Preparation method of magnetic covalent organic framework material for simultaneously enriching angiotensin I, angiotensin II, angiotensin III and aldosterone

Abstract

The invention discloses a preparation method of a magnetic covalent organic framework material for simultaneously enriching angiotensin I, II and III and aldosterone, and belongs to the field of polypeptide and hormone enrichment. The preparation method comprises the following steps: firstly, synthesizing ferroferric oxide (Fe3O4) magnetic nanoparticles; the preparation method comprises the following steps: coating Fe3O4 with COFs (TpPa) by using a solvothermal method; and finally, introducing a silanization reagent on Fe3O4 (at) TpPa by a heating reflux method. According to the material prepared by the invention, the magnetic nanoparticles have the characteristics of quick response to an external magnetic field, easiness in separation and the like, and a target object is simply and quickly extracted and separated by combining a magnetic solid-phase extraction technology. Meanwhile, by introducing a silanization reagent with quaternary ammonium groups, the adsorption capacity of the material is enhanced, and the material has hydrophobic and electrostatic dual adsorption effects. The method is simple to prepare, has excellent sensitivity, binding capacity and reusability, shows excellent enrichment capacity, and has potential application value in the aspects of medical detection, clinical diagnosis, drug research and development and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide and hormone enrichment, and specifically relates to a method for preparing a magnetic covalent skeleton material with hydrophobic and electrostatic dual adsorption effects, and applies the method to enrich angiotensin I, II, III and aldosterone. Background Art

[0002] Primary aldosteronism (PA) is caused by excessive secretion of aldosterone in the body, and is typically manifested by symptoms such as hypertension, high aldosterone, hyponatremia, and low renin. In the blood pressure regulation system, angiotensin is a type of polypeptide substance that can constrict blood vessels. After renin secreted by glomerular cells enters the blood, it reacts with angiotensinogen produced by the liver to hydrolyze it into angiotensin I. Angiotensin I can be cleaved into angiotensin II by angiotensin converting enzyme, and then converted into angiotensin III by aminopeptidase. Angiotensin II can stimulate the adrenal cortex to produce aldosterone, and together with angiotensin II I and aldosterone, regulate the balance of blood pressure in the body. Therefore, simultaneous detection of angiotensin I, II, III and aldosterone and accurate quantification play a vital role in the diagnosis of PA. At present, the commonly used detection methods for hormones and polypeptides are radioimmunoassay, enzyme-linked immunosorbent assay, and chemiluminescence immunoassay. However, these methods are all based on antigen-antibody reactions, which can easily cause interference and overestimation of the results. Therefore, there is an urgent need to find more sensitive and accurate methods to detect aldosterone and angiotensin. The emergence of liquid chromatography-tandem mass spectrometry has solved this problem. It has the characteristics of high separation of liquid chromatography and high sensitivity of mass spectrometry, high accuracy and precision of analysis results, and short analysis time. However, liquid-mass spectrometry instruments are difficult to directly detect some analytes whose lower limit of quantification must reach the picogram level or even higher sensitivity. Therefore, enrichment of angiotensin and aldosterone in complex biological samples before liquid-mass analysis is the key to the successful identification of PA.

[0003] The commonly used techniques for liquid sample pretreatment include liquid-liquid extraction, solid phase extraction, liquid membrane extraction, purge trapping, liquid phase microextraction, etc. Among them, solid phase extraction technology, as a fast and simple sample pretreatment technology, can achieve the enrichment of analytes and improve the accuracy of analytical detection, and suitable solid phase extraction materials can improve the sensitivity and selectivity of the method. Covalent organic framework (COFs) materials, as a new type of solid phase extraction material, have the advantages of large specific surface area, good thermal and chemical stability, and functional modification. They are widely used in food inspection, biomedicine and other pretreatments. However, the direct use of COFs to adsorb polypeptide substances has the disadvantages of cumbersome centrifugal adsorption and desorption, as well as relatively low affinity. Therefore, it is crucial to develop new COFs materials with strong magnetic response and affinity to improve the enrichment efficiency of angiotensin and aldosterone. Summary of the invention

[0004] The purpose of the present invention is to prepare a magnetic covalent organic framework material with dual hydrophobic and electrostatic adsorption effects, and to use it for the separation and enrichment of angiotensin I, II, III and aldosterone. The present invention uses a magnetic covalent organic framework material to overcome the disadvantages of the cumbersome centrifugation of traditional COFs to enrich the target. At the same time, the single enrichment effect of the magnetic covalent organic framework material is improved by post-synthetic modification of the quaternary ammonium group. The positively charged magnetic covalent organic framework material of the present invention is simple to prepare, the conditions are mild, and it has good reusability. The present invention uses human plasma to demonstrate the excellent performance of the synthesized material for the enrichment of angiotensin and aldosterone.

