CN113720894B - A direct sampling ionization analysis system, method and application - Google Patents

Abstract

The invention discloses a direct sampling ionization analysis system, comprising: the sampling module is used for collecting a sample to be detected in a contact manner and collecting components to be detected; the reagent box module is used for fixing the sampling module and eluting the component to be detected to obtain an elution solution; the reaction module is used for carrying out high-flux chemical reaction on the elution solution; the sample injection module is used for fixing the reagent box module at a sample injection end of the mass spectrometer, so that the sample injection module and the mass spectrometer are matched for sample injection; the sampling module is a metal probe with one end fixed with a porous membrane, and the porous membrane is used for collecting a sample to be detected in a contact manner. The direct sampling ionization analysis system provided by the invention is convenient to operate and can quickly obtain a test result by sampling with the metal probe with one end fixed with the porous membrane, is suitable for the applications of on-site quick detection, clinical quick detection and the like, meets the requirements of quick sampling analysis, and greatly simplifies the sample pretreatment process of mass spectrometry. The invention also provides a direct sampling ionization analysis method using the system.

Description

A direct sampling ionization analysis system, method and application

technical field

The invention belongs to the technical field of mass spectrometry, and in particular relates to a direct sampling ionization analysis system, method and application.

Background technique

In recent years, mass spectrometers have played an important role in the analysis of complex mixtures such as biological tissues, body fluids, and environmental samples due to their high sensitivity, high accuracy, and high throughput. The mass spectrometer can detect the vast majority of compounds in the sample to be tested, so as to realize the structure identification of target compounds, quantitative analysis of low-level drugs and biomarkers in vivo, etc. In addition to on-site rapid detection of drugs and illegal drugs, there is also a great demand for rapid detection in the medical field. Disease markers detected by mass spectrometers are playing an increasing role in clinical diagnosis. At present, relevant studies have shown that compared with normal people, the proportion of carbon-carbon double bond isomers of unsaturated phospholipids is quite different in patients with diabetes and breast cancer.

However, the traditional sample pretreatment method takes a long time and does not match the rapid detection capability of the mass spectrometer, which is not conducive to improving the detection efficiency and cannot provide the analysis results in time. In recent years, methods such as direct sampling with metal probes have been developed. The amount of adsorbed substances to be tested is limited, making it difficult to detect low-content target substances. At the same time, the solid analyte carried by them may block the sampling path and cause trouble for analysis. When sampling by solid-phase microextraction, the sampling process takes a long time, and the recovery rate of extraction is low and selective, which may lead to the loss of some key chemical information. At the same time, the above methods cannot achieve high-throughput biological sample sampling, reaction and analysis, and cannot obtain the required analysis results accurately and efficiently. Therefore, it is necessary to develop a new sampling technology that can be used in conjunction with mass spectrometers to achieve micro and quantitative sampling, and at the same time to preserve the complete chemical information of the samples to meet the rapid sampling and analysis needs of mass spectrometers.

SUMMARY OF THE INVENTION

Aiming at the above-mentioned technical problems such as long time consumption and loss of chemical information in the sample sampling process in the prior art, the purpose of the present invention is to provide a direct sampling ionization analysis system, method and application.

In order to achieve the above purpose, the present invention proposes a direct sampling ionization analysis system, comprising: a sampling module for contacting the sample to be tested and collecting the components to be tested; a kit module for fixing the sampling module to elute the components to be tested, Obtain the elution solution; the reaction module is used to perform high-throughput chemical reactions on the elution solution; the sample injection module is used to fix the kit module on the sample injection end of the mass spectrometer, so that the sample injection module and the mass spectrometer cooperate with each other. The sampling module is a metal probe with a porous membrane fixed at one end, and the porous membrane is used to contact and collect the sample to be tested.

The reaction module of the present invention performs a high-throughput chemical reaction on the elution solution, which is convenient for subsequent monitoring. In addition, the reaction module can be a multi-chamber structure, so as to simultaneously perform chemical reactions on the elution solutions in multiple kits. The injection module is a device that can fix the kit module on the injection end of the mass spectrometer, and is used for coordinating injection with the mass spectrometer to improve the analysis efficiency.

Further, the porous membrane also includes chemical reagents modified thereon for improving contact acquisition conditions.

