A composite polymer microsphere for extracting and purifying chloropolyfluoroalkyl ether sulfonic acid from urine, its preparation method and application.

Abstract

This invention discloses a composite polymer microsphere for extracting and purifying chloropolyfluoroalkyl ether sulfonic acid (CFA) from urine, its preparation method, and its application. The composite polymer microsphere comprises: a spherical core layer composed of iron(III) oxide; a first shell layer covering the core layer, composed of zirconium-titanium composite oxide; and a second shell layer covering the first shell layer, composed of naphthidine-short peptide bifunctionalized polymer, the outer surface of which has fibrous short peptide chains. The composite polymer microsphere of this invention can rapidly, sensitively, and accurately detect the concentration of trace amounts of CFA in urine samples.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a composite polymer microsphere for extracting and purifying chlorinated polyfluoroalkyl ether sulfonic acid from urine, its preparation method, and its application. Background Technology

[0002] Chlorinated polyfluoroalkyl ether sulfonic acids are a new class of chlorinated perfluoroalkyl compounds. Two common chlorinated polyfluoroalkyl ether sulfonic acids have the following chemical structural formulas: Figure 1 As shown. In recent years, chlorinated polyfluoroalkyl ether sulfonic acids (PFAS) have been widely used in industry, such as as key components for waterproofing and oil repellency, industrial lubricants, corrosion inhibitors, and as alternatives to traditional PFAS (perfluoroalkyl and polyfluoroalkyl substances) fire extinguishing foams. However, chlorinated PFAS are environmentally persistent, bioaccumulative, and biotoxic. They can enter the human body and accumulate through multiple pathways, posing potential health hazards, including liver damage, impaired immune system, endocrine disorders, and developmental abnormalities. Long-term exposure may also lead to chronic diseases.

[0003] Currently, most research focuses on the detection of chloroperfluoroalkyl ether sulfonic acids in plasma samples. For example, patent document CN111624274A discloses a high-throughput rapid detection method for perfluorinated and polyfluorinated compounds in serum, using liquid-liquid extraction to detect perfluoroalkyl compounds such as 6:2 chloroperfluoropolyether sulfonic acid in serum. This method uses highly toxic methyl tert-butyl ether as the extractant and MPFACs-Mix as an internal standard for quantitative detection. Another example is patent document CN118549564A, which discloses a one-step determination method for perfluorinated / polyfluoroalkyl compounds in serum. This method uses a stepped split-type HMR purification tube, employing a pass-through purification process to complete sample pretreatment within the purification tube.

[0004] Compared to plasma samples, urine has a more complex matrix, containing large amounts of uric acid, inorganic salts, and other organic interfering substances. Existing testing methods cannot effectively eliminate matrix interference during the detection process, severely affecting the accuracy of determining chlorinated polyfluoroalkyl ether sulfonic acid (PFAS) in urine. Furthermore, the detection limits for perfluoroalkyl compounds in plasma are generally higher than 0.01 μg / L, offering no reference value for detecting extremely low concentrations (ng / L level) of PFAS in human biological samples. Therefore, existing methods cannot accurately monitor the concentration of trace amounts of PFAS in human urine samples. Summary of the Invention

[0005] The present invention aims to address the problems of existing technologies in detecting chlorinated polyfluoroalkyl ether sulfonic acid in urine, which suffer from poor detection specificity, inadequate purification effect, low quantitative accuracy, and difficulty in detecting low concentrations of target substances.

[0006] To address the aforementioned technical problems, a first aspect of the present invention provides composite polymer microspheres for extracting and purifying chlorinated polyfluoroalkyl ether sulfonic acid from urine, comprising:

[0007] A spherical core layer, the core layer being composed of iron(III) oxide;

[0008] A first shell layer, which covers the outside of the core layer, is composed of zirconium-titanium composite oxide;

[0009] The second shell is wrapped around the outside of the first shell. The second shell is composed of naphthidine-short peptide bifunctionalized polymer, and the outer surface of the second shell has fibrous short peptide chains.

[0010] The composite polymer microspheres of this invention have a spherical core-shell-shell structure. The core layer is made of iron oxide, which gives it magnetism, facilitating rapid solid-liquid phase magnetic separation by magnetic force and greatly improving sample purification efficiency. The first shell layer, zirconium-titanium composite oxide, can effectively adsorb impurities such as phospholipids, uric acid, and inorganic salts in urine. The second shell layer, naphthidine-short peptide bifunctional polymer, has fibrous short peptide chains on its outer surface that work with naphthidine to effectively improve the selectivity and detection sensitivity of chloropolyfluoroalkyl ether sulfonic acid.

[0011] Furthermore, the short peptide in the second shell is a caseinase-hydrolyzable peptide. This caseinase-hydrolyzable peptide can synergistically interact with naphthidine to exhibit better adsorption selectivity during the extraction and purification of chloropolyfluoroalkyl ether sulfonic acid in urine, thereby further improving the purification efficiency and sensitizing effect on the target compound.

