Rapid detection kit for diazepam and metabolite thereof, preparation method of kit and detection method

Abstract

The invention discloses a rapid detection kit for diazepam and metabolites thereof, a preparation method of the kit and a detection method, the kit comprises a kit body, the kit body is internally provided with a detection card, a gold-labeled antibody enzyme-labeled hole, an extracting agent, a reconstitution fluid and normal hexane, and the preparation method comprises the following steps: preparing the gold-labeled antibody enzyme-labeled hole; preparing a detection card; preparing an extracting agent; preparing a reconstitution fluid; the detection method comprises the following steps: treating a sample to obtain a to-be-detected solution; putting the to-be-detected solution into a gold-labeled antibody enzyme-labeled hole for reaction, and then transferring the to-be-detected solution into a detection card for reaction to obtain a detection result; samples capable of being detected comprise aquatic products and culture water, the detection range is wide, point card detection can be achieved through simple sample extraction and concentration steps, the professional requirement is not high, operation is easy, the detection time is short, the result can be detected within 30 min, the lowest detection limit is 0.35 microgram / kg, the price is low, detection can be conducted indoors and outdoors at the temperature of 10-35 DEG C, and the method is suitable for popularization and application. And the method is suitable for on-site rapid screening by vast base-level personnel.

Description

Technical Field

[0001] This invention relates to diazepam detection technology, specifically a rapid detection kit for diazepam and its metabolites, a method for preparing the kit, and a detection method. Background Technology

[0002] Diazepam (D) is a benzodiazepine sedative-hypnotic prescription drug. Long-term use of diazepam can lead to dependence and drug tolerance. After discontinuation, symptoms such as irritability, agitation, and auditory hallucinations may occur, and it is more toxic to pregnant women and fetuses. The National Food Safety Standard GB31650-2019, "Maximum Residue Limits for Veterinary Drugs in Food," stipulates that diazepam is a veterinary drug permitted for therapeutic use but not detectable in animal-derived foods. The "Aquaculture Drug Information Sheet Nos. 1 and 2 of 2022" stipulates that diazepam and other veterinary drugs for livestock and poultry have not been reviewed and approved for use in aquatic animals in my country and are prohibited from use in aquaculture. Diazepam is metabolized in the liver, undergoing demethylation and hydroxylation to form phase I metabolites norazepam (ND) and temazepam (TZ), which are further metabolized to oxazepam (OZ). Based on existing studies on the decomposition kinetics of diazepam and its metabolites in urine, it has been found that, apart from the original diazepam, the content of phase I and phase II diazepam metabolites in urine showed an increasing trend over time on day 34 and day 3, respectively. In actual testing, all target substances exist simultaneously in the same matrix, and the changes in the content of each target substance involve complex situations such as mutual transformation and mutual influence. Therefore, while detecting diazepam residues, it is also necessary to pay attention to the content of its metabolites.

[0003] The existing testing standards for aquatic tissues mainly include: GB29697-2013 National Food Safety Standard for Determination of Multiple Residues of Diazepam and Methamphetamine in Animal-Derived Foods by Gas Chromatography-Mass Spectrometry, which has a relatively complex testing process, is expensive, and is not convenient for rapid on-site screening; and KJ202105 Rapid Detection of Diazepam Residues in Aquatic Products by Colloidal Gold Immunochromatography, which uses column chromatography for pretreatment and has a detection limit of 0.5 μg / kg. Moreover, this method only targets aquatic tissues, does not include aquaculture water in the test sample, and the target analyte is only diazepam, without involving its metabolites. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention provides a rapid detection kit for diazepam and its metabolites. The kit can be used to detect samples including aquatic products and aquaculture water. The detection process is convenient, requires minimal expertise, is simple to operate, and has a short detection time (results within 30 minutes). It has a detection limit of 0.35 μg / kg, is inexpensive, and can be performed both indoors and outdoors at temperatures ranging from 10 to 35°C, making it suitable for rapid on-site screening by grassroots personnel.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution: a rapid detection kit for diazepam and its metabolites, comprising a box body, wherein the box body contains an aluminum foil bag, an extraction reagent, a reconstitution solution and n-hexane, and the aluminum foil bag contains a detection card and a gold-labeled antibody enzyme-labeled well.

