CN117110484A - Sample injection probe device based on membrane auxiliary strategy and analysis method - Google Patents
Sample injection probe device based on membrane auxiliary strategy and analysis method Download PDFInfo
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Abstract
The invention discloses a sample injection probe device and a sample injection method based on a membrane auxiliary strategy. The device adopts a membrane auxiliary strategy to realize in-situ pretreatment, sample introduction, transmission, separation and detection of micro complex samples under the pressure condition. The main advantages of the invention include: the system integrates the pretreatment of the micro-sample with the sample introduction, chromatographic separation and detection systems, has simple structure, rapid and automatic operation, high flexibility, convenient use and wide applicability, and is particularly suitable for analyzing the micro-sample. The sample can be introduced after in-situ pretreatment based on the membrane auxiliary strategy, a new sample introduction mode is provided for analysis of complex samples, and a new approach is provided for integration and miniaturization of a chromatographic analysis system. The invention has wide application prospect in the fields of biological analysis, organic synthesis, biological engineering, clinical mass spectrum, single cell analysis, micro complex sample analysis, multi-group chemical analysis, biological imaging analysis and the like.
Description
Technical Field
The invention relates to the field of sample pretreatment and chromatographic analysis, in particular to a sample injection probe device with a trace complex sample pretreatment function based on a membrane auxiliary strategy and an analysis method.
Background
Chromatographic analysis technology has strong separation capability and has been widely used in analysis in biomedical, chemical, food, environmental and other fields. For complex sample analysis, chromatography is often combined with sample pretreatment to further reduce matrix interference in the sample.
The procedure for applying sample pretreatment and chromatographic techniques to complex samples generally involves: (1) Sample pretreatment, including operations such as filtration, centrifugation, enrichment and the like, to remove impurities or interference in complex samples; (2) Introducing the treated sample into a sample injection valve in a liquid or gas form; (3) Separating, namely separating components with different properties in a sample in a chromatographic column by using a pressure driving system and taking liquid, gas or supercritical fluid as a mobile phase; (4) The components after detection and separation are detected in a detector, and analysis of complex samples is completed.
Currently, mass spectrometric detectors are the most commonly used in conjunction with chromatographic techniques. Compared with other detection technologies, the mass spectrum detection technology has the characteristics of high sensitivity, high analysis speed, capability of analyzing molecular structure information and the like, and is widely applied. Although the sensitivity of mass spectrometry detection techniques is high, it can be used directly for detection of simple samples. However, this method is not suitable for analysis of complex samples, because the actual biological samples have complex components and large differences in abundance, and have serious matrix effects, which interfere with the accuracy and sensitivity of mass spectrometry detection. Thus, in the face of complex samples, pretreatment and chromatographic separation of the sample prior to mass spectrometry detection are often required. However, the existing sample pretreatment method is mostly aimed at conventional sample analysis, has complex operation, also involves steps such as sample transfer, and is easy to cause sample loss, and is not suitable for micro complex sample analysis.
Disclosure of Invention
The invention aims to provide a sample injection probe device based on a membrane auxiliary strategy and a use method thereof, which are used for analyzing micro complex samples. The device adopts a membrane auxiliary strategy to realize in-situ pretreatment, sample introduction, transmission, separation and detection of micro complex samples under the pressure condition.
The invention also discloses a method for analyzing the sample by adopting the sample injection probe device based on the membrane auxiliary strategy.
A membrane assisted strategy based sample injection probe device comprising:
a sample carrier assembly for holding a sample;
the sample injection probe is provided with a fluid inlet channel, a fluid outlet channel and a sample injection channel port, wherein the sample injection channel port is communicated with the fluid inlet channel and the fluid outlet channel, and the sample injection channel port is opposite to the sample during use;
a fluid inlet pipeline which is arranged in the fluid introduction channel in a sealing way and is used for introducing fluid;
the liquid outlet fluid pipeline is arranged in the fluid extraction channel or in sealing butt joint with the fluid extraction channel, and the other end of the liquid outlet fluid pipeline is connected with a sample detection device for analyzing a sample;
the fluid driving device is connected with the inlet end of the liquid inlet fluid pipeline and used for controlling fluid;
A moving stage for adjusting the relative position of the sample carrying assembly and the probe;
the sample carrier assembly is composed of a sample chip for placing a sample and a membrane assembly covering the sample chip.
The fluid driving device is used for controlling fluid; the fluid is various fluids required in the detection process, such as a fluid for dissolving a sample, a fluid for carrying a mobile phase for conveying the sample, or a fluid for specific chemical or physical reaction, etc., and can be determined according to actual detection needs.
The sample detection device is used for analyzing a sample, and according to the invention, the detection device comprises, but is not limited to, an optical detector, or an electrochemical detector, or a mass spectrum detector, or a combination of two or more of the above.
The probe is used for realizing sample injection. The sample injection capillary is used for introducing fluid into the probe; the fluid-introducing capillary is connected to the fluid driving device. The membrane is used to pretreat a sample. The mobile station is used for adjusting the relative positions of the probe and the sample chip, and can be connected with the probe to adjust the position of the probe and also can be connected with the sample chip or the sample bearing assembly to adjust the position of the sample bearing assembly.
According to the invention, the probe is of an integral structure design, the internal channel is of a V-shaped or approximately V-shaped or T-shaped structure, and an interface for sealing the channel, a fluid inlet channel, a sample inlet channel port and a fluid outlet channel are integrated on the probe. The fluid introducing channel is connected with a capillary tube for introducing the fluid into the probe; the two sides of the sample inlet channel are respectively provided with a fluid inlet channel and a fluid outlet channel; the fluid leading-out channel is connected with the separation analysis capillary; the separation analysis capillary is combined with a detection device.
Further, as the preferable fluid inlet channel and the preferable fluid outlet channel are arranged in a V shape, the bottom ends of the fluid inlet channel and the fluid outlet channel are in butt joint, and the butt joint position is provided with the sample inlet channel port.
