A methamphetamine test card for preventing urine sample fraud and its preparation method and application

Abstract

The present invention relates to the field of biological detection technology, and in particular to a methamphetamine detection card for preventing urine sample fraud, and a preparation method and application thereof. The methamphetamine detection card of the present invention comprises a temperature sensing area, a methamphetamine detection area and a urine property detection area; wherein the temperature sensing area is used to detect urine temperature, the methamphetamine detection area is used to detect the content of methamphetamine, and the urine property detection area is used to detect the physical and chemical properties of urine; the detection card can simultaneously detect the content of methamphetamine in urine samples and detect fraud such as whether the urine samples are diluted, and has high specificity and sensitivity for the fraud detection of urine samples and the content detection of methamphetamine, can effectively prevent missed detection caused by fraud such as dilution of urine samples, improve the accuracy of methamphetamine detection, and has important application value in methamphetamine detection.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a methamphetamine detection card for preventing urine sample counterfeiting, and a preparation method and application thereof. Background Art

[0002] Methamphetamine (methamphetamine, deoxyephedrine, ice) is a new type of drug and the most widely abused drug in the world in the 21st century. Due to the strong toxicity of ice, long-term inhalation will reduce the body's immunity, damage the body's organs, and in severe cases, cause death. In addition, taking ice will cause strong stimulation to the human central nervous system. Large doses will cause mental disorders in the human body, manifested as emotional impulses, paranoia, delusions, and easily lead to violent behavior.

[0003] The detection methods of methamphetamine mainly include chromatography, enzyme-linked immunosorbent (ELISA), high-performance capillary electrophoresis, and colloidal gold. High-performance liquid chromatography and gas chromatography-mass spectrometry have the advantages of good repeatability, high sensitivity, and the ability to quantitatively detect, and are the gold standard for drug detection, but because of their cumbersome operation process and high detection cost, they are not suitable as conventional detection methods. ELISA is a commonly used method for methamphetamine detection, and has the advantages of high sensitivity, low cost, and simple operation, but this method requires certain sample processing, and the entire detection process requires 2-3 hours, which cannot meet the needs of on-site rapid detection. Immunochromatography such as colloidal gold has the advantages of simple operation, the result can be interpreted within ten minutes, the test results are obtained, no supporting equipment is required, the reagents are simple to store, and the detection cost is low, and has become an ideal method for on-site rapid detection of methamphetamine.

[0004] The composition and detection principle of the existing methamphetamine rapid test card (colloidal gold method) are as follows: a water absorbent pad, an Nc film sprayed with methamphetamine-BSA antigen (test line) and goat anti-mouse secondary antibody (quality control line), a gold label pad sprayed with methamphetamine antibody and a sample absorption pad are stacked and fixed on a reaction support plate in sequence, and then cut into test strips and loaded into a plastic module to obtain a methamphetamine determination test card. A urine sample to be tested is added to the sample well, and the chromatography process begins. The gold nano-labeled methamphetamine antibody on the gold label pad is released, and the methamphetamine in the sample will compete with the methamphetamine-BSA antigen of the test line to bind to the colloidal gold-labeled methamphetamine antibody, so that the color reaction of the test line is light or colorless, and the unbound gold nano-labeled methamphetamine antibody particles will bind to the goat anti-mouse secondary antibody at the quality control line to produce a color reaction, and the test result is obtained by visual inspection, thereby realizing the rapid detection of drugs in the sample.

[0005] However, in the actual detection process, when the urine sample of a suspected drug user is tested, if the drug user evades the inspection by diluting or switching urine samples, the accuracy of the test results will be affected. Although the existing methamphetamine rapid test card (colloidal gold method) can achieve rapid detection, it cannot effectively avoid the missed detection caused by the suspected drug user diluting or switching urine samples. Therefore, it is of great significance to develop a methamphetamine rapid test card that prevents urine sample fraud. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the purpose of the present invention is to provide a test card that can simultaneously detect whether a urine sample is fake and the methamphetamine content therein. Another purpose of the present invention is to provide a preparation method and application of the test card.

[0007] To achieve the above purpose, the present invention creatively introduces a urine temperature sensing area and a urine property detection area. Among them, the urine temperature sensing area can determine whether the urine sample has been adulterated or switched by dilution by detecting the urine temperature, and the urine property detection area can determine whether the urine sample has been adulterated by dilution by detecting the characteristic physical and chemical indicators of urine. The combination of the two can achieve functional complementarity and coordination, and effectively improve the detection rate of urine sample fraud. Further, by optimizing the urine temperature sensing area, the urine property detection area and the methamphetamine detection area, mutual interference between the detection areas is effectively avoided, and the specificity and sensitivity of methamphetamine detection are guaranteed while ensuring the specificity and sensitivity of urine sample fraud detection.

[0008] Specifically, the technical solution of the present invention is as follows:

[0009] First, the present invention provides a methamphetamine detection card, which includes a temperature sensing area and a reaction area; the reaction area includes a methamphetamine detection area and a urine property detection area; wherein the temperature sensing area is used to detect the temperature of a urine sample; the methamphetamine detection area is used to detect the content of methamphetamine in a urine sample; the urine property detection area is used to detect the physicochemical properties of a urine sample, and the physicochemical properties of the urine are selected from one or more of urine microalbumin, urine creatinine, urine calcium, protein, leukocytes, occult blood, glucose, ketone bodies, urobilinogen, specific gravity, bilirubin, pH, vitamin C, and nitrite.

[0010] The methamphetamine test card is used to test a urine sample, and the content of methamphetamine in the urine sample is determined based on the test results of the methamphetamine test area; and whether the urine sample to be tested is fake is determined based on the test results of the temperature sensing area and the urine property test area.

[0011] In the present invention, the detection index of the physicochemical property of urine can specifically be: urine creatinine, or urine creatinine and urine calcium.

[0012] Through extensive research and practice, the present invention has determined a urine physicochemical property detection index that is relatively more suitable for complementing the temperature sensing area, preferably urine creatinine content or urine creatinine content and urine calcium content. The above physicochemical property index combined with temperature sensing can better improve the sensitivity and specificity of urine sample fraud detection.

