A sample injection probe device and analysis method based on a membrane auxiliary strategy

Abstract

This invention discloses a sample introduction probe device and method based on a membrane-assisted strategy. The device employs a membrane-assisted strategy to achieve in-situ pretreatment, injection, transfer, separation, and detection of trace amounts of complex samples under pressure. The main advantages of this invention include: integrating trace sample pretreatment with injection, chromatographic separation, and detection systems; a simple system structure; rapid and automated operation; high flexibility; ease of use; and wide applicability, particularly suitable for the analysis of trace samples. The membrane-assisted strategy enables in-situ sample pretreatment followed by injection, providing a new injection mode for the analysis of complex samples and offering a new approach to the integration and miniaturization of chromatographic analysis systems. This invention has broad application prospects in fields such as bioanalysis, organic synthesis, bioengineering, clinical mass spectrometry, single-cell analysis, analysis of trace complex samples, multi-omics analysis, and bioimaging analysis.

Description

Technical Field

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

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

[0003] The process of applying sample pretreatment and chromatographic analysis techniques to complex samples generally includes: (1) Sample pretreatment, including operations such as filtration, centrifugation, and enrichment, to remove impurities or interferences from complex samples; (2) Injection, introducing the pretreated sample into the injection valve in liquid or gas form; (3) Separation, using a pressure-driven system with liquid, gas, or supercritical fluid as the mobile phase, to separate components of different properties in the sample within the chromatographic column; (4) Detection, the separated components are detected in the detector, completing the analysis of the complex sample.

[0004] Currently, mass spectrometry is the most commonly used detector coupled with chromatographic analysis. Compared to other detection techniques, mass spectrometry offers advantages such as high sensitivity, fast analysis speed, and the ability to resolve molecular structure information, leading to its widespread application. While mass spectrometry boasts high sensitivity and can be directly used for simple samples, it is not suitable for analyzing complex samples. This is because real-world biological samples are complex in composition and exhibit significant abundance variations, resulting in severe matrix effects that can interfere with the accuracy and sensitivity of mass spectrometry. Therefore, when dealing with complex samples, sample pretreatment and chromatographic separation are often necessary before mass spectrometry detection. However, current sample pretreatment methods are mostly designed for routine sample analysis, are cumbersome, and involve sample transfer steps, which can easily lead to sample loss, making them unsuitable for analyzing trace amounts of complex samples. Summary of the Invention

[0005] The purpose of this invention is to provide a membrane-assisted sample introduction probe device and its usage method for the analysis of trace amounts of complex samples. This device employs a membrane-assisted strategy to achieve in-situ pretreatment, introduction, transport, separation, and detection of trace amounts of complex samples under pressure conditions.

[0006] The present invention also discloses a method for sample analysis using the above-mentioned membrane-assisted strategy-based sample probe device.

[0007] A sample introduction probe device based on a membrane-assisted strategy includes:

[0008] Sample carrier assembly for placing samples;

[0009] A sample probe with a fluid inlet channel, a fluid outlet channel, and a sample inlet. The sample inlet is connected to both the fluid inlet and outlet channels. When in use, the sample inlet should be positioned directly facing the sample.

[0010] A sealed inlet fluid line for introducing fluid is placed inside the fluid inlet channel;

[0011] A liquid outlet fluid pipeline is installed inside or sealed to the liquid outlet channel, and the other end of the liquid outlet fluid pipeline is connected to a sample detection device for analyzing samples.

[0012] A fluid drive device connected to the inlet end of the liquid inlet pipeline for controlling the fluid;

[0013] A stage used to adjust the relative position of the sample carrier assembly and the probe;

[0014] The sample carrier assembly consists of a sample chip for placing the sample and a membrane assembly covering the sample chip.

[0015] The fluid drive device is used to control the fluid; the fluid can be any fluid required in the detection process, such as a fluid for dissolving the sample, a mobile phase fluid for carrying and transporting the sample, or a fluid for a specific chemical or physical reaction, etc., which can be determined according to the actual detection needs.

[0016] The sample detection device is used for analyzing samples. According to the present invention, the detection device includes, but is not limited to, an optical detector, an electrochemical detector, a mass spectrometer, or a combination of two or more of the above detectors.

[0017] The probe is used for sample introduction. The sample introduction capillary is used to introduce fluid into the probe; the fluid introduction capillary is connected to the fluid driving device. The membrane is used for sample pretreatment. The stage is used to adjust the relative position of the probe and the sample chip; the stage can be connected to the probe to adjust the probe position, or it can be connected to the sample chip or sample carrier assembly to adjust the sample carrier assembly position.

[0018] According to the present invention, the probe is an integral structure with an internal channel designed as a V-shape, approximately V-shape, or T-shape. The probe integrates an interface for sealing the channel, a fluid inlet channel, a sample inlet, and a fluid outlet channel. The fluid inlet channel is connected to a capillary that introduces fluid into the probe; the sample inlet has a fluid inlet channel on one side and a fluid outlet channel on the other; the fluid outlet channel is connected to a separation and analysis capillary; and the separation and analysis capillary is integrated with the detection device.

[0019] Furthermore, preferably, the fluid inlet channel and the fluid outlet channel are arranged in a V-shape, with their bottom ends joined together, and the sample inlet is provided at the joint position.

[0020] Alternatively, as a preferred embodiment, the fluid inlet channel and the fluid outlet channel are arranged in a T-shape, with the sample inlet located at the bottom of the fluid outlet channel; a liquid inlet gap is left between the bottom of the liquid outlet pipeline and the inner wall of the fluid outlet channel; the liquid outlet of the liquid inlet pipeline is connected to the liquid inlet gap to form a liquid inlet channel.