[0012] Furthermore, the molar ratio of naphthidine to short peptide in the second shell is 0.5:1 to 2:1. This ratio allows naphthidine and short peptide to exert the best synergistic effect, ensuring good adsorption selectivity and detection sensitivity for chloropolyfluoroalkyl ether sulfonic acid in urine.

[0013] Furthermore, the molar ratio of zirconium to titanium in the first shell is 0.5:1 to 2:1. At this ratio, the zirconium-titanium composite oxide exhibits the best removal capability for impurities in urine. Compared to single metal oxides or other ratios, it can remove impurities such as phospholipids more efficiently, thereby improving the purification efficiency of urine samples and ensuring accurate subsequent detection of chloropolyfluoroalkyl ether sulfonic acid.

[0014] A second aspect of the present invention provides a method for preparing the above-mentioned composite polymer microspheres, comprising the following steps:

[0015] S1. Preparation of magnetic microspheres of iron oxide;

[0016] S2. The surface of the magnetic microspheres obtained in step S1 is modified with zirconium-titanium composite oxide by solvothermal reaction and then calcined at high temperature to obtain zirconium-titanium composite magnetic microspheres.

[0017] S3. Double bonds are modified on the surface of the zirconium-titanium composite magnetic microspheres obtained in step S2 using covalent coupling technology, and then naphthidine-quaternary ammonium zirconium-titanium composite magnetic microspheres are synthesized by polymerization reaction.

[0018] S4. Using directional self-assembly technology, the quaternary ammonium groups of the naphthidine-quaternary ammonium zirconium titanium composite magnetic microspheres obtained in step S3 are coupled with short peptides to obtain naphthidine-short peptide bifunctionalized zirconium titanium composite magnetic microspheres.

[0019] This invention prepares each structural layer sequentially through clearly defined steps, ensuring the structural and performance stability of the composite polymer microspheres, resulting in microspheres with excellent adsorption, separation, and detection properties. The rational connection of each step and the specific process ensure that the microspheres can effectively remove impurities and improve the selectivity and sensitivity to target substances.

[0020] Further, step S2 specifically includes: dispersing magnetite magnetic microspheres in a solvent, adding tetrabutyl titanate and zirconium oxychloride to the dispersion, reacting the mixture at 180-220℃ for 10-15 h, collecting the product, washing and drying it, and calcining it at 500-800℃ for 2-5 h to obtain zirconium-titanium composite magnetic microspheres. This step enables the zirconium-titanium composite oxide to form a uniform and stable coating layer on the surface of the magnetite magnetic microspheres, ensuring the adsorption performance of the first shell layer on impurities in urine, giving the prepared zirconium-titanium composite magnetic microspheres good impurity removal ability, thereby improving the purification efficiency of the entire composite polymer microsphere.

[0021] Further, step S3 specifically includes: dispersing zirconium-titanium composite magnetic microspheres in a solvent, adding ammonia and sodium thiopropane sulfonate solution under nitrogen protection, reacting at 60-80℃ for 8-16 h, collecting the product, washing and drying to obtain zirconium-titanium composite magnetic microspheres modified with double bonds; then dispersing the zirconium-titanium composite magnetic microspheres modified with double bonds in a solvent, adding 3-allyl-1,5-naphthidine, dimethyldiallyl ammonium chloride and a catalyst, heating to 78-82℃, maintaining for 30 min, then heating to 81-82℃, refluxing for 2-5 h, collecting the product, washing and drying to obtain naphthidine-quaternary ammonium zirconium-titanium composite magnetic microspheres. This step can accurately modify double bonds on the surface of zirconium-titanium composite magnetic microspheres and synthesize naphthidine-quaternary ammonium zirconium-titanium composite magnetic microspheres, laying the foundation for subsequent coupling with short peptides and ensuring the accurate construction of the second shell structure.

[0022] Further, step S4 specifically includes: dissolving 20 parts bovine serum albumin and 0.5 parts trypsin in PBS buffer solution, mixing, and enzymatically hydrolyzing at 35-40℃ for 14-18 h; adding naphthidine-quaternary ammonium zirconium titanium composite magnetic microspheres to the mixed solution, allowing for self-assembly reaction for 1-2 h; collecting the product, washing, and drying to obtain naphthidine-short peptide bifunctional zirconium titanium composite magnetic microspheres. This step ensures accurate coupling of the short peptide with the quaternary ammonium group, forming naphthidine-short peptide bifunctional zirconium titanium composite magnetic microspheres with good performance, guaranteeing the functional integrity of the second shell, thereby achieving efficient adsorption and detection of the target compound.

