Rapid detection method for diazepam residue in aquatic products based on immunomagnetic materials

Abstract

This invention provides a rapid detection method for diazepam residues in aquatic products based on immunomagnetic materials. The method utilizes immunomagnetic materials to achieve high-level enrichment and rapid separation of diazepam, thereby improving the sensitivity of the detection method. Combined with colloidal gold detection, it can be used for rapid on-site detection of diazepam residues in aquatic products. This method uses immunomagnetic beads for selective adsorption and separation of diazepam residues in aquatic products. It is simple to operate, highly sensitive, with an average recovery rate of 75.9%–96.0%, a relative standard deviation of 2.96%–8.99%, and a detection limit of 1.0 μg / kg for diazepam. Methodological validation shows that this method has high specificity and good stability, and can be used for rapid on-site detection of diazepam residues in aquatic products, providing technical support for food safety supervision.

Description

Technical Field

[0001] This invention belongs to the field of drug residue detection technology in aquatic products, and relates to a rapid detection method for diazepam residues in aquatic products based on immunomagnetic materials. Background Technology

[0002] Diazepam (also known as Valium) is a commonly used growth promoter in animal husbandry, possessing sedative, hypnotic, and anticonvulsant pharmacological effects. In the aquaculture, fishing, and transportation processes, some unscrupulous vendors illegally and excessively use diazepam to reduce the risk of fish injuring each other, leading to excessive diazepam residues in aquatic products. Studies have shown that long-term ingestion of animal-derived foods containing diazepam residues may cause serious adverse reactions such as memory impairment, confusion, and even damage to white blood cells and motor nerves, posing a significant potential threat to human health. In 2002, my country's Ministry of Agriculture issued the "Maximum Residue Limits for Veterinary Drugs in Animal-Derived Foods" (Announcement No. 235), which clearly stipulates that diazepam can only be used for therapeutic purposes and must not be detected in animal-derived foods.

[0003] Currently, methods for detecting diazepam residues mainly include high-performance liquid chromatography (HPLC), gas chromatography-tandem mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA). While these methods offer good sensitivity, accuracy, and reliability, the complex matrix of aquatic product samples and the trace amounts of the target analyte make direct detection difficult. Therefore, there is an urgent need to develop an efficient, simple method for detecting diazepam residues in aquatic products that can meet the requirements for rapid, large-scale on-site testing.

[0004] Immunomagnetic materials, by coupling antibodies to the surface of magnetic particles, enable specific immunobinding of target analytes, and under the influence of an external magnetic field, achieve the enrichment and separation of the target analytes. This technology has advantages such as simple operation and specific binding to target analytes. Currently, immunomagnetic materials are widely used in the sorting of cells, bacteria, and viruses in the medical field, as well as the separation of pathogenic microorganisms in food. However, research in the field of rapid detection of drug residues in food is still in its early stages. Colloidal gold immunochromatography uses colloidal gold as a tracer marker in antigen-antibody reactions. It is simple, rapid, provides intuitive results, and requires no complex operation or special equipment, making it an effective means of rapid detection of food safety. A rapid detection method for colloidal gold based on immunomagnetic materials may be helpful for the large-scale rapid detection of diazepam residues in aquatic products on-site. Summary of the Invention

[0005] This invention addresses the aforementioned shortcomings by establishing a rapid colloidal gold detection method for diazepam residues in aquatic products based on immunomagnetic materials. The method utilizes immunomagnetic materials to achieve high-level enrichment and rapid separation of diazepam, thereby improving the sensitivity of the detection method. Combined with colloidal gold detection, it can be used for rapid on-site detection of diazepam residues in actual aquatic products.

[0006] To achieve this objective, the specific solution adopted by the present invention is as follows:

[0007] The present invention provides a rapid detection method for diazepam residues in aquatic products based on immunomagnetic materials, comprising the following steps:

[0008] 1. Preparation of magnetic materials

[0009] FeCl3 and FeSO4 are added to water in a molar ratio of 1:2 to obtain Fe 3+ A solution with a concentration of 0.07~0.08 mol / L (preferably 0.072 mol / L) was prepared by adding concentrated ammonia to adjust the pH to 10 with stirring, and reacting at 80℃ for 30~50 min. Then, a mixed solution of tetraethyl orthosilicate and methanol was added dropwise, and after reacting for 2 h, the mixture was washed five times with water and methanol using magnetic separation to obtain Fe3O4-SiO2 magnetic material. The volume ratio of tetraethyl orthosilicate to methanol was 3:2, and the volume ratio of their mixed solution to water was 1:2.