Or preferably, the fluid inlet channel and the fluid outlet channel are arranged in a T shape, and the sample inlet channel port is arranged at the position of the bottom end of the fluid outlet channel; a liquid inlet gap is reserved between the bottom of the liquid outlet fluid pipeline and the inner wall of the fluid outlet channel; and a liquid outlet of the liquid inlet fluid pipeline is in butt joint and conduction with the liquid inlet gap to form a liquid inlet channel.
According to the invention, the probe is made of polymer material such as polyether ether ketone (PEEK), metal material such as stainless steel and titanium alloy, inorganic material such as glass, or organic-inorganic composite material.
According to the invention, the probe sample inlet channel port is formed by intersecting a fluid inlet channel and a fluid outlet channel, the channel port is rectangular, elliptical, circular or the like, the cross section of the channel port has a long diameter of 1-1000 microns, and a short diameter of 0.5-500 microns.
The invention introduces a membrane auxiliary strategy into sample analysis, wherein the strategy takes a membrane as an intermediate medium, a sample injection channel port of a probe is contacted with the membrane covered on a sample chip and exerts acting force, and the sealing between the sample injection probe and the sample chip in the device is realized by utilizing the acting force and the elasticity of the membrane.
Preferably, the membrane is an elastic membrane.
According to the invention, the membrane-assisted strategy is to implement in-situ pretreatment operations of the sample in the device by means of different types of membranes, including but not limited to pretreatment operations such as filtration, solid phase extraction, liquid membrane extraction, affinity separation, dialysis, ultrafiltration, electrodialysis, reverse osmosis, etc.
According to the present invention, the auxiliary membrane material includes, but is not limited to, cellulose ester membrane (CE), regenerated cellulose membrane (RC), polyvinylidene fluoride membrane (PVDF), polytetrafluoroethylene (PTFE) and other polymer material membrane, porous glass membrane, glass fiber membrane and other inorganic material membrane, organic material membrane, or composite membrane of multiple materials (such as solid phase extraction membrane), etc. The class of membranes includes, but is not limited to, microporous membranes, filtration membranes, ultrafiltration membranes, nanofiltration membranes, reverse osmosis membranes, solid phase extraction membranes, and the like.
According to the invention, the surface of the membrane can be modified differently, including but not limited to loading titanium dioxide, magnetic microspheres, silica gel particles, molecular imprinting materials and other enriched materials, hydrophilic modification of polar groups such as hydroxyl, carboxyl, nitrile groups and the like can be introduced into the membrane materials or the enriched materials, or nonpolar groups C can be introduced into the membrane materials or the enriched materials 8 、C 18 And hydrophobic modification of hydrocarbon groups, halogen atoms, nitro groups and the like.
According to the invention, the membrane is a porous or through-hole structure, the pore size is in the range of 1 nanometer to 10 micrometers, the thickness is 10 nanometers to 1 millimeter, and the membrane can be selected appropriately according to the sample requirement. Preferably, to avoid lateral leakage of the sample through the membrane pores, a through-hole membrane should be used instead of a porous membrane. Preferably, the membrane is a through-hole structure with a hole size ranging from 1 nm to 10 microns and a thickness ranging from 10 nm to 1 mm.
According to the invention, the sample chip can be made of inorganic materials such as glass, capillary tubes and the like, or metal materials such as stainless steel or titanium alloy and the like, or polymer materials such as PEEK and the like, or other hard materials, or composite materials composed of the above materials.
According to the invention, the surface of the sample chip for loading the sample can be flat, or can be pits, convex columns or channels. Preferably, the sample chip is provided with a sample micro-pit, and the sample is placed in the micro-pit during detection.
According to the invention, the loaded trace sample form of the sample chip is solid, liquid, solid-liquid coexisting, gas and supercritical fluid.
According to the invention, the micro sample loaded by the sample chip is from single cell or micro cell sample, biological body fluid sample, biological tissue slice sample, organic synthesis reaction liquid, biological engineering fermentation liquid, or other micro samples, etc., and can be subjected to in-situ sample pretreatment operation on the chip according to the requirement by utilizing a membrane auxiliary strategy, thereby reducing the loss caused by transfer and realizing sample injection.
According to the invention, the fluid pipeline can be an empty capillary only used for fluid flow, or a capillary with internal surface modified fixing liquid, or a capillary chromatographic column of a monolithic column, or a capillary chromatographic column filled with stationary phase, or a pipeline prepared by various modification of capillary based on capillary, such as a filling capillary, and hard materials such as stainless steel tubes.
Further, the liquid inlet fluid pipeline is an empty capillary or a capillary with inner surface modified with fixing liquid; the liquid outlet fluid pipeline is an empty capillary or capillary chromatographic column. The capillary chromatographic column is a filling capillary column, an open-tube capillary column or an integral capillary column. When the capillary chromatographic column is adopted, one end of the capillary chromatographic column is inserted into the fluid extraction channel and aligned with the sample injection channel port, and is sealed with the fluid extraction channel, and the other end of the capillary chromatographic column is connected with the sample detection device in a sealing way.
Alternatively, the separation analysis task of the sample components can be completed without using a separation analysis capillary, and the separation analysis task of the sample components can be completed directly by using a fluid extraction channel on the sample injection probe. If the chromatographic stationary phase is filled or formed in the fluid extraction channel, or the surface of the fluid extraction channel is modified to have a separation function, or the fluid extraction channel is directly used as a sample transmission channel, the sample is transmitted to the detector for detection.
The application method of the sample injection probe device based on the membrane auxiliary strategy utilizes the characteristics (such as elasticity) of the membrane material, and adopts a method of clamping a membrane between the sample injection probe and the sample chip to realize the sealing of the sample injection channel port of the sample injection probe and the sample chip.
The invention utilizes the fluid driving device to drive the fluid in the device under the sealing state of the device, the fluid inlet channel of the probe flows through the sample inlet channel opening, contacts with the sample carried on the sample chip through the membrane hole, and then the fluid flows out of the probe through the fluid outlet channel. In the process, the interaction among the fluid, the sample and the membrane is utilized to complete the in-situ pretreatment operation of the sample and the subsequent sample injection operation.