[0013] In order to better ensure the rapidity and convenience of the detection, the methamphetamine detection area is an immunochromatography detection area, preferably a colloidal gold immunochromatography.

[0014] By designing and optimizing the positional relationship of each detection area, it is helpful to reduce the mutual interference of detection between the areas. Preferably, the structure of the detection card is as follows: the temperature sensing area is provided with an independent sample addition area; the methamphetamine detection area and the urine property detection area share a sample addition area, and the methamphetamine detection area and the urine property detection area are arranged in sequence along the chromatography direction of the urine sample.

[0015] Preferably, the structure of the methamphetamine detection card of the present invention is as follows: the temperature sensing area includes a thermochromic plastic; the urine property detection area includes a urine property detection block; and a reaction liquid for detecting the physical and chemical properties of urine is fixed on the urine property detection block.

[0016] The decolorization temperature range of the thermochromic plastic is preferably 33°C to 37°C.

[0017] As a preferred embodiment of the present invention, the test card comprises a backing and a thermochromic plastic, a sample pad, a conjugate release pad, a methamphetamine reaction membrane, an absorption pad and a urine property detection block sequentially attached to the backing along the long axis of the test card from one end to the other; any two adjacent ones of the sample pad, the conjugate release pad, the methamphetamine reaction membrane, the absorption pad and the urine property detection block are partially overlapped and attached to the backing. The thermochromic plastic is directly attached to the backing without overlapping.

[0018] The conjugate release pad is coated with a labeled methamphetamine antibody; the methamphetamine reaction membrane is fixed with a methamphetamine antigen and a secondary antibody; the methamphetamine antigen and the secondary antibody respectively form a detection line (T line) and a quality control line (C line); the detection line is close to the conjugate release pad, and the quality control line is close to the absorption pad.

[0019] Preferably, a distance of 2 to 4 mm is provided between the thermochromic plastic and the sample pad; 1 / 3 to 1 / 2 of the conjugate release pad is covered under the sample absorption pad.

[0020] Preferably, the detection line and the quality control line are parallel to each other and perpendicular to the long axis direction of the detection card. Optionally, the detection card is packaged in a card shell, and a sample addition hole is provided at a corresponding position of the thermochromic plastic; and a sample addition hole is provided at a corresponding position of the sample pad. The detection card may also be provided with two visual observation windows, which are respectively located at the corresponding positions of the detection line and the quality control line and the corresponding position of the urine property detection block.

[0021] In the temperature sensing area, by using a thermochromic plastic that can sense temperature and change color, the temperature range of urine can be simply and quickly determined. The thermochromic plastic is prepared from a raw material including a reversible thermochromic compound; the reversible thermochromic compound includes the following components in parts by weight: cryptagent: 0.3-0.8 parts of thermosensitive rose red or crystal violet lactone; developer: 2-5 parts of bisphenol A or benzyl parahydroxybenzoate; solvent: 25-35 parts of tetradecanol. The use of the reversible thermochromic compound in the temperature sensing area can better cooperate with the urine property detection area to achieve false detection of urine samples, while not affecting the detection of the methamphetamine detection area.

[0022] As a preferred embodiment of the present invention, the mass ratio of the leuco agent, the developer and the solvent is 1:6:60.

[0023] The raw materials for preparing the reversible thermochromic compound also include plastic raw materials; in the raw materials, the mass percentage of the reversible thermochromic compound is 20% to 30%.

[0024] As an embodiment of the present invention, the urine physicochemical property is the urine creatinine content, and the reaction solution for detecting the physicochemical property of urine includes an enzyme substrate and a chromogen. The enzyme substrate includes the following components in units of enzyme activity: 5 to 25 parts of creatinase, 5 to 25 parts of peroxidase: 5 to 25 parts of creatinine enzyme: 15 to 75 parts of sarcosine oxidase. By controlling the ratio of key reaction enzymes in the urine creatinine detection reagent within the above range, it is more conducive to improving the sensitivity and specificity of the detection card for urine sample fraud detection.

[0025] The chromogen is 4-aminoantipyrine and one selected from sodium N-ethyl-N(2-hydroxy-3-propylsulfonyl)-3,5-dimethylaniline, phenol, p-chlorophenol, p-hydroxybenzoic acid and sodium 3,5-dichloro-2-hydroxybenzenesulfonate.

[0026] Preferably, the reaction solution for detecting the physical and chemical properties of urine comprises the following components:

[0027] As a preferred embodiment of the present invention, in the reaction solution for detecting the physical and chemical properties of urine, the enzyme activity ratio of creatinase, creatinase, sarcosine oxidase, and peroxidase is 1:1:3:1. In the reaction solution for detecting the physical and chemical properties of urine, the molar ratio of 4-aminoantipyrine to sodium N-ethyl-N(2-hydroxy-3-propanesulfonyl)-3,5-dimethylaniline, phenol, p-chlorophenol, p-hydroxybenzoic acid, or sodium 3,5-dichloro-2-hydroxybenzenesulfonate is 1:1.

[0028] In the methamphetamine detection zone, the labeled methamphetamine antibody is preferably a colloidal gold labeled methamphetamine antibody; the methamphetamine antigen is preferably methamphetamine coupled to a carrier protein.

[0029] The carrier protein includes but is not limited to BSA, KLH, etc., preferably BSA. The type of the secondary antibody is determined according to the source of the methamphetamine antibody, for example, it can be a goat anti-mouse secondary antibody.

[0030] Preferably, during colloidal gold labeling, the ratio of the methamphetamine antibody to the colloidal gold is (1-10) μg of methamphetamine antibody / ml of colloidal gold; the concentration of the methamphetamine antigen is 0.2-2 mg / ml; and the concentration of the secondary antibody is 0.2-0.5 mg / ml.

[0031] The methamphetamine detection card of the present invention can be prepared by using the following materials: sample pad: glass cellulose membrane; conjugate release pad: glass fiber membrane or polyester membrane; methamphetamine reaction membrane: nitrocellulose membrane or cellulose acetate membrane; absorption pad: absorbent paper; urine property detection block: filter paper / nylon membrane; backing: PVC.