[0021] According to the present invention, the probe is made of polymer materials such as polyetheretherketone (PEEK), or metal materials such as stainless steel and titanium alloy, or inorganic materials such as glass, or organic-inorganic composite materials.

[0022] According to the present invention, the probe injection channel is formed by the intersection of a fluid inlet channel and a fluid outlet channel. The shape of the channel opening is rectangular, elliptical, or circular, etc., and the major axis of the channel opening cross-section is 1 micrometer to 1000 micrometers, and the minor axis is 0.5 micrometers to 500 micrometers.

[0023] This invention introduces a membrane-assisted strategy into sample analysis. This strategy uses a membrane as an intermediate medium, with the probe's injection channel contacting and applying force to the membrane covering the sample chip. This force and the membrane's elasticity are used to achieve a seal between the injection probe and the sample chip in the device.

[0024] Preferably, the membrane is an elastic membrane.

[0025] According to the present invention, the membrane-assisted strategy is to perform in-situ sample pretreatment operations in the device by means of different types of membranes. These operations include, but are not limited to, pretreatment operations such as filtration, solid phase extraction, liquid membrane extraction, affinity separation, dialysis, ultrafiltration, electrodialysis, and reverse osmosis.

[0026] According to the present invention, the auxiliary membrane materials include, but are not limited to, polymeric membranes such as cellulose ester membranes (CE), regenerated cellulose membranes (RC), polyvinylidene fluoride membranes (PVDF), and polytetrafluoroethylene (PTFE), or inorganic membranes such as porous glass membranes and glass fiber membranes, or organic membranes, or composite membranes of multiple materials (such as solid-phase extraction membranes). The types of membranes include, but are not limited to, microporous filter membranes, filtration membranes, ultrafiltration membranes, nanofiltration membranes, reverse osmosis membranes, and solid-phase extraction membranes.

[0027] According to the present invention, the surface of the membrane can be modified in various ways, including but not limited to enrichment materials such as loaded titanium dioxide, magnetic microspheres, silica gel particles, and molecularly imprinted materials. The membrane material or enrichment material can be modified with hydrophilic polar groups such as hydroxyl, carboxyl, and nitrile groups, or with non-polar C8 and C9 groups. 18Hydrophobic modifications such as hydrocarbon groups, halogen atoms, and nitro groups.

[0028] According to the present invention, the membrane has a porous or through-pore structure with a pore size ranging from 1 nanometer to 10 micrometers and a thickness ranging from 10 nanometers to 1 millimeter, and a suitable membrane can be selected according to the sample requirements. Preferably, to avoid lateral leakage of the sample through the membrane pores, a through-pore membrane should be used instead of a porous membrane. Preferably, the membrane has a through-pore structure with a pore size ranging from 1 nanometer to 10 micrometers and a thickness ranging from 10 nanometers to 1 millimeter.

[0029] According to the present invention, the sample chip can be made of inorganic materials such as glass and capillary tubes, or metallic materials such as stainless steel or titanium alloys, or polymeric materials such as PEEK, or other hard materials, or composite materials composed of the above materials.

[0030] According to the present invention, the surface of the sample chip for loading the sample can be flat, or it can be a pit, a protrusion, or a channel. Preferably, the sample chip has sample micropits, and the sample is placed in the micropits during detection.

[0031] According to the present invention, the sample chip is loaded with trace amounts of sample in the form of solid, liquid, solid-liquid coexistence, gas, and supercritical fluid.

[0032] According to the present invention, the trace samples loaded on the sample chip are derived from sources such as single-cell or trace-cell samples, biological fluid samples, biological tissue section samples, organic synthesis reaction solutions, bioengineering fermentation broths, or other trace samples. Membrane-assisted strategies can be used to perform in-situ sample pretreatment operations on the chip as needed, reducing losses caused by transfer and enabling sample introduction.

[0033] According to the present invention, the fluid conduit may be an empty capillary for fluid flow only, or a capillary with a stationary liquid modified on its inner surface, or a capillary chromatography column of monolithic column, or a capillary chromatography column filled with a stationary phase, or a conduit made of various modified capillary materials such as stainless steel tubes.

[0034] Furthermore, the inlet fluid line is an empty capillary or a capillary with a stationary phase modified on its inner surface; the outlet fluid line is an empty capillary or a capillary column. The capillary column can be a packed capillary column, an open capillary column, or a monolithic capillary column. When using a capillary column, one end is inserted into the fluid outlet channel and aligned with the injection channel, and sealed to the fluid outlet channel; the other end is sealed to the sample detection device.

[0035] As an alternative, instead of using a separation capillary, the separation and analysis of sample components can be performed directly using the fluid extraction channel on the injection probe. This can be achieved by filling or forming a chromatographic stationary phase within the fluid extraction channel, modifying the surface of the fluid extraction channel to enable separation, or directly using the fluid extraction channel as a sample transport channel to transfer the sample to the detector for detection.

[0036] The present invention relates to a method for using a sample probe device based on a membrane-assisted strategy. This method utilizes the inherent properties of the membrane material (such as elasticity) and employs a membrane sandwich method between the sample probe and the sample chip to achieve a seal between the sample probe's injection channel and the sample chip.

[0037] This invention utilizes a fluid-driven device to drive fluid within the device in a sealed state. Fluid enters the probe through the fluid inlet channel, flows through the sample inlet, and contacts the sample on the sample chip via the membrane pores. The fluid then exits the probe through the fluid outlet channel. In this process, the interaction between the fluid, sample, and membrane enables in-situ pretreatment of the sample and subsequent sample introduction.