[0005] The invention first uses a solvothermal method to synthesize ferrosoferric oxide (Fe3O4), then synthesizes a magnetic covalent organic framework (Fe3O4@TpPa) material according to the Schiff base condensation method, and finally introduces positively charged quaternary ammonium groups by heating and reflux to obtain a magnetic covalent organic framework material with both hydrophobic and electrostatic adsorption effects.

[0006] In order to achieve the above object, the present invention is implemented according to the following technical solutions:

[0007] A method for preparing a positively charged magnetic covalent organic framework material for enriching angiotensin and aldosterone, characterized by comprising the following steps:

[0008] (1) Dissolve ferric chloride hexahydrate (FeCl3·6H2O) and anhydrous sodium acetate (CH3COONa) in ethylene glycol, and then transfer the mixed solution to a reactor for reaction. After the reaction is completed, remove the ethylene glycol by magnetic separation, and wash the product with water and anhydrous ethanol respectively. Finally, the obtained black Fe3O4 is vacuum dried.

[0009] (2) Fe3O4 magnetic nanoparticles and 1,4-phenylenediamine (Pa) are dissolved in anhydrous ethanol and mixed evenly. 2,4,6-triformylpyrogallol (Tp) is also dissolved in anhydrous ethanol. The two solutions are mixed and transferred to a reactor for reaction. After the reaction is completed, the red product (Fe3O4@TpPa) is washed with N,N-dimethylformamide and acetone respectively and then dried in vacuum.

[0010] (3) Fe3O4@TpPa was added to toluene, and then dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (Si-QAC) was added. The mixture was refluxed for reaction. After the reaction, the obtained red product (Fe3O4@TpPa@Si-QAC) was ultrasonically washed with ethanol and then vacuum dried.

[0011] (4) Furthermore, in the above step (1), the molar ratio of FeCl3·6H2O to CH3COONa is 1:1 to 1:10; the reaction temperature is 150 to 200°C; and the reaction time is 10 to 15 hours.

[0012] (5) Furthermore, in the above step (2), the molar ratio of Fe3O4, Tp and Pa is 1:2:3 to 3:4:9; the reaction temperature is 100 to 150°C; and the reaction time is 10 to 15 hours.

[0013] (6) Furthermore, in the above step (3), the mass ratio of Fe3O4@TpPa and Si-QAC is 1:1-20:1; the reaction temperature is 100-150°C; and the reaction time is 10-20h.

[0014] (7) Furthermore, the role of introducing the positively charged quaternary ammonium group in the above step (3) is to increase the electrostatic adsorption between the material and the target.

[0015] (8) Furthermore, the prepared Fe3O4@TpPa@Si-QAC is a three-dimensional material with magnetic properties and can respond rapidly to external magnetic fields.

[0016] (9) The prepared Fe3O4 is uniformly spherical with a diameter ranging from 400 to 600 nm; the prepared Fe3O4@TpPa grows nanorods on the spheres.

[0017] (10) Furthermore, the prepared Fe3O4@TpPa@Si-QAC is a porous organic material with benzene rings, which has a large specific surface area and more binding sites and has a good enrichment efficiency.

[0018] The advantages of the present invention are:

[0019] (1) This material relies on the hydrophobicity of the benzene ring in COFs and the electrostatic interaction of the quaternary ammonium group in the further modified Si-QAC to enrich angiotensin and aldosterone. This method has mild reaction conditions when enriching the target and does not destroy the structure of angiotensin and aldosterone, which has high versatility.

[0020] (2) The magnetic nanoparticles in this material can respond quickly to external magnetic fields and are easy to separate, saving time.

[0021] (3) This material is combined with magnetic solid phase extraction technology and liquid chromatography-tandem mass spectrometry to establish a detection method for angiotensin I, II, III and aldosterone with high sensitivity and low detection limit.

[0022] (4) The material has good reusability. After five cycles of extraction of angiotensin I, II, III and aldosterone, the peak areas of the four target substances were basically the same as those of the first extraction.

[0023] (5) The material has good reproducibility. The material was used to extract angiotensin I, II, III and aldosterone from human plasma, and the coefficient of variation of the results of three groups of parallel samples was less than 15%.

[0024] (6) The present invention can be successfully applied to the separation and enrichment of angiotensin and aldosterone standards, as well as the enrichment and identification of angiotensin and aldosterone in complex biological samples such as human plasma.

[0025] (7) The material prepared by the present invention has high stability and enrichment effect, and has potential application value in medical testing, clinical diagnosis, drug development, etc. DETAILED DESCRIPTION

[0026] The present invention is described in detail below with reference to examples, but they are not intended to limit the present invention.

