Agricultural product organophosphorus pesticide multi-residue detection kit and preparation method thereof

Abstract

The application discloses a kind of agricultural product organophosphorus pesticide multi-residue detection kit and preparation method thereof, it is related to detection kit preparation technical field, including: enzyme freeze-dried powder, substrate liquid, color reagent liquid, sample extraction liquid and colorimetric tube.The modified beta-cyclodextrin is added in the substrate liquid, it has stronger molecular inclusion, electrostatic adsorption and heavy metal chelating multiple capacity, can efficiently shield endogenous interference impurities in onion and garlic agricultural products, significantly reduce the probability of false positive occurrence during detection, while can protect the structure of detection substrate, inhibit substrate from self-hydrolysis metamorphic, improve reagent long-term storage stability, effectively resist external interference, such as heavy metal, fundamentally improve the detection accuracy and anti-interference ability of kit;Modified tetradecyl sulfobetaine is added in color reagent liquid, which can improve the sensitivity of pesticide detection kit, can stabilize the color reaction system, inhibit the oxidation of color product and fade, and greatly reduce the light value drift after standing.

Description

Technical Field

[0001] This invention relates to the field of detection kit preparation technology, and in particular to a detection kit for multiple residues of organophosphorus pesticides in agricultural products and its preparation method. Background Technology

[0002] A test kit is a commercial system that pre-packages various reagents (such as chromogenic agents, buffer solutions, standards, etc.), consumables, and accompanying instructions required for a specific test. Users can quickly and accurately perform qualitative and quantitative analysis of target substances (such as biochemical substances, pathogens, genes, etc.) in samples through standardized operating procedures without having to prepare them themselves.

[0003] Traditional multi-residue detection kits for organophosphorus pesticides in agricultural products suffer from insufficient enzyme protection. Enzyme activity is easily and significantly reduced after long-term low-temperature storage and freeze-thaw cycles, resulting in short shelf lives, baseline shifts, and poor consistency between batches. Furthermore, they cannot effectively shield against endogenous interfering substances and trace heavy metals inherent in onion and garlic products, leading to a high probability of false positives. They also cannot inhibit substrate hydrolysis and deterioration, resulting in weak reagent storage stability and insufficient detection accuracy. Moreover, the colorimetric system lacks signal enrichment, amplification, and steady-state maintenance, resulting in low overall detection sensitivity. The colorimetric products are prone to oxidation and fading, and optical values ​​fluctuate significantly after standing. During storage, component aggregation, turbidity, and stratification can occur, and the colorimetric gradation becomes blurred, severely affecting the stability and reliability of the detection results. Therefore, this invention provides a multi-residue detection kit for organophosphorus pesticides in agricultural products and its preparation method. Summary of the Invention

[0004] The main objective of this invention is to provide a multi-residue detection kit for organophosphorus pesticides in agricultural products with high enzyme activity retention and low false positive rate in onion and garlic matrix, which is applied in a multi-residue detection kit for organophosphorus pesticides in agricultural products and its preparation method.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a multi-residue detection kit for organophosphorus pesticides in agricultural products, which consists of enzyme lyophilized powder, substrate solution, chromogenic reagent solution, sample extraction solution and colorimetric tubes.

[0006] The enzyme lyophilized powder consists of acetylcholinesterase stock solution and a protectant.

[0007] The substrate solution consists of mannitol, phosphate buffer, disodium EDTA, tris(hydroxymethyl)aminomethane, modified β-cyclodextrin, and thioacetylcholine iodide.

[0008] The colorimetric reagent solution is composed of sodium bicarbonate, phosphate buffer, modified tetradecyl sulfobetaine, hydroxypropyl-β-cyclodextrin, L-theanine and dithiobisnitrobenic acid.

[0009] The sample extract and colorimetric tubes are both existing technologies.

[0010] The sample extraction solution was a mixed powder buffer (Weifang Ruige Test Instrument Co., Ltd.).

[0011] The colorimetric tube is a thickened, shock-resistant colorimetric tube.

[0012] The phosphate buffer solution used in this invention has a pH of 7.45, a concentration of 0.05 mol / L, is used at a temperature of 4°C, and is sterile and biochemical grade.

[0013] All operations in this invention are performed under light-protected conditions.

[0014] The argon gas introduced in this invention is of high purity and is introduced by liquid surface purging, which only covers the surface of the liquid layer to form a protective atmosphere, thus preventing the gas from being directly introduced into the liquid and causing bubbling.

[0015] All solid raw materials are of biochemical reagent grade / analytical purity.

[0016] Secondly, the present invention provides a method for preparing a multi-residue detection kit for organophosphorus pesticides in agricultural products, the method comprising the following steps: S1. To prepare the enzyme lyophilized powder, add the acetylcholinesterase stock solution to the protectant and stir at 120 rpm for 8-12 minutes. Let it stand at 4℃ for 30 minutes, filter it using a 0.22 μm filter membrane to remove impurities, and obtain a mixture. Place the mixture into lyophilized vials and freeze-dry it in a freeze dryer. Set the temperature to -40℃ and freeze-dry for 3-5 hours. Set the vacuum degree to 10Pa-20Pa and the temperature to -30℃ and freeze-dry for 12 hours. Set the vacuum degree to 5Pa-10Pa and the temperature to 10℃ and freeze-dry for 6 hours to obtain the enzyme lyophilized powder.

[0017] S2. Prepare the substrate solution by adding mannitol to phosphate buffer and stirring, introducing argon gas, setting the temperature to 4℃ and the speed to 150 rpm, stirring for 2-4 minutes, adding disodium ethylenediaminetetraacetate and stirring for 1-3 minutes, adding tris(hydroxymethyl)aminomethane and stirring for 1-3 minutes, adding modified β-cyclodextrin and stirring, setting the speed to 120 rpm, stirring for 4-6 minutes, adding thioacetylcholine iodide and stirring for 7-9 minutes, filtering with a 0.22 μm filter membrane to remove particles, and obtaining the substrate solution.

[0018] S3. To prepare the colorimetric reagent solution, add sodium bicarbonate to phosphate buffer and stir. Purge with argon gas, set the temperature to 4℃, and the stirring speed to 120 rpm for 7-9 minutes. Add modified tetradecyl sulfobetaine and stir for 8-12 minutes. Add hydroxypropyl-β-cyclodextrin and L-theanine sequentially and stir for 8-12 minutes. Add dithiobisnitrobenzic acid and stir for 25-35 minutes. Filter using a 0.22 μm filter membrane to remove particles and obtain the colorimetric reagent solution.

