An analysis method for selectively enriching important biomarkers related to oxidative stress and application

Abstract

This invention discloses an analytical method and application for selectively enriching important biomarkers related to oxidative stress, belonging to the field of biomarker detection technology. The method disclosed in this invention achieves the purification and enrichment of biomarkers in the sample solution through centrifugal microsolid phase extraction; this method establishes a new approach for effectively enriching and highly sensitively analyzing important biomarkers related to oxidative stress, through poly(VIM- co The introduction of coordination polymers on the surface of the EDMA centrifuge column improves the extraction and enrichment of target analytes by the adsorbent, thus solving the technical problem of low throughput in existing detection methods.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomarker detection, and particularly relates to an analysis method for selectively enriching important biomarkers related to oxidative stress and application. BACKGROUND

[0002] Oxidative stress is defined as an adverse stress response resulting from an imbalance between the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and the ability of the organism to counteract their harmful effects. Oxidative stress-related free radicals mainly include ROS and RNS. Studies have shown that oxidative stress is closely related to physiological and pathological processes such as neurodegenerative diseases, tumors, metabolic syndrome, aging, etc. Therefore, quantitative evaluation of the oxidative stress state of the organism is particularly important for the study of the pathogenesis of the organism, disease diagnosis, drug development, etc.

[0003] 3-nitro-L-tyrosine (3-NT) is produced by the reaction of tyrosine with active nitrogen species (such as peroxynitrite and nitrochloride), and is a recognized biomarker of oxidative stress. 3-chloro-L-tyrosine (3-CT) and 3-bromo-L-tyrosine (3-BT) are formed by the reaction of tyrosine with active oxygen species hypochlorous acid and hypobromous acid, respectively, and are considered to be stable endogenous biomarkers of oxidative stress. A biomarker is an index that can be objectively measured and evaluated. Quantitative determination of the oxidative stress-related biomarkers 3-CT, 3-NT and 3-BT can objectively evaluate the different physiological or pathological states of the organism, and has important significance for the study of the pathological mechanism of related diseases, drug efficacy, etc.

[0004] Due to the complexity of biological sample matrix and the low content of target analyte, it is not possible to directly and accurately quantify the target analyte in the biological sample quickly. Therefore, a suitable sample pretreatment method is needed to selectively enrich the target analyte in the sample and remove impurities. At present, the sample pretreatment methods for the determination of 3-CT, 3-NT and 3-BT in complex samples mainly include derivatization and solid phase extraction technology. The derivatization method is complex and time-consuming, and may have the problems of incomplete derivatization and by-product generation. The solid phase extraction technology mainly uses commercially available solid phase extraction columns such as C-18 Bond Elutcartridges, C18 POLAR RP cartridges and aminopropyl Isolute-cartridges, which are relatively expensive and have relatively poor selectivity for target analytes, and usually need to be combined with derivatization methods. In summary, in the quantitative analysis of 3-CT, 3-NT and 3-BT, it is urgent to establish a new sample pretreatment method that is cost-effective, simple and rapid, and has high throughput. SUMMARY

[0005] The application aims to provide an analysis method and application for selectively enriching an important biomarker related to oxidative stress, so as to solve the technical problem of low throughput of the existing detection method.

[0006] In order to achieve the above-mentioned purpose, the application adopts the following technical solutions:

[0007] The application discloses an analysis method for selectively enriching an important biomarker related to oxidative stress, comprising the following steps:

[0008] The poly(VIM-EDMA) centrifugal column is prepared by using monomers, cross-linking agents, pore-forming agents and initiators as raw materials; co

[0009] The surface of the poly(VIM-EDMA) centrifugal column is sequentially washed by a methanol solution of zinc nitrate, methanol, a methanol solution of imidazole analogues and methanol, so as to obtain a MOFs@poly(VIM-EDMA) centrifugal column; the surface of the MOFs@poly(VIM-EDMA) centrifugal column is provided with a coordination polymer; co co co

[0010] The biomarker in the sample solution to be measured is selectively extracted and enriched by using the MOFs@poly(VIM-EDMA) centrifugal column, and the content of the biomarker is determined. co

[0011] Further, the specific steps for preparing the poly(VIM-EDMA) centrifugal column are as follows: co The monomers, cross-linking agents, pore-forming agents and initiators are mixed to obtain a prepolymer solution;

[0012] The prepolymer solution is injected into an empty centrifugal column, and a polymerization reaction is carried out under a sealed condition by heating, so as to obtain the poly(VIM-EDMA) centrifugal column.

[0013] co

[0014] Further, the mass ratio of the monomers, cross-linking agents, pore-forming agents and initiators is (40-50):(95-115):(320-380):(1.2-1.8).

[0015] Further, the monomers are 1-vinylimidazole; the cross-linking agents are ethylene glycol dimethacrylate; the pore-forming agents are propanol / 1,4-butanediol; and the initiators are azobisisobutyronitrile.