As an embodiment, the membrane has a pretreatment function of in-situ filtering a sample, and the sample can be carried by fluid to perform filtration (such as microporous membrane, ultrafiltration membrane or dialysis membrane filtration) according to the size exclusion principle, and then sample injection operation of the filtered sample.
As a further alternative embodiment, the membrane has the pretreatment function of enriching the sample in situ, and the sample can be carried by fluid to complete the sample enrichment (such as solid phase extraction, liquid membrane extraction and affinity separation) operation of selectively combining specific components in the sample with the surface of the membrane; thereafter, the enriched sample on the membrane is eluted by an eluting fluid (which has a composition different from the composition of the fluid at the time of enrichment), and is carried by the eluting fluid to complete the sample introduction operation of the eluted sample.
The invention uses fluid to transfer the sample pretreated by the membrane to the fluid leading-out channel and the capillary chromatographic column connected with the fluid leading-out channel to realize the chromatographic sample injection operation of the sample; driving a mobile phase fluid to separate a sample loaded on a chromatographic column stationary phase by using a fluid driving device; and detecting the separated sample components by using the detection device.
In the present invention, the membrane functions mainly in two ways: firstly, sealing between a sample injection probe and a sample chip is realized by utilizing the characteristics (such as elastic characteristics) of a membrane material; and secondly, the membrane has a porous property, so that fluid can flow through the membrane holes to communicate a sample inlet of a sample inlet probe with a sample on a sample chip, and then various sample pretreatment functions are completed, including but not limited to pretreatment operations such as filtration, solid phase extraction, liquid membrane extraction, affinity separation, dialysis, ultrafiltration, electrodialysis, reverse osmosis and the like.
A method for sample analysis using the membrane-assisted strategy-based sample probe device of any of the above claims, comprising: and the fluid in the device is driven by the fluid driving device in the device sealing state, the fluid inlet channel of the probe flows through the sample inlet channel port, the in-situ pretreatment of the sample is completed by the membrane, the pretreated sample is contacted with the sample carried on the sample chip through the membrane hole, then the fluid flows out of the probe through the fluid outlet channel, and the sample analysis is realized by the capillary chromatographic column and the sample detection device.
Preferably, the method for analyzing the sample by the sample injection probe device based on the membrane auxiliary strategy comprises the following steps:
(1) Placing a sample onto the sample chip, and covering the sample chip with a membrane;
(2) The sample inlet of the control probe is contacted with the membrane and aligned with the sample, and an extrusion (or compaction) acting force is applied to form a sandwich membrane structure of the sample inlet-membrane-sample, so that the device is sealed;
(3) Starting a fluid driving device, introducing fluid through a capillary tube, carrying a sample through a membrane hole, finishing in-situ pretreatment of the sample by using the membrane, loading the sample subjected to in-situ pretreatment onto a capillary chromatographic column to finish sample injection operation, introducing mobile phase fluid by using the fluid driving device, and performing isocratic or gradient elution separation and detection on the sample loaded onto the capillary chromatographic column;
Alternatively, the analysis of different samples or the analysis of different location areas of the same sample can be accomplished by repeating steps (1) through (3).
Furthermore, the application method of the sample injection probe device based on the membrane auxiliary strategy comprises the following steps:
(a) Loading a sample onto a sample chip;
(b) Fixing the sample chip to the mobile station;
(c) Covering the sample chip with a film;
(d) The moving platform is controlled to enable the sample injection passage opening of the probe to be in contact with the membrane, and extrusion (or compaction) acting force is applied to form a sandwich membrane structure of the sample injection passage opening-the membrane-the sample, so that the device is sealed;
(e) Starting a fluid driving device, introducing fluid to carry a sample through a membrane hole, completing in-situ filtering operation of the sample, loading the filtered sample onto a capillary chromatographic column, completing sample injection operation, introducing mobile phase fluid by using the fluid driving device, and performing isocratic or gradient elution separation and detection on the sample loaded onto the capillary chromatographic column;
(f) Repeating steps (a) to (e) to complete analysis of different samples on the sample chip or analysis of samples on different sample chips; or analysis of different location areas of the same sample on a sample chip (spatial imaging analysis).
As another embodiment, the method for using the sample probe device based on the membrane auxiliary strategy comprises the following steps:
(a) Loading a sample onto a sample chip;
(b) Fixing the sample chip to the mobile station;
(c) Covering the sample chip with a film;
(d) The moving platform is controlled to enable the sample injection passage opening of the probe to be in contact with the membrane, and extrusion (or compaction) acting force is applied to form a sandwich membrane structure of the sample injection passage opening-the membrane-the sample, so that the device is sealed;
(e) Starting a fluid driving device, introducing fluid to carry a sample through the membrane hole, and finishing the in-situ enrichment operation of the sample on the membrane; introducing an eluting fluid by using a fluid driving device to elute the sample enriched on the membrane and loading the eluted sample on the capillary chromatographic column to complete the sample injection operation of the sample; introducing mobile phase fluid by using a fluid driving device, and performing isocratic or gradient elution separation and detection on a sample loaded on a capillary chromatographic column;
(f) Repeating steps (a) to (e) to complete analysis of different samples on the sample chip or analysis of samples on different sample chips; or analysis of different location areas of the same sample on a sample chip (spatial imaging analysis).
As another embodiment, unlike the above technical solution, a sample probe device based on a membrane-assisted strategy, the sample probe is replaced by a separate liquid inlet fluid pipe and liquid outlet fluid pipe; when the device is used, the liquid inlet fluid pipe and the liquid outlet fluid pipe are respectively arranged at two sides of the sample bearing assembly, the outlet end of the liquid inlet fluid pipe and the inlet end of the liquid outlet fluid pipe are opposite to the sample, and the liquid inlet fluid pipe and the liquid outlet fluid pipe are respectively in sealing butt joint with the sample chip side and the membrane side of the sample bearing assembly.
