Composite polymer microsphere for extracting and purifying chloro-polyfluoroalkyl ether sulfonic acid in urine as well as preparation method and application of composite polymer microsphere

Abstract

The invention discloses a composite polymer microsphere for extracting and purifying chloro-polyfluoroalkyl ether sulfonic acid in urine and a preparation method and application thereof, the composite polymer microsphere comprises a spherical core layer, the component of the core layer is ferroferric oxide; the first shell layer is coated outside the core layer, and the component of the first shell layer is zirconium-titanium composite oxide; and the first shell layer is coated with the first shell layer, the second shell layer is coated outside the first shell layer, the component of the second shell layer is naphthyridine-oligopeptide bifunctional polymer, and the outer surface of the second shell layer is provided with a fibrous oligopeptide chain. The composite polymer microspheres provided by the invention can be used for rapidly, highly sensitively and accurately detecting the concentration of ultra-trace chloro-polyfluoroalkyl ether sulfonic acid in a urine sample.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection technologies, and particularly to a composite polymer microsphere for extracting and purifying chlorinated polyfluoroalkyl ether sulfonic acid in urine, a preparation method thereof, and an application thereof. Background Art

[0002] Chlorinated polyfluoroalkyl ether sulfonic acid is a new type of chlorine-containing perfluoroalkyl compound. The chemical structural formulas of two common chlorinated polyfluoroalkyl ether sulfonic acids are as Figure 1 shown. In recent years, chlorinated polyfluoroalkyl ether sulfonic acid has been widely used in the industrial field, such as being used as a key component for waterproofing and oil repellency, an industrial lubricant, an anti-corrosion agent, and as a substitute for traditional PFAS (perfluoro- and polyfluoroalkyl substances) fire extinguishing foams. However, chlorinated polyfluoroalkyl ether sulfonic acid has environmental persistence, bioaccumulation, and biological toxicity, and can enter the human body through various channels and accumulate, causing potential harm to health, including liver damage, impaired immune system, endocrine disorders, abnormal development, etc. Long-term exposure may also trigger chronic diseases.

[0003] Currently, most studies mainly focus on the detection of chlorinated polyfluoroalkyl ether sulfonic acid in plasma samples. For example, patent document CN111624274A discloses a high-throughput and rapid detection method for perfluoro- and polyfluoroalkyl compounds in serum, which uses a liquid-liquid extraction method to detect perfluoroalkyl compounds such as 6:2 chlorinated perfluoropolyether sulfonic acid in serum. This method uses highly toxic methyl tert-butyl ether as an extraction agent and MPFACs-Mix as an internal standard for quantitative detection. Another example is that patent document CN118549564A discloses a one-step determination method for perfluoro / polyfluoroalkyl compounds in serum. By using a stepped split-type HMR purification tube, a through-type purification process is adopted for the sample to be loaded, and the pretreatment of the sample is completed in the purification tube.

[0004] Compared with plasma samples, the urine matrix is complex, containing a large amount of uric acid, inorganic salts, and other organic interfering substances. The existing test methods cannot effectively eliminate the matrix interference effect during the detection process, seriously affecting the determination accuracy of chlorinated polyfluoroalkyl ether sulfonic acid in urine. Moreover, the detection limits of perfluoroalkyl compounds in plasma are generally higher than 0.01 μg / L, which has no reference value for the detection of extremely low concentrations (ng / L level) of chlorinated polyfluoroalkyl ether sulfonic acid in human biological samples, and the concentration of extremely trace chlorinated polyfluoroalkyl ether sulfonic acid in human urine samples cannot be accurately monitored by existing methods. Summary of the Invention

[0005] The problem to be solved by the present invention is that when the existing technology detects chlorinated polyfluoroalkyl ether sulfonic acid in urine, there are problems such as poor targeting of the detection method, poor purification effect, low quantitative accuracy, and difficulty in detecting low-concentration target substances.

[0006] To solve the above technical problems, a first aspect of the present invention provides a composite polymer microsphere for extracting and purifying chlorinated polyfluoroalkyl ether sulfonic acid in urine, comprising: A spherical core layer, the component of the core layer being magnetite; A first shell layer, the first shell layer being coated on the outside of the core layer, the component of the first shell layer being a zirconium-titanium composite oxide; A second shell layer, the second shell layer being coated on the outside of the first shell layer, the component of the second shell layer being a naphthyridine-short peptide bifunctional polymer, and the outer surface of the second shell layer having fibrous short peptide chains.

[0007] The composite polymer microsphere of the present invention has a spherical core-shell-shell structure. The core layer of magnetite endows it with magnetism, facilitating rapid solid-liquid phase magnetic separation by magnetic force and greatly improving the sample purification efficiency. The first shell layer of zirconium-titanium composite oxide can effectively adsorb impurities such as phospholipids, uric acid, and inorganic salts in urine. The second shell layer of naphthyridine-short peptide bifunctional polymer, with its outer surface fibrous short peptide chains cooperating with naphthyridine, can effectively improve the selectivity and detection sensitivity of chlorinated polyfluoroalkyl ether sulfonic acid.

