Perfluorosulfonyl fluoride hapten as well as preparation method and application thereof

Abstract

The invention relates to a perfluorosulfonyl fluoride hapten and a preparation method and application thereof, the structure of the perfluorosulfonyl fluoride hapten is as shown in a formula I: # imgabs0 #, the perfluorosulfonyl fluoride hapten and the perfluorosulfonyl fluoride antigen provided by the invention are simple in synthesis method, high in purity and high in yield, and the preparation method of the perfluorosulfonyl fluoride hapten and the perfluorosulfonyl fluoride hapten can be used for preparing a perfluorosulfonyl fluoride antibody. And the method has great value in perfluorosulfonyl fluoride drug residue detection. The detection limit of the perfluorosulfonyl fluoride enzyme linked immunosorbent assay kit developed by the invention in animal-derived food is 2.0 mu g / kg, the linear range is 0.5-27 mu g / L, and the recovery rate is 82.76%-115.98%. The invention provides a high-sensitivity and low-cost technical scheme for rapid detection of perfluorosulfonyl fluoride, and is suitable for screening food residues.

Description

Technical Field

[0001] The invention relates to a perfluorosulfonyl fluoride hapten and antigen, a preparation method and application thereof, and belongs to the field of rapid food safety detection. Background Art

[0002] Perfluorinated compounds are a class of organic compounds in which all hydrogen atoms on the carbon chain are replaced by fluorine atoms. They have characteristics such as high stability and low surface energy and are widely used in many fields such as industrial manufacturing and consumer products. Perfluorosulfonyl fluoride is an important class of perfluorinated substituted alkylsulfonyl compounds. Due to the high reactivity of the sulfonyl fluoride group in the structure, it plays a key role in organic synthesis and material preparation. However, as research deepens, it is found that this type of substance has potential environmental and health risks such as bioaccumulation, persistence, and potential endocrine disruption effects, which will cause long-term adverse effects on the ecological environment and human health. In order to ensure environmental safety and human health, it is necessary to establish an effective detection method to monitor the content of perfluorosulfonyl fluoride compounds in the environment, food, biological samples, etc.

[0003] Currently, the detection of perfluorosulfonyl fluoride compounds primarily relies on instrumental analysis methods, such as high-performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS). While these methods offer the advantages of high sensitivity and accuracy, they are expensive, complex, and require high operator skills. Furthermore, the sample pretreatment process is cumbersome and time-consuming, making rapid on-site detection difficult and unable to meet the needs of rapid screening of large numbers of samples.

[0004] Immunoassays based on antigen-antibody specific reactions offer advantages such as high sample throughput, simple operation, good specificity, and relatively low cost. They are suitable for rapid on-site screening of large numbers of samples and are an effective complement to instrumental analysis methods. The key to building an efficient immunoassay system lies in obtaining antibodies with high specificity and affinity, making the preparation of haptens a key breakthrough. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a perfluorosulfonyl fluoride hapten and a preparation method and application thereof.

[0006] The first object of the present invention is to provide a perfluorosulfonyl fluoride hapten, the structure of which is shown in Formula I:

[0007]

[0008] A second object of the present invention is to provide a method for preparing the perfluorosulfonyl fluoride hapten of formula I, comprising the steps of:

[0009] 564.8 mg of N-tert-butyloxycarbonyl-1,4-butanediamine was added to 20 ml of dichloromethane, stirred until dissolved, and then 675.6 μL of triethylamine was added. After stirring at room temperature for 10 min, 1200 μL of perfluorosulfonyl fluoride was added. The mixture was refluxed and stirred at 40°C for 16 hours. The mixture was concentrated under reduced pressure and subjected to silica gel column chromatography to obtain an oily substance. The oily substance was dissolved in ethyl acetate, and a hydrogen chloride ethyl acetate solution was added for reaction. After solid-liquid separation, washing, and drying, the perfluorosulfonyl fluoride hapten represented by Formula I was obtained.