[0006] Preferably, the test card includes a PVC base plate, a filter membrane, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper. The nitrocellulose membrane is fixed at the center of the PVC base plate. The conjugate pad and absorbent paper are respectively connected to both sides of the nitrocellulose membrane. The sample pad is disposed above the conjugate pad, and the filter membrane is disposed above the sample pad. The nitrocellulose membrane has a T-line and a C-line. The T-line is located on the side of the nitrocellulose membrane facing the conjugate pad, and the C-line is located on the side of the nitrocellulose membrane facing the absorbent paper.

[0007] Preferably, the extractant includes extractant 1 and extractant 2.

[0008] To achieve the above technical objectives, the present invention adopts the following technical solution: a method for preparing a reagent kit for diazepam and its metabolites, comprising the following steps:

[0009] Preparation of gold-labeled antibody enzyme-labeled wells;

[0010] Prepare test cards;

[0011] Preparation of extractant;

[0012] Prepare complex solutions.

[0013] Preferably, the preparation of gold-labeled antibody enzyme-labeled wells includes the following sub-steps:

[0014] Preparation of colloidal gold solution: 1% chloroauric acid aqueous solution and 1% trisodium citrate aqueous solution were added at a volume ratio of 1:1-2.5 and gold was boiled down to obtain a colloidal gold solution with a particle size of 20 nm, which was used as seed gold. A second gold boiling was carried out, in which seed gold, 1% chloroauric acid, 1% trisodium citrate and 10% hydroquinone were added at a volume ratio of 1:0.5:1:0.3. After the reaction was completed, the mixture was cooled to room temperature, the pH was adjusted to 8.0 with sodium hydroxide solution, and centrifuged. The precipitated flower-shaped colloidal gold was redissolved with 0.2% trisodium citrate to obtain a flower-shaped colloidal gold solution with a particle size of 50-60 nm.

[0015] Preparation of gold-labeled antibody ELISA wells: Adjust the pH of colloidal gold using 0.1-0.2 mol / L potassium carbonate solution, select the optimal pH, add 5-8 μg / mL diazepam monoclonal antibody, spin-label for 30-60 minutes, add 10% bovine serum albumin for blocking, block at room temperature for 30 minutes, then centrifuge at 11000 rpm for 30 minutes at 4°C, discard the supernatant precipitate, reconstitute with 1×PBS reconstitution solution 10 times, add 2-5 μL / well to the gold-labeled antibody ELISA wells treated with 5-10% sucrose, and dry at 37°C overnight.

[0016] Preferably, the preparation of the test card includes the following sub-steps:

[0017] Preparation of nitrocellulose membranes: Dilute diazepam-BSA antigen to 0.2-0.5 mg / mL with 0.01-0.02 mol / L, pH 7.4 PBS buffer, and coat the membrane at the T-line using a coating apparatus at a rate of 0.75 μL / cm; dilute goat anti-mouse secondary antibody to 0.3-0.65 mg / mL with 0.01-0.02 mol / L, pH 7.4 PBS buffer, and coat the membrane at the C-line using a coating apparatus at a rate of 0.75 μL / cm; dry at 37°C overnight;

[0018] Preparation of the binding pad: Prepare 1L of 4%-8% sucrose solution, immerse the glass fiber SB08 pad in it, remove it after 5 minutes, and dry it at 37℃ overnight;

[0019] Preparation of sample pads: Weigh 2.9g disodium hydrogen phosphate dodecahydrate, 8.0g sodium chloride, 2.4g potassium chloride, and 2.0g potassium dihydrogen phosphate, dissolve them in 800mL of deionized water, then add 0.5%–1% bovine serum albumin, 0.1%–0.3% sodium casein, 0.5%–1% S17, and 0.5% Tween 20. Mix well and bring the volume to 1L. Adjust the pH to 8.0 with 1mol / L sodium hydroxide solution to obtain the sample pad treatment solution. Then immerse the glass fiber SB08 in the sample pad treatment solution, remove it after 5 minutes, and dry it overnight at 37℃.

[0020] Preferably, the preparation of the extractant includes the following sub-steps:

[0021] Preparation of extractant 1: Weigh 0.5-1g of neutral alumina and 0.3-0.6g of sodium chloride;

[0022] Preparation of extractant 2: Mix acetonitrile and ethyl acetate in a volume ratio of 1:1 to form an acetonitrile / ethyl acetate mixed solution. Add 5%-10% ammonia water to the acetonitrile / ethyl acetate mixed solution and mix well.

