Analysis method for selective enrichment of important biomarkers related to oxidative stress and application

Abstract

The invention discloses an analysis method and application of selective enrichment oxidative stress related important biomarkers, and belongs to the technical field of biomarker detection. According to the method disclosed by the invention, the purification and enrichment of the biomarker in the sample solution to be detected are realized in a centrifugal micro solid-phase extraction manner; according to the method, a novel method for effectively enriching and highly sensitively analyzing the important biomarkers related to the oxidative stress is established, a coordination polymer is introduced to the surface of a poly (VIM-co-EDMA) centrifugal column, the extraction and enrichment effects of an adsorbent on a target analyte are improved, and the technical problem that an existing detection method is low in flux is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomarker detection, and specifically relates to an analysis method and application of selectively enriching important biomarkers related to oxidative stress. Background Art

[0002] Oxidative stress is defined as an unbalanced oxidation and antioxidant system, in which excessive free radicals continue to produce adverse stress reactions that exceed their elimination capacity. Oxidative stress-related free radicals mainly include reactive oxygen species and reactive nitrogen species. Studies have shown that oxidative stress is closely related to physiological and pathological processes such as neurodegenerative diseases, tumors, metabolic syndrome, and aging. Therefore, the quantitative evaluation of the body's oxidative stress state is particularly important for the study of the body's pathogenesis, disease diagnosis, and drug development.

[0003] 3-Nitro-L-tyrosine (3-NT) is produced by the reaction of tyrosine with reactive nitrogen species (such as pernitrite 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 reactive oxygen species, hypochlorous acid and hypobromous acid, respectively, and are considered to be stable endogenous biomarkers of oxidative stress. Biomarkers are indicators that can be objectively measured and evaluated. The quantitative determination of oxidative stress-related biomarkers 3-CT, 3-NT and 3-BT can objectively evaluate the different physiological or pathological states of the body, which is of great significance for studying the pathological mechanisms of related diseases and the efficacy of drugs.

[0004] Due to the complexity of biological sample matrices and the low content of target analytes, it is impossible to directly quantify the target analytes in biological samples quickly and accurately. Therefore, it is necessary to select appropriate sample pretreatment methods to selectively enrich the target analytes in the samples and achieve the purpose of removing 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 techniques. The derivatization method is complicated and time-consuming, and there may be problems such as incomplete derivatization and the generation of by-products. Solid phase extraction technology mostly 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. It is usually necessary to use it in combination with a derivatization method. In summary, in the quantitative analysis of 3-CT, 3-NT and 3-BT, it is urgent to establish a new cost-effective, simple and fast, and high-throughput sample pretreatment method. Summary of the invention

[0005] The purpose of the present invention is to provide an analytical method and application for selectively enriching important biomarkers related to oxidative stress, so as to solve the technical problem of low throughput in existing detection methods.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention discloses an analytical method for selectively enriching important biomarkers related to oxidative stress, comprising the following steps: Poly(VIM- co -EDMA) centrifugal column; Poly(VIM- co -EDMA) centrifugal column surface to obtain MOFs@poly(VIM- co -EDMA) centrifugal column; the MOFs@poly(VIM- co -EDMA) spin column has coordination polymer on its surface; Using MOFs@poly(VIM- co -EDMA) centrifugal column is used to selectively extract and enrich the biomarkers in the sample solution to be tested, and the content of the biomarkers is determined.

[0007] Furthermore, the preparation of poly(VIM- co -EDMA) centrifugal column specific steps are: After mixing the monomer, the cross-linking agent, the porogen and the initiator, a prepolymer solution is obtained; The prepolymer solution was injected into an empty centrifugal column and heated under closed conditions to perform polymerization reaction to obtain poly(VIM- co -EDMA) spin column.

[0008] Furthermore, the mass ratio of the monomer, the cross-linking agent, the porogen and the initiator is (40-50): (95-115): (320-380): (1.2-1.8).

[0009] Further, the monomer is 1-vinyl imidazole; the crosslinking agent is ethylene glycol dimethacrylate; the porogen is propanol / 1,4-butanediol; the initiator is azobisisobutyronitrile; In the porogen, the weight ratio of propanol to 1,4-butanediol is (125-155): (195-225).

[0010] Further, the volume of the prepolymer solution is greater than or equal to 150 μL; The polymerization reaction temperature is 65-75° C. and the reaction time is 10-14 h.