[0010] The preparation method of the above-mentioned perfluorosulfonyl fluoride hapten has the following synthetic route:

[0011]

[0012] The third object of the present invention is to provide a perfluorosulfonyl fluoride antigen, including an immunogen and a coating, which is obtained by coupling the compound of formula I with a carrier protein.

[0013] The compound of formula I was coupled with bovine serum albumin (BSA) by glutaraldehyde method at a coupling molar ratio of 4.66:1. After purification by dialysis, perfluorosulfonyl fluoride immunogen was obtained at a concentration of 6.04 mg / mL.

[0014] The same method was used to couple the hapten with thyroglobulin (BTG) to prepare the perfluorosulfonyl fluoride coating with a concentration of 9.51 mg / mL.

[0015] The fourth object of the present invention is to provide a perfluorosulfonyl fluoride antibody obtained by immunizing an animal with the perfluorosulfonyl fluoride antigen.

[0016] Furthermore, the antibody is selected from a monoclonal antibody, a polyclonal antibody or an antiserum.

[0017] A fifth object of the present invention is to provide any of the following uses of the perfluorosulfonyl fluoride hapten represented by formula I:

[0018] (i) Use in the preparation of perfluorosulfonyl fluoride antigens or perfluorosulfonyl fluoride antibodies;

[0019] (ii) use in the preparation of a device / reagent for detecting perfluorosulfonyl fluoride;

[0020] (iii) Other related uses in the detection of perfluorosulfonyl fluoride.

[0021] The equipment and / or reagents for detecting perfluorosulfonyl fluoride include but are not limited to enzyme-linked immunosorbent assay kits and immunochromatographic detection cards.

[0022] The sixth object of the present invention is to provide a method for detecting perfluorosulfonyl fluoride using the above-mentioned perfluorosulfonyl fluoride antibody.

[0023] Furthermore, the detection method includes enzyme-linked immunosorbent assay, colloidal gold immunochromatography, time-resolved immunochromatography, chemiluminescence immunoassay, etc.

[0024] The present invention lays the foundation for the development of an efficient, sensitive and specific immunoassay method for perfluorosulfonyl fluoride compounds, realizes the rapid and accurate detection of perfluorosulfonyl fluoride compounds in samples such as animal-derived foods, and provides strong technical support for food safety testing in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the mass spectrum of the perfluorosulfonyl fluoride hapten represented by formula I obtained in Example 1;

[0026] Figure 2 is the MALDI-TOF-MS image of BSA in Example 2;

[0027] Figure 3 This is the MALDI-TOF-MS spectrum of the perfluorosulfonyl fluoride-BSA complex in Example 2. DETAILED DESCRIPTION

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] Example 1. Preparation of perfluorosulfonyl fluoride hapten

[0031] In a 50 mL round-bottom flask, accurately weigh 564.8 mg of N-tert-butyloxycarbonyl-1,4-butanediamine and add 20 mL of dichloromethane. Stir at room temperature until completely dissolved, then slowly add 675.6 μL of triethylamine. Stir the reaction at room temperature for 10 minutes, then add 1200 μL of perfluorosulfonyl fluoride. Reflux and stir at 40°C for 16 hours. After the reaction, remove the solvent by vacuum distillation, dissolve the residue in dichloromethane, and add 3000 mg of 100-200 mesh silica gel. Separate the mixture by column chromatography using 200-300 mesh silica gel with a 5:1 (volume) ratio of petroleum ether to ethyl acetate as the eluent. Thin-layer chromatography (TLC) is performed to collect 2000 mg of the main product as an oily compound.

[0032] 2000 mg of the oily compound obtained above was placed in a 50 mL round-bottom flask. 15 mL of ethyl acetate was added and stirred until completely dissolved. Subsequently, 15 mL of a 4 M hydrogen chloride-ethyl acetate solution was added and stirred at room temperature for 120 min. During the reaction, a large amount of white solid precipitated. The reaction mixture was filtered, and the resulting solid was washed several times with ethyl acetate and finally dried in a 40°C forced air drying oven to constant weight, yielding 775 mg of amino-modified perfluorosulfonyl fluoride hapten.

[0033]

[0034] Figure 1 This is the mass spectrum of the perfluorosulfonyl fluoride hapten represented by formula I obtained in Example 1, and the results show that its molecular weight is correct.