[0023] Preferably, the preparation of the complex solution includes the following sub-steps:

[0024] Weigh 2.9g of disodium hydrogen phosphate dodecahydrate, 8.0g of sodium chloride, 2.4g of potassium chloride, and 2.0g of potassium dihydrogen phosphate. Dissolve them completely in 800mL of deionized water and bring the volume to 1L. Add 10%-20% methanol and mix well.

[0025] To achieve the above technical objectives, the present invention adopts the following technical solution: a method for detecting diazepam and its metabolites using a reagent kit, comprising the following steps:

[0026] Process the sample to obtain the test solution:

[0027] The edible parts of aquatic products such as fish and shrimp are crushed using a food processor; 5g of the processed sample is weighed into a 50mL centrifuge tube; extractant 1 and 7mL of sample extractant 2 are added, and the mixture is shaken vigorously for 3 minutes and centrifuged at 4000 rpm for 5 minutes at room temperature; 5mL of the supernatant is taken into a 10mL centrifuge tube and dried under nitrogen (air) at 65℃; 500μL of n-hexane and 300μL of reconstitution solution are added, mixed well, and allowed to stand or centrifuged. The lower clear liquid is the test solution; the aquaculture water is collected directly into a clean glass container or centrifuge tube. If the aquaculture water is turbid, it needs to be centrifuged at 4000rpm for 10 minutes or filtered through a 0.45μm filter membrane to form the test solution.

[0028] After reacting the test solution in the gold-labeled antibody ELISA wells, transfer it to the test card for further reaction to obtain the test results: Before testing, allow the test card and test solution to return to room temperature; remove the test card and gold-labeled antibody ELISA wells from the aluminum foil bag and use them within half an hour; use a pipette to transfer 120 μL of the test solution into the gold-labeled antibody ELISA wells, and repeatedly pipette until the red substance in the gold-labeled antibody ELISA wells is completely dissolved, and wait for the reaction to proceed for 2 minutes; place the test card flat and add all the incubated test solution onto the filter membrane of the test card; react for 8-10 minutes and interpret the results; results interpreted after 20 minutes are invalid.

[0029] Preferably, the detection results include: the T line being darker or equivalent to the C line; the T line being non-discolored or weaker than the C line; and the C line being non-discolored. A T line being darker or equivalent to the C line indicates a negative result, meaning the concentration of diazepam and its metabolites in the sample is below the detection limit. A T line being non-discolored or weaker than the C line indicates a positive result, meaning the concentration of diazepam and its metabolites in the sample is equal to or higher than the detection limit. A C line being non-discolored indicates an invalid result, meaning the reagent is faulty or the operation is incorrect.

[0030] In summary, the present invention has achieved the following technical effects:

[0031] 1. The rapid detection kit for diazepam and its metabolites, the preparation method of the kit, and the detection method of the present invention are applicable to aquatic products and aquaculture water, with a wide detection range and good adaptability;

[0032] 2. The rapid detection kit for diazepam and its metabolites, the preparation method of the kit, and the detection method of the present invention are convenient to use. The detection steps are simple, requiring only a simple sample extraction and concentration process, and can be performed by spot testing. It does not require high levels of expertise, is easy to operate, and has a short detection time, providing results within just 30 minutes.

[0033] 3. The rapid detection kit for diazepam and its metabolites, the preparation method of the kit, and the detection method of the present invention have a minimum detection limit of 0.35 μg / kg, are inexpensive, and can be performed indoors and outdoors at temperatures ranging from 10 to 35°C, making them suitable for rapid on-site screening by grassroots personnel. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the detection card structure of the rapid detection kit for diazepam and its metabolites of the present invention;

[0035] Figure 2 This is a schematic diagram of the detection results of the rapid detection kit for diazepam and its metabolites of the present invention;

[0036] Instruction manual diagram markings: 1. PVC base plate; 2. Filter membrane; 3. Sample pad; 4. Conjugate pad; 5. T-line; 6. C-line; 7. Nitrocellulose membrane; 8. Absorbent paper. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the accompanying drawings.