[0016] In the pore-forming agents, the weight ratio of propanol to 1,4-butanediol is (125-155):(195-225).​​​​​​​

[0017] Further, the volume of the pre-polymer solution is greater than or equal to 150 µL;

[0018] The temperature of the polymerization reaction is 65~75℃, and the time is 10~14 h.

[0019] Further, the concentration of the methanol solution of zinc nitrate is 5~15 mM, and the volume is 1~1.5 mL;

[0020] The imidazole analogues include one of 2-methylimidazole, benzimidazole, 5,6-dimethylbenzimidazole, naphtho[2,3-d]imidazole, 9,10-phenanthroimidazole, 4-phenylimidazole and 4,5-diphenylimidazole; the concentration of the methanol solution of the imidazole analogues is 5~15 mM, and the volume is 2~3 mL.

[0021] Further, the number of layers of the coordination polymer is greater than or equal to one layer.

[0022] Further, the MOFs@poly(VIM-EDMA) centrifugal column is used for selective extraction and enrichment of the biomarker in the sample solution to be measured, and the content of the biomarker is determined. co -EDMA) centrifugal column in a centrifugal micro-solid phase extraction mode, the biomarker in the sample solution to be measured is selectively extracted and enriched, and further desorption is performed by using an elution solvent, and the eluate is collected to determine the content of the biomarker.

[0023] According to the centrifugal micro-solid phase extraction mode, the MOFs@poly(VIM-EDMA) centrifugal column is used for selective extraction and enrichment of the biomarker in the sample solution to be measured, and further desorption is performed by using an elution solvent, and the eluate is collected to determine the content of the biomarker. co The pH value of the sample solution to be measured is 8.0; the volume of the sample solution to be measured is 4.0 mL; and 10 mM sodium chloride solution and acetonitrile with a volume fraction of 40% are added to the sample solution to be measured before use.

[0024] Further, the elution solvent is methanol containing 10% formic acid.

[0025] The application further discloses application of the analysis method for selective enrichment of the important biomarker related to oxidative stress in purification and enrichment of endogenous 3-CT, 3-NT and 3-BT of Escherichia coli.

[0026] Compared with the prior art, the application has the following beneficial effects:

[0027] The application discloses an analysis method for selective enrichment of important biomarkers related to oxidative stress.

[0028] The application discloses an analysis method for selective enrichment of important biomarkers related to oxidative stress. co-EDMA) centrifugal column, followed by the reaction of zinc nitrate methanol solution, imidazole analog methanol solution in sequence, to obtain MOFs@poly(VIM- co -EDMA) centrifugal column; the purification and enrichment of biomarkers in the sample solution to be tested are realized by means of centrifugal micro solid phase extraction; the method establishes a new method for effectively enriching and highly sensitively analyzing important biomarkers related to oxidative stress, and the poly(VIM- co -EDMA) centrifugal column is introduced with coordination polymers, so that the extraction and enrichment effect of the adsorbent on target analytes is improved, and the technical problem of low throughput existing in the existing detection method is solved.

[0029] Further, the purification and enrichment of endogenous 3-CT, 3-NT and 3-BT in E. coli are realized by means of centrifugal micro solid phase extraction; the method establishes a new method for effectively enriching and highly sensitively analyzing important biomarkers 3-CT, 3-NT and 3-BT related to oxidative stress, and the poly(VIM- co -EDMA) centrifugal column is introduced with coordination polymers, so that the extraction and enrichment effect of the adsorbent on target analytes is improved; in addition, the method adopts the miniaturized solid phase extraction technology - centrifugal micro solid phase extraction, compared with the traditional solid phase extraction technology, the method needs less sample and organic solvent, and adopts the centrifugal mode for extraction and elution, so that the operation is quick and simple, and the extraction of multiple samples can be completed at one time, high-throughput analysis is realized; further, the related experimental results show that the method exhibits high sensitivity and ideal matrix tolerance, and is suitable for rapid and accurate determination of target analytes in complex biological samples; under the optimized conditions, the method has a wide linear range, good precision and low detection limit, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0030] co The poly(VIM- co -EDMA) centrifugal column and the MOFs@poly(VIM- co -EDMA) centrifugal column are scanning electron micrographs;

[0031] Wherein: a, b: scanning electron micrographs of the poly(VIM- co -EDMA) centrifugal column at 10 mu m and 1 mu m; c, d: scanning electron micrographs of the MOFs@poly(VIM- co -EDMA) centrifugal column at 10 mu m and 1 mu m;

[0032] co The effect of eluent type on centrifugal micro solid phase extraction;

[0033] coEffect of pH of sample solution to be tested on centrifugal micro solid phase extraction

[0034] co Effect of salt concentration of sample solution to be tested on centrifugal micro solid phase extraction

[0035] co Effect of acetonitrile content in sample solution to be tested on centrifugal micro solid phase extraction

[0036] co Effect of sample solution loading volume on centrifugal micro solid phase extraction

[0037] co Content of endogenous 3-NT in E. coli of wild type group and wild type plus drug group p <0.001). DETAILED DESCRIPTION

[0038] To enable persons skilled in the art to understand the features and effects of the present application, the following is a general description and definition of the terms and phrases mentioned in the specification and claims. Unless otherwise specified, all technical and scientific words used in the text are of the usual meaning understood by those skilled in the art of the present application, and in case of conflict, the definition in the specification shall prevail.