[0035] Example 2: Preparation and structural identification of perfluorosulfonyl fluoride artificial antigen

[0036] 1. Synthesis of immunogens

[0037] (1) Add 47.2 mg of perfluorosulfonyl fluoride hapten to 2 mL of DMF and stir until completely dissolved.

[0038] (2) Weigh 50 mg of BSA and dissolve it in 5 mL of 0.1 M sodium bicarbonate solution. Add the reaction solution prepared in step (1) and then add 335.8 μL of freshly prepared 1% glutaraldehyde. React overnight.

[0039] (3) The reaction product was placed in a dialysis bag rinsed with distilled water and dialyzed against 1 L of 0.01 M PBS (1×, pH 7.2) at 4°C with stirring (100 rpm) for 2 days. The solution was changed three times a day. The dialyzed product was centrifuged at 5000 rpm for 6 min, aliquoted, and stored at -20°C for later use.

[0040] 2. Synthesis of coating precursor

[0041] (1) Add 18.9 mg of perfluorosulfonyl fluoride hapten to 2 mL of DMF and stir until completely dissolved.

[0042] (2) Weigh 20 mg of BTG and dissolve it in 5 mL of 0.1 M sodium bicarbonate solution. Add the reaction solution prepared in step (1) and then add 134.3 μL of freshly prepared 1% glutaraldehyde. React overnight.

[0043] (3) The reaction product was placed in a dialysis bag rinsed with distilled water and dialyzed against 1 L of 0.01 M PBS (1×, pH 7.2) at 4°C with stirring (100 rpm) for 2 days. The solution was changed three times a day. The dialyzed product was centrifuged at 5000 rpm for 6 min, aliquoted, and stored at -20°C for later use.

[0044] 3. Identification of Antigens

[0045] (1) The concentrations of the synthesized immunogen and coating agent were determined by ultraviolet absorption method. The results of the immunogen and coating agent were 6.04 mg / mL and 9.51 mg / mL respectively.

[0046] (2) Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify the immunogen. Figure 2 (Carrier protein BSA MALD-TOF detection results) and Figure 3 (Immunogen perfluorosulfonyl fluoride-BSA MALDl-TOF detection results) shown; the calculated molar ratio of perfluorosulfonyl fluoride hapten (Formula I) and bovine serum albumin (BSA) coupling is: R = (69949.50 -66674.86) / 702.73 = 4.66.

[0047] Example 3: Immunizing Animals with Perfluorosulfonyl Fluoride Artificial Antigen to Prepare Monoclonal Antibodies

[0048] Perfluorosulfonyl fluoride monoclonal antibody was prepared according to conventional methods. The specific steps are as follows:

[0049] 1. Animal Immunization

[0050] The immunogen prepared in Example 2 was diluted to 1 mg / mL with PBS (10 mmol / L, pH 7.4) and then emulsified with Freund's adjuvant. Mice were immunized using multiple subcutaneous injections at the back of the neck at a dose of 0.2 mL per mouse. Freund's complete adjuvant was used for the first immunization, and booster immunizations were performed every three weeks after the first immunization. Incomplete Freund's adjuvant was used for the booster immunizations. Orbital venous blood was collected from the mice, and the serum was tested by ic-ELISA to screen the mice for cell fusion.

[0051] 2. Cell Fusion and Cloning

[0052] Mouse spleen cells with high serum titers and high sensitivity were selected for fusion with tumor cells to produce hybridoma cells. Mouse spleen cells and SP2 / 0 cells were fused using electrofusion. After 5-7 days of fusion, the cell supernatant (50 μL / well) was transferred to a coated ELISA plate for detection. Single cell clusters with high OD values ​​and high inhibition rates were selected for subcloning. After four subcloning cycles, when the cell positive rate reached 100%, single cell clusters with high cell supernatant sensitivity were transferred to new culture plates for expansion, thus obtaining a stable hybridoma cell line.

