Hypercrosslinked polymer solid-phase microextraction probe, preparation method thereof and application of hypercrosslinked polymer solid-phase microextraction probe in amino acid metabonomics

Abstract

The invention relates to a super-crosslinked polymer solid-phase microextraction probe, a preparation method thereof and application of the super-crosslinked polymer solid-phase microextraction probe in amino acid metabonomics. The super-crosslinked polymer is prepared by taking tryptophan and biphenyl benzyl dichloride as building monomers through a Friedel-Crafts alkylation reaction and a nucleophilic substitution reaction, and the molecular structure of the super-crosslinked polymer is shown as a formula (I); # imgabs0 # is represented by formula (I). The prepared solid-phase microextraction probe can efficiently adsorb amino acid through rich intermolecular acting force, and meanwhile, the rigid structure of biphenyl can improve the porosity of the material, increase the specific surface area of the material and improve the adsorption capacity of the material.

Description

Technical Field

[0001] The present invention relates to the technical field of amino acid metabolomics, and particularly to a hypercrosslinked polymer solid-phase microextraction probe, a preparation method thereof, and an application in amino acid metabolomics. Background Art

[0002] Metabolomics is a research method for quantitatively characterizing small molecule compounds in biological cells, tissues, organs or organisms and finding the relative relationship between metabolites and physiological and pathological changes, and is an important part of systems biology. Amino acid metabolism is widely involved in the life activities of organisms and is an indispensable part of normal physiological metabolism of organisms. Amino acid metabolomics aims to obtain amino acids in organisms in a high-throughput manner, helping researchers obtain the metabolic characteristics of amino acids, and then enabling them to master the physiological state of organisms. By analyzing the changes in the types and abundances of amino acids in organisms exposed to environmental pollutants, the correlation between pollutants and amino acid metabolic pathways can be revealed, and further the impact of pollutants on the normal physiological activities of organisms can be discovered; on the other hand, through amino acid metabolomics, we can also study the impact of pollutant exposure on the nutritional components of aquatic products, providing a reliable basis for food safety and nutritional health assessment.

[0003] However, most of the existing amino acid extraction methods are limited to lethal, ex vivo solvent sampling, which may not reflect the true state of organisms. This sampling method has defects such as a cumbersome operation process, a large consumption of organic solvents, and a large consumption of experimental animals, and does not meet the requirements of current researchers for green analytical chemistry. The solid-phase microextraction (SPME) in vivo sampling technology, as an advanced sample pretreatment technology with simple operation and no need for a large amount of solvents, is expected to provide a more accurate and effective strategy for the real-time monitoring of the abundance of amino acids in vivo.

[0004] The SPME probe coating material is a key factor affecting the solid-phase microextraction effect. Commercial SPME coatings, such as materials like PDMS and PDMS / DVB, are mainly optimized for volatile / semi-volatile organic compounds and lack specific adsorption sites for polar compounds. Due to the complex composition of biological matrices, and amino acid metabolomics requires capturing as many amino acids as possible in organisms, the existing SPME probes have insufficient affinity and specific interaction for strongly hydrophilic amino acids and cannot meet the analysis requirements of amino acid metabolomics.

[0005] For example, patent application CN112547030A proposes a super-crosslinked polymer nanoparticle solid-phase microextraction biocompatible probe and its application in in vivo analysis. The surface coating contains super-crosslinked polymer nanoparticles formed from divinylbenzene and 4-chloromethylstyrene monomers and a poly-noradrenaline biocompatible coating. In the coating structure of the super-crosslinked polymer nanoparticles, a large number of benzene rings can improve the corresponding enrichment factor with herbicides having benzene rings through π-π interactions. However, amino acids are small molecular compounds with high polarity and high water solubility, and all have amino and carboxyl groups in their structures, and some amino acids also have benzene ring structures. To perform high-throughput extraction analysis and detection of amino acids, it is usually necessary to synergistically promote the adsorption of amino acids by the material through various intermolecular forces. This type of SPME probe mainly uses the rich aromatic ring structure of the coating to only produce a strong enrichment effect on herbicides with benzene rings through π-π stacking, and the enrichment ability for substances such as amino acids fails to meet the requirements. Therefore, it is necessary to develop a new type of SPME probe with strong anti-matrix interference ability, stable structure and high adsorption capacity for amino acids to promote the development and application of the amino acid SPME in vivo sampling method in amino acid metabolomics analysis. Summary of the Invention

[0006] The object of the present invention is to overcome the defects and deficiencies of the existing SPME probes and provide a super-crosslinked polymer (Hyper-crosslinked porous polymer, HPP) that can efficiently adsorb amino acids and has strong anti-matrix interference ability.

