An integrated chromatographic analysis probe assembly, device and chromatographic analysis method

Abstract

The present invention discloses an integrated chromatographic analysis probe assembly, including a probe body with a mobile phase channel and a chromatographic column channel, one end of the chromatographic column channel being a sampling port; a mobile phase capillary arranged on the probe body and communicating with the mobile phase channel; a chromatographic column detachably fixed in the chromatographic column channel, the inlet of the chromatographic column corresponding to the sampling port; a mobile phase inlet gap is left between the chromatographic column channel and the outer wall of the chromatographic column, and the gap is communicated with the outlet of the mobile phase channel and the sampling port at the same time. The present invention also discloses an analysis device and method. The device of the present invention has high integration, high strength and hardness, simple structure, small size, is suitable for batch processing, and is easy to operate. It is mainly aimed at trace samples with small size, complex composition, and low concentration, and minimizes the loss of trace samples during transfer and transportation in pipelines.

Description

Technical Field

[0001] The invention belongs to the field of chromatographic analysis, and in particular relates to an integrated chromatographic analysis probe component, a device and a chromatographic analysis method. Background Art

[0002] Chromatographic analysis technology has long been widely concerned by scientific researchers for its powerful separation and analysis capabilities for complex samples, and has broad development potential and application prospects. At present, high performance liquid chromatography and gas chromatography have become one of the most important analytical technologies in the fields of chemistry, biomedicine, agronomy, environmental science, and food science.

[0003] Traditional HPLC injection methods usually rely on automatic samplers or injection valves, and injection is achieved through the coordination of quantitative loops, valve switching and liquid pumps. In recent years, with the increasing demand for trace sample analysis in various research fields, more and more work has put forward more trace injection requirements for the sample introduction link of liquid chromatography methods. Different from traditional liquid chromatography injection methods, micro-chromatography injection methods are usually aimed at trace samples with extremely small volume, extremely low concentration and complex composition. At present, the main application areas of micro-chromatography injection technology include single-cell proteomics analysis, metabolomics analysis, mass spectrometry imaging, spatial multi-omics analysis, micro-high-throughput screening, etc.

[0004] At present, in the field of micro-chromatographic injection, the literature usually uses droplet microextraction, microfluidic chip method, probe injection method, and in-situ reaction method to transfer trace samples into the chromatographic column. However, these methods have limitations, including: the inability to achieve complete sample transfer, sample residual loss; difficulty in adding sample reagents, and incompatibility with complex pre-treatment operations; when using the probe injection method, the device based on the capillary processing probe body has a structural strength that cannot withstand high pressure, and the lack of working durability and reliability of the device limits its practical application. Summary of the invention

[0005] The present invention provides an integrated chromatographic analysis probe assembly and a chromatographic analysis method, which integrates chromatographic sampling, injection and separation functions into one probe, and can realize chromatographic micro-injection and separation analysis with extremely low loss.

[0006] An integrated chromatographic analysis probe assembly, comprising:

[0007] A probe body with a mobile phase channel and a chromatographic column channel, one end of the chromatographic column channel being a sampling port;

[0008] A mobile phase capillary disposed on the probe body and communicating with the mobile phase channel;

[0009] A chromatographic column detachably fixed in the chromatographic column channel, the inlet of the chromatographic column corresponding to the sampling port;

[0010] A mobile phase inlet gap is left between the chromatographic column channel and the outer wall of the chromatographic column, and the gap is communicated with the mobile phase channel outlet and the sampling port at the same time.

[0011] The sampling port is used to cover the sample area and to form a sealed space with the sample chip;

[0012] The mobile phase inlet gap enables the connection between the mobile phase channel outlet, the sampling port and the chromatographic column inlet, thereby achieving the entry of the mobile phase and the injection of the sample.

[0013] The integrated chromatographic analysis probe assembly adopts a modular structure. Preferably, the integrated chromatographic analysis probe assembly can use split structures with different integration levels, until adopting an integrated structure in which all parts are integrated and processed into the probe body.

[0014] Furthermore, the body of the integrated chromatographic analysis probe is processed with a mobile phase capillary fixing interface, a mobile phase channel, a sampling port, a chromatographic column channel, and a chromatographic column fixing interface, and the mobile phase capillary and the chromatographic column are fixed to the integrated chromatographic analysis probe body by a capillary fixing assembly.

[0015] As a further preference, the integrated chromatography analysis probe uses a more integrated structure, and the mobile phase capillary fixing interface, the mobile phase channel, the sampling port, the chromatographic column channel, and the chromatographic column are integrated and processed on the probe body.

[0016] As a preferred embodiment, one end of the mobile phase channel and the chromatographic column channel is provided with a fixed interface, and a fixing component for fixing the mobile phase capillary and the chromatographic column to the corresponding fixed interface.

[0017] The fixed interface of the mobile phase channel cooperates with the capillary fixing assembly to connect the mobile phase capillary with the mobile phase channel on the probe body and keep it sealed. The other end of the mobile phase capillary is connected to the fluid driving device to introduce the mobile phase into the integrated chromatographic analysis probe.

[0018] The fixed interface of the chromatographic column channel cooperates with the chromatographic column fixing component to connect the chromatographic column with the chromatographic column channel of the probe and keep it sealed to prevent the mobile phase from leaking from the chromatographic column.

[0019] As a further preference, the fixing assembly includes a chromatographic column fixing assembly and a mobile phase capillary fixing assembly, and the structures of the two can be the same, or they can be adaptively adjusted according to the different structures of the chromatographic column and the mobile phase capillary. As a scheme, the fixing assembly includes: a positioning sleeve for positioning the chromatographic column or the mobile phase capillary and a locking member for fixing the sleeve and the probe body to each other. A positioning protrusion is provided on the outer wall of the positioning sleeve. The locking member can be a threaded locking member; for example, the locking member is a hollow member, one end of which is provided with an inner folding edge for clamping the positioning protrusion, and the other end is provided with an internal thread; in this case, an external thread that cooperates with the locking member can be provided at the channel interface corresponding to the probe body.

[0020] Preferably, the apparatus further comprises a sample chip for carrying the sample and a moving device for controlling the displacement of the sample chip. The position of the sample chip can be adjusted by using the moving device.