[0032] The present invention also provides a methamphetamine detection kit, which comprises the methamphetamine detection card and a urine creatinine reaction colorimetric card; the surface of the urine creatinine reaction colorimetric card is provided with different urine creatinine concentration value markings and a standard color block of the urine property detection block corresponding to the urine creatinine concentration value markings.

[0033] Optionally, the different urine creatinine concentration values ​​are indicated as 0, 0.9, 4.4, 8.8, 17.7, and 26.5 mmol / L.

[0034] The detection principle of the methamphetamine detection card of the present invention is as follows: a newly collected urine sample is added to the sample adding hole in the temperature sensing area, and the urine is temperature-sensitive in the temperature sensing area. The temperature of a normal urine sample is about 37°C, and the thermochromic plastic undergoes a color change reaction (the color-reducing temperature range is 33°C to 37°C), and the color becomes lighter. However, the temperature of a urine sample that has been adulterated or switched by dilution is generally lower than 33°C, and the thermochromic plastic does not change color and still maintains the original color. The urine sample is added from the sample adding hole in the reaction area, and the urine sample is moved from the sample pad along the sample pad. When the chromatography direction reaches the gold label pad, the methamphetamine antibodies labeled with gold nanoparticles are released. The methamphetamine in the urine sample competes with the methamphetamine antigen coated on the test line (T line) for binding to the colloidal gold-labeled methamphetamine antibodies, resulting in the methamphetamine antigen on the test line being unable to bind to the colloidal gold-labeled methamphetamine antibodies, making the test line light or colorless; when the remaining colloidal gold-labeled methamphetamine antibodies continue to chromatographically reach the quality control line (C line), they specifically bind to the secondary antibody coated on the quality control line and develop color. A negative result for methamphetamine is when both the test line and the quality control line are colored; a positive result is when the test line becomes lighter or disappears and the quality control line is colored; if the quality control line does not show color, it is invalid; finally, the urine sample reaches the urine property detection block and undergoes a color reaction with the fixed reaction liquid components on the reaction block (among which, the principle of urine creatinine detection is: creatinine in urine will produce hydrogen peroxide under the catalysis of related enzymes, and hydrogen peroxide and 4-aminoantipyrine will undergo a color reaction under the catalysis of peroxidase). If the urine sample is diluted or adulterated, the concentration of urine creatinine will decrease, and compared with the colorimetric card, it will be lower than the normal value.

[0035] On the other hand, the present invention also provides a method for preparing the methamphetamine detection card, comprising the following steps:

[0036] (1) Preparation of thermochromic plastic: After the solvent is completely melted, a leucochromic agent and a developer are added to react to obtain a reversible thermochromic compound; the reversible thermochromic compound is added to a plastic raw material at a mass ratio of 20% to 30%, and molded to obtain a plastic sheet with grooves;

[0037] (2) Preparation of a conjugate release pad coated with a labeled methamphetamine antibody: soaking a glass fiber membrane or a polyester membrane in a buffer solution of pH 7.0 to 8.0 for 2 to 3 hours and drying; spraying the labeled methamphetamine antibody at 5 to 25 μl / cm on the soaked and dried glass fiber membrane or polyester membrane;

[0038] (3) Preparation of methamphetamine reaction membrane: Spray the methamphetamine antigen solution and the secondary antibody solution at 5-10 μL / cm and 5-15 μL / cm respectively on the nitrocellulose membrane or cellulose acetate membrane to form the detection line and quality control line respectively;

[0039] (4) Preparation of urine property detection block: prepare a reaction solution for detecting the physical and chemical properties of urine according to a certain proportion, and soak the filter paper or nylon membrane in the reaction solution for 2 to 3 hours;

[0040] (5) Assembly of the test card: overlap and paste the sample pad, conjugate release pad, methamphetamine reaction membrane, absorption pad and urine property detection block on the backing in sequence; directly paste the thermochromic plastic on the backing without overlapping and cut.

[0041] The present invention also provides any of the following applications of the methamphetamine detection card or the methamphetamine detection kit:

[0042] (1) Application in the detection of methamphetamine content;

[0043] (2) Application in the detection of methamphetamine adulteration in urine samples.

[0044] The present invention also provides a method for detecting methamphetamine in a urine sample and eliminating the falsification of the urine sample, comprising: taking a urine sample to be tested, using the methamphetamine detection card or the methamphetamine detection kit to perform detection, judging the content of methamphetamine in the urine sample to be tested according to the detection result of the methamphetamine detection area; judging whether the urine sample to be tested is falsified according to the detection results of the temperature sensing area and the urine property detection area.

[0045] Specifically, the detection method comprises: adding 80 to 130 μl of freshly collected urine sample along the sample wells of the temperature sensing area and the sample wells of the reaction area, reacting for 1 to 2 minutes, observing the detection result of the temperature sensing area through the observation window of the temperature sensing area, and reacting for 3 to 8 minutes to read the result through the observation window of the reaction area; the result interpretation method is: negative result (the urine sample is not falsified and the methamphetamine test is negative): the color of the temperature sensing area is decolorized, the T line and the C line are colored, and the color of the urine creatinine reaction block is compared with the urine creatinine reaction ratio The color card is normal when compared; positive result (urine sample is not falsified and methamphetamine test is positive): the temperature sensing area is colorless, the T line is light or colorless, the C line is purple-red, and the color of the urine creatinine reaction block is normal when compared with the urine creatinine reaction color card; falsified result (urine sample is falsified): the temperature sensing area is not colorless or the color of the urine creatinine reaction block is lighter than the normal value of the urine creatinine reaction color card, or the temperature sensing area is not colorless and the color of the urine creatinine reaction block is lighter than the normal value of the urine creatinine reaction color card; invalid result: the C line is not colored.