[0008] Further, the short peptide in the second shell layer is a trypsinolytic peptide. The trypsinolytic peptide can cooperate with naphthyridine to play a better adsorption selectivity in the process of extracting and purifying chlorinated polyfluoroalkyl ether sulfonic acid in urine, further improving the purification efficiency and sensitization effect of the target compound.

[0009] Further, the molar ratio of naphthyridine to short peptide in the second shell layer is 0.5:1 to 2:1. This ratio can enable naphthyridine and short peptide to exert the best synergistic effect, ensuring good adsorption selectivity and detection sensitivity for chlorinated polyfluoroalkyl ether sulfonic acid in urine.

[0010] Further, the molar ratio of zirconium to titanium elements in the first shell layer is 0.5:1 to 2:1. Under this ratio, the zirconium-titanium composite oxide has the best ability to remove impurities in urine, and can remove impurities such as phospholipids more efficiently than single metal oxides or other ratios, thereby improving the purification efficiency of urine samples and providing guarantee for the subsequent accurate detection of chlorinated polyfluoroalkyl ether sulfonic acid.

[0011] A second aspect of the present invention provides a preparation method of the above composite polymer microsphere, comprising the following steps: S1. Prepare magnetite magnetic microspheres; S2. Modify zirconium-titanium composite oxide on the surface of the magnetite magnetic microspheres obtained in step S1 by solvothermal reaction and perform high-temperature calcination to obtain zirconium-titanium composite-based magnetic microspheres; S3. Modify double bonds on the surface of the zirconium-titanium composite magnetic microspheres obtained in step S2 by using covalent bond coupling technology, and then synthesize naphthyridine-quaternized zirconium-titanium composite magnetic microspheres through polymerization reaction; S4. Use the directed self-assembly technology to couple the quaternary ammonium groups of the naphthyridine-quaternized zirconium-titanium composite magnetic microspheres obtained in step S3 with short peptides to obtain naphthyridine-short peptide bifunctionalized zirconium-titanium composite magnetic microspheres.

[0012] In the present invention, each structural layer is prepared successively through clear steps to ensure the stability of the structure and performance of the composite polymer microspheres, so that the prepared microspheres have good adsorption, separation and detection performances; the reasonable connection and specific processes of each step ensure that the microspheres can achieve technical effects such as effectively removing impurities, improving the selectivity and sensitivity to the target substance.

[0013] Further, step S2 specifically includes: dispersing the magnetic microspheres of magnetite in a solvent, adding tetrabutyl titanate and zirconium oxychloride to the dispersion liquid, reacting the mixed liquid at 180-220 °C for 10-15 h, collecting the product, washing and drying, and calcining at 500-800 °C for 2-5 h to obtain zirconium-titanium composite magnetic microspheres. This step forms a uniform and stable coating layer of zirconium-titanium composite oxide on the surface of the magnetic microspheres of magnetite, ensures the adsorption performance of the first shell layer for impurities in urine, enables the prepared zirconium-titanium composite magnetic microspheres to have good impurity removal ability, and further improves the purification efficiency of the entire composite polymer microspheres.

[0014] Further, step S3 specifically includes: dispersing the zirconium-titanium composite magnetic microspheres in a solvent, adding ammonia water and sodium 3-(propylsulfanyl)propane-1-sulfonate solution under nitrogen protection, reacting at 60-80 °C for 8-16 h, collecting the product, washing and drying to obtain zirconium-titanium composite magnetic microspheres with double bond modification; then dispersing the zirconium-titanium composite magnetic microspheres with double bond modification in a solvent, adding 3-allyl-1,5-naphthyridine, dimethyldiallylammonium chloride and a catalyst, heating to 78-82 °C, maintaining for 30 min, then heating to 81-82 °C, and carrying out condensation reflux for 2-5 h, collecting the product, washing and drying to obtain naphthyridine-quaternized zirconium-titanium composite magnetic microspheres. This step can accurately modify double bonds on the surface of the zirconium-titanium composite magnetic microspheres and synthesize naphthyridine-quaternized zirconium-titanium composite magnetic microspheres, laying a foundation for subsequent coupling with short peptides and ensuring the accurate construction of the structure of the second shell layer.

[0015] Further, the step S4 specifically includes: dissolving 20 parts of bovine serum albumin enzyme and 0.5 part of trypsin in PBS buffer solution, enzymatically digesting at 35-40°C for 14-18 h after mixing, adding naphthyridine-quaternized zirconium titanate composite magnetic microspheres into the mixed solution, performing self-assembly reaction for 1-2 h, collecting the product, washing and drying to obtain naphthyridine-peptide bifunctionalized zirconium titanate composite magnetic microspheres. This step enables the peptide to accurately couple with the quaternary ammonium group, forming naphthyridine-peptide bifunctionalized zirconium titanate composite magnetic microspheres with good performance, ensuring the functional integrity of the second shell layer, and thus realizing the efficient adsorption and detection of target compounds.

