Sample injection probe device based on membrane-assisted strategy, and analysis method

Abstract

A sample injection probe (3) device based on a membrane (9)-assisted strategy, and a sample injection method. The sample injection probe (3) device uses the membrane (9)-assisted strategy to achieve in-situ pretreatment, sample injection, transmission, separation and detection of trace complex samples (8) under a pressure condition. The device has the following advantages: by integrating a trace sample (8) pretreatment system with sample injection, chromatographic separation and detection systems, the integrated system has a simple structure, fast and automatic operation, high flexibility, easy use and wide applicability, and is particularly suitable for analysis of the trace samples (8). On the basis of the membrane (9)-assisted strategy, sample injection after in-situ pretreatment of the samples (8) can be achieved, providing a new sample injection mode for the analysis of the complex samples (8), and also providing a new approach for integration and miniaturization of a chromatographic analysis system. The present invention has wide application prospects in the fields of biological analysis, organic synthesis, bioengineering, clinical mass spectrometry, single cell analysis, trace complex sample analysis, multi-omics analysis, bioimaging analysis and the like.

Description

A sampling probe device and analysis method based on membrane-assisted strategy Technical Field

[0001] The present invention relates to the fields of sample pretreatment and chromatographic analysis, and in particular to a membrane-assisted strategy-based sampling probe device and an analysis method with the function of pretreatment of trace complex samples. Background Art

[0002] Chromatographic analysis, with its powerful separation capabilities, has been widely used in biomedical, chemical, food, environmental, and other fields. When used for complex sample analysis, chromatographic analysis is often combined with sample pretreatment to further reduce matrix interference.

[0003] The process of applying sample pretreatment and chromatographic analysis technology to complex samples generally includes: (1) sample pretreatment, including filtration, centrifugation, enrichment and other operations to remove impurities or interferences in complex samples; (2) injection, introducing the treated sample into the injection valve in the form of liquid or gas; (3) separation, with the help of a pressure-driven system, using liquid, gas or supercritical fluid as the mobile phase, the components of different properties in the sample are separated in the chromatographic column; (4) detection, the separated components are detected in the detector to complete the analysis of the complex sample.

[0004] Currently, the most commonly used detector in conjunction with chromatographic analysis technology is mass spectrometry. Compared with other detection technologies, mass spectrometry has the characteristics of high sensitivity, fast analysis speed, and the ability to analyze molecular structure information, and has been widely used. Although mass spectrometry has high sensitivity and can be directly used for the detection of simple samples, this method is not suitable for the analysis of complex samples because the actual biological samples have complex components and large differences in abundance, and there are serious matrix effects that interfere with the accuracy and sensitivity of mass spectrometry detection. Therefore, when faced with complex samples, it is often necessary to pretreat and chromatographically separate the samples before mass spectrometry detection. However, current sample pretreatment methods are mostly targeted at routine sample analysis, and the operation is cumbersome. In addition, they involve steps such as sample transfer, which can easily cause sample loss and are not suitable for the analysis of trace complex samples.

[0005] Summary of the Invention

[0006] The present invention aims to provide a membrane-assisted sampling probe device and method for use in analyzing trace amounts of complex samples. The device employs a membrane-assisted strategy to achieve in-situ pretreatment, sampling, transmission, separation, and detection of trace amounts of complex samples under pressure.

[0007] At the same time, the present invention also discloses a method for performing sample analysis using the sampling probe device based on the membrane-assisted strategy.

[0008] A sampling probe device based on a membrane-assisted strategy, comprising:

[0009] a sample holding assembly for placing a sample;

[0010] An injection probe with a fluid introduction channel, a fluid outlet channel, and an injection channel port, wherein the injection channel port is connected to the fluid introduction channel and the fluid outlet channel, and when in use, the injection channel port is placed facing the sample;

[0011] a sealing fluid inlet pipeline disposed in the fluid introduction channel for introducing fluid;

[0012] An outlet fluid pipeline is provided in the fluid outlet channel or is sealed and docked with the fluid outlet channel, and the other end of the outlet fluid pipeline is connected to a sample detection device for analyzing the sample;

[0013] a fluid driving device connected to the inlet end of the inlet fluid pipeline for controlling the fluid;

[0014] A movable stage for adjusting the relative position of the sample holding assembly and the probe;

[0015] The sample holding component consists of a sample chip for placing samples and a membrane component covering the sample chip.

[0016] The fluid driving device is used to manipulate fluid; the fluid is various fluids required in the detection process, such as fluid for dissolving samples, mobile phase fluid for carrying and transporting samples, or fluid for specific chemical or physical reactions, etc., which can be determined according to actual detection needs.

[0017] The sample detection device is used to analyze samples. According to the present invention, the detection device includes but is not limited to an optical detector, an electrochemical detector, a mass spectrometer detector, or a combination of two or more of the above detectors.

[0018] The probe is used for sample injection. The injection capillary is used to introduce fluid into the probe; the capillary for introducing fluid is connected to the fluid drive device. The membrane is used to pretreat the sample. The movable stage is used to adjust the relative position of the probe and the sample chip. The movable stage can be connected to the probe to adjust the probe position, or it can be connected to the sample chip or sample support assembly to adjust the position of the sample support assembly.

[0019] According to the present invention, the probe has an integrated structural design, with an internal channel designed in a V-shaped, or approximately V-shaped, or T-shaped structure. The probe integrates a sealing interface, a fluid introduction channel, an injection channel port, and a fluid extraction channel. The fluid introduction channel is connected to a capillary tube that introduces fluid into the probe; the injection channel port is flanked by a fluid introduction channel and a fluid extraction channel; the fluid extraction channel is connected to a separation and analysis capillary tube; and the separation and analysis capillary tube is integrated with a detection device.

[0020] Furthermore, preferably, the fluid introduction channel and the fluid outlet channel are arranged in a V-shape, with their bottom ends butted against each other, and the injection channel port is provided at the butting position.