[0046] The invention has the beneficial effects that: the methamphetamine detection card of the invention comprises a temperature sensing area, a methamphetamine detection area and a urine property detection area, and can simultaneously realize the detection of the methamphetamine content in a urine sample and the detection of whether the urine sample has been diluted or other falsifications on one detection card, and has high specificity and sensitivity for the detection of the falsification of urine samples and the detection of the methamphetamine content; the methamphetamine detection card of the invention can effectively prevent missed detection caused by the suspected drug addicts diluting or adulterating urine samples and other falsifications, improve the accuracy of methamphetamine detection, and has important application value in methamphetamine detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The diagram is a schematic diagram of the structure of the methamphetamine detection card in Examples 1, 2 and 3 of the present invention.

[0048] Figure 2 1 is a top view of the methamphetamine detection card in Examples 1, 2 and 3 of the present invention.

[0049] Figure 1 and Figure 2 In the figure, 1: thermochromic plastic, 2: sample pad, 3: gold label pad, 4: methamphetamine reaction membrane, 5: detection line (T), 6: quality control line (C), 7: absorption pad, 8: backing, 9: urine property detection block, 10: temperature sensing area sample well, 11: reaction area sample well.

[0050] Figure 3 This is a graph showing the test results of the methamphetamine test cards of Example 1 and Comparative Example 1 in Experimental Example 1 of the present invention; wherein the concentration unit of the urine creatinine colorimetric card is mmol / L.

[0051] Figure 4 This is a test result diagram of the methamphetamine test card in Experimental Example 8 of the present invention; wherein the concentration unit of the urine creatinine colorimetric card is mmol / L. DETAILED DESCRIPTION

[0052] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0054] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Among them, methamphetamine antibody was purchased from Longji Biotechnology Co., Ltd. with the catalog number MD00101; methamphetamine-BSA antigen was purchased from Longji Biotechnology Co., Ltd. with the catalog number AD00102; and goat anti-mouse secondary antibody was purchased from Feipeng Biotechnology Co., Ltd.

[0055] Example 1 Methamphetamine Detection Card (1)

[0056] This embodiment provides a methamphetamine detection card, and its structural schematic diagram is as follows Figure 1 As shown, the top view is Figure 2 As shown, the preparation method is as follows:

[0057] (1) Preparation of thermochromic plastic: Weigh 30g of tetradecanol solvent, stir and melt at 70°C, add 0.5g of thermosensitive rose red and 3g of bisphenol A at the same time after complete melting, stir and react to obtain a reversible thermochromic compound. After natural cooling, add 30% by mass to 78g of PE raw material, and form a plastic sheet with grooves by molding;

[0058] (2) Preparation of colloidal gold: Heat 50 mL of 0.01% chloroauric acid aqueous solution to boiling, add 750 μl of 1% trisodium citrate solution, and continue boiling for 10 minutes after the solution changes from golden yellow to purple red. After cooling naturally, dilute to 50 ml;

[0059] (3) Preparation of gold label pad: soak the glass fiber membrane in 50 mM pH 7.4 PBS buffer (containing 0.1% Tween 20, 0.9% NaCl, 0.1% BSA and 0.5% trehalose) for 2 h, and dry at 37°C for 2 h for use; adjust the pH of 50 ml colloidal gold solution to 7.8, add 100 μg of methamphetamine antibody under stirring, then add 250 μl 5% PEG20000, continue stirring for 30 minutes, and finally add 2.5 ml 5% BSA solution for blocking for 1 hour; centrifuge at 3000 rpm for 15 minutes to obtain colloidal gold antibody precipitate, resuspend it in a re-solution (50 mM pH 7.4 PBS buffer containing 0.1% Tween 20, 0.1% BSA and 1% sucrose) to 50 ml; spray it on the glass fiber membrane according to the parameter of 25 μl / cm, dry it at 37°C for 3 h, and place it in a dry environment for use;

[0060] (4) Preparation of methamphetamine reaction membrane: 0.5 mg / mL methamphetamine-BSA antigen was prepared with 5 ml of 50 mM pH 7.4 PBS buffer, and sprayed on the nitrocellulose membrane at a parameter of 10 μl / cm to form a 0.7 mm wide detection line; 0.2 mg / mL goat anti-mouse secondary antibody was prepared with 10 ml of 50 mM pH 7.4 PBS buffer, and coated on the nitrocellulose membrane at a parameter of 15 μl / cm to form a 0.7 mm wide quality control line; the coated nitrocellulose membrane was placed at 37°C to dry for 3 h and placed in a dry environment for use;

[0061] (5) Preparation of urine creatinine reaction block: Prepare a reaction solution for urine creatinine detection, the formula is as follows (50 ml): 50 mM pH 7.0 PBS buffer (solvent), Tween 20 0.1%, sucrose 0.5%, proclin 300 0.1%, vitamin C oxidase 5 U / mL, creatinase 5 U / mL, creatinine enzyme 5 U / mL, peroxidase 5 U / mL, sarcosine oxidase 15 U / mL, 4-aminoantipyrine 2 mmol / L, N-ethyl-N (2-hydroxy-3-propylsulfonyl) -3,5-dimethylaniline sodium 2 mmol / L; soak the filter paper in the prepared reaction solution for 2 h, dry it at 37 ° C for 3 h, and place it in a dry environment for use;

[0062] (6) Assembly of the test card: In order from one end of the long axis of the test card to the other end, overlap the thermochromic plastic 1, sample pad 2, gold label pad 3, methamphetamine reaction membrane 4, absorption pad 7, and urine creatinine reaction block 9 and stick them on the backing 8, wherein 1 / 3 of the conjugate release pad is covered under the sample pad, and the remaining adjacent parts overlap to cover a length of 1 mm. The thermochromic plastic does not need to be overlapped and directly attached to the PVC backing. Cut the spliced ​​test paper into 4 mm width and assemble the card; the upper card shell is provided with a sample addition hole 10 above the thermochromic plastic and a sample addition hole 11 above the sample pad, and two visual observation windows are provided above the test line 5 and the quality control line 6 and above the urine creatinine reaction block; after the test card is assembled, it is put into an aluminum foil bag, sealed with a desiccant, and placed in a dry environment at 4 to 30 ° C for 24 months.