[0021] Preferably, all components of the integrated chromatographic analysis probe, including the probe body, mobile phase capillary, chromatographic column, and sample chip, are made of solvent-inert inorganic materials or high molecular polymer materials, such as metal, glass, quartz, polyetheretherketone, polytetrafluoroethylene, or composites of two or more of the above, to counteract negative effects such as swelling caused by long-term liquid contact.

[0022] Furthermore, the integrated chromatography analysis probe body is made of hard metal material, high molecular polymer material or other materials.

[0023] Preferably, in order to improve the sealing, stability, life and pressure resistance of the integrated chromatographic analysis probe, the material of the sample chip needs to be different from the material of the integrated chromatographic analysis probe body.

[0024] Preferably, the hardness of the probe body material corresponding to the sampling port is greater than or equal to the hardness of the sample chip material.

[0025] Preferably, the sample chip is made of a material that is less hard than the probe body material corresponding to the sampling port and has relative elasticity.

[0026] Furthermore, the integrated chromatographic analysis probe body and its components are processed from a whole piece of material in a subtractive processing mode to ensure the pressure resistance, structural strength and service life of the probe.

[0027] Furthermore, the integrated chromatography analysis probe is made of stainless steel or titanium alloy material and is processed using program-controlled automated machine tool (CNC) technology, so that the pressure resistance of the probe inner channel reaches more than 30 MPa, while improving the strength and service life of the integrated chromatography analysis probe and making it suitable for mass production.

[0028] The channel configuration in the integrated chromatographic analysis probe is a T-shaped structure or a Y-shaped structure, or a structure similar to the T-shaped structure or the Y-shaped structure. The bottom of the T-shaped structure or the Y-shaped structure is provided with a sampling port. The mobile phase channel is connected to the chromatographic column channel and is connected to the chromatographic column inlet, and the intersection is the sampling port. Further, the sampling port can be provided at the inlet of the chromatographic column.

[0029] According to the present invention, the inner diameter (diameter) or inner side length of the mobile phase channel, chromatographic column channel, chromatographic column, and sampling port on the integrated chromatographic analysis probe ranges from 0.05 micrometers to 5 millimeters. The diameter of the chromatographic column channel is greater than the outer diameter of the chromatographic column. Preferably, the difference between the diameter of the chromatographic column channel and the outer diameter of the chromatographic column is reduced to reduce the dead volume in the probe channel.

[0030] The mobile phase capillary fixing interface and the chromatographic column fixing interface are processed with external threads, or internal threads, or buckles, or a combination of the above types for use in sealing the capillary fixing assembly.

[0031] Preferably, the outer surface of the sampling port and the portion of the sample chip in contact with the outer surface of the sampling port are polished or otherwise planarized.

[0032] Preferably, the mobile phase capillary fixing interface and the chromatographic column fixing interface are processed with external threads. This solution combined with a split capillary fixing component processed with internal threads can provide a good sealing effect, so that the probe has a higher ability to withstand hydraulic pressure in the channel.

[0033] In the present invention, the chromatographic column tube and the mobile phase capillary are made of glass, quartz, metal, or high molecular polymer, or other materials. The above materials have tolerance to the liquid and mobile phase flowing in the probe channel.

[0034] Preferably, the mobile phase capillary is a quartz capillary with a coating on the surface, which has good pressure resistance, corrosion resistance and toughness.

[0035] In the present invention, the chromatographic column type is a packed column, or an open tubular column, or a chemically synthesized monolithic column, or a microfabricated monolithic column, or a composite configuration of the above configurations.

[0036] Preferably, the outlet of the chromatographic column is processed with an electrospray tip for connection with an electrospray mass spectrometry detection system, and the sample flows out through the tip at the outlet of the chromatographic column to form an electrospray.

[0037] According to the present invention, the sample chip is made of a material with a hardness lower than that of the probe body, and the material type is inorganic material, organic material, or high molecular polymer material.

[0038] According to the present invention, the hardness and elasticity matching between the integrated chromatography analysis probe material and the sample chip material needs to be screened and optimized to obtain a satisfactory sealing effect and service life.

[0039] Preferably, the sample chip is made of a relatively elastic material, which can significantly improve the sealing ability between the probe sampling port and the sample chip.

[0040] Preferably, the sample chip is made of polyetheretherketone (PEEK) material or a material with similar hardness and elasticity, and matched with an integrated chromatography analysis probe made of stainless steel or titanium alloy. While achieving a sealing effect that meets the analysis requirements of a high-performance liquid chromatography device (with a pressure resistance of more than 30 MPa), the number of uses and life of the sample chip are significantly improved.

[0041] According to the present invention, the shape of the sample chip is a circular sheet with thickness, a square sheet with thickness, a polygonal sheet with thickness, a cylinder, a cube, a cylinder with steps, a cube with steps, or other three-dimensional shapes.

[0042] Preferably, the sample chip is made by one-time CNC processing and has a cylindrical shape with steps, which facilitates the fixation of the sample chip and the positioning of the pits.

[0043] According to the present invention, the sample chip is processed with one or more pits for carrying trace samples, the volume range of the pits is 1 attoliter to 1 milliliter; the diameter range of the pits is 1 micron to 10 centimeters; the depth range of the pits is 1 micron to 1 centimeter.

[0044] Furthermore, the sample-carrying pits on the sample chip are cylindrical, cross-shaped, hemispherical, or other shaped microstructures, which play a role in fixing the position of the sample on the surface of the sample chip.

[0045] According to the present invention, the initial state of the sample carried by the sample chip is a liquid sample, a solid sample or a solid-liquid mixed sample, and the final state of the sample can be a liquid sample or a solid-liquid mixed sample or a solid sample formed after the liquid sample is dried.

[0046] According to the present invention, a moving device for controlling the displacement of the sample chip drives the sample chip to contact and squeeze the outer surface of the sampling port of the integrated chromatographic analysis probe, and the squeezing force is used to achieve sealing between the sample chip and the outer surface of the sampling port of the integrated chromatographic analysis probe.

[0047] Further, the extrusion force acting on the sample chip and the probe ranges from 0.01 kg to 100 kg. Preferably, according to actual experimental needs, the extrusion force output by the pressure driving device is adjusted by theoretical calculation to the highest hydraulic pressure of the fluid driving device when the system does not leak.