[0011] Furthermore, the concentration of the zinc nitrate methanol solution is 5-15 mM and the volume is 1-1.5 mL; The imidazole analogue includes one of 2-methylimidazole, benzimidazole, 5,6-dimethylbenzimidazole, naphtho[2,3-d]imidazole, 9,10-phenimidazole, 4-phenylimidazole and 4,5-diphenylimidazole; the methanol solution of the imidazole analogue has a concentration of 5-15 mM and a volume of 2-3 mL.

[0012] Furthermore, the number of layers of the coordination polymer is greater than or equal to one layer.

[0013] Furthermore, using MOFs@poly(VIM- co -EDMA) centrifugal column to selectively extract and enrich the biomarkers in the sample solution to be tested, and determine the content of the biomarkers, which specifically includes the following steps: According to the centrifugal micro solid phase extraction method, MOFs@poly(VIM- co -EDMA) centrifugal column to selectively extract and enrich the biomarkers in the sample solution to be tested, and further use an elution solvent to desorb, and collect the eluate to determine the content of the biomarker; 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; and 10 mM sodium chloride solution and 40% by volume acetonitrile are added to the sample solution to be tested before use.

[0014] Furthermore, the elution solvent is methanol containing 10% by volume of formic acid.

[0015] The present invention also discloses the application of the above analytical method for selectively enriching important biomarkers related to oxidative stress in the purification and enrichment of endogenous 3-CT, 3-NT and 3-BT in Escherichia coli.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses an analytical method for selectively enriching important biomarkers related to oxidative stress, which uses specific monomers, cross-linking agents, pore-forming agents, and initiators to react to obtain poly(VIM- co -EDMA) centrifugal column, followed by the introduction of a methanol solution of zinc nitrate and a methanol solution of an imidazole analog to react and obtain MOFs@poly(VIM- co-EDMA) centrifugal column; the biomarkers in the sample solution to be tested were purified and enriched by centrifugal micro-solid phase extraction; this method established a new method for effective enrichment and highly sensitive analysis of important biomarkers related to oxidative stress, by co -EDMA) centrifugal column surface, which improves the extraction and enrichment effect of the adsorbent on the target analyte and solves the technical problem of low throughput of existing detection methods.

[0017] Furthermore, the purification and enrichment of endogenous 3-CT, 3-NT and 3-BT of Escherichia coli were achieved by centrifugal micro-solid phase extraction. This method established a new method for effective enrichment and highly sensitive analysis of important biomarkers related to oxidative stress, 3-CT, 3-NT and 3-BT. co -EDMA) centrifugal column surface, which improves the extraction and enrichment effect of the adsorbent on the target analyte; in addition, the method adopts a miniaturized solid phase extraction technology - centrifugal micro solid phase extraction. Compared with the traditional solid phase extraction technology, the required sample amount and organic solvent amount are small, and the extraction and elution are carried out by centrifugation, which is quick and simple to operate. At the same time, multiple samples can be extracted at one time to achieve high-throughput analysis; further, relevant experimental results show that this method exhibits high sensitivity and ideal matrix tolerance, and is suitable for the rapid and accurate determination of target analytes in complex biological samples; under optimized conditions, this method has a wide linear range, good precision, and low detection limit, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The poly(VIM- co -EDMA) centrifugal column and MOFs@poly(VIM- co -EDMA) centrifugal column scanning electron microscopy; Where: a, b: poly(VIM- co -EDMA) centrifugal column at 10 μm and 1 μm; c, d: MOFs@poly(VIM- co -EDMA) SEM images of the centrifugal column at 10 μm and 1 μm; Figure 2 The effect of elution solvent type on the effect of centrifugal micro solid phase extraction; Figure 3 The effect of pH of the sample solution on the centrifugal micro solid phase extraction effect; Figure 4 The effect of the salt concentration of the sample solution on the centrifugal micro solid phase extraction effect; Figure 5The effect of acetonitrile content in the sample solution on the centrifugal micro solid phase extraction effect; Figure 6 The influence of the sample volume on the centrifugal micro solid phase extraction effect; Figure 7 is the content of endogenous 3-NT in Escherichia coli in the wild group and the wild group with drug addition ( p <0.001). DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0020] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0021] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0022] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0023] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0024] The present invention discloses an analytical method for selectively enriching important biomarkers related to oxidative stress, comprising the following steps: S1: After mixing the monomer, crosslinking agent, porogen and initiator, the prepolymer solution is injected into an empty centrifugal column and heated in a sealed manner to cause polymerization reaction to obtain poly(VIM- co -EDMA) centrifugal column; S2: Use zinc nitrate methanol solution to wash the centrifugal column so that the zinc ions are fixed on the poly(VIM- co -EDMA) material surface; further use methanol to wash the centrifugal column to remove unreacted zinc ions; then use a methanol solution of imidazole analogue to wash the centrifugal column so that the imidazole analogue can be fixed on the material surface by forming a coordination bond with the zinc ions; finally, use methanol to wash the material to obtain MOFs@poly(VIM- co -EDMA) centrifugal column. The above steps are the operating steps for preparing one layer of coordination polymer. If multiple layers are required, the steps can be repeated; S3: According to the centrifugal micro solid phase extraction method, MOFs@poly(VIM- co -EDMA) centrifugal column is used to selectively extract and enrich the target analyte in the actual biological sample (Escherichia coli), and then the elution solvent is used for desorption, and the eluate is collected for further processing.