Further, a sample injection probe device based on a membrane assisted strategy, comprising:
a sample carrier assembly for holding a sample;
a liquid inlet fluid line and a liquid outlet fluid line for introducing a fluid;
the sample detection device is connected with the outlet end of the liquid outlet pipeline and is used for analyzing a sample;
the fluid driving device is connected with the inlet end of the liquid inlet fluid pipeline and used for controlling fluid;
the sample bearing component consists of a sample chip for placing a sample and a membrane covering the sample chip;
during detection, the outlet end of the liquid inlet fluid pipeline and the inlet end of the liquid outlet fluid pipeline are respectively in sealing butt joint with the sample chip side and the membrane side of the sample bearing assembly.
In the above technical scheme, 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 the porous membrane structure during detection.
When the technical scheme is adopted for sample analysis, the method comprises the following steps:
(1) Fixing a sample at a corresponding position of the sample bearing assembly, aligning the liquid inlet fluid pipeline and the liquid outlet fluid pipeline up and down, and extruding the middle sample bearing assembly to realize sealing;
(2) Starting a fluid driving device, introducing fluid to carry a sample through the membrane hole, and finishing the in-situ enrichment operation of the sample on the membrane; introducing an eluting fluid by using a fluid driving device to elute the sample enriched on the membrane, and completing sample injection operation of the sample by using a liquid outlet fluid pipeline; when the liquid outlet fluid pipeline adopts a capillary chromatographic column, a fluid driving device is utilized to introduce mobile phase fluid, and the sample loaded on the capillary chromatographic column is subjected to isocratic or gradient elution separation and detection;
or starting a fluid driving device, introducing fluid to carry a sample through the membrane hole, completing in-situ filtering operation of the sample, completing sample injection operation of the filtered sample by utilizing a liquid outlet fluid pipeline, and introducing mobile phase fluid by utilizing the fluid driving device; when the liquid outlet fluid pipeline adopts a capillary chromatographic column, the sample loaded on the capillary chromatographic column is subjected to isocratic or gradient elution separation and detection;
Repeating the steps to finish the analysis of different samples on the sample chip or the analysis of the samples on the different sample chips; or analysis of different location areas of the same sample on a sample chip (spatial imaging analysis).
When the filtration operation is carried out, insoluble substances larger than the pore diameter are intercepted by utilizing the difference of the pore diameters of the membranes, so that the filtration of the sample is realized. When the enrichment operation is carried out, the polarity difference of the sample is utilized, during the process of passing through the membrane, one part of components can be adsorbed on the surface of the membrane material due to interaction force, and the other part of insoluble components are trapped below the membrane, so that other soluble components in the sample pass through the membrane; removing the trapped insoluble components and the permeated soluble components, eluting the components adsorbed on the surface of the membrane material by using eluent, and finally loading the eluted target components into a chromatographic column to complete the whole enrichment and elution operation.
When the membrane is adopted to perform in-situ enrichment operation of the sample, different sampling probes can be adopted to respectively complete enrichment of the sample on the membrane and elution and separation operation of the sample enriched on the membrane.
According to the invention, the method for realizing the sealing of the device by the contact between the sample inlet of the probe and the membrane is that the mobile station is controlled to form a sandwich structure of the probe, the membrane and the sample, and the pressure required by sealing is borne by the membrane by utilizing the elasticity of the membrane, so that the sealing of the device without obvious liquid leakage under the pressure of more than 60MPa can be realized.
According to the invention, the relative movement of the sample injection probe and the sample chip relative to the membrane provides the pressure for sealing the device; the sample chip may be fixed on a mobile station, or the sample probe may be fixed on a mobile station, or the sample chip and the sample probe may be fixed on different mobile stations, respectively.
According to the membrane auxiliary strategy, the direct contact between the probe and the sample is isolated by adopting the membrane, so that the step of cleaning the probe is omitted, and the operation steps are simplified.
According to the invention, the portion of the membrane that is in contact with the sample is in a single use. I.e. the portion of the membrane in contact with the sample is discarded after sample pretreatment of the membrane in contact with the sample. For new samples, sample pretreatment is performed with a different membrane or with other non-sample-contacting areas of the membrane.
According to the invention, a porous supporting sheet or a membrane is additionally arranged between the sample chip and the membrane so as to improve the strength of the membrane and avoid the deformation of the membrane in the direction of the sample chip.
According to the present invention, pretreatment of different samples can be performed on different regions of one membrane, respectively, in order to improve the utilization efficiency of the membrane and to improve the treatment flux.
According to the present invention, the sample pretreatment operation and the sample introduction operation of the probe may be performed in an integrated order or separately. In the case of the separation method, the pretreatment operation of the sample is performed by using the probe and the membrane, and then the membrane is removed and stored. And combining the membrane with a sample injection probe at required time to perform sample injection operation. In this way, the probe used for sample pretreatment and the probe used for sample introduction may be the same probe or different probes.
According to the invention, the mode of adding the switching valve in the device is adopted to realize simultaneous sample injection and chromatographic separation of different samples, thereby reducing the time of the whole analysis flow and improving the analysis flux.
According to the invention, the mobile station can move in three dimensions, and can realize array sample injection and imaging analysis.
It should be noted that the application field of the sample injection probe device based on the membrane auxiliary strategy is not limited to chromatographic analysis, but can be applied to other flow analysis fields including but not limited to continuous flow analysis, flow injection analysis, microfluidic analysis, capillary electrophoresis analysis, etc.
The main advantages of the invention include: the system integrates the pretreatment of the micro-sample with the sample introduction, chromatographic separation and detection systems, has simple structure, rapid and automatic operation, high flexibility, convenient use and wide applicability, and is particularly suitable for analyzing the micro-sample. The sample can be introduced after in-situ pretreatment based on the membrane auxiliary strategy, a new sample introduction mode is provided for analysis of complex samples, and a new approach is provided for integration and miniaturization of a chromatographic analysis system.
The invention has wide application prospect in the fields of biological analysis, organic synthesis, biological engineering, clinical mass spectrum, single cell analysis, micro complex sample analysis, multi-group chemical analysis, biological imaging analysis and the like.