[0038] As one possible implementation, the membrane has an in-situ sample pretreatment function, and the sample can be filtered under fluid conditions according to the principle of size exclusion (such as microporous membrane, ultrafiltration membrane or dialysis membrane filtration), as well as the subsequent sample injection operation.

[0039] As another possible implementation, the membrane has a pretreatment function for in-situ enrichment of samples. The sample can be enriched by selectively binding specific components in the sample to the membrane surface (such as solid phase extraction, liquid membrane extraction, affinity separation) under the transport of fluid. Subsequently, the enriched sample on the membrane is eluted by an elution fluid (whose composition is different from that of the fluid used for enrichment) and is carried by the elution fluid to complete the injection of the eluted sample.

[0040] This invention utilizes a fluid to transport a pre-treated sample through a membrane to a fluid outlet channel and a capillary column connected thereto to achieve chromatographic injection of the sample; a fluid driving device drives the mobile phase fluid to separate the sample loaded on the stationary phase of the chromatographic column; and the detection device is used to detect the components of the separated sample.

[0041] In this invention, the membrane has two main functions: first, it uses the inherent properties of the membrane material (such as elasticity) to achieve a seal between the sample probe and the sample chip; second, the membrane itself has porous properties, allowing fluid to flow through the membrane pores and connect the sample inlet of the sample probe with the sample on the sample chip, thereby completing various sample pretreatment functions, including but not limited to filtration, solid-phase extraction, liquid membrane extraction, affinity separation, dialysis, ultrafiltration, electrodialysis, and reverse osmosis.

[0042] A method for sample analysis using a membrane-assisted probe device according to any of the above technical solutions includes: using a fluid driving device to drive fluid within the device in a sealed state; the fluid is introduced through the probe via a fluid inlet channel and flows through the injection channel port; the membrane performs 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 via a fluid outlet channel; and the sample analysis is achieved using a capillary chromatographic column and a sample detection device.

[0043] Preferably, the method for sample analysis using the membrane-assisted probe device includes the following steps:

[0044] (1) Place the sample onto the sample chip and cover the sample chip with the membrane;

[0045] (2) Control the probe's injection channel to contact the membrane and align it with the sample, and apply a squeezing (or pressing) force to form a "sandwich" membrane structure of injection channel-membrane-sample to achieve device sealing;

[0046] (3) Start the fluid drive device. The fluid is introduced through the capillary and carries the sample through the membrane pores. The membrane is used to complete the in-situ pretreatment of the sample. The in-situ pretreated sample is loaded onto the capillary column to complete the injection operation. The fluid drive device is used to introduce the mobile phase fluid to perform isocratic or gradient elution separation and detection on the sample loaded onto the capillary column.

[0047] Alternatively, steps (1) to (3) can be repeated to complete the analysis of different samples or different regions of the same sample.

[0048] Furthermore, the method of using the sample introduction probe device based on the membrane-assisted strategy includes the following steps:

[0049] (a) Loading the sample onto the sample chip;

[0050] (b) Fix the sample chip onto the moving stage;

[0051] (c) Cover the sample chip with the membrane;

[0052] (d) Control the moving stage to bring the probe's sample inlet into contact with the membrane and apply a squeezing (or pressing) force to form a "sandwich" membrane structure of the sample inlet-membrane-sample, thereby sealing the device.

[0053] (e) Start the fluid drive device, introduce fluid through the membrane pores to carry the sample, complete the in-situ filtration of the sample, and load the filtered sample onto the capillary column to complete the injection operation. Use the fluid drive device to introduce the mobile phase fluid to perform isocratic or gradient elution separation and detection on the sample loaded onto the capillary column.

[0054] (f) Repeat steps (a) to (e) to complete the analysis of different samples on the sample chip, or the analysis of samples on different sample chips; or the analysis of different regions of the same sample on the sample chip (spatial imaging analysis).

[0055] As another implementation, another method of using the sample probe device based on the membrane-assisted strategy includes the following steps:

[0056] (a) Loading the sample onto the sample chip;

[0057] (b) Fix the sample chip onto the moving stage;

[0058] (c) Cover the sample chip with the membrane;

[0059] (d) Control the moving stage to bring the probe's sample inlet into contact with the membrane and apply a squeezing (or pressing) force to form a "sandwich" membrane structure of the sample inlet-membrane-sample, thereby sealing the device.

[0060] (e) Start the fluid drive device to introduce fluid through the membrane pores to carry the sample and complete the in-situ enrichment of the sample on the membrane; use the fluid drive device to introduce elution fluid to elute the sample enriched on the membrane and load it onto the capillary column to complete the sample injection operation; use the fluid drive device to introduce mobile phase fluid to perform isocratic or gradient elution separation and detection of the sample loaded onto the capillary column.

[0061] (f) Repeat steps (a) to (e) to complete the analysis of different samples on the sample chip, or the analysis of samples on different sample chips; or the analysis of different regions of the same sample on the sample chip (spatial imaging analysis).

[0062] As another implementation, a sample introduction probe device based on a membrane-assisted strategy differs from the above technical solution in that the sample introduction probe is replaced by separate inlet and outlet fluid tubes. In use, the inlet and outlet fluid tubes are respectively arranged on both sides of the sample carrier assembly, with the outlet end of the inlet fluid tube and the inlet end of the outlet fluid tube facing the sample, and respectively sealing and docking with the sample chip side and membrane side of the sample carrier assembly.

[0063] Furthermore, a sample introduction probe device based on a membrane-assisted strategy includes:

[0064] Sample carrier assembly for placing samples;

[0065] Inlet and outlet fluid lines are used to introduce fluid.

