Method for rapidly detecting sulfonamides in meat through improved QuEChERS binding enzyme inhibition colorimetric method

Abstract

The invention discloses a method for rapidly detecting sulfonamides in meat by an improved QuEChERS combined enzyme inhibition colorimetric method, which uses a natural eutectic solvent instead of a toxic organic solvent as an extraction agent, reduces the harm to the environment and human body, improves the extraction efficiency, and is beneficial to protecting the enzyme activity, so that the detection is more sensitive and stable; the magnetic COF (TpPa-Cl) coated Fe3O4 synthesized by adopting a simple and rapid stirring method not only has good chemical stability and can be repeatedly used, but also has excellent adsorption capacity and strong magnetism, and when the magnetic COF (TpPa-Cl) coated Fe3O4 is used as an adsorbent for improving QuEChERS, interfering substances in a sample can be effectively removed in a small amount, the pretreatment time is shortened, and the purification process is simplified; in the enzyme inhibition colorimetric detection process, a digital image colorimetric method of a smart phone is combined for quantitative analysis. The whole method has the remarkable advantages of greenness, high efficiency, short time consumption, low cost, simplicity in operation, high sensitivity and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and specifically discloses a method for quickly detecting sulfonamide drugs in meat by using an improved QuEChERS combined with enzyme inhibition colorimetric method. Background Art

[0002] In recent years, the abuse of veterinary drugs has brought serious problems to humans. Sulfonamides are a broad-spectrum veterinary drug that is commonly used in animal husbandry for treatment and prevention. In the process of treating livestock and poultry, the widespread use of sulfonamides inevitably leads to their accumulation in animal food tissues. These residues can enter the human body and cause many harms to consumers' health. The European Union has stipulated that the maximum residue limit (MRL) of the total content of sulfonamides in animal-derived foods is 100 μg kg -1 . Take pork as an example. Pork is one of the most consumed meats in the world because it is rich in protein, lipids, minerals and vitamins, providing nutrition for human growth and development. According to the Food and Agriculture Organization of the United Nations, global pork production reached 124.5 million tons in 2023. In recent years, incidents of sulfonamide residues in pork have occurred continuously. Therefore, the development of sensitive, practical, green and reliable analysis methods for sulfonamide residues is crucial to ensure food safety, public health and human health.

[0003] The QuEChERS method was proposed by Anastassiades et al. in 2003. It has attracted much attention due to its advantages such as large sample throughput, low solvent consumption, low cost, simplicity, effectiveness, and high recovery rate. Although the QuEChERS method has shown great advantages, it still needs to be further improved. The traditional QuEChERS method uses acetonitrile as an extractant, and ethylenediamine-N-propylsilane (PSA), graphitized carbon black (GCB), octadecylsilane (C18), multi-walled carbon nanotubes (MWCNTs) and graphene as purification adsorbents. The main toxicity of acetonitrile comes from its effect on the central nervous system, and its use violates the principles of green chemistry. At the same time, traditional adsorbents are expensive and require additional centrifugation and filtration, which increases the pretreatment time. Therefore, it is urgent to develop new green, environmentally friendly, low-cost extractants and adsorbents to improve extraction efficiency and purification efficiency. Summary of the invention

[0004] The present invention provides a method for rapidly detecting sulfonamides in meat by combining an improved QuEChERS method with an enzyme inhibition colorimetric method, so as to overcome the defects of a traditional QuEChERS method.

[0005] The method for rapid detection of sulfonamides in meat by the improved QuEChERS combined with enzyme inhibition colorimetric method, wherein the sulfonamides to be detected include one or more of sulfadiazine, sulfonamide, sulfathiazole, sulfamethoxazine and sulfadimethoxine, comprises the following steps:

[0006] S1, using any one of ethylene glycol, propylene glycol, octanoic acid and butyric acid as a hydrogen bond donor, using any one of choline chloride, betaine, thymol, vanillin and tetrabutylammonium chloride as a hydrogen bond acceptor, heating in a water bath to form a clear and transparent liquid, and obtaining six different natural deep eutectic solvents as extractants: ethylene glycol-choline chloride, ethylene glycol-betaine, propylene glycol-betaine, octanoic acid-thymol, butyric acid-vanillin, butyric acid-tetrabutylammonium chloride;

[0007] S2, adding any one of the natural low eutectic solvents prepared in step S1 to the meat sample, vortexing and mixing, centrifuging, and extracting the liquid to obtain an extract a;

[0008] S3, adding an adsorbent to the extract a obtained in step S2, and obtaining an extract b after separation and impurity removal;

[0009] The adsorbents are ethylenediamine-N-propylsilane, octadecylsilane, graphitized carbon black, graphene, multi-walled carbon nanotubes, Fe 3 O 4 or COF(TpPa-Cl)@Fe 3 O 4 Any of;