[0016] The third aspect of the present invention provides a kit, including the above-mentioned composite polymer microspheres, aqueous formic acid solution, standard series working solutions, and centrifuge tubes.

[0017] The kit of the present invention can realize on-site sampling, processing and preservation of urine samples, has good timeliness, can quickly obtain results accurately, reduces the changes and losses of chlorinated polyfluoroalkyl ether sulfonic acid during sampling and transportation, and improves the accuracy of detection results.

[0018] The fourth aspect of the present invention provides an application of the above-mentioned composite polymer microspheres, using the composite polymer microspheres to detect the concentration of chlorinated polyfluoroalkyl ether sulfonic acid in urine, and the detection method includes the following steps: pipetting 1 ml of urine sample into a centrifuge tube, adding 1 mL of 2% aqueous formic acid solution and 8 mL of pure water, mixing well; adding 10-50 mg of composite polymer microspheres, vortex extracting, performing magnetic separation, and removing the upper layer liquid; adding aqueous formic acid solution and methanol aqueous solution for washing; adding ammonia-methanol solution for elution, vortex extracting, performing magnetic separation, and removing the supernatant; drying with nitrogen, redissolving with methanol, and injecting for LC-MS / MS analysis.

[0019] The present invention applies the composite polymer microspheres to the detection of the concentration of chlorinated polyfluoroalkyl ether sulfonic acid in urine. Through specific detection steps, by utilizing the characteristics of the composite polymer microspheres, it effectively removes interfering impurities in urine, eliminates the matrix interference effect during the detection process, realizes the rapid, highly sensitive and accurate detection of chlorinated polyfluoroalkyl ether sulfonic acid in urine, and the detection method has a good linear relationship, low detection limit and quantification limit, as well as good accuracy and precision.

[0020] Compared with the prior art, the beneficial effects of the present invention include: (1)Efficient impurity removal and precise detection: The zirconium-titanium composite base design enables the composite polymer microspheres to efficiently remove interfering substances such as phospholipids in urine, with small batch-to-batch differences; The naphthyridine-short peptide bifunctional modification greatly improves the selectivity and detection sensitivity for chlorinated polyfluoroalkyl ether sulfonic acid. The quaternary amine modification precisely controls the amount of short peptide modification, further enhancing the purification and sensitization effects, and enabling high-accuracy and high-sensitivity quantitative detection without relying on matrix matching or internal standard methods.

[0021] (2)Enhanced purification and working efficiency: The synergistic effect of the zirconium-titanium composite base makes its performance in removing phospholipids far exceed that of single zirconia and titania, significantly improving the purification efficiency of urine samples; The magnetic component cooperates with the short peptide modification, and the automatic magnetic solid-phase extraction instrument can be used to realize the automatic processing of urine samples, greatly improving the working efficiency of monitoring a large number of urine samples.

[0022] (3)Good recyclability: The performance of the naphthyridine-quaternized zirconium-titanium composite magnetic microspheres shows no obvious decline after being recycled 6 times, effectively saving the usage cost.

[0023] (4)Convenient on-site sampling and detection: The kit combined with a small vortex mixer can realize on-site sampling, processing and preservation of urine samples, reducing the changes and losses of chlorinated polyfluoroalkyl ether sulfonic acid during sampling and transportation. It has good timeliness and accurate results, and can be detected after simple processing in the laboratory. Description of the Drawings

[0024] Figure 1 They are the chemical structural formulas of two kinds of chlorinated polyfluoroalkyl ether sulfonic acids.

[0025] Figure 2 They are the TEM images of the composite polymer microspheres measured in Experimental Example 1.

[0026] Figure 3 They are the TEM-mapping images of the composite polymer microspheres measured in Experimental Example 1.

[0027] Figure 4 They are the SEM images of the composite polymer microspheres measured in Experimental Example 1.

[0028] Figure 5 They are the EDS spectra of the composite polymer microspheres measured in Experimental Example 1.

[0029] Figure 6 They are the test result images of the influence of the amount of composite polymer microspheres on the extraction effect of the target substance measured in Experimental Example 2.

[0030] Figure 7 They are the test result images of the influence of the zirconium-titanium molar ratio in the composite polymer microspheres on the extraction effect of the target substance measured in Experimental Example 3.

[0031] Figure 8Test result graph of the influence of the molar ratio of naphthyridine to short peptide in the composite polymer microspheres measured in Experimental Example 4 on the extraction effect of the target substance.

[0032] Figure 9 Test result graph of the recycling performance of the composite polymer microspheres measured in Experimental Example 5. Detailed implementation manners

[0033] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention will be given in conjunction with the accompanying drawings.

[0034] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0036] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and embodiments of this application are only exemplary.