The porous membrane of the present invention can collect substances to be tested in solid tissues or biological fluids waiting to be tested. The porous membrane has a porous structure, and its porous structure can collect more substances to be tested, and the eluate obtained by subsequent elution It can be used for mass spectrometry analysis, so that the corresponding analyte can be detected and the corresponding analysis can be completed. According to the needs of analysis, chemical modification is performed on the porous structure of the porous membrane to improve the sampling conditions and better sample the object to be tested. Chemical modification is to add other chemical reagents to the porous structure of the porous membrane.

Further, the pore size of the porous membrane is 0.1-10 μm.

Further, a nano-spray glass tube is fixed on the kit module, the nano-spray glass tube is filled with an elution solvent for eluting the components to be tested, and the metal probe is inserted into the nano-spray glass tube after the collection is completed; a voltage is applied to the metal probe. After that, electrospray was formed at the tip of the nanospray glass tube.

Further, the metal probe is used to fix the porous membrane, and the outer diameter of the metal probe is 0.2-1 mm.

Further, the sampling module includes a single mass spectrometry sampling device and a sequential mass spectrometry sampling device.

The present invention also provides a direct sampling ionization analysis method, comprising the following steps:

(1) Contact the porous film of the metal probe with the sample to be tested, and absorb the component to be tested;

(2) placing the metal probe in the cleaning solution to remove the adhered matrix components;

(3) insert the metal probe into the nano-spray glass tube containing the elution solvent, stand or shake to elute to obtain the elution solution;

(4) In the reaction module, a high-throughput chemical reaction is performed on the elution solution;

(5) Perform mass spectrometry analysis on the reacted elution solution to obtain chemical molecule information in the sample to be tested.

Further, when the sample to be tested is solid, the contact method between the porous film and the sample to be tested is as follows: inserting the porous film into the sample to be tested or rolling the porous film on the surface of the sample to be tested; when the sample to be tested is liquid, the porous film is connected to the sample to be tested. The contact mode of the sample to be tested is to insert the porous membrane into the sample to be tested.

Further, the methods of using the metal probe for sampling include sampling directly with the metal probe and sampling with the metal probe and the kit.

Among them, the specific method of using the metal probe and the kit for sampling is: first take out half of the kit with the metal probe fixed, then contact the porous membrane of the metal probe with the sample to be tested for a period of time, and then put the metal probe Push it to a suitable position to facilitate subsequent elution, mass spectrometry and other operations, and finally splicing the two parts of the kit to form a complete kit.

Further, the application of direct sampling ionization analysis methods, including qualitative analysis and quantitative analysis, in terms of qualitative analysis, direct sampling ionization analysis methods are used to determine the composition of elements, sn isomers of phospholipids and carbon-carbon double bond position isomers ; In terms of quantitative analysis, the direct sampling ionization analysis method is used to achieve quantitative detection of the components to be measured by adding an internal standard on the porous membrane or in the elution solution.

Compared with the prior art, the technical effect of the present invention is as follows: the direct sampling ionization analysis system involved in the present invention uses a metal probe with a porous membrane fixed at one end for sampling, the operation is convenient, and the test results can be obtained quickly, and it is suitable for field use. Applications such as rapid detection and clinical rapid detection meet the needs of rapid sampling and analysis, greatly simplifying the sample pretreatment process for mass spectrometry analysis; in addition, high-throughput reaction and analysis methods improve the overall analysis efficiency. The invention also relates to a direct sampling ionization analysis method using the above system, and the application of the direct sampling ionization analysis method.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of embodiments taken in conjunction with the accompanying drawings, wherein:

Fig. 1 is the overall structure schematic diagram of the direct sampling ionization analysis system of the present invention;

2 is a schematic structural diagram of the direct sampling ionization analysis system of the present invention and a mass spectrometer;

Fig. 3 is the structural schematic diagram of the metal probe of the present invention;

Fig. 4 is the electron microscope picture before and after the modification of the porous membrane of the present invention;

Fig. 5 is the operation flow chart of the direct sampling ionization analysis method of the present invention;

6 is a schematic diagram of a method for sampling using a metal probe;

Fig. 7 is the positive ion mode mass spectrogram of mouse brain tissue;

Fig. 8 is the high-quality terminal negative ion mode mass spectrogram of mouse brain tissue;

Figure 9 is a low mass end negative ion mode mass spectrogram of mouse brain tissue;

Fig. 10 is the lipid signal comparison diagram of using the extraction method of the present invention and the traditional extraction method to extract the same mouse brain tissue;

Fig. 11 is the mass spectrum of fatty acid carbon-carbon double bond isomers in rat brain tissue;

Figure 12 is a mass spectrum of the sn isomer and carbon-carbon double bond isomer of lipids in rat brain tissue.