[0021] Alternatively, preferably, the fluid introduction channel and the fluid outlet channel are arranged in a T-shape, and the injection channel opening is provided at the bottom end of the fluid outlet channel; a liquid inlet gap is left between the bottom of the liquid outlet fluid pipeline and the inner wall of the fluid outlet channel; the liquid outlet of the liquid inlet fluid pipeline is connected to the liquid inlet gap to form a liquid inlet channel.

[0022] According to the present invention, the probe is made of a polymer material such as polyetheretherketone (PEEK), or a metal material such as stainless steel or titanium alloy, or an inorganic material such as glass, or an organic-inorganic composite material.

[0023] According to the present invention, the probe injection channel port is formed by the intersection of a fluid introduction channel and a fluid outlet channel, and the channel port shape is rectangular, elliptical, circular, etc. The major diameter of the channel port cross section is 1 micron to 1000 microns, and the minor diameter is 0.5 micron to 500 microns.

[0024] The present invention introduces a membrane-assisted strategy into sample analysis. This strategy uses a membrane as an intermediate medium. The sampling channel port of the probe contacts and applies a force to the membrane covering the sample chip. The force and the elasticity of the membrane are used to achieve sealing between the sampling probe and the sample chip in the device.

[0025] Preferably, the membrane is an elastic membrane.

[0026] According to the present invention, the membrane-assisted strategy is to use different types of membranes to realize in situ sample pretreatment operations in the device. These operations include but are not limited to filtration, solid phase extraction, liquid membrane extraction, affinity separation, dialysis, ultrafiltration, electrodialysis, reverse osmosis and other pretreatment operations.

[0027] According to the present invention, the auxiliary membrane material includes, but is not limited to, polymer membranes such as cellulose ester membrane (CE), regenerated cellulose membrane (RC), polyvinylidene fluoride membrane (PVDF), polytetrafluoroethylene (PTFE), or inorganic membranes such as porous glass membrane and glass fiber membrane, or organic membrane, or composite membranes of multiple materials (such as solid phase extraction membranes). The types of membranes include, but are not limited to, microporous membranes, filtration membranes, ultrafiltration membranes, nanofiltration membranes, reverse osmosis membranes, solid phase extraction membranes, etc.

[0028] According to the present invention, the surface of the membrane can be modified in various ways, including but not limited to loading titanium dioxide, magnetic microspheres, silica gel particles, molecular imprinting materials and other enrichment materials, and the membrane material or enrichment material can be modified by introducing polar groups such as hydroxyl, carboxyl, and nitrile groups, or introducing non-polar groups such as C8, C 18 Hydrophobic modifications such as hydrocarbon groups, halogen atoms, and nitro groups.

[0029] According to the present invention, the membrane is a porous or through-hole structure, and its pore size ranges from 1 nm to 10 μm, and its thickness ranges from 10 nm to 1 mm. The appropriate membrane can be selected according to the sample requirements. Preferably, to avoid lateral leakage of the sample through the membrane pores, a through-hole membrane should be used rather than a porous membrane. Preferably, the membrane is a through-hole structure, and its pore size ranges from 1 nm to 10 μm, and its thickness ranges from 10 nm to 1 mm.

[0030] According to the present invention, the sample chip can be made of inorganic materials such as glass and capillaries, or metal materials such as stainless steel or titanium alloy, or polymer materials such as PEEK, or other hard materials, or composite materials composed of the above materials.

[0031] According to the present invention, the sample chip can also be a thin layer chromatography plate based on a glass, plastic or other material substrate, and the types of adsorbents include but are not limited to silica gel, alumina, microcrystalline cellulose and other materials, and non-polar groups C2, C8, C 18 Hydrophobic modifications such as hydrocarbon groups, halogen atoms, and nitro groups.

[0032] According to the present invention, the surface of the sample chip for loading the sample can be flat, or can be a pit, a convex column or a channel. Preferably, the sample chip is provided with a sample micro-pit, and the sample is placed in the micro-pit during detection.

[0033] According to the present invention, the sample chip can be loaded with trace samples in the form of solid, liquid, solid-liquid coexistence, gas and supercritical fluid.

[0034] According to the present invention, the sample chip carries trace samples such as single cells or trace cell samples, biological fluid samples, biological tissue slice samples, organic synthesis reaction liquid, bioengineering fermentation liquid, or other trace samples. A membrane-assisted strategy can be used to perform in-situ sample pretreatment operations on the chip as needed, thereby reducing losses caused by transfer and achieving sample injection.

[0035] According to the present invention, the fluid pipeline can be an empty capillary used only for fluid flow, or a capillary with a fixed liquid modified on the inner surface, or a capillary chromatographic column of a monolithic column, or a capillary chromatographic column filled with a stationary phase, or a filled capillary, etc., based on various modifications of capillaries, and pipelines made of hard materials such as stainless steel tubes.

[0036] Furthermore, the inlet fluid line is a hollow capillary tube or a capillary tube with a stationary liquid modified on its inner surface; the outlet fluid line is a hollow capillary tube or a capillary chromatographic column. The capillary chromatographic column can be a packed capillary column, an open capillary column, or a monolithic capillary column. When a capillary chromatographic column is used, one end of the capillary chromatographic column is inserted into the fluid outlet channel, aligned with the inlet channel opening, and sealed to the fluid outlet channel, while the other end is sealedly connected to the sample detection device.

[0037] Alternatively, the sample components can be separated and analyzed directly through the fluid extraction channel on the injection probe, rather than using a separation and analysis capillary. This can be accomplished by filling or forming a chromatographic stationary phase within the fluid extraction channel, modifying the surface of the fluid extraction channel to impart separation capabilities, or directly using the fluid extraction channel as a sample transport channel to transfer the sample to a detector for detection.

[0038] The present invention uses a membrane-assisted strategy to use a sampling probe device. By utilizing the properties of the membrane material (such as elasticity), the membrane is sandwiched between the sampling probe and the sample chip to achieve sealing between the sampling channel of the sampling probe and the sample chip.

[0039] The present invention utilizes a fluid-driven mechanism to propel fluid within the sealed device. Fluid flows from the probe's fluid inlet channel through the injection port, passes through the membrane pores, and contacts the sample carried on the sample chip. The fluid then exits the probe through the fluid outlet channel. This process utilizes the interaction between the fluid, sample, and membrane to achieve in-situ sample pretreatment and subsequent sample injection.