[0010] S4, mixing the extract b obtained in step S3 with the freshly prepared carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution in a container, incubating the mixture, placing the container in an opaque white box, the top of the white box is evenly illuminated by an LED light panel, capturing the color change of the mixture in the container with a smart phone, and then using Adobe Photoshop CC software to read the channel values ​​of the red, green, and blue channels of the active site in RGB mode, and calculating the intensity of the sulfonamide drugs to be detected in the meat sample;

[0011] S5. Prepare standard samples of different sulfonamides, mix them with freshly prepared carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution in a container, incubate the mixture, place the container in an opaque white box, and evenly illuminate the top of the white box with an LED light panel. Use a smart phone to capture the color change of the mixture in the container, and then use Adobe Photoshop CC software to read the channel values ​​of the red, green, and blue channels of the active site in RGB mode, and calculate the intensity of the standard. Draw standard working curves for different sulfonamides with the standard concentration as the horizontal axis and the corresponding intensity as the vertical axis. Substitute the intensity of the sulfonamide to be detected in the meat sample obtained in step S4 into the standard working curve to obtain the concentration of the sulfonamide in the meat sample.

[0012] Specifically, in step S1, the mixing ratio of the hydrogen bond donor and the hydrogen bond acceptor is 1:1 to 1:6 in molar ratio, the water bath temperature is 80° C., and the water content of the natural deep eutectic solvent is 0%.

[0013] Preferably, in step S2, the natural low eutectic solvent selected is propylene glycol-betaine, and the mixing ratio of propylene glycol-betaine is 1:4 in molar ratio.

[0014] Specifically, in step S2, the meat sample and the natural low eutectic solvent are mixed according to 0.5-3 g: 200-800 μL; the vortex time is 0.5-2 min; the centrifugation time is 90 s, and the rotation speed is 5000 r / min.

[0015] Preferably, in step S3, the adsorbent selected is COF(TpPa-Cl)@Fe 3 O 4 , extract a and COF(TpPa-Cl)@Fe 3 O 4 The mixture was mixed in a ratio of 1 mL:10 mg, and after vortexing, impurities were removed by adsorption using an external magnetic field to obtain extract b.

[0016] Specifically, in step S3, COF(TpPa-Cl)@Fe 3 O 4 The method is prepared by stirring at room temperature, comprising the following steps:

[0017] Fe was added at a ratio of 1.2 mg:1 mL. 3 O 4Add dimethyl sulfoxide and disperse it evenly by ultrasonication. Then add 1,3,5-triformyl benzenetriol and 4-chloro-m-phenylenediamine respectively. Every 1 mL of dimethyl sulfoxide corresponds to 0.63 mg of 1,3,5-triformyl benzenetriol and 0.9626 mg of 4-chloro-m-phenylenediamine. Perform ultrasonication again. Then stir and add glacial acetic acid. Every 1 mL of dimethyl sulfoxide corresponds to 0.02 mL of glacial acetic acid. After the reaction, let it stand to obtain a brown-yellow product. Take the supernatant and rinse it with methanol and water for several times until it is colorless. Dry it in a vacuum drying oven to obtain COF(TpPa-Cl)@Fe 3 O 4 .

[0018] Specifically, in steps S4 and S5, the concentration of the carbonic anhydrase solution is 100 to 350 U·mL -1 ; Phosphate buffer concentration is 10.0 mM, pH is 6.5-9; p-nitrophenyl acetate solution concentration is 0.02-0.12 mg·mL -1 ;

[0019] In step S4, the volume ratio of the extract b, the carbonic anhydrase solution, the phosphate buffer solution, and the p-nitrophenyl acetate solution is 1:0.5:2:1;

[0020] In step S5, the volume ratio of the standard, carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution is 1:0.5:2:1.

[0021] Specifically, in steps S4 and S5, the incubation temperature is 20 to 70° C., and the incubation time is 1 to 6 minutes.

[0022] Specifically, in steps S4 and S5, the intensity I=280-G, where G is the channel value of the green channel.

[0023] Preferably, in step S2, the meat sample and propylene glycol-betaine are mixed at a ratio of 1 g: 500 μL, vortexed for 1 min and centrifuged for 90 s; in steps S4 and S5, the concentration of the carbonic anhydrase solution is 300 U·mL -1 ; Phosphate buffer concentration is 10.0 mM, pH is 8.5; p-nitrophenyl acetate solution concentration is 0.1 mg·mL -1 ; The incubation temperature is 40℃ and the incubation time is 3min.

[0024] Compared with the prior art, the present invention has the following beneficial effects.

[0025] 1. The method provided by the present invention for rapid detection of sulfonamides in meat by improved QuEChERS combined with enzyme inhibition colorimetry is based on a natural deep eutectic solvent and an adsorbent COF(TpPa-Cl)@Fe 3 O 4The modified QuEChERS combined with carbonic anhydrase smartphone colorimetric method for the detection of sulfonamide residues in pork was developed. This method is rapid, sensitive, simple and accurate, which not only provides a useful tool for daily monitoring, but also lays the foundation for further food safety risk assessment.