[0038] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

[0039] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0040] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0041] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0042] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0043] like Figure 1 As shown, a rapid detection kit for diazepam and its metabolites includes a box containing an aluminum foil bag, an extraction reagent, a reconstitution solution, and n-hexane. The aluminum foil bag contains a detection card and gold-labeled antibody wells. The detection card includes a PVC base plate 1, a filter membrane 2, a sample pad 3, a conjugate pad 4, a nitrocellulose membrane 7, and absorbent paper 8. The nitrocellulose membrane 7 is fixed to the center of the PVC base plate 1. The conjugate pad 4 and absorbent paper 8 are connected to opposite sides of the nitrocellulose membrane 7. The sample pad 3 is positioned above the conjugate pad 4, and the filter membrane 2 is positioned above the sample pad 3. The nitrocellulose membrane 7 has a T-line 5 and a C-line 6. The T-line 5 is located on the side of the nitrocellulose membrane 7 facing the conjugate pad 4, and the C-line 6 is located on the side of the nitrocellulose membrane 7 facing the absorbent paper 8. The extraction reagent includes extraction reagent 1 and extraction reagent 2.

[0044] Example 1

[0045] A method for preparing a reagent kit for diazepam and its metabolites includes the following steps:

[0046] Preparation of colloidal gold solution: 1% chloroauric acid aqueous solution and 1% trisodium citrate aqueous solution were added at a volume ratio of 1:1 and gold was boiled down to obtain a colloidal gold solution with a particle size of 20 nm, which was used as seed gold. The gold was boiled down again, and seed gold, 1% chloroauric acid, 1% trisodium citrate and 10% hydroquinone were added at a volume ratio of 1:0.5:1:0.3. After the reaction was completed, the mixture was cooled to room temperature, the pH was adjusted to 8.0 with sodium hydroxide solution, and centrifuged. The precipitated flower-shaped colloidal gold was redissolved with 0.2% trisodium citrate to obtain a flower-shaped colloidal gold solution with a particle size of 50-60 nm.

[0047] Preparation of gold-labeled antibody ELISA wells: The pH of the colloidal gold was adjusted using 0.2 mol / L potassium carbonate solution. The optimal pH was selected, and 5 μg / mL diazepam monoclonal antibody was added. The mixture was rotated for 60 minutes, then blocked with 10% bovine serum albumin at room temperature for 30 minutes. The mixture was then centrifuged at 11,000 rpm for 30 minutes at 4°C. The supernatant was discarded, and the precipitate was reconstituted 10-fold with 1×PBS. After reconstitution, 2 μL of the solution was added to each well of the 5% sucrose-treated gold-labeled antibody ELISA well. The wells were dried overnight at 37°C. 1×PBS was 0.01 mol / L.

[0048] Preparation of nitrocellulose membrane 7: Dilute diazepam-BSA antigen to 0.2 mg / mL with 0.01 mol / L, pH 7.4 PBS buffer, and coat the membrane at position 5 (T line) using a coating apparatus at a rate of 0.75 μL / cm; dilute goat anti-mouse secondary antibody to 0.3 mg / mL with 0.01 mol / L, pH 7.4 PBS buffer, and coat the membrane at position 6 (C line) using a coating apparatus at a rate of 0.75 μL / cm; dry at 37°C overnight;

[0049] Preparation of binding pad 4: Prepare 1L of 4% sucrose solution, immerse the glass fiber SB08 pad in it, remove it after 5 minutes, and dry it at 37℃ overnight;

[0050] Preparation of Sample Pad 3: Weigh 2.9g disodium hydrogen phosphate dodecahydrate, 8.0g sodium chloride, 2.4g potassium chloride, and 2.0g potassium dihydrogen phosphate, dissolve them in 800mL deionized water, then add 1% bovine serum albumin, 0.3% casein sodium salt, 1% S17, and 0.5% Tween 20. Mix well and bring the volume to 1L. Adjust the pH to 8.0 with 1mol / L sodium hydroxide solution to obtain the sample pad 3 treatment solution. Then, immerse glass fiber SB08 in the sample pad 3 treatment solution, remove it after 5 minutes, and dry it overnight at 37℃. S17 is a surfactant.

[0051] Preparation of extractant 1: Weigh 1g of neutral alumina and 0.3g of sodium chloride;

[0052] Preparation of extractant 2: Mix acetonitrile and ethyl acetate solutions at a volume ratio of 1:1 to form an acetonitrile / ethyl acetate mixed solution. Add 10% ammonia water to the acetonitrile / ethyl acetate mixed solution and mix well.

[0053] Preparation of the reconstituted solution: Weigh 2.9 g disodium hydrogen phosphate dodecahydrate, 8.0 g sodium chloride, 2.4 g potassium chloride, and 2.0 g potassium dihydrogen phosphate. Dissolve them completely in 800 mL of deionized water, then bring the volume to 1 L. Add 10% methanol and mix well.