Description of reference numbers:

Sampling module 1 , reagent box module 2 , reaction module 3 , sample injection module 4 , mass spectrometer 5 , metal probe 6 , porous membrane 7 , sample to be tested 8 , and reagent box 9 .

Detailed ways

The following describes in detail the embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary, and are intended to explain the present invention and should not be construed as limiting the present invention.

The following describes the direct sampling ionization analysis system and method according to the embodiments of the present invention with reference to the accompanying drawings.

As shown in FIG. 1-FIG. 4, the direct sampling ionization analysis system includes: a sampling module 1, used to contact the sample to be tested 8 and collect the components to be tested; a kit module 2, used to fix the sampling module 1, to elute the sample to be tested components, to obtain the elution solution; the reaction module 3, used to perform high-throughput chemical reactions on the elution solution; the sample injection module 4, used to fix the kit module 2 on the sample injection end of the mass spectrometer 5, so that the sample can be injected The module 4 cooperates with the mass spectrometer 5 to inject samples; the sampling module 1 is a metal probe 6 with a porous membrane 7 fixed at one end, and the porous membrane 7 is used to contact and collect the sample 8 to be tested.

The module is a metal probe 6 with a porous membrane 7 fixed at one end, which is used to directly contact the sample 8 to be tested and collect the components to be tested. The metal probe 6 is used to fix the porous membrane 7, the outer diameter of the metal probe 6 is 0.2-1 mm, and the metal probe 6 is a metal wire such as platinum wire or stainless steel wire. The porous membrane 7 is an integrated membrane material fixed on the metal probe 6. The pore size of the porous membrane 7 is 0.1-10 μm, and its material is nylon, polyethersulfone, polytetrafluoroethylene, mixed cellulose or polypropylene. Adsorb the sample 8 to be tested. The porous membrane 7 can collect the substance to be tested in the solid tissue or biological fluid waiting for the test sample 8. The porous membrane 7 is a porous structure, and its porous structure can collect more substances to be tested, and the eluate obtained by subsequent elution It can be used for mass spectrometry analysis, so that the corresponding analyte can be detected and the corresponding analysis can be completed. According to the analysis requirements, chemical modification is performed on the porous structure of the porous membrane 7 to improve the sampling conditions and better sample the object to be tested. The chemical modification is to add other chemical structures to the porous structure of the porous membrane 7 . Taking polypropylene membrane as an example, polydopamine (PDA) and cysteine (Cys) were modified on the porous structure of polypropylene membrane, as shown in Figure 4, Figure 4(a), Figure 4(c), Figure 4 4(e) are the electron microscope images of the modified polypropylene film at 5000X, 500X, and 50X magnification, respectively. The electron microscope image at 50X magnification, comparing Figure 4(a) and Figure 4(b), it can be seen that polydopamine and cysteine have been modified on the polypropylene film, as shown in the part framed by the dotted line in Figure 4(a). Show. In addition, it can be seen from the electron microscope images of different magnifications of the polypropylene film before and after modification that the polypropylene film has uniform pore size and good shape, which is suitable for the present invention.

The kit module 2 is adapted to the sampling module 1, and is used for fixing the sampling module 1 to elute the components to be tested to obtain an elution solution. A nano-spray glass tube is fixed on the kit module 2. The nano-spray glass tube is filled with an elution solvent. After the metal probe 6 is collected, it is inserted into the nano-spray glass tube. Elution is carried out to obtain an elution solution, and the elution solution is contained in a nano-spray glass tube. The mass spectrometer 5 provides a voltage, which is applied to the metal probe 6, so that an electrospray is formed at the tip of the nanospray glass tube. The reaction module 3 is used for high-throughput chemical reaction of the elution solution. The reaction module 3 can be a multi-chamber structure, and one reagent cartridge 9 can be placed in one chamber to perform chemical reactions on the elution solutions in multiple reagent cartridges 9 at the same time. The chemical reactions in the reaction module 3 include photochemical reactions, peroxidation reactions and epoxidation reactions. The chemical reaction in the reaction module 3 is not limited to the above-mentioned reaction types. In practical applications, whether to perform the chemical reaction can be selected according to the properties and requirements of the sample 8 to be tested. The sampling module 4 is used to fix the kit module 2 on the sampling end of the mass spectrometer 5, so that the sampling module 4 and the mass spectrometer 5 cooperate to inject samples, and the sampling module 4 includes a single mass spectrometry sampling device and a sequence mass spectrometry analysis device. injection device.