[0040] As an implementable embodiment, the membrane has the pretreatment function of in situ filtration of samples. The samples can be carried by fluid to complete the filtration operation based on the size exclusion principle (such as microporous membrane, ultrafiltration membrane or dialysis membrane filtration) and the subsequent injection operation of the filtered samples.

[0041] As another possible implementation plan, the membrane has a pretreatment function for in situ enrichment of samples. The sample can be carried by the fluid to complete the sample enrichment (such as solid phase extraction, liquid membrane extraction, affinity separation) operation in which specific components in the sample selectively bind to the membrane surface; thereafter, the enriched sample on the membrane is eluted by the elution fluid (whose composition is different from the fluid composition during enrichment) and is carried by the elution fluid to complete the injection operation of the eluted sample.

[0042] The present invention utilizes fluid to transfer the sample after membrane pretreatment to the fluid outlet channel and the capillary chromatographic column connected thereto to realize the chromatographic injection operation of the sample; utilizes a fluid driving device to drive the mobile phase fluid to separate the sample loaded on the fixed phase of the chromatographic column; and utilizes the detection device to detect the sample components after separation.

[0043] In the present invention, the membrane has two main functions: one is to utilize the properties of the membrane material itself (such as elastic properties) to achieve a seal between the sampling probe and the sample chip; the other is that the membrane itself has a porous nature, so that the fluid can flow through the membrane pores and connect the sampling channel port of the sampling probe with the sample on the sample chip, thereby completing a variety of sample pretreatment functions, including but not limited to filtration, solid phase extraction, liquid membrane extraction, affinity separation, dialysis, ultrafiltration, electrodialysis, reverse osmosis and other pretreatment operations.

[0044] A method for performing sample analysis using a sampling probe device based on a membrane-assisted strategy as described in any of the above technical solutions, comprising: using a fluid driving device to drive the fluid in the device when the device is in a sealed state, the fluid flows through the sampling channel port from the probe's fluid introduction channel, the membrane completes in-situ pretreatment of the sample, the pretreated sample contacts the sample carried on the sample chip through the membrane pores, and then the fluid flows out of the probe through the fluid introduction channel, and the sample analysis is achieved using a capillary chromatographic column and a sample detection device.

[0045] Preferably, the method for performing sample analysis using the sampling probe device based on the membrane-assisted strategy comprises the following steps:

[0046] (1) Place the sample on the sample chip and cover the sample chip with a membrane;

[0047] (2) Control the probe's injection channel port to contact the membrane and align with the sample, and apply a squeezing (or pressing) force to form a "sandwich" membrane structure of the injection channel port-membrane-sample to achieve device sealing;

[0048] (3) starting the fluid driving device, introducing the fluid through the capillary and carrying the sample through the membrane pores, and using the membrane to complete the in-situ pretreatment of the sample, and loading the in-situ pretreated sample onto the capillary chromatographic column to complete the injection operation, using the fluid driving device to introduce the mobile phase fluid, and performing isocratic or gradient elution separation and detection on the sample loaded onto the capillary chromatographic column;

[0049] Optionally, steps (1) to (3) may be repeated to complete the analysis of different samples or the analysis of different locations of the same sample.

[0050] Furthermore, the method for using the sampling probe device based on the membrane-assisted strategy comprises the following steps:

[0051] (a) Loading the sample onto the sample chip;

[0052] (b) Fixing the sample chip on the moving stage;

[0053] (c) covering the membrane on the sample chip;

[0054] (d) controlling the movable stage to bring the sampling channel port of the probe into contact with the membrane, and applying a squeezing (or pressing) force to form a "sandwich" structure of the sampling channel port, the membrane, and the sample, thereby achieving sealing of the device;

[0055] (e) starting the fluid drive device, introducing fluid to carry the sample through the membrane pores, completing an in-situ filtration operation of the sample, and loading the filtered sample onto the capillary chromatographic column to complete the injection operation, introducing a mobile phase fluid using the fluid drive device, and performing isocratic or gradient elution separation and detection on the sample loaded onto the capillary chromatographic column;

[0056] (f) Repeating steps (a) to (e) can complete the analysis of different samples on the sample chip, or the analysis of samples on different sample chips; or the analysis of different position areas of the same sample on the sample chip (spatial imaging analysis).

[0057] As another embodiment, another method for using the sampling probe device based on the membrane-assisted strategy comprises the following steps:

[0058] (a) Loading the sample onto the sample chip;

[0059] (b) Fixing the sample chip on the moving stage;

[0060] (c) covering the membrane on the sample chip;

[0061] (d) controlling the movable stage to bring the sampling channel port of the probe into contact with the membrane, and applying a squeezing (or pressing) force to form a "sandwich" structure of the sampling channel port, the membrane, and the sample, thereby achieving sealing of the device;

[0062] (e) starting the fluid driving device to introduce fluid to carry the sample through the membrane pores, thereby completing an in-situ enrichment operation of the sample on the membrane; introducing an elution fluid using the fluid driving device to elute the sample enriched on the membrane and load it onto a capillary chromatographic column to complete the sample injection operation; introducing a mobile phase fluid using the fluid driving device to perform isocratic or gradient elution separation and detection on the sample loaded on the capillary chromatographic column;

[0063] (f) Repeating steps (a) to (e) can complete the analysis of different samples on the sample chip, or the analysis of samples on different sample chips; or the analysis of different position areas of the same sample on the sample chip (spatial imaging analysis).

[0064] As another implementation scheme, a sampling probe device based on a membrane-assisted strategy is different from the above-mentioned technical scheme. The sampling probe is replaced by a separate liquid inlet fluid tube and liquid outlet fluid tube; when in use, the liquid inlet fluid tube and the liquid outlet fluid tube are respectively arranged on both sides of the sample-carrying component, and the outlet end of the liquid inlet fluid tube and the inlet end of the liquid outlet fluid tube are arranged facing the sample, and are sealed and docked with the sample chip side and the membrane side of the sample-carrying component respectively.