[0048] Preferably, the outer surface of the sampling port of the integrated chromatographic analysis probe and the portion of the sample chip in contact with the outer surface of the sampling port of the integrated chromatographic analysis probe are polished or otherwise flattened to ensure airtightness between the sample chip and the integrated chromatographic analysis probe.

[0049] According to the present invention, the operation of bringing the sample chip into contact with the outer surface of the sampling port of the integrated chromatographic analysis probe and forming an extrusion seal can also be carried out by using a moving device to drive the integrated chromatographic analysis probe to contact the sample chip, or by using a moving device to simultaneously drive the integrated chromatographic analysis probe and the sample chip toward each other to achieve contact and extrusion sealing between the two.

[0050] Preferably, the driving device of the integrated chromatography analysis probe and the sample chip is machined with precision grooves for fixing the sample chip; or, the driving device has a mechanical structure, an adhesive structure, or an integrated sample chip for precise positioning of the sample holding area.

[0051] According to the present invention, when the sample chip contacts the outer surface of the sampling port of the integrated chromatographic analysis probe and forms an extrusion seal, the inlet of the chromatographic column in the probe is higher than, flush with, or lower than the sealing surface of the sample chip.

[0052] Preferably, when the sample chip contacts the outer surface of the sampling port of the integrated chromatographic analysis probe and forms an extrusion seal, the inlet of the chromatographic column in the probe is lower than the chip sealing surface, that is, the inlet end of the chromatographic column is inserted into the pit of the sample chip, which is beneficial to reduce the dead volume, speed up the elution rate of the sample in the pit, and improve the analysis speed.

[0053] Preferably, the sample chip is processed with one or more pits for carrying trace samples.

[0054] A chromatographic analysis device comprises any one of the above-mentioned integrated chromatographic analysis probe components, a fluid driving device and a detection device.

[0055] The mobile phase channel is connected to a fluid driving device providing a chromatographic mobile phase through a mobile phase capillary to control the mobile phase to flow into an integrated chromatographic analysis probe, and the chromatographic column outlet is connected to a detection device;

[0056] Preferably, when the outlet of the chromatographic column is connected to an electrospray mass spectrometry detection device, a mass spectrometry electrospray nozzle tip is integrated and processed at the outlet of the chromatographic column to realize the integration of four functions of chromatographic sampling, injection, separation, and mass spectrometry electrospray on the probe.

[0057] A method for performing chromatographic analysis using the integrated chromatographic analysis probe assembly described in any one of the above technical solutions comprises:

[0058] (1) moving the sample chip carrying the sample to the target position, aligning the sampling port of the integrated chromatographic analysis probe assembly with the sample position, and making the outer surface of the sampling port contact with the sample chip to form an extrusion seal;

[0059] (2) using a fluid driving device to introduce the mobile phase into the mobile phase inlet gap of the chromatographic column channel through the mobile phase capillary and the mobile phase channel, and then to the sampling port, carrying the sample on the sample chip into the chromatographic column;

[0060] (3) Driven by the subsequent mobile phase, the sample components entering the chromatographic column are separated in the chromatographic column and flow out from the chromatographic column outlet into the detector for detection.

[0061] The present invention is suitable for use in fields requiring rapid and high-throughput chromatographic analysis of trace samples, such as single cell analysis, multidimensional chromatographic analysis, ultra-micro sample analysis, and high-throughput screening, and is also suitable for on-site analysis requiring a small volume or portable chromatographic analysis device.

[0062] A method for using an integrated chromatographic analysis probe assembly, comprising:

[0063] (a) adding a sample into a recess of a sample chip and directly performing a subsequent sample injection operation, or performing an in-situ pretreatment of the sample in the recess;

[0064] (b) fixing the sample chip on a moving device for controlling the displacement of the sample chip;

[0065] (c) using a moving device for controlling the displacement of the sample chip to drive the sample chip so that the sampling port of the integrated chromatographic analysis probe is accurately aligned with a recess on the sample chip carrying the target sample, and then the outer surface of the sampling port of the integrated chromatographic analysis probe is brought into contact with the sample chip to form an extrusion seal;

[0066] (d) using a fluid driving device to introduce a mobile phase into the integrated chromatographic analysis probe through a mobile phase capillary and a mobile phase channel inlet, and the mobile phase flows through a probe sampling port, carries a liquid sample in a recess of the sample chip or dissolves and elutes a solid sample in the recess, and enters an inlet of a chromatographic column;

[0067] (e) driven by the subsequent mobile phase, the sample components entering the chromatographic column are separated in the chromatographic column and flow out from the chromatographic column outlet into the detector for detection;

[0068] (f) Repeat steps (a) to (e) to complete the sampling, injection and analysis of different samples on the sample chip, or samples on different sample chips, or different spatial locations of the same sample.

[0069] Preferably, the extrusion force acting on the sample chip and the integrated chromatography analysis probe ranges from 0.01 kg to 100 kg. Preferably, the gas pressure generated by the sample chip moving device (for example, the gas pressure generated by the air pump configured in the sample chip moving device) is used to drive the sealing operation of the sample chip and the integrated chromatography analysis probe sampling port; or the sample chip is fixed on a bracket with a certain elasticity on the moving device that controls the displacement of the sample chip, so as to compensate for the deformation of the sample chip in real time, so as to achieve the sealing operation of the sample chip and the integrated chromatography analysis probe sampling port. Both of the above methods can compensate for the pressure changes caused by the deformation of the sample chip in real time, and provide continuous thrust to ensure the long-term sealing of the sample chip and the integrated chromatography analysis probe sampling port.

[0070] Preferably, when the sample chip contacts the outer surface of the sampling port of the integrated chromatographic analysis probe and forms an extrusion seal, the inlet of the chromatographic column in the probe is higher, flush, or lower than the sealing surface of the sample chip. Preferably, the inlet of the chromatographic column in the probe is lower than the chip sealing surface, that is, the inlet end of the chromatographic column is inserted into the concave pit of the sample chip to reduce dead volume and improve analysis speed.