[0053] mAbs were prepared using the in vivo ascites induction method. One week in advance, sterile liquid paraffin was injected into the peritoneal cavity of mice (0.5 mL / mouse). Hybridoma cells were harvested by centrifugation at 800 rpm for 5 minutes. The cell pellet was resuspended in incomplete DMEM medium, counted, and injected into the peritoneal cavity of the mouse. Approximately 7-10 days after inoculation, the ascites was collected and centrifuged to obtain a pale yellow clear liquid, which was the mAb. The aliquots were stored in a -20°C refrigerator.

[0054] After cell fusion, hybridoma cell screening and subcloning, several hybridoma cell lines that can secrete perfluorosulfonyl fluoride monoclonal antibodies were obtained. Among them, the cell line with the highest antibody titer and the best inhibition was 3A6, IC 50 It is 1.26ng / mL.

[0055] The cross-reactivity rate of the monoclonal antibody prepared by the present invention against structural / functional analogs of perfluorosulfonyl fluoride, including perfluorooctanesulfonyl fluoride, perfluorobutylsulfonyl fluoride, perfluorodecanoic acid, perfluorooctanoic acid, perfluorohexanesulfonic acid, perfluorooctanesulfonamide, and perfluorobutanecarboxylic acid, was tested by indirect competitive ELISA. The results are shown in Table 2. Cross-reactivity rate (%) = IC 50(全氟磺酰氟) / IC 50(类似物) ×100%.

[0056] Table 1 Perfluorosulfonyl fluoride monoclonal antibody cross-reactivity test results

[0057] name Cross-reactivity rate Perfluorosulfonyl fluoride 100％ Perfluorooctanesulfonyl fluoride 86.3％ Perfluorobutylsulfonyl fluoride 31.4％ Perfluorodecanoic acid 4.6％ Perfluorohexanesulfonic acid 1.5％ Perfluorooctanesulfonamide 1.3％ Perfluorooctanoic acid <1％ Perfluorobutanecarboxylic acid <0.1％

[0058] As shown in Table 2, the antibody obtained from the perfluorosulfonyl fluoride hapten provided by the present invention has cross-reactivity rates of 86.3% for perfluorooctanesulfonyl fluoride and 31.4% for perfluorobutylsulfonyl fluoride, respectively. It has lower cross-reactivity rates for perfluorodecanoic acid, perfluorooctanoic acid, perfluorohexanesulfonic acid, perfluorooctanesulfonamide, and perfluorobutanecarboxylic acid. This indicates that the antibody has high specificity for perfluorosulfonyl fluoride and also has some recognition ability for other structurally similar perfluorochemicals, although the degree of recognition varies. This discovery is of great significance for the further research and development of detection methods and technologies for perfluorochemicals, especially in the field of residue detection in animal-derived foods.

[0059] Example 4: Establishment of perfluorosulfonyl fluoride enzyme-linked immunosorbent assay

[0060] The optimal dosage of coating source, perfluorosulfonyl fluoride monoclonal antibody, and the optimal coating solution were determined by the checkerboard method. Eight concentration gradients were set up, and the coating source was diluted at 1:4000, 1:6000, 1:8000, 1:16000, 1:32000, and 1:64000 before coating the ELISA plate. The antibody was diluted at 1:8000, 1:10000, 1:20000, 1:40000, and 1:80000, respectively. A standard curve was established and the IC was calculated. 50 The specific operations are as follows:

[0061] (1) Antigen coating: Dilute the perfluorosulfonyl fluoride coating agent appropriately with CB buffer, add 100 μL / well to the ELISA plate, and incubate at 4°C overnight.

[0062] (2) Washing: Pour off the liquid in the wells, wash once with washing solution (PBST), 280 μL / well, and pat dry on absorbent paper.

[0063] (3) Blocking: Add 150 μL / well of blocking solution (2.5% casein), incubate at 37°C for 2 h, and pat dry directly after removal.

[0064] (4) Sample addition: Add 50 μL / well of a series of concentrations of perfluorosulfonyl fluoride standard (0 μg / L, 0.5 μg / L, 1.5 μg / L, 3 μg / L, 9 μg / L, 27 μg / L), add 50 μL of enzyme marker working solution to each well; then add 50 μL of perfluorosulfonyl fluoride monoclonal antibody working solution to each well; react at room temperature for 30 minutes.