[0065] Furthermore, a sampling probe device based on a membrane-assisted strategy comprises:

[0066] a sample holding assembly for placing a sample;

[0067] an inlet fluid line and an outlet fluid line for introducing fluid;

[0068] a sample detection device connected to the outlet end of the liquid outlet fluid pipeline for analyzing the sample;

[0069] a fluid driving device connected to the inlet end of the inlet fluid pipeline for controlling the fluid;

[0070] The sample holding assembly comprises a sample chip for placing the sample and a film covering the sample chip;

[0071] During detection, the outlet end of the inlet fluid pipeline and the inlet end of the outlet fluid pipeline are sealed and docked with the sample chip side and the membrane side of the sample supporting component respectively.

[0072] In the above technical solution, a through-hole membrane or a porous membrane is provided on the sample chip, and the sample is pre-loaded on the through-hole membrane or the porous membrane structure during detection.

[0073] When the above technical solution is used for sample analysis, the following steps are included:

[0074] (1) Fix the sample in the corresponding position of the sample holding assembly, align the inlet fluid line and the outlet fluid line up and down, and squeeze the middle sample holding assembly to achieve a seal;

[0075] (2) starting the fluid driving device, introducing fluid to carry the sample through the membrane pores, and completing the in-situ enrichment operation of the sample on the membrane; using the fluid driving device to introduce the elution fluid to elute the sample enriched on the membrane and using the outlet fluid pipeline to complete the sample injection operation; when the outlet fluid pipeline adopts a capillary chromatographic column, using the fluid driving device to introduce the mobile phase fluid, and perform isocratic or gradient elution separation and detection on the sample loaded on the capillary chromatographic column;

[0076] Alternatively, the fluid driving device is activated to introduce fluid to carry the sample through the membrane pores to complete the in-situ filtration operation of the sample, and the filtered sample is injected through the outlet fluid pipeline to complete the injection operation, and the mobile phase fluid is introduced through the fluid driving device; when the outlet fluid pipeline adopts a capillary chromatographic column, the sample loaded on the capillary chromatographic column is separated and detected by isocratic or gradient elution;

[0077] By repeating the above steps, the analysis of different samples on the sample chip, or the analysis of samples on different sample chips, or the analysis of different position areas of the same sample on the sample chip (spatial imaging analysis) can be completed.

[0078] During filtration, the different pore sizes of the membrane are utilized to intercept insoluble substances larger than the pore size, thus achieving sample filtration. During enrichment, the different polarities of the samples are utilized. As the sample passes through the membrane, some components will be adsorbed onto the membrane surface due to interaction forces, while other insoluble components will be retained underneath the membrane. The remaining soluble components in the sample will pass through the membrane. The retained insoluble components and the soluble components that pass through are removed, and then the components adsorbed onto the membrane surface are eluted with an eluent. Finally, the eluted target components are loaded into the chromatographic column, completing the entire enrichment and elution operation.

[0079] When a membrane is used for in-situ enrichment of a sample, different injection probes can be used to respectively complete the enrichment of the sample on the membrane and the elution and separation of the sample enriched on the membrane.

[0080] According to the present invention, the method for achieving device sealing by contacting the sampling channel port of the probe with the membrane is to control the moving stage to form a "sandwich" structure of probe-membrane-sample. By utilizing the elasticity of the membrane, the pressure required for sealing is borne by the membrane, and the device sealing without obvious liquid leakage under a hydraulic pressure exceeding 60MPa can be achieved.

[0081] According to the present invention, the relative movement of the sampling probe and the sample chip relative to the membrane provides the sealing pressure of the device; the sample chip can be fixed on the mobile stage, or the sampling probe is fixed on the mobile stage, or the sample chip and the sampling probe are fixed on different mobile stages respectively.

[0082] According to the present invention, the membrane-assisted strategy uses a membrane to isolate the probe from direct contact with the sample, eliminating the probe cleaning step and simplifying the operation steps.

[0083] According to the present invention, the portion of the membrane that contacts the sample is disposable. That is, after the membrane contacts the sample for sample pretreatment, the portion of the membrane that contacts the sample is discarded. For new samples, a different membrane is used, or another area of ​​the membrane that has not contacted the sample is used for sample pretreatment.

[0084] According to the present invention, a porous support sheet or membrane is installed between the sample chip and the membrane to improve the strength of the membrane and prevent the membrane from deforming toward the sample chip.

[0085] According to the present invention, in order to improve the utilization efficiency of the membrane and increase the processing flux, different samples can be pretreated on different areas of a membrane.

[0086] According to the present invention, the sample pretreatment and sample injection operations of the probe can be performed sequentially and integrated, or separately. When separate procedures are employed, the sample pretreatment operation is first performed using the probe and membrane, and then the membrane is removed and stored. At the desired time, the membrane is then combined with the sample injection probe for sample injection. In this method, the probe used for sample pretreatment and the probe used for sample injection can be the same probe or different probes.

[0087] According to the present invention, a switching valve is added to the device to achieve simultaneous injection and chromatographic separation of different samples, thereby reducing the time of the entire analysis process and improving the analysis throughput.

[0088] According to the present invention, the mobile stage can move in three dimensions, and can realize arrayed sampling and imaging analysis.

[0089] It should be noted that the application field of the sampling probe device based on the membrane-assisted strategy is not limited to chromatographic analysis, but can also be applied to other flow analysis fields, including but not limited to continuous flow analysis, flow injection analysis, microfluidic analysis, capillary electrophoresis analysis, etc.

[0090] The main advantages of this invention include the integration of trace sample pretreatment with injection, chromatographic separation, and detection systems. This system offers a simple structure, rapid and automated operation, high flexibility, ease of use, and wide applicability, making it particularly suitable for the analysis of trace samples. The membrane-assisted strategy allows for in-situ sample pretreatment followed by injection, providing a new injection mode for the analysis of complex samples and a novel approach to the integration and miniaturization of chromatographic analysis systems.

