage layered embedded coaxial outlet through the inner sheath liquid 1.6. S3: Under the action of a high-voltage electric field, a liquid cone spray 1.8 is formed at the triple-layer embedded coaxial outlet. Through droplet fragmentation and solvent evaporation, ionization of sample molecules is achieved. S4: Adjust the liquid cone spray through a multi-axis linkage micro-positioning module to guide the ions into the mass spectrometer for analysis to achieve the best mass spectrometry signal response.

[0045] The working principle of the present invention is mainly reflected in the following aspects: Firstly, spray formation (formation of Taylor cone). The present invention eliminates the traditional metal spray needle or capillary needle tip formed by laser ablation and drawing of capillaries, avoiding the blockage problem caused by salt deposition or impurity contamination of the spray needle. The output high voltage is directly applied to the inner sheath liquid 1.6, so that the high-voltage electric field is applied through the inner sheath liquid 1.6 over a long distance to directly form a liquid cone spray 1.8 at the liquid outlet end. The present invention further improves the available voltage value through the tip spray formed by the liquid cone itself, and effectively avoids the problems of tip discharge of the spray needle and nozzle blockage caused by burning due to too high voltage.

[0046] Secondly, droplet fragmentation and solvent evaporation. Under the action of the electric field, the spray droplets are charged and rapidly reduce in volume. As the solvent of the droplets evaporates, the surface charge density of the droplets continuously increases, and finally exceeds the limit of the surface tension of the droplets (the so-called Rayleigh limit), resulting in the droplets breaking into smaller charged droplets (referred to as "Coulomb explosion"). The action of the high voltage ensures the continuous progress of this series of processes, splitting the liquid into charged micro-droplets. In the present invention, the outer sheath liquid uses an organic reagent, which not only reduces the surface tension of the droplets, but also accelerates the evaporation of the droplet solvent, further improving the ionization efficiency.

[0047] Thirdly, molecular ionization. The droplets gradually evaporate under the action of the high-voltage electric field until finally only the molecules originally dissolved in the solution remain. The complete evaporation of the solvent keeps the molecules in the solution charged, generating charged gas-phase ions (such as [M+H]⁺, [M-H]⁻, etc.). The high-voltage electric field ensures sufficient ionization efficiency, thereby converting the sample molecules into charged ions that can be detected by the mass spectrometer.

[0048] Finally, accelerating the migration of charged ions to the mass spectrometer. The high-voltage electric field can also provide an electric field force to accelerate the migration of charged gas-phase ions from the spray region to the inlet of the vacuum system of the mass spectrometer. This is very important for improving the sensitivity and detection efficiency of the mass spectrometer.

[0049] The stability of the dual-sheath liquid system is one of the core advantages of the present invention. It relies on the synergistic effect of the inner sheath liquid and the outer sheath liquid, as well as the precise coordination of hydrodynamic optimization and design structure. The inner sheath liquid is usually an acidified aqueous solution (such as 0.1% formic acid). By increasing the protonation ability, it provides a stable ionization environment for sample molecules. This environment not only improves the ionization efficiency but also provides a basic hydrodynamic balance for the sample during the liquid cone formation process. The inner sheath liquid wraps the sample liquid and forms a uniform liquid flow at the three-stage hierarchical embedded coaxial outlet, effectively alleviating the unstable influence that the sample liquid flow rate fluctuation may have on the spray cone.

[0050] The outer sheath liquid has multiple functions: First, the outer sheath liquid uses a low surface tension solvent (such as acetonitrile) to reduce the influence of liquid surface tension at the three-stage hierarchical embedded coaxial outlet, making it easier for the liquid cone to form a stable spray cone (Taylor cone) under the action of an electric field; Second, the outer sheath liquid wraps the inner sheath liquid and the sample liquid at a high flow rate, diluting the high salt or other complex matrix components in the sample and reducing the adverse effect of matrix effects on liquid spraying; In addition, the high-speed flow of the outer sheath liquid forms a protective barrier between the three-stage hierarchical embedded coaxial outlet and the external air, reducing the influence of external disturbances such as environmental air flow and temperature fluctuations on spray stability.