[0037] Example 1 In this example, a composite polymer microsphere for extracting and purifying chlorinated polyfluoroalkyl ether sulfonic acid in urine is prepared, and its preparation method includes the following steps: Step 1: Prepare magnetic iron oxide microspheres Using the solvothermal method, weigh 0.8 g of sodium citrate and 1.5 g of ferric chloride hexahydrate into a 250 mL flask, add 80 mL of ethylene glycol, stir mechanically at room temperature for 30 min to dissolve, then add 5 g of sodium acetate and continue stirring for 30 min. Transfer the above mixed solution into a 100 mL autoclave, react at 200 °C for 10 h, and cool to room temperature. Then wash with pure water and ethanol three times respectively, and dry overnight in vacuum at 60 °C to obtain magnetic iron oxide microspheres (Fe3O4).

[0038] Step 2: Prepare zirconium-titanium composite-based magnetic microspheres Disperse 5.0 g of magnetic iron oxide microspheres ultrasonically in a mixed solution of 90 mL of N,N-dimethylformamide (DMF) and 270 mL of isopropanol. Then add 3.4 g of tetrabutyl titanate (TBOT) and 3.2 g of zirconium oxychloride (ZrOCl2) within 10 min under ultrasonic conditions. Transfer the mixed solution to an autoclave, then react at a constant temperature of 200 °C for 12 h. Wash the reaction product several times with absolute ethanol and then dry overnight at 60 °C, and then calcine at 600 °C for 5 h to obtain zirconium-titanium composite-based magnetic microspheres (Fe3O4@Zr x Ti y O z ).

[0039] Step 3: Prepare naphthyridine-quaternized zirconium-titanium composite-based magnetic microspheres Disperse 5.0 g of zirconium-titanium composite-based magnetic microspheres in 35 mL of 95% ethanol containing 6.0 g of cetyltrimethylammonium bromide (CTAB), and ultrasonically disperse for 15 min. Then add 2 mL of ammonia water dropwise with stirring under nitrogen protection, and gradually add 15 mL of an ethanol solution of 3-mercapto-1-propanesulfonic acid (MPS). React at 60 °C for 12 h, and wash with pure water and ethanol three times to obtain zirconium-titanium composite-based magnetic microspheres with double-bond modification (Fe3O4@Zr x Ti y O z -MPS).

[0040] Disperse 5.0 g of zirconium-titanium composite-based magnetic microspheres with double-bond modification ultrasonically in 200 mL of isopropanol, place them in a 500 mL three-necked flask, and place the three-necked flask in a microwave reactor, and stir mechanically for 10 min. Dissolve 1.7 g of 3-allyl-1,5-naphthyridine, 4.9 g of dimethyldiallylammonium chloride and 0.5 g of azobisisobutyronitrile (AIBN) in 250 mL of acetonitrile, and add it to the above dispersion. Under ultrasonic-assisted mechanical stirring, heat up to 78 - 82 °C and maintain for 30 min, then quickly heat up to 81 - 82 °C and carry out reflux condensation for 2 h. Wash the product obtained from the reaction three times with pure water and ethanol to obtain naphthyridine-quaternized zirconium-titanium composite-based magnetic microspheres (Fe3O4@Zr x Tiy O z -NAD / QA).

[0041] Step 4. Synthesis of naphthyridine - short peptide bifunctionalized zirconium - titanium composite magnetic microspheres Accurately weigh 20 mg of casein and dissolve it in 10 mL of phosphate - buffered saline (PBS) with pH = 7.4. Meanwhile, add 5 mL of PBS solution containing 0.5 mg of trypsin. After mixing, enzymatically hydrolyze at 37 °C for 16 h. Add 2.5 g of naphthyridine - quaternized zirconium - titanium composite magnetic microspheres to the above - mentioned mixed solution, place it on a rotary mixer for self - assembly reaction for 1 h, wash it twice with water and twice with 0.1% ammonia - methanol solution in sequence, and then wash it twice with water and dry it to obtain naphthyridine - short peptide bifunctionalized zirconium - titanium composite magnetic microspheres (Fe₃O₄@Zr x Ti y O z -NAD / SP).

[0042] Example 2 This example provides a kit, which includes the composite polymer microspheres of Example 1 (Fe₃O₄@Zr x Ti y O z -NAD / SP), aqueous formic acid solution, standard series working solutions, and centrifuge tubes.

[0043] Example 3 This example uses the kit of Example 2 to detect the concentrations of chlorinated polyfluoroalkyl ether sulfonic acids (9 - chlorohexadecafluoro - 3 - oxoalkane - 1 - sulfonic acid (9Cl - PF₃ONS), 11 - chloroeicosafluoro - 3 - oxododecane - 1 - sulfonic acid (11Cl - PF₃OUdS)) in urine samples. The detection method includes the following steps: a. Accurately transfer 1 ml of urine sample into a centrifuge tube, add 1 mL of 2% aqueous formic acid solution and 8 mL of pure water, and mix well.