[0071] According to the present invention, when analyzing a sample, after adding a liquid sample or a solid sample to the pit of the sample chip, the sample chip is installed in a positioning device of a moving device that controls the displacement of the sample chip, in order to achieve precise positioning of the sample chip pit relative to the integrated chromatography analysis probe sampling port.

[0072] Furthermore, after adding liquid samples or solid samples to the pits of the sample chip, a multi-step, complex pretreatment operation is performed in the pits, including pretreatment reagent addition operations and reaction steps, and then the sample chip is installed in a positioning device of a moving device that controls the displacement of the sample chip.

[0073] Preferably, for ultra-trace biological samples with complex compositions, such as single cells, subcellular organelles, biological tissue samples, or other trace samples that require pre-processing operations, the method of adding the original sample to the pit, performing in-situ sample pre-treatment in the pit, and performing subsequent in-situ sampling can greatly reduce the loss of samples due to transfer during pre-treatment and sampling, and even achieve completely lossless sampling of all samples, thereby improving sample utilization and detection sensitivity.

[0074] Preferably, in order to reduce the dead volume inside the integrated chromatographic analysis probe device, when making the chromatographic column, the stationary phase is filled in the chromatographic column, the chromatographic column channel connected thereto, and even the probe sampling port, which helps to reduce the band broadening and dilution effect caused by the sample entering the sampling port and the interior of the chromatographic column.

[0075] According to the present invention, the integrated chromatography analysis probe sampling port contacts the sample chip and achieves sealing operation, and a tight fit can be achieved by utilizing the thrust provided by a pressure drive device, or the thrust propelled by a motor, or by threaded tightening, or by snap-on locking, or other methods.

[0076] Preferably, the air pressure generated by the air pump configured in the sample chip moving device is used to drive the airtight operation of the sample chip and the integrated chromatographic analysis probe sampling port, which can provide continuous thrust to ensure the long-term airtightness of the sample chip and the integrated chromatographic analysis probe sampling port. Because the sample chip has a certain degree of deformability, it is easy to deform during a long period of extrusion, which will cause the pressure between the two to decrease, the airtightness to deteriorate, and the fluid to flow out or leak from the closed interface. When using methods such as motor-propelled thrust, it is difficult to compensate for the pressure changes caused by the deformation of the sample chip in real time, and leakage of the interface is likely to occur.

[0077] Preferably, the sample chip is fixed on a bracket with a certain elasticity on a moving device for controlling the displacement of the sample chip, and the elasticity of the bracket can be used to compensate for the pressure change caused by the deformation in real time.

[0078] As a further preference, by using a smaller contact area between the sampling port and the sample chip, the size of the squeezing force can be significantly reduced, thereby reducing the loss of the sample chip material and the integrated chromatographic analysis probe body material.

[0079] According to the present invention, the sealing effect achieved by the integrated chromatography analysis probe sampling port and the sample chip is positively correlated with the ultimate pressure resistance performance of the system. Improving the ultimate pressure resistance performance can make the device suitable for chromatographic columns with finer inner diameters, finer filler particle sizes, and longer lengths but higher back pressures, which is beneficial to improving the separation efficiency, separation degree and detection sensitivity of the samples.

[0080] According to the present invention, during the sampling and injection process, the fluid driving device controls the mobile phase to be injected into the integrated chromatographic analysis probe. When flowing through the pits carrying the samples, the mobile phase is used to carry all or most of the samples into the chromatographic column for separation and analysis at one time, or the mobile phase gradually elutes the samples carried on the pits into the chromatographic column for separation and analysis, or mobile phases of different compositions and concentrations are used to gradually elute components of different properties carried on the pits into the chromatographic column for separation and analysis.

[0081] According to the present invention, the composition, concentration and flow rate of the mobile phase for carrying or eluting the sample into the chromatographic column may be the same as or different from the mobile phase conditions used in the subsequent separation and analysis. The main consideration is how to make the device obtain a better sampling effect. Alternatively, a gas mobile phase can be used to complete the sampling and injection operations, and a liquid mobile phase can be used for the separation operation.

[0082] According to the present invention, preferably, the sample chip can be reused. After completing the sampling, injection and separation analysis of a sample, before analyzing the next sample or the next batch of samples, a cleaning solution is used to thoroughly clean the surface of the sample to reduce cross contamination between different samples.

[0083] According to the present invention, the detection method adopted by the detection device of the chromatographic system is optical analysis, or electrochemical analysis, or mass spectrometry, or a combination of two or more of the above detection methods. The optical analysis method includes ultraviolet visible spectrophotometry, fluorescence spectroscopy, infrared spectroscopy, Raman spectroscopy, nuclear magnetic resonance spectroscopy, optical imaging analysis, optical sensor analysis, atomic spectroscopy, etc. The electrochemical analysis method includes potentiometric analysis, electrolysis and coulometric analysis, voltammetry, conductivity, electrochemical sensor analysis, etc. The mass spectrometry includes electron bombardment mass spectrometry, field desorption mass spectrometry, fast atom bombardment mass spectrometry, matrix-assisted laser desorption mass spectrometry, electrospray mass spectrometry, in-situ ionization mass spectrometry, inductively coupled plasma mass spectrometry, etc.

[0084] According to the present invention, an array injection system composed of multiple integrated chromatographic analysis probes is used to perform sample analysis in parallel, which is beneficial to improving the analysis throughput of the system;

[0085] According to the present invention, the integrated chromatography analysis probe is suitable for liquid chromatography analysis, gas chromatography analysis, or supercritical fluid chromatography analysis of trace samples.

[0086] It should be noted that the application field of the integrated chromatography analysis probe is not limited to the field of chromatography analysis, but can also be applied to other flow analysis fields, such as direct injection ionization mass spectrometry, microfluidics analysis, flow injection analysis, continuous flow analysis, capillary electrophoresis analysis, bubble interval flow analysis, or the combined use of the above analysis systems for analysis.