[0066] A sample detection device for analyzing samples, connected to the outlet end of the liquid fluid pipeline;

[0067] A fluid drive device connected to the inlet end of the liquid inlet pipeline for controlling the fluid;

[0068] The sample carrier assembly consists of a sample chip for placing the sample and a membrane covering the sample chip;

[0069] During testing, the outlet end of the inlet fluid pipeline and the inlet end of the outlet fluid pipeline are respectively sealed and connected to the sample chip side and membrane side of the sample carrier component.

[0070] In the above technical solution, the sample chip is provided with a through-hole membrane or a porous membrane, and the sample is preloaded on the through-hole membrane or porous membrane structure during detection.

[0071] When using the above technical solution for sample analysis, the following steps are included:

[0072] (1) Fix the sample in the corresponding position of the sample carrier component, align the inlet fluid line and the outlet fluid line vertically, and squeeze the sample carrier component in the middle to achieve a seal;

[0073] (2) Start the fluid drive device, introduce fluid through the membrane pores to carry the sample, and complete the in-situ enrichment of the sample on the membrane; use the fluid drive device to introduce elution fluid to elute the sample enriched on the membrane and use the effluent fluid line to complete the sample injection operation; when the effluent fluid line uses a capillary column, use the fluid drive device to introduce mobile phase fluid to perform isocratic or gradient elution separation and detection of the sample loaded on the capillary column.

[0074] Alternatively, the fluid drive device can be activated to introduce fluid through the membrane pores to carry the sample, complete the in-situ filtration of the sample, and then use the effluent fluid line to complete the injection operation. The fluid drive device can be used to introduce the mobile phase fluid. When the effluent fluid line uses a capillary column, the sample loaded on the capillary column can be separated and detected by isocratic or gradient elution.

[0075] 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 regions of the same sample on the sample chip (spatial imaging analysis) can be completed.

[0076] During filtration, the difference in membrane pore size is utilized to retain insoluble substances larger than the pore size, thus achieving sample filtration. During enrichment, the difference in sample polarity is utilized; as the sample passes through the membrane, some components are adsorbed onto the membrane surface due to interaction forces, while other insoluble components are retained below the membrane. Other soluble components in the sample pass through the membrane. The process removes the retained insoluble components and the permeated soluble components, then uses an eluent to elute the components adsorbed onto the membrane surface. Finally, the eluted target components are loaded onto the chromatographic column, completing the entire enrichment and elution process.

[0077] When using membranes for in-situ sample enrichment, different injection probes can be used to complete the enrichment of samples on the membrane and the elution and separation of the enriched samples on the membrane.

[0078] According to the present invention, the method of sealing the device by contacting the sample inlet 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 device sealing without significant liquid leakage can be achieved under hydraulic pressure exceeding 60MPa.

[0079] According to the present invention, the relative movement of the injection probe and the sample chip relative to the membrane provides pressure for sealing the device; the sample chip may be fixed on the moving stage, or the injection probe may be fixed on the moving stage, or the sample chip and the injection probe may be fixed on different moving stages respectively.

[0080] According to the present invention, the membrane-assisted strategy uses a membrane to isolate the probe from direct contact with the sample, eliminating the need for probe cleaning and simplifying the operation.

[0081] According to the present invention, the portion of the membrane in contact with the sample is for single use only. That is, after sample pretreatment by contacting the membrane with the sample, the portion of the membrane in contact with the sample is discarded. For new samples, sample pretreatment is performed using a different membrane or other areas of the membrane that have not been in contact with the sample.

[0082] According to the present invention, a porous support sheet or membrane is added between the sample chip and the membrane to improve the strength of the membrane and prevent the membrane from deforming towards the sample chip.

[0083] According to the present invention, in order to improve the utilization efficiency of the membrane and increase the processing throughput, different samples can be pretreated separately on different regions of a single membrane.

[0084] According to the present invention, the sample pretreatment and injection operations of the probe can be performed sequentially or separately. When performed separately, the sample is first pretreated using the probe and membrane, and then the membrane is removed and stored. At the required time, the membrane is then reattached to the injection probe for injection. In this method, the probe used for sample pretreatment and the probe used for injection can be the same probe or different probes.

[0085] According to the present invention, by adding a switching valve to the device, the injection and chromatographic separation of different samples can be carried out simultaneously, thereby reducing the time of the entire analysis process and increasing the throughput of the analysis.

[0086] According to the present invention, the mobile stage can move in three dimensions, enabling array-based sample introduction and imaging analysis.

[0087] It should be noted that the application fields of the membrane-assisted injection probe device are 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.

[0088] The main advantages of this invention include: integrating trace sample pretreatment with injection, chromatographic separation, and detection systems; the system has a simple structure, operates quickly and automatically, is highly flexible, easy to use, and widely applicable, especially suitable for the analysis of trace samples. Based on a membrane-assisted strategy, in-situ sample pretreatment followed by injection can be achieved, providing a new injection mode for the analysis of complex samples and offering a new approach to the integration and miniaturization of chromatographic analysis systems.

[0089] This invention has broad application prospects in fields such as bioanalysis, organic synthesis, bioengineering, clinical mass spectrometry, single-cell analysis, analysis of complex trace samples, multi-omics analysis, and bioimaging analysis.

[0090] Compared with the prior art, the main advantages of the present invention are:

[0091] (1) The system integrates the micro sample pretreatment with the injection, chromatographic separation and detection system. The system has a simple structure, fast and automatic operation, high flexibility, is easy to use and has wide applicability. It is especially suitable for the analysis of micro samples.