[0025] Preferably, in S1, the monomer is 1-vinylimidazole; the cross-linking agent is ethylene glycol dimethacrylate; the porogen is propanol / 1,4-butanediol; and the initiator is azobisisobutyronitrile.

[0026] Preferably, the mass ratio of the monomer, the cross-linking agent, the porogen and the initiator is (40-50): (95-115): (320-380): (1.2-1.8).

[0027] Preferably, in S1, the volume of the prepolymer solution is greater than or equal to 150 μL.

[0028] Preferably, in S1, the polymerization reaction is carried out at a temperature of 65-75° C. and for a time of 10-14 h.

[0029] Preferably, in S2, the concentration of the methanol solution of zinc nitrate is 5-15 mM and the volume is 1-1.5 mL.

[0030] Preferably, in S2, the imidazole analogs include 2-methylimidazole, benzimidazole, 5,6-dimethylbenzimidazole, naphtho[2,3-d]imidazole, 9,10-phenimidazole, 4-phenylimidazole and 4,5-diphenylimidazole; the concentration of the imidazole analogs is 5-15 mM, and the volume is 2-3 mL.

[0031] Preferably, in S2, the number of layers of the coordination polymer is greater than or equal to one layer.

[0032] 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.

[0033] Preferably, in S3, the volume of the Escherichia coli sample solution to be tested is 4.0 mL.

[0034] Preferably, in S3, the elution solvent is methanol containing 10% formic acid.

[0035] The entire process of centrifugal micro-solid phase extraction includes four steps: activation, loading, washing and elution; (a) Activation: 1 mL of methanol, 0.5 mL of water, and 0.5 mL of sample solvent (10 mM ammonium formate, containing 40% acetonitrile and 10 mM sodium chloride solution, pH 8.0) are passed through the centrifugal column in sequence; (b) Loading: 4 mL of the sample solution to be tested is loaded and extracted so that the target analyte is retained on the adsorbent; (3) Washing: The adsorbent is rinsed with 0.5 mL of water to remove impurities that are not retained on the adsorbent; (4) Elution: The target analyte retained on the adsorbent is eluted with 0.5 mL of elution solvent.

[0036] Subsequently, ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) was used for detection and analysis: the eluent was dried by nitrogen, redissolved in 0.1 mL 2% ACN, and analyzed by UHPLC-MS / MS. Chromatographic conditions: 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 volume 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 parameters were as follows: the electrospray ion source was in negative ion mode, the voltage was 3500 V; the gas temperature was 350 °C; the drying 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 deprotonated quasi-molecular ion of the target compound was selected as the parent ion, and the ion with the highest abundance in the secondary mass spectrometry was selected as the daughter ion. The m / z channels of the target analyte and internal standard were detected at 214.0→117.0 (3-CT), 225.1→163.1 (3-NT), 258.0→79.0 (3-BT), 228.1→166.1 (3-NT-d 3 , 3-nitro-L-tyrosine-d 3 ) for quantitative analysis.

[0037] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0038] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples are conventional commercial products unless otherwise specified, and their specifications are conventional specifications in the art. In the specification of the present invention and the following examples, unless otherwise specified, "%" means weight percentage, "part" means weight part, and ratio means weight ratio.