The present invention also provides a direct sampling ionization analysis method, the operation flow chart of which is shown in Figure 5, including the following steps:

(1) Contact the porous membrane 7 of the metal probe 6 with the sample to be tested 8, and absorb the component to be tested;

(2) placing the metal probe 6 in the cleaning solution to remove the adhered matrix components;

(3) insert the metal probe 6 into the nano-spray glass tube equipped with the elution solvent, stand or shake to elute to obtain the elution solution;

(4) in the reaction module 3, a high-throughput chemical reaction is carried out to the elution solution;

(5) Perform mass spectrometry analysis on the reacted elution solution to obtain chemical molecule information in the sample 8 to be tested.

Wherein, when the sample to be tested 8 is solid, the contact between the porous film 7 and the sample to be tested 8 is as follows: inserting the porous film 7 into the sample to be tested 8 or rolling the porous film 7 on the surface of the sample to be tested 8; the sample to be tested 8 When it is a liquid, the contact mode between the porous membrane 7 and the sample to be tested 8 is to insert the porous membrane 7 into the sample to be tested 8 . As shown in FIG. 6 , the method of using the metal probe 6 for sampling includes sampling directly with the metal probe 6 and sampling with the metal probe 6 and the reagent kit 9 together. The specific method of using the metal probe 6 and the reagent kit 9 for sampling is as follows: first, take out half of the reagent box 9 with the metal probe 6 fixed thereon, and then contact the porous membrane 7 of the metal probe 6 with the sample 8 to be tested for a period of time. After the time has elapsed, the metal probe 6 is pushed to a suitable position to facilitate subsequent elution, mass spectrometry and other operations.

In addition, the application of direct sampling ionization analysis method, including qualitative analysis and quantitative analysis, in terms of qualitative analysis, the direct sampling ionization analysis method is used to determine the composition of elements, sn isomers of phospholipids and carbon-carbon double bond position isomers; In terms of quantitative analysis, the direct sampling ionization analysis method is used to achieve quantitative detection of the component to be measured by adding an internal standard on the porous membrane or in the elution solution. Specifically, in terms of qualitative detection, the element composition can be determined, and the structure of phospholipids such as sn isomer and carbon-carbon double bond position isomer can be determined by means of photochemical derivatization reaction or epoxidation reaction combined with tandem mass spectrometry analysis; In terms of quantitative detection, the absolute quantitative analysis of the substance to be tested can be achieved by adding an internal standard substance on the porous membrane or in the elution solution.

Using the method of the present invention to monitor the sample rat brain tissue, Figure 7, Figure 8, Figure 9 are the positive ion mode mass spectrogram of the rat brain tissue, the high quality end negative ion mode mass spectrogram of the rat brain tissue, and the low ion mode mass spectrogram of the rat brain tissue. Mass spectrum in negative ion mode. The positive ion mode spectral peaks of mouse brain tissue lipids can be obtained from FIG. 7 , the negative ion mode spectral peaks of mouse brain tissue lipids can be obtained from FIG. 8 , and the spectral peaks of mouse brain tissue fatty acids can be obtained from FIG. 9 . It can be proved that the method can effectively obtain information on lipids and fatty acids in mouse brain tissue, and the sample pretreatment system of the present invention can effectively extract lipids, which is convenient for subsequent identification and analysis. The lipid signals in a piece of homogenized rat brain tissue extracted by the metal probe 6 sampling method of the present invention and the traditional lipid extraction method were respectively counted. The comparison diagram of the rat brain signals is shown in Figure 10, where PC is phosphatidylcholine; PE is phosphatidylethanolamine; PS is phosphatidylserine; PG is phosphatidylglycerol; PI is phosphatidylinositol; When the ratio is around 1, the lipid species and abundance extracted by the two methods have no significant difference, and the stability is good, which proves that the method has universal applicability.