[0023] A third aspect of the present invention provides a kit comprising the above-mentioned composite polymer microspheres, formic acid aqueous solution, standard series working solutions, and centrifuge tubes.

[0024] The kit of this invention enables on-site sampling, processing and preservation of urine samples, with good timeliness, rapid and accurate results, and reduces the changes and losses of chloropolyfluoroalkyl ether sulfonic acid during sampling and transportation, thereby improving the accuracy of test results.

[0025] A fourth aspect of this invention provides an application of the above-mentioned composite polymer microspheres, wherein 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 into a centrifuge tube, adding 1 mL of 2% formic acid aqueous solution and 8 mL of pure water, and mixing well; adding 10-50 mg of composite polymer microspheres, vortexing extraction, magnetic separation, and removing the supernatant; washing with formic acid aqueous solution and methanol aqueous solution; eluting with ammonia-methanol solution, vortexing extraction, magnetic separation, and removing the supernatant; drying with nitrogen, reconstituted with methanol, and analyzing by LC-MS / MS.

[0026] This invention applies composite polymer microspheres to the detection of chlorinated polyfluoroalkyl ether sulfonic acid concentration in urine. Through specific detection steps, the properties of the composite polymer microspheres are utilized to effectively remove interfering impurities in urine and eliminate matrix interference effects during the detection process. This enables rapid, highly sensitive, and accurate detection of chlorinated polyfluoroalkyl ether sulfonic acid in urine. Furthermore, the detection method exhibits good linearity, low limits of detection and quantitation, as well as excellent accuracy and precision.

[0027] Compared with the prior art, the beneficial effects of the present invention include:

[0028] (1) High efficiency in removing impurities and accurate detection: The zirconium-titanium composite matrix design enables the composite polymer microspheres to efficiently remove interfering substances such as phospholipids in urine with small batch-to-batch differences; the naphthidine-short peptide bifunctional modification greatly improves the selectivity and detection sensitivity of chloropolyfluoroalkyl ether sulfonic acid; the quaternary ammonium modification precisely controls the amount of short peptide modification, further enhancing the purification and sensitization effect; high accuracy and sensitivity of quantitative detection can be achieved without relying on matrix matching or internal standard method.

[0029] (2) Improve purification and work efficiency: The synergistic effect of zirconium-titanium composite makes its phospholipid removal performance far exceed that of zirconium dioxide and titanium dioxide alone, greatly improving the purification efficiency of urine samples; the combination of magnetic components and short peptide modification can realize the automated processing of urine samples with the help of a fully automatic magnetic solid phase extractor, greatly improving the work efficiency of monitoring large batches of urine samples.

[0030] (3) Good recyclability: The performance of the naphthidine-quaternary ammonium zirconium titanium composite magnetic microspheres did not decrease significantly after being recycled 6 times, effectively saving the cost of use.

[0031] (4) Convenient on-site sampling and testing: The kit, together with a small vortex apparatus, enables on-site sampling, processing and preservation of urine samples, reducing the changes and losses of chloropolyfluoroalkyl ether sulfonic acid during sampling and transportation. It has good timeliness and accurate results, and can be tested after simple processing in the laboratory. Attached Figure Description

[0032] Figure 1 These are the chemical structural formulas of two chlorinated polyfluoroalkyl ether sulfonic acids.

[0033] Figure 2 The image shown is a TEM image of the composite polymer microspheres obtained in Experiment Example 1.

[0034] Figure 3 The image shows the TEM mapping of the composite polymer microspheres obtained in Experiment Example 1.

[0035] Figure 4 The image shows the SEM image of the composite polymer microspheres obtained in Experiment Example 1.

[0036] Figure 5 The image shows the EDS spectrum of the composite polymer microspheres obtained in Experiment Example 1.

[0037] Figure 6 The figure shows the test results of the effect of the amount of composite polymer microspheres on the extraction effect of the target substance, as measured in Experiment Example 2.

[0038] Figure 7 The figure shows the test results of the effect of the zirconium-titanium molar ratio in the composite polymer microspheres on the extraction effect of the target analyte, as measured in Experiment Example 3.

[0039] Figure 8The figure shows the test results of the effect of the molar ratio of naphthidine to short peptide in the composite polymer microspheres on the extraction effect of the target analyte, as measured in Experiment Example 4.

[0040] Figure 9 The figure shows the test results of the recyclability of the composite polymer microspheres obtained in Experiment Example 5. Detailed Implementation

[0041] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This application specification and embodiments are merely exemplary.