[0051] Another advantage of the present invention lies in hydrodynamic optimization. The stability of the dual-sheath liquid system is closely related to the flow rate ratio. In the present invention, the flow rate ratios of the sample liquid, the inner sheath liquid, and the outer sheath liquid have been experimentally optimized (such as 1:2:4), forming a good hydrodynamic balance. The flow rate of the inner sheath liquid is slightly higher than that of the sample liquid to ensure that the inner sheath liquid wraps the sample liquid and provides a uniform flow path. The flow rate of the outer sheath liquid is significantly higher than that of the inner sheath liquid and the sample liquid, and its shear force effectively stabilizes the shape of the liquid cone and further dilutes the interference of the high salt matrix sample on the spray cone.

[0052] Example Three The present invention also provides the application of the dual-sheath liquid micro-nano liquid cone electrospray mass spectrometry ion source system described above, or the method using the above dual-sheath liquid micro-nano liquid cone electrospray mass spectrometry ion source system, in the mass spectrometry analysis of high salt matrix samples, biological samples, or single cells.

[0053] The following further illustrates the present invention through specific application cases.

[0054] Application Case One Establishment of the method for the dual-sheath liquid cone electrospray ion source system: To verify the performance advantages of the dual-sheath liquid cone electrospray ion source of the present invention in trace sample detection and its optimization method, the experimental scheme selected glutamine, phenylalanine, and the mass spectrometry calibration solution of Thermo (containing caffeine and MRFA) as reference substances, and successively carried out concentration gradient linear detection and ionization condition optimization experiments.

[0055] First, prepare the experimental samples and standard solutions. The experimental target substances include glutamine, phenylalanine, caffeine in the Thermo mass spectrometry calibration solution, and MRFA (tetrapeptide: Met-Arg-Phe-Ala). Accurately weigh the target compounds and prepare a standard solution with a mother liquor concentration of 10 mM, using 50% acetonitrile / water (v / v) as the solvent, and add 0.1% formic acid to improve the ionization efficiency. Use the serial dilution method to successively dilute the mother liquor to standard working solutions of 10 aM, 100 aM, 10 fM, 100 fM, and 10 pM.

[0056] The experiment uses the dual-sheath liquid micro-nano liquid cone electrospray mass spectrometry ion source system proposed in the present invention, combined with a ThermoFisher mass spectrometer (such as Q-Exactive) for signal detection. The mass spectrometry mode uses the positive ion mode (ESI+), the scanning range is m / z = 100 - 1500, the resolution is 70000, and the S-lens is 40.

[0057] The flow rate range of the sample liquid is 0.1 μL / min - 2 μL / min; the flow rate range of the inner sheath liquid is 0.1 μL / min - 5 μL / min; the flow rate range of the outer sheath liquid is 0.1 μL / min - 10 μL / min.

[0058] The optimization design of key parameters includes voltage optimization, types of outer sheath liquid, flow rate ratio, and comparison with or without outer sheath liquid. In terms of voltage optimization, set the spray voltage range to 3.5 kV, 4.0 kV, 5.0 kV, 5.5 kV, and 6.0 kV, and respectively test their effects on the liquid cone shape and mass spectrometry signal intensity. In terms of types of outer sheath liquid, when the flow rates of the sample liquid, inner sheath liquid, and outer sheath liquid are 0.5 μL / min, 1 μL / min, and 2 μL / min respectively, test acetonitrile, water, and isopropanol as the outer sheath liquid, and analyze the differences in spray stability and ionization efficiency among different solvents. In terms of flow rate ratio, the inner sheath liquid uses an aqueous solution of 0.1% formic acid and is delivered at flow rates of 0.5 μL / min, 1.0 μL / min, and 2.0 μL / min; the flow rates of the outer sheath liquid are 1.0 μL / min, 2.0 μL / min, 5.0 μL / min, and 10.0 μL / min respectively, and acetonitrile is tested; the flow rates of the sample liquid are 0.5 μL / min and 1 μL / min respectively, and the target working solution is directly delivered. In terms of comparison with or without outer sheath liquid, test the spray stability and mass spectrometry response respectively under the conditions of no outer sheath liquid and with outer sheath liquid (acetonitrile).

[0059] Under optimized conditions (spray voltage 5.5 kV), with acetonitrile as the sheath liquid and a flow rate ratio of 0.5:1:2 μL / min (sample liquid: inner sheath liquid: outer sheath liquid), gradient detection was performed on glutamine, phenylalanine, caffeine, and MRFA. The concentration range was 10 aM to 10 pM. The mass spectrometry signal intensity of each target was measured, and a concentration-signal linear relationship curve was plotted.