[0019] S4. Prepare sample extract and colorimetric tubes.

[0020] Modified β-cyclodextrin, along with disodium EDTA, mannitol, and thioacetylcholine iodide, forms a highly efficient anti-interference and substrate-stabilizing synergistic system. Modified cyclodextrin encapsulates organic interfering substances and organosulfur compounds, disodium EDTA chelates heavy metal ions, and mannitol inhibits spontaneous substrate hydrolysis and stabilizes the system's osmotic pressure. The complementary and synergistic effects of these three components not only eliminate detection interference caused by the complex matrix of agricultural products but also provide long-term protection against substrate degradation and failure, ensuring that the substrate solution retains its performance after frozen storage, with low detection blank values ​​and a stable linear range.

[0021] The modified trehalose stock solution, together with bovine serum albumin, mannitol, and glycerol, forms a multi-component synergistic protective system. The modified trehalose stock solution primarily locks the internal conformation of the enzyme molecule, while bovine serum albumin forms a protective protein film on the surface. Mannitol and glycerol regulate the osmotic pressure of the system and buffer against low-temperature freeze-thaw damage. The complementary and synergistic effects of the multiple components further enhance the acetylcholinesterase's resistance to lyophilization, dilution, and temperature fluctuations, significantly reducing enzyme activity attenuation during dispensing, lyophilization, storage, and reconstitution. This results in a dual improvement in the enzyme activity stability, repeatability, and shelf life of the kit.

[0022] Modified tetradecyl sulfobetaine forms a synergistic system of sensitization and color stabilization with dithiobisnitrosobenzoic acid, L-theanine, and hydroxypropyl-β-cyclodextrin. Modified tetradecyl sulfobetaine enriches trace amounts of chromogenic products through micelles and amplifies the chromogenic absorbance signal. Hydroxypropyl-β-cyclodextrin assists in color enhancement and solubilization and prevents the chromogenic components from being separated. L-theanine stabilizes the balance of the chromogenic system and prevents the chromogenic products from dissociating and fading at low concentrations. Sodium bicarbonate maintains the optimal weakly alkaline chromogenic environment. The synergistic effect of multiple components achieves sensitive chromogenic reaction, clear color gradient, and stable interpretation even at low concentrations of pesticides. At the same time, it improves the storage stability of the chromogenic solution, preventing stratification, oxidation, and baseline drift.

[0023] Furthermore, the volume ratio of acetylcholinesterase stock solution to protectant in S1 is 1:4.

[0024] Further, the preparation of the protective agent includes the following steps: adding mannitol to phosphate buffer and stirring, introducing argon gas, setting the temperature to 4°C, stirring at 150 rpm for 3 minutes, adding bovine serum albumin and stirring at 120 rpm for 5 minutes, adding glycerol and stirring at 120 rpm for 2 minutes, adding modified trehalose stock solution and stirring at 120 rpm for 8 minutes, filtering with a 0.22 μm filter membrane to remove impurities, and obtaining the protective agent.

[0025] The acetylcholinesterase stock solution was obtained by extraction and chromatography purification from the electric organ of electric rays. It is of biochemical grade, with an enzyme activity ≥1000U / mL. The temperature should be controlled at 4℃ during use.

[0026] Furthermore, the mass-to-volume ratio of mannitol, phosphate buffer, bovine serum albumin, glycerol, and modified trehalose stock solution in the protective agent is 30g:780mL:5g:20mL:200mL.

[0027] Furthermore, the preparation of the modified trehalose stock solution includes the following steps: A1. Soak silicon dioxide in a weak alkaline solution of sodium bicarbonate for 30 minutes. Wash the soaked silicon dioxide three times with deionized water to obtain activated silicon dioxide.

[0028] A2. Add trehalose to phosphate buffer and stir at 4°C and 200 rpm for 15 minutes. Add 6-carboxyhexanoic acid and stir at 4°C and 150 rpm for 12 hours. Add Tween 80 and stir at 150 rpm for 3 minutes. Add activated silica and ultrasonically disperse at 40 kHz and 100 W for 5 minutes. Stop dispersion, start stirring at 4°C and 180 rpm for 20 minutes. Add β-hydroxyproline and let stand at 4°C for 30 minutes to obtain material A. Place material A in a dialysis bag with a molecular weight cutoff of 3500 Da for dialyzing. Control the ambient temperature at 4°C and change the dialysate every 6 hours for 36 hours. Vacuum concentrate the dialyzed material A at 40°C and -0.09 MPa for 4 hours to obtain modified trehalose stock solution.

[0029] The ratio of the volume of the dialysis fluid inside the dialysis bag to the volume of the dialysis fluid outside the dialysis tank is 1:20.

[0030] The concentration of the sodium bicarbonate weak alkaline solution is 0.05 mol / L, and the pH of the sodium bicarbonate weak alkaline solution is 8.4.

[0031] The dialysate was a sterile phosphate buffer solution at 4°C and pH 7.45 with a concentration of 0.05 mol / L.

[0032] The mass-to-volume ratio of activated silica, trehalose, phosphate buffer, 6-carboxyhexanoic acid, Tween 80, and β-hydroxyproline in A2 is 0.95 g: 60 g: 800 mL: 1.91 g: 1 mL: 0.63 g.

[0033] The silica has a particle size of 10 nm and is of high purity and hydrophilic type.

[0034] Furthermore, the mass-to-volume ratio of mannitol, phosphate buffer, disodium EDTA, tris(hydroxymethyl)aminomethane, modified β-cyclodextrin, and thioacetylcholine iodide in S2 is 5 g: 1000 mL: 0.5 g: 0.242 g: 5 g: 2.86 g.

[0035] Furthermore, the preparation of the modified β-cyclodextrin includes the following steps: B1. Mix β-cyclodextrin and phosphate buffer, introduce argon gas, set the temperature to 4℃, the speed to 150 rpm, stir for 10 minutes, add N-acetyl-L-cysteine ​​and stir for 2 hours to obtain material C.

[0036] B2. Mix β-cyclodextrin and phosphate buffer, introduce argon gas, set the temperature to 4℃, the speed to 120 rpm, stir for 12 minutes, add gallic acid and stir for 2 hours to obtain material D.