[0087] The main advantages of the present invention include: the integrated chromatographic analysis probe integrates the three main steps of sampling, injection, and separation in chromatographic analysis, combines the advantages of microfluidic chips and probe injection methods, and directly covers the sample area sealed on the sample chip for trace samples with small volume, complex composition, and low concentration, thereby avoiding the loss of trace samples due to transfer and transportation. The integrated chromatographic analysis probe device has a small volume, short pipeline, small dead volume, and fast injection speed, which can significantly improve the analysis throughput. The integrated probe is a detachable design, and the parts are easy to replace, which helps to extend the service life of the integrated chromatographic analysis probe body and reduce costs. Using the device described in the present invention, sample pretreatment is completed in situ on the sample chip, and the combination of in-situ pretreatment and in-situ analysis can be achieved.

[0088] The main advantages of the present invention are:

[0089] (1) The device has high integration, high strength and hardness, simple structure, small size, suitable for batch processing, and easy operation. The sampling, injection, and separation functions in the chromatographic analysis process are integrated into the chromatographic analysis probe, shortening the pipeline length of the injection module and the distance between the sample and the chromatographic column inlet, significantly shortening the sampling and injection time, and improving the analysis speed and analysis throughput;

[0090] (2) The device adopts the strategy of atmospheric pressure in-situ reactor pretreatment-high pressure integrated chromatographic analysis probe sealed injection analysis, realizing the combination of sample pretreatment and online separation analysis, and realizing zero transfer of the entire process from sample pretreatment to injection, solving the sample transfer problem that is difficult to solve by the automatic sample injector of the conventional chromatographic system and the problem of micro-volume and ultra-small volume sample injection, minimizing the loss of micro-volume samples during transfer and transportation in the pipeline, and improving the analytical sensitivity;

[0091] (3) The integrated chromatographic analysis probe is small in size and suitable for automation, and proposes a solution for miniaturization of conventional liquid chromatography systems; by further reducing the size of the sampling port, it can be applied to high spatial resolution in situ imaging analysis of biological, environmental, and cultural relic samples;

[0092] (4) The ultra-high strength and hardness of the integrated probe and the extremely high pressure resistance of the device have successfully adapted to the common ultra-high pressure liquid chromatography pumps on the market, providing favorable conditions for the promotion and adaptation of the device and method.

[0093] (5) Wide applicability: The system of the present invention is expected to achieve important applications in the fields of multidimensional chromatography analysis of trace complex samples, high-throughput drug screening analysis, single-cell omics analysis, biological tissue imaging analysis, spatial multi-omics analysis, and on-site analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 It is a schematic diagram of the structure of the integrated chromatographic analysis probe assembly made of polyetheretherketone in Example 1.

[0095] Figure 2a It is a schematic diagram of the structure of the integrated chromatographic analysis probe made of stainless steel in Example 2.

[0096] Figure 2b It is a schematic diagram of the sample carrying structure on the various sampling ports and sample chips used in Example 2 and Example 4.

[0097] Figure 3 This is the result of liquid chromatography-electrospray mass spectrometry analysis of a mixed solution sample containing four standard peptide segments in gradient elution mode using the analysis device of Example 2.

[0098] Figure 4 It is a schematic diagram of the structure of the integrated chromatographic analysis probe of Example 4 which is processed in a Y-shaped configuration.

[0099] In the above drawings, 1-integrated chromatographic analysis probe body, 2-mobile phase channel, 3-sampling port, 4-chromatographic column, 5-chromatographic column channel, 6-mobile phase capillary fixing interface, 7-chromatographic column fixing interface, 8-capillary fixing assembly, 9-chromatographic column outlet, 10-sample chip, 11-pressure moving device for controlling displacement of the sample chip, 12-sample, 13-detection device, 14-fluid driving device, 15-mobile phase capillary, 16-sample chip pit. DETAILED DESCRIPTION

[0100] The technical solution of the present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0101] With reference to the accompanying drawings, preferred embodiments according to the present invention will be described in detail below.

[0102] Example 1

[0103] Figure 1It is a schematic diagram of the structure of the integrated chromatographic analysis probe assembly established according to the present invention. It is composed of a probe body 1 with a mobile phase channel 2 and a chromatographic column channel 5, one end of the chromatographic column channel is a sampling port 3; a mobile phase capillary 15 arranged on the probe body and connected to the mobile phase channel; a chromatographic column 4 detachably fixed in the chromatographic column channel, the inlet of the chromatographic column corresponds to the sampling port; a mobile phase inlet gap is left between the chromatographic column channel and the outer wall of the chromatographic column, and the gap is simultaneously connected to the mobile phase channel outlet, the chromatographic column inlet and the sampling port. The mobile phase channel and the chromatographic column channel are both provided with fixed interfaces at one end, namely a mobile phase capillary fixed interface 6 and a chromatographic column fixed interface 7, the mobile phase capillary fixed interface 6 is provided with a capillary fixed component 8 for fixing the mobile phase capillary 15, and the chromatographic column fixed interface 7 is provided with a chromatographic column fixed component 8a for fixing the chromatographic column 4.

[0104] The capillary fixing assembly 8 and the chromatographic column fixing assembly 8a have similar structures, both of which include a positioning sleeve for positioning the chromatographic column or mobile phase capillary and a locking member for fixing the sleeve to the probe body. The outer wall of the positioning sleeve is provided with a positioning protrusion. The locking member can be a threaded locking member; in the embodiment, the locking member is a hollow member, that is, a sleeve structure, one end of which is provided with an inner folding edge that is clamped with the positioning protrusion, which is used to limit the positioning sleeve axially, and the other end of the hollow member is provided with an internal thread; at this time, an external thread that cooperates with the locking member can be provided at the fixed interface corresponding to the probe body. Figure 1 In the embodiment, the locking piece of the capillary fixing assembly 8 is in a locked state, the locking piece of the chromatographic column fixing assembly 8a is in an open state, and the chromatographic column 4 is also in a state of not being installed in place.

[0105] The probe body 1 is made of a polyetheretherketone monolithic material, and in actual use, it can also be matched with a sample chip 10 and a pressure moving device 11 for fixing the sample chip 10 (see Figure 2a ), and can also be matched with a liquid chromatography pump fluid driving device 14 and an electrospray mass spectrometer as the detection device 13. Figure 2a The arrow in the middle shows the flow direction of the chromatographic mobile phase.