[0054] Example 2

[0055] A method for preparing a reagent kit for diazepam and its metabolites includes the following steps:

[0056] Preparation of colloidal gold solution: 1% chloroauric acid aqueous solution and 1% trisodium citrate aqueous solution were added at a volume ratio of 1:2.5 and gold was boiled down to obtain a colloidal gold solution with a particle size of 20 nm, which was used as seed gold. A second gold boiling was carried out, in which seed gold, 1% chloroauric acid, 1% trisodium citrate and 10% hydroquinone were added at a volume ratio of 1:0.5:1:0.3. After the reaction was completed, the mixture was cooled to room temperature, the pH was adjusted to 8.0 with sodium hydroxide solution, and centrifuged. The precipitated flower-shaped colloidal gold was redissolved with 0.2% trisodium citrate to obtain a flower-shaped colloidal gold solution with a particle size of 50-60 nm.

[0057] Preparation of gold-labeled antibody ELISA wells: The pH of colloidal gold was adjusted using 0.1 mol / L potassium carbonate solution. The optimal pH was selected, and 8 μg / mL diazepam monoclonal antibody was added. The mixture was rotated and labeled for 30 minutes. 10% bovine serum albumin was added for blocking. The mixture was blocked at room temperature for 30 minutes, followed by centrifugation at 11,000 rpm for 30 minutes at 4°C. The supernatant precipitate was discarded, and the precipitate was reconstituted 10 times with 1×PBS. After reconstitution, 5 μL of the solution was added to the 10% sucrose-treated gold-labeled antibody ELISA wells and dried at 37°C overnight.

[0058] Preparation of nitrocellulose membrane 7: Dilute diazepam-BSA antigen to 0.5 mg / mL with 0.01 mol / L, pH 7.4 PBS buffer, and coat the membrane at position 5 (T line) using a coating apparatus at a rate of 0.75 μL / cm; dilute goat anti-mouse secondary antibody to 0.65 mg / mL with 0.01 mol / L, pH 7.4 PBS buffer, and coat the membrane at position 6 (C line) using a coating apparatus at a rate of 0.75 μL / cm; dry at 37°C overnight;

[0059] Preparation of binding pad 4: Prepare 1L of 8% sucrose solution, immerse the glass fiber SB08 pad in it, remove it after 5 minutes, and dry it at 37℃ overnight;

[0060] Preparation of Sample Pad 3: Weigh 2.9g disodium hydrogen phosphate dodecahydrate, 8.0g sodium chloride, 2.4g potassium chloride, and 2.0g potassium dihydrogen phosphate, dissolve them in 800mL deionized water, then add 0.5% bovine serum albumin, 0.1% casein sodium salt, 0.5% S17, and 0.5% Tween 20. Mix well and bring the volume to 1L. Adjust the pH to 8.0 with 1mol / L sodium hydroxide solution to obtain the sample pad 3 treatment solution. Then, immerse glass fiber SB08 in the sample pad 3 treatment solution, remove it after 5 minutes, and dry it overnight at 37℃. S17 is a surfactant.

[0061] Preparation of extractant 1: Weigh 0.5g of neutral alumina and 0.6g of sodium chloride;

[0062] Preparation of extractant 2: Mix acetonitrile and ethyl acetate solutions at a volume ratio of 1:1 to form an acetonitrile / ethyl acetate mixed solution. Add 10% ammonia water to the acetonitrile / ethyl acetate mixed solution and mix well.

[0063] Preparation of the reconstituted solution: Weigh 2.9 g disodium hydrogen phosphate dodecahydrate, 8.0 g sodium chloride, 2.4 g potassium chloride, and 2.0 g potassium dihydrogen phosphate. Dissolve them completely in 800 mL of deionized water, then bring the volume to 1 L. Add 20% methanol and mix well.

[0064] Example 3

[0065] A method for preparing a reagent kit for diazepam and its metabolites includes the following steps:

[0066] Preparation of colloidal gold solution: 1% chloroauric acid aqueous solution and 1% trisodium citrate aqueous solution were added at a volume ratio of 1:1.7 and gold was boiled down to obtain a colloidal gold solution with a particle size of 20 nm, which was used as seed gold. A second gold boiling was carried out, in which seed gold, 1% chloroauric acid, 1% trisodium citrate and 10% hydroquinone were added at a volume ratio of 1:0.5:1:0.3. After the reaction was completed, the mixture was cooled to room temperature, the pH was adjusted to 8.0 with sodium hydroxide solution, and centrifuged. The precipitated flower-shaped colloidal gold was redissolved with 0.2% trisodium citrate to obtain a flower-shaped colloidal gold solution with a particle size of 50-60 nm.