Compared with the prior art, the invention has the main advantages that:
(1) The system integrates the pretreatment of the micro-sample with the sample introduction, chromatographic separation and detection systems, has simple structure, rapid and automatic operation, high flexibility, convenient use and wide applicability, and is particularly suitable for analyzing the micro-sample;
(2) The sample can be introduced after in-situ pretreatment based on a membrane auxiliary strategy, a new sample introduction mode is provided for analysis of complex samples, and a new approach is provided for integration and miniaturization of a chromatographic analysis system;
(3) The flexibility of the film auxiliary strategy is high, and the used film can be replaced according to the pretreatment requirement;
(4) The whole device is modularized, flexible to assemble and convenient to use, the hard materials selected by the probe can be repeatedly used, and automatic operation is realized;
(5) The sample chip can be subjected to in-situ pretreatment, so that the loss of sample transfer is reduced, even the nondestructive sample pretreatment can be realized, and the probe is not in direct contact with the sample, so that the cleaning step is reduced, and the method is suitable for analyzing trace samples;
(6) The membrane auxiliary strategy can realize in-situ pretreatment of different modes, including but not limited to pretreatment operations such as filtration, solid phase extraction, liquid membrane extraction, affinity separation, dialysis, ultrafiltration, electrodialysis, reverse osmosis and the like, so that the complexity of the sample is reduced, and the sectional analysis of the sample can be realized;
(7) The device has wide application prospect, and can be applied to the fields of organic synthesis, bioengineering, clinical mass spectrometry, micro complex sample analysis, single cell analysis, multi-group chemical analysis, biological imaging analysis, on-site rapid analysis and the like.
Drawings
FIG. 1 is a schematic diagram of a membrane assisted strategy based V-type sampling probe apparatus of example 1. In the figure, 1-fluid driving device, 2-capillary for introducing fluid, 3-sample probe, 4-interface for sealing channel, 5-fluid introduction channel, 6-fluid extraction channel, 7-sample introduction channel port, 8-sample, 9-membrane, 10-sample chip, 11-mobile station, 12-capillary chromatographic column, 13-fluid, 14-eluting fluid, 15-mobile phase fluid, 16-electrospray mass spectrometer detection device.
FIG. 2 is the result of chromatographic analysis of dry spots of a mixed sample of three peptide fragments in isocratic elution mode using the apparatus of example 1 and method of use thereof.
FIG. 3 shows the results of chromatographic analysis of the trypsin-cleaved product of protein cytochrome C in a gradient elution mode using the device of example 1 and the method of using the same.
FIG. 4 is a sample introduction after in situ pretreatment filtration of a sample using the apparatus of example 1 and method of use thereof in example 2. (a) Mass spectrometry results for protein cytochrome C without the use of a membrane-assisted strategy; (b) Results of the cytochrome C chromatography of the protein using a membrane-assisted strategy; (c) And carrying out in-situ trypsin enzymolysis on the sample after sample introduction, and analyzing the peptide fragments of the products after enzymolysis under the condition of using a membrane auxiliary strategy, wherein the mass spectrum detection result of one peptide fragment of the products is marked as the signal of the peptide fragment.
FIG. 5 is a schematic diagram of a T-type sample probe device incorporating a solid phase extraction membrane in accordance with example 3. In the figure, the 1-fluid driving device, the 2-capillary for introducing fluid, the 3-sample introduction probe, the 4-interface for sealing the channel, the 5-fluid introduction channel, the 6-fluid extraction channel, the 7-sample introduction channel port, the 8-sample, the 9-membrane, the 10-sample chip, the 11-mobile station, the 12-capillary chromatographic column, the 13-fluid, the 14-elution fluid, the 15-mobile phase fluid, the 16-electrospray mass spectrometer detection device, the 17-diversion capillary, the 18-switching valve and the 19-sample introduction gap.
Fig. 6 is an enlarged view of a portion of the probe of fig. 5.
FIG. 7 is a schematic illustration of sample injection from an array sample injection probe device incorporating a solid phase extraction membrane of example 4. In the figure, the sample probe 3-sample, the membrane with solid phase extraction function 9-sample chip with micro-pits 10-sample chip with micro-pits 11-mobile station 15-mobile phase fluid 21-sample micro-pits 22-sample micro-pits 23-sample probe.
FIG. 8 is a schematic diagram of the sample extraction and injection mode of the channel sample combined with the solid phase extraction membrane for dry blood spot sample of example 5. In the figure, 2-capillary tube 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 Description
The technical scheme of the present invention is further described below with reference to specific embodiments, but the scope of the present invention is not limited thereto.
Preferred embodiments according to the present invention will be described in detail below with reference to the accompanying drawings.
Example 1
Fig. 1 is a schematic diagram of a sample injection probe device based on the membrane assisted strategy of example 1. The system consists of a set of fluid driving device 1, a PEEK V-shaped sample injection probe 3, a capillary tube 2 for introducing a mobile phase, an RC membrane 9, a sample chip 10, a mobile station 11 for moving the sample chip, a capillary chromatographic column 12 and an electrospray mass spectrometer detection device 16.
The sampling probe 3 is processed by PEEK material and comprises an interface 4 of a sealing channel, a fluid inlet channel 5, a fluid outlet channel 6 and a sampling channel port 7. The capillary 2 for introducing the mobile phase is arranged in the fluid introducing channel 5, the capillary chromatographic column 12 is arranged in the fluid extracting channel, and the capillary 2 and the capillary chromatographic column 12 can be sealed with the corresponding fluid introducing channel and the fluid extracting channel by using conical sealing elements; the sample inlet 7 is arranged at the bottom of the probe, and the two sides are respectively provided with the fluid inlet channel 5 and the fluid outlet channel 6, namely, the butt joint positions of the bottom ends of the fluid inlet channel 5 and the fluid outlet channel 6 are provided with the sample inlet 7. The inlet of the fluid inlet channel 5 and the outlet of the fluid outlet channel 6 are respectively provided with the interface 4, so that the fluid inlet channel and the fluid outlet channel are respectively in sealed connection with the fluid driving device 1 and the electrospray mass spectrometer detection device 16.