[0039] Theories or mechanisms described and disclosed herein, whether correct or not, should not be considered limiting on the scope of the present application, i.e., the present application can be practiced without regard to any particular theory or mechanism.

[0040] In this text, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents and concentrations, are for the sake of brevity and convenience. Therefore, the description of numerical ranges or percentage ranges should be considered to have encompassed and specifically disclosed all possible sub-ranges and individual numerical values within the range (including integers and fractions).

[0041] In this text, unless otherwise specified, "comprise", "include", "contain", "have" or similar terms encompass the meaning of "consist of" and "consist essentially of", for example, "A comprises a" encompasses the meaning of "A comprises a and other" and "A comprises only a".

[0042] In this text, for the sake of brevity, all possible combinations of the technical features in each embodiment or example are not described. Therefore, as long as the combinations of the technical features do not conflict, the technical features in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered as the scope disclosed in the specification.

[0043] The application discloses an analysis method for selectively enriching an important biomarker related to oxidative stress, and comprises the following steps.

[0044] S1: monomers, cross-linking agents, pore-forming agents and initiators are mixed, the prepolymer solution is injected into an empty centrifugal column, and a polymerization reaction is generated by closed heating to obtain a poly(VIM-EDMA) centrifugal column; co

[0045] S2: the centrifugal column is flushed with a methanol solution of zinc nitrate, so that zinc ions are fixed on the surface of the poly(VIM-EDMA) material; further flushing the centrifugal column with methanol to remove unreacted zinc ions; then flushing the centrifugal column with a methanol solution of imidazole analogs to fix the imidazole analogs on the material surface by forming a coordination bond with zinc ions; finally, flushing the material with methanol to obtain a MOFs@poly(VIM-EDMA) centrifugal column. The above steps are the operation steps for preparing a layer of coordination polymer, and the steps can be repeated to prepare multiple layers; co co

[0046] S3: in a centrifugal micro-solid phase extraction mode, the MOFs@poly(VIM-EDMA) centrifugal column is used for selectively extracting and enriching target analytes in an actual biological sample (E. coli), and further desorption is performed by using an elution solvent, and the eluate is collected. co

[0047] Preferably, in S1, the monomer is 1-vinylimidazole; the cross-linking agent is ethylene glycol dimethacrylate; the pore-forming agent is propanol / 1,4-butanediol; and the initiator is azobisisobutyronitrile.

[0048] Preferably, the mass ratio of the monomer, the cross-linking agent, the pore-forming agent and the initiator is (40-50):(95-115):(320-380):(1.2-1.8).

[0049] Preferably, in S1, the volume of the prepolymer solution is greater than or equal to 150 µL.

[0050] Preferably, in S1, the reaction temperature of the polymerization reaction is 65-75 ℃, and the time is 10-14 h.

[0051] Preferably, in S2, the concentration of the methanol solution of zinc nitrate is 5-15 mM, and the volume is 1-1.5 mL.

[0052] ​​​​Preferably, in S2, the imidazole analogs include 2-methylimidazole, benzimidazole, 5,6-dimethylbenzimidazole, naphtho[2,3-d]imidazole, 9,10-phenanthroimidazole, 4-phenylimidazole, and 4,5-diphenylimidazole; the concentration of the imidazole analogs is 5-15 mM, and the volume is 2-3 mL.

[0053] Preferably, in S2, the number of layers of the coordination polymer is greater than or equal to one layer.

[0054] Preferably, in S3, the pH value of the sample solution to be tested is adjusted to 8.0, and 10 mM sodium chloride solution and 40% acetonitrile are added.

[0055] Preferably, in S3, the volume of the sample solution of the E. coli to be tested is 4.0 mL.

[0056] Preferably, in S3, the elution solvent is 10% formic acid in methanol.

[0057] The entire process of the centrifugal micro-solid phase extraction includes four steps of activation, sample loading, washing, and elution; (a) activation: sequentially passing 1 mL of methanol, 0.5 mL of water, and 0.5 mL of a sample solvent (10 mM ammonium formate, containing 40% acetonitrile and 10 mM sodium chloride solution, pH 8.0) through the centrifugal column; (b) sample loading: loading 4 mL of the sample solution to be tested for extraction, so that the target analyte is retained on the adsorbent; (3) washing: using 0.5 mL of water to flush the adsorbent to remove impurities that are not retained on the adsorbent; (4) elution: using 0.5 mL of an elution solvent to elute the target analyte retained on the adsorbent.