[0092] (2) Based on the membrane-assisted strategy, in-situ pretreatment of samples can be achieved before injection, which provides a new injection mode for the analysis of complex samples and also provides a new way for the integration and miniaturization of chromatographic analysis systems.

[0093] (3) The membrane-assisted strategy is highly flexible, and the membrane used can be changed according to the pretreatment requirements;

[0094] (4) The whole device is modularly designed, flexible in assembly and easy to use. The probe is made of hard material that can be reused and can be automated.

[0095] (5) It can perform in-situ pretreatment on the sample chip, reducing sample transfer loss and even achieving non-destructive sample pretreatment. Furthermore, since the probe does not directly contact the sample, the cleaning steps are reduced, making it suitable for the analysis of trace samples.

[0096] (6) Membrane-assisted strategies can achieve in-situ pretreatment in different modes, including but not limited to filtration, solid phase extraction, liquid membrane extraction, affinity separation, dialysis, ultrafiltration, electrodialysis, reverse osmosis and other pretreatment operations, which reduce the complexity of samples and enable segmented analysis of samples.

[0097] (7) The device has broad application prospects and can be applied to fields such as organic synthesis, bioengineering, clinical mass spectrometry, analysis of trace complex samples, single-cell analysis, multi-omics analysis, bioimaging analysis, and rapid on-site analysis. Attached Figure Description

[0098] Figure 1 This is a schematic diagram of the V-shaped injection probe device based on the membrane-assisted strategy in Example 1. In the figure, 1-fluid driving device, 2-capillary tube for introducing fluid, 3-injection probe, 4-sealed channel interface, 5-fluid inlet channel, 6-fluid outlet channel, 7-injection channel port, 8-sample, 9-membrane, 10-sample chip, 11-moving stage, 12-capillary column, 13-fluid, 14-elution fluid, 15-mobile phase fluid, 16-electrospray mass spectrometer detection device.

[0099] Figure 2 The results are obtained by chromatographic analysis of a mixed sample of three peptide segments in isocratic elution mode using the apparatus and method described in Example 1.

[0100] Figure 3 The results are obtained by performing chromatographic analysis on the product of trypsin digestion of protein cytochrome C using the apparatus and method described in Example 1, in gradient elution mode.

[0101] Figure 4Example 2 uses the apparatus and method of Example 1 for in-situ sample pretreatment and filtration before injection. (a) Mass spectrometry analysis results of protein cytochrome C without using a membrane-assisted strategy; (b) Chromatographic analysis results of protein cytochrome C using a membrane-assisted strategy; (c) In-situ trypsin digestion of the above-injected sample, analysis of the digested peptides using a membrane-assisted strategy, and mass spectrometry detection results of one of the peptides, with * indicating the peptide signal.

[0102] Figure 5 This is a schematic diagram of the T-type injection probe device combined with a solid-phase extraction membrane in Example 3. In the figure, 1-fluid drive device, 2-capillary tube for introducing fluid, 3-injection probe, 4-sealed channel interface, 5-fluid inlet channel, 6-fluid outlet channel, 7-injection channel port, 8-sample, 9-membrane, 10-sample chip, 11-moving stage, 12-capillary column, 13-fluid, 14-elution fluid, 15-mobile phase fluid, 16-electrospray mass spectrometer detection device, 17-guided capillary, 18-switching valve, 19-injection gap.

[0103] Figure 6 for Figure 5 A magnified view of a portion of the probe.

[0104] Figure 7 This is a schematic diagram of sample introduction using the array of sample introduction probes combined with solid-phase extraction membranes in Example 4. In the figure, 3-sample introduction probe, 9-membrane with solid-phase extraction function, 10-sample chip with micropits, 11-moving stage, 15-mobile phase fluid, 21-sample micropit number one, 22-sample micropit number two, 23-sample introduction probe number two.

[0105] Figure 8 This is a schematic diagram of the channel sample extraction and injection mode of the solid-phase extraction membrane in Example 5 for the extraction and injection of dried blood spot samples. 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, 13-fluid. Detailed Implementation

[0106] The technical solution of the present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0107] Referring to the accompanying drawings, preferred embodiments of the present invention will be described in detail below.

[0108] Example 1

[0109] Figure 1This is a schematic diagram of the membrane-assisted injection probe device of Example 1. The system consists of a fluid drive device 1, a V-shaped injection probe 3 made of PEEK material, a capillary 2 for introducing the mobile phase, an RC membrane 9, a sample chip 10, a stage 11 for moving the sample chip, a capillary column 12, and an electrospray mass spectrometer detection device 16.

[0110] The injection probe 3 is made of PEEK material and includes a sealed channel interface 4, a fluid inlet channel 5, a fluid outlet channel 6, and an injection port 7. The fluid inlet channel 5 houses the capillary tube 2 for introducing the mobile phase, and the fluid outlet channel houses the capillary column 12. Both the capillary tube 2 and the capillary column 12 can be sealed with their respective fluid inlet and outlet channels using conical seals. The injection port 7 is located at the bottom of the probe, with the fluid inlet channel 5 and the fluid outlet channel 6 on either side, i.e., at the bottom joint of the fluid inlet channel 5 and the fluid outlet channel 6. The interface 4 is located at the inlet of the fluid inlet channel 5 and the outlet of the fluid outlet channel 6, respectively, to achieve sealed connections with the fluid drive device 1 and the electrospray mass spectrometer detection device 16.