[0065] (5) Washing: Pour out the liquid in the wells, add 260 μL of washing solution to each well, wash thoroughly 4 times, pour out the liquid in the wells, and pat dry on absorbent paper.

[0066] (6) Color development: Add freshly prepared TMB substrate solution, 100 μL / well, and react at 37°C in the dark for 15 min.

[0067] (7) Termination: Add 2 mol / L H2SO4 as the stop solution, 50 μL / well.

[0068] (8) Determination: Read the OD of each well using a microplate reader 450 nm Value (dual wavelength: 630nm is the reference filter wavelength).

[0069] result : The optimal dilution of perfluorosulfonyl fluoride coating material was 1:16000 and the optimal dilution of perfluorosulfonyl fluoride monoclonal antibody was 1:20000 by the checkerboard method test.

[0070] Example 5: Application of perfluorosulfonyl fluoride enzyme-linked immunosorbent assay

[0071] 1. Sample pretreatment

[0072] The animal-derived food samples were peeled and defatted, and the samples to be tested were homogenized using a homogenizer. 3.00±0.05 g of the homogenized sample to be tested was weighed and placed in a 15 mL centrifuge tube. 4 mL of an extract containing acetonitrile:water (8:2, v / v) was accurately added to the centrifuge tube, followed by 2 mL of a 2% NaCl solution. The tube was vigorously shaken for 3 minutes and centrifuged at 4000 rpm for 3 minutes. 2 mL of the supernatant was added to a 5 mL centrifuge tube and nitrogen purged. 1 mL of n-hexane was added to the dried centrifuge tube, which was vigorously shaken, followed by 0.3 mL of PBS buffer. The tube was thoroughly mixed for 30 seconds and allowed to stand for 1 minute until distinct layers were separated. 120 μL of the lower layer solution was aspirated for testing.

[0073] 2. Detection steps

[0074] The test kit and method of Example 4 were used for detection.

[0075] Calculate the percentage absorbance value: divide the average absorbance value of each standard (or sample to be tested) by the absorbance value of the zero standard (standard with a concentration of 0 μg / L) and multiply by 100% to obtain the percentage of absorbance corresponding to each standard, i.e. the percentage absorbance value;

[0076] Prepare a standard curve: use the percentage absorbance of each standard as the ordinate and the corresponding perfluorosulfonyl fluoride concentration as the abscissa to draw a standard curve;

[0077] Calculate the perfluorosulfonyl fluoride content in the sample: Substitute the percentage absorbance value of the sample to be tested into the standard curve equation to obtain the corresponding concentration of the sample to be tested. Multiply it by the dilution factor of the corresponding sample to obtain the actual content of perfluorosulfonyl fluoride in the original sample to be tested.

[0078] Example 6. Evaluation and application of perfluorosulfonyl fluoride enzyme-linked immunosorbent assay

[0079] 1. Determination of minimum detection limit

[0080] Take blank samples of fish, shrimp, eggs and pork for testing, calculate the measured value according to the standard curve, calculate the average value, and add 3 times the standard deviation to obtain the minimum detection limit (LOD).

[0081] Table 2 Statistics of blank sample determination results (μg / kg)

[0082]

[0083] The results are shown in Table 2. To prevent false positives, the detection limit of perfluorosulfonyl fluoride in animal-derived foods such as fish, shrimp, eggs, and pork is 2 μg / kg.

[0084] 3. Determination of method accuracy and precision

[0085] Accuracy refers to the degree of agreement between the measured value and the true value. In ELISA assays, accuracy is often expressed as recovery, while precision is often expressed as the coefficient of variation. Blank animal-derived food samples were spiked with 2.0 μg / kg and 4.0 μg / kg perfluorosulfonyl fluoride standards, with five replicates for each spike. Three batches of the kit were used for the assay, and the spiked recoveries and intra- and inter-batch coefficients of variation were calculated.