[0037] B3. Mix β-cyclodextrin and phosphate buffer at 200 rpm for 15 minutes. Add material C and stir. Purge with argon gas at 4°C and stir at 150 rpm for 8 hours. Add 2-hydroxyethyldimethylammonium chloride and stir at 180 rpm for 6 hours. Add material D and stir at 120 rpm for 4 hours to obtain material E. Dialyze material E in a 1000 Da dialysis bag with a diameter of 44 mm, ensuring the liquid volume is ≤1 / 4 and the external liquid is 20 times the internal liquid. Replace the external liquid every 6 hours and dialyze for 36 hours. Then, place the dialyzed material E into an ultrafiltration machine for ultrafiltration at 4°C, 0.25 MPa, and a membrane molecular weight cutoff of 1000 Da for 6 hours to obtain modified β-cyclodextrin.

[0038] The ratio of the volume of the dialysis fluid inside the dialysis bag to the volume of the dialysis fluid outside the dialysis tank is 1:20.

[0039] The dialysate was a sterile phosphate buffer solution at 4°C and pH 7.45 with a concentration of 0.05 mol / L.

[0040] The mass-to-volume ratio of β-cyclodextrin, phosphate buffer, and N-acetyl-L-cysteine ​​in B1 is 5g:50mL:4g.

[0041] The mass-to-volume ratio of β-cyclodextrin, phosphate buffer, and gallic acid in B2 is 15g:100mL:3g.

[0042] The mass-to-volume ratio of β-cyclodextrin, phosphate buffer, material C, 2-hydroxyethyldimethylammonium chloride, and material D in B3 is 70g:1000mL:50mL:3g:100mL.

[0043] Furthermore, the mass-to-volume ratio of sodium bicarbonate, phosphate buffer, modified tetradecyl sulfobetaine, hydroxypropyl-β-cyclodextrin, L-theanine, and dithiobisnitrobenzene in S3 is 1.5g:1000mL:0.8g:1.2g:0.6g:0.2g.

[0044] Furthermore, the preparation of the modified tetradecyl sulfobetaine includes the following steps: C1. Silica was ultrasonically dispersed in a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution at a frequency of 40 kHz and a power of 100 W for 10 minutes. Dopamine hydrochloride was added and stirred at a speed of 150 rpm for 24 hours to obtain material F. Material F was centrifuged at a speed of 8000 rpm for 10 minutes. The precipitate was collected and washed three times with deionized water. The washed precipitate was then vacuum dried at a temperature of 40℃ and a vacuum of -0.095 MPa for 16 hours. The dried precipitate was then ground through a 200-mesh sieve to obtain the silica-polydopamine complex.

[0045] C2. Tetradecyl sulfobetaine was added to phosphate buffer and stirred. Argon gas was introduced, and the temperature was set at 4℃ and the speed at 150 rpm for 5 minutes. N-acetyl-D-glucosamine was added and stirred for 10 minutes. Silica-polydopamine complex was added and ultrasonically dispersed at a frequency of 40 kHz and a power of 100 W for 3 minutes. 2-Aminobenzimidazole was added and stirred at a speed of 150 rpm for 15 minutes. The mixture was filtered through a 0.22 μm filter membrane to remove particles, yielding material G. Material G was then concentrated under vacuum at low temperature at 40℃ and a vacuum of -0.09 MPa for 6 hours to obtain modified tetradecyl sulfobetaine.

[0046] The tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution has a pH of 8.5, a concentration of 0.05 mol / L, and is of sterile analytical grade.

[0047] The silica has a particle size of 10 nm and is of high purity and hydrophilic type.

[0048] The mass-to-volume ratio of silica, tris(hydroxymethyl)aminomethane-hydrochloric acid buffer, and dopamine hydrochloride in C1 is 6.67 g: 500 mL: 3.33 g.

[0049] The mass-to-volume ratio of tetradecyl sulfobetaine, phosphate buffer, N-acetyl-D-glucosamine, silica-polydopamine complex, and 2-aminobenzimidazole in C2 is 7.42 g: 100 mL: 1.08 g: 0.576 g: 0.36 g.

[0050] The present invention has the following beneficial effects: 1. In this invention, a modified trehalose stock solution is added, wherein 6-carboxyhexanoic acid serves as a functional modifier, attaching a carboxyl polar site to the basic trehalose to enhance the hydrogen bonding ability between trehalose and acetylcholinesterase, thus strengthening the protective film structure on the enzyme protein surface; 10-nanometer hydrophilic silica serves as an inorganic framework modifier, providing rigid support for trehalose molecules, improving overall structural stability, preventing molecular stacking and aggregation, and optimizing the water distribution environment; β-hydroxyproline serves as a biosynergistic modifier, improving the biocompatibility and osmotic pressure regulation ability of the material, helping to lock the enzyme's natural conformation, buffering the activity damage caused by low temperature and long-term storage; the modified trehalose stock solution has excellent enzyme protein conformation protection ability, outstanding resistance to freeze-drying and low-temperature freeze-thaw, and can stably protect the biological activity of the detection enzyme for a long time, significantly delaying the denaturation and activity decay of the enzyme protein during storage, effectively extending the shelf life of the kit, stabilizing the detection blank baseline, reducing batch-to-batch detection data deviation, and improving the consistency and reliability of the overall detection results.

[0051] 2. In this invention, modified β-cyclodextrin is added. N-acetyl-L-cysteine ​​serves as a functional modifier, introducing a thiol functional group into β-cyclodextrin, expanding the hydrophobic cavity space, and significantly enhancing the inclusion and capture ability of sulfur-containing endogenous interfering substances in onions and garlic. Gallic acid serves as a polyphenol chelating modifier, endowing cyclodextrin with the function of complexing polyphenol impurities and chelating trace heavy metal ions, eliminating the interference of heavy metals and endogenous plant impurities on detection. 2-hydroxyethyldimethylammonium chloride serves as a cationic modifier, introducing a positive charge onto the surface of cyclodextrin, enhancing the electrostatic adsorption of polar interfering substances in the system. Modified β-cyclodextrin possesses stronger molecular inclusion, electrostatic adsorption, and heavy metal chelation capabilities, which can effectively shield endogenous interfering impurities in onion and garlic agricultural products, significantly reducing the probability of false positives. At the same time, it can protect the structure of the detection substrate, inhibit the self-hydrolysis and deterioration of the substrate, improve the long-term storage stability of the reagent, and effectively resist external interference such as heavy metals, fundamentally improving the detection accuracy and anti-interference ability of the kit.