[0044] b. Add 10 - 50 mg of composite polymer microspheres (Fe₃O₄@Zr x Ti y O z -NAD / SP), vortex - extract for 5 min, place it on a magnetic stand for magnetic separation for 10 s, and pour out the upper layer liquid.

[0045] c. Add 4 mL of 2% aqueous formic acid solution and 4 mL of 50% methanol - aqueous solution for washing, vortex for 5 min, magnetic separation for 10 s, and pour out the upper layer liquid.

[0046] d. Add 4 mL of 0.1% ammonia - methanol solution for elution, vortex for 5 min, magnetic separation for 10 s, and aspirate the supernatant.

[0047] e. Dry it with nitrogen, re-dissolve it in 1 mL of methanol, and perform injection analysis by LC-MS / MS.

[0048] The LC-MS / MS instrument conditions are as follows: (1) Liquid phase conditions Chromatographic conditions: Chromatographic column: ACQUITYTM PREMIER BEH C 18 Column (1.7 μm * 100 mm * 2.1 mm); Injection volume: 5.0 μL; Mobile phase: A is methanol, B is 0.5 mmol / L ammonium fluoride solution; Column temperature: 40 °C; Flow rate: 0.3 mL / min; Elution gradient: Gradient elution: 0 - 2.0 min, 20% B - 60% B; 2.0 - 10.0 min, 60% B - 95% B; 10.0 - 12.0 min, 95% B; 12.0 - 12.1 min, 95% B - 20% B; 12.1 - 15.0 min, 20% B.

[0049] (2) Mass spectrometry conditions Ion source: Electrospray ionization source (ESI source); Detection mode: Multiple reaction monitoring (MRM); Scanning mode: Negative ion mode scanning; Electrospray voltage (IS): Negative ion (-4500 V); Nebulizing gas pressure (GS1): 50.0 psi; Auxiliary gas flow rate (GS2): 50.0 psi; Curtain gas pressure (CUR): 40.0 psi; Collision gas (CAD): 6.0 psi; Ion source temperature (TEM): 500 °C. Other mass spectrometry parameters are listed in Table 1.

[0050] Table 1 Retention time, parent ion, fragment ion, declustering voltage and collision energy of the target compound Experimental Example 1 Characterization of the composite polymer microspheres The morphology and elemental composition of the composite polymer microspheres synthesized in Example 1 were characterized by TEM, TEM-mapping, SEM and EDS respectively. The TEM characterization results are as Figure 2 shown. The composite polymer microspheres exhibit a significant core-shell-shell structure, where the inner core is a magnetic iron oxide microsphere, the middle shell layer is a zirconium-titanium composite oxide, and the outermost layer is a naphthyridine-short peptide bifunctional polymer. Figure 3 The distribution images of Fe, O, Ti, Zr, C, and N in the material verified the rationality of the material synthesis route. Figure 4 The SEM image of the composite polymer microspheres shows that they are spherical as a whole and the surface contains fibrous short peptide chains. Figure 5The EDS analysis results of the composite polymer microspheres show that the material contains Fe, O, Ti, Zr, C, and N elements on the surface. Based on the above characterization analysis results, the composite polymer microspheres are Fe3O4@Zr x Ti y O z -NAD / SP has been successfully synthesized.

[0051] Experimental Example 2 Effect of the dosage of composite polymer microspheres on the extraction efficiency Add 1 mL of urine spiked sample to each of 6 polypropylene centrifuge tubes. The spiked concentrations of two chlorinated polyfluoroalkyl ether sulfonic acids (9Cl-PF3ONS, 11Cl-PF3OUdS) are both 10.0 μg / L. Add 1 mL of 2% formic acid aqueous solution and 8 mL of pure water, vortex extract for 5 min, then add 1 mg, 5 mg, 10 mg, 20 mg, 50 mg, and 100 mg of the composite polymer microspheres prepared in Example 1 respectively, vortex extract for 5 min, then place on a magnetic stand for magnetic separation for 10 s, and pour out the upper layer liquid. Then add 4 mL of 2% formic acid aqueous solution and 4 mL of 50% methanol aqueous solution for washing, vortex for 5 min, magnetic separation for 10 s, and pour out the upper layer liquid. Then add 4 mL of 0.1% ammonia methanol solution for elution, vortex for 5 min, magnetic separation for 10 s, aspirate the supernatant, dry with nitrogen, redissolve with 1 mL of methanol, and inject for LC-MS / MS analysis. The results are as Figure 6 shown.

[0052] From Figure 6 it can be seen that when the dosage of the composite polymer microspheres is small, the purification effect is poor, and the peak areas of the two chlorinated polyfluoroalkyl ether sulfonic acids are small; when the dosage of the composite polymer microspheres is 10 mg, the peak areas of both reach the maximum value; further increasing the dosage of the composite polymer microspheres, the peak areas of both basically remain unchanged. Therefore, the preferred dosage of the composite polymer microspheres in the detection method is 10 - 50 mg.