[0067] Preparation of gold-labeled antibody ELISA wells: The pH of colloidal gold was adjusted using 0.15 mol / L potassium carbonate solution. The optimal pH was selected, and 6.5 μg / mL diazepam monoclonal antibody was added. The mixture was rotated and labeled for 45 minutes. 10% bovine serum albumin was added for blocking. The mixture was blocked at room temperature for 30 minutes, followed by centrifugation at 11,000 rpm for 30 minutes at 4°C. The supernatant precipitate was discarded, and the precipitate was reconstituted 10 times with 1×PBS. After reconstitution, 3.5 μL of the gold-labeled antibody was added to each well treated with 5-10% sucrose and dried overnight at 37°C.

[0068] Preparation of nitrocellulose membrane 7: Dilute diazepam-BSA antigen to 0.35 mg / mL with 0.015 mol / L, pH 7.4 PBS buffer, and coat the membrane at position 5 (T line) using a membrane scrubbing apparatus at a rate of 0.75 μL / cm; dilute goat anti-mouse secondary antibody to 0.5 mg / mL with 0.015 mol / L, pH 7.4 PBS buffer, and coat the membrane at position 6 (C line) using a membrane scrubbing apparatus at a rate of 0.75 μL / cm; dry at 37°C overnight;

[0069] Preparation of binding pad 4: Prepare 1L of 6% sucrose solution, immerse the glass fiber SB08 pad in it, remove it after 5 minutes, and dry it at 37℃ overnight;

[0070] Preparation of Sample Pad 3: Weigh 2.9g disodium hydrogen phosphate dodecahydrate, 8.0g sodium chloride, 2.4g potassium chloride, and 2.0g potassium dihydrogen phosphate, dissolve them in 800mL deionized water, then add 0.75% bovine serum albumin, 0.2% casein sodium salt, 0.75% S17, and 0.5% Tween 20. Mix well and bring the volume to 1L. Adjust the pH to 8.0 with 1mol / L sodium hydroxide solution to obtain the sample pad 3 treatment solution. Then, immerse glass fiber SB08 in the sample pad 3 treatment solution, remove it after 5 minutes, and dry it overnight at 37℃. S17 is a surfactant.

[0071] Preparation of extractant 1: Weigh 0.75g of neutral alumina and 0.45g of sodium chloride;

[0072] Preparation of extractant 2: Mix acetonitrile and ethyl acetate solutions at a volume ratio of 1:1 to form an acetonitrile / ethyl acetate mixed solution. Add 7.5% ammonia water to the acetonitrile / ethyl acetate mixed solution and mix well.

[0073] Preparation of the complex solution: Weigh 2.9g disodium hydrogen phosphate dodecahydrate, 8.0g sodium chloride, 2.4g potassium chloride, and 2.0g potassium dihydrogen phosphate, dissolve them completely in 800mL deionized water, and then bring the volume to 1L. Add 15% methanol and mix well.

[0074] A method for detecting diazepam and its metabolites using a kit, comprising the following steps:

[0075] The edible parts of aquatic products such as fish and shrimp are crushed using a food processor; 5g of the processed sample is weighed into a 50mL centrifuge tube; extractant 1 and 7mL of sample extractant 2 are added, and the mixture is vigorously shaken for 3 minutes and centrifuged at 4000 rpm for 5 minutes at room temperature; 5mL of the supernatant is taken into a 10mL centrifuge tube and dried under nitrogen (air) at 65℃; 500μL of n-hexane and 300μL of reconstitution solution are added, mixed well, and allowed to stand or centrifuged. The lower clear liquid is the test solution; the aquaculture water is collected directly into a clean glass container or centrifuge tube. If the aquaculture water is turbid, it needs to be centrifuged at 4000rpm for 10 minutes or filtered through a 0.45μm filter membrane 2 to form the test solution.

[0076] Before testing, bring the test card and test solution to room temperature; remove the test card and gold-labeled antibody enzyme-labeled wells from the aluminum foil bag and use them within half an hour; use a pipette to transfer 120 μL of the test solution into the gold-labeled antibody enzyme-labeled wells, and repeatedly pipette until the red substance in the gold-labeled antibody enzyme-labeled wells is completely dissolved, and wait for the reaction to proceed for 2 minutes; lay the test card flat and add all the incubated test solution to filter membrane 2 of the test card; react for 8-10 minutes and read the results; results read after 20 minutes are invalid.