[0045] Example 1

[0046] This embodiment prepares a composite polymer microsphere for extracting and purifying chlorinated polyfluoroalkyl ether sulfonic acid from urine. The preparation method includes the following steps:

[0047] Step 1: Preparation of ferric oxide magnetic microspheres

[0048] Using a solvothermal method, 0.8 g of sodium citrate and 1.5 g of ferric chloride hexahydrate were weighed into a 250 mL flask, and 80 mL of ethylene glycol was added. The mixture was mechanically stirred at room temperature for 30 min to dissolve, followed by the addition of 5 g of sodium acetate and stirring for another 30 min. The resulting solution was then transferred to a 100 mL high-pressure reactor and reacted at 200 °C for 10 h, followed by cooling to room temperature. The mixture was then washed three times with pure water and ethanol, and dried under vacuum at 60 °C overnight to obtain magnetite microspheres (Fe3O4).

[0049] Step 2: Preparation of zirconium-titanium composite magnetic microspheres

[0050] 5.0 g of magnetite (Fe3O4@ZrO4) magnetic microspheres were ultrasonically dispersed in a mixed solution of 90 mL N,N-dimethylformamide (DMF) and 270 mL isopropanol. Then, under ultrasonic conditions, 3.4 g of tetrabutyl titanate (TBOT) and 3.2 g of zirconium oxychloride (ZrOCl2) were added over 10 min. The mixture was transferred to an autoclave and reacted at 200 °C for 12 h. The reaction product was washed several times with anhydrous ethanol, dried overnight at 60 °C, and then calcined at 600 °C for 5 h to obtain the prepared zirconium-titanium composite magnetic microspheres (Fe3O4@ZrO4). x Ti y O z ).

[0051] Step 3: Preparation of naphthidine-quaternary zirconium titanium composite magnetic microspheres

[0052] 5.0 g of zirconium-titanium composite magnetic microspheres were dispersed in 350 mL of 95% ethanol containing 6.0 g of cetyltrimethylammonium bromide (CTAB) and ultrasonically dispersed for 15 min. Then, under nitrogen protection, 2 mL of ammonia water was added while stirring, followed by dropwise addition of 15 mL of ethanol solution of sodium 3-mercapto-1-propanesulfonate (MPS). The reaction was carried out at 60 °C for 12 h. The microspheres were washed three times with pure water and ethanol to obtain zirconium-titanium composite magnetic microspheres (Fe3O4@Zr) with double bond modification. x Ti y O z -MPS).

[0053] 5.0 g of zirconium-titanium composite magnetic microspheres modified with double bonds were ultrasonically dispersed in 200 mL of isopropanol and placed in a 500 mL three-necked flask. The flask was then placed in a microwave reactor and mechanically stirred for 10 min. 1.7 g of 3-allyl-1,5-naphthidine, 4.9 g of dimethyldiallylammonium chloride, and 0.5 g of azobisisobutyronitrile (AIBN) were dissolved in 250 mL of acetonitrile and added to the dispersion. Under ultrasonic-assisted mechanical stirring, the mixture was heated to 78–82 °C and maintained for 30 min, then rapidly heated to 81–82 °C and refluxed for 2 h. The product was washed three times with pure water and ethanol to obtain naphthidine-quaternary ammonium zirconium-titanium composite magnetic microspheres (Fe3O4@Zr). x Ti y O z -NAD / QA).

[0054] Step 4: Synthesis of naphthidine-short peptide bifunctionalized zirconium-titanium composite magnetic microspheres

[0055] Accurately weigh 20 mg of casein and dissolve it in 10 mL of phosphate-buffered saline (PBS) at pH 7.4. Simultaneously, add 5 mL of PBS solution containing 0.5 mg of trypsin. Mix and incubate at 37°C for 16 h. Add 2.5 g of naphthidine-quaternary ammonium zirconium titanium composite magnetic microspheres to the above mixture and allow them to self-assemble on a rotary mixer for 1 h. Wash twice with water and twice with 0.1% ammonia-methanol solution, then wash twice with water and dry to obtain naphthidine-short peptide bifunctionalized zirconium titanium composite magnetic microspheres (Fe3O4@Zr). x Ti y O z -NAD / SP).

[0056] Example 2

[0057] This embodiment provides a kit, including the composite polymer microspheres (Fe3O4@Zr) from Example 1. x Ti y O z -NAD / SP), formic acid aqueous solution, standard series working solutions, centrifuge tubes.

[0058] Example 3

[0059] This embodiment uses the kit from Example 2 to detect the concentration of chloropolyfluoroalkyl ether sulfonic acids (9-chlorohexadecylfluoro-3-oxoalkylone-1-sulfonic acid (9Cl-PF3ONS) and 11-chloroeicosicofluoro-3-oxododecane-1-sulfonic acid (11Cl-PF3OUdS)) in urine samples. The detection method includes the following steps:

[0060] a. Accurately transfer 1 ml of urine sample into a centrifuge tube, add 1 mL of 2% formic acid aqueous solution and 8 mL of pure water, and mix well.

