Sample pad processing method for drug abuse detection, detection method and chromatographic test paper