[0039] Example 1 An analytical method for selectively enriching important biomarkers related to oxidative stress, comprising the following steps: S1: 45 mg of monomer 1-vinylimidazole, 105 mg of crosslinker ethylene glycol dimethacrylate, 140 mg of propanol and 210 mg of 1,4-butanediol, and 1.5 mg of initiator azobisisobutyronitrile were mixed in a centrifuge tube to form a prepolymer solution, and ultrasonicated for 15 minutes to fully mix the prepolymer solution and remove the gas therein; then 250 μL of the prepolymer solution was added to an empty centrifuge column, which was sealed and placed at 70°C for polymerization reaction for 12 h. After the polymerization reaction was completed, the unreacted monomer, crosslinker and porogen were removed by washing with methanol to obtain poly(VIM- co -EDMA) centrifugal column; S2: First, use 0.5 mL of methanol to precipitate the prepared poly(VIM- co -EDMA) for washing; further use 1.25 mL of zinc nitrate methanol solution (10 mmol / L) to wash the centrifugal column. In this step, zinc ions form coordination bonds with imidazole, so that zinc ions are fixed on the surface of the material; then use 0.5 mL of methanol to wash the material to remove unreacted zinc ions; then use 2.5 mL of 2-methylimidazole methanol solution (10 mmol / L) to wash the centrifugal column to form coordination bonds between 2-methylimidazole and zinc ions, so that 2-methylimidazole is fixed on the surface of the material; finally, use 0.5 mL of methanol to wash the material to remove unreacted 2-methylimidazole, completing MOFs@poly(VIM- co -EDMA) centrifugal column preparation; S3: Wild-type K12 MG1655 E. coli was grown in LB medium to an OD of 600To reach 0.2~0.3, then add a certain volume of kanamycin solution to make its concentration reach 6.5 μg / mL, and culture at 37°C, 200 rpm for 1h; centrifuge 200mL of bacterial solution at 4000 rpm for 10 min, discard the upper culture medium, add 1.5 mL of PBS solution to the E. coli block at the bottom for ultrasonic lysis 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 the Bradford kit. Further add protease E (protease E mass: protein mass = 1:30) and place at 50°C for 16h to fully lyse the protein in the supernatant. Take 20 µL of lysate and dilute to 4 mL with sample solvent, add internal standard 3-NT-d 3 The final concentration was made to reach 10 nM to prepare the wild-type medicated group Escherichia coli sample solution; the wild-type medicated group Escherichia coli sample solution was subjected to centrifugal micro-solid phase extraction using the prepared MOFs@poly(VIM-co-EDMA) centrifugal column, and the eluate was collected for UHPLC-MS / MS analysis to determine the concentrations of endogenous 3-CT, 3-NT and 3-BT of Escherichia coli in the wild-type medicated group.

[0040] Example 2 An analytical method for selectively enriching important biomarkers related to oxidative stress, comprising the following steps: S1: 45 mg of monomer 1-vinylimidazole, 105 mg of crosslinker ethylene glycol dimethacrylate, 140 mg of propanol and 210 mg of 1,4-butanediol, and 1.5 mg of initiator azobisisobutyronitrile were mixed in a centrifuge tube to form a prepolymer solution, and ultrasonicated for 15 minutes to fully mix the prepolymer solution and remove the gas therein; then 250 μL of the prepolymer solution was added to an empty centrifuge column, which was sealed and placed at 70°C for polymerization reaction for 12 h. After the polymerization reaction was completed, the unreacted monomer, crosslinker and porogen were removed by washing with methanol to obtain poly(VIM- co -EDMA) centrifugal column; S2: First, use 0.5 mL of methanol to precipitate the prepared poly(VIM- co-EDMA) for washing; further use 1.25 mL of zinc nitrate methanol solution (10 mmol / L) to wash the centrifugal column. In this step, zinc ions form coordination bonds with imidazole, so that zinc ions are fixed on the surface of the material; then use 0.5 mL of methanol to wash the material to remove unreacted zinc ions; then use 2.5 mL of 2-methylimidazole methanol solution (10 mmol / L) to wash the centrifugal column to form coordination bonds between 2-methylimidazole and zinc ions, so that 2-methylimidazole is fixed on the surface of the material; finally, use 0.5 mL of methanol to wash the material to remove unreacted 2-methylimidazole, completing MOFs@poly(VIM- co -EDMA) centrifugal column preparation; S3: Wild-type K12 MG1655 E. coli was grown in LB medium to an OD of 600 Reach 0.2~0.3, then add sterile water of the same volume as the kanamycin solution in Example 1, and culture at 37°C, 200 rpm for 1h; centrifuge 200 mL of bacterial solution at 4000 rpm for 10 min, discard the upper culture solution, add 1.5 mL of PBS solution to the E. coli blocks at the bottom for ultrasonic lysis 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 kit. Further add protease E (protease E mass: protein mass = 1:30) and place at 50°C for 16h to fully lyse the protein in the supernatant. Take 20 µL of the lysate and dilute it to 4 mL with the sample solvent, add the internal standard 3-NT-d 3 The final concentration was made to reach 10 nM to prepare a wild group Escherichia coli sample solution; the wild group Escherichia coli sample solution was subjected to centrifugal micro-solid phase extraction using the prepared MOFs@poly(VIM-co-EDMA) centrifugal column, and the eluate was collected for UHPLC-MS / MS analysis to determine the concentrations of endogenous 3-CT, 3-NT and 3-BT of Escherichia coli in the wild group.