[0027] Example 1: Application of magnetic covalent organic framework material to extract angiotensin I, II, III and aldosterone standard solutions.

[0028] (1) Preparation process of magnetic covalent organic framework materials

[0029] (a) 15 g FeCl3·6H2O and 30 g CH3COONa were dissolved in ethylene glycol, and the mixed solution was transferred to a reactor at 180°C for 8 h. After the reaction, the ethylene glycol was removed by magnetic separation, and the product was washed with 20 mL water and 20 mL anhydrous ethanol respectively. Finally, the obtained black Fe3O4 was dried in vacuum.

[0030] (b) 400 mg Fe3O4 magnetic nanoparticles and 360 mg Pa were dissolved in 60 mL anhydrous ethanol and mixed evenly. Another 400 mg Tp was dissolved in 60 mL anhydrous ethanol. The two solutions were mixed and transferred to a reactor at 110 °C for 15 h. After the reaction, the obtained red Fe3O4@TpPa was washed with 20 mL N, N-dimethylformamide and 20 mL acetone, respectively, and then vacuum dried.

[0031] (c) 1 g of Fe3O4@TpPa was added to toluene, followed by 0.5 g of Si-QAC. The mixture was refluxed at 110 °C for 12 h. After the reaction, the obtained red Fe3O4@TpPa@Si-QAC was ultrasonically washed with 20 mL of anhydrous ethanol and then vacuum dried.

[0032] (2) Preparation of the loading solution containing angiotensin I, II, III and aldosterone standards: Use a pipette to take 20 μL of the mixed standard stock solution (using 20% ​​acetonitrile, 1% formic acid and 79% aqueous solution to prepare angiotensin I, II, III and aldosterone at concentrations of 20, 0.2, 0.5 and 2 ng / mL), 180 μL of bovine serum albumin solution (the mass ratio of bovine serum albumin to water is 1:100) and 200 μL of working buffer solution (the mass ratio of ethylenediaminetetraacetic acid, tris(hydroxymethylaminomethane), phenylmethylsulfonyl fluoride, soybean trypsin inhibitor to water is 80000:135000:70:1:1000000) in a 1.5 mL centrifuge tube, vortex and incubate in a 37°C oven for 3 h. Then, 300 μL of internal standard working solution (angiotensin I, II, III and aldosterone were prepared with 1% ammonia and 99% anhydrous ethanol solution to concentrations of 0.5, 0.2, 0.5, and 5 ng / mL) was added to the incubated solution. The mixed solution was centrifuged and set aside for use.

[0033] (3) Extraction of the sample solution containing angiotensin I, II, III and aldosterone standards: 30 mg of the prepared composite material Fe3O4@TpPa@Si-QAC was weighed, evenly dispersed in 550 μL of the sample solution and vortexed for extraction. After magnetic separation, the supernatant was removed, and the captured angiotensin I, II, III and aldosterone were vortexed and eluted with 150 μL of 90% acetonitrile 1% formic acid 9% aqueous solution and 150 μL of anhydrous ethanol in turn, and the supernatant was collected. The eluted liquid nitrogen containing angiotensin I, II, III and aldosterone was re-dissolved by LC-MS / MS detection and analysis (a total of 3 parallel experiments). The results showed that the four targets could be enriched simultaneously without solvent interference, indicating that the prepared Fe3O4@TpPa@Si-QAC has a good enrichment ability for angiotensin I, II, III and aldosterone. 1% ammonia 99% anhydrous ethanol solution, 20% acetonitrile 1% formic acid 79% aqueous solution, 90% acetonitrile 1% formic acid 9% water are all in volume ratio.

[0034] (4) Investigation of the binding capacity of magnetic covalent organic framework materials: Weigh different masses of Fe3O4@TpPa@Si-QAC (10 mg to 50 mg) and disperse them in 550 μL of sample solution and vortex extract. After magnetic separation, remove the supernatant, vortex elute with 150 μL of 90% acetonitrile 1% formic acid 9% aqueous solution and 150 μL of anhydrous ethanol, and collect the supernatant. The eluted liquid nitrogen of angiotensin I, II, III and aldosterone was blown for 1 hour and then re-dissolved and analyzed by LC-MS / MS. The results show that as the amount of material increases, the peak area of ​​angiotensin I, II, III and aldosterone enriched also increases, and reaches the maximum value at 30 mg and remains relatively unchanged, indicating that the invented Fe3O4@TpPa@Si-QAC material has excellent enrichment performance.

[0035] (5) Investigation of the reusability of magnetic covalent organic framework materials

[0036] (a) Enrichment process of angiotensin I, II, III and aldosterone: the same as the enrichment process in (3) of Example 1.