[0111] The specific usage method of the device in Example 1 is as follows: (1) The sample chip 10 is fixed on the moving stage, and the membrane 9 covers the sample chip 10. By controlling the movement of the moving stage 11, the injection port 7 of the injection probe 3 is brought into contact with the membrane 9, and the injection port 7 and the membrane 9 are sealed by the applied squeezing force to prevent the mobile phase from leaking from the injection port 7; (2) In the sealed state, the fluid drive device 1 is turned on, and the chromatographic mobile phase is injected from the capillary 2 that introduces the mobile phase. After passing through the fluid introduction channel 5, the injection port 7 and the membrane 9, the sample 8 is dissolved. After in-situ pretreatment, the sample passes through the membrane 9 and the injection port 7 in sequence. The sample enters the capillary chromatographic column 12 through the fluid outlet channel 6, changes the composition of the mobile phase to elute the sample, forms an electrospray under the action of the electric field, and enters the detection device 16 of the electrospray mass spectrometer for detection; (3) The fluid drive device 1 is paused, and the moving stage 11 is moved to the next sample 8. The steps in (2) are repeated to realize the analysis of different samples 8.

[0112] Figure 2 The results are obtained by mass spectrometry analysis of a mixture of three peptides in isocratic elution mode using the apparatus and method described in Example 1, with an RC membrane having a molecular weight cutoff of 2000.

[0113] Specifically, three peptide samples, GE-11, GF-9-NH2, and TL-9-NH2, were used, each at a concentration of 0.1 μmol / L, to form sample 8. 10 nmol of sample 8 was added dropwise to sample chip 10 and allowed to air dry to form a dry spot. The sample was eluted isocratically using a 30% acetonitrile aqueous solution (containing 0.1% formic acid) as the mobile phase. The 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.

[0114] The results of this analysis indicate that the system can perform chromatographic analysis of trace samples and has good system reproducibility.

[0115] Figure 3 The results are obtained by mass spectrometry analysis of the product of trypsin digestion of protein cytochrome C in gradient elution mode using the apparatus and method of Example 1.

[0116] Specifically, the product of enzymatic hydrolysis of protein cytochrome C in the conventional system was used as sample 8 and added to sample chip 10. Sample 8 was analyzed using the system, with the following chromatographic gradient: 0–10 minutes, 0% solution B (the remainder being 100% solution A); 10–20 minutes, 15% solution B (the remainder being 85% solution A); 20–35 minutes, 30% solution B (the remainder being 70% solution A); 35–40 minutes, 80% solution B (the remainder being 20% ​​solution A). Solution A was a 0.1% formic acid aqueous solution, and solution B was a 0.1% formic acid acetonitrile solution.

[0117] The results of this analysis demonstrate that the system can be used for the analysis of complex samples in minute quantities.

[0118] Example 2

[0119] Figure 4 Example 2 uses the apparatus and method of Example 1. (a) Mass spectrometry analysis results of protein cytochrome C without using a membrane-assisted strategy; (b) Chromatographic analysis results of protein cytochrome C using a membrane-assisted strategy; (c) Mass spectrometry detection results of one of the digestion products after in-situ trypsin digestion of the above-mentioned sample using a membrane-assisted strategy.

[0120] Specifically, in (a) a system without a membrane-assisted strategy (i.e., no membrane covering the sample, with the sample inlet in direct contact with the sample) was used to analyze the protein cytochrome C sample dropped onto the sample chip, yielding 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 the protein cytochrome C sample dropped onto the sample chip, and no signal of protein cytochrome C was detected. In (c) the sample injected in (b) was digested in situ with trypsin on the sample chip, and the reaction products were analyzed using a system with a membrane-assisted strategy (i.e., the apparatus and method of use in Example 1).

[0121] Figure 4 (a) The results demonstrate that the protein cytochrome C can be detected by the electrospray mass spectrometry detection device; Figure 4 (b) The results showed that the selected membrane pore size was smaller than that of protein cytochrome C. Under membrane-assisted conditions, protein cytochrome C was blocked by the membrane and could not pass through the membrane, thus it could not be detected by the detection device. Figure 4 (c) The results show that after the original protein cytochrome C is digested into peptide samples and then analyzed, the samples can pass through the membrane and be identified by the detection device. This indicates that the system can achieve in-situ size exclusion filtration of sample introduction by selecting an appropriate membrane.

[0122] The results of this analysis demonstrate that the system employs a membrane-assisted strategy, which blocks proteins larger than the membrane pore size while allowing peptides obtained after enzymatic digestion to pass through, proving its suitability for the analysis of trace amounts of complex samples.

[0123] Example 3

[0124] Figure 5 This is a schematic diagram of the T-type injection probe device combined with a solid-phase extraction membrane in Example 3. The system consists of a fluid drive device 1, a switching valve 18, a flow guide capillary 17, a capillary 2 for introducing the mobile phase, a T-type injection probe 3, a connector 4 with an integrated sealed channel inside the probe, a fluid inlet channel 5, a fluid outlet channel 6, an injection port 7, a solid-phase extraction membrane 9, a sample chip 10, a stage 11 for moving the sample chip, a capillary column 12, and an electrospray mass spectrometer detection device 16.

[0125] The T-type injection probe 3 is made of stainless steel and includes a sealed channel interface 4, a fluid inlet channel 5, and an injection port 7. The fluid inlet channel 5 houses the capillary tube 2 for introducing the mobile phase, the fluid outlet channel 6 houses the capillary column 12, and the injection port 7 is located at the bottom of the probe, with the fluid inlet channel 5 above it.