[0061] b. Add 10-50 mg of composite polymer microspheres (Fe3O4@Zr) x Ti y O z -NAD / SP), vortex extraction for 5 min, place on a magnetic rack for magnetic separation for 10 s, and pour off the supernatant.

[0062] c. Add 4 mL of 2% formic acid aqueous solution and 4 mL of 50% methanol aqueous solution for washing, vortex for 5 min, magnetically separate for 10 s, and pour off the supernatant.

[0063] d. Add 4 mL of 0.1% ammonia-methanol solution for elution, vortex for 5 min, perform magnetic separation for 10 s, and collect the supernatant.

[0064] e. Dry with nitrogen, add 1 mL of methanol to reconstitute, and analyze by LC-MS / MS.

[0065] The LC-MS / MS instrument conditions are as follows:

[0066] (1) Liquid phase conditions

[0067] Chromatographic conditions: Column: ACQUITY™ PREMIER BEH C 18 Column (1.7μm*100mm*2.1 mm); Injection volume: 5.0μL; Mobile phase: A is methanol, B is 0.5mmol / L ammonium fluoride solution; Column temperature: 40℃; Flow rate: 0.3mL / min; Elution gradient: Gradient elution: 0~2.0min, 20%B~60%B; 2.0~10.0min, 60%B~95%B; 10.0~12.0min, 95%B; 12.0~12.1min, 95%B~20%B; 12.1~15.0min, 20%B.

[0068] (2) Mass spectrometry conditions

[0069] Ion source: Electrospray ionization (ESI) source; Detection method: Multiple reaction monitoring (MRM); Scanning method: Negative ion mode scan; Electrospray voltage (IS): Negative ions (-4500V); Nebulizer gas pressure (GS1): 50.0 psi; Assist 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℃. Other mass spectrometry parameters are listed in Table 1.

[0070] Table 1 Retention time, parent ion, fragment ion, declustering voltage and collision energy of the target compound

[0071]

[0072] Example 1: Characterization of composite polymer microspheres

[0073] The morphology and elemental composition of the composite polymer microspheres synthesized in Example 1 were characterized using TEM, TEM-mapping, SEM, and EDS, respectively. The TEM characterization results are as follows: Figure 2 As shown, the composite polymer microspheres exhibit a significant core-shell-shell structure, with the core being a magnetic microsphere of iron oxide, the middle shell being a zirconium-titanium composite oxide, and the outermost layer being a naphthidine-short peptide bifunctional polymer. Figure 3 The distribution images of Fe, O, Ti, Zr, C, and N in the material were provided, verifying the rationality of the material synthesis route. Figure 4 The image shows a SEM image of the composite polymer microspheres, which are spherical in shape and contain fibrous short peptide chains on the surface. Figure 5 The EDS analysis results for the composite polymer microspheres show that the material contains Fe, O, Ti, Zr, C, and N elements. In summary, the characterization results indicate that the composite polymer microspheres are Fe3O4@Zr... x Ti y O z -NAD / SP has been successfully synthesized.

[0074] Experimental Example 2: Effect of Composite Polymer Microsphere Dosage on Extraction Efficiency

[0075] Add 1 mL of urine spiked sample to each of six polypropylene centrifuge tubes. The spiked concentrations of the two chloropolyfluoroalkyl ether sulfonic acids (9Cl-PF3ONS and 11Cl-PF3OUdS) were both 10.0 μg / L. Add 1 mL of 2% formic acid aqueous solution and 8 mL of pure water, vortex for 5 min, and 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 for 5 min, then place on a magnetic rack for magnetic separation for 10 s, and decant the supernatant. 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 decant the supernatant. Then add 4 mL of 0.1% ammonia-methanol solution for elution, vortex for 5 min, magnetic separation for 10 s, collect the supernatant, dry under nitrogen, redissolve in 1 mL of methanol, and analyze by LC-MS / MS. The results are as follows: Figure 6 As shown.

[0076] Depend on Figure 6It can be seen that when the amount of composite polymer microspheres is small, the purification effect is poor, and the peak areas of the two chloropolyfluoroalkyl ether sulfonic acids are small. When the amount of composite polymer microspheres is 10 mg, the peak areas of both reach their maximum values. Further increasing the amount of composite polymer microspheres does not change the peak areas of both. Therefore, the preferred amount of composite polymer microspheres in the detection method is 10-50 mg.

[0077] Experimental Example 3: Effect of the zirconium-titanium molar ratio of composite polymer microspheres on extraction efficiency

[0078] Four types of composite polymer microspheres were prepared using the preparation method of Example 1, labeled as adsorbent a, adsorbent b, adsorbent c, and adsorbent d. The differences were as follows: in step two of the preparation process for adsorbent a, tetrabutyl titanate and zirconium oxychloride were not added; in step two of the preparation process for adsorbent b, 20 mmol of tetrabutyl titanate was added; in step two of the preparation process for adsorbent c, 10 mmol of tetrabutyl titanate and 10 mmol of zirconium oxychloride were added; and in step two of the preparation process for adsorbent d, 20 mmol of zirconium oxychloride was added. All other preparation steps and process parameters were the same.