[0052] 3. In this invention, modified tetradecyl sulfobetaine is added, wherein N-acetyl-D-glucosamine acts as a hydrogen-bonding modifier, attaching polyhydroxy groups to tetradecyl sulfobetaine, thereby strengthening the stability of the micelle network structure and improving its solubilization, enrichment, and binding locking ability for the chromogenic product; the nano-silica-polydopamine complex acts as a dispersing and coating modifier, which can be uniformly dispersed in the system, inhibiting surfactant molecule aggregation, precipitation, and stratification, and maintaining the long-term uniformity and transparency of the chromogenic system; 2-aminobenzimidazole acts as a charge and acid-base regulating modifier. It balances the surface potential of the system, buffers slight pH fluctuations, stabilizes the equilibrium of the colorimetric chemical reaction, and inhibits the oxidative degradation and spontaneous fading of the colorimetric products. Modified tetradecyl sulfobetaine has a good colorimetric signal enrichment and amplification effect, which can improve the sensitivity of the kit for pesticide detection. It can stabilize the colorimetric reaction system, inhibit the oxidative fading of the colorimetric products, significantly reduce the drift of the photometric values ​​after standing, and at the same time avoid the aggregation and precipitation of system components, so that the reagents can remain clear and uniform for a long time. The colorimetric scale is clear and easy to distinguish, effectively improving the repeatability of detection and the accuracy of result interpretation. Detailed Implementation

[0053] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0054] It should be noted that all raw materials used in the following experiments are commercially available.

[0055] The preparation methods of the modified trehalose stock solution, modified β-cyclodextrin, and modified tetradecyl sulfobetaine used in the following embodiments and comparative examples of the present invention are described below: I. Preparation of Modified Trehalose Stock Solution A1. Soak silicon dioxide in a weak alkaline solution of sodium bicarbonate for 30 minutes. Wash the soaked silicon dioxide three times with deionized water to obtain activated silicon dioxide.

[0056] A2. Add 60g of trehalose to 800mL of phosphate buffer and stir at 4℃ and 200rpm for 15 minutes. Add 1.91g of 6-carboxyhexanoic acid and stir at 4℃ and 150rpm for 12 hours. Add 1mL of Tween 80 and stir at 150rpm for 3 minutes. Add 0.95g of activated silica and ultrasonically disperse at 40kHz and 100W for 5 minutes. Stop dispersion, start stirring at 4℃ and 180rpm for 20 minutes. Add 0.63g of β-hydroxyproline and let stand at 4℃ for 30 minutes to obtain material A. Place material A in a dialysis bag with a molecular weight cutoff of 3500Da for dialyzing. Control the ambient temperature at 4℃ and change the dialysate every 6 hours for 36 hours. Vacuum concentrate the dialyzed material A at 40℃ and -0.09MPa for 4 hours to obtain modified trehalose stock solution.

[0057] II. Preparation of Modified β-Cyclodextrin B1. Mix 5g of β-cyclodextrin and 50mL of phosphate buffer, introduce argon gas, set the temperature to 4℃, and the speed to 150rpm, stir for 10 minutes, add 4g of N-acetyl-L-cysteine ​​and stir for 2 hours to obtain material C.

[0058] B2. Mix 15g of β-cyclodextrin and 100mL of phosphate buffer, introduce argon gas, set the temperature to 4℃, and the speed to 120rpm. Stir for 12 minutes, add 3g of gallic acid and stir for 2 hours to obtain material D.

[0059] B3. Mix 70g of β-cyclodextrin and 1000mL of phosphate buffer at 200rpm for 15 minutes. Add 50mL of material C and stir. Purge with argon gas at 4℃ and stir at 150rpm for 8 hours. Add 3g of 2-hydroxyethyldimethylammonium chloride and stir at 180rpm for 6 hours. Add 100mL of material D and stir at 120rpm for 4 hours to obtain material E. Dialyze material E in a 1000Da dialysis bag with a diameter of 44mm, ensuring the liquid volume is ≤1 / 4 and the external liquid is 20 times the internal liquid. Replace the external liquid every 6 hours and dialyze for 36 hours. Then, ultrafilter material E in an ultrafiltration machine at 4℃ and 0.25MPa, with a membrane molecular weight cutoff of 1000Da for 6 hours to obtain modified β-cyclodextrin.

[0060] III. Preparation of Modified Tetradecyl Sulfobetaine C1. 6.67g of silica was ultrasonically dispersed in 500mL of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer at a frequency of 40kHz and a power of 100W for 10 minutes. Then, 3.33g of dopamine hydrochloride was added and stirred at a speed of 150rpm for 24 hours to obtain material F. Material F was centrifuged at a speed of 8000rpm for 10 minutes. The precipitate was collected and washed three times with deionized water. The washed precipitate was then vacuum dried at a temperature of 40℃ and a vacuum of -0.095MPa for 16 hours. The dried precipitate was then ground through a 200-mesh sieve to obtain the silica-polydopamine complex.

[0061] C2. Add 7.42 g of tetradecyl sulfobetaine to 100 mL of phosphate buffer and stir. Purge with argon gas, set the temperature to 4℃, and the stirring speed to 150 rpm for 5 minutes. Add 1.08 g of N-acetyl-D-glucosamine and stir for 10 minutes. Add 0.576 g of silica-polydopamine complex and disperse ultrasonically at a frequency of 40 kHz and a power of 100 W for 3 minutes. Add 0.36 g of 2-aminobenzimidazole and stir at a speed of 150 rpm for 15 minutes. Filter using a 0.22 μm filter membrane to remove particles and obtain material G. Concentrate material G under vacuum at low temperature at a temperature of 40℃ and a vacuum degree of -0.09 MPa for 6 hours to obtain modified tetradecyl sulfobetaine.

[0062] Example 1: A multi-residue detection kit for organophosphorus pesticides in agricultural products. The kit consists of enzyme lyophilized powder, substrate solution, chromogenic reagent solution, sample extraction solution, and colorimetric tubes.