[0041] 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 of Escherichia coli in the wild group were determined.

[0042] Example 3 The difference from Example 1 is that benzimidazole is used in this example to replace the 2-methylimidazole in Example 1, and the remaining steps are the same as those in Example 1.

[0043] Example 4 The difference from Example 1 is that 5,6-dimethylbenzimidazole is used in this example to replace the 2-methylimidazole in Example 1, and the remaining steps are the same as those in Example 1.

[0044] Example 5 The difference from Example 1 is that naphtho[2,3-d]imidazole is used in this example to replace the 2-methylimidazole in Example 1, and the remaining steps are the same as those in Example 1.

[0045] Example 6 The difference from Example 1 is that 9,10-phenimidazole is used in this example to replace the 2-methylimidazole in Example 1, and the remaining steps are the same as those in Example 1.

[0046] Example 7 The difference from Example 1 is that 4-phenylimidazole is used in this example to replace the 2-methylimidazole in Example 1, and the remaining steps are the same as those in Example 1.

[0047] Example 8 The difference from Example 1 is that 4,5-diphenylimidazole is used in this example to replace the 2-methylimidazole in Example 1, and the remaining steps are the same as those in Example 1.

[0048] Example 9 An analytical method for selectively enriching important biomarkers related to oxidative stress, comprising the following steps: S1: 40 mg of monomer 1-vinylimidazole, 95 mg of crosslinker ethylene glycol dimethacrylate, 125 mg of propanol and 195 mg of 1,4-butanediol, and 1.2 mg of initiator azobisisobutyronitrile were mixed in a centrifuge tube to form a prepolymer solution, and ultrasonicated for 15 minutes to fully mix the prepolymer solution and remove the gas therein; then 150 μL of the prepolymer solution was added to an empty centrifuge column, which was sealed and placed at 65°C for polymerization reaction for 14 h; after the polymerization reaction, the unreacted monomer, crosslinker and porogen were removed by washing with methanol to obtain poly(VIM- co -EDMA) centrifugal column; S2: First, use 0.5 mL of methanol to precipitate the prepared poly(VIM- co-EDMA) for washing; further use 1 mL of zinc nitrate methanol solution (15 mmol / L) to wash the centrifugal column. In this step, zinc ions form coordination bonds with imidazole, so that zinc ions are fixed on the surface of the material; then use 0.5 mL of methanol to wash the material to remove unreacted zinc ions; then use 2 mL of 2-methylimidazole methanol solution (15 mmol / L) to wash the centrifugal column to form coordination bonds between 2-methylimidazole and zinc ions, so that 2-methylimidazole is fixed on the surface of the material; finally, use 0.5 mL of methanol to wash the material to remove unreacted 2-methylimidazole, completing MOFs@poly(VIM- co -EDMA) centrifugal column preparation; S3: Wild-type K12 MG1655 E. coli was grown in LB medium to an OD of 600 To reach 0.2~0.3, then add a certain volume of kanamycin solution to make the concentration reach 6.5 μg / mL, and culture at 37°C, 200 rpm for 1h; centrifuge 200mL of bacterial solution at 4000 rpm for 10 min, discard the upper culture medium, add 1.5 mL of PBS solution to the E. coli block at the bottom for ultrasonic lysis 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 the Bradford kit; further add protease E (protease E mass: protein mass = 1:30) and place at 50°C for 16h to fully lyse the protein in the supernatant; take 20 μL of lysate and dilute it to 4 mL with sample solvent, add internal standard 3-NT-d 3 The final concentration was made to reach 10 nM to prepare the wild-type medicated group Escherichia coli sample solution; the wild-type medicated group Escherichia coli sample solution was subjected to centrifugal micro-solid phase extraction using the prepared MOFs@poly(VIM-co-EDMA) centrifugal column, and the eluate was collected for UHPLC-MS / MS analysis to determine the concentrations of endogenous 3-CT, 3-NT and 3-BT of Escherichia coli in the wild-type medicated group.