The specific method of use of the device of example 1 is as follows: (1) The sample chip 10 is fixed on a mobile table, the membrane 9 covers the sample chip 10, the movement of the mobile table 11 is controlled to enable the sample injection passage opening 7 of the sample injection probe 3 to be in contact with the membrane 9, and the sealing of the sample injection passage opening 7 and the membrane 9 is realized through the applied extrusion acting force, so that the mobile phase is prevented from leaking from the sample injection passage opening 7; (2) In a sealed state, the fluid driving device 1 is started, a chromatographic mobile phase is injected from the capillary 2 which is introduced into the mobile phase, the sample 8 is dissolved after passing through the fluid introduction channel 5, the sample introduction channel opening 7 and the membrane 9, the sample after in-situ pretreatment passes through the membrane 9 and the sample introduction channel opening 7 in sequence, the sample enters the capillary chromatographic column 12 through the fluid introduction channel 6, the elution sample is formed by changing the mobile phase, electrospray is formed under the action of an electric field, and the electrospray enters the detection device 16 of the electrospray mass spectrometer for detection; (3) The fluid driving device 1 is stopped, the mobile station 11 is controlled to move to the next sample 8, and the analysis of different samples 8 can be realized by repeating the step (2).
FIG. 2 is the result of mass spectrometry in isocratic elution mode of a sample of a mixture of three peptide fragments using an RC membrane having a molecular weight cut-off of 2000 using the apparatus of example 1 and methods of use thereof.
Specifically, three polypeptide samples are GE-11, GF-9-NH2 and TL-9-NH2, respectively, the concentrations of which are 0.1 micromoles per liter, and are mixed to form sample 8; dripping 10nL of sample 8 on the sample chip 10, and naturally drying to form a dry point; the sample was eluted isocratically using a chromatographic mobile phase of 30% acetonitrile in water (0.1% formic acid). The analysis of sample 8 was repeated three times with three peptide fragments having a retention time of 0.6%,1.0% and 1.1% RSD, respectively.
The analysis result shows that the system can realize the chromatographic analysis of the trace sample and has good system reproducibility.
FIG. 3 shows the results of mass spectrometry of the trypsin-digested product of protein cytochrome C in a gradient elution mode using the device of example 1 and the method of using the same.
Specifically, the product of enzymatic hydrolysis of protein cytochrome C in a conventional system is used as sample 8 and is dropped on sample chip 10. Sample 8 was analyzed using the system, with a chromatographic gradient of: 0% solution B (the remaining 100% is solution A) 0-10 minutes; 10 to 20 minutes, 15% solution B (the remaining 85% is solution A); 20-35 minutes, 30% solution B (the rest 70% is solution A); 35-40 minutes, 80% solution B (the remaining 20% is solution A). Wherein the solution A is 0.1% formic acid aqueous solution, and the solution B is 0.1% formic acid acetonitrile solution.
The analysis result proves that the system can be used for analyzing micro complex samples.
Example 2
FIG. 4 is a mass spectrometry analysis of protein cytochrome C using the device of example 1 and method (a) of its use of example 2 without the use of a membrane-assisted strategy; (b) Results of the cytochrome C chromatography of the protein using a membrane-assisted strategy; (c) And carrying out in-situ trypsin enzymolysis on the sample after sample introduction under the condition of using a membrane auxiliary strategy, and detecting a product by adopting mass spectrum, wherein the mass spectrum of one enzymolysis product is detected.
Specifically, in (a), a system without using a membrane auxiliary strategy (i.e. the sample is not covered by a membrane, and the sample inlet is in direct contact with the sample), and the protein cytochrome C sample dripped on the sample chip is analyzed to obtain a mass spectrum of the protein cytochrome C. (b) In the system using the membrane-assisted strategy (i.e., the apparatus and method of use of example 1), protein cytochrome C samples that were dropped onto the sample chip were analyzed and no signal of protein cytochrome C was detected. (c) The samples after sample introduction in (b) were enzymatically hydrolyzed in situ on the sample chip using trypsin, and the reaction products were analyzed using the system of the membrane-assisted strategy (i.e., the apparatus and method of use in example 1).
FIG. 4 (a) shows that protein cytochrome C can be detected by the electrospray mass spectrometry detection device; FIG. 4 (b) shows that the size of the selected membrane pore is smaller than that of the protein cytochrome C, and the protein cytochrome C is blocked by the membrane under the condition of membrane assistance and cannot pass through the membrane and cannot be detected by the detection device; the results in fig. 4 (C) show that the original protein cytochrome C is hydrolyzed into peptide sample and then analyzed, and the sample can penetrate the membrane and be further identified by the detection device, so that the system can realize in-situ sample introduction according to size exclusion filtration by selecting an appropriate membrane.
The analysis result proves that the system adopts a membrane auxiliary strategy, blocks proteins with the size larger than that of a membrane pore canal, and can pass peptide fragments obtained after enzymolysis, so that the system is suitable for analysis of micro complex samples.
Example 3
FIG. 5 is a schematic diagram of a T-type sample probe device incorporating a solid phase extraction membrane in accordance with example 3. The system consists of a set of fluid driving device 1, a switching valve 18, a diversion capillary 17, a capillary 2 for introducing a mobile phase, a T-shaped sampling probe 3, a joint 4 of an integrated sealing channel in the probe, a fluid inlet channel 5, a fluid outlet channel 6, a sampling channel port 7, a solid-phase extraction membrane 9, a sample chip 10, a mobile table 11 for moving the sample chip, a capillary chromatographic column 12 and an electrospray mass spectrometer detection device 16.
The T-shaped sampling probe 3 is formed by processing stainless steel materials and comprises an interface 4 of a sealing channel, a fluid introducing channel 5 and a sampling channel port 7. The fluid inlet channels 5 are respectively provided with capillaries 2 for introducing mobile phases, the fluid outlet channels 6 are provided with capillary chromatographic columns 12, the sample inlet channel ports 7 are arranged at the bottom of the probe, and the fluid inlet channels 5 are arranged above the sample inlet channels.