[0007] Another object of the present invention is to provide a preparation method of the super-crosslinked polymer.

[0008] Another object of the present invention is to provide a solid-phase microextraction probe based on the super-crosslinked polymer.

[0009] Another object of the present invention is to provide a preparation method of the solid-phase microextraction probe based on the super-crosslinked polymer.

[0010] Another object of the present invention is to provide an application of the solid-phase microextraction probe based on the super-crosslinked polymer in amino acid metabolomics analysis.

[0011] The present invention protects a super-crosslinked polymer, which is formed from tryptophan and biphenyl dichlorobenzyl as building monomers through Friedel-Crafts alkylation reaction and nucleophilic substitution reaction. The molecular structure of the super-crosslinked polymer is shown in formula (Ⅰ); Formula (Ⅰ); Wherein, n is the degree of polymerization.

[0012] The hypercrosslinked polymer of the present invention has a delocalized π-bond structure of biphenyl and indole, as well as carboxyl and secondary amine functional groups, and has intermolecular forces such as hydrogen bonds, π-π stacking interactions and van der Waals forces with amino acids, enabling efficient adsorption of amino acids. At the same time, the rigid structure of the biphenyl segment can also increase the porosity of the material, increase the specific surface area of the material, and improve the adsorption capacity of the material.

[0013] The present invention protects a preparation method of a hypercrosslinked polymer, which includes the following steps: under the condition of a Lewis acid catalyst, tryptophan and biphenyldichlorobenzyl are uniformly mixed in a halogenated hydrocarbon solvent, and the hypercrosslinked polymer is obtained based on the Friedel-Crafts alkylation reaction and the nucleophilic substitution reaction.

[0014] In some embodiments, the reaction temperature is 75-85 °C, and the reaction time is 7-9 h. Preferably, the reaction temperature is 78-82 °C.

[0015] In some embodiments, the molar ratio of tryptophan to biphenyldichlorobenzyl is 1:(2-3); preferably 1:(2.4-2.8).

[0016] In some embodiments, the Lewis acid catalyst is selected from at least one of ferric chloride, ferric bromide, aluminum trichloride or zinc chloride. Preferably, it is ferric chloride.

[0017] In some embodiments, the halogenated hydrocarbon solvent is selected from dichloromethane and / or dichloroethane.

[0018] The present invention protects a solid-phase microextraction probe based on a hypercrosslinked polymer, and its solid-phase microextraction coating is made of the hypercrosslinked polymer described in claim 1.

[0019] In some embodiments, the length of the solid-phase microextraction coating is 1-3 cm, and the thickness is 15-60 μm.

[0020] The present invention protects a preparation method of a solid-phase microextraction probe based on a hypercrosslinked polymer, which includes the following steps: S1. The hypercrosslinked polymer and polyacrylonitrile are fully mixed, a solvent is added and heated until the polyacrylonitrile is completely dissolved to obtain an HPP-PAN slurry; S2. The stainless steel wire is vertically placed into the HPP-PAN slurry to make the HPP-PAN slurry evenly distributed on the surface of the stainless steel wire, and dried; the above operation in step S2 is repeated 2-5 times to obtain a solid-phase microextraction probe based on the hypercrosslinked polymer.

[0021] The present invention protects the application of a solid-phase microextraction probe based on a hypercrosslinked polymer in amino acid metabolomics analysis.

[0022] In some of these embodiments, after using the solid-phase microextraction probe based on hypercrosslinked polymer to perform in vivo sampling on an organism, non-targeted amino acid metabolomics analysis is carried out on the sample.

[0023] Compared with the prior art, it has the following beneficial effects: The present invention provides a hypercrosslinked polymer, and the solid-phase microextraction probe prepared therefrom can efficiently adsorb amino acids through rich intermolecular forces; at the same time, the hypercrosslinked polymer has a dense porous structure, high porosity and specific surface area, which is conducive to improving the adsorption capacity of the material for small molecule metabolites, especially amino acids, and isolating the entry of biological macromolecules such as proteins. The solid-phase microextraction probe has the advantages of strong anti-matrix interference ability, high extraction capacity for metabolites such as amino acids, high coating mechanical strength, and good biocompatibility.