[0049] Example 10 An analytical method for selectively enriching important biomarkers related to oxidative stress, comprising the following steps: S1: 50 mg of monomer 1-vinylimidazole, 115 mg of crosslinker ethylene glycol dimethacrylate, 155 mg of propanol and 225 mg of 1,4-butanediol, and 1.8 mg of initiator azobisisobutyronitrile were mixed in a centrifuge tube to form a prepolymer solution, and ultrasonicated for 15 minutes to fully mix the prepolymer solution and remove the gas therein; then 400 μL of the prepolymer solution was added to an empty centrifuge column, which was sealed and placed at 75°C for polymerization reaction for 10 h; after the polymerization reaction, the unreacted monomer, crosslinker and porogen were removed by washing with methanol to obtain poly(VIM- co -EDMA) centrifugal column; S2: First, use 0.5 mL of methanol to precipitate the prepared poly(VIM- co -EDMA) for washing; further use 1.5 mL of zinc nitrate methanol solution (5 mmol / L) to wash the centrifugal column. In this step, zinc ions form coordination bonds with imidazole, so that zinc ions are fixed on the surface of the material; then use 0.5 mL of methanol to wash the material to remove unreacted zinc ions; then use 3 mL of 2-methylimidazole methanol solution (5 mmol / L) to wash the centrifugal column to form coordination bonds between 2-methylimidazole and zinc ions, so that 2-methylimidazole is fixed on the surface of the material; finally, use 0.5 mL of methanol to wash the material to remove unreacted 2-methylimidazole, completing MOFs@poly(VIM- co -EDMA) centrifugal column preparation; S3: Wild-type K12 MG1655 E. coli was grown in LB medium to an OD of 600 The concentration of kanamycin was 0.2-0.3, and then a certain volume of kanamycin solution was added to make the concentration reach 6.5 μg / mL, and cultured at 37℃ and 200 rpm for 1h; 200mL of bacterial solution was centrifuged at 4000 rpm for 10 min, the upper culture medium was discarded, and 1.5 mL of PBS solution was added to the E. coli block at the bottom for ultrasonic lysis 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 the Bradford kit; protease E (protease E mass: protein mass = 1:30) was further added and placed at 50℃ for 16h to fully lyse the protein in the supernatant; 20 μL of lysate was taken and diluted to 4 mL with sample solvent, and internal standard 3-NT-d was added 3 The final concentration was 10 nM to prepare the wild-type drug-treated E. coli sample solution. After the wild-type drug-treated E. coli sample solution was subjected to centrifugal micro-solid phase extraction, 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-treated E. coli.

[0050] In the above embodiments, the sample solution to be tested can be selected under the optimal conditions of pH 8.0, 10 mM sodium chloride solution and 40% acetonitrile, a volume of 4.0 mL, and an elution solvent of methanol containing 10% formic acid.

[0051] To investigate MOFs@poly(VIM- co -EDMA) centrifugal column, and the poly(VIM- co -EDMA) centrifugal column and the final synthesized MOFs@poly(VIM- co -EDMA) centrifugal column morphology was determined. Figure 1 a- Figure 1 As shown in Figure d, the scanning electron microscopy spectrum shows that the prepared material is spherical particles, which are cross-linked to form clusters and exhibit a porous structure. These morphological features give the centrifugal column a large surface area, providing sufficient binding sites to selectively extract target analytes from complex matrices; at the same time, the centrifugal column has good permeability, which is conducive to the smooth progress of the extraction operation.