[0106] A homemade chromatographic column with an inner diameter of 75 microns and an outer diameter of 360 microns and an integrated nozzle tip is fixed in the chromatographic column channel of the probe body 1. A quartz mobile phase capillary 15 with an inner diameter of 75 microns, which is sealed and connected to the mobile phase channel, is also fixed on the probe body 1. The two are fixed by fixing the capillary fixing component 8 and the chromatographic column fixing component 8a respectively.

[0107] In this embodiment, the pressure moving device 11 may be a three-dimensional moving platform, or a moving platform with an air pump (which may be a common one-dimensional moving platform, two-dimensional moving platform or three-dimensional moving platform), etc. The fluid driving device may be a chromatographic pump.

[0108] When processing the integrated chromatographic analysis probe body, polyetheretherketone is used as the probe manufacturing material, and CNC technology is used for processing, so that the internal chromatographic column channel and the mobile phase channel are in a T-shaped structure, and a capillary fixing interface 6 for fixing the mobile phase capillary and a chromatographic column fixing interface 7 for fixing the chromatographic column 4 are processed; a cylindrical chromatographic column channel 5 with a diameter of 400 microns and a length of 9 mm for installing the chromatographic column 4 is processed at the outlet of the tube chromatographic column fixing interface and is connected to the sampling port. A circular sampling port is processed at the outlet of the chromatographic column channel, and a conical cavity is processed in the center. When using the integrated chromatographic analysis probe, the circular surface of the sampling port is the contact surface, which is in a position where the mobile phase cannot flow through; the conical cavity is used to cover the sample, and is in a position where the mobile phase flows through, which is easy to clean to avoid cross contamination.

[0109] The two-ended cylindrical sample chip is made of polyetheretherketone material and adopts a single-hole chip solution. The pit used to carry the sample is processed on the top, and the pit has a diameter of 700 microns and a depth of 300 microns.

[0110] The specific method of using the device of Example 1 is as follows: (1) fix the sample chip 10 in a pressure moving device 11 with a groove; (2) start the air pump, and the pressure moving device carries the sample chip to the sampling port 3 of the integrated chromatographic analysis probe, so that the sampling port and the sample carrying surface of the sample chip are attached and sealed; (3) keep the sealing between the sampling port of the integrated chromatographic analysis probe and the sample chip, start the fluid driving device 14, i.e., the liquid chromatography pump, and continuously inject the chromatographic mobile phase along the mobile phase channel 2 of the integrated chromatographic analysis probe assembly into the mobile phase inlet gap in the integrated chromatographic analysis probe body, and reach the sampling port and enter the chromatographic column.

[0111] After a mobile phase with a pressure of 30 MPa was injected into the system for 8 hours, no obvious mobile phase leakage was observed near the sampling port 3, the mobile phase channel fixed interface (mobile phase capillary fixed interface 6), and the chromatographic column fixed interface 7, proving that the device of this embodiment maintained good sealing stability during a long period of high-pressure mobile phase experiment.

[0112] Example 2

[0113] Figure 2a Embodiment 2 is a schematic diagram of the structure of an integrated chromatographic analysis probe made of stainless steel material, which is used to improve the system strength and pressure resistance; the chromatographic column and mobile phase channel are replaced with a thinner quartz capillary with an inner diameter of 50 microns. A pit 16 is processed on the sample chip 10.

[0114] The specific method of using the device of Example 2 is as follows: (1) fix the sample chip 10 in the pressure moving device 11 with a groove; (2) start the air pump, and the pressure moving device carries the sample chip to the sampling port 3 of the integrated chromatographic analysis probe, so that the sampling port and the sample carrying surface of the sample chip are attached and sealed; (3) keep the sealing between the sampling port of the integrated chromatographic analysis probe and the sample chip, start the liquid chromatography pump of the fluid driving device 14, and move the chromatographic mobile phase along the mobile phase of the integrated chromatographic analysis probe. The channel continuously injects the integrated chromatographic analysis probe, and flows through the mobile phase inlet gap in the chromatographic column channel to reach the sampling port, carrying the sample 12 into the chromatographic column; (3) adjusting the mobile phase ratio delivered by the liquid chromatography pump of the fluid driving device, and the mobile phase flows through the sample to separate it on the chromatographic column; (4) starting the electrospray mass spectrometer detection device, the sample separated on the chromatographic column and reaching the chromatographic column outlet 9 is ionized under the action of the electric field, and enters the mass spectrometer detection device for detection; (5) repeating steps (1)1 to (4) to complete the sampling, injection and analysis of samples in different sample chips.

[0115] Using the chromatographic analysis device and the method of using the same of Example 2, the mobile phase pressure of the injection device was gradually increased to 120 MPa within one minute, and then the mobile phase pressure of the device was gradually reduced to 0 MPa within one minute. No obvious mobile phase leakage was observed near the sampling port, the mobile phase channel fixed interface, and the chromatographic column fixed interface, which proved that the device maintained good sealing stability during the short-term ultra-high pressure mobile phase experiment process, and the system had an ultimate pressure resistance capability of at least 120 MPa of mobile phase pressure.

[0116] Figure 2b Schematic diagram of various sampling ports and sample carrying structures on sample chips that can be used in Embodiment 2 and Embodiment 4. Figure 2b In the configuration b1, the probe is a T-shaped structure as a whole, the sampling port 3 is an inverted cone, the pit 16 on the sample chip 10 is cylindrical, and the inlet end of the capillary chromatographic column 4 is flush with the upper surface of the sample chip 10;

[0117] Figure 2b The configuration of b2 is that the probe is a T-shaped structure as a whole, the probe sampling port 3 is a circular tube shape which is the same as the chromatographic column channel 5, the pit 16 on the sample chip 10 is a cylindrical shape, and the inlet end of the capillary chromatographic column 4 is flush with the upper surface of the sample chip 10;

[0118] Figure 2b In the configuration b3, the probe is a T-shaped structure as a whole, the probe sampling port 3 is a configuration with a minimum dead volume, the chromatographic column channel 5 is a tapered cone, the pit 16 on the sample chip 10 is a hemispherical shape, and the inlet end of the capillary chromatographic column 4 is flush with the upper surface of the sample chip 10;