[0024] The present invention provides an application of a solid-phase microextraction probe based on hypercrosslinked polymer in amino acid metabolomics analysis. Using a specific solid-phase microextraction probe can facilitate the efficient enrichment of small molecule metabolites, especially amino acids, by the SPME in vivo sampling technique. It overcomes the deficiencies of the traditional sample pretreatment methods for metabolomics analysis, such as destructive sampling and cumbersome operation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a result graph of the morphology and structure characterization of the HPP material and the HPP probe prepared in Example 1 of the present invention. Among them, Figure 1 Figure (a) is a scanning electron microscope image (SEM) of the HPP material; Figure 1 Figure (b) is the SEM image of the HPP probe; Figure 1 Figure (c) is Figure 1 The SEM image of the surface morphology of the HPP probe in Figure (b) at a high magnification; Figure 1 Figure (d) is the infrared absorption spectrum of the HPP material and its preparation monomer; Figure 1 Figure (e) is the nitrogen adsorption-desorption isotherm curve of the HPP material; Figure 1 Figure (f) is the pore size distribution diagram of the HPP material.

[0026] Figure 2 It is a molecular feature map detected in the HPP probe prepared in Example 1 of the present invention and the C18 commercial in vivo SPME extract. Figure 2 Figure (a) is the molecular feature map detected in the in vivo SPME extract of the HPP probe in the positive ion mode; Figure 2 Figure (b) is the molecular feature map detected in the in vivo SPME extract of the HPP probe in the negative ion mode; Figure 2 Figure (c) is the molecular feature map detected in the in vivo SPME extract of the C18 commercial probe in the positive ion mode;Figure 2 Figure (d) is the molecular feature map detected in the in-vivo SPME extract of a C18 commercial probe in negative ion mode.

[0027] Figure 3 This is the principal component analysis (PCA) map in different ionization modes obtained by multivariate unsupervised principal component analysis of the extractable metabolites data of the sulfonamide drug group and the blank group in Example 2 of the present invention. Among them, Figure 3 Figure (a) in it is the PCA map in positive ion mode; Figure 3 Figure (b) in it is the PCA map in negative ion mode.

[0028] Figure 4 This is the result map obtained by volcano plot analysis of the extractable metabolites of the sulfonamide drug group and the blank group in Example 2 of the present invention; among them, Figure 4 Figure (a) in it is the volcano plot detected in positive ion mode; Figure 4 Figure (b) in it is the volcano plot detected in negative ion mode. Detailed implementation manners

[0029] The present invention will be further described below in conjunction with the detailed implementation manners, but the embodiments do not limit the present invention in any form.

[0030] Example 1 A preparation method of a solid-phase microextraction probe based on hypercrosslinked polymer, comprising the following steps: S1, preparing hypercrosslinked polymer (HPP); Weigh tryptophan (0.50 mmol, 102.16 mg), biphenyldichlorobenzyl (1.39 mmol, 349.1 mg) and anhydrous ferric chloride (3.47 mmol, 562.8 mg) respectively into a 100 mL round-bottom flask, and add 40 mL of dichloroethane. After ultrasonic mixing evenly, react at 80 °C for 8 h. Collect the dark green product generated by the reaction, wash it 5 times with absolute ethanol, and dry it at 60 °C to obtain the hypercrosslinked polymer.

[0031] S2, pretreatment of the substrate; Cut the stainless steel wire (SS) with a diameter of 300 μm into small sections with a length of 8 cm, then ultrasonically treat it in saturated concentrated hydrochloric acid, water and methanol for 15 min in sequence, and dry it.

[0032] S3, preparing HPP-PAN slurry; 100 mg of hypercrosslinked polymer and 100 mg of polyacrylonitrile powder (PAN, Mw = 85000) were thoroughly mixed in a 2 mL sample vial. Subsequently, 0.95 mL of N,N-dimethylformamide (DMF) was added, and the mixture was heated to 90 °C until PAN was completely dissolved, obtaining a brown viscous HPP-PAN slurry system.