[0091] The present invention has broad application prospects in the fields of biological analysis, organic synthesis, bioengineering, clinical mass spectrometry, single-cell analysis, trace complex sample analysis, multi-omics analysis, biological imaging analysis, etc.

[0092] Compared with the prior art, the main advantages of the present invention are:

[0093] (1) The system integrates trace sample pretreatment with sample injection, chromatographic separation, and detection systems. The system has a simple structure, fast and automatic operation, high flexibility, ease of use, and wide applicability, making it particularly suitable for the analysis of trace samples.

[0094] (2) The membrane-assisted strategy can realize in-situ pretreatment of samples before injection, providing a new injection mode for the analysis of complex samples and a new approach for the integration and miniaturization of chromatographic analysis systems;

[0095] (3) The membrane-assisted strategy is highly flexible, and the membrane used can be replaced according to the needs of pretreatment;

[0096] (4) The entire device has a modular design, flexible assembly, and is easy to use. The hard material used for the probe can be reused and can be operated automatically.

[0097] (5) In-situ pretreatment can be performed on the sample chip, reducing the loss of sample transfer and even achieving non-destructive sample pretreatment. Moreover, since the probe does not directly contact the sample, the cleaning steps are reduced, making it suitable for the analysis of trace samples.

[0098] (6) Membrane-assisted strategies can achieve different modes of in situ pretreatment, including but not limited to filtration, solid phase extraction, liquid membrane extraction, affinity separation, dialysis, ultrafiltration, electrodialysis, reverse osmosis and other pretreatment operations, reducing the complexity of the sample and enabling segmented analysis of the sample;

[0099] (7) The device has broad application prospects and can be applied to organic synthesis, bioengineering, clinical mass spectrometry, trace complex sample analysis, single cell analysis, multi-omics analysis, biological imaging analysis, on-site rapid analysis and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 is a schematic diagram of a V-shaped sampling probe device based on a membrane-assisted strategy according to Example 1. In the figure, 1 - fluid driving device, 2 - capillary for introducing fluid, 3 - sampling probe, 4 - interface of the sealing channel, 5 - fluid introduction channel, 6 - fluid outlet channel, 7 - sampling channel port, 8 - sample, 9 - membrane, 10 - sample chip, 11 - moving stage, 12 - capillary chromatographic column, 13 - fluid, 14 - elution fluid, 15 - mobile phase fluid, and 16 - electrospray mass spectrometer detection device.

[0101] FIG2 shows the chromatographic analysis results of a dry-spot analysis of a mixed sample of three peptide segments in an isocratic elution mode using the apparatus and its use method of Example 1.

[0102] FIG3 shows the results of chromatographic analysis of the product of trypsin hydrolysis of protein cytochrome C in gradient elution mode using the device and its use method of Example 1.

[0103] Figure 4 illustrates the device and method of use of Example 1 for in situ sample pretreatment and filtration prior to sample injection in Example 2. (a) Mass spectrometry analysis of protein cytochrome C without a membrane-assisted strategy; (b) Chromatographic analysis of protein cytochrome C using a membrane-assisted strategy; (c) In situ trypsin digestion of the sample after injection, followed by analysis of the resulting peptide fragments using a membrane-assisted strategy. The mass spectrometry result for one of the resulting peptide fragments is shown, with * indicating the peptide fragment signal.

[0104] Figure 5 is a schematic diagram of a T-shaped sampling probe device combined with a solid-phase extraction membrane according to Example 3. In the figure, 1 - fluid driving device, 2 - capillary for introducing fluid, 3 - sampling probe, 4 - interface of the sealing channel, 5 - fluid introduction channel, 6 - fluid outlet channel, 7 - sampling channel port, 8 - sample, 9 - membrane, 10 - sample chip, 11 - moving stage, 12 - capillary chromatographic column, 13 - fluid, 14 - elution fluid, 15 - mobile phase fluid, 16 - electrospray mass spectrometer detection device, 17 - flow guide capillary, 18 - switching valve, 19 - sampling gap.

[0105] FIG6 is a partial enlarged view of the probe in FIG5 .

[0106] Figure 7 is a schematic diagram of the sample injection process of the array sampling probe device combined with a solid phase extraction membrane according to Example 4. In the figure, 3 - sampling probe, 9 - membrane with solid phase extraction function, 10 - sample chip with microwells, 11 - moving stage, 15 - mobile phase fluid, 21 - sample microwell No. 1, 22 - sample microwell No. 2, and 23 - sampling probe No. 2.

[0107] Figure 8 is a schematic diagram of the channel sample extraction and injection mode combined with a solid-phase extraction membrane chip for extraction and injection of a dried blood spot sample according to Example 5. In the figure, 2 - capillary through which fluid passes, 8 - dried blood spot sample, 9 - membrane with solid-phase extraction function, 10 - sample chip with dried blood spot sample, and 13 - fluid. DETAILED DESCRIPTION

[0108] The technical solution of the present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0109] With reference to the accompanying drawings, preferred embodiments according to the present invention will be described in detail below.

[0110] Example 1

[0111] Figure 1 is a schematic diagram of a membrane-assisted sampling probe device according to Example 1. The system comprises a fluid drive device 1, a V-shaped sampling probe 3 made of PEEK, a capillary 2 for introducing the mobile phase, an RC membrane 9, a sample chip 10, a moving stage 11 for moving the sample chip, a capillary chromatographic column 12, and an electrospray mass spectrometer detection device 16.

[0112] The injection probe 3 is made of PEEK material and includes a sealing channel interface 4, a fluid introduction channel 5, a fluid outlet channel 6, and an injection channel port 7. The fluid introduction channel 5 is provided with a capillary 2 for introducing the mobile phase, and the fluid outlet channel is provided with a capillary chromatographic column 12. Both the capillary 2 and the capillary chromatographic column 12 can be sealed with the corresponding fluid introduction channel and fluid outlet channel using conical seals; the injection channel port 7 is provided at the bottom of the probe, with the fluid introduction channel 5 and the fluid outlet channel 6 on both sides, that is, the position where the bottom ends of the fluid introduction channel 5 and the fluid outlet channel 6 are connected, and the injection channel port 7 is provided. The inlet of the fluid introduction channel 5 and the outlet of the fluid outlet channel 6 are both provided with the interface 4, which respectively realizes a sealed connection with the fluid drive device 1 and the electrospray mass spectrometer detection device 16.