[0058] Subsequently, liquid chromatography-mass spectrometry (UHPLC-MS / MS) detection analysis was performed: the eluent was blown dry by nitrogen, and was redissolved in 0.1 mL 2% ACN, and UHPLC-MS / MS analysis was performed. The chromatographic conditions were as follows: the mobile phase was 0.001% formic acid / water solution (A) and acetonitrile (B), the flow rate was 0.3 mL / min, and the injection amount was 20 µL. The gradient elution program was set as follows: solution B 0~2.5 min, 2~95%; 2.5~3.5 min, 95%; 3.5~3.7 min, 95~2%; 3.7~5 min, 2%. The ion source related parameters were as follows: the electrospray ion source was in negative ion mode, the voltage was 3500V; the gas temperature was 350°C; the dry gas flow rate was 12 L / min; the sheath gas temperature was 380°C; and the sheath gas flow rate was 12 L / min. The quasi-molecular ion after deprotonation of the target compound was selected as the parent ion, and the ion with the highest abundance in the secondary mass spectrum was selected as the daughter ion, and the m / z channels of the target analyte and the internal standard were 214.0→117.0 (3-CT), 225.1→163.1 (3-NT), 258.0→79.0 (3-BT), 228.1→166.1 (3-NT-d3, 3-nitro-L-tyrosine-d3) for quantitative analysis.

[0059] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application but not used to limit the scope of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content of the application, and these equivalent forms also fall within the scope of the appended claims.

[0060] The following examples use the instruments and equipment that are conventional in the art. The experimental methods in the following examples that are not specified in terms of specific conditions are generally performed according to conventional conditions, or according to the conditions recommended by the manufacturers. The various raw materials used in the following examples are, unless otherwise specified, conventional commercially available products, and the specifications thereof are conventional specifications in the art. In the specification of the application and in the following examples, unless otherwise specified, "%" means "percent by weight", "parts" means "parts by weight", and the ratio means "ratio by weight".

[0061] Example 1

[0062] An analysis method for selectively enriching important biomarkers related to oxidative stress, comprising the following steps:

[0063] S1: 45 mg of monomer 1-vinylimidazole, 105 mg of crosslinking agent ethylene glycol dimethacrylate, 140 mg of porogen propanol and 210 mg of 1,4-butanediol, and 1.5 mg of initiator azobisisobutyronitrile (AIBN) were mixed in a centrifuge tube to form a prepolymer solution. The mixture was sonicated for 15 minutes to thoroughly mix the prepolymer solution and remove any gas. Then, 250 µL of the prepolymer solution was added to an empty centrifuge column, sealed, and placed at 70 °C for polymerization for 12 h. After the polymerization reaction was completed, unreacted monomers, crosslinking agents, and porogens were removed by rinsing with methanol to obtain poly(VIM- co -EDMA) centrifuge column;

[0064] S2: First, use 0.5 mL of methanol to react with the prepared poly(VIM- co The column was rinsed with 2-methylimidazole (-EDMA); then further rinsed with 1.25 mL of a methanol solution of zinc nitrate (10 mmol / L), in which zinc ions form coordination bonds with imidazole, thus fixing the zinc ions on the material surface; next, the material was rinsed with 0.5 mL of methanol to remove unreacted zinc ions; then, the column was rinsed with 2.5 mL of a methanol solution of 2-methylimidazole (10 mmol / L) to allow 2-methylimidazole to form coordination bonds with zinc ions, thus fixing 2-methylimidazole on the material surface; finally, the material was rinsed with 0.5 mL of methanol to remove unreacted 2-methylimidazole, completing the MOFs@poly(VIM- co Preparation of EDMA centrifuge columns;

[0065] S3: Wild-type K12 MG1655 Escherichia coli grown to OD on LB medium. 600The concentration of the bacteria was adjusted to 0.2-0.3, then a certain volume of kanamycin solution was added to make its concentration reach 6.5 μg / mL, and it was cultured at 37°C, 200 rpm for 1 h; 200 mL of bacterial solution was centrifuged at 4000 rpm for 10 min, and the supernatant was discarded. The bottom E. coli bacterial block was added with 1.5 mL of PBS solution for ultrasonic lysis to release the cell contents. The suspension after sufficient lysis was centrifuged at 12000 rpm for 10 min, and the supernatant was collected; the concentration of protein in the supernatant was determined using the Bradford kit. Further addition of protease E (mass of protease E: mass of protein = 1:30) at 50°C for 16 h made the protein in the supernatant fully lysed. 20 μL of the lysate was diluted to 4 mL with a sample solvent, and an internal standard 3-NT-d3 was added to make its final concentration reach 10 nM, to prepare a wild type drug group E. coli sample solution; after the wild type drug group E. coli sample solution was subjected to centrifugal micro-solid phase extraction using the prepared MOFs@poly(VIM-co-EDMA) centrifugal column, the eluate was collected for UHPLC-MS / MS analysis, and the concentrations of endogenous 3-CT, 3-NT and 3-BT in the wild type drug group E. coli were determined.