[0053] Experimental Example 3 Effect of the zirconium-titanium molar ratio of composite polymer microspheres on the extraction efficiency Using the preparation method of Example 1, four kinds of composite polymer microspheres were prepared and labeled as adsorbent a, adsorbent b, adsorbent c, and adsorbent d. The difference is that in step two of the preparation process of adsorbent a, tetrabutyl titanate and zirconium oxychloride are not added; in step two of the preparation process of adsorbent b, 20 mmol of tetrabutyl titanate is added; in step two of the preparation process of adsorbent c, 10 mmol of tetrabutyl titanate and 10 mmol of zirconium oxychloride are added; in step two of the preparation process of adsorbent d, 20 mmol of zirconium oxychloride is added, and the other preparation steps and process parameters are the same.

[0054] Add 1 mL of urine spiked samples into 4 polypropylene centrifuge tubes respectively. The spiked concentrations of two kinds of chlorinated polyfluoroalkyl ether sulfonic acids (9Cl-PF3ONS, 11Cl-PF3OUdS) are both 10.0 μg / L. Adopt the detection method of Experimental Example 3, and detect the concentrations of chlorinated polyfluoroalkyl ether sulfonic acids with adsorbent a, adsorbent b, adsorbent c and adsorbent d. The dosage of adsorbent for each group of experiments is 10 mg. The test results are as Figure 7 shown that when zirconium oxychloride and tetrabutyl titanate (adsorbent a) are not used in the preparation process of the composite polymer microspheres, their purification performance for the two chlorinated polyfluoroalkyl ether sulfonic acids in urine is poor; when only one of the metal sources (adsorbent b and adsorbent d) is adopted, the purification performance of the two chlorinated polyfluoroalkyl ether sulfonic acids in urine is worse than that when both metal sources are used (adsorbent c), but the purification performance is better than that of adsorbent a. Thus, it can be seen that the polymer microspheres containing zirconium-titanium composite base can significantly improve the purification performance for urine samples, and the purification performance is the best when the molar ratio of zirconium to titanium is close.

[0055] Experimental Example 4 Influence of the molar ratio of naphthyridine to short peptide of the composite polymer microspheres on the extraction efficiency Adopt the preparation method of Example 1 to prepare four kinds of composite polymer microspheres respectively, marked as adsorbent 1, adsorbent 2, adsorbent 3 and adsorbent 4. The difference lies in that steps three and four are not carried out in the preparation process of adsorbent 1; 20 mmol of 3-allyl-1,5-naphthyridine is added in step three of the preparation process of adsorbent 2; 10 mmol of 3-allyl-1,5-naphthyridine and 10 mmol of dimethyldiallylammonium chloride are added in step two of the preparation process of adsorbent 3; 20 mmol of dimethyldiallylammonium chloride is added in step two of the preparation process of adsorbent 4, and other preparation steps and process parameters are the same.

[0056] Add 1 mL of urine spiked samples into 4 polypropylene centrifuge tubes respectively. The spiked concentrations of two kinds of chlorinated polyfluoroalkyl ether sulfonic acids (9Cl-PF3ONS, 11Cl-PF3OUdS) are both 10.0 μg / L. Adopt the detection method of Experimental Example 3, and detect the concentrations of chlorinated polyfluoroalkyl ether sulfonic acids with adsorbent 1, adsorbent 2, adsorbent 3 and adsorbent 4 respectively. The dosage of adsorbent for each group of experiments is 10 mg. The test results are as Figure 8As shown in the figure, when the composite polymer microspheres do not contain naphthyridine (NAD) and short peptide (SP) functional groups (adsorbent 1), the peak areas of the two chlorinated polyfluoroalkyl ether sulfonic acids are extremely low, indicating that adsorbent 1 has poor adsorption capacity for chlorinated polyfluoroalkyl ether sulfonic acids in urine, that is, the naphthyridine - short peptide functional groups play a key role in the adsorption of both. In addition, the peak areas of adsorbent 2 (NAD / SP = 1:0) and adsorbent 4 (NAD / SP = 0:1) are lower than those of adsorbent 3 (NAD / SP = 1:1). It can be seen that the bifunctional groups NAD and SP play a synergistic role in the extraction and purification of chlorinated polyfluoroalkyl ether sulfonic acids in urine.

[0057] Experimental Example 5 Recycling Performance Test Add 1 mL of urine spiked samples into 6 polypropylene centrifuge tubes respectively. The spiked concentrations of the two chlorinated polyfluoroalkyl ether sulfonic acids (9Cl - PF3ONS, 11Cl - PF3OUdS) are both 10.0 μg / L. Using the detection method of Experimental Example 3, the 10 mg of composite polymer microspheres prepared in Example 1 were used for 6 cycles, and the test results are as Figure 9 shown. The test results show that for the composite polymer microspheres (Fe3O4@Zr x Ti y O z -NAD / SP), after 6 cycles, the peak areas of the two chlorinated polyfluoroalkyl ether sulfonic acids in urine did not show a significant decrease. It can be seen that the composite polymer microspheres developed in the present invention can be recycled multiple times, which is beneficial to reducing the detection cost and is also more environmentally friendly.