[0126] The capillary tube 2 and the capillary column 12 are respectively sealed and fixed in the fluid inlet channel 5 and the fluid outlet channel 6 by conical seals 20. Specifically, in the fluid inlet channel 5, the conical seal 20 ensures that the capillary tube 2 is only connected to the inlet gap 19 in the fluid outlet channel 6, while simultaneously sealing it against the inner wall of the fluid inlet channel 5 to prevent fluid from entering the gap. In the fluid outlet channel 6, an injection gap 19 is provided between the bottom outer wall of the capillary column 12 and the inner wall of the fluid outlet channel 6. This injection gap 19 is connected to the capillary tube 2 to allow for sample injection; simultaneously, the portion of the capillary column 12 above the injection gap 19 is sealed against the inner wall of the fluid outlet channel 6 by the conical seal 20, preventing fluid from entering the gap formed between the two.

[0127] Also refer to Figure 6 The specific usage method of the device in Example 3 is as follows: (1) The sample chip 10 is fixed on the moving stage, and the solid phase extraction membrane 9 covers the sample chip 10. By controlling the movement of the moving stage 11, the injection port 7 is aligned with the sample position on the sample chip 10, and the applied squeezing force is used to make the injection port 7 of the injection probe 3 contact and seal with the membrane 9 to prevent the mobile phase from leaking from the injection port 7; (2) In the sealed state, the fluid drive device 1 is turned on, and the switching valve 18 is controlled so that the chromatographic mobile phase is introduced from the mobile phase. (2) The sample 8 is injected through the capillary 2, and after passing through the fluid introduction channel 5, the liquid inlet gap 19, the sample inlet 7 and the membrane 9, it is dissolved and carried onto the membrane 9; (3) The composition of the mobile phase is changed, the sample 8 is eluted from the membrane 9, and it is transported through the capillary column 12. Under the action of the electric field, an electrospray is formed and it enters the detection device 16 of the electrospray mass spectrometer for detection; (4) The switching valve 18 is controlled so that the mobile phase flows out from 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 achieve the analysis of different samples 8.

[0128] Example 4

[0129] Figure 7 This is a schematic diagram of sample introduction using the array of sample probes combined with a solid-phase extraction membrane in Example 4. In the figure, 11-moving stage, 10-sample chip with micropits, 9-solid-phase extraction membrane, 21-sample micropit number one, 3-sample probe number one, 15-mobile phase fluid, 22-sample micropit number two, 23-sample probe number two, 14-elution fluid.

[0130] The array injection probe device uses two injection probes to load the sample onto the solid phase extraction membrane and to elute the sample from the solid phase extraction membrane, respectively. Compared with Example 3, it realizes in-situ sample pretreatment and detection in parallel, reducing the analysis time.

[0131] The specific usage method of the array sample introduction probe device in Example 4 is as follows: (1) The sample chip 10 is fixed on the moving stage 11, and the solid phase extraction membrane 9 covers the sample chip 10. By controlling the movement of the moving stage 11, the first sample introduction probe 3 contacts and seals the solid phase extraction membrane 9 above the first sample micro-pit 21; (2) In the sealed state, the mobile phase fluid 15 is introduced, passes through the solid phase extraction membrane 9, enters the first sample micro-pit 21, and loads the sample onto the solid phase extraction membrane 9; (3) The first sample introduction probe 3 is moved to contact the solid phase extraction membrane 9 above the second sample micro-pit 22. (3) Seal the sample and move the second sample probe 23 to the solid phase extraction membrane 9 above the first sample micropit to contact and seal it; (4) In the sealed state, introduce the mobile phase fluid 6 into the first sample probe 3, pass through the solid phase extraction membrane 9, enter the second sample micropit 23, and load the sample onto the solid phase extraction membrane 9; at the same time, introduce the elution fluid 14 into the second sample probe 23 to elute the sample on the solid phase extraction membrane 9 above the first sample micropit 21 for subsequent analysis; (5) Repeat the steps in (4) to realize the parallel analysis of sample injection by arraying solid phase extraction membranes.

[0132] Example 5

[0133] Figure 8 This is a schematic diagram of the channel sample introduction mode combined with the solid phase extraction membrane in Example 5 for dried blood spot sample introduction. In the figure, 2-capillary tube for introducing fluid, 13-fluid, 8-dried blood spot sample, 10-sample chip with dried blood spot, 9-solid phase extraction membrane.

[0134] This sample introduction mode follows the membrane-assisted strategy, combining solid-phase extraction membranes to achieve in-situ pretreatment and injection of samples on dried blood spot filter paper. Unlike Examples 1-4 above, Example 5 does not use the method of integrating the sample introduction probe to complete the sample pretreatment and injection operation, but instead uses the method of performing sample pretreatment and injection operation separately.

[0135] The specific method for injecting dried blood spot in Example 5 is as follows: (1) Cover the solid phase extraction membrane 9 with the paper chip 10 (the blood sample is loaded in the mesh structure of the filter paper on the paper chip) containing the dried blood spot sample, align the two capillaries 2 for introducing fluid, and squeeze the sample chip 10 and the solid phase extraction membrane 9 in the middle to achieve a seal; (2) Introduce fluid 13 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) Remove the solid phase extraction membrane with the enriched sample, combine it with the injection probe, and use a method similar to Example 3 to sequentially complete the operation of eluting the target substance adsorbed on the solid phase extraction membrane 9, injecting the sample, chromatographic separation and detection.