[0066] Example 2

[0067] An analysis method for selectively enriching important biomarkers related to oxidative stress, comprising the following steps:

[0068] S1: 45 mg of monomer 1-vinylimidazole, 105 mg of crosslinking agent ethylene glycol dimethacrylate, 140 mg of porogen propanol and 210 mg of 1,4-butanediol, 1.5 mg of initiator azobisisobutyronitrile were mixed in a centrifuge tube to form a pre-polymer solution, and ultrasonic was applied for 15 minutes to mix the pre-polymer solution thoroughly and remove the gas therein; then 250 μL of the pre-polymer solution was added to an empty centrifugal column, which was sealed and placed at 70°C for polymerization reaction for 12 h. After the polymerization reaction was completed, methanol was used to rinse to remove unreacted monomers, crosslinking agents and porogens, and a poly(VIM-co-EDMA) centrifugal column was prepared. co

[0069] S2: First, 0.5 mL of methanol was used to prepare the prepared poly(VIM-co-EDMA) centrifugal column, and then 1 mL of the prepared E. coli solution was added to the prepared poly(VIM-co-EDMA) centrifugal column, which was sealed and placed at 37°C, 200 rpm for 1 h; after centrifugal micro-solid phase extraction, the eluate was collected for UHPLC-MS / MS analysis. co ​-EDMA) were flushed with 1.25 mL of a methanolic zinc nitrate solution (10 mmol / L), which allowed the zinc ions to form a coordination bond with the imidazole, thus immobilizing the zinc ions on the surface of the material; then the material was flushed with 0.5 mL of methanol to remove the unreacted zinc ions; then the centrifugal column was flushed with 2.5 mL of a methanolic 2-methylimidazole solution (10 mmol / L) to allow the 2-methylimidazole to form a coordination bond with the zinc ions, thus immobilizing the 2-methylimidazole on the surface of the material; finally, the material was flushed with 0.5 mL of methanol to remove the unreacted 2-methylimidazole, thus completing the preparation of the MOFs@poly(VIM-co-EDMA) centrifugal column. co Preparation of the MOFs@poly(VIM-co-EDMA) centrifugal column;

[0070] S3: Wild-type K12 MG1655 E. coli was grown in LB medium to OD 600 to 0.2~0.3, then an equal volume of sterile water was added, and the culture was incubated at 37°C, 200 rpm for 1 h; 200 mL of the bacterial solution was centrifuged at 4000 rpm for 10 min, and the supernatant was discarded. The E. coli pellet at the bottom was added with 1.5 mL of PBS solution for ultrasonic lysis to release the cell contents. The well-lysed suspension was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The concentration of the protein in the supernatant was determined using a Bradford kit. Further, protease E (mass of protease E: mass of protein = 1:30) was added to the supernatant, which was placed at 50°C for 16 h to fully lyse the protein in the supernatant. 20 μL of the lysate was diluted with a sample solvent to 4 mL, and an internal standard 3-NT-d3 was added to a final concentration of 10 nM to prepare a wild-type E. coli sample solution. After centrifugal micro-solid phase extraction of the wild-type E. coli sample solution using the prepared MOFs@poly(VIM-co-EDMA) centrifugal column, the eluate was collected for UHPLC-MS / MS analysis to determine the concentrations of endogenous 3-CT, 3-NT and 3-BT in the wild-type E. coli.

[0071] Different from Example 1, in this example, the kanamycin solution was replaced with an equal volume of sterile water, and the remaining steps were the same as in Example 1. The concentrations of endogenous 3-CT, 3-NT and 3-BT in the wild-type E. coli were determined.

[0072] Example 3

[0073] Different from Example 1, in this example, benzimidazole was used to replace 2-methylimidazole in Example 1, and the remaining steps were the same as in Example 1.

[0074] Example 4

[0075] Different from example 1, in this example, 5,6-dimethylbenzimidazole is used to replace 2-methylimidazole in example 1, and the remaining steps are the same as in example 1.

[0076] Example 5

[0077] Different from example 1, in this example, naphtho[2,3-d]imidazole is used to replace 2-methylimidazole in example 1, and the remaining steps are the same as in example 1.

[0078] Example 6

[0079] Different from example 1, in this example, 9,10-phenanthroimidazole is used to replace 2-methylimidazole in example 1, and the remaining steps are the same as in example 1.

[0080] Example 7

[0081] Different from example 1, in this example, 4-phenylimidazole is used to replace 2-methylimidazole in example 1, and the remaining steps are the same as in example 1.

[0082] Example 8

[0083] Different from example 1, in this example, 4,5-diphenylimidazole is used to replace 2-methylimidazole in example 1, and the remaining steps are the same as in example 1.

[0084] Example 9

[0085] An analysis method for selectively enriching an important biomarker related to oxidative stress, comprising the following steps:

[0086] S1: 40 mg of monomer 1-vinylimidazole, 95 mg of crosslinking agent ethylene glycol dimethacrylate, 125 mg of porogen propanol and 195 mg of 1,4-butanediol, 1.2 mg of initiator azobisisobutyronitrile are mixed in a centrifuge tube to form a pre-polymerization liquid, and ultrasonic treatment is performed for 15 minutes to fully mix the pre-polymerization liquid and remove the gas therein; then 150 μL of the pre-polymerization liquid is added into an empty centrifuge column, which is sealed and placed in a 65°C polymerization reaction for 14 h; after the polymerization reaction is completed, the unreacted monomer, crosslinking agent and porogen are removed by washing with methanol, and a poly(VIM-EDMA) centrifuge column is prepared. co