[0058] Experimental Example 6 Matrix Effect Evaluation Accurately pipette 1 mL of urine blank sample into a polypropylene centrifuge tube respectively, add 1 mL of 2% formic acid aqueous solution and 8 mL of pure water, vortex extract for 5 min, and then add 10 mg of the composite polymer microspheres prepared in Example 1 (Fe3O4@Zr x Ti y O z-NAD / SP), vortex extraction for 5 min, then placed on a magnetic stand for magnetic separation for 10 s, and the upper layer liquid was poured off. Then 4 mL of 2% formic acid aqueous solution and 4 mL of 50% methanol aqueous solution were added for washing, vortex for 5 min, magnetic separation for 10 s, and the upper layer liquid was poured off. Then 4 mL of 0.1% ammonia methanol solution was added for elution, vortex for 5 min, magnetic separation for 10 s, the supernatant was aspirated, dried under nitrogen, and re-dissolved in 1 mL of methanol. Then appropriate amounts of standard stock solutions of chlorinated polyfluoroalkyl ether sulfonic acid were added respectively, and matrix-matched standard solutions with concentrations of 0.005 - 20.0 μg / L were prepared with blank urine purification solution. At the same time, the matrix-matched working curve purified by a commercial Waters WAX solid-phase extraction cartridge and the solvent standard series solutions prepared with methanol were compared. Using the formula

matrix effect η = (slope K of the matrix-matched standard curve a - slope K of the solvent standard curve b ) / slope K of the solvent standard curve b

[0059] As can be seen from Table 2, the naphthyridine - short peptide bifunctionalized zirconium - titanium composite magnetic microspheres of the present invention can effectively eliminate the matrix effect during the detection of two chlorinated polyfluoroalkyl ether sulfonic acids in urine samples, and the matrix effect η1 is 0, indicating no matrix effect. That is, during the quantification process, there is no need to prepare a matrix-matched working curve or use isotope internal standards. For the commercial Waters WAX solid-phase extraction cartridge, the absolute values of the matrix effects η2 of the two chlorinated polyfluoroalkyl ether sulfonic acids are 50% and 100% respectively, which are strong matrix enhancement effects. It is necessary to use a matrix-matched working curve or isotope internal standards for quantitative analysis, increasing the complexity of the experimental operation and the running cost of the detection method. Therefore, using the kit based on composite polymer microspheres of the present invention can effectively remove interfering impurities in urine, effectively eliminate the matrix interference effect during the detection of chlorinated polyfluoroalkyl ether sulfonic acids, improve the accuracy of detection, and has the advantages of rapidity, simplicity, and accuracy.

[0060] Table 2 Comparison of the anti-matrix interference capabilities between the examples and the commercial Waters WAX solid-phase extraction cartridge Experimental Example 7 Linear equation, correlation coefficient (R 2 ), accuracy, precision, method detection limit and quantification limit Using the method of Example 3, the residual amounts of two chlorinated polyfluoroalkyl ether sulfonic acids in urine were detected simultaneously. As shown in Table 3, the two chlorinated polyfluoroalkyl ether sulfonic acids showed a good linear relationship in the concentration range of 0.005 - 20.0 μg / L (correlation coefficient R 2 > 0.999). In addition, by testing a series of low-concentration spiked urine samples, the method detection limit and quantification limit were defined as signal-to-noise ratio S / N≥3 and S / N≥9 respectively. The method detection limit and quantification limit of the two chlorinated polyfluoroalkyl ether sulfonic acids in urine were 0.0002 μg / L and 0.0006 μg / L. The detection method established in the present invention can achieve ultra-high sensitive and accurate detection of the two chlorinated polyfluoroalkyl ether sulfonic acids in urine in the concentration range of 0.005 - 20.0 μg / L.

[0061] Table 3 Linear range, linear equation, correlation coefficient, detection limit and quantification limit of the detection method of the present invention The accuracy and precision of the detection method of Example 3 were investigated. The spiked levels of the two chlorinated polyfluoroalkyl ether sulfonic acids in the urine samples were controlled at 0.1, 1.0, 10.0 and 20.0 μg / L respectively, and the detection was carried out using the method of Example 3. The results are shown in Table 4. The test results showed that the spiked recoveries of the two chlorinated polyfluoroalkyl ether sulfonic acids by the detection method of the present invention were between 99.0% - 117% and 88.0% - 104% respectively, and the relative standard deviations (RSDs) were 1.2% - 5.8%. It can be seen that the detection method based on the composite polymer microspheres of the present invention has the advantages of rapidity, sensitivity and accuracy.