[0079] One mL of urine sample was added to each of four polypropylene centrifuge tubes. The spiking concentrations of the two chloropolyfluoroalkyl ether sulfonic acids (9Cl-PF3ONS and 11Cl-PF3OUdS) were both 10.0 μg / L. The concentrations of the chloropolyfluoroalkyl ether sulfonic acids were determined using the detection method described in Example 3, with adsorbents a, b, c, and d. The amount of adsorbent used in each experiment was 10 mg. The test results are as follows: Figure 7 As shown, when zirconium oxychloride and tetrabutyl titanate (adsorbent a) were not used in the preparation of the composite polymer microspheres, their purification performance for the two chloropolyfluoroalkyl ether sulfonic acids in urine was poor. When only one of the metal sources (adsorbent b and adsorbent d) was used, the purification performance for the two chloropolyfluoroalkyl ether sulfonic acids in urine was worse than that when both metal sources were used (adsorbent c), but the purification performance was better than that of adsorbent a. Therefore, polymer microspheres containing zirconium-titanium composite groups can significantly improve the purification performance of urine samples, and the purification performance is optimal when the molar ratio of zirconium to titanium is similar.

[0080] Experimental Example 4: Effect of the molar ratio of naphthidine to short peptide in composite polymer microspheres on extraction efficiency

[0081] Four types of composite polymer microspheres were prepared using the preparation method of Example 1, labeled as Adsorbent 1, Adsorbent 2, Adsorbent 3, and Adsorbent 4. The differences were as follows: Adsorbent 1 was prepared without steps three and four; Adsorbent 2 was prepared with 20 mmol of 3-allyl-1,5-naphthidine added in step three; Adsorbent 3 was prepared with 10 mmol of 3-allyl-1,5-naphthidine and 10 mmol of dimethyldiallyl ammonium chloride added in step two; and Adsorbent 4 was prepared with 20 mmol of dimethyldiallyl ammonium chloride in step two. All other preparation steps and process parameters were the same.

[0082] One mL of urine sample was added to each of four polypropylene centrifuge tubes. The spiking concentrations of the two chloropolyfluoroalkyl ether sulfonic acids (9Cl-PF3ONS and 11Cl-PF3OUdS) were both 10.0 μg / L. The concentrations of the chloropolyfluoroalkyl ether sulfonic acids were determined using the detection method described in Example 3, with adsorbents 1, 2, 3, and 4, each using 10 mg of adsorbent. The test results are as follows. Figure 8 As shown, when the composite polymer microspheres do not contain naphthidine (NAD) and short peptide (SP) functional groups (adsorbent 1), the peak areas of the two chloropolyfluoroalkyl ether sulfonic acids are extremely low, indicating that adsorbent 1 has poor adsorption capacity for chloropolyfluoroalkyl ether sulfonic acids in urine, meaning that the naphthidine-short peptide functional groups play a key role in the adsorption of both. Furthermore, 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). Therefore, it can be concluded that the bifunctionalized groups NAD and SP play a synergistic role in the extraction and purification of chloropolyfluoroalkyl ether sulfonic acids in urine.

[0083] Experiment Example 5: Cyclic Use Performance Test

[0084] One mL of urine spiked sample was added to each of six polypropylene centrifuge tubes. The spiked concentrations of the two chloropolyfluoroalkyl ether sulfonic acids (9Cl-PF3ONS and 11Cl-PF3OUdS) were both 10.0 μg / L. The detection method of Example 3 was used, and 10 mg of the composite polymer microspheres prepared in Example 1 were used for six cycles. The test results are as follows: Figure 9 As shown. Test results indicate that the composite polymer microspheres (Fe3O4@Zr) x Ti y O z After six cycles of use, the peak areas of the two chloropolyfluoroalkyl ether sulfonic acids in urine did not show a significant decrease, indicating that the composite polymer microspheres developed in this invention can be reused multiple times, which helps reduce detection costs and is more environmentally friendly.