[0087] S2: First, 0.5 mL of methanol is used to wash the prepared poly(VIM-EDMA) centrifuge column, and then 0.5 mL of methanol is used to elute the target compound. co ​-EDMA) were flushed with 1 mL of methanol; further, the column was flushed with 1 mL of zinc nitrate in methanol (15 mmol / L), in which zinc ions formed coordination bonds with imidazole, so that the zinc ions were fixed on the surface of the material; then, the material was flushed with 0.5 mL of methanol to remove the unreacted zinc ions; then, the column was flushed with 2 mL of 2-methylimidazole in methanol (15 mmol / L) to form coordination bonds between 2-methylimidazole and zinc ions, so that the 2-methylimidazole was fixed on the surface of the material; finally, the material was flushed with 0.5 mL of methanol to remove the unreacted 2-methylimidazole, and the preparation of the MOFs@poly(VIM-co-EDMA) column was completed. co Preparation of the MOFs@poly(VIM-co-EDMA) column;

[0088] S3: Wild-type K12 MG1655 E. coli was grown in LB medium to OD 600 to 0.2-0.3, and then a certain volume of kanamycin solution was added to make the concentration reach 6.5 μg / mL, and the culture was incubated at 37°C, 200 rpm for 1 h; 200 mL of bacterial solution was centrifuged at 4000 rpm for 10 min, and the supernatant was discarded, and the bottom E. coli block was added with 1.5 mL of PBS solution for ultrasonic lysis to release the cell contents. The suspension after sufficient lysis was centrifuged at 12000 rpm for 10 min, and the supernatant was collected; the concentration of protein in the supernatant was determined using a Bradford kit; further, protease E (mass of protease E: mass of protein = 1:30) was added to the supernatant and placed at 50°C for 16 h to fully lyse the protein; 20 μL of the lysate was diluted to 4 mL with a sample solvent, and an internal standard 3-NT-d3 was added to make the final concentration reach 10 nM, to prepare a wild-type drug-added group E. coli sample solution; after the wild-type drug-added group E. coli sample solution was subjected to centrifugal micro-solid phase extraction using the prepared MOFs@poly(VIM-co-EDMA) column, the eluate was collected for UHPLC-MS / MS analysis to determine the concentrations of endogenous 3-CT, 3-NT and 3-BT in the wild-type drug-added group E. coli.

[0089] Example 10

[0090] An analysis method for selectively enriching important biomarkers related to oxidative stress, comprising the following steps:

[0091] S1: 50 mg of monomer 1-vinylimidazole, 115 mg of crosslinking agent ethylene glycol dimethacrylate, 155 mg of porogen propanol and 225 mg of 1,4-butanediol, and 1.8 mg of initiator azobisisobutyronitrile (AIBN) were mixed in a centrifuge tube to form a prepolymer solution. The mixture was sonicated for 15 minutes to thoroughly mix the prepolymer solution and remove any gas. Then, 400 µL of the prepolymer solution was added to an empty centrifuge column, sealed, and placed at 75 °C for polymerization for 10 h. After the polymerization reaction, unreacted monomers, crosslinking agents, and porogens were removed by rinsing with methanol to obtain poly(VIM- co -EDMA) centrifuge column;

[0092] S2: First, use 0.5 mL of methanol to react with the prepared poly(VIM- co The column was rinsed with 2-methylimidazole (-EDMA); then further rinsed with 1.5 mL of a methanol solution of zinc nitrate (5 mmol / L), in which zinc ions form coordinate bonds with imidazole, thus fixing the zinc ions on the material surface; next, the material was rinsed with 0.5 mL of methanol to remove unreacted zinc ions; then, the column was rinsed with 3 mL of a methanol solution of 2-methylimidazole (5 mmol / L) to allow 2-methylimidazole to form coordinate bonds with zinc ions, thus fixing 2-methylimidazole on the material surface; finally, the material was rinsed with 0.5 mL of methanol to remove unreacted 2-methylimidazole, completing the MOFs@poly(VIM- co Preparation of EDMA centrifuge columns;

[0093] S3: Wild-type K12 MG1655 Escherichia coli grown to OD on LB medium. 600 The concentration was increased to 0.2-0.3, and then a certain volume of kanamycin solution was added to achieve a concentration of 6.5 μg / mL. The mixture was incubated at 37℃ and 200 rpm for 1 h. 200 mL of the bacterial suspension was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the bottom E. coli bacterial block was subjected to sonication with 1.5 mL of PBS solution to release the cell contents. The fully lysed suspension was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The protein concentration in the supernatant was determined using a Bradford assay kit. Proteinase E (proteinase E mass: protein mass = 1:30) was further added, and the mixture was incubated at 50℃ for 16 h to fully lyse the proteins in the supernatant. 20 µL of the lysate was taken and diluted to 4 mL with sample solvent, and internal standard 3-NT-d3 was added to achieve a final concentration of 10 nM, thus obtaining the wild-type drug-treated E. coli sample solution. After centrifugation and microsolid phase extraction, the eluent of the wild-type treated Escherichia coli sample solution was collected for analysis by UHPLC-MS / MS to determine the concentrations of endogenous 3-CT, 3-NT and 3-BT in the wild-type treated Escherichia coli.