[0077] like Figure 2 As shown, the test results include: T line 5 being darker or equivalent to C line 6; T line 5 not developing or weaker than C line 6; C line 6 not developing; and T line 5 being darker or equivalent to C line 6 indicating a negative result, meaning the concentration of diazepam and its metabolites in the sample is below the detection limit. T line 5 not developing or weaker than C line 6 indicating a positive result, meaning the concentration of diazepam and its metabolites in the sample is equal to or higher than the detection limit. C line 6 not developing indicates an invalid result, meaning the reagent is invalid or the operation is incorrect.

[0078] Precautions for using this kit:

[0079] ① The test card is for single use at room temperature. Avoid direct sunlight and direct airflow from electric fans during testing.

[0080] ② Tap water, distilled water, or deionized water cannot be used as negative controls.

[0081] ③ If the sample moves slowly after adding the sample, you can use a dropper to gently press the sample pad in the sample well to accelerate the flow of the sample.

[0082] ④ The test card is a screening method. If a positive result is found, it can be confirmed by instrumental methods.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

Claims

1. A rapid detection kit for diazepam and its metabolites, characterized in that, The device includes a box containing an aluminum foil bag, an extractant, a reconstitution solution, and n-hexane. The aluminum foil bag contains a test card and a gold-labeled antibody enzyme-linked well.

2. The rapid detection kit for diazepam and its metabolites according to claim 1, characterized in that, The test card includes a PVC base plate, a filter membrane, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper. The nitrocellulose membrane is fixed at the center of the PVC base plate. The conjugate pad and absorbent paper are respectively connected to both sides of the nitrocellulose membrane. The sample pad is placed above the conjugate pad, and the filter membrane is placed above the sample pad. The nitrocellulose membrane has a T-line and a C-line. The T-line is located on the side of the nitrocellulose membrane facing the conjugate pad, and the C-line is located on the side of the nitrocellulose membrane facing the absorbent paper.

3. A rapid detection kit for diazepam and its metabolites according to claim 2, characterized in that, The extractant includes extractant 1 and extractant 2.

4. A method for preparing a reagent kit for diazepam and its metabolites, applicable to the rapid detection kit as described in any one of claims 1-3, characterized in that, Includes the following steps: Preparation of gold-labeled antibody enzyme-labeled wells; Prepare test cards; Preparation of extractant; Prepare complex solutions.

5. The method for preparing a reagent kit for diazepam and its metabolites according to claim 4, characterized in that, The preparation of gold-labeled antibody enzyme-labeled wells includes the following sub-steps: Preparation of colloidal gold solution: 1% chloroauric acid aqueous solution and 1% trisodium citrate aqueous solution were added at a volume ratio of 1:1-2.5 and gold was boiled down to obtain a colloidal gold solution with a particle size of 20 nm, which was used as seed gold. A second gold boiling was carried out, in which seed gold, 1% chloroauric acid, 1% trisodium citrate and 10% hydroquinone were added at a volume ratio of 1:0.5:1:0.

3. After the reaction was completed, the mixture was cooled to room temperature, the pH was adjusted to 8.0 with sodium hydroxide solution, and centrifuged. The precipitated flower-shaped colloidal gold was redissolved with 0.2% trisodium citrate to obtain a flower-shaped colloidal gold solution with a particle size of 50-60 nm. Preparation of gold-labeled antibody ELISA wells: Adjust the pH of colloidal gold using 0.1-0.2 mol / L potassium carbonate solution, select the optimal pH, add 5-8 μg / mL diazepam monoclonal antibody, spin-label for 30-60 minutes, add 10% bovine serum albumin for blocking, block at room temperature for 30 minutes, then centrifuge at 11000 rpm for 30 minutes at 4°C, discard the supernatant precipitate, reconstitute with 1×PBS reconstitution solution 10 times, add 2-5 μL / well to the gold-labeled antibody ELISA wells treated with 5-10% sucrose, and dry at 37°C overnight.