[0026] 2. The improved QuEChERS combined with enzyme inhibition colorimetry provided by the present invention is a method for rapid detection of sulfonamides in meat, which improves the traditional QuEChERS method and uses a new green solvent natural low eutectic solvent instead of traditional organic solvents such as acetonitrile as an extractant. The natural low eutectic solvent has the advantages of simple preparation, low melting point, strong volatility, low cost, and strong solubility for hydrophilic and hydrophobic substances. It can achieve green and environmentally friendly extraction of sulfonamides in pork. At the same time, the natural low eutectic solvent is safe and stable, and can protect the activity of the enzyme in the subsequent detection process.

[0027] 3. The present invention uses COF(TpPa-Cl)@Fe 3 O 4 Instead of the traditional QuEChERS adsorbent, COF(TpPa-Cl)@Fe 3 O 4 Combining covalent organic framework materials and magnetic nanoparticles Fe 3 O 4 The advantages of COF(TpPa-Cl)@Fe make it an ideal adsorbent for QuEChERS, and its magnetic separation speed is 10 times faster than the centrifugation process of traditional QuEChERS adsorbents. 3 O 4 It is prepared by a simple and convenient stirring method at room temperature. It has the advantages of large specific surface area, strong magnetism, good stability, low cost, and reusability. A small amount can achieve the purification effect. The improved QuEChERS can achieve rapid, green and environmentally friendly extraction and purification of sulfonamides in pork.

[0028] 4. The method provided by the present invention can directly detect sulfonamides by color change based on enzyme inhibition colorimetry without complicated detection process. Directly using natural carbonic anhydrase to detect sulfonamides has the advantages of high sensitivity, short response time, simple operation, low cost and good selectivity. Smartphones are the most convenient image acquisition tools due to their portability, multiple information transmission functions and storage capacity, and are currently the most promising field monitoring and portable detection platform. Combining enzyme inhibition colorimetric detection with smartphones greatly improves the accuracy of test results and shortens detection time.

[0029] 5. The method provided by the present invention can process a large number of samples at one time, and the amount of reagents used to process the samples is small, which is green and environmentally friendly.

[0030] 6. The method provided by the present invention is time-saving, and the entire sample pretreatment and detection process only takes 5 minutes.

[0031] 7. The method provided by the present invention does not require large-scale instruments for detection. It is a simple, rapid and green method, which is applied to the analysis of sulfonamides in pork samples. The recovery rate is 98.42% to 101.6%, and the relative standard deviation is less than 3.6%. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 Flow chart of the method for rapid detection of sulfonamides in meat by improved QuEChERS combined with enzyme inhibition colorimetric method;

[0034] Figure 2 This is a diagram showing the effect of natural deep eutectic solvent types on extraction results;

[0035] Figure 3 This is the effect of the molar ratio of natural deep eutectic solvent on the extraction effect;

[0036] Figure 4 This is a graph showing the effect of the volume of natural deep eutectic solvent on the extraction effect;

[0037] Figure 5 This is a graph showing the effect of water content in natural deep eutectic solvent on extraction effect;

[0038] Figure 6 This is the effect of adsorbent type on extraction effect;

[0039] Figure 7 This is the effect of adsorbent dosage on extraction effect;

[0040] Figure 8 is the effect of carbonic anhydrase concentration on colorimetric detection intensity (I);

[0041] Fig. 9 is the effect of substrate concentration on colorimetric detection intensity (I);

[0042] Fig.10 is the effect of phosphate buffer pH on colorimetric detection intensity (I);

[0043] Fig.11 is the effect of reaction temperature on colorimetric detection intensity (I);

[0044] Fig.12 Graph showing the effect of reaction time on colorimetric detection intensity (I). DETAILED DESCRIPTION

[0045] To date, a variety of methods have been used to detect sulfonamides, including high performance liquid chromatography, spectroscopy, capillary electrophoresis, mass spectrometry, and electrochemical methods. In general, these traditional methods have their limitations, including expensive equipment, specialized laboratory personnel, and time-consuming processes. In addition, due to the large size of the equipment and the difficulty of carrying it, it is not possible to conduct on-site detection. In contrast, carbonic anhydrase can hydrolyze p-nitrophenylacetate to p-nitrophenol, and sulfonamide can inhibit the hydrolysis process of carbonic anhydrase. Sulfonamide-based enzyme inhibition can directly detect sulfonamides through color changes without the need for a complex detection process. Digital image colorimetry is an emerging colorimetric method, which is an image acquisition tool for recording colors and uses image processing software to analyze the colors in the acquired images. Smartphones have become the most convenient image acquisition tool due to their portability, multiple information transmission functions, and storage capacity. They are currently the most promising on-site monitoring and portable detection platform, which can be used for quantitative and qualitative analysis of various samples, greatly improving the accuracy of the detection results and becoming a common alternative method for colorimetric detection of analytes. This example is the first to use a magnetic covalent organic framework material as an adsorbent and a natural deep eutectic solvent as an extraction solvent to improve QuEChERS and combine it with enzyme inhibition smartphone digital image colorimetry to detect sulfonamide drug residues in pork.