[0060] Through the within-day precision experiment, a 10 fM working solution was used as the test sample, and 6 independent injections were performed on the same day. The working solution was reinjected before each measurement to ensure the reproducibility of the spray at each startup of the device. The mass spectrometry signal intensities of glutamine, phenylalanine, caffeine, and MRFA were recorded, and the relative standard deviation (RSD) was calculated to evaluate the within-day precision. For the between-day precision experiment, a 10 fM working solution was used as the test sample, and 3 independent injections were performed each day for three consecutive days. The spray parameters were reconfigured and the working solution was injected before each daily measurement. The mass spectrometry signal intensities (peak areas) of glutamine, phenylalanine, caffeine, and MRFA were recorded, and the relative standard deviation (RSD) was calculated to evaluate the between-day precision.

[0061] The experimental results showed that at 5.0 kV, the liquid cone stability was the best, the signal response intensity was the highest, and the signal noise was the lowest. As Figure 6 shown, when below 5 kV and above 5.5 kV, the electric field effect was too strong, resulting in liquid cone fluctuations and unstable signals. In the experiment on the type of sheath liquid, acetonitrile and isopropanol performed better during the spraying process due to their high volatility and low surface tension. In contrast, as Figure 7 shown, when acetonitrile was used as the sheath liquid compared to isopropanol, the liquid cone was more stable and the signal intensity was higher. In the flow rate ratio experiment, as Figure 8 shown, when the flow rate ratio was (0.5:1:2 μL / min), the synergistic effect between the inner and outer sheath liquids was the best, the liquid cone was stable, and the mass spectrometry response was the best. As Figure 9 shown, in the comparison experiment with or without the sheath liquid, without the sheath liquid, it was difficult to maintain the shape of the liquid cone and the signal was unstable during the spraying process; when the sheath liquid (acetonitrile) was present, the spraying stability and signal intensity were significantly improved.

[0062] In the linear detection experiment, as Figures 10 - 11 shown, gradient concentration detection (10 / 50 / 100 / 150 / 200 fM) of glutamine, phenylalanine, caffeine, and MRFA was performed under optimized conditions and the detection limit analysis was carried out in the low concentration range (1 aM to 200 fM). The results showed that the R2 of each linear equation was greater than 0.98, presenting good linearity and a low detection limit under a wide low concentration range. As Figures 12 - 13As shown, in the reproducibility experiments, the RSDs of all target substances were below 15% in the intra-day and inter-day precision tests, indicating that the device had good reproducibility and stability in experiments conducted at different times. As Figure 14 shown, in the salt tolerance experiment, 1 μg / mL bovine serum albumin standard solutions were prepared using phosphate buffer solutions (PBS) with different concentrations, and then detected using the dual-sheath liquid ion source, nano-spray ion source, and conventional electrospray ion source of the present invention, respectively, to obtain the relative signal abundances of BSA. The results showed that the dual-sheath liquid ion source of the present invention was superior to the nano-spray ion source and significantly superior to the conventional electrospray ion source, indicating that the dual-sheath liquid ion source of the present invention had better salt tolerance and obvious detection and analysis advantages in high-salt biological samples.

[0063] Application Case 2 Single-cell metabolomics analysis: Another important application of the present invention is single-cell metabolomics research. By selecting four myeloma-related cells, such as human multiple myeloma peripheral blood B lymphocytes (8226) (RPMI-8226), human acute myeloid leukemia cells-3 (OCI-Aml-3), human myeloma cells-H929 (NCI-H929), and human monocytic leukemia cells-1 (THP1), high-sensitivity detection of single-cell small molecule metabolites was achieved using the device of the present invention to reveal the metabolic characteristics and heterogeneity of different cell types.