[0062] Table 4 Spiked recoveries and precision of the detection method of the present invention (n = 6) Although the present invention is disclosed as above, the protection scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will all fall within the protection scope of the present invention.

Claims

1. A composite polymer microsphere for extracting and purifying chlorinated polyfluoroalkyl ether sulfonic acid in urine, characterized in that, include: A spherical core layer, wherein the core layer is composed of ferroferric oxide; A first shell layer, the first shell layer is coated on the outside of the core layer, and the first shell layer is composed of zirconium-titanium composite oxide; The second shell layer is coated on the outside of the first shell layer. The second shell layer is composed of a naphthyridine-short peptide bifunctional polymer. The outer surface of the second shell layer has a fibrous short peptide chain.

2. The composite polymer microspheres according to claim 1, characterized in that, The short peptides in the second shell are casein-hydrolyzed peptides.

3. The composite polymer microspheres according to claim 1, characterized in that, The molar ratio of naphthyridine to short peptide in the second shell layer is 0.5:1 to 2:

1.

4. The composite polymer microspheres according to claim 1, characterized in that, The molar ratio of zirconium to titanium in the first shell layer is 0.5:1 to 2:

1.

5. A method for preparing a composite polymer microsphere according to any one of claims 1-4, characterized in that, The following steps are involved: S1, preparing ferroferric oxide magnetic microspheres; S2, modifying the surface of the ferroferric oxide magnetic microspheres obtained in step S1 with zirconium-titanium composite oxide by a solvothermal reaction, and calcining at a high temperature to obtain zirconium-titanium composite-based magnetic microspheres; S3, using a covalent coupling technique to modify double bonds on the surface of the zirconium-titanium composite magnetic microspheres obtained in step S2, and then using a polymerization reaction to synthesize naphthyridine-quaternary ammonium zirconium-titanium composite magnetic microspheres; S4, using directed self-assembly technology to couple the quaternary ammonium groups of the naphthyridine-quaternary ammonium zirconium titanium composite-based magnetic microspheres obtained in step S3 with short peptides to obtain naphthyridine-short peptide bifunctional zirconium titanium composite-based magnetic microspheres.

6. The preparation method according to claim 5, characterized in that, The step S2 specifically includes: dispersing ferroferric oxide magnetic microspheres in a solvent, adding tetrabutyl titanate and zirconium oxychloride to the dispersion, reacting the mixture at 180-220° C. for 10-15 hours, collecting the product, washing, and drying it, and calcining it at 500-800° C. for 2-5 hours to obtain zirconium-titanium composite-based magnetic microspheres.

7. The preparation method according to claim 5, characterized in that The step S3 specifically includes: dispersing the zirconium-titanium composite magnetic microspheres in a solvent, adding ammonia water and sodium thiopropane sulfonate solution under nitrogen protection, reacting at 60-80° C. for 8-16 hours, collecting the product, washing, and drying to obtain zirconium-titanium composite magnetic microspheres containing double bonds; then dispersing the zirconium-titanium composite magnetic microspheres containing double bonds in a solvent, adding 3-allyl-1,5-naphthyridine, dimethyldiallylammonium chloride, and a catalyst, heating to 78-82° C., maintaining for 30 minutes, then heating to 81-82° C., condensing and reflux for 2-5 hours, collecting the product, washing, and drying to obtain naphthyridine-quaternized zirconium-titanium composite magnetic microspheres.

8. The preparation method according to claim 5, characterized in that The step S4 specifically includes: dissolving 20 parts of bovine serum albumin and 0.5 parts of trypsin in a PBS buffer solution, mixing and enzymolyzing at 35-40° C. for 14-18 hours, adding naphthyridine-quaternary ammonium zirconium titanium composite magnetic microspheres to the mixed solution, performing a self-assembly reaction for 1-2 hours, collecting the product, washing, and drying to obtain naphthyridine-short peptide bifunctional zirconium titanium composite magnetic microspheres.

9. A kit, characterized in that The method comprises the composite polymer microspheres according to any one of claims 1 to 4, a formic acid aqueous solution, a standard series working solution, and a centrifuge tube.

10. A use of the composite polymer microspheres according to any one of claims 1 to 4, characterized in that: The composite polymer microspheres are used to detect the concentration of chloropolyfluoroalkyl ether sulfonic acid in urine. The detection method includes the following steps: transferring 1 ml of urine sample to a centrifuge tube, adding 1 ml of 2% formic acid aqueous solution and 8 ml of pure water, and mixing; adding 10-50 mg of the composite polymer microspheres, vortex extraction, magnetic separation, and removing the supernatant; adding formic acid aqueous solution and methanol aqueous solution for washing; adding ammonia methanol solution for elution, vortex extraction, magnetic separation, and removing the supernatant; blowing dry with nitrogen, adding methanol for re-dissolution, and LC-MS / MS sampling analysis.

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