[0052] In order to investigate MOFs@poly(VIM- co -EDMA) centrifugal column, the present invention uses 3-CT, 3-NT and 3-BT standards as model compounds for centrifugal micro solid phase extraction, and systematically optimizes the relevant factors affecting the extraction efficiency, 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. In order to obtain more reliable experimental conditions, three parallel groups were set up for each condition.

[0053] 1. Effect of elution solvent type on centrifugal micro solid phase extraction Selecting a suitable elution solvent is the key to eluting the target analyte. The present invention compares the elution effects of methanol, methanol containing 1% formic acid, methanol containing 5% formic acid, and methanol containing 10% formic acid. Figure 2 As shown in the figure, with the increase of formic acid content in methanol, the extraction efficiency gradually increases. In order to fully elute the target analytes, methanol containing 10% formic acid was finally selected as the elution solvent.

[0054] 2. Effect of pH of sample solution on centrifugal micro-solid phase extraction like Figure 3 As shown, the present invention investigates the influence of the sample base pH in the range of 3.0 to 10.0 on the extraction effect. The results show that when the sample pH is 8.0, the extraction effect is the best. Therefore, the pH of the sample solution is controlled at 8.0 for subsequent research.

[0055] 3. Effect of Salt Concentration of Sample Solution on Centrifugal Micro-SPE 3-CT, 3-NT and 3-BT are both acidic and alkaline 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 effect of the salt concentration of the sample solution on the centrifugal micro-solid phase extraction, different concentrations of sodium chloride (0~100 mM) were added to the sample solution. Figure 4 As shown in the figure, the extraction efficiency was best when the sample solution contained 10 mM sodium chloride. Therefore, 10 mM sodium chloride was added to the sample solution in subsequent studies.

[0056] 4. Effect of organic solvent content in sample solution on centrifugal micro solid phase extraction like Figure 5 As shown in the figure, adding a small amount of acetonitrile to the sample solution is not conducive to the extraction and enrichment of the target analyte, but when the acetonitrile content of the sample solution exceeds 20%, the extraction effect of the target analyte is significantly improved and then tends to be stable. Therefore, in order to reduce the amount of organic solvent used, 40% acetonitrile was added to the sample solution for further study.

[0057] 5. Effect of sample volume on centrifugal micro solid phase extraction The sample volume not only affects the enrichment factor of centrifugal micro-solid phase extraction, but also affects the total time of sample pretreatment. Figure 6 As shown in Figure 2, the extraction efficiency increases linearly with the increase of sample volume, indicating that the prepared MOFs@poly(VIM- co -EDMA) centrifugal columns have a higher loading capacity for target analytes. Taking into account the enrichment multiple of the target analytes and the sample preparation time, the volume of the sample solution was finally set to 4.0 mL.

[0058] Based on the developed centrifugal micro-solid phase extraction technology, the present invention further established a highly sensitive analytical method combining centrifugal micro-solid phase extraction with UHPLC-MS / MS for the quantitative detection of 3-CT, 3-NT, and 3-BT, and conducted a detailed investigation of the linear range, quantitative limit, precision, accuracy, recovery rate, and matrix effect of the method. The results are shown in Table 1. In the range of 0.08 to 40 nM, 3-CT, 3-NT, and 3-BT have a good linear relationship; R 2The values ​​were between 0.9986 and 0.9998. The limits of quantification 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% to 17.3%, and the accuracy of the method was between 99.1% and 113.1%. Furthermore, spike experiments at low, medium and high concentrations were carried out with wild-type Escherichia coli as the matrix to test the recovery of the established method. After calculation, the spike recovery of 3-CT was between 90.9% and 107.5%, the spike recovery of 3-NT was between 93.1% and 98.6%, and the spike recovery of 3-BT was between 97.0% and 103.7%. The matrix effect was evaluated by the post-extraction addition method, and the results showed that the matrix effect of this method was between 87.6% and 108.3%. In summary, this method has high sensitivity, good precision and accuracy, and no obvious matrix effect interference, which is suitable for the analysis of endogenous 3-CT, 3-NT and 3-BT in Escherichia coli samples.