[0064] First, sample preparation was carried out. The four types of cells, RPMI-8226, OCI-Aml-3, NCI-H929, and THP1, were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and the culture conditions were 37 °C and 5% CO2. After replacing the medium with sterile PBS, the cell suspension was centrifuged (500 g, 5 min), the supernatant was discarded, and the cells were washed 3 times with PBS buffer to remove residual components of the culture medium. The cells were observed under an inverted microscope, and a sample tube (quartz capillary with an inner diameter of 50 μm and an outer diameter of 250 μm) was connected to the injection pump module, and a single target cell was precisely aspirated into the tube for testing under the microscope. Additionally, approximately 5×10 4 cells were centrifuged to remove PBS, and pre-cooled methanol solution was added to quickly quench the metabolic activity and extract small molecule metabolites. The extract was centrifuged (14000 g, 4 °C, 10 min), and the supernatant was collected for subsequent mass spectrometry detection.

[0065] In terms of mass spectrometry detection, the dual-sheath liquid micro-nano liquid cone electrospray mass spectrometry ion source system of the present invention is used in combination with a ThermoFisher Q-Exactive mass spectrometer for cell extract metabolite and single-cell metabolomics analysis. The dual-sheath liquid cone ion source system is set as follows: the sample liquid is a single-cell extract with a flow rate of 0.5 μL / min; the inner sheath liquid is a 1% formic acid-aqueous solution with a flow rate of 1 μL / min; the outer sheath liquid is acetonitrile with a flow rate of 2.0 μL / min. The spray voltage is set at 5.0 kV, and the high-voltage electric field is conducted through the inner sheath liquid to the three-stage layered embedded coaxial outlet to form a stable liquid cone to ensure efficient spraying. The mass spectrometry acquisition is in the positive ion mode, the scanning range is 100 - 1500 m / z, the S-lens radio frequency voltage is 40, the resolution is 70000, the longest injection time is 200 milliseconds, and the automatic gain control (AGC) target value is 5×10 5 .

[0066] In terms of data analysis, the mass-to-charge ratio (m / z) and signal intensity of metabolites collected by mass spectrometry are used to obtain the mass spectrometry information of small molecule metabolites at the single-cell level. The ThermoXcalibur software is used to perform peak extraction and screening of characteristic metabolites on the original data. The background signal of the mass spectrometry data is corrected to ensure accurate reflection of the characteristic peaks of sample metabolites. In combination with the HMDB (Human Metabolome Database), the metabolite identity is confirmed based on the mass-to-charge ratio and secondary fragment information. The metabolic profile characteristics of each type of cell are extracted, and data statistical analysis is performed on the categories of metabolites. A pie chart is used to present the proportion of metabolites in 4 cell types. Principal component analysis (PCA) is used to explore the differences in metabolic characteristics between cells.

[0067] As Figures 15 - 18 shown, the experimental results show that using the device of the present invention, a variety of small molecule metabolites in single cells are successfully detected and matched through searching the HMDB library. Among them, 151 metabolites are matched in RPMI-8226 cells, 174 metabolites are matched in OCI-Aml-3 cells, 181 metabolites are matched in NCI-H929 cells, and 189 metabolites are matched in THP1 cells. The mass spectrometry signals are clear and the peak intensities are good, demonstrating the high sensitivity and high signal-to-noise ratio of the dual-sheath liquid cone ion source.

[0068] As Figures 19 - 22 shown, the results show that lipids and lipid-like molecules account for more than 60% in various cells, and organic acids account for about 12%. The proportions of organic heterocyclic compounds, benzene ring-type compounds, oxygen-containing organic compounds, and nucleosides, nucleotides and their analogs are different, and there are significant differences between different cells.

[0069] As Figure 23As shown, principal component analysis (PCA) shows that the metabolomes of different cell types can be clearly distinguished in the omics space. The reason for the overlap of metabolomic features may be the similar metabolic pathway activities, such as amino acid metabolism. Both RPMI-8226 and OCI-Aml-3 cells show a high dependence on glutamine, which is a metabolic characteristic of many rapidly proliferating cells (such as tumor cells). In the PCA analysis, the distinct separation of NCI-H929 and THP1 cells from RPMI-8226 and OCI-Aml-3 cells reflects the significant differences in their metabolic characteristics due to different cell functions, metabolic demands, and tumor metabolic reprogramming pathways.

[0070] In the present invention, the three-level hierarchical embedded dual-sheath liquid micro / nano liquid cone electrospray ionization mass spectrometry ion source system, as an innovative technology, provides an efficient, sensitive, and stable mass spectrometry analysis platform for the field of single-cell metabolomics and has broad application prospects. The research demonstrates the excellent performance of the dual-sheath liquid cone electrospray ionization source in single-cell metabolome analysis, providing technical support for the study of tumor heterogeneity, metabolic pathway reprogramming, and disease mechanisms.