[0063] The preparation method of the urine creatinine reaction colorimetric card used in conjunction with the methamphetamine detection card is as follows: using a urine creatinine reaction block to respectively detect urine samples with creatinine concentrations of 0, 0.9, 4.4, 8.8, 17.7, and 26.5 mmol / L, taking photos to record the color development results, matching each concentration value with a color, and printing to produce a colorimetric card.

[0064] Example 2 Methamphetamine Detection Card (2)

[0065] This embodiment provides a methamphetamine detection card, and its structural schematic diagram is as follows Figure 1 As shown, the top view is Figure 2 As shown, the preparation method is as follows:

[0066] (1) Preparation of thermochromic plastic: Weigh 30g of tetradecanol solvent, stir and melt at 70°C, add 0.5g of thermosensitive rose red and 3g of bisphenol A at the same time after complete melting, stir and react to obtain a reversible thermochromic compound. After natural cooling, add 20% by mass to 134g of PE raw material, and form a plastic sheet with grooves by molding;

[0067] (2) Preparation of colloidal gold: Heat 50 mL of 0.03% chloroauric acid aqueous solution to boiling, add 500 μl of 1.5% trisodium citrate solution, and continue boiling for 15 minutes after the solution changes from golden yellow to purple red. After cooling naturally, dilute to 50 ml;

[0068] (3) Preparation of gold label pad: soak the glass fiber membrane in 50 mM pH 7.6 HEPES buffer (containing 0.1% Tween 80, 0.9% NaCl, 0.2% BSA and 0.5% trehalose) for 2 h and dry; adjust the pH of 50 ml colloidal gold solution to 8.0, add 200 μg methamphetamine antibody under stirring, then add 250 μl of 10% PEG 4000, continue stirring for 60 min, finally add 2.5 ml of 5% BSA solution, block for 1 h, centrifuge at 5000 rpm for 15 min to obtain colloidal gold antibody precipitate, resuspend to 50 ml with resol (containing 50 mM pH 7.6 HEPES buffer containing 0.3% BSA, 0.1% Tween 80 and 1% sucrose), spray on the glass fiber membrane at 15 μl / cm, dry at 37°C for 4 h, and place in a dry environment for use;

[0069] (4) Preparation of methamphetamine reaction membrane: 1 mg / mL methamphetamine-BSA antigen was prepared with 10 ml 50 mM pH 7.6 HEPES buffer, and sprayed on the nitrocellulose membrane at a parameter of 7 μl / cm to form a 0.8 mm wide detection line; 0.3 mg / mL goat anti-mouse secondary antibody was prepared with 20 ml 50 mM pH 7.6 HEPES buffer, and sprayed on the nitrocellulose membrane at a parameter of 7 μL / cm to form a 0.8 mm wide quality control line; the coated nitrocellulose membrane was dried at 37°C for 4 h and placed in a dry environment for use;

[0070] (5) Preparation of urine creatinine reaction block: Prepare a reaction solution for urine creatinine detection, the formula is as follows (50 ml): 50 mM pH 7.6 HEPES buffer (solvent), Tween 80 0.15%, sucrose 0.75%, proclin 300 0.1%, vitamin C oxidase 10 U / ml, creatinase 10 U / mL, creatinine enzyme 10 U / mL, peroxidase 10 U / mL, sarcosine oxidase 30 U / mL, 4-aminoantipyrine 4 mmol / L, N-ethyl-N (2-hydroxy-3-propylsulfonyl) -3,5-dimethylaniline sodium 4 mmol / L; soak the filter paper in the prepared reaction solution for 2 h, dry it at 37 ° C for 4 h, and place it in a dry environment for use;

[0071] (6) Assembly of the test card: overlap the thermochromic plastic 1, sample pad 2, gold label pad 3, methamphetamine reaction membrane 4, absorption pad 7, and urine creatinine reaction block 9 on the backing 8 in the order from one end of the long axis of the test card to the other end, wherein 1 / 3 of the conjugate release pad is covered under the sample absorption pad, and the remaining adjacent parts overlap to cover a length of 1 mm. The thermochromic plastic does not need to be overlapped and is directly attached to the PVC backing. Cut the spliced ​​test paper into 4 mm width and assemble the card; the upper card shell is provided with a sample addition hole 10 above the thermochromic plastic and a sample addition hole 11 above the sample pad, and two visual observation windows are provided above the test line 5 and the quality control line 6 and above the urine creatinine reaction block; after the test card is assembled, it is put into an aluminum foil bag, sealed with a desiccant, and placed in a dry environment at 4 to 30 ° C for 24 months.

[0072] The preparation method of the urine creatinine reaction colorimetric card used in conjunction with the methamphetamine detection card is as follows: using a urine creatinine reaction block to respectively detect urine samples with creatinine concentrations of 0, 0.9, 4.4, 8.8, 17.7, and 26.5 mmol / L, taking photos to record the color development results, matching each concentration value with a color, and printing to produce a colorimetric card.

[0073] Example 3 Methamphetamine Detection Card (3)

[0074] This embodiment provides a methamphetamine detection card, and its structural schematic diagram is as follows Figure 1 As shown, the top view is Figure 2 As shown, the preparation method is as follows:

[0075] (1) Preparation of thermochromic plastic: 30 g of tetradecanol was weighed and stirred to melt at 70° C. After complete melting, 0.5 g of crystal violet lactone and 3 g of benzyl p-hydroxybenzoate were added simultaneously and stirred to react to obtain a reversible thermochromic compound; after natural cooling, the mixture was added to 100.5 g of PE raw material at a mass ratio of 25%, and a plastic sheet with grooves was prepared by molding;

[0076] (2) Preparation of colloidal gold: Heat 50 ml of 0.05% chloroauric acid aqueous solution to boiling, add 750 μl of chloroauric acid aqueous solution to a volume of 2% trisodium citrate solution, and continue boiling for 20 minutes after the solution changes from golden yellow to purple red. After cooling naturally, dilute to 50 ml;