The fatty acid FA 18:1 in the rat brain tissue was selected for the identification of carbon-carbon double bond isomers, and lipid PC 34:1 was used for the identification of sn isomerism and carbon-carbon double bond isomers. The identification spectra are shown in Figure 11. and shown in Figure 12. In reaction module 3, the lipid and fatty acid are subjected to photochemical derivatization reaction, and the position information of lipid carbon-carbon double bond is obtained by tandem mass spectrometry analysis, so as to analyze the fine structure of lipid. It can be seen from Figure 11 and Figure 12 that, FA 18:1 has three carbon-carbon double bonds: Δ8 (m/z 246.2, m/z 248.2), Δ9 (m/z 232.2, m/z 262.2), Δ11 (m/z 204.2, m/z 290.2) isomers; while PC 34:1 has two sn isomers (m/z 380.4, m/z 396.1, m/z 466.2) and two carbon-carbon double bond isomers (m/z 489.2, m/z z 517.2, m/z 578.5, m/z 606.5), where m/z is the mass-to-charge ratio, which can prove that this method can analyze the fine structure of lipids.

In the description of this specification, description with reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples", etc., mean specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine the different embodiments or examples described in this specification, as well as the features of the different embodiments or examples, without conflicting each other.

In addition, the terms "first" and "second" are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with "first", "second" may expressly or implicitly include at least one of that feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise expressly and specifically defined.

Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, The scope of the invention is defined by the claims and their equivalents.

Claims (8)

1. a direct sampling ionization analysis system, is characterized in that, comprises: The sampling module is used to contact the sample to be tested and collect the components to be tested; a kit module, used to fix the sampling module, elute the component to be tested, and obtain an elution solution; a reaction module for carrying out a high-throughput chemical reaction on the elution solution; a sample injection module, used to fix the kit module on the sample injection end of the mass spectrometer, so that the sample injection module and the mass spectrometer cooperate to inject samples; The sampling module is a metal probe with a porous membrane fixed at one end. The porous membrane is used to contact and collect the sample to be tested. The porous membrane also includes chemical reagents modified on it to improve the contact and collection conditions. A nano-spray glass tube is fixed on the kit module, the nano-spray glass tube is filled with an elution solvent for eluting the component to be tested, and the metal probe is inserted into the nano-spray glass tube after collecting; After a voltage is applied to the metal probe, an electrospray is formed at the tip of the nanospray glass tube. 2 . The direct sampling ionization analysis system according to claim 1 , wherein the pore size of the porous membrane is 0.1-10 μm. 3 . 3 . The direct sampling ionization analysis system according to claim 1 , wherein the metal probe is used to fix the porous membrane, and the outer diameter of the metal probe is 0.2-1 mm. 4 . 4 . The direct sampling ionization analysis system according to claim 1 , wherein the sampling module comprises a single mass spectrometry sampling device and a sequential mass spectrometry sampling device. 5 . 5. the direct sampling ionization analysis method based on the direct sampling ionization analysis system as described in any one of claim 1-4, is characterized in that, comprises the following steps: (1) Contact the porous film of the metal probe with the sample to be tested, and absorb the component to be tested; (2) placing the metal probe in a cleaning solution to remove the adhered matrix components; (3) inserting the metal probe into a nano-spray glass tube equipped with an elution solvent, and standing or shaking to elute to obtain an elution solution; (4) in the reaction module, a high-throughput chemical reaction is carried out to the elution solution; (5) Perform mass spectrometry analysis on the reacted elution solution to obtain chemical molecule information in the sample to be tested. 6 . The direct sampling ionization analysis method according to claim 5 , wherein when the sample to be tested is a solid, the contact mode between the porous membrane and the sample to be tested is as follows: inserting the porous membrane into the The inside of the sample to be tested or the porous membrane is rolled on the surface of the sample to be tested; when the sample to be tested is liquid, the contact between the porous membrane and the sample to be tested is to insert the porous membrane into the sample to be tested. inside the sample to be tested. 7 . The method for direct sampling ionization analysis according to claim 5 , wherein the method of using the metal probe for sampling comprises directly sampling with the metal probe and sampling with the metal probe and a kit. 8 . . 8. the application of direct sampling ionization analysis method as claimed in claim 5 is characterized in that, comprises qualitative analysis and quantitative analysis, in described qualitative analysis aspect, described direct sampling ionization analysis method is used to determine the composition of element, phospholipid Sn isomers and carbon-carbon double bond position isomers; in terms of quantitative analysis, the direct sampling ionization analysis method is used to achieve by adding an internal standard on the porous membrane or in the elution solution Quantitative detection of the components to be tested.

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