[0010] A suitable amount of Fe3O4-SiO2 magnetic material was dispersed in methanol at a mass-to-volume ratio of 1:30-40. 3-Aminopropyltriethoxysilane (APTS) was added, and the mixture was reacted at 60°C for 10-15 h (preferably 12 h). Finally, excess citric acid was added, and the reaction was carried out for 20 h. After magnetic separation and washing, the Fe3O4-SiO2-COOH composite material, i.e., carboxyl magnetic beads, was obtained. The volume ratio of APTS to methanol was 2:15-20.

[0011] 2. Preparation and optimization of immunomagnetic materials

[0012] Take an appropriate amount of carboxyl magnetic beads, wash and resuspend them with 2-morpholinoethanesulfonic acid (MES) solution, then add 1-ethyl-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution (EDC and NHS mass ratio of 1:1) for activation for 30 min; after magnetic separation to remove the supernatant, add diazepam monoclonal antibody at a ratio of 40-50 μg diazepam monoclonal antibody per mg of magnetic beads, and carry out the coupling reaction for 60-90 min at pH 6-9. After the coupling is completed, block with ethanolamine solution for 1 h, and finally wash repeatedly with phosphate buffer and store. The optimal coupling ratio is 40 μg diazepam monoclonal antibody per mg of magnetic beads, and the coupling is carried out for 60 min at pH 7.4.

[0013] 3. Sample processing and testing

[0014] Add the sample to ethyl acetate at a mass-to-volume ratio of 2:5, vortex and mix thoroughly. After vigorous shaking for 2 min, centrifuge at 4000 r / min for 5 min, and take the supernatant to dry under nitrogen. Dissolve the dried material in PBS buffer, add immunomagnetic beads, vortex for 5 min, remove the supernatant by magnetic adsorption, add methanol and vortex for 5 min, and after magnetic separation, add PBS buffer to the supernatant to make up the volume. Perform sample testing according to the colloidal gold test card instructions. If the result is positive, the presence of residual diazepam in the sample is determined.

[0015] The volume of the sample was the same as the volume of the PBS buffer; the amount of immunomagnetic beads added was 1 / 4000 of the weight of the sample to be tested; and the amount of methanol added was 1 / 5 of the volume of the PBS buffer.

[0016] Invention Function and Effect

[0017] This invention establishes a rapid colloidal gold detection method for diazepam residues in aquatic products based on immunomagnetic materials. The method utilizes immunomagnetic materials to achieve high-level enrichment and rapid separation of diazepam, thereby improving the sensitivity of the detection method. Combined with colloidal gold detection, it can be used for rapid on-site detection of diazepam residues in actual aquatic products.

[0018] In terms of stability, the average recoveries of diazepam in five substrates—grass carp, crucian carp, common carp, whiteleg shrimp, and Chinese mitten crab—were 75.9%–96.0%, with relative standard deviations of 2.96%–8.99%. The high recovery rates and good stability and repeatability meet the technical requirements for the detection of diazepam residues in aquatic products.

[0019] In terms of sensitivity, the immunomagnetic bead-colloidal gold rapid detection method used in this invention detects diazepam, with a detection limit of 1.0 μg / kg.

[0020] Regarding specificity, four benzodiazepine drugs with structures similar to diazepam—flurazepam, temazepam, oxazepam, and flunitrazepam—were selected for an anti-interference test. The results showed that the colorimetric results of the test card were stable for both negative and spiked positive samples, and there was no cross-reaction between positive and negative sample test results, indicating that the detection method has good specificity and can effectively avoid interference from other drugs. Attached Figure Description

[0021] Figure 1 The optimization of the conjugation conditions for immunomagnetic beads (n=3) was shown: a. conjugation rate and conjugation amount under different antibody addition amounts; b. conjugation rate and conjugation amount under different pH values; c. conjugation rate and conjugation amount under different conjugation times.

[0022] Figure 2 The optimization of sample pretreatment conditions (n=3) is shown: a. Effect of different extractants on diazepam recovery; b. Adsorption of diazepam by immunomagnetic beads at different times; c. Effect of different methanol volumes on diazepam elution efficiency; d. Elution efficiency of diazepam at different elution times.

[0023] Figure 3 The results of the stability evaluation of the diazepam immunomagnetic bead-colloidal gold rapid detection method are shown. Detailed Implementation

[0024] The implementation of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] The detection method in this invention includes several steps: preparation of magnetic materials, preparation and optimization of immunomagnetic materials, sample processing and detection, while also evaluating the detection efficiency.