The capillary tube 2 and the capillary chromatographic column 12 are respectively sealed and fixed in the fluid inlet channel 5 and the fluid outlet channel 6 by conical sealing elements 20. Further, in the fluid introduction passage 5, the capillary tube 2 is made to communicate only with the liquid inlet gap 19 in the fluid extraction passage 6 by means of the tapered seal 20, while being sealed with the inner wall of the fluid introduction passage 5, preventing the fluid from entering the gap between them. In the fluid extraction channel 6, a sample injection gap 19 is reserved between the outer wall of the bottom of the capillary chromatographic column 12 and the inner wall of the fluid extraction channel 6, and the sample injection gap 19 is communicated with the capillary 2 to realize sample injection; and the part of the capillary chromatographic column 12 above the sample injection gap 19 is sealed with the inner wall of the fluid extraction channel 6 by using a conical sealing piece 20, so that the fluid is prevented from entering a gap formed by the conical sealing piece and the fluid extraction channel.
Referring also to fig. 6, the specific method of using the device of example 3 is as follows: (1) The sample chip 10 is fixed on a moving table, the solid-phase extraction membrane 9 covers the sample chip 10, the movement of the moving table 11 is controlled to enable the sample injection passage opening 7 to be opposite to the sample position on the sample chip 10, and the applied extrusion acting force is utilized to enable the sample injection passage opening 7 of the sample injection probe 3 to be in contact with the membrane 9 and be sealed, so that the mobile phase is prevented from leaking from the sample injection passage opening 7; (2) In a sealed state, the fluid driving device 1 is opened, the switching valve 18 is controlled, so that the chromatographic mobile phase is injected from the capillary tube 2 into which the mobile phase is introduced, and the sample 8 is dissolved and carried onto the membrane 9 after passing through the fluid introduction channel 5, the liquid inlet gap 19, the sample introduction channel opening 7 and the membrane 9; (3) Changing the composition of the mobile phase, eluting the sample 8 from the membrane 9, transmitting the sample through a capillary chromatographic column 12, forming electrospray under the action of an electric field, and detecting the electrospray by an electrospray mass spectrometer detection device 16; (4) The switching valve 18 is controlled so that the mobile phase flows out of the diversion capillary 17, the mobile station 11 is controlled to move to the next sample 8, and analysis of different samples 8 can be realized by repeating the steps (2) to (3).
Example 4
FIG. 7 is a schematic illustration of sample injection from an array sample injection probe device incorporating a solid phase extraction membrane of example 4. In the figure, the sample chip with micro pits is 11-mobile station, 10-solid phase extraction membrane, 21-first sample micro pits, 3-first sample injection probe, 15-mobile phase fluid, 22-second sample micro pits, 23-second sample injection probe and 14-eluting fluid.
The array sample-injection probe device uses two sample-injection probes for respectively loading and eluting the sample on and from the solid-phase extraction membrane, and compared with the embodiment 3, the in-situ sample pretreatment and detection parallelism are realized, and the analysis time is reduced.
Example 4 the specific method of using the array sampling probe device is as follows: (1) The sample chip 10 is fixed on the moving table 11, the solid-phase extraction membrane 9 is covered on the sample chip 10, and the first sample injection probe 3 is contacted with the solid-phase extraction membrane 9 above the first sample micro-pit 21 and sealed by controlling the movement of the moving table 11; (2) Introducing a mobile phase fluid 15 in a sealed state, passing through the solid phase extraction membrane 9, entering a first sample micro-pit 21, and loading a sample on the solid phase extraction membrane 9; (3) The first sample introduction probe 3 is moved to the solid phase extraction membrane 9 above the second sample micro-pit 22 to be contacted and sealed, and the second sample introduction probe 23 is moved to the solid phase extraction membrane 9 above the first sample micro-pit to be contacted and sealed; (4) In a sealed state, introducing a mobile phase fluid 6 into the first sample injection probe 3, passing through the solid phase extraction membrane 9, entering a second sample micro-pit 23, and loading a sample onto the solid phase extraction membrane 9; simultaneously, introducing an eluting fluid 14 into the second sample introduction probe 23, eluting the sample on the solid phase extraction membrane 9 above the first sample micro-pit 21 for subsequent analysis; (5) And (3) repeating the step (4) to realize the parallel analysis of the arrayed sample injection combined with the solid-phase extraction membrane.
Example 5
FIG. 8 is a schematic diagram of the channel sample injection mode of example 5 in combination with a solid phase extraction membrane for dry blood spot sample injection. In the figure, 2-capillary tube into which fluid is introduced, 13-fluid, 8-dried blood spot sample, 10-sample chip with dried blood spot, 9-solid phase extraction membrane.
The sample injection mode adopts a membrane auxiliary strategy and combines a solid phase extraction membrane to realize in-situ pretreatment and sample injection of a sample on the dry blood spot filter paper, and is different from the above embodiments 1-4 in that embodiment 5 does not adopt a method for integrating sample pretreatment and sample injection by a sample injection probe, but adopts a method for respectively carrying out sample pretreatment and sample injection.
The specific method of dry blood spot sampling in example 5 is as follows: (1) Covering a paper chip 10 with a dried blood spot sample (the blood sample is loaded in a mesh structure of filter paper on the paper chip) on a solid-phase extraction membrane 9, aligning two capillaries 2 for introducing fluid up and down, and extruding the sample chip 10 and the solid-phase extraction membrane 9 in the middle to realize sealing; (2) Introducing a fluid 13 to dissolve the dried blood spot sample 8 and flow downwards, wherein when the dried blood spot sample passes through the solid-phase extraction membrane 9, the target substances in the dried blood spot sample 8 are adsorbed by the solid-phase extraction membrane 9, and other substances pass through the solid-phase extraction membrane 9 to complete enrichment of the sample; (3) The sample-enriched solid phase extraction membrane is taken down and combined with a sample-feeding probe, and the operations of eluting the sample target substance adsorbed on the solid phase extraction membrane 9, sample-feeding, chromatographic separation and detection are sequentially completed by adopting the method similar to the method of the embodiment 3.