[0046] The present invention comprises four parts: a low eutectic solvent and a magnetic adsorbent COF(TpPa-Cl)@Fe 3 O 4 The first step is to prepare the samples, the second step is to improve the QuEChERS method for sample extraction and purification, and optimize the conditions to obtain the best extraction conditions, the third step is to detect the samples by enzyme inhibition colorimetry, and optimize the conditions to obtain the best detection conditions, and the fourth step is to use the best extraction, purification and detection conditions to measure the meat samples.

[0047] Example 1: Deep eutectic solvent and magnetic adsorbent COF(TpPa-Cl)@Fe 3 O 4 Preparation

[0048] Choline chloride, betaine, thymol, vanillin and tetrabutylammonium chloride were selected as hydrogen bond acceptors, ethylene glycol, propylene glycol, caprylic acid and butyric acid were selected as hydrogen bond donors, and heated in a water bath at 80°C until a clear and transparent liquid was formed. Six different natural low eutectic solvents, i.e., extractants, were synthesized.

[0049] COF(TpPa-Cl)@Fe 3O 4 The stirring method was used to prepare the mixture at room temperature. First, 120 mg Fe 3 O 4 Add 100 mL of dimethyl sulfoxide, sonicate for 10 minutes to make it evenly dispersed, then add 1,3,5-triformyl benzenetriol (63 mg) and 4-chloro-m-phenylenediamine (96.26 mg) respectively, and sonicate for another 10 minutes. Under electric stirring, add 2 mL of glacial acetic acid, react for 30 minutes, and stand for 35 minutes to obtain a brown-yellow product. The supernatant was rinsed with methanol and water several times until it was almost colorless. After drying in a vacuum oven for 12 hours, COF(TpPa-Cl)@Fe 3 O 4 .

[0050] Example 2 Parameter Optimization

[0051] The influencing factors of the present invention are many, and a series of optimization experiments need to be performed. Therefore, the type of natural deep eutectic solvent, the molar ratio of natural deep eutectic solvent, the volume of natural deep eutectic solvent, the water content in natural deep eutectic solvent, the type of adsorbent, the amount of adsorbent, carbonic anhydrase concentration, substrate (p-nitrophenyl acetate) concentration, phosphate buffer pH, reaction temperature and reaction time were optimized by single factors. Samples were purchased in local supermarkets to verify the reliability of the test results of the method of the present invention in practical applications. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0052] This embodiment uses pork as an example. The original amount of sulfonamide drugs is 0. After adding sulfonamide drugs according to a preset amount, it is used as a sample to be tested, and the test is performed and the recovery rate is calculated.

[0053] 1. Optimization of natural deep eutectic solvent types

[0054] Step 1: In a 5 mL centrifuge tube, 1 g of meat sample was vortexed with 0.5 mL of different natural low eutectic solvents for 1 min and centrifuged for 90 s, and the liquid was extracted to obtain extract a and placed in a centrifuge tube.

[0055] Step 2: Then add 2 mg COF(TpPa-Cl)@Fe to the centrifuge tube containing 0.2 ml of extract a. 3 O 4 After vortexing for 30 seconds, the extract b was quickly separated using an external magnet.

[0056] Step 3: 100 μL of extract b, 50 μL of freshly prepared carbonic anhydrase solution (300 units / mL), 200 μL of phosphate buffer (10.0 mM, pH 8.5), and 100 μL of p-nitrophenyl acetate solution (0.1 mg mL -1) were mixed in a 0.5 mL centrifuge tube. The mixture was then incubated at 40°C for 3 min. The centrifuge tube was placed in an opaque white box with even illumination from the LED light panels around the top of the box. The color changes were captured with a smartphone, and then Adobe Photoshop CC software was used to read the red (R), green (G), and blue (B) channel values ​​of the active site in RGB mode and calculate the intensity (I), using the formula I = 280-G.

[0057] Step 4, substituting the strength of the sulfonamide drugs to be detected obtained in step 3 into the standard working curve of sulfonamide drugs, obtaining the concentration of sulfonamide drugs in the meat sample, and calculating the recovery rate.

[0058] Results Figure 1 The results showed that the natural deep eutectic solvents synthesized by propylene glycol and betaine had the highest extraction recovery rate, which may be due to the stronger hydrogen bonding and electrostatic interactions between them and sulfonamide drugs than other deep eutectic solvents. Therefore, propylene glycol and betaine were selected as the optimal natural deep eutectic solvents.