[0071] It can be seen from the above embodiments that the three-level hierarchical embedded multi-functional dual-sheath liquid micro / nano liquid cone electrospray ionization mass spectrometry ion source system provided by the present invention not only effectively solves the problems faced by traditional electrospray ionization sources in the analysis of complex samples, such as low ionization efficiency, poor spray stability, and weak anti-pollution ability, but also demonstrates excellent performance in single-cell metabolomics research. Through the innovative needleless design, the synergistic effect of the dual-sheath liquid, the optimized high-voltage electric field distribution, and the precise hydrodynamic control, the system has achieved a significant improvement in the efficiency and scope of application of mass spectrometry analysis, providing strong technical support for the analysis of complex samples and biomedical research.

[0072] The above embodiments are only used to illustrate the technical concept and features of the present invention, and their purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly, and it should not be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. Dual-sheath liquid micro / nano liquid cone electrospray mass spectrometry ion source system, characterized in that, Comprising: A double sheath liquid pipeline module, the double sheath liquid pipeline module includes a sample quartz capillary tube, an inner sheath liquid quartz capillary tube, and an outer sheath liquid stainless steel tube arranged coaxially. Among them, the sample quartz capillary tube is located at the central position, the inner sheath liquid quartz capillary tube wraps the sample quartz capillary tube, and the outer sheath liquid stainless steel tube wraps the inner sheath liquid quartz capillary tube; the outlet ends of the sample quartz capillary tube, the inner sheath liquid quartz capillary tube, and the outer sheath liquid stainless steel tube are arranged in sequence to form a three-stage layered embedded coaxial outlet with a concave-convex structure; An integrated micro-flow pump control module, the integrated micro-flow pump control module is connected to the double sheath liquid pipeline module, and is used to respectively transport the sample liquid to the sample quartz capillary tube, transport the inner sheath liquid to the inner sheath liquid quartz capillary tube, and transport the outer sheath liquid to the outer sheath liquid stainless steel tube; A high-voltage potential loading module, the output end of the high-voltage potential loading module is connected to the inner sheath liquid quartz capillary tube, and is used to transfer the high-voltage electric field to the three-stage layered embedded coaxial outlet through the inner sheath liquid, so that the high-voltage electric field is evenly distributed at the three-stage layered embedded coaxial outlet; A liquid cone is formed at the three-stage layered embedded coaxial outlet under the action of the high-voltage electric field, generating charged ions.

2. The dual-sheath liquid micro / nano liquid cone electrospray mass spectrometry ion source system according to claim 1, wherein The nozzle of the sample quartz capillary tube at the three-stage layered embedded coaxial outlet is indented compared with the nozzle of the inner sheath liquid quartz capillary tube, and the nozzle of the inner sheath liquid quartz capillary tube protrudes from the nozzle of the outer sheath liquid stainless steel tube.

3. The dual-sheath liquid micro / nano liquid cone electrospray mass spectrometry ion source system according to claim 1, wherein The double sheath liquid pipeline module further includes: PEEK three-way joints, which are used to connect and fix the sample quartz capillary tube, the inner sheath liquid quartz capillary tube, and the outer sheath liquid stainless steel tube; among them, the PEEK three-way joint includes two T-shaped PEEK three-way joints, which are used to realize the independent and co-directional transportation of the inner sheath liquid and the outer sheath liquid; the T-shaped PEEK three-way joint is divided into a first T-shaped three-way joint and a second T-shaped three-way joint. The sample quartz capillary tube is inserted into the main pipeline input port of the first T-shaped three-way joint, and sequentially passes through the main pipeline output port of the first T-shaped three-way joint, the main pipeline input port of the second T-shaped three-way joint, and the main pipeline output port of the second T-shaped three-way joint; One end of the inner sheath liquid quartz capillary tube wraps the sample quartz capillary tube and is connected to the main pipeline output port of the first T-shaped three-way joint, and the other end sequentially passes through the main pipeline input port of the second T-shaped three-way joint and the main pipeline output port of the second T-shaped three-way joint; One end of the outer sheath liquid stainless steel tube wraps the inner sheath liquid quartz capillary tube extending from the main pipeline output port of the second T-shaped three-way joint and is connected to the main pipeline output port of the second T-shaped three-way joint; The sample quartz capillary tube, the inner sheath liquid quartz capillary tube, and the outer sheath liquid stainless steel tube are all connected to the T-shaped PEEK three-way joint through joints.