[0119] Figure 2b In the configuration of b4, the probe is a T-shaped structure as a whole, the sampling port 3 of the probe is an inverted cone, the pit 16 on the sample chip 10 is cylindrical, and the inlet end of the capillary chromatographic column 4 is lower than the upper surface of the sample chip 10, that is, the capillary chromatographic column 4 is inserted into the pit 16 on the sample chip 10 to reduce the dead volume of injection;

[0120] Figure 2b In the configuration of b5, the probe is a T-shaped structure as a whole, the sampling port 3 of the probe is an inverted cone, the sample chip 10 is not processed with a pit 16, the surface of the sample chip 10 is directly used to carry the sample, and the inlet end of the capillary chromatographic column 4 is slightly higher than the upper surface of the sample chip 10;

[0121] Figure 2b In the configuration of b6, the probe is a T-shaped structure as a whole, the sampling port 3 of the probe is an inverted cone, the pit 16 on the sample chip 10 is a cone, and the inlet end of the capillary chromatographic column 4 is flush with the upper surface of the sample chip 10;

[0122] Figure 2b The configuration of b7 is that the probe is a V-shaped structure as a whole, the sampling port 3 of the probe is an inverted cone, the pit 16 on the sample chip 10 is cylindrical, and the capillary chromatographic column 4 is tilted, and its inlet end is substantially flush with the upper surface of the sample chip 10;

[0123] exist Figure 2b In b1, b2, b3, b4, b5, b6, and b7, the diameter or side length of the probe sampling port 3 is greater than the diameter or side length of the pit 16 on the sample chip 10.

[0124] Figure 2b The configuration of b8 is that the probe is a V-shaped structure as a whole, the probe sampling port 3 is a V-shaped channel configuration, the pit 16 on the sample chip 10 is cylindrical, the side length of the probe sampling port 3 is equal to the diameter of the pit 16 on the sample chip 10, and the capillary chromatographic column 4 is tilted, and its inlet end is far from the upper surface of the sample chip 10;

[0125] Figure 2b The configuration of b9 is that the probe is a V-shaped structure as a whole, the probe sampling port 3 is a V-shaped channel configuration, the pit 16 on the sample chip 10 is cylindrical, the diameter of the pit 16 is larger than the side length of the sampling port 3, the capillary chromatographic column 4 is tilted, and its inlet end is far away from the upper surface of the sample chip 10.

[0126] Figure 3 The chromatographic analysis device and the method for using the same in Example 2 are used to achieve the liquid chromatography-electrospray mass spectrometry analysis results of a mixed solution sample containing four standard peptide segments in a gradient elution mode.

[0127] Specifically, the mixed solution sample of the four standard peptides contained 10 ng / μL of each of the four peptides, the sample droplet volume was 200 nanoliters, and six injections were performed in parallel. The chromatographic mobile phases used were: (1) mobile phase A: aqueous solution containing 0.01% formic acid (phase A); (2) mobile phase B: acetonitrile solution containing 0.01% formic acid (phase B). The sampling mobile phase ratio was 100% mobile phase A, the time was 4.5 minutes, and it was combined in the elution gradient. The mobile phase gradient of all chromatograms is: 0% mobile phase B (the rest 100% is mobile phase A) from 0 to 4.5 minutes; 0% mobile phase B (the rest 100% is mobile phase A) from 4.5 to 9 minutes; 50% mobile phase B (the rest 100% is mobile phase A) from 9 to 10 minutes; 50% mobile phase B (the rest 50% is mobile phase A) from 10 to 14 minutes; 65% mobile phase B (the rest 35% is mobile phase A) from 10 to 14.1 minutes; 65% mobile phase B (the rest 35% is mobile phase A) from 14.1 to 15 minutes; the electrospray flow rate in the mass spectrometer detection device is 1.9 microliters per minute, and the spray voltage is 2.2 kilovolts.

[0128] In the six mixed sample separation analyses performed using the method in this embodiment, the relative standard deviations of the retention times of the four peptides were 0.5%, 0.6%, 0.8%, and 0.3%, respectively, and the relative standard deviations of the peak areas of the four peptides were 4.9%, 9.7%, 6.0%, and 8.7%, respectively, demonstrating that the device has good stability and reproducibility when analyzing simple mixed samples.

[0129] The chromatographic analysis device and the method of use of Example 2 were used to implement liquid chromatography-electrospray mass spectrometry analysis of trace HeLa protein digests.

[0130] Specifically, the sample concentration of the HeLa protein digest solution used was 1 ng / μl, and a sample droplet of 200 nanoliters was accurately added to the pit on the sample chip, and five parallel injections were continuously performed, and the control group was set to pure water. The analysis time was 40 minutes, and the chromatographic mobile phases used were: mobile phase A: aqueous solution containing 0.01% formic acid (phase A); mobile phase B: 20% water-80% acetonitrile solution containing 0.01% formic acid (phase B). The electrospray flow rate in the mass spectrometer detection device was 100 nanoliters per minute, and the spray voltage was 1.75 kilovolts.

[0131] The five HeLa protein digestion sample analyses performed using the method in this example identified an average of 3909 peptides and 866 proteomes, and a total of 620 proteins were detected in five consecutive analyses, accounting for 50.4% of the total number of identifications; in comparison, the commercial autosampler identified an average of 3481 peptides and 842 proteomes in five parallel analyses. The results show that the number of proteomes identified using the method in this example is slightly higher than that of the autosampler, and the time consumed for the analysis is only half of that of the commercial autosampler, proving that the device is suitable for the analysis of trace complex mixed samples and has advantages in throughput.

[0132] Example 3

[0133] By using the integrated chromatography analysis probe and the use method in the preferred embodiment 2, the combination of in situ 293 single cell pretreatment on the sample chip and in situ liquid chromatography-electrospray mass spectrometry separation and analysis is achieved.

[0134] Specifically, six single 293 cells were sequentially aspirated and added to the wells of the sample chip array, and pre-treatment steps such as cell rupture and protein enzymatic hydrolysis were performed by sequentially adding cell lysis reagent RapiGest, protein reduction reagent TCEP, protein alkylation reagent IAA, and protein enzymatic hydrolysis reagent Trypsin and Lys-C to the wells. The sample chip was then taken out and transferred to the pressure-driven device for analysis. The analysis time was 40 minutes.