[0077] (3) Preparation of gold label pad: Soak polyester film in 50 mM pH 8.0 Tris buffer (containing 0.5% TritonX, 0.8% NaCl, 0.5% BSA and 1% sucrose) for 3 h, and dry at 37°C for 5 h for use; adjust the pH of 50 ml colloidal gold solution to 8.2, add 500 μg of methamphetamine antibody under stirring, then add 250 μl PVP, continue stirring for 30 minutes, add 500 μl 10% BSA solution, and block for 2 hours; centrifuge at 7000 rpm for 20 minutes to obtain colloidal gold antibody precipitate, resuspend in resol (containing 0.5% BSA, 0.5% TritonX and 5% trehalose in 50 mM pH 8.0 Tris buffer) to 50 ml; spray on glass fiber membrane or polyester membrane at 5 μl / cm2, dry at 37°C for 2 h, and place in a dry environment for use;

[0078] (4) Preparation of methamphetamine reaction membrane: 2 mg / mL methamphetamine-BSA antigen was prepared with 15 ml 50 mM pH 8.0 Tris buffer, and sprayed on the nitrocellulose membrane at 5 μl / cm to form a 1 mm wide detection line; 0.5 mg / mL goat anti-mouse secondary antibody was prepared with 15 ml 50 mM pH 8.0 Tris buffer, and coated on the nitrocellulose membrane at 5 μL / cm to form a 1 mm wide quality control line; the coated nitrocellulose membrane was placed at 37°C to dry for 5 h and placed in a dry environment for use;

[0079] (5) Preparation of urine creatinine reaction block: Prepare a reaction solution for urine creatinine detection, the formula is as follows (50 ml): 50 mM pH 8.0 Tris buffer (solvent), TritonX 0.5%, trehalose 1%, NaN3 0.5%, vitamin C oxidase 15 U / mL, creatinase 25 U / mL, creatininase 25 U / mL, peroxidase 25 U / mL, sarcosine oxidase 75 U / mL, 4-aminoantipyrine 8 mmol / L, phenol 8 mmol / L; soak the nylon membrane in the prepared reaction solution for 3 h, dry it at 37°C for 5 h, and place it in a dry environment for use;

[0080] (6) Assembly of the test card: overlap the thermochromic plastic 1, sample pad 2, gold label pad 3, methamphetamine reaction membrane 4, absorption pad 7, and urine creatinine reaction block 9 on the backing 8 in the order from one end of the long axis of the test card to the other end, wherein 1 / 3 of the conjugate release pad is covered under the sample absorption pad, and the remaining adjacent parts overlap to cover a length of 1 mm. The thermochromic plastic does not need to be overlapped and is directly attached to the PVC backing. Cut the spliced ​​test paper into 4 mm width and assemble the card; the upper card shell is provided with a sample addition hole 10 above the thermochromic plastic and a sample addition hole 11 above the sample pad, and two visual observation windows are provided above the test line 5 and the quality control line 6 and above the urine creatinine reaction block; after the test card is assembled, it is put into an aluminum foil bag, sealed with a desiccant, and placed in a dry environment at 4 to 30 ° C for 24 months.

[0081] The preparation method of the urine creatinine reaction colorimetric card used in conjunction with the methamphetamine detection card is as follows: using a urine creatinine reaction block to respectively detect urine samples with creatinine concentrations of 0, 0.9, 4.4, 8.8, 17.7, and 26.5 mmol / L, taking photos to record the color development results, matching each concentration value with a color, and printing to produce a colorimetric card.

[0082] Embodiment 4 The detection method of methamphetamine

[0083] The test cards prepared in Examples 1 to 3 were restored to room temperature, taken out of the aluminum foil bag, and 100 μl of freshly collected urine samples were added to the sample wells in the temperature sensing area and the sample wells in the reaction area, respectively. The reaction was performed for 1 minute, and the results in the temperature sensing area were observed. The reaction was performed for 5 minutes, and the methamphetamine and urine creatinine test results were read through the observation window.

[0084] The result interpretation method is as follows: Negative result (urine sample is not falsified and methamphetamine test is negative): the temperature sensing area is colorless (the color becomes significantly lighter or colorless), the T line and C line are colored, and the color of the urine creatinine reaction block is normal compared with the urine creatinine reaction colorimetric card (4.4-17.7mmol / L); Positive result (urine sample is not falsified and methamphetamine test is positive): the temperature sensing area is colorless, the T line is light or colorless, the C line is purple-red, and the urine is The color of the creatinine reaction block is normal when compared with the urine creatinine reaction colorimetric card (4.4-17.7mmol / L); Falsified result (falsified urine sample): the temperature sensing area does not fade (still retains the original rose red or blue-purple color) or the color of the urine creatinine reaction block is lighter than the normal value of the urine creatinine reaction colorimetric card (4.4-17.7mmol / L), or the temperature sensing area does not fade and the color of the urine creatinine reaction block is lighter than the normal value of the urine creatinine reaction colorimetric card; Invalid result: the C line does not show color.

[0085] Comparative Example 1

[0086] This comparative example provides a methamphetamine detection card, which differs from the detection card of Example 1 only in that it lacks a urine creatinine reaction block and only retains a temperature sensing area and a methamphetamine detection area.

[0087] Experimental Example 1: Analysis of the accuracy of methamphetamine test card anti-counterfeiting detection

[0088] The urine samples were tested using the methamphetamine test cards of Examples 1 to 3 and Comparative Example 1, respectively, and the specific method is as follows:

[0089] (1) Preparation of urine samples: Sample 1 with a methamphetamine concentration of 1000 ng / ml was prepared using methamphetamine-negative urine and methamphetamine standard, and sample 2 was obtained by diluting sample 1 10 times;

[0090] (2) Samples 1 and 2 were heated to 37° C. and immediately tested using the test cards of Example 1 and Comparative Example 1. The test method was the same as that of Example 4.

[0091] The test results are as follows: for sample 1, the test card results of Example 1 and Comparative Example 1 both showed positive for methamphetamine; for sample 2, the test card result of the urine creatinine reaction block of Comparative Example 1 was negative, and the color of the urine creatinine reaction block of Example 1 was lighter than the color corresponding to the normal concentration range (4.4-17.7mmol / L), and the color was close to the color corresponding to 0.9mmol / L, which was determined to be a false negative result ( Figure 3 ). The results show that the synergistic effect of the urine creatinine reaction block and the temperature sensing area can better prevent fraud.