[0033] S4, preparing a solid-phase microextraction probe based on hypercrosslinked polymer; The stainless steel wire obtained in step S2 was vertically immersed 1.5 cm into the brown viscous HPP-PAN slurry, and rotated to evenly distribute the mucus loaded with HPP on the SS surface; subsequently, the SS was slowly and uniformly withdrawn; it was dried in an oven at 80 °C for 30 min to volatilize DMF and allow HPP-PAN to form a coating attached to the SS. The above operation was repeated 3 times to ensure uniform thickness, obtaining a solid-phase microextraction probe based on hypercrosslinked polymer (HPP probe).

[0034] The prepared HPP material and HPP probe were characterized by scanning electron microscope (SEM), Fourier transform infrared spectrometer, and BET analyzer respectively.

[0035] As Figure 1 shown in Figure (a) in

[0036] As Figure 1 shown in Figure (b) in

[0037] Further, as Figure 1 shown in Figure (c) in

[0038] As Figure 1 shown in the infrared absorption spectrum diagram in Figure (d) in-1 The absorption peak corresponds to the stretching vibration of the carboxyl C=O in tryptophan. The absorption peak at 1724 cm -1 can also be observed in the infrared absorption spectrum of the HPP material, indicating that the carboxyl group did not participate in the hypercrosslinking polymerization reaction and its structure was still retained in the HPP material. The doublet at 3387 cm -1 and 3343 cm -1 appearing in tryptophan, as well as the absorption peak at 1660 cm -1 correspond to the stretching vibration and in-plane deformation vibration of the primary amino group respectively. Both of these absorption peaks disappeared in the absorption spectrum of HPP, proving that the primary amino group of tryptophan participated in the hypercrosslinking polymerization.

[0039] Furthermore, the characteristic multi-substituted absorption peaks of the benzene rings in dichlorodibenzyl and HPP at 1380 - 1650 cm -1 could both be observed. The three sharp absorption peaks with wave numbers between 563 - 729 cm -1 in the absorption spectrum of dichlorodibenzyl were generated by the stretching vibration of C-Cl, while the absorption peak at 818 cm -1 corresponded to the para-phenyl substitution structure of dichlorodibenzyl. The former disappeared in the absorption spectrum of HPP, while the latter remained, proving that the chlorine atom was removed during the hypercrosslinking reaction due to the Friedel-Crafts alkylation reaction and nucleophilic substitution reaction. Therefore, the C-Cl stretching vibration peak disappeared, while the para-phenyl substitution structure still existed. In summary, the results of the infrared absorption spectrum proved the successful preparation of the hypercrosslinked polymer material of tryptophan and dichlorodibenzyl.

[0040] As shown in the nitrogen adsorption - desorption isotherm curve of HPP in (e) of Figure 1 , the HPP material in this example had a large specific surface area. The results calculated according to the Brunauer–Emmett–Teller (BET) model showed that the specific surface area of the HPP material reached 996.20 m 2 / g.

[0041] As shown in the pore size distribution diagram of HPP in (f) of Figure 1 , the pore size distribution of the HPP material in this example was relatively wide, and it had both microporous and mesoporous structures. The above results indicated that the HPP material had a large specific surface area and a rich pore structure, and the abundant accessible active sites enabled it to be an ideal adsorption material.

[0042] Example 2 Application of a solid-phase microextraction probe based on hypercrosslinked polymer in amino acid metabolomics analysis This example provides a method for SPME in vivo sampling and amino acid metabolomics analysis based on the HPP probe of Example 1, and applies it to the study of amino acid metabolomics analysis of tilapia under the exposure of sulfonamide drugs (sulfamethoxazole). The specific operations are as follows: 1. Pretreatment: Obtain juvenile tilapia from a local fish farm and culture them in the laboratory for 8 months (the culture water is dechlorinated tap water, disinfected by ultraviolet light, fully oxygenated, and filtered through activated carbon). Before the formal experiment, divide the tilapia into two groups, namely the blank group (Blank) and the sulfonamide drug group (Sulfonamides, SAs), with 4 fish in each group. Place them in a glass water tank containing 50 L of dechlorinated tap water for acclimation for one week to ensure that the fish adapt to the experimental environment.