[0059] 2. Optimization of the molar ratio of natural deep eutectic solvent

[0060] The molar ratio of hydrogen bond acceptors to hydrogen bond donors will affect the polarity, solubility and viscosity of the natural deep eutectic solvent, thereby affecting the extraction of sulfonamide drugs. In the experiment, propylene glycol and betaine were selected to synthesize natural deep eutectic solvents according to the molar ratios of 1:1, 1:2, 1:3, 1:4, 1:5 and 1:6, and other operations were the same as the optimization of the natural deep eutectic solvent type in 1 above.

[0061] Results Figure 2 , the results show that when the molar ratio of propylene glycol to betaine is 1:1 to 1:4, the extraction recovery rate shows an upward trend, and when the molar ratio is 1:4 to 1:6, the extraction recovery rate decreases. This effect is due to the reduction and instability of the hydrogen bonds of the natural deep eutectic solvent. Finally, the molar ratio of propylene glycol to betaine is 1:4.

[0062] 3. Optimization of the volume of natural deep eutectic solvent

[0063] The volume of the extractant is an important factor that plays a vital role in the extraction efficiency in the QuEChERS method. Insufficient volume of the extractant will lead to insufficient extraction of the target analytes, while excessive volume will reduce sensitivity. Different amounts of extractants ranging from 200 to 800 μL were selected in the experiment, and other operations were the same as the optimization of the natural deep eutectic solvent type in the above 1.

[0064] Results Figure 3 ,The results show that the extraction efficiency is the highest when 500μL, but as the volume of the extractant increases, the extraction efficiency gradually decreases. Therefore, 500μL is selected as the optimal volume of the extractant.

[0065] 4. Optimization of water content in natural deep eutectic solvents

[0066] The water content affects the viscosity and polarity of the natural deep eutectic solvent, and the optimal water content also depends on the physical and chemical properties of the specific compound. Different water contents of 0%, 20%, 40%, 60% and 80% were used in the experiment, and the other operations were the same as the optimization of the natural deep eutectic solvent type in the above 1.

[0067] Results Figure 4 , as the water content in the natural deep eutectic solvent gradually increases, the extraction recovery rate gradually decreases. The addition of water may destroy the hydrogen bonds between hydrogen bond donors and hydrogen bond acceptors, thereby affecting the extraction. Therefore, a natural deep eutectic solvent without water was used in the experiment.

[0068] 5. Optimization of adsorbent types

[0069] The use of adsorbents reduces matrix interference and improves the selectivity of sulfonamides. Different types of materials were selected as adsorbents in the experiment, including ethylenediamine-N-propylsilane (PSA), octadecylsilane (C18), graphitized carbon black (GCB), graphene, multi-walled carbon nanotubes (MWCNTs), Fe 3 O 4 and COF(TpPa-Cl)@Fe 3 O 4 The other operations are the same as the optimization of the natural deep eutectic solvent type in the above 1. 3 O 4 Synthesized by solvothermal reaction.

[0070] Results Figure 5 The results showed that the recovery rate of sulfonamides by traditional adsorbents such as ethylenediamine-N-propylsilane (PSA), octadecylsilane (C18), graphitized carbon black (GCB), graphene, and multi-walled carbon nanotubes (MWCNTs) was reduced, while the recovery rate of sulfonamides by magnetic COF (TpPa-Cl)@Fe 3 O 4 High recovery rate for sulfonamide. 3 O 4 The disadvantage is that it is easy to agglomerate into blocks and has fewer surface functional groups, which leads to a decrease in the adsorption capacity of impurities. 3 O 4 It not only has a large specific surface area, but also has abundant benzene rings and pore structures, which can provide a large number of adsorption sites to adsorb impurities through π-π stacking interactions and hydrophobic interactions. 3 O 4, just by adding a magnet, the extract and the adsorbent can be successfully separated in a few seconds. Compared with traditional centrifugal separation, magnetic separation is more convenient and faster.

[0071] 6. Optimization of adsorbent dosage

[0072] In the QuEChERS method, the amount of adsorbent plays a crucial role in obtaining satisfactory analyte recovery and higher purification effect. In order to select the optimal amount of adsorbent, COF(TpPa-Cl)@Fe 3 O 4 The adsorption effect is within the range of 1 to 5 mg. Other operations are the same as the optimization of the natural deep eutectic solvent type in the above 1.

[0073] Results Figure 6 The results show that when the adsorbent dosage is 2 mg, the active sites are more easily exposed to interacting impurities and the recovery rate of the analyte is the highest. When the adsorbent mass is greater than 2 mg, the adsorption surface area decreases due to the aggregation of adsorbent particles, resulting in a decrease in the recovery rate. Finally, 2 mg was determined as the optimal adsorbent dosage for the experiment.