[0030] I. Rapid Detection of Diazepam Residue Colloidal Gold in Aquatic Products Based on Immunomagnetic Materials

[0031] 1. Preparation and Characterization of Magnetic Materials

[0032] Add 100 mL of water to a three-necked flask, then add FeCl3 and FeSO4 in a 1:2 molar ratio to obtain Fe 3+ A 0.072 mol / L solution was stirred and concentrated ammonia was added to adjust the pH to 10. The reaction was carried out at 80℃ for 40 min. Then, a mixed solution of 30 mL tetraethyl orthosilicate and 20 mL methanol was added dropwise. After reacting for 2 h, the mixture was washed five times with water and methanol using magnetic separation to obtain Fe3O4-SiO2 magnetic material. An appropriate amount of Fe3O4-SiO2 magnetic material was dispersed in 150 mL of methanol at a mass-to-volume ratio of 1:30. 20 mL of 3-aminopropyltriethoxysilane (APTS) was added, and the mixture was reacted at 60℃ for 12 h. Finally, 35 g of citric acid was added, and the reaction was carried out for 20 h. After magnetic separation and washing, Fe3O4-SiO2-COOH composite material was obtained, which is the carboxyl magnetic bead.

[0033] 2. Preparation and optimization of immunomagnetic materials

[0034] Take an appropriate amount of carboxyl magnetic beads, wash and resuspend them with 2-morpholinoethanesulfonic acid (MES) solution, then add 1-ethyl-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution to activate for 30 min. After magnetic separation to remove the supernatant, add diazepam monoclonal antibody for conjugation reaction. After conjugation, block with ethanolamine solution for 1 h, and finally wash repeatedly with phosphate buffer and store.

[0035] This study systematically investigated the effects of antibody dosage, conjugation pH, and conjugation time on the conjugation efficiency. The protein content in the supernatant after conjugation was determined using a BCA protein concentration assay kit, and the antibody conjugation amount and conjugation rate were then calculated.

[0036] Diazepam monoclonal antibody at concentrations of 10 μg, 25 μg, 40 μg, 50 μg, 75 μg, and 100 μg was conjugated to carboxylated magnetic beads. With increasing antibody dosage, the conjugation amount gradually increased, while the conjugation rate gradually decreased, indicating that the binding of the magnetic beads to the antibody gradually approached saturation. Figure 1 a) The pH value of the reaction system affects the reaction efficiency between the amino group of the antibody and the carboxyl group of the magnetic beads; excessively acidic or alkaline conditions will destroy antibody activity. Coupling experiments between magnetic beads and antibodies were conducted under five conditions: pH 4.8, 6.0, 7.4, 8.3, and 9.0 to determine the optimal coupling pH. The results showed that the coupling rate and coupling amount were lowest at pH 4.8, and reached their maximum values ​​at pH 7.4. Figure 1 b) The effect of antibody-magnetic bead coupling time is as follows: Figure 2 As shown in c, the coupling rate and coupling amount gradually increase with time. When the coupling time is 60-90 min, the growth trend of the coupling rate tends to level off.

[0037] Taking into account both detection effectiveness and time cost, the optimal conjugation time was determined to be 60 min. The final optimal conjugation conditions were: 40 μg of diazepam monoclonal antibody per mg of magnetic beads, pH 7.4, and conjugation time of 60 min.

[0038] 3. Research on sample pretreatment

[0039] First, the effect of different extractants on the extraction efficiency of diazepam was investigated. 10 ng of diazepam was added to 4 g of negative-labeled fish meat sample, followed by extraction with 10 mL of different extractants. The extraction efficiency was determined using liquid chromatography-tandem mass spectrometry. Diazepam is weakly polar, readily soluble in acetone and chloroform, almost insoluble in water, and soluble in organic solvents such as methanol, acetonitrile, and ethyl acetate. Due to the high toxicity of chloroform and acetone, and their potential use as precursors for drug manufacturing, they were not preferred extractants. Methanol, acetonitrile, ethyl acetate, and n-hexane were all effective extractants for diazepam from fish meat tissue. The extraction efficiencies of the four solvents were as follows: Figure 2 As shown in a. Methanol, acetonitrile, and ethyl acetate all yielded high extraction rates. Considering the time efficiency of subsequent nitrogen blowing, ethyl acetate, which has higher volatility, was ultimately chosen as the extractant.