4. The dual-sheath liquid micro / nano liquid cone electrospray mass spectrometry ion source system according to claim 1, characterized in that The integrated micro-flow pump control module includes: an integrated micro-flow pump control module one, an integrated micro-flow pump control module two, and an integrated micro-flow pump control module three with the same structure; the integrated micro-flow pump control module one transports the outer sheath liquid to the outer sheath liquid stainless steel tube, the integrated micro-flow pump control module two transports the inner sheath liquid to the inner sheath liquid quartz capillary tube, and the integrated micro-flow pump control module three transports the sample liquid to the sample quartz capillary tube; The integrated micro-flow pump control module one, the integrated micro-flow pump control module two, and the integrated micro-flow pump control module three all include: a stepper motor, which is used to independently control the flow rate of the sample liquid, the inner sheath liquid, or the outer sheath liquid; A lead screw and a lead screw nut, the lead screw is connected to the stepper motor, and the lead screw nut is arranged on the lead screw for converting the rotational motion of the stepper motor into a linear motion; A luer connector glass syringe for storing sample liquid, inner sheath liquid or outer sheath liquid; A micro linear slide rail and a slider for ensuring the stable pushing motion of the push rod of the luer connector glass syringe; one end of the slider clamps the outer shell of the luer connector glass syringe and is arranged on the micro linear slide rail, and the other end of the slider is connected to the lead screw nut; A fixed block is further arranged on the micro linear slide rail, and the push rod of the luer connector glass syringe abuts against the fixed block.

5. The dual-sheath liquid micro / nano liquid cone electrospray mass spectrometry ion source system according to claim 1, wherein It further includes: A multi-axis linkage micro-positioning module, the multi-axis linkage micro-positioning module includes a spatial position fine-tuning component and a digital microscope component, the spatial position fine-tuning component is used to realize the fine-tuning of the spatial position between the liquid cone and the mass spectrometry inlet, and the digital microscope component is used to display the positions of the liquid cone and the mass spectrometry inlet and the spray state of the liquid cone in real time; A mass spectrometry interface connection module for fixing the double-sheath liquid micro-nano liquid cone electrospray mass spectrometry ion source system on the mass spectrometer.

6. The dual sheath liquid micro / nano liquid cone electrospray mass spectrometry ion source system according to claim 1, characterized in that, The inner sheath liquid is an aqueous solution of a volatile organic acid or an aqueous solution of an organic base, which is used to provide an ionization environment for sample molecules and load a high-voltage potential over a long distance; the outer sheath liquid is an organic solvent, which is used to reduce the surface tension of the liquid and accelerate the evaporation of the liquid droplets.

7. A method of using the dual sheath liquid micro / nano liquid cone electrospray mass spectrometry ion source system according to any one of claims 1-6, characterized in that, It includes the following steps: S1: Through the integrated micro-flow pump control module, the sample liquid is transported to the sample quartz capillary, the inner sheath liquid is transported to the inner sheath liquid quartz capillary, and the outer sheath liquid is transported to the outer sheath liquid stainless steel tube based on a preset flow rate ratio; S2: Through the high-voltage potential loading module, a high-voltage electric field is applied to the inner sheath liquid, so that the high-voltage electric field is conducted to the three-stage layered embedded coaxial outlet through the inner sheath liquid; S3: Under the action of the high-voltage electric field, a liquid cone spray is formed at the three-stage layered embedded coaxial outlet, and the ionization of sample molecules is realized through droplet cleavage and solvent evaporation; S4: The liquid cone spray is adjusted through the multi-axis linkage micro-positioning module to guide the ions into the mass spectrometer for analysis.

8. The method according to claim 7, characterized in that In step S1, the flow rate ratio of the sample liquid, the inner sheath liquid and the outer sheath liquid is 0.5-1:0.5-2:1-10.

9. Application of the double-sheath liquid micro-nano liquid cone electrospray mass spectrometry ion source system according to any one of claims 1-6, or the method according to any one of claims 7-8 in the mass spectrometry analysis of high-salt matrix samples, biological samples or single cells.

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