[0074] 7. Optimization of carbonic anhydrase concentration

[0075] The enzyme content is crucial to the construction of the colorimetric process and the detection sensitivity. In the experiment, the carbonic anhydrase content was 100-350 U·mL -1 The other operations were the same as the optimization of the natural deep eutectic solvent type in 1 above. The results are shown in Figure 7 The results showed that the intensity (I) gradually increased with the increase of carbonic anhydrase content. When the carbonic anhydrase concentration exceeded 300 U·mL -1 When the intensity (I) did not change significantly. Finally, 300U·mL -1 Carbonic anhydrase is the best enzyme content in the experiment. The reason may be that after the combination of carbonic anhydrase and p-nitrophenyl acetate reaches saturation, increasing the amount of carbonic anhydrase will not have a significant change.

[0076] 8. Optimization of substrate concentration

[0077] Substrate concentration is a key parameter affecting the precision of colorimetric detection. The experiment studied the content of p-nitrophenyl acetate in 0.02-0.12 mg·mL -1 The effect on the colorimetric detection of sulfonamide drugs, other operations are the same as the optimization of the type of natural deep eutectic solvent in the above 1.

[0078] Results Figure 8 The results show that with the increase of p-nitrophenyl acetate content, the intensity (I) gradually increases. When the p-nitrophenyl acetate content exceeds 0.1 mg·mL -1When , the intensity (I) no longer increases significantly. The reason for this phenomenon may be that p-nitrophenyl acetate binds to carbonic anhydrase, occupies part of the binding sites between carbonic anhydrase and sulfonamide drugs, and hinders the inhibitory effect of sulfonamide drugs on carbonic anhydrase. Finally, 0.1 mg·mL -1 It is the optimal substrate concentration in the experiment.

[0079] 9. Optimization of pH of phosphate buffer

[0080] In order to study the detection performance of the colorimetric method under different buffer conditions, the pH value of the buffer was optimized, and other operations were the same as the optimization of the natural low eutectic solvent type in the above 1.

[0081] The results of intensity (I) changes at different pH values ​​are shown in Fig. 9 , the results showed that with the increase of pH value, the intensity (I) gradually increased and reached the maximum value at pH 8.5; the color of the solution also changed from yellow to colorless. When the pH value exceeded 8.5, the activity of carbonic anhydrase weakened and the intensity (I) gradually decreased. Finally, the experiments were all carried out at a pH value of 8.5. This may be because the excessively high pH value affected the binding of carbonic anhydrase to the substrate, resulting in a weakened colorimetric detection effect.

[0082] 10. Optimization of reaction temperature

[0083] In order to study the effect of reaction temperature on colorimetric detection, sulfonamides were added to carbonic anhydrase and placed at different temperatures for a few minutes. Then, after adding buffer and substrate, the effect of incubation temperature of 20, 30, 40, 50, 60, and 70°C on colorimetric detection was studied. Other operations were the same as the optimization of the natural deep eutectic solvent type in 1 above.

[0084] Results Fig.10 The results showed that from 20℃ to 40℃, as the incubation temperature increased, the intensity (I) also increased, and then decreased after 40℃. The reason is that high temperature inhibits the activity of carbonic anhydrase. Finally, the experiment selected 40℃ as the best incubation temperature. At this temperature, sulfonamides have a good inhibitory effect on carbonic anhydrase, which promotes the application of sulfonamide colorimetry in on-site real-time detection.

[0085] 11. Optimization of response time

[0086] The reaction time also affects the strength (I). The experiment studied the reaction time of 1 to 6 minutes, and the other operations were the same as the optimization of the type of natural deep eutectic solvent in the above 1.

[0087] Results Fig.11, the results show that the intensity (I) increases steadily and tends to saturation after 3 minutes. As time goes by, the yellow color turns to colorless and remains stable. Finally, the optimal reaction time is 3 minutes.

[0088] Example 3 Effect Verification

[0089] In this embodiment, untreated pork is used as the sample to be tested, and the sulfonamides in meat are detected by using a modified QuEChERS combined with enzyme inhibition colorimetric method for rapid detection of sulfonamides in meat.

[0090] (1) Preparation of natural deep eutectic solvents

[0091] Propylene glycol and betaine are mixed in a molar ratio of 1:4, and continuously heated in a water bath at 80° C. until a clear and transparent liquid is formed, thereby obtaining a natural low eutectic solvent, which is then stored at room temperature for later use.

[0092] (2) Adsorbent COF(TpPa-Cl)@Fe 3 O 4 Preparation

[0093] (3) Preparation of sample solution

[0094] Accurately weigh 1 g of the pulverized and homogenized pork sample to be tested, add 0.5 mL of the natural deep eutectic solvent, vortex for 1 min and centrifuge for 90 s for extraction to obtain extract a.

[0095] (4) Purification and collection

[0096] Take out 0.2 ml of extract a from step (3) above and add 2 mg COF(TpPa-Cl)@Fe 3 O 4 After vortexing for 30 seconds, the extract b was quickly separated using an external magnet.