[0040] Immunomagnetic material was added to 1 mL of an aqueous solution containing 100 ng diazepam, and its maximum adsorption capacity and adsorption time were investigated. The results are as follows: Figure 2As shown in b. The study found that the immunomagnetic material reached its maximum adsorption capacity for diazepam (86.2 ng / mg) within 5 minutes. Methanol was used as the eluent for the immunomagnetic material, and the effects of eluent volume and elution time on elution efficiency were investigated. The results are shown in Figure b. Figure 2 As shown in c and d, the results indicate that when eluting with 100 μL–500 μL of pure methanol, the elution rate gradually increased from 85.1% to 94.1%, but remained relatively stable after the methanol volume increased to 200 μL. To reduce the amount of organic solvent used, 200 μL of methanol was ultimately determined as the optimal eluent volume. With prolonged elution time, the elution rate gradually increased, reaching 97.2% at 5 min. Further extending the elution time resulted in a stable elution rate; therefore, the optimal elution time was determined to be 5 min.

[0041] 4. Establishment of a rapid detection method using immunomagnetic beads and colloidal gold

[0042] A rapid detection method for immunomagnetic beads-colloidal gold was established. 4.0 g of sample was weighed, added to 10 mL of ethyl acetate, vortexed, and shaken vigorously for 2 min. The mixture was then centrifuged at 4000 r / min for 5 min, and the supernatant was dried under nitrogen. 1 mL of PBS buffer was added to a test tube, followed by 1 mg of immunomagnetic beads. The mixture was vortexed for 5 min, and the supernatant was removed by magnetic separation. 200 μL of methanol was added, and the mixture was vortexed for 5 min. After magnetic separation, PBS buffer was added to the supernatant to bring the volume to 1 mL. The sample was then loaded and detected according to the instructions of the colloidal gold detection card.

[0043] II. Performance Evaluation

[0044] 2.1 Stability

[0045] Five substrates were selected: grass carp, crucian carp, common carp, whiteleg shrimp, and Chinese mitten crab. The spiked recoveries of the pretreatment method were investigated using liquid chromatography-tandem mass spectrometry (LC-MS / MS) at spiked levels of 1.0 μg / kg, 5.0 μg / kg, and 10.0 μg / kg. The results are shown in Table 1. The average recoveries of diazepam in the five substrates ranged from 75.9% to 96.0%, with relative standard deviations ranging from 2.96% to 8.99%. The results indicate that this method has high recovery, good stability and repeatability, and can meet the technical requirements for the detection of diazepam residues in aquatic products.

[0046] Table 1. Recovery rates and relative standard deviations of diazepam (n=5) spiking standard

[0047]

[0048] 2.2 Sensitivity

[0049] To evaluate the sensitivity of the detection method, negative samples from grass carp, crucian carp, common carp, whiteleg shrimp, and Chinese mitten crab were selected. Diazepam was detected using the immunomagnetic bead-colloidal gold rapid detection method at spiked levels of 0, 0.5 μg / kg, 1.0 μg / kg, and 2.0 μg / kg. The limit of detection was 1.0 μg / kg. The specific results are shown in Table 2.

[0050] Table 2. Results of detection limit determination for diazepam immunomagnetic bead-colloidal gold rapid detection method (n=5)

[0051]

[0052] 2.3 Specificity

[0053] To verify the specificity of the method, four benzodiazepine drugs with structures similar to diazepam—flurazepam, temazepam, oxazepam, and flunitrazepam—were selected for an anti-interference test. Standard solutions of the above interfering substances were added to the samples at a spiking level of 1.0 μg / kg. The results are shown in Table 3. The colorimetric results of the test card were stable for both negative and spiked positive samples, and there was no cross-reaction between positive and negative sample test results. This indicates that the detection method has good specificity and can effectively avoid interference from other drugs.

[0054] Table 3. Specificity evaluation results of the rapid detection method for diazepam using immunomagnetic beads-colloidal gold (n=5)

[0055]

[0056] 2.4 Stability

[0057] To verify the stability of the method, 10 negative samples each of grass carp, crucian carp, common carp, whiteleg shrimp, and Chinese mitten crab were selected and tested for diazepam using the immunomagnetic bead-colloidal gold rapid detection method. All results were negative, indicating a false positive rate of 0. Positive tests were then performed on the aforementioned 50 samples by adding 1.0 μg / kg diazepam; all results were positive, indicating a false negative rate of 0. See below for detailed results. Figure 3 .