[0094] In the above embodiments, the sample solutions to be tested can be selected from the optimal conditions of pH 8.0, the addition of 10 mM sodium chloride solution and 40% acetonitrile, a volume of 4.0 mL, and methanol containing 10% formic acid as the elution solvent.

[0095] To examine MOFs@poly(VIM- co The morphology of the -EDMA) centrifuge column was analyzed using scanning electron microscopy to examine the poly(VIM-) synthesized in the first step. co -EDMA) centrifuge column and the finally synthesized MOFs@poly(VIM- co The morphology of the EDMA centrifuge column was determined. co a- co As shown in Figure d, the scanning electron microscopy (SEM) images reveal that the prepared material consists of spherical particles that are cross-linked and clustered together, exhibiting a porous structure. These morphological characteristics result in a centrifuge column with a large surface area, providing ample binding sites for the selective extraction of target analytes from complex matrices; simultaneously, they also ensure good permeability of the centrifuge column, facilitating the smooth execution of the extraction operation.

[0096] To examine MOFs@poly(VIM- co This invention relates to the extraction performance of EDMA centrifugal columns. Using 3-CT, 3-NT, and 3-BT standards as model compounds, centrifugal microsolid phase extraction was performed, and the relevant factors affecting extraction efficiency were systematically optimized, including: the type of elution solvent, the pH value of the sample solution, the salt concentration of the sample solution, the organic solvent content of the sample solution, and the volume of the sample solution. To obtain more reliable experimental conditions, three replicates were established for each condition.

[0097] I. The Influence of Elution Solvent Type on Centrifugal Microsolid Phase Extraction

[0098] Choosing a suitable elution solvent is crucial for eluting the target analyte. This invention compares the elution effects of methanol, methanol containing 1% formic acid, methanol containing 5% formic acid, and methanol containing 10% formic acid. co As shown, the extraction efficiency gradually increases with the increase of formic acid content in methanol. To fully elute the target analyte, methanol containing 10% formic acid was ultimately chosen as the elution solvent.

[0099] II. The effect of sample solution pH on centrifugal microsolid phase extraction

[0100] like coAs shown, the present application investigated the influence of sample substrate pH in the range of 3.0-10.0 on the extraction effect. The results showed that when the sample pH was 8.0, the extraction effect was the best. Therefore, the pH of the sample solution was controlled at 8.0 for subsequent research.

[0101] III. Influence of salt concentration of sample solution on centrifugal micro-solid phase extraction

[0102] 3-CT, 3-NT and 3-BT are amphoteric chemicals, so the salt concentration in the sample solution will affect the extraction efficiency of centrifugal micro-solid phase extraction. In order to evaluate the influence of salt concentration of sample solution on centrifugal micro-solid phase extraction, different concentrations of sodium chloride (0-100 mM) were added to the sample solution. As shown, co , the extraction efficiency was the best when the sample solution contained 10 mM sodium chloride. Therefore, 10 mM sodium chloride was added to the sample solution in the subsequent research.

[0103] IV. Influence of organic solvent content in sample solution on centrifugal micro-solid phase extraction

[0104] As shown, co , the addition of a small amount of acetonitrile in the sample solution was not conducive to the extraction and enrichment of the target analyte, but when the acetonitrile content in the sample solution exceeded 20%, the extraction effect of the target analyte was significantly improved, and then tended to be stable. Therefore, in order to reduce the amount of organic solvent, 40% acetonitrile was added to the sample solution for further research.

[0105] V. Influence of sample solution loading volume on centrifugal micro-solid phase extraction

[0106] The sample solution loading volume not only affects the enrichment factor of centrifugal micro-solid phase extraction, but also affects the total sample preparation time. As shown, co , the extraction efficiency increased linearly with the increase of the loading volume, indicating that the prepared MOFs@poly(VIM- co -EDMA) centrifugal column had a high sample loading capacity for the target analyte. Considering the enrichment factor of the target analyte and the sample preparation time, the volume of the sample solution was finally set to 4.0 mL.

[0107] Based on the developed centrifugal micro-solid phase extraction technology, the present application further established a high-sensitivity analysis method combining centrifugal micro-solid phase extraction with UHPLC-MS / MS for the quantitative detection of 3-CT, 3-NT and 3-BT, and the linear range, limit of quantification, precision, accuracy, recovery rate, and matrix effect of the method were investigated in detail. The results are shown in Table 1. In the range of 0.08-40 nM, 3-CT, 3-NT and 3-BT had good linear relationship; R 2The values were between 0.9986 and 0.9998; the quantification limits of the three target analytes were 0.08 nM. The intra-day and inter-day precisions at low, medium and high concentrations were in the range of 3.9-17.3%, and the method accuracy was between 99.1% and 113.1%. Further, the wild group of E. coli was used as a matrix for the low, medium and high concentration spiked experiments to test the recovery rate of the established method. After calculation, the 3-CT spiked recovery rate was between 90.9 and 107.5%, the 3-NT spiked recovery rate was between 93.1 and 98.6%, and the 3-BT spiked recovery rate was between 97.0 and 103.7%; the matrix effect was evaluated by the extraction followed by addition method, and the results showed that the matrix effect of the method was between 87.6% and 108.3%. In summary, the method has high sensitivity, good precision and accuracy, and no obvious matrix effect interference, and is suitable for the analysis of endogenous 3-CT, 3-NT and 3-BT in E. coli samples.