[0059] Table 1 Methodological data for the analysis of 3-CT, 3-NT and 3-BT by centrifugal micro-solid phase extraction coupled with UHPLC-MS / MS

[0060] Among them: Low concentration solution: contains 0.5 nM 3-CT / 3-NT / 3-BT; Medium concentration solution: contains 5 nM 3-CT / 3-NT / 3-BT; High concentration solution: contains 40 nM 3-CT / 3-NT / 3-BT.

[0061] The target analytes 3-CT and 3-BT were not detected in E. coli in the wild group and the wild group with drug addition. The content of 3-NT in E. coli in different groups was as follows Figure 7 As shown; the 3-NT content in the wild group was 5.8 pmol / mg protein; the 3-NT content in the wild-type drug-added group was 14.3 pmol / mg protein; the significant increase in the endogenous 3-NT content of Escherichia coli under the action of kanamycin indicated that the oxidative stress of bacteria was activated during the antibiotic-mediated killing process.

[0062] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. An analytical method for selectively enriching important biomarkers related to oxidative stress, characterized in that: The following steps are involved: Poly(VIM- co -EDMA) centrifugal column; Poly(VIM- co -EDMA) centrifugal column surface to obtain MOFs@poly(VIM- co -EDMA) centrifugal column; the MOFs@poly(VIM- co -EDMA) spin column has coordination polymer on its surface; Using MOFs@poly(VIM- co -EDMA) centrifugal column is used to selectively extract and enrich the biomarkers in the sample solution to be tested, and the content of the biomarkers is determined.

2. The method for selectively enriching important biomarkers related to oxidative stress according to claim 1, characterized in that: The preparation of poly(VIM- co -EDMA) centrifugal column specific steps are: After mixing the monomer, the cross-linking agent, the porogen and the initiator, a prepolymer solution is obtained; The prepolymer solution was injected into an empty centrifugal column and heated under closed conditions to perform polymerization reaction to obtain poly(VIM- co -EDMA) spin column.

3. The method for selectively enriching important biomarkers related to oxidative stress according to claim 2, characterized in that: The mass ratio of the monomer, the cross-linking agent, the porogen and the initiator is (40-50): (95-115): (320-380): (1.2-1.8).

4. The method for selectively enriching important biomarkers related to oxidative stress according to claim 2, characterized in that: 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 porogen, the weight ratio of propanol to 1,4-butanediol is (125-155): (195-225).

5. The method for selectively enriching important biomarkers related to oxidative stress according to claim 2, characterized in that: The volume of the prepolymer solution is greater than or equal to 150 μL; The polymerization reaction temperature is 65-75° C. and the reaction time is 10-14 h.

6. The method for selectively enriching important biomarkers related to oxidative stress according to claim 1, characterized in that: The zinc nitrate methanol solution has a concentration of 5-15 mM and a volume of 1-1.5 mL; The imidazole analogue includes one of 2-methylimidazole, benzimidazole, 5,6-dimethylbenzimidazole, naphtho[2,3-d]imidazole, 9,10-phenimidazole, 4-phenylimidazole and 4,5-diphenylimidazole; the methanol solution of the imidazole analogue has a concentration of 5-15 mM and a volume of 2-3 mL.

7. The method for selectively enriching important biomarkers related to oxidative stress according to claim 1, characterized in that: The number of layers of the coordination polymer is greater than or equal to one layer.

8. The method for selectively enriching important biomarkers related to oxidative stress according to claim 1, characterized in that: Using MOFs@poly(VIM- co -EDMA) centrifugal column to selectively extract and enrich the biomarkers in the sample solution to be tested, and determine the content of the biomarkers, which specifically includes the following steps: According to the centrifugal micro solid phase extraction method, MOFs@poly(VIM- co -EDMA) centrifugal column to selectively extract and enrich the biomarkers in the sample solution to be tested, and further use an elution solvent for desorption, and collect the eluate to determine the content of the biomarker; 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; and 10 mM sodium chloride solution and 40% by volume acetonitrile are added to the sample solution to be tested before use.

9. The method for selectively enriching important biomarkers related to oxidative stress according to claim 8, characterized in that: The elution solvent is methanol containing 10% formic acid by volume.

10. Application of the analytical method for selectively enriching important biomarkers related to oxidative stress according to any one of claims 1 to 9 in the purification and enrichment of endogenous 3-CT, 3-NT and 3-BT in Escherichia coli.

Images