[0063] Enzyme lyophilized powder consists of acetylcholinesterase stock solution and a protectant.

[0064] The substrate solution consisted of mannitol, phosphate buffer, disodium EDTA, tris(hydroxymethyl)aminomethane, modified β-cyclodextrin, and thioacetylcholine iodide.

[0065] The colorimetric reagent solution consists of sodium bicarbonate, phosphate buffer, modified tetradecyl sulfobetaine, hydroxypropyl-β-cyclodextrin, L-theanine, and dithiobisnitrobenic acid.

[0066] A method for preparing a multi-residue detection kit for organophosphorus pesticides in agricultural products, comprising the following steps: S1. To prepare the enzyme lyophilized powder, add the acetylcholinesterase stock solution to the protectant and stir at 120 rpm for 8 minutes. Let it stand at 4°C for 30 minutes, filter it using a 0.22 μm filter membrane to remove impurities, and obtain a mixture. Place the mixture into a lyophilized vial and freeze-dry it in a freeze dryer. Set the temperature to -40°C and freeze-dry for 3 hours. Set the vacuum degree to 10 PaPa and freeze-dry at -30°C for 12 hours. Set the vacuum degree to 5 PaPa and freeze-dry at 10°C for 6 hours to obtain the enzyme lyophilized powder.

[0067] S2. To prepare the substrate solution, add mannitol to phosphate buffer and stir. Purge with argon gas, set the temperature to 4°C, and stir at 150 rpm for 2 minutes. Add disodium ethylenediaminetetraacetate and stir for 1 minute. Add tris(hydroxymethyl)aminomethane and stir for 1 minute. Add modified β-cyclodextrin and stir at 120 rpm for 4 minutes. Add thioacetylcholine iodide and stir for 7 minutes. Filter using a 0.22 μm filter membrane to remove particles and obtain the substrate solution.

[0068] S3. To prepare the colorimetric reagent solution, add sodium bicarbonate to phosphate buffer and stir. Purge with argon gas, set the temperature to 4℃, and the stirring speed to 120 rpm for 7 minutes. Add modified tetradecyl sulfobetaine and stir for 8 minutes. Add hydroxypropyl-β-cyclodextrin and L-theanine sequentially and stir for 8 minutes. Add dithiobisnitrobenzic acid and stir for 25 minutes. Filter using a 0.22 μm filter membrane to remove particles and obtain the colorimetric reagent solution.

[0069] S4. Prepare sample extract and colorimetric tubes.

[0070] The volume ratio of acetylcholinesterase stock solution to protectant in S1 is 1:4.

[0071] The preparation of the protective agent includes the following steps: mannitol is added to phosphate buffer and stirred, argon gas is introduced, the temperature is set to 4℃, the speed is 150 rpm, and the stirring time is 3 minutes; bovine serum albumin is added and stirred, the speed is set to 120 rpm, and the stirring time is 5 minutes; glycerol is added and stirred, the speed is set to 120 rpm, and the stirring time is 2 minutes; modified trehalose stock solution is added and stirred, the speed is set to 120 rpm, and the stirring time is 8 minutes; the mixture is filtered through a 0.22 μm filter membrane to remove impurities, and the protective agent is obtained.

[0072] The mass-to-volume ratio of mannitol, phosphate buffer, bovine serum albumin, glycerol, and modified trehalose stock solution in the protective agent is 30 g: 780 mL: 5 g: 20 mL: 200 mL.

[0073] The mass-to-volume ratio of mannitol, phosphate buffer, disodium EDTA, tris(hydroxymethyl)aminomethane, modified β-cyclodextrin, and thioacetylcholine iodide in S2 is 5 g: 1000 mL: 0.5 g: 0.242 g: 5 g: 2.86 g.

[0074] The mass-to-volume ratio of sodium bicarbonate, phosphate buffer, modified tetradecyl sulfobetaine, hydroxypropyl-β-cyclodextrin, L-theanine, and dithiobisnitrobenzic acid in S3 is 1.5 g: 1000 mL: 0.8 g: 1.2 g: 0.6 g: 0.2 g.

[0075] Example 2: A multi-residue detection kit for organophosphorus pesticides in agricultural products. The kit consists of enzyme lyophilized powder, substrate solution, chromogenic reagent solution, sample extraction solution, and colorimetric tubes.

[0076] Enzyme lyophilized powder consists of acetylcholinesterase stock solution and a protectant.

[0077] The substrate solution consisted of mannitol, phosphate buffer, disodium EDTA, tris(hydroxymethyl)aminomethane, modified β-cyclodextrin, and thioacetylcholine iodide.

[0078] The colorimetric reagent solution consists of sodium bicarbonate, phosphate buffer, modified tetradecyl sulfobetaine, hydroxypropyl-β-cyclodextrin, L-theanine, and dithiobisnitrobenic acid.

[0079] A method for preparing a multi-residue detection kit for organophosphorus pesticides in agricultural products, comprising the following steps: S1. To prepare the enzyme lyophilized powder, add the acetylcholinesterase stock solution to the protectant and stir at 120 rpm for 10 minutes. Let it stand at 4°C for 30 minutes, filter it through a 0.22 μm filter membrane to remove impurities, and obtain a mixture. Place the mixture into a lyophilized vial and freeze-dry it in a freeze dryer. Set the temperature to -40°C and freeze-dry for 4 hours. Set the vacuum degree to 15 Pa and freeze-dry at -30°C for 12 hours. Set the vacuum degree to 7.5 Pa and freeze-dry at 10°C for 6 hours to obtain the enzyme lyophilized powder.

[0080] S2. To prepare the substrate solution, add mannitol to phosphate buffer and stir. Purge with argon gas, set the temperature to 4°C, and stir at 150 rpm for 3 minutes. Add disodium ethylenediaminetetraacetate and stir for 2 minutes. Add tris(hydroxymethyl)aminomethane and stir for 2 minutes. Add modified β-cyclodextrin and stir at 120 rpm for 5 minutes. Add thioacetylcholine iodide and stir for 8 minutes. Filter using a 0.22 μm filter membrane to remove particles and obtain the substrate solution.