[0097] (5) Colorimetric detection of sulfonamides

[0098] According to Example 2, extract b, carbonic anhydrase solution, phosphate buffer and p-nitrophenyl acetate solution were mixed in a 0.5 mL centrifuge tube. The mixture was then incubated at 40°C for 3 min. The centrifuge tube was placed in an opaque white box, and the LED light panels around the top of the box were evenly illuminated. The color changes were captured with a smartphone, and then the red (R), green (G), and blue (B) channel values ​​of the active site in RGB mode were read using Adobe Photoshop CC software, and the intensity (I) was calculated using the formula I = 280-G.

[0099] (6) Testing of actual samples

[0100] This example uses the optimal conditions obtained in Example 2 to evaluate the application of the modified QuEChERS combined with enzyme inhibition colorimetry to detect sulfonamides in pork. Under the optimal conditions, the established method was verified in terms of linear range, detection limit, quantification limit, determination coefficient and precision. The detection limit and quantification limit were estimated according to the 3σ / K and 10σ / K formulas, where σ represents the standard deviation of 10 blank samples and K is the slope of the calibration standard curve. The detection limits of all analytes were between 0.53 and 0.82 μg kg -1 The detection limit is in line with the EU MRL of 100 μg kg for the total content of sulfonamides in animal food tissues. -1 The limit of quantification is 1.52~1.98μg·kg -1 . Intra-day and inter-day analysis were used to evaluate the precision, accuracy and repeatability of the established method. The intra-day and inter-day precision were tested in parallel within one day and three days, respectively (n=3). The results are shown in Table 1. The linear correlation was good, and the determination coefficient was between 0.9956 and 0.9991. The intra-day and inter-day relative standard deviations were 1.96% to 3.42% and 1.32 to 3.58%, respectively. In order to investigate the reproducibility of the method, spiked experiments were carried out in three samples, five sets of parallel experiments were performed and the relative standard deviations were calculated. The results are shown in Table 2. The results showed that the recovery rate was in the range of 98.42% to 101.60%, and the relative standard deviation was between 1.2% and 3.6%. The results showed that the established method had good reproducibility and was suitable for the detection of sulfonamides in food.

[0101] Table 1 Method evaluation of sulfonamides in real samples

[0102]

[0103] Table 2 Spiked recovery and relative standard deviation of actual samples

[0104]

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for rapid detection of sulfonamides in meat by using a modified QuEChERS combined with enzyme inhibition colorimetry, wherein the sulfonamides to be detected include one or more of sulfadiazine, sulfonamide, sulfathiazole, sulfamethoxazine and sulfadimethoxine, characterized in that: The steps include: S1, using any one of ethylene glycol, propylene glycol, octanoic acid and butyric acid as a hydrogen bond donor, using any one of choline chloride, betaine, thymol, vanillin and tetrabutylammonium chloride as a hydrogen bond acceptor, heating in a water bath to form a clear and transparent liquid, and obtaining six different natural deep eutectic solvents as extractants: ethylene glycol-choline chloride, ethylene glycol-betaine, propylene glycol-betaine, octanoic acid-thymol, butyric acid-vanillin, butyric acid-tetrabutylammonium chloride; S2, adding any one of the natural low eutectic solvents prepared in step S1 to the meat sample, vortexing and mixing, centrifuging, and extracting the liquid to obtain an extract a; S3, adding an adsorbent to the extract a obtained in step S2, and obtaining an extract b after separation and impurity removal; The adsorbent is any one of ethylenediamine-N-propylsilane, octadecylsilane, graphitized carbon black, graphene, multi-walled carbon nanotubes, Fe3O4 or COF(TpPa-Cl)@Fe3O4; S4, the extract b obtained in step S3 is placed in a container and mixed with freshly prepared carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution. After incubating the mixture, the container is placed in an opaque white box, the top of the white box is evenly illuminated by an LED light panel, and the color change of the mixture in the container is captured by a smart phone, and then the channel values ​​of the red, green, and blue channels of the active site in RGB mode are read using Adobe Photoshop CC software, and the intensity of the sulfonamide drugs to be detected in the meat sample is calculated; S5, standard samples of different sulfonamide drugs are prepared, and the standard samples are placed in a container and mixed with freshly prepared carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution. After incubating the mixture, the container is placed in an opaque white box, the top of the white box is evenly illuminated by an LED light panel, and the color change of the mixture in the container is captured by a smart phone, and then Adobe Photoshop CC software is used to read the channel values ​​of the red, green, and blue channels of the active site in RGB mode, and the intensity of the sulfonamide drugs to be detected in the meat sample is calculated. The CC software reads the channel values ​​of the red, green and blue channels of the active part in the RGB mode, and calculates the intensity of the standard. The standard working curves of different sulfonamide drugs are drawn with the concentration of the standard as the horizontal axis and the corresponding intensity as the vertical axis. The intensity of the sulfonamide drugs to be detected in the meat sample obtained in step S4 is substituted into the standard working curve to obtain the concentration of the sulfonamide drugs in the meat sample.