[0043] 2. SPME in vivo sampling: Set the SAs exposure concentration in the sulfonamide drug group to 1 mg / L. Use the SPME in vivo sampling technique to enrich and extract the metabolites in the fish muscle tissue, and focus on observing the changes in amino acids. The total duration is 24 h, and the sampling point is set at the 24th h after the fish is exposed to the target spiked pollutant. The in vivo sampling process is as follows: Insert the HPP probe under the guidance of a stainless-steel needle, then withdraw the needle to expose the HPP coating in the fish muscle. After 60 min of extraction, insert the needle back and then remove the HPP probe together. Then rinse the surface of the HPP probe coating with deionized water and dry its moisture with lint-free paper. Subsequently, desorb the metabolites extracted by the probe coating in 100 μL of ACN / water (v / v = 8:2) at 1000 rpm for 60 min. To compare the metabolite broad-spectrum extraction ability with the C18 commercial coating SPME probe, perform the above experimental operations synchronously using the C18 commercial probe.

[0044] 3. Liquid chromatography and mass spectrometry detection: Perform amino acid metabolomics analysis by detecting the eluate through ultra-high performance liquid chromatography combined with functional inorganic-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS, Agilent).

[0045] Separation was carried out using a Poroshell C18 column (2.1×150 mm², particle size 2.7 μm, Agilent). In the positive ion mode, mobile phase A was an aqueous solution containing 0.1% (v / v) formic acid (Formic acid, FA), and mobile phase B was an acetonitrile solution containing 0.1% (v / v) formic acid (Formic acid, FA). In the negative ion mode, mobile phase A was pure water and mobile phase B was acetonitrile. The flow rate was 0.2 mL / min, and the column oven temperature was 30 °C. The injection volume was 5 μL, and scanning analysis was performed in the positive ion mode and the negative ion mode respectively. The mobile phase gradient program was as follows: 0 min, 10% B; 1 min, 10% B; 12 min, 90% B; 18 min, 90% B; 22 min, 10% B; 25 min, 10% B.

[0046] The mass spectrometry scanning range was 50 - 1500 m / z, and the scanning time was 100 ms. The ion source temperature and the desolvation gas temperature were 150 °C and 600 °C respectively. The desolvation gas flow rate was 1100 L / h, and the curtain gas flow rate was 150 L / h. Except for the collision gas being argon, other gases were nitrogen. In the positive ion mode, the capillary voltage was 1 kV; in the negative ion mode, the capillary voltage was 2 kV.

[0047] After every 5 sample analyses, a quality control sample (QC) was injected. This sample was made from an equal mixture of all samples and was used to monitor the system stability and performance. All samples were analyzed in a random order. Mass calibration was performed by continuously injecting the standard calibration substance Leucine enkephalin widely used in mass spectrometry analysis.

[0048] As Figure 2 shown in the molecular feature scatter plot (including both positive and negative ionization modes) of the substances extracted from fish meat by the HPP probe and the C18 commercial probe of Example 1 under the same operating conditions. Each scatter point represents a molecular feature. In the positive ion mode, the metabolite desorption solution of the C18 commercial probe could extract 1073 molecular features ( Figure 2 Figure (c) in Figure 2 ), among which 555 substances could be identified by the mass-to-charge ratio; in the negative ion mode, the metabolite desorption solution of the C18 commercial probe could extract 357 molecular features ( Figure 2In (a) figure, 1233 substances can be identified by mass-to-charge ratio; in the negative ion mode, 452 molecular features can be extracted from the metabolite desorption solution of the HPP probe ( Figure 2 In (b) figure, 274 substances can be identified by mass-to-charge ratio. This proves that compared with the commercial C18 probe, the self-made probe based on HPP material in the present invention can effectively extract a wider spectrum of metabolites through more abundant intermolecular forces (hydrogen bonds, π-π stacking interactions, van der Waals forces, etc.) and more abundant pore structures.

[0049] Combining the results of the two ionization modes, the amino acids extracted and enriched by the HPP probe are shown in Table 1.

[0050] Table 1 Amino acids extracted and enriched by the HPP probe

[0051] *: Non-protein amino acids.

[0052] The results show that the HPP probe of the present invention can enrich and extract 34 amino acids, including 17 standard protein amino acids and 17 non-protein amino acids (see Table 1).

[0053] Among them, leucine and isoleucine cannot distinguish isomers on the first-order mass spectrum, so the results can only be given in one molecular form, while the other two un-detected standard protein amino acids (cysteine and aspartic acid) can both find their derivatives in fish through in vivo SPME sampling of the HPP probe: acetylcysteine and N-methylaspartic acid.