[0081] S3. To prepare the colorimetric reagent solution, add sodium bicarbonate to phosphate buffer and stir. Purge with argon gas, set the temperature to 4℃, and the stirring speed to 120 rpm for 8 minutes. Add modified tetradecyl sulfobetaine and stir for 10 minutes. Then add hydroxypropyl-β-cyclodextrin and L-theanine sequentially and stir for 10 minutes. Finally, add dithiodinitrobenzene and stir for 30 minutes. Filter using a 0.22 μm filter membrane to remove particles and obtain the colorimetric reagent solution.

[0082] S4. Prepare sample extract and colorimetric tubes.

[0083] The volume ratio of acetylcholinesterase stock solution to protectant in S1 is 1:4.

[0084] The preparation of the protective agent includes the following steps: mannitol is added to phosphate buffer and stirred, argon gas is introduced, the temperature is set to 4℃, the speed is 150 rpm, and the stirring time is 3 minutes; bovine serum albumin is added and stirred, the speed is set to 120 rpm, and the stirring time is 5 minutes; glycerol is added and stirred, the speed is set to 120 rpm, and the stirring time is 2 minutes; modified trehalose stock solution is added and stirred, the speed is set to 120 rpm, and the stirring time is 8 minutes; the mixture is filtered through a 0.22 μm filter membrane to remove impurities, and the protective agent is obtained.

[0085] The mass-to-volume ratio of mannitol, phosphate buffer, bovine serum albumin, glycerol, and modified trehalose stock solution in the protective agent is 30 g: 780 mL: 5 g: 20 mL: 200 mL.

[0086] The mass-to-volume ratio of mannitol, phosphate buffer, disodium EDTA, tris(hydroxymethyl)aminomethane, modified β-cyclodextrin, and thioacetylcholine iodide in S2 is 5 g: 1000 mL: 0.5 g: 0.242 g: 5 g: 2.86 g.

[0087] The mass-to-volume ratio of sodium bicarbonate, phosphate buffer, modified tetradecyl sulfobetaine, hydroxypropyl-β-cyclodextrin, L-theanine, and dithiobisnitrobenzic acid in S3 is 1.5 g: 1000 mL: 0.8 g: 1.2 g: 0.6 g: 0.2 g.

[0088] Example 3: A multi-residue detection kit for organophosphorus pesticides in agricultural products. The kit consists of enzyme lyophilized powder, substrate solution, chromogenic reagent solution, sample extraction solution, and colorimetric tubes.

[0089] Enzyme lyophilized powder consists of acetylcholinesterase stock solution and a protectant.

[0090] The substrate solution consisted of mannitol, phosphate buffer, disodium EDTA, tris(hydroxymethyl)aminomethane, modified β-cyclodextrin, and thioacetylcholine iodide.

[0091] The colorimetric reagent solution consists of sodium bicarbonate, phosphate buffer, modified tetradecyl sulfobetaine, hydroxypropyl-β-cyclodextrin, L-theanine, and dithiobisnitrobenic acid.

[0092] A method for preparing a multi-residue detection kit for organophosphorus pesticides in agricultural products, comprising the following steps: S1. To prepare the enzyme lyophilized powder, add the acetylcholinesterase stock solution to the protectant and stir at 120 rpm for 12 minutes. Let it stand at 4°C for 30 minutes, filter it through a 0.22 μm filter membrane to remove impurities, and obtain a mixture. Place the mixture into a lyophilized vial and freeze-dry it in a freeze dryer. Set the temperature to -40°C and freeze-dry for 5 hours. Set the vacuum degree to 20 Pa and the temperature to -30°C and freeze-dry for 12 hours. Set the vacuum degree to 10 Pa and the temperature to 10°C and freeze-dry for 6 hours to obtain the enzyme lyophilized powder.

[0093] S2. To prepare the substrate solution, add mannitol to phosphate buffer and stir. Purge with argon gas, set the temperature to 4°C, and stir at 150 rpm for 4 minutes. Add disodium ethylenediaminetetraacetate and stir for 3 minutes. Add tris(hydroxymethyl)aminomethane and stir for 3 minutes. Add modified β-cyclodextrin and stir at 120 rpm for 6 minutes. Add thioacetylcholine iodide and stir for 9 minutes. Filter using a 0.22 μm filter membrane to remove particles and obtain the substrate solution.

[0094] S3. To prepare the colorimetric reagent solution, add sodium bicarbonate to phosphate buffer and stir. Purge with argon gas, set the temperature to 4℃, and the stirring speed to 120 rpm for 9 minutes. Add modified tetradecyl sulfobetaine and stir for 12 minutes. Add hydroxypropyl-β-cyclodextrin and L-theanine sequentially and stir for 12 minutes. Add dithiobisnitrobenzic acid and stir for 35 minutes. Filter using a 0.22 μm filter membrane to remove particles and obtain the colorimetric reagent solution.

[0095] S4. Prepare sample extract and colorimetric tubes The volume ratio of acetylcholinesterase stock solution to protectant in S1 is 1:4.

[0096] The preparation of the protective agent includes the following steps: mannitol is added to phosphate buffer and stirred, argon gas is introduced, the temperature is set to 4℃, the speed is 150 rpm, and the stirring time is 3 minutes; bovine serum albumin is added and stirred, the speed is set to 120 rpm, and the stirring time is 5 minutes; glycerol is added and stirred, the speed is set to 120 rpm, and the stirring time is 2 minutes; modified trehalose stock solution is added and stirred, the speed is set to 120 rpm, and the stirring time is 8 minutes; the mixture is filtered through a 0.22 μm filter membrane to remove impurities, and the protective agent is obtained.

[0097] The mass-to-volume ratio of mannitol, phosphate buffer, bovine serum albumin, glycerol, and modified trehalose stock solution in the protective agent is 30 g: 780 mL: 5 g: 20 mL: 200 mL.

[0098] The mass-to-volume ratio of mannitol, phosphate buffer, disodium EDTA, tris(hydroxymethyl)aminomethane, modified β-cyclodextrin, and thioacetylcholine iodide in S2 is 5 g: 1000 mL: 0.5 g: 0.242 g: 5 g: 2.86 g.

[0099] The mass-to-volume ratio of sodium bicarbonate, phosphate buffer, modified tetradecyl sulfobetaine, hydroxypropyl-β-cyclodextrin, L-theanine, and dithiobisnitrobenzic acid in S3 is 1.5 g: 1000 mL: 0.8 g: 1.2 g: 0.6 g: 0.2 g.