Claims

1. A method for sample analysis using a membrane-assisted probe device, characterized in that, include: The fluid-driven device drives the fluid inside the device in a sealed state. The fluid enters the probe through the fluid inlet channel and flows through the sample inlet. The membrane performs in-situ pretreatment of the sample. The pretreated sample comes into contact with the sample on the sample chip through the membrane pores. Then the fluid flows out of the probe through the fluid outlet channel. The sample is analyzed by the sample detection device. The inlet fluid line is an empty capillary or a capillary with a stationary liquid modified on the inner surface. The outlet fluid line is a capillary chromatographic column. Specifically, the steps include the following: (1) Place the sample onto the sample chip and cover the sample chip with the membrane; (2) Control the probe's injection channel to contact the membrane and align it with the sample, and apply a squeezing force to form a "sandwich" membrane structure of injection channel-membrane-sample to achieve device sealing; (3) Start the fluid drive device. The fluid is introduced through the capillary and carries the sample through the membrane pores. The membrane is used to complete the in-situ pretreatment of the sample. The in-situ pretreated sample is loaded onto the capillary column to complete the injection operation. The fluid drive device is used to introduce the mobile phase fluid to perform isocratic or gradient elution separation and detection on the sample loaded onto the capillary column. By repeating steps (1) to (3), the analysis of different samples or different regions of the same sample can be completed; The sample introduction probe device based on the membrane-assisted strategy includes: Sample carrier assembly for placing samples; A sample probe with a fluid inlet channel, a fluid outlet channel, and a sample inlet. The sample inlet is connected to both the fluid inlet and outlet channels. When in use, the sample inlet should be positioned directly facing the sample. A sealed inlet fluid line for introducing fluid is placed inside the fluid inlet channel; A liquid outlet fluid pipeline is installed inside or sealed to the liquid outlet channel, and the other end of the liquid outlet fluid pipeline is connected to a sample detection device for analyzing samples. A fluid drive device connected to the inlet end of the inlet fluid pipeline for controlling the fluid; A stage used to adjust the relative position of the sample carrier assembly and the probe; The sample carrier assembly consists of a sample chip for placing the sample and a membrane covering the sample chip. The membrane is an elastic membrane; the membrane has a porous structure; the membrane is a functional membrane with one or more functions including filtration, solid-phase extraction, liquid membrane extraction, affinity separation, dialysis, ultrafiltration, electrodialysis, and reverse osmosis.

2. The method for sample analysis using a sample introduction probe device based on a membrane-assisted strategy according to claim 1, characterized in that, The fluid inlet channel and the fluid outlet channel are arranged in a V-shape, with their bottom ends joined together, and the sample inlet is located at the joint position.

3. The method for sample analysis using a sample introduction probe device with a membrane-based assisted strategy according to claim 1, wherein, The fluid inlet channel and fluid outlet channel are arranged in a T-shape, and the sample inlet is located at the bottom of the fluid outlet channel; there is an inlet gap between the bottom of the liquid outlet pipeline and the inner wall of the fluid outlet channel; the outlet of the liquid inlet pipeline is connected to the inlet gap to form an inlet channel.

4. The method for sample analysis using a membrane-assisted probe device according to claim 1, characterized in that, The membrane has a pore size ranging from 1 nanometer to 10 micrometers and a thickness ranging from 10 nanometers to 1 millimeter; the membrane is a cellulose ester membrane, a regenerated cellulose membrane, a polyvinylidene fluoride membrane, a polytetrafluoroethylene membrane, a porous glass membrane, or a glass fiber membrane.

5. The method for sample analysis using a sample introduction probe device based on a membrane-assisted strategy according to claim 1, wherein, The sample chip has a sample carrying area, which can be a micropit, a plane, or a convex pillar. During detection, the sample is placed in the sample carrying area.

6. The method of sample analysis using a sample introduction probe device based on a membrane assisted strategy according to any one of claims 1.4.5, characterized in that, The sample probe is replaced by separate inlet and outlet fluid tubes. In use, the inlet and outlet fluid tubes are respectively set on both sides of the sample carrier assembly, with the outlet end of the inlet fluid tube and the inlet end of the outlet fluid tube facing the sample, and respectively sealing and docking with the sample chip side and membrane side of the sample carrier assembly.

7. The method of sample analysis using a membrane-assisted strategy based injection probe device of claim 1, wherein, Includes the following steps: (1) Place the sample onto the sample chip and cover the sample chip with the membrane; (2) Control the probe's injection channel to contact the membrane and align it with the sample, and apply a squeezing force to form a "sandwich" membrane structure of injection channel-membrane-sample to achieve device sealing; (3) Start the fluid drive device. The fluid is introduced through the inlet fluid pipeline and carries the sample through the membrane pores. The membrane is used to complete the sample filtration operation. The filtered sample is loaded onto the capillary column to complete the injection operation. The fluid drive device is used to introduce the mobile phase fluid to perform isocratic or gradient elution separation and detection on the sample loaded onto the capillary column. By repeating steps (1) to (3), the analysis of different samples or different regions of the same sample can be completed.

8. The method for sample analysis using the membrane-assisted probe device according to claim 1, characterized in that, Includes the following steps: (1) Place the sample onto the sample chip and cover the sample chip with the membrane; (2) Control the probe's injection channel to contact the membrane and align it with the sample, and apply a squeezing force to form a "sandwich" membrane structure of injection channel-membrane-sample to achieve device sealing; (3) Start the fluid drive device. The fluid is introduced through the liquid inlet fluid line and carries the sample through the membrane pores. The sample enrichment operation is completed by the membrane. The elution fluid is introduced by the fluid drive device to elute the sample enriched on the membrane and load it onto the capillary column to complete the sample injection operation. The mobile phase fluid is introduced by the fluid drive device to perform isocratic or gradient elution separation and detection on the sample loaded onto the capillary column. By repeating steps (1) to (3), the analysis of different samples or different regions of the same sample can be completed.

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