[0113] The specific method of using the device of Example 1 is as follows: (1) The sample chip 10 is fixed on the moving stage, and the membrane 9 is covered on the sample chip 10. By controlling the movement of the moving stage 11, the sampling channel port 7 of the sampling probe 3 is brought into contact with the membrane 9, and the sampling channel port 7 and the membrane 9 are sealed by the applied squeezing force to prevent the mobile phase from leaking from the sampling channel port 7; (2) In the sealed state, the fluid driving device 1 is turned on, and the chromatographic mobile phase is injected from the capillary 2 for introducing the mobile phase. After passing through the fluid introduction channel 5, the sampling channel port 7 and the membrane 9, the sample 8 is dissolved. After the in-situ pretreatment, the sample passes through the membrane 9 and the sampling channel port 7 in turn. The sample enters the capillary chromatographic column 12 through the fluid outlet channel 6, the mobile phase composition is changed to elute the sample, and an electrospray is formed under the action of the electric field, and enters the electrospray mass spectrometer detection device 16 for detection; (3) The fluid driving device 1 is paused, and the moving stage 11 is controlled to move to the next sample 8. The steps in (2) are repeated to achieve the analysis of different samples 8.

[0114] FIG2 shows the mass spectrometry analysis results of a mixture sample of three peptide segments in isocratic elution mode using the device and its use method of Example 1 and an RC membrane with a molecular weight cutoff of 2000.

[0115] Specifically, three peptide samples, GE-11, GF-9-NH2, and TL-9-NH2, were mixed at a concentration of 0.1 micromolar per liter to form Sample 8. 10 nL of Sample 8 was dropwise applied to Sample Chip 10 and allowed to dry naturally to form a dry spot. The sample was then isocratically eluted using a 30% acetonitrile aqueous solution (containing 0.1% formic acid) as the mobile phase. Analysis of Mixed Sample 8 was repeated three times, and the RSDs of the retention times of the three peptides were 0.6%, 1.0%, and 1.1%, respectively.

[0116] The results of this analysis show that the system can realize chromatographic analysis of trace samples and has good system reproducibility.

[0117] FIG3 shows the results of mass spectrometry analysis of the product of trypsin hydrolysis of protein cytochrome C in gradient elution mode using the device and its use method of Example 1.

[0118] Specifically, the product of enzymatic hydrolysis of protein cytochrome C in a conventional system was added as sample 8 onto sample chip 10. Sample 8 was analyzed using this system using the following chromatographic gradient: 0% Solution B (the remaining 100% was Solution A) from 0 to 10 minutes; 15% Solution B (the remaining 85% was Solution A) from 10 to 20 minutes; 30% Solution B (the remaining 70% was Solution A) from 20 to 35 minutes; and 80% Solution B (the remaining 20% ​​was Solution A) from 35 to 40 minutes. Solution A consisted of 0.1% formic acid in water, and Solution B consisted of 0.1% formic acid in acetonitrile.

[0119] The results of this analysis demonstrate that the system can be used to analyze trace amounts of complex samples.

[0120] Example 2

[0121] Figure 4 shows the mass spectrometry analysis results of protein cytochrome C using the device and method of use of Example 1 in Example 2 (a) without using a membrane-assisted strategy; (b) using a membrane-assisted strategy, the chromatographic analysis results of protein cytochrome C; (c) using a membrane-assisted strategy, in situ trypsin hydrolysis of the sample after injection, and mass spectrometry detection of the product, including the mass spectrometry detection result of one of the hydrolysis products.

[0122] Specifically, in (a), a system without a membrane-assisted strategy (i.e., the sample was not covered with a membrane, and the injection channel was in direct contact with the sample) was used to analyze a protein cytochrome C sample dripped onto the sample chip, resulting in a mass spectrum of protein cytochrome C. In (b), a system using a membrane-assisted strategy (i.e., the apparatus and method of use in Example 1) was used to analyze a protein cytochrome C sample dripped onto the sample chip, but no protein cytochrome C signal was detected. In (c), the sample injected in (b) was in situ enzymatically digested using trypsin on the sample chip, and the reaction products were analyzed using a system using a membrane-assisted strategy (i.e., the apparatus and method of use in Example 1).

[0123] The results in Figure 4(a) prove that protein cytochrome C can be detected by the electrospray mass spectrometry detection device; the results in Figure 4(b) show that the pore size of the selected membrane is smaller than the size of protein cytochrome C. Under membrane-assisted conditions, protein cytochrome C is blocked by the membrane and cannot pass through the membrane and cannot be detected by the detection device; the results in Figure 4(c) show that after the original protein cytochrome C is enzymatically hydrolyzed into peptide samples and then analyzed, the sample can pass through the membrane and be identified by the detection device, indicating that the system can achieve in situ sample injection based on size exclusion filtration by selecting an appropriate membrane.

[0124] The results of this analysis demonstrate that the system uses a membrane-assisted strategy to block proteins larger than the membrane pore size, while allowing the peptides obtained after enzymatic hydrolysis to pass through, demonstrating its suitability for the analysis of trace complex samples.

[0125] Example 3

[0126] Figure 5 is a schematic diagram of a T-type sampling probe device combined with a solid-phase extraction membrane according to Example 3. The system comprises a fluid drive device 1, a switching valve 18, a flow-guiding capillary 17, a capillary 2 for introducing the mobile phase, a T-type sampling probe 3, a connector 4 with an integrated sealing channel within the probe, a fluid introduction channel 5, a fluid outlet channel 6, an injection channel port 7, a solid-phase extraction membrane 9, a sample chip 10, a movable stage 11 for moving the sample chip, a capillary chromatographic column 12, and an electrospray mass spectrometer detection device 16.