[0100] Comparative Example 1: The difference between this comparative example and Example 1 is that: In this comparative example, the modified trehalose stock solution in the protective agent was replaced with unmodified trehalose.

[0101] Comparative Example 2: The difference between this comparative example and Example 1 is that: Unmodified β-cyclodextrin was used in this comparative example.

[0102] Comparative Example 3 differs from Example 1 in that: Unmodified tetradecyl sulfobetaine was used in this comparative example.

[0103] Performance testing: The multi-residue detection kit for organophosphorus pesticides in agricultural products prepared in Examples 1, 2, 3, Comparative Examples 1, 2, and 3 were tested.

[0104] Performance testing: The relevant performance of the multi-residue detection kit for organophosphorus pesticides in agricultural products and its preparation method provided in Examples 1-3 and Comparative Examples 1-3 were tested respectively. The test data are recorded in Table 1 below: Table 1 - Performance Testing of the Detection Kit for Multiple Residues of Organophosphorus Pesticides in Agricultural Products

[0105] Based on the above data, the following conclusions can be drawn: Among them, the 6-month enzyme activity retention rate of the multi-residue detection kits for organophosphorus pesticides in agricultural products prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3 was tested using the test method in NY / T4716-20256.3.2.

[0106] The false positive rate of the onion and garlic matrix of the detection kits for multiple residues of organophosphorus pesticides in agricultural products prepared in Examples 1, 2, 3, Comparative Examples 1, 2, and 3 was tested using the test method in NY / T4716-20256.4.2.

[0107] The absorbance drift rate of the multi-residue detection kits for organophosphorus pesticides in agricultural products prepared in Examples 1, 2, 3, Comparative Examples 1, 2, and 3 was tested using the test method in NY / T4716-20256.3.4 after 30 minutes of color development.

[0108] The modified trehalose stock solution, through trehalose bulk, 6-carboxyhexanoate hydrogen bond modification, nano-silica framework support, and β-hydroxyproline biosynergistic modification, can form a dense hydrogen bond protective film on the surface of acetylcholinesterase protein, filling the water molecule vacancies on the protein surface, firmly locking the enzyme molecule's natural spatial conformation, and inhibiting enzyme protein folding, aggregation, denaturation, and inactivation during long-term storage at 4°C, low-temperature freeze-thaw, and lyophilization. At the same time, it can precisely regulate the system's osmotic pressure, buffering the natural decline in enzyme activity caused by temperature changes and long-term storage, reducing enzyme molecule conformational dissociation and active site failure, and continuously maintaining the stability of enzyme catalytic function, thereby significantly improving the enzyme's long-term storage capacity. Modified β-cyclodextrin, through ternary functional modification with N-acetyl-L-cysteine, gallic acid, and 2-hydroxyethyldimethylammonium chloride, not only expands the inclusion volume of the hydrophobic cavity of β-cyclodextrin but also endows it with multiple functions such as molecular electrostatic adsorption and polyphenol chelation. Allium vegetables release sulfur-containing volatile substances, endogenous alkaloids, and polyphenolic impurities. These substances non-specifically inhibit acetylcholinesterase, causing enzyme inhibition even in the absence of organophosphorus pesticides, leading to false positives. Ternary modified β-cyclodextrin can efficiently encapsulate sulfur-containing interfering substances in allium vegetables, electrostatically adsorb charged endogenous impurities, and chelate polyphenolic interfering components, thus isolating and capturing matrix interfering substances in advance, preventing them from contacting and inhibiting enzyme activity, eliminating non-specific inhibition caused by the allium matrix from the root, and significantly reducing the false positive rate of allium matrix in the kit. Modified tetradecyl sulfobetaine, through N-acetyl-D-glucosamine hydrogen bond modification, nano-silica-polydopamine composite dispersion, and 2-aminobenzimidazole charge-regulated ternary modification, can form a highly stable micellar microenvironment in the colorimetric system. This microenvironment can both micellize and solubilize the colored products generated by the dithiobisnitrobenzic acid colorimetric reaction, preventing spontaneous degradation, sedimentation, and oxidative fading of small color molecules, and maintain the uniformity and stability of the entire colorimetric system through nano-dispersion, preventing component aggregation and stratification. At the same time, it buffers the disruption of colorimetric equilibrium by slight pH fluctuations, locks in the steady state at the endpoint of the colorimetric reaction, and ensures that the absorbance changes little within 30 minutes after color development, effectively controlling absorbance drift and ensuring a stable colorimetric baseline and stable color gradation.

[0109] A higher enzyme activity retention rate at 6 months is better, indicating less activity loss and greater conformational stability of acetylcholinesterase during long-term storage, thus enhancing the kit's storage performance and detection reliability. A lower false positive rate for onion and garlic matrices is better, representing better shielding and inhibition of endogenous interfering substances in onions and garlic, lower probability of non-specific misjudgments, and higher accuracy of detection results. A lower absorbance drift rate at 30 minutes of color development is better, indicating more stable chromogenic products, less susceptibility to oxidation, fading, and system stratification fluctuations, a more stable colorimetric baseline, and more accurate and repeatable interpretation of subsequent results.

[0110] Based on the above demonstrations, the present invention is significantly superior to the control group in terms of enzyme activity retention rate at 6 months, false positive rate of onion and garlic matrix, and absorbance drift rate at 30 minutes of color development.

[0111] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0112] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A multi-residue detection kit for organophosphorus pesticides in agricultural products, characterized in that, The detection kit for multiple residues of organophosphorus pesticides in agricultural products consists of enzyme lyophilized powder, substrate solution, chromogenic solution, sample extraction solution and colorimetric tubes. The lyophilized enzyme powder consists of acetylcholinesterase stock solution and a protectant. The substrate solution consists of mannitol, phosphate buffer, disodium EDTA, tris(hydroxymethyl)aminomethane, modified β-cyclodextrin, and thioacetylcholine iodide. The colorimetric reagent solution is composed of sodium bicarbonate, phosphate buffer, modified tetradecyl sulfobetaine, hydroxypropyl-β-cyclodextrin, L-theanine, and dithiobisnitrobenic acid.