[0108] Table 1 Methodology data of centrifugal micro-solid phase extraction combined with UHPLC-MS / MS for analyzing 3-CT, 3-NT and 3-BT

[0109]

[0110] Among them: low concentration solution: containing 0.5 nM 3-CT / 3-NT / 3-BT;

[0111] Medium concentration solution: containing 5 nM 3-CT / 3-NT / 3-BT;

[0112] High concentration solution: containing 40 nM 3-CT / 3-NT / 3-BT.

[0113] The target analytes 3-CT and 3-BT were not detected in the E. coli of the wild group and the wild medicated group, and the content of 3-NT in the E. coli of different groups was as shown in co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co The content of 3-NT in the wild group was 5.8 pmol / mg protein; the content of 3-NT in the wild medicated group was 14.3 pmol / mg protein; the significant increase in the endogenous 3-NT content of E. coli under the action of kanamycin indicated that the oxidative stress of bacteria was activated in the process of antibiotic-mediated killing.

[0114] The above content only illustrates the technical idea of the present application, and cannot limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical scheme falls within the protection scope of the claims of the present application.

Claims

1. An analytical method for selectively enriching important biomarkers related to oxidative stress, characterized in that, Includes the following steps: Poly(VIM-) was prepared using monomers, crosslinking agents, pore-forming agents, and initiators as raw materials. co -EDMA) centrifuge column; The preparation of poly(VIM- co The specific steps for using an EDMA centrifuge column are as follows: The monomer, crosslinking agent, porogen, and initiator are mixed to obtain a prepolymer solution; The prepolymer solution was injected into an empty centrifuge column, and polymerization was carried out under closed conditions by heating to obtain poly(VIM- co -EDMA) centrifuge column; The mass ratio of the monomer, crosslinking agent, porogen, and initiator is (40~50):(95~115):(320~380):(1.2~1.8); the monomer is 1-vinylimidazole; the crosslinking agent is ethylene glycol dimethacrylate; the porogen is propanol / 1,4-butanediol; and the initiator is azobisisobutyronitrile. In the pore-forming agent, the weight ratio of propanol to 1,4-butanediol is (125~155):(195~225). Poly(VIM-) was rinsed sequentially with a methanol solution of zinc nitrate, methanol, a methanol solution of an imidazole analog, and methanol. co -EDMA) centrifuge column surface, to obtain MOFs@poly(VIM- co -EDMA) centrifuge column; the MOFs@poly(VIM- co The surface of the EDMA centrifuge column has a coordination polymer; The concentration of the zinc nitrate methanol solution is 5~15 mM, and the volume is 1~1.5 mL; The imidazole analogues include one of 2-methylimidazolium, benzimidazole, 5,6-dimethylbenzimidazole, naphtho[2,3-d]imidazolium, 9,10-phenanthreneimidazole, 4-phenylimidazolium, and 4,5-diphenylimidazolium; the methanol solution concentration of the imidazole analogues is 5-15 mM, and the volume is 2-3 mL; Using MOFs@poly(VIM- co -EDMA centrifugal column is used to selectively extract and enrich biomarkers in the sample solution to determine the content of biomarkers; Using MOFs@poly(VIM- co -EDMA centrifugal column selectively extracts and enriches biomarkers in the sample solution, and then determines the content of biomarkers, specifically including the following steps: Following the centrifugal microsolid phase extraction method, MOFs@poly(VIM- co -EDMA) centrifugal column selectively extracts and enriches biomarkers in the sample solution, and further desorbs them using an elution solvent. The eluent is then collected for biomarker content determination. The pH value of the sample solution to be tested is 8.0; the volume of the sample solution to be tested is 4.0 mL; the sample solution to be tested contains 10 mM sodium chloride solution and 40% acetonitrile by volume before use; The biomarkers are endogenous 3-CT, 3-NT, and 3-BT in Escherichia coli.

2. The analytical method for selectively enriching important biomarkers related to oxidative stress according to claim 1, characterized in that, The volume of the prepolymer liquid is greater than or equal to 150 µL; The polymerization reaction is carried out at a temperature of 65-75°C for 10-14 hours.

3. The analytical method for selectively enriching important biomarkers related to oxidative stress according to claim 1, characterized in that, The coordination polymer has one or more layers.

4. The analytical method for selectively enriching important biomarkers related to oxidative stress according to claim 1, characterized in that, The elution solvent is methanol containing 10% formic acid by volume.

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