Claims (10)
1. Sample introduction probe device based on membrane auxiliary strategy, characterized by comprising:
a sample carrier assembly for holding a sample;
the sample injection probe is provided with a fluid inlet channel, a fluid outlet channel and a sample injection channel port, wherein the sample injection channel port is communicated with the fluid inlet channel and the fluid outlet channel, and the sample injection channel port is opposite to the sample during use;
a fluid inlet pipeline which is arranged in the fluid introduction channel in a sealing way and is used for introducing fluid;
the liquid outlet fluid pipeline is arranged in the fluid extraction channel or in sealing butt joint with the fluid extraction channel, and the other end of the liquid outlet fluid pipeline is connected with a sample detection device for analyzing a sample;
the fluid driving device is connected with the inlet end of the liquid inlet fluid pipeline and used for controlling fluid;
a moving stage for adjusting the relative position of the sample carrying assembly and the probe;
the sample carrier assembly is composed of a sample chip for placing a sample and a membrane covering over the sample chip.
2. The sample injection probe device based on the membrane auxiliary strategy according to claim 1, wherein the fluid inlet channel and the fluid outlet channel are arranged in a V shape, the bottom ends of the fluid inlet channel and the fluid outlet channel are in butt joint, and the butt joint position is provided with the sample injection channel port.
3. The sample injection probe device based on the membrane auxiliary strategy according to claim 1, wherein the fluid inlet channel and the fluid outlet channel are arranged in a T shape, and the sample injection channel port is arranged at the position of the bottom end of the fluid outlet channel; a liquid inlet gap is reserved between the bottom of the liquid outlet fluid pipeline and the inner wall of the fluid outlet channel; and a liquid outlet of the liquid inlet fluid pipeline is in butt joint and conduction with the liquid inlet gap to form a liquid inlet channel.
4. The membrane assisted strategy based sampling probe device of claim 1, wherein the membrane is an elastic membrane; the membrane is of a through hole structure, the size of the through hole ranges from 1 nanometer to 10 micrometers, and the thickness of the through hole ranges from 10 nanometers to 1 millimeter; the membrane is a functional membrane with one or more functions of filtration, solid phase extraction, liquid membrane extraction, affinity separation, dialysis, ultrafiltration, electrodialysis and reverse osmosis; the membrane is a cellulose ester membrane, a regenerated cellulose membrane, a polyvinylidene fluoride membrane, polytetrafluoroethylene, a porous glass membrane, a glass fiber membrane and a solid phase extraction membrane.
5. The membrane-assisted strategy-based sampling probe device of claim 1, wherein the fluid inlet pipeline is an empty capillary or a capillary with an internal surface modified fixing fluid; the liquid outlet fluid pipeline is an empty capillary and a capillary chromatographic column; or the fluid extraction channel is filled or formed with a chromatographic stationary phase, or the surface of the fluid extraction channel is modified to have a separation function.
6. The membrane-assisted strategy-based sampling probe device according to claim 1, wherein a sample carrying area is provided on the sample chip, the carrying area is a micro-pit, a plane, or a convex column configuration, and the sample is placed in the sample carrying area during detection.
7. The membrane assisted strategy based sampling probe apparatus of any one of claims 1.4.5.6, wherein said sampling probe is replaced with separate feed and discharge fluid tubes; when the device is used, the liquid inlet fluid pipe and the liquid outlet fluid pipe are respectively arranged at two sides of the sample bearing assembly, the outlet end of the liquid inlet fluid pipe and the inlet end of the liquid outlet fluid pipe are opposite to the sample, and the liquid inlet fluid pipe and the liquid outlet fluid pipe are respectively in sealing butt joint with the sample chip side and the membrane side of the sample bearing assembly.
8. A method of sample analysis using the membrane assisted strategy based sample probe device of any of claims 1 to 6, comprising: and the fluid in the device is driven by the fluid driving device in the device sealing state, the fluid inlet channel of the probe flows through the sample inlet channel port, the in-situ pretreatment of the sample is completed by the membrane, the pretreated sample is contacted with the sample carried on the sample chip through the membrane hole, then the fluid flows out of the probe through the fluid outlet channel, and the sample analysis is realized by the sample detection device.
9. The method for sample analysis based on membrane assisted strategy of claim 8, comprising the steps of:
(1) Placing a sample onto the sample chip, and covering the sample chip with a membrane;
(2) Controlling the sampling passage opening of the probe to be in contact with the membrane and aligned with the sample, and applying extrusion acting force to form a sandwich membrane structure of the sampling passage opening-the membrane-the sample, so as to realize the sealing of the device;
(3) Starting a fluid driving device, introducing fluid through a fluid inlet pipeline, carrying a sample through a membrane hole, completing the filtering operation of the sample by utilizing a membrane, loading the filtered sample on a capillary chromatographic column to complete the sample injection operation, introducing mobile phase fluid by utilizing the fluid driving device, and performing isocratic or gradient elution separation and detection on the sample loaded on the capillary chromatographic column;
alternatively, the analysis of different samples or the analysis of different location areas of the same sample can be accomplished by repeating steps (1) through (3).
10. The method for sample analysis based on membrane assisted strategy of claim 8, comprising the steps of:
(1) Placing a sample onto the sample chip, and covering the sample chip with a membrane;
(2) Controlling the sampling passage opening of the probe to be in contact with the membrane and aligned with the sample, and applying extrusion acting force to form a sandwich membrane structure of the sampling passage opening-the membrane-the sample, so as to realize the sealing of the device;
(3) Starting a fluid driving device, introducing fluid through a fluid inlet pipeline, carrying a sample through a membrane hole, finishing the enrichment operation of the sample by using the membrane, eluting the sample enriched on the membrane by introducing elution fluid through the fluid driving device, loading the eluted sample on a capillary chromatographic column, finishing the sample injection operation of the sample, introducing mobile phase fluid through the fluid driving device, and performing isocratic or gradient elution separation and detection on the sample loaded on the capillary chromatographic column;
alternatively, the analysis of different samples or the analysis of different location areas of the same sample can be accomplished by repeating steps (1) through (3).
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