[0086] Table 3 Accuracy and precision test results

[0087]

[0088] The results are shown in Table 3. The recoveries of animal-derived food samples at various spiked concentrations ranged from 82.76% to 115.98%, the intra-batch coefficient of variation was less than 10%, and the inter-batch coefficient of variation was less than 15%.

[0089] 4. Actual sample determination using perfluorosulfonyl fluoride enzyme-linked immunosorbent assay

[0090] Twenty blind samples of animal-derived food were analyzed using both ELISA and UPLC-MS / MS. The results were compared, with the detection limit of the kit used as the negative / positive value. Kit results below the detection limit were marked as "-," while instrument results were marked as "ND." Results above the detection limit were expressed as the actual value. The results, shown in Table 4, showed good overall consistency between the ELISA results and the LC-MS / MS results.

[0091] Table 4 Actual sample determination by perfluorosulfonyl fluoride enzyme-linked immunosorbent assay

[0092] sample ELISA (μg / kg) LC-MS / MS (μg / kg) Fish 1# - ND Fish 2# - ND Fish 3# - ND Fish 4# - ND Fish 5# - ND Shrimp meat 1# - ND Shrimp Meat 2# - ND Shrimp Meat 3# - ND Shrimp Meat 4# - ND Shrimp Meat 5# 3.8 3.5 Egg 1# - ND Egg 2# - ND Egg 3# - ND Egg 4# - ND Egg 5# - ND Pork 1# - ND Pork 2# - ND Pork #3 4.8 5.2 Pork 4# - ND Pork 5# - ND

Claims

1. A perfluorosulfonyl fluoride hapten, characterized in that The structure is shown in Formula I:

2. The method for preparing the perfluorosulfonyl fluoride hapten according to claim 1, wherein The following steps are involved: Under the catalysis of triethylamine, N-tert-butyloxycarbonyl-1,4-butanediamine and perfluorosulfonyl fluoride are refluxed and reacted at 40°C for 16 hours, and an intermediate oily substance is obtained by vacuum distillation and silica gel column chromatography. The intermediate is dissolved in ethyl acetate, and a hydrogen chloride ethyl acetate solution is added for reaction. After solid-liquid separation, washing, and drying, a hapten represented by formula I is obtained.

3. The method for preparing the perfluorosulfonyl fluoride hapten according to claim 2, characterized in that: The usage ratio of N-tert-butyloxycarbonyl-1,4-butanediamine, triethylamine and perfluorosulfonyl fluoride is 564.8 mg:675.6 μL:1200 μL.

4. The method for preparing the perfluorosulfonyl fluoride hapten according to claim 2, wherein: The silica gel column chromatography used 200-300 mesh silica gel, and the eluent was petroleum ether-ethyl acetate in a volume ratio of 5:

1.

5. A perfluorosulfonyl fluoride antigen, characterized in that The antibody is obtained by coupling the perfluorosulfonyl fluoride hapten according to claim 1 or 2 with a carrier protein, and comprises an immunogen and a coating agent.

6. The perfluorosulfonyl fluoride antigen according to claim 5, characterized in that The immunogen is a conjugate of perfluorosulfonyl fluoride hapten and bovine serum albumin (BSA), and the conjugation molar ratio is 3-6:

1.

7. The perfluorosulfonyl fluoride antigen according to claim 6, characterized in that The coupling molar ratio is 4.66:

1.

8. A perfluorosulfonyl fluoride antibody, characterized in that The method is prepared by immunizing an animal with the perfluorosulfonyl fluoride antigen according to any one of claims 5 to 7.

9. The perfluorosulfonyl fluoride antibody according to claim 8, characterized in that The antibody is a monoclonal antibody, and its IC 50 The cross-reactivity rate to perfluorooctanesulfonyl fluoride was 86.3%, and the cross-reactivity rate to perfluorobutanesulfonyl fluoride was 31.4%.

10. Use of the perfluorosulfonyl fluoride hapten according to claim 1 or 2, characterized in that: Used for preparing perfluorosulfonyl fluoride antigen, perfluorosulfonyl fluoride antibody or perfluorosulfonyl fluoride detection reagent.

Images