[0085] Experimental Example 6: Evaluation of Matrix Effect

[0086] Accurately pipette 1 mL of urine blank sample into each polypropylene centrifuge tube, 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 (Fe3O4@Zr) prepared in Example 1. x Ti y O z -NAD / SP), vortex extraction for 5 min, followed by magnetic separation on a magnetic rack for 10 s, and decant the supernatant. 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 decant the supernatant. Then, add 4 mL of 0.1% ammonia-methanol solution for elution, vortex for 5 min, magnetic separation for 10 s, collect the supernatant, blow dry with nitrogen, and redissolve with 1 mL of methanol. Then, add an appropriate amount of chloropolyfluoroalkyl ether sulfonic acid standard stock solution to prepare matrix-matched standard solutions of 0.005~20.0 μg / L using blank urine purification solution. At the same time, compare the matrix-matched working curve purified by commercial Waters WAX solid phase extraction column and the solvent standard series solutions prepared with methanol. The formula [Matrix effect η = (Slope of matrix-matched standard curve K)] is used. a -Slope K of the solvent standard curve b Slope K of the solvent standard curve b Evaluation of Fe3O4@Zr a Ti b O c The matrix effect of NAD / SP extraction and purification of two chloropolyfluoroalkyl ether sulfonic acids in urine is shown in Table 2.

[0087] As shown in Table 2, the naphthidine-short peptide bifunctionalized zirconium-titanium composite magnetic microspheres of this invention can effectively eliminate the matrix effect in the detection of two chloropolyfluoroalkyl ether sulfonic acids (PFAS) in urine samples, and the matrix effect η1 is 0, indicating no matrix effect. This means that no matrix-matching working curve or isotope internal standard is needed during quantification. For commercially available Waters WAX solid-phase extraction columns, the absolute values ​​of the matrix effect η2 for the two PFAS are 50% and 100%, respectively, indicating a strong matrix enhancement effect. This necessitates the use of a matrix-matching working curve or isotope internal standard for quantification, increasing the complexity of experimental procedures and raising the operating cost of the detection method. Therefore, the kit based on composite polymer microspheres of this invention can effectively remove interfering impurities in urine, effectively eliminate matrix interference effects in the detection of PFAS, and improve detection accuracy, offering the advantages of speed, simplicity, and accuracy.

[0088] Table 2 Comparison of matrix interference resistance between the examples and commercial Waters WAX solid phase extraction columns

[0089]

[0090] Experiment Example 7: Linear Equation and Correlation Coefficient (R²) 2 Accuracy, precision, limit of detection and limit of quantitation

[0091] The method described in Example 3 was used to simultaneously detect the residual amounts of two chlorinated polyfluoroalkyl ether sulfonic acids in urine. 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). Furthermore, tests were conducted on a series of low-concentration spiked urine samples, with the limits of detection (LOD) and quantitation (LOQ) defined by signal-to-noise ratios (S / N) ≥ 3 and S / N ≥ 9, respectively. The LOD and LOQ for the two chloropolyfluoroalkyl ether sulfonic acids in urine were 0.0002 μg / L and 0.0006 μg / L, respectively. The detection method established in this invention can achieve ultra-high sensitivity and accuracy in detecting the two chloropolyfluoroalkyl ether sulfonic acids in urine within a concentration range of 0.005–20.0 μg / L.

[0092] Table 3. Linear range, linear equation, correlation coefficient, limit of detection, and limit of quantitation of the detection method of the present invention.

[0093]

[0094] The accuracy and precision of the detection method in Example 3 were investigated. The spiking levels of the two chloropolyfluoroalkyl ether sulfonic acids in urine samples were controlled at 0.1, 1.0, 10.0, and 20.0 μg / L, respectively. The method in Example 3 was used for detection, and the results are shown in Table 4. The test results show that the recoveries of the two chloropolyfluoroalkyl ether sulfonic acids using the detection method of this invention are between 99.0% and 117% and 88.0% and 104%, respectively, with relative standard deviations (RSDs) of 1.2% to 5.8%. It is evident that the detection method based on the composite polymer microspheres of this invention has the advantages of being rapid, sensitive, and accurate.

[0095] Table 4. Spike recovery and precision of the detection method of the present invention (n=6)

[0096]

[0097] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the scope of protection of this invention.

Claims

1. A composite polymer microsphere for extracting a chlorinated polyfluoroalkyl ether sulfonic acid in purified urine, characterized by comprising a polymer having a functional group capable of binding to a chlorinated polyfluoroalkyl ether sulfonic acid, and a compound having a functional group capable of binding to a chlorinated polyfluoroalkyl ether sulfonic acid. The chloro polyfluoroalkyl ether sulfonic acid is selected from at least one of 9-chloro hexadecafluoro-3-oxoalkanone-1-sulfonic acid and 11-chloro eicosanfluoro-3-oxododecan-1-sulfonic acid, and the composite high molecular microspheres comprise: a spherical core layer, a component of the core layer being ferroferric oxide; a first shell layer, the first shell layer being coated on the outside of the core layer, a component of the first shell layer being zirconium-titanium composite oxide; a second shell layer, the second shell layer being coated on the outside of the first shell layer, a component of the second shell layer being naphthyridine-short peptide bifunctionalized high molecule, an outer surface of the second shell layer having fibrous short peptide chains, and the short peptide in the second shell layer being casein enzymatic peptide; a preparation method of the composite high molecular microspheres comprising the following steps: S1, preparing ferroferric oxide magnetic microspheres; S2, modifying zirconium-titanium composite oxide on the surface of the ferroferric oxide magnetic microspheres obtained in step S1 by using a solvothermal reaction, and performing high-temperature calcination to obtain zirconium-titanium composite-based magnetic microspheres; S3, modifying a double bond on the surface of the zirconium-titanium composite-based magnetic microspheres obtained in step S2 by using a covalent bond coupling technology, and then synthesizing naphthyridine-quaternized zirconium-titanium composite-based magnetic microspheres by using a polymerization reaction; S4, coupling quaternary ammonium groups of the naphthyridine-quaternized zirconium-titanium composite-based magnetic microspheres obtained in step S3 with short peptides by using a directional self-assembly technology to obtain naphthyridine-short peptide bifunctionalized zirconium-titanium composite-based magnetic microspheres.