[0127] The T-shaped injection probe 3 is made of stainless steel and includes a sealing channel interface 4, a fluid introduction channel 5, and an injection channel port 7. The fluid introduction channel 5 houses the capillary 2 for introducing the mobile phase, while the fluid outlet channel 6 houses the capillary chromatographic column 12. The injection channel port 7 is located at the bottom of the probe, with the fluid introduction channel 5 located above it.

[0128] The capillary tube 2 and the capillary chromatographic column 12 are sealed and fixed in the fluid introduction channel 5 and the fluid outlet channel 6, respectively, by means of a conical seal 20. Furthermore, in the fluid introduction channel 5, the conical seal 20 is used to ensure that the capillary tube 2 is only connected to the liquid inlet gap 19 in the fluid outlet channel 6, while being sealed to the inner wall of the fluid introduction channel 5 to prevent fluid from entering the gap between the two. In the fluid outlet channel 6, an injection gap 19 is left between the outer wall of the bottom of the capillary chromatographic column 12 and the inner wall of the fluid outlet channel 6. This injection gap 19 is connected to the capillary tube 2 to enable injection. At the same time, the portion of the capillary chromatographic column 12 located above the injection gap 19 is sealed to the inner wall of the fluid outlet channel 6 by means of a conical seal 20 to prevent fluid from entering the gap formed between the two.

[0129] 6 , the specific method of using the device of Example 3 is as follows: (1) The sample chip 10 is fixed on the moving stage, and the solid phase extraction membrane 9 is covered on the sample chip 10. By controlling the movement of the moving stage 11, the injection channel port 7 is aligned with the sample position on the sample chip 10, and the injection channel port 7 of the injection probe 3 is brought into contact with the membrane 9 and sealed by the applied squeezing force, thereby preventing the mobile phase from leaking from the injection channel port 7; (2) In the sealed state, the fluid driving device 1 is opened, and the switching valve 18 is controlled so that the chromatographic mobile phase is introduced into the flow channel 7. The mobile phase is injected into the capillary 2, and the sample 8 is dissolved and brought to the membrane 9 after passing through the fluid introduction channel 5, the liquid inlet gap 19, the injection channel port 7 and the membrane 9; (3) the composition of the mobile phase is changed, the sample 8 is eluted from the membrane 9, and is transmitted through the capillary chromatographic column 12, where an electrospray is formed under the action of the electric field and enters the electrospray mass spectrometer detection device 16 for detection; (4) the switching valve 18 is controlled to make the mobile phase flow out of the guide capillary 17, and the moving stage 11 is controlled to move to the next sample 8. The steps (2) to (3) are repeated to realize the analysis of different samples 8.

[0130] Example 4

[0131] Figure 7 is a schematic diagram of the sample injection process of the array sampling probe device combined with the solid phase extraction membrane according to Example 4. In the figure, 11 is the mobile stage, 10 is the sample chip with micro-wells, 9 is the solid phase extraction membrane, 21 is the sample micro-well No. 1, 3 is the sampling probe No. 1, 15 is the mobile phase fluid, 22 is the sample micro-well No. 2, 23 is the sampling probe No. 2, and 14 is the elution fluid.

[0132] The array sampling probe device uses two sampling probes for loading the sample onto the solid phase extraction membrane and eluting the sample from the solid phase extraction membrane respectively. Compared with Example 3, it realizes in situ sample pretreatment and detection in parallel, reducing analysis time.

[0133] The specific method of using the array sampling probe device of Example 4 is as follows: (1) The sample chip 10 is fixed on the moving platform 11, and the solid phase extraction membrane 9 is covered on the sample chip 10. By controlling the movement of the moving platform 11, the No. 1 sampling probe 3 is brought into contact with the solid phase extraction membrane 9 above the No. 1 sample micro-pit 21 and sealed; (2) In the sealed state, the mobile phase fluid 15 is introduced, passes through the solid phase extraction membrane 9, enters the No. 1 sample micro-pit 21, and the sample is loaded onto the solid phase extraction membrane 9; (3) The No. 1 sampling probe 3 is moved to the solid phase extraction membrane 9 above the No. 2 sample micro-pit 22 to contact and seal, and at the same time move the No. 2 sampling probe 23 to the solid phase extraction membrane 9 above the No. 1 sample micro-pit to contact and seal; (4) in the sealed state, introduce the mobile phase fluid 6 into the No. 1 sampling probe 3, pass through the solid phase extraction membrane 9, enter the No. 2 sample micro-pit 23, and load the sample onto the solid phase extraction membrane 9; at the same time, introduce the elution fluid 14 into the No. 2 sampling probe 23 to elute the sample on the solid phase extraction membrane 9 above the No. 1 sample micro-pit 21 for subsequent analysis; (5) repeat the steps in (4) to achieve arrayed sample injection and parallel analysis combined with the solid phase extraction membrane.

[0134] Example 5

[0135] Figure 8 is a schematic diagram of the channel injection mode combined with the solid phase extraction membrane for dried blood spot injection in Example 5. In the figure, 2 is the capillary for introducing fluid, 13 is the fluid, 8 is the dried blood spot sample, 10 is the sample chip with the dried blood spot, and 9 is the solid phase extraction membrane.

[0136] This injection mode adopts the membrane-assisted strategy and combines it with the solid-phase extraction membrane to realize the in-situ pretreatment and injection of samples on the dried blood spot filter paper. Unlike the above-mentioned Examples 1-4, Example 5 does not use the method of integrating the injection probe to complete the sample pretreatment and injection operations, but instead uses a method of performing sample pretreatment and injection operations separately.

[0137] The specific method of dried blood spot sampling in Example 5 is as follows: (1) The paper chip 10 with the dried blood spot sample (the blood sample is loaded in the mesh structure of the filter paper on the paper chip) is covered on the solid phase extraction membrane 9, the two capillaries 2 for introducing fluid are aligned up and down, and the sample chip 10 and the solid phase extraction membrane 9 in the middle are squeezed to achieve sealing; (2) The fluid 13 is introduced to dissolve the dried blood spot sample 8 and flow downward. When passing through the solid phase extraction membrane 9, the target substance in the dried blood spot sample 8 is adsorbed by the solid phase extraction membrane 9, and other substances pass through the solid phase extraction membrane 9 to complete the enrichment of the sample; (3) The solid phase extraction membrane enriched with the sample is removed and combined with the sampling probe. Using a method similar to that of Example 3, the sample target substance adsorbed on the solid phase extraction membrane 9 is eluted, sampled, chromatographically separated and detected.