[0135] The six single-cell analyses performed using the method in this example identified a maximum of 1508 peptides and 256 proteomes, with a total of 3140 peptides and 418 proteomes. The proteomes identified in all groups accounted for 14.4% of the total. This proves that the device can analyze biological samples at the single-cell level and has the potential for single-cell proteomics research.

[0136] Example 4

[0137] Figure 4 Schematic diagram of an integrated chromatographic analysis probe processed with a Y-shaped configuration in Example 4. The system consists of an integrated chromatographic analysis probe body 1 made of a hard material with a Y-shaped configuration, a pressure moving device 11 for fixing a sample chip 10 and providing a high squeezing force, a fluid driving device 14 (liquid chromatography pump), and a detection device 13 (electrospray mass spectrometer). Figure 4 The arrow in the middle shows the flow direction of the chromatographic mobile phase. The other numbers are the same as Figure 1 And Figure 2, see Example 1 and Example 2.

[0138] When processing the Y-type integrated chromatographic analysis probe body, CNC technology is used for processing, and the internal chromatographic column channel and mobile phase channel thereof are in a Y-type structure; a cylindrical chromatographic column channel 5 with a diameter of 400 microns and a length of 5 mm for installing the chromatographic column 4 is processed at the outlet of the capillary fixed interface for fixing the chromatographic column and is connected to the sampling port. The use of a shorter chromatographic column channel is conducive to reducing the dead volume of the device and reducing the band broadening and dilution effect caused by the sample entering the sampling port and the interior of the chromatographic column. When using the integrated chromatographic analysis probe, the assembled Y-type integrated chromatographic analysis probe is tilted, the sampling port is vertically downward, and the probe is fixed, and the axis of the sampling port is aligned with the axis of the sample chip.

[0139] The specific method of using the device of Example 4 is as follows: (1) fix the sample chip in a pressure-driven device with a groove; (2) start the air pump, and the pressure-driven device carries the sample chip to the sampling port of the integrated chromatographic analysis probe, so that the sampling port and the sample carrying surface of the sample chip are attached and sealed; (3) keep the sealing between the sampling port of the integrated chromatographic analysis probe and the sample chip, start the liquid chromatography pump of the fluid-driven device, and continuously inject the chromatographic mobile phase into the integrated chromatographic analysis probe along the mobile phase channel of the integrated chromatographic analysis probe, and flow through the chromatographic column channel to reach the sampling port, carrying the sample into the chromatographic column; (3) adjust the proportion of the mobile phase delivered by the liquid chromatography pump of the fluid-driven device, and the mobile phase flows through the sample to separate it on the chromatographic column; (4) start the electrospray mass spectrometer detection device, and the sample separated on the chromatographic column and reaching the outlet of the chromatographic column is ionized under the action of the electric field and enters the mass spectrometer detection device for detection; (5) repeat steps (1) to (4) to complete the sampling, injection and analysis of samples in different sample chips.

Claims

1. An integrated chromatographic analysis probe assembly, characterized in that: include: A probe body with a mobile phase channel and a chromatographic column channel, one end of the chromatographic column channel being a sampling port; A mobile phase capillary disposed on the probe body and communicating with the mobile phase channel; A chromatographic column detachably fixed in the chromatographic column channel, the inlet of the chromatographic column corresponding to the sampling port; A mobile phase inlet gap is left between the chromatographic column channel and the outer wall of the chromatographic column, and the gap is connected to the mobile phase channel outlet and the sampling port at the same time; The channel configuration in the integrated chromatographic analysis probe is a T-shaped structure or a Y-shaped structure, and a sampling port is provided at the bottom of the T-shaped structure or the Y-shaped structure; One end of the mobile phase channel and the chromatographic column channel is provided with a fixed interface, and a fixing component used to fix the mobile phase capillary and the chromatographic column to the corresponding fixed interface respectively.

2. The integrated chromatography analysis probe assembly according to claim 1, characterized in that: The invention also comprises a sample chip for carrying the sample and a moving device for controlling the displacement of the sample chip.

3. The integrated chromatography analysis probe assembly according to claim 2, characterized in that: The hardness of the probe body material corresponding to the sampling port is greater than or equal to the hardness of the sample chip material.

4. The integrated chromatography analysis probe assembly according to claim 3, characterized in that: The sample chip is made of a material that is less hard than the probe body material corresponding to the sampling port and has relative elasticity.

5. The integrated chromatography analysis probe assembly according to claim 2, characterized in that: The outer surface of the sampling port and the portion of the sample chip in contact with the outer surface of the sampling port are polished or otherwise flattened.

6. The integrated chromatography analysis probe assembly according to claim 2, characterized in that: The sample chip is processed with one or more pits for carrying trace samples.

7. A chromatographic analysis device, characterized in that: It comprises the integrated chromatographic analysis probe assembly as claimed in any one of claims 1 to 6, a fluid driving device and a detection device.

8. A method for performing chromatographic analysis using the chromatographic analysis device according to claim 7, characterized in that: include: (1) moving the sample chip carrying the sample to the target position, aligning the sampling port of the integrated chromatographic analysis probe assembly with the sample position, and making the outer surface of the sampling port contact with the sample chip to form an extrusion seal; (2) using a fluid driving device to introduce the mobile phase into the mobile phase inlet gap of the chromatographic column channel through the mobile phase capillary and the mobile phase channel, and then to the sampling port, carrying the sample on the sample chip into the chromatographic column; (3) Driven by the subsequent mobile phase, the sample components entering the chromatographic column are separated in the chromatographic column and flow out from the outlet of the chromatographic column into the detection device for detection.

9. The method for performing chromatographic analysis using the integrated chromatographic analysis probe assembly according to claim 8, characterized in that: In step (1), the inlet of the chromatographic column is lower than the sealing surface of the sample chip, that is, the inlet end of the chromatographic column is inserted into the recess of the sample chip.

10. The method for performing chromatographic analysis using the integrated chromatographic analysis probe assembly according to claim 8, characterized in that: The gas pressure generated by the sample chip moving device is used to drive the sealing operation of the sample chip and the sampling port of the integrated chromatographic analysis probe assembly, or it also includes an elastic member for sealing the sample chip and the sampling port of the integrated chromatographic analysis probe assembly.

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