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

1.

3. The composite polymeric microspheres of claim 1, wherein, The molar ratio of zirconium to titanium elements in the first shell layer is 0.5:1 to 2:

1.

4. A method for preparing the composite polymeric microspheres according to any one of claims 1 to 3, characterized by, comprising the following steps: S1, preparing ferroferric oxide magnetic microspheres; S2, modifying zirconium-titanium composite oxide on the surface of the ferroferric oxide magnetic microspheres obtained in step S1 by using a solvothermal reaction, and performing high-temperature calcination to obtain zirconium-titanium composite-based magnetic microspheres; S3, modifying a double bond on the surface of the zirconium-titanium composite-based magnetic microspheres obtained in step S2 by using a covalent bond coupling technology, and then synthesizing naphthyridine-quaternized zirconium-titanium composite-based magnetic microspheres by using a polymerization reaction; S4, coupling quaternary ammonium groups of the naphthyridine-quaternized zirconium-titanium composite-based magnetic microspheres obtained in step S3 with short peptides by using a directional self-assembly technology to obtain naphthyridine-short peptide bifunctionalized zirconium-titanium composite-based magnetic microspheres.

5. The preparation method according to claim 4, characterized in that, The step S2 specifically comprises: dispersing ferroferric oxide magnetic microspheres in a solvent, adding tetrabutyl titanate and zirconium oxychloride in the dispersion liquid, reacting the mixed liquid at 180-220℃ for 10-15h, collecting the product, washing and drying, and calcining at 500-800℃ for 2-5h to obtain zirconium-titanium composite-based magnetic microspheres.

6. The preparation method according to claim 4, characterized in that, The step S3 specifically comprises: dispersing the zirconium-titanium composite-based magnetic microspheres in a solvent, adding ammonia water and sodium thio propanesulfonate solution under nitrogen protection, reacting at 60-80℃ for 8-16h, collecting the product, washing and drying to obtain the zirconium-titanium composite-based magnetic microspheres containing double bond modification; then dispersing the zirconium-titanium composite-based magnetic microspheres containing double bond modification in a solvent, adding 3-allyl-1,5-naphthyridine, dimethyl diallyl ammonium chloride and a catalyst, heating to 78-82℃ for 30min, then heating to 81-82℃, condensing and refluxing for 2-5h, collecting the product, washing and drying to obtain the naphthyridine-quaternary amine zirconium-titanium composite-based magnetic microspheres.

7. The preparation method according to claim 4, characterized in that, The step S4 specifically comprises: dissolving 20mg casein and 0.5mg trypsin in a PBS buffer solution, mixing and then enzymolysis at 35-40℃ for 14-18h, adding the naphthyridine-quaternary amine zirconium-titanium composite-based magnetic microspheres into the mixed solution, self-assembling for 1-2h, collecting the product, washing and drying to obtain the naphthyridine-short peptide bifunctional zirconium-titanium composite-based magnetic microspheres.

8. A kit characterized in that, The kit comprises the composite polymer microspheres as claimed in any one of claims 1-3, a formic acid aqueous solution, a standard series working solution and a centrifugal tube.

9. Use of the composite polymeric microspheres according to any one of claims 1 to 3, characterized in that, The composite polymer microspheres are used for detecting the concentration of chlorinated polyfluoroalkyl ether sulfonic acid in urine, the chlorinated polyfluoroalkyl ether sulfonic acid being at least one selected from 9-chlorohexadecafluoro-3-oxoalkanone-1-sulfonic acid and 11-chloroicosafluoro-3-oxododecan-1-sulfonic acid, and the detection method comprises the following steps: taking 1ml of urine sample into a centrifugal tube, adding 1mL of 2% formic acid aqueous solution and 8mL of pure water, mixing; adding 10-50mg of the composite polymer microspheres, vortex extraction, magnetic separation, 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 then absorbing the supernatant; nitrogen blowing drying, adding methanol for redissolution, and then LC-MS / MS sample analysis.

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