[0092] The test results of the test cards prepared in Examples 2 and 3 for samples 1 and 2 are the same as those in Example 1.

[0093] Experimental Example 2 Sensitivity Analysis of Methamphetamine Detection Card

[0094] The standard substance used for sensitivity assessment is methamphetamine standard (batch number 171212-201605), which was purchased from the China Food and Biological Products Control Institute.

[0095] Urine samples with a final concentration of 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 ng / ml of methamphetamine were prepared using methamphetamine-negative urine and methamphetamine standards. The urine samples were tested using the test card prepared in Example 1. The test method was the same as in Example 4, and each concentration was tested 5 times. The test results showed that urine samples with a concentration of 500 to 1000 ng / ml were all positive for methamphetamine in 5 tests.

[0096] Samples with methamphetamine concentrations of 425, 450, and 475 ng / ml were further prepared, and the urine samples were tested using the test card prepared in Example 1. The test method was the same as in Example 4, and the test was repeated 5 times for each concentration. The test results showed that the results were all positive when the concentration was 475 ng / ml, the results were all weakly positive when the concentration was 450 ng / ml, and the results were all negative when the concentration was 425 ng / ml. The above results show that the minimum detection limit of the test card is 450 ng / ml.

[0097] The minimum detection limit results of the test cards prepared in Examples 2 and 3 are the same as those in Example 1.

[0098] Experimental Example 3 Negative Reference Conformity Analysis

[0099] Aspirin, paracetamol, chlorpheniramine maleate, caffeine, pseudoephedrine, buprenorphine, ephedrine, tramadol, N-benzyl isopropylamine, and dimethyl sulfone standards were added to the negative urine to make the final concentration of each standard 100 μg / ml. The prepared urine samples were tested using the test cards prepared in Examples 1 to 3, respectively. The detection method was the same as that in Example 4, and the detection was repeated 5 times for each concentration.

[0100] The test results showed that no positive results were detected in the test cards of Examples 1 to 3, and the conformity rate of the negative reference was 100%.

[0101] Experimental Example 4: Analysis of the Conformity Rate of Positive Reference Products

[0102] The standard substance used for the compliance evaluation of the positive reference substance is: methamphetamine standard (batch number 171212-201605), which was purchased from the China Food and Biological Products Control Institute.

[0103] Urine samples with a final concentration of 500 ng / ml, 1000 ng / ml, and 1500 ng / ml of methamphetamine were prepared using methamphetamine negative urine and standard substances, and tested using the test cards of Examples 1 to 3, respectively. The test method was the same as that of Example 4, and each sample was tested 10 times. The test results were all positive, and the positive reference product compliance rate was 100%.

[0104] Experimental Example 5 Repeatability Analysis of Methamphetamine Test Card

[0105] The standard substance used for repeatability evaluation was methamphetamine standard (batch number 171212-201605), which was purchased from the China Food and Biological Products Control Institute.

[0106] The test cards of Examples 1 to 3 of the same batch were taken and the urine samples with methamphetamine concentrations of 500 and 1000 ng / ml were tested 10 times using the test method of Example 4. The test results were all positive, indicating that the test cards of Examples 1 to 3 had good test repeatability.

[0107] Experimental Example 6: Verification of the Anti-Counterfeiting Detection Capability of Methamphetamine Detection Card

[0108] Sample 1 with a methamphetamine concentration of 1000 ng / ml was prepared using negative urine, and sample 1 was diluted stepwise by 5, 10, 20, 50, and 100 times to obtain samples 2 to 6, respectively. The sample temperature was maintained at 37° C. and the test cards of Examples 1 to 3 and the test method of Example 4 were used for testing, respectively. Each sample was tested 10 times.

[0109] The test results show that when all samples were tested using the test cards of Examples 1 to 3, the temperature sensing areas were all colorless, the methamphetamine tests of samples 2 to 6 were all negative, and the urine creatinine reaction blocks of samples 3 to 6 were all lighter in color than the normal value of the urine creatinine reaction colorimetric card, indicating that the minimum dilution of urine that can be detected by the test cards of Examples 1 to 3 is 10 times dilution, with no upper limit.

[0110] Experimental Example 7 Analysis of the stability of the effective period of methamphetamine test card

[0111] The test cards of Examples 1 to 3 of the same batch were stored at room temperature, and the sensitivity, positive-negative coincidence rate, repeatability and other properties of each test card were tested using the test methods of Experimental Examples 2, 4 and 5 after 3, 6, 12, 18, 20, 22, 24 and 25 months of storage, respectively.

[0112] The test results show that when stored for 3 to 25 months, the sensitivity, positive reference compliance rate and repeatability of the test card all meet the standards, indicating that the methamphetamine test card of the present invention can be stably stored at room temperature for 24 months and still maintain high accuracy and sensitivity.

[0113] Experimental Example 8 Application of Methamphetamine Test Card in Detection of Fake Urine Samples

[0114] Basic information of the person to be tested: Female, 24 years old. The drug inspection department conducted a surprise inspection on a certain entertainment venue. The newly collected urine sample provided by the person was tested using the test card of Example 1 and the test method of Example 4. The test results are as follows: Figure 4As shown, the thermochromic plastic did not fade, the methamphetamine test result was negative, and the urine creatinine concentration was lower than the normal value (4.4-17.7mmol / L) by comparing with the urine creatinine reaction colorimetric card, indicating that the sample was a fake sample. A fresh urine sample was collected for testing (retest), the thermochromic plastic faded, the methamphetamine test result was positive, and the urine creatinine concentration was within the normal range (4.4-17.7mmol / L), indicating that the person under test had drug abuse.