2. A method for preparing a multi-residue detection kit for organophosphorus pesticides in agricultural products according to claim 1, characterized in that, The preparation method of the above-mentioned detection kit for multiple residues of organophosphorus pesticides in agricultural products includes the following steps: S1. To prepare the enzyme lyophilized powder, add the acetylcholinesterase stock solution to the protectant, stir, let stand, filter with a filter membrane to obtain a mixture, put the mixture into a lyophilized vial and freeze dry in a lyophilizer to obtain the enzyme lyophilized powder. S2. To prepare the substrate solution, add mannitol to phosphate buffer and stir, add disodium ethylenediaminetetraacetate and stir, add tris(hydroxymethyl)aminomethane and stir, add modified β-cyclodextrin and stir, add thioacetylcholine iodide and stir, and filter using a filter membrane to obtain the substrate solution. S3. To prepare the colorimetric reagent solution, add sodium bicarbonate to phosphate buffer and stir, add modified tetradecyl sulfobetaine and stir, then add hydroxypropyl-β-cyclodextrin and L-theanine in sequence and stir, then add dithiodinitrobenzic acid and stir, and filter using a filter membrane to obtain the colorimetric reagent solution. S4. Prepare sample extract and colorimetric tubes.

3. The preparation method of the multi-residue detection kit for organophosphorus pesticides in agricultural products according to claim 2, characterized in that, The volume ratio of acetylcholinesterase stock solution to protective agent in S1 is 1:

4.

4. The preparation method of the multi-residue detection kit for organophosphorus pesticides in agricultural products according to claim 2, characterized in that, The preparation of the protective agent includes the following steps: Mannitol was added to phosphate buffer and stirred, bovine serum albumin was added and stirred, glycerol was added and stirred, modified trehalose stock solution was added and stirred, and the mixture was filtered through a filter membrane to obtain the protective agent.

5. The preparation method of the multi-residue detection kit for organophosphorus pesticides in agricultural products according to claim 4, characterized in that, The mass-to-volume ratio of mannitol, phosphate buffer, bovine serum albumin, glycerol, and modified trehalose stock solution in the protective agent is 30g:780mL:5g:20mL:200mL.

6. The preparation method of the multi-residue detection kit for organophosphorus pesticides in agricultural products according to claim 4, characterized in that, The preparation of the modified trehalose stock solution includes the following steps: A1. Soak silicon dioxide in a weak alkaline solution of sodium bicarbonate, and wash the soaked silicon dioxide with deionized water to obtain activated silicon dioxide; A2. Add trehalose to phosphate buffer and stir, add 6-carboxyhexanoic acid and stir, add Tween 80 and stir, add activated silica and disperse by ultrasonication, stop dispersion, turn on stirring, add β-hydroxyproline and let stand to obtain material A, put material A into a dialysis bag for dialyzing, and concentrate the dialyzed material A under vacuum to obtain modified trehalose stock solution. The mass-to-volume ratio of activated silica, trehalose, phosphate buffer, 6-carboxyhexanoic acid, Tween 80, and β-hydroxyproline in A2 is 0.95 g: 60 g: 800 mL: 1.91 g: 1 mL: 0.63 g.

7. The preparation method of the multi-residue detection kit for organophosphorus pesticides in agricultural products according to claim 2, characterized in that, The mass-to-volume ratio of mannitol, phosphate buffer, disodium EDTA, tris(hydroxymethyl)aminomethane, modified β-cyclodextrin, and thioacetylcholine iodide in S2 is 5 g: 1000 mL: 0.5 g: 0.242 g: 5 g: 2.86 g.

8. The preparation method of the multi-residue detection kit for organophosphorus pesticides in agricultural products according to claim 7, characterized in that, The preparation of the modified β-cyclodextrin includes the following steps: B1. Mix β-cyclodextrin and phosphate buffer, add N-acetyl-L-cysteine ​​and stir to obtain material C; B2. Mix β-cyclodextrin and phosphate buffer, stir, add gallic acid and stir to obtain material D; B3. Mix β-cyclodextrin and phosphate buffer, add material C and stir, add 2-hydroxyethyldimethylammonium chloride and stir, add material D and stir to obtain material E, put material E into a dialysis bag for dialysis, and put the dialyzed material E into an ultrafiltration machine for ultrafiltration to obtain modified β-cyclodextrin. The mass-to-volume ratio of β-cyclodextrin, phosphate buffer, and N-acetyl-L-cysteine ​​in B1 is 5g:50mL:4g. The mass-to-volume ratio of β-cyclodextrin, phosphate buffer, and gallic acid in B2 is 15g:100mL:3g; The mass-to-volume ratio of β-cyclodextrin, phosphate buffer, material C, 2-hydroxyethyldimethylammonium chloride, and material D in B3 is 70g:1000mL:50mL:3g:100mL.

9. The preparation method of the multi-residue detection kit for organophosphorus pesticides in agricultural products according to claim 2, characterized in that, The mass-to-volume ratio of sodium bicarbonate, phosphate buffer, modified tetradecyl sulfobetaine, hydroxypropyl-β-cyclodextrin, L-theanine, and dithiobisnitrobenzene in S3 is 1.5 g: 1000 mL: 0.8 g: 1.2 g: 0.6 g: 0.2 g.

10. The preparation method of the multi-residue detection kit for organophosphorus pesticides in agricultural products according to claim 9, characterized in that, The preparation of the modified tetradecyl sulfobetaine includes the following steps: C1. Disperse silica in tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution by ultrasonication, add dopamine hydrochloride and stir to obtain material F. Centrifuge material F in a centrifuge, collect the precipitate, wash the precipitate with deionized water, vacuum dry the washed precipitate, grind the dried precipitate to obtain silica-polydopamine complex. C2. Tetradecyl sulfobetaine was added to phosphate buffer and stirred. N-acetyl-D-glucosamine was added and stirred. The silica-polydopamine complex was added and ultrasonically dispersed. 2-Aminobenzimidazole was added and stirred. The mixture was filtered through a filter membrane to obtain material G. Material G was concentrated under vacuum at low temperature to obtain modified tetradecyl sulfobetaine. The mass-to-volume ratio of silica, tris(hydroxymethyl)aminomethane-hydrochloric acid buffer, and dopamine hydrochloride in C1 is 6.67 g: 500 mL: 3.33 g; The mass-to-volume ratio of tetradecyl sulfobetaine, phosphate buffer, N-acetyl-D-glucosamine, silica-polydopamine complex, and 2-aminobenzimidazole in C2 is 7.42 g: 100 mL: 1.08 g: 0.576 g: 0.36 g.

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