[0037] (b) The Fe3O4@TpPa@Si-QAC material eluted in (a) was collected and vortex-washed twice with 300 μL of anhydrous ethanol. After removing the supernatant, it was used for the enrichment of angiotensin I, II, III and aldosterone in the next cycle. This process was repeated five times, and the eluted liquid nitrogen was blown and redissolved each time for LC-MS / MS analysis. After investigation, compared with the first use, the peak areas of angiotensin I, II, III and aldosterone detected after repeated use for three and five times were basically the same.

[0038] Example 2: Magnetic covalent organic framework materials for the enrichment of angiotensin I, II, III and aldosterone in human plasma

[0039] (1) Preparation process of magnetic covalent organic framework material Fe3O4@TpPa@Si-QAC: same as (1) in Example 1.

[0040] (2) Processing of human plasma samples: 200 μL of plasma sample was added to 200 μL of working buffer solution (the mass ratio of ethylenediaminetetraacetic acid, tris(hydroxymethylaminomethane), phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and water was 80000:135000:70:1:1000000), mixed evenly and incubated in a 37°C incubator for 3 h.

[0041] (3) 400 μL of the mixed human plasma sample after incubation in Example 2 (2) was mixed with 300 μL of the internal standard working solution (angiotensin I, II, III and aldosterone were dissolved in 1% ammonia 99% anhydrous ethanol solution to concentrations of 0.5, 0.2, 0.5 and 5 ng / mL). 550 μL of the centrifuged solution was placed in a centrifuge tube containing 30 mg of Fe3O4@TpPa@Si-QAC and vortexed at room temperature. After magnetic separation, the supernatant was removed and vortexed with 150 μL of 90% acetonitrile 1% formic acid 9% aqueous solution and 150 μL of anhydrous ethanol, and the supernatant was collected. The eluted liquid nitrogen containing angiotensin I, II, III and aldosterone was blown for 1 hour and then re-dissolved and analyzed by LC-MS / MS (a total of 3 parallel tests were performed).

[0042] (4) The obtained LC-MS / MS data were processed by Masslynx software. The four targets can be detected simultaneously, indicating that the invented Fe3O4@TpPa@Si-QAC composite material exhibits excellent enrichment ability in complex biological samples.

[0043] (5) Investigation of the reproducibility of magnetic covalent organic framework materials:

[0044] (a) Human plasma samples were treated at intervals of 0 min, 40 min, and 24 h, respectively: the same as in Example 2 (2).

[0045] (b) Enrichment process of angiotensin I, II, III and aldosterone in plasma: the same as the enrichment process in (3) of Example 2.

[0046] (6) The obtained LC-MS / MS data were processed by Masslynx software. The coefficient of variation of the results of the four target substances measured three times was less than 15%, indicating that the invented Fe3O4@TpPa@Si-QAC has good sample result reproducibility.

Claims

1. A method for preparing a magnetic covalent organic framework material for simultaneously enriching angiotensin I, II, III and aldosterone, characterized in that: The following steps are involved: (1) Dissolving ferric chloride hexahydrate and anhydrous sodium acetate in ethylene glycol, and then transferring the mixed solution into a reactor for reaction; after the reaction is completed, washing the product with deionized water and anhydrous ethanol; finally, vacuum drying the obtained ferroferric oxide, i.e., Fe3O4 magnetic nanoparticles; the molar ratio of FeCl3·6H2O to CH3COONa is 1:1 to 1:10; the reaction temperature is 150 to 200°C; and the reaction time is 10 to 15 hours; (2) Fe3O4 magnetic nanoparticles and 1,4-phenylenediamine Pa are dissolved in anhydrous ethanol and mixed evenly; 2,4,6-triformylpyrogallol Tp is dissolved in anhydrous ethanol; the two solutions are mixed and transferred to a reactor for reaction; after the reaction, the red product Fe3O4@TpPa is washed with N,N-dimethylformamide and acetone respectively and then vacuum dried; the molar ratio of Fe3O4, Tp, and Pa is 1:2:3 to 3:4:9; the reaction temperature is 100 to 150°C; the reaction time is 10 to 15 hours; (3) Fe3O4@TpPa was added to toluene, and then dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (abbreviated as Si-QAC) was added; the mixture was refluxed for reaction; after the reaction, the obtained red product Fe3O4@TpPa@Si-QAC was ultrasonically washed with anhydrous ethanol and then vacuum dried; In step (3), the mass ratio of Fe3O4@TpPa and Si-QAC is 1:1-20:1; the reaction temperature is 100-150°C; and the reaction time is 10-20h.

2. A magnetic covalent organic framework material prepared according to the method of claim 1.

3. The magnetic covalent organic framework material according to claim 2, characterized in that It is used for separation and enrichment of angiotensin and aldosterone standards, or enrichment and identification of angiotensin and aldosterone in complex biological samples such as human plasma.