[0054] As Figure 3 shown, after SPME in vivo sampling and detection by UPLC-QTOF-MS before and after 1-day exposure to SAs, the data of the extractable metabolites were analyzed by multivariate unsupervised principal component analysis, and the principal component analysis (PCA) map of the extractable metabolites in the dorsal muscle of tilapia was obtained. It can be seen that after 24h of SAs exposure, the metabolites detected in the two ionization modes showed an obvious clustering trend, indicating that the metabolism of tilapia was significantly disturbed.

[0055] As Figure 4 shown by the volcano plot obtained through SPME in vivo sampling metabolomics analysis, the number of differential metabolites obtained based on the results of this volcano plot is shown in Table 2.

[0056] Table 2 Differential metabolites detected under different treatment groups

[0057] As can be seen from Table 2, under the positive ion mode, a total of 288 significantly expressed metabolites were detected, among which 128 metabolites were significantly up-regulated and 160 metabolites were significantly down-regulated; while under the negative ion mode, a total of 61 differentially expressed metabolites were monitored, among which 20 metabolites were significantly up-regulated and 41 metabolites were significantly down-regulated. Among them, the expressions of serine, glutamate, threonine, aspartic acid, and citrulline were significantly down-regulated, indicating that the stress of sulfonamides would cause a significant decrease in the contents of some amino acids in tilapia.

[0058] The experimental results show that the HPP probe of the present invention can achieve efficient enrichment and extraction of amino acids in fish bodies, and can realize real-time tracking and monitoring of metabolic changes in fish bodies under pollutant exposure, and has great application prospects in the in vivo sampling analysis of amino acid metabolomics.

[0059] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A hypercrosslinked polymer, characterized in that, It is formed by using tryptophan and biphenyl dichlorobenzyl as building monomers through Friedel-Crafts alkylation reaction and nucleophilic substitution reaction, and the molecular structure of the hypercrosslinked polymer is shown in formula (I); Formula (I).

2. The preparation method of the hypercrosslinked polymer according to claim 1, characterized in that, It includes the following steps: Under the condition of a Lewis acid catalyst, tryptophan and biphenyl dichlorobenzyl are uniformly mixed in a halogenated hydrocarbon solvent, and the hypercrosslinked polymer is obtained based on the Friedel-Crafts alkylation reaction and nucleophilic substitution reaction.

3. The preparation method of the hypercrosslinked polymer according to claim 2, wherein The reaction temperature is 75 - 85 °C, and the reaction time is 7 - 9 h.

4. The preparation method of the hypercrosslinked polymer according to claim 2, characterized in that, The molar ratio of the tryptophan to the biphenyl dichlorobenzyl is 1:(2 - 3).

5. The preparation method of the hypercrosslinked polymer according to claim 2, wherein The Lewis acid catalyst is selected from at least one of ferric chloride, ferric bromide, aluminum trichloride or zinc chloride.

6. A solid-phase microextraction probe based on hypercrosslinked polymer, characterized in that, Its solid-phase microextraction coating is made of the hypercrosslinked polymer described in claim 1.

7. The solid-phase microextraction probe based on hypercrosslinked polymer according to claim 6, wherein The length of the solid-phase microextraction coating is 1 - 3 cm, and the thickness is 15 - 60 μm.

8. A method for preparing the solid-phase microextraction probe based on hypercrosslinked polymer according to claim 6 or 7, characterized in that, It includes the following steps: S1, fully mix the hypercrosslinked polymer described in claim 1 with polyacrylonitrile, add a solvent and heat it until the polyacrylonitrile is completely dissolved to obtain an HPP-PAN slurry; S2, vertically place a stainless steel wire into the HPP-PAN slurry to make the HPP-PAN slurry uniformly distributed on the surface of the stainless steel wire, and dry it; repeat the above operation in step S2 for 2 - 5 times to obtain a solid-phase microextraction probe based on the hypercrosslinked polymer.

9. Use of the solid-phase microextraction probe based on the hypercrosslinked polymer described in claim 6 or 7 in amino acid metabolomics analysis.

10. The application according to claim 9, characterized in that, After in vivo sampling of an organism using the solid-phase microextraction probe based on the hypercrosslinked polymer, non-targeted amino acid metabolomics analysis is performed on the sample.

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