6. The method for preparing a reagent kit for diazepam and its metabolites according to claim 4, characterized in that, The preparation of the test card includes the following sub-steps: Preparation of nitrocellulose membranes: Dilute diazepam-BSA antigen to 0.2-0.5 mg / mL with 0.01-0.02 mol / L, pH 7.4 PBS buffer, and coat the membrane at the T-line using a coating apparatus at a rate of 0.75 μL / cm; dilute goat anti-mouse secondary antibody to 0.3-0.65 mg / mL with 0.01-0.02 mol / L, pH 7.4 PBS buffer, and coat the membrane at the C-line using a coating apparatus at a rate of 0.75 μL / cm; dry at 37°C overnight; Preparation of the binding pad: Prepare 1L of 4%-8% sucrose solution, immerse the glass fiber SB08 pad in it, remove it after 5 minutes, and dry it at 37℃ overnight; Preparation of sample pads: Weigh 2.9g disodium hydrogen phosphate dodecahydrate, 8.0g sodium chloride, 2.4g potassium chloride, and 2.0g potassium dihydrogen phosphate, dissolve them in 800mL of deionized water, then add 0.5%–1% bovine serum albumin, 0.1%–0.3% sodium casein, 0.5%–1% S17, and 0.5% Tween 20. Mix well and bring the volume to 1L. Adjust the pH to 8.0 with 1mol / L sodium hydroxide solution to obtain the sample pad treatment solution. Then immerse the glass fiber SB08 in the sample pad treatment solution, remove it after 5 minutes, and dry it overnight at 37℃.

7. The method for preparing a reagent kit for diazepam and its metabolites according to claim 4, characterized in that, The preparation of the extractant includes the following sub-steps: Preparation of extractant 1: Weigh 0.5-1g of neutral alumina and 0.3-0.6g of sodium chloride; Preparation of extractant 2: Mix acetonitrile and ethyl acetate in a volume ratio of 1:1 to form an acetonitrile / ethyl acetate mixed solution. Add 5%-10% ammonia water to the acetonitrile / ethyl acetate mixed solution and mix well.

8. The method for preparing a reagent kit for diazepam and its metabolites according to claim 4, characterized in that, The preparation of the complex solution includes the following sub-steps: Weigh 2.9g of disodium hydrogen phosphate dodecahydrate, 8.0g of sodium chloride, 2.4g of potassium chloride, and 2.0g of potassium dihydrogen phosphate. Dissolve them completely in 800mL of deionized water and bring the volume to 1L. Add 10%-20% methanol and mix well.

9. A method for detecting diazepam and its metabolites using a reagent kit, applied to the rapid detection kit as described in any one of claims 1-3, characterized in that, Includes the following steps: To prepare the test solution, the edible parts of aquatic products such as fish and shrimp were crushed using a blender. 5g of the prepared sample was weighed into a 50mL centrifuge tube. Extraction reagent 1 and 7mL of sample extraction reagent 2 were added, and the mixture was vigorously shaken for 3 minutes. The mixture was then centrifuged at 4000 rpm for 5 minutes at room temperature. 5mL of the supernatant was collected into a 10mL centrifuge tube and dried under nitrogen (air) at 65℃. 500μL of n-hexane and 300μL of reconstitution solution were added, mixed, and allowed to stand or centrifuged. The clear lower layer was the test solution. The aquaculture water was collected directly into a clean glass container or centrifuge tube. If the aquaculture water was turbid, it needed to be centrifuged at 4000 rpm for 10 minutes or filtered through a 0.45μm filter membrane to prepare the test solution. After reacting the test solution in the gold-labeled antibody ELISA wells, transfer it to the test card for further reaction to obtain the test results: Before testing, allow the test card and test solution to return to room temperature; remove the test card and gold-labeled antibody ELISA wells from the aluminum foil bag and use them within half an hour; use a pipette to transfer 120 μL of the test solution into the gold-labeled antibody ELISA wells, and repeatedly pipette until the red substance in the gold-labeled antibody ELISA wells is completely dissolved, and wait for the reaction to proceed for 2 minutes; place the test card flat and add all the incubated test solution onto the filter membrane of the test card; react for 8-10 minutes and interpret the results; results interpreted after 20 minutes are invalid.

10. The detection method of a reagent kit for diazepam and its metabolites according to claim 9, characterized in that, The detection results include: the T line being darker or equivalent to the C line; the T line being non-discolored or weaker than the C line; and the C line being non-discolored. A T line being darker or equivalent to the C line indicates a negative result, meaning the concentration of diazepam and its metabolites in the sample is below the detection limit. A T line being non-discolored or weaker than the C line indicates a positive result, meaning the concentration of diazepam and its metabolites in the sample is equal to or higher than the detection limit. A C line being non-discolored indicates an invalid result, meaning the reagent is faulty or the operation was incorrect.

Images