Claims

1. A sampling probe device based on a membrane-assisted strategy, characterized in that: include: a sample holding assembly for placing a sample; An injection probe with a fluid introduction channel, a fluid outlet channel and an injection channel port, wherein the injection channel port is connected to the fluid introduction channel and the fluid outlet channel, and the injection channel port is arranged facing the sample when in use; A sealing fluid inlet pipeline disposed in the fluid introduction channel for introducing fluid; An outlet fluid pipeline is arranged in the fluid outlet channel or is sealed and connected to the fluid outlet channel, and the other end of the outlet fluid pipeline is connected to a sample detection device for analyzing the sample; A fluid driving device connected to the inlet end of the inlet fluid pipeline for controlling the fluid; A moving stage for adjusting the relative position of the sample holding assembly and the probe; The sample holding component consists of a sample chip for placing the sample and a film covering the sample chip.

2. The sampling probe device based on the membrane-assisted strategy according to claim 1, characterized in that: The fluid introduction channel and the fluid outlet channel are arranged in a V shape, the bottom ends of the two are butted, and the injection channel opening is arranged at the butted position.

3. The sampling probe device based on the membrane-assisted strategy according to claim 1, characterized in that: The fluid introduction channel and the fluid outlet channel are arranged in a T shape, and the injection channel opening is provided at the bottom end of the fluid outlet channel; a liquid inlet gap is left between the bottom of the liquid outlet fluid pipeline and the inner wall of the fluid outlet channel; the liquid outlet of the liquid inlet fluid pipeline is connected to the liquid inlet gap to form a liquid inlet channel.

4. The sampling probe device based on membrane-assisted strategy according to claim 1, characterized in that: The membrane is an elastic membrane; the membrane is a through-pore structure with a pore size ranging from 1 nanometer to 10 micrometers and a thickness of 10 nanometers to 1 millimeter; the membrane is a functional membrane having one or more functions of filtration, solid phase extraction, liquid membrane extraction, affinity separation, dialysis, ultrafiltration, electrodialysis, and reverse osmosis; the membrane is a cellulose ester membrane, a regenerated cellulose membrane, a polyvinylidene fluoride membrane, polytetrafluoroethylene, a porous glass membrane, a glass fiber membrane, or a solid phase extraction membrane.

5. The sampling probe device based on membrane-assisted strategy according to claim 1, characterized in that: The inlet fluid pipeline is an empty capillary, or a capillary with a fixed liquid modified on the inner surface; the outlet fluid pipeline is an empty capillary or a capillary chromatographic column; or the fluid outlet channel is filled with or forms a chromatographic fixed phase, or the surface of the fluid outlet channel is modified to give it a separation function.

6. The sampling probe device based on the membrane-assisted strategy according to claim 1, characterized in that: The sample chip is provided with a sample carrying area, which is in the shape of a micro-pit, or a plane, or a convex column. During detection, the sample is placed in the sample carrying area.

7. The sampling probe device based on the membrane-assisted strategy according to any one of claims 1.4.5.6, characterized in that: The injection probe is replaced by a separate inlet fluid tube and outlet fluid tube; when in use, the inlet fluid tube and outlet fluid tube are respectively arranged on both sides of the sample-carrying component, and the outlet end of the inlet fluid tube and the inlet end of the outlet fluid tube are arranged facing the sample, and are respectively corresponding to the sample chip side and the membrane side of the sample-carrying component.

8. A method for sample analysis using the sampling probe device based on the membrane-assisted strategy according to any one of claims 1 to 6, characterized in that: include: A fluid driving device is used to drive the fluid in the device in a sealed state. The fluid is introduced into the probe through the injection channel, and the membrane completes the in-situ pretreatment of the sample. The pretreated sample contacts the sample carried on the sample chip through the membrane pores. Then the fluid flows out of the probe through the fluid outlet channel, and the sample is analyzed using a sample detection device.

9. The method for performing sample analysis using a sampling probe device based on a membrane-assisted strategy according to claim 8, characterized in that: The steps include: (1) placing a sample on a sample chip and covering the sample chip with a membrane; (2) Controlling the injection channel port of the probe to contact the membrane and align with the sample, and applying a squeezing force to form a "sandwich" membrane structure of the injection channel port-membrane-sample to achieve device sealing; (3) starting the fluid driving device, introducing the fluid through the inlet fluid pipeline and carrying the sample through the membrane pores, and completing the filtration operation of the sample by using the membrane, and loading the filtered sample onto the capillary chromatographic column to complete the injection operation, and introducing the mobile phase fluid by using the fluid driving device, and performing isocratic or gradient elution separation and detection on the sample loaded onto the capillary chromatographic column; Optionally, steps (1) to (3) may be repeated to complete the analysis of different samples, or the analysis of different locations of the same sample.

10. The method for performing sample analysis using a sampling probe device based on a membrane-assisted strategy according to claim 8, characterized in that: The steps include: (1) placing a sample on a sample chip and covering the sample chip with a membrane; (2) Controlling the injection channel port of the probe to contact the membrane and align with the sample, and applying a squeezing force to form a "sandwich" membrane structure of the injection channel port-membrane-sample to achieve device sealing; (3) starting the fluid driving device, introducing the fluid through the inlet fluid pipeline and carrying the sample through the membrane pores, and completing the sample enrichment operation by using the membrane, introducing the elution fluid by using the fluid driving device to elute the sample enriched on the membrane and load it onto the capillary chromatographic column to complete the sample injection operation, and introducing the mobile phase fluid by using the fluid driving device to perform isocratic or gradient elution separation and detection on the sample loaded onto the capillary chromatographic column; Optionally, steps (1) to (3) may be repeated to complete the analysis of different samples, or the analysis of different locations of the same sample.

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