2. The method for rapid detection of sulfonamides in meat by using the improved QuEChERS combined with enzyme inhibition colorimetry according to claim 1, characterized in that: In step S1, the mixing ratio of the hydrogen bond donor and the hydrogen bond acceptor is 1:1 to 1:6 in molar ratio, and the water bath temperature is 80° C.; The water content of natural deep eutectic solvents is 0%.

3. The method for rapid detection of sulfonamides in meat by the improved QuEChERS combined with enzyme inhibition colorimetry according to claim 2, wherein in step S2, the natural low eutectic solvent selected is propylene glycol-betaine, and the mixing ratio of propylene glycol-betaine is 1:4 in molar ratio.

4. The method for rapid detection of sulfonamides in meat by the improved QuEChERS combined with enzyme inhibition colorimetric method according to claim 3, wherein in step S2, the meat sample and the natural deep eutectic solvent are mixed according to 0.5-3 g: 200-800 μL; The vortex time is 0.5 to 2 minutes; The centrifugation time was 90 s and the rotation speed was 5000 r / min.

5. The method for rapid detection of sulfonamides in meat by using the improved QuEChERS combined with enzyme inhibition colorimetry according to claim 4, characterized in that: In step S3, the selected adsorbent is COF(TpPa-Cl)@Fe3O4, and the extract a and COF(TpPa-Cl)@Fe3O4 are mixed in a ratio of 1 mL:10 mg. After vortexing, the impurities are removed by adsorption through an external magnetic field to obtain the extract b.

6. The method for rapid detection of sulfonamides in meat by using the improved QuEChERS combined with enzyme inhibition colorimetry according to claim 1 or 5, characterized in that: In step S3, COF(TpPa-Cl)@Fe3O4 is prepared at room temperature by a stirring method, comprising the following steps: Fe3O4 was added to dimethyl sulfoxide at a ratio of 1.2 mg:1 mL, and ultrasonic dispersion was performed to obtain a uniform dispersion. Then, 1,3,5-triformyl benzenetriol and 4-chloro-m-phenylenediamine were added respectively, with 0.63 mg of 1,3,5-triformyl benzenetriol and 0.9626 mg of 4-chloro-m-phenylenediamine corresponding to each 1 mL of dimethyl sulfoxide. Ultrasound was performed again, and then stirred and glacial acetic acid was added, with 0.02 mL of glacial acetic acid corresponding to each 1 mL of dimethyl sulfoxide. After the reaction, the mixture was allowed to stand to obtain a brown-yellow product. The supernatant was washed with methanol and water several times until it was colorless, and then dried in a vacuum drying oven to obtain COF(TpPa-Cl)@Fe3O4.

7. The method for rapid detection of sulfonamides in meat by using the improved QuEChERS combined with enzyme inhibition colorimetry according to claim 6, characterized in that: In steps S4 and S5, the concentration of the carbonic anhydrase solution is 100 to 350 U·mL -1 ; The concentration of phosphate buffer is 10.0 mM, and the pH is 6.5-9; The concentration of p-nitrophenyl acetate solution is 0.02~0.12mg·mL -1 ; In step S4, the volume ratio of the extract b, the carbonic anhydrase solution, the phosphate buffer solution, and the p-nitrophenyl acetate solution is 1:0.5:2:1; In step S5, the volume ratio of the standard, carbonic anhydrase solution, phosphate buffer, and p-nitrophenyl acetate solution is 1:0.5:2:

1.

8. The method for rapid detection of sulfonamides in meat by using the improved QuEChERS combined with enzyme inhibition colorimetry according to claim 7, characterized in that: In steps S4 and S5, the incubation temperature is 20 to 70° C., and the incubation time is 1 to 6 minutes.

9. The method for rapid detection of sulfonamides in meat by using the improved QuEChERS combined with enzyme inhibition colorimetry according to claim 8, characterized in that: In steps S4 and S5, the intensity I=280-G, where G is the channel value of the green channel.

10. The method for rapid detection of sulfonamides in meat by using the improved QuEChERS combined with enzyme inhibition colorimetry according to claim 9, characterized in that: In step S2, the meat sample and propylene glycol-betaine were mixed at 1 g:500 μL, vortexed for 1 min, and centrifuged for 90 s; In steps S4 and S5, the concentration of the carbonic anhydrase solution is 300 U·mL -1 ; Phosphate buffer concentration was 10.0 mM, pH 8.5; The concentration of p-nitrophenyl acetate solution is 0.1 mg mL -1 ; The incubation temperature was 40°C and the incubation time was 3 min.

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