[0058] In summary, this study successfully established a rapid detection method for diazepam residues in aquatic products based on immunomagnetic bead-colloidal gold immunochromatography. This method utilizes immunomagnetic beads for selective adsorption and separation of diazepam residues in aquatic products. It is simple to operate, highly sensitive, with an average recovery rate of 75.9%–96.0%, a relative standard deviation of 2.96%–8.99%, and a limit of detection of 1.0 μg / kg for diazepam. Methodological validation demonstrated that this method is highly specific and stable, and can be used for rapid on-site detection of diazepam residues in aquatic products, providing technical support for food safety supervision.

[0059] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. A rapid detection method for diazepam residues in aquatic products based on immunomagnetic materials, characterized in that, Includes the following steps: A. Preparation of magnetic materials FeCl3 and FeSO4 are added to water in a molar ratio of 1:2 to obtain Fe 3+ A solution with a concentration of 0.07~0.08 mol / L was added with concentrated ammonia to adjust the pH of the system to 10 under stirring, and the reaction was carried out at 80℃ for 30~50 min; then a mixed solution of tetraethyl orthosilicate and methanol was added dropwise. After the reaction, the mixture was repeatedly washed with water and methanol by magnetic separation to obtain Fe3O4-SiO2 magnetic material. An appropriate amount of Fe3O4-SiO2 magnetic material was dispersed in methanol, and 3-aminopropyltriethoxysilane (APTS) was added. The mixture was reacted at 60℃ for 10-15 h. Finally, a certain amount of citric acid was added, and the reaction was carried out for 20 h. After magnetic separation and washing, the Fe3O4-SiO2-COOH composite material was obtained, which is the carboxyl magnetic bead. B. Preparation of Immunomagnetic Materials Carboxyl magnetic beads were first washed and resuspended with 2-morpholinoethanesulfonic acid (MES) solution, then activated for 30 min with 1-ethyl-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution. After magnetic separation to remove the supernatant, diazepam monoclonal antibody was added at a ratio of 40-50 μg of diazepam monoclonal antibody per mg of magnetic beads. The coupling reaction was carried out at pH 6-9 for 60-90 min. After coupling, the beads were blocked with ethanolamine solution for 1 h. Finally, the beads were repeatedly washed with phosphate buffer and stored. C. Sample processing and testing Add the sample to be tested to ethyl acetate at a mass-to-volume ratio of 2:5, vortex and mix well, shake vigorously, centrifuge at 4000 r / min, and take the supernatant liquid nitrogen to dry. After dissolving the dried material in PBS buffer, add immunomagnetic beads, vortex adsorption, and then magnetically separate and remove the supernatant. Add methanol, vortex elute, and magnetically separate. Add PBS buffer to the supernatant to make up the volume. Perform sample testing according to the colloidal gold test card instructions. If the result is positive, the presence of residual diazepam in the sample is determined.

2. The rapid detection method for diazepam residues in aquatic products based on immunomagnetic materials according to claim 1, characterized in that: in, In step A, the volume ratio of tetraethyl orthosilicate to methanol is 3:2, and the volume ratio of the mixed solution to water is 1:

2. After reacting for 2 hours, the solution is washed repeatedly with water and methanol 5 times for later use.

3. The rapid detection method for diazepam residues in aquatic products based on immunomagnetic materials according to claim 1, characterized in that: in, In step A, the mass-to-volume ratio of Fe3O4-SiO2 magnetic material to methanol is 1:30~40. The volume ratio of APTS to methanol is 2:15~20.

4. The rapid detection method for diazepam residues in aquatic products based on immunomagnetic materials according to claim 1, characterized in that: in, In step B, 40 μg of diazepam monoclonal antibody was optimally conjugated per mg of magnetic beads, and the conjugation was carried out at pH 7.4 for 60 min. The volume ratio of EDC to NHS is 1:

1.

5. The rapid detection method for diazepam residues in aquatic products based on immunomagnetic materials according to claim 1, characterized in that: in, In step C, the sample to be tested is added to ethyl acetate and vortexed to mix, then shaken vigorously for 2 min; the centrifugation time, the vortex adsorption time after adding immunomagnetic beads, and the vortex elution time after adding methanol are all 5 min.

6. The rapid detection method for diazepam residues in aquatic products based on immunomagnetic materials according to claim 1, characterized in that: in, In step C, the volume of the final volume is the same as the volume of the PBS buffer; the amount of immunomagnetic beads added is 1 / 4000 of the weight of the sample to be tested; and the amount of methanol added is 1 / 5 of the volume of the PBS buffer.

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