[0115] Although the present invention has been described in detail above by means of general description, specific implementation methods and tests, it is obvious to those skilled in the art that some modifications or improvements may be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A methamphetamine detection card, characterized in that: It comprises a temperature sensing area and a reaction area; the reaction area comprises a methamphetamine detection area and a urine property detection area; the temperature sensing area is used to detect the temperature of a urine sample; the methamphetamine detection area is used to detect the content of methamphetamine in a urine sample; the urine property detection area is used to detect the physical and chemical properties of a urine sample, and the physical and chemical properties are selected from one or more of urine microalbumin, urine creatinine, urine calcium, protein, leukocytes, occult blood, glucose, ketone bodies, urobilinogen, specific gravity, bilirubin, pH, vitamin C and nitrite; The temperature sensing area includes a thermochromic plastic; the urine property detection area includes a urine property detection block; the urine property detection block is fixed with a reaction liquid for detecting the physical and chemical properties of urine; The reaction solution for detecting the physical and chemical properties of urine comprises the following components: Tween20, Tween80 or TritonX 0.05%~0.5%; Sucrose or trehalose 0.5%~1%; proclin300 or NaN3 0.1%~0.5%; Creatinase 5~25U / mL; Creatinase 5~25U / mL; Peroxidase 5~25U / mL; Sarcosine oxidase 15~75U / mL; 4-aminoantipyrine 2~8mmol / L; Sodium N-ethyl-N(2-hydroxy-3-propanesulfonyl)-3,5-dimethylaniline, phenol, p-chlorophenol, p-hydroxybenzoic acid or sodium 3,5-dichloro-2-hydroxybenzenesulfonate 2~8mmol / L; Vitamin C oxidase 2-15 U / mL; pH 7.0~8.

0.

2. The methamphetamine test card according to claim 1, characterized in that: The physicochemical property is urine creatinine, or urine creatinine and urine calcium.

3. The methamphetamine detection card according to claim 1 or 2, characterized in that: The methamphetamine detection area is an immunochromatographic detection area; the temperature sensing area is provided with an independent sample addition area; the methamphetamine detection area and the urine property detection area share a sample addition area, and the methamphetamine detection area and the urine property detection area are arranged in sequence along the chromatography direction of the urine sample.

4. The methamphetamine detection card according to claim 1 or 2, characterized in that: The test card comprises a backing and a thermochromic plastic, a sample pad, a conjugate release pad, a methamphetamine reaction membrane, an absorption pad and a urine property detection block sequentially attached to the backing along one end to the other end of the long axis of the test card; any two adjacent ones of the sample pad, the conjugate release pad, the methamphetamine reaction membrane, the absorption pad and the urine property detection block are partially overlapped; The conjugate release pad is coated with a labeled methamphetamine antibody; the methamphetamine reaction membrane is fixed with a methamphetamine antigen and a secondary antibody; the methamphetamine antigen and the secondary antibody form a detection line and a quality control line respectively; the detection line is close to the conjugate release pad, and the quality control line is close to the absorption pad.

5. The methamphetamine detection card according to claim 1 or 2, characterized in that: The thermochromic plastic is prepared from raw materials including a reversible thermochromic compound; the reversible thermochromic compound includes the following components in parts by weight: a leucochromic agent: 0.3-0.8 parts of thermosensitive rose red or crystal violet lactone; a developer: 2-5 parts of bisphenol A or benzyl p-hydroxybenzoate; Solvent: 25-35 parts of tetradecanol.

6. The methamphetamine detection card according to claim 4, characterized in that: The labeled methamphetamine antibody is colloidal gold labeled methamphetamine antibody; the methamphetamine antigen is methamphetamine coupled to a carrier protein.

7. The methamphetamine detection card according to claim 6, characterized in that: 1-10 µg of methamphetamine antibody is labeled with colloidal gold per ml; the concentration of methamphetamine antigen is 0.2-2 mg / ml; and the concentration of the secondary antibody is 0.2-0.5 mg / ml.

8. The method for preparing the methamphetamine test card according to any one of claims 1 to 7, characterized in that: include: (1) Preparation of thermochromic plastic: After the solvent is completely melted, a leucochromic agent and a developer are added to react to obtain a reversible thermochromic compound; the reversible thermochromic compound is added to a plastic raw material at a mass ratio of 20% to 30%, and molded to obtain a plastic sheet with grooves; (2) Preparation of a conjugate release pad coated with labeled methamphetamine antibodies: soak a glass fiber membrane or polyester membrane in a pH 7.0-8.0 buffer for 2-3 hours and dry it; spray the labeled methamphetamine antibodies onto the soaked and dried glass fiber membrane or polyester membrane at a rate of 5-25 µl / cm; (3) Preparation of methamphetamine reaction membrane: Spray the methamphetamine antigen solution and secondary antibody solution on nitrocellulose membrane or cellulose acetate membrane at 5-10 µL / cm and 5-15 µL / cm, respectively, to form the detection line and quality control line; (4) Preparation of urine property test block: prepare a reaction solution for detecting the physical and chemical properties of urine according to a certain proportion, and soak the filter paper or nylon membrane in the reaction solution for 2 to 3 hours; (5) Assembly of the test card: overlap and stick the two adjacent parts of the sample pad, conjugate release pad, methamphetamine reaction membrane, absorption pad and urine property test block on the backing in sequence; directly stick the thermochromic plastic on the backing without overlapping and cut.

9. A methamphetamine detection kit, characterized in that: Comprising the methamphetamine detection card and urine creatinine reaction colorimetric card according to any one of claims 1 to 7; The surface of the urine creatinine reaction colorimetric card is provided with different urine creatinine concentration value markings and standard color block of the urine property detection block corresponding to the urine creatinine concentration value markings.

10. Any of the following uses of the methamphetamine test card according to any one of claims 1 to 7 or the methamphetamine test kit according to claim 9: (1) Application in the detection of methamphetamine content; (2) Application in the detection of fake urine samples for methamphetamine.

11. A method for detecting methamphetamine in a urine sample and eliminating fraudulent urine samples, characterized in that: Take a urine sample to be tested, and use the methamphetamine test card according to any one of claims 1 to 7 or the methamphetamine test kit according to claim 9 to perform a test, and judge the content of methamphetamine in the urine sample to be tested according to the test result of the methamphetamine detection area; and judge whether the urine sample to be tested is fake according to the test results of the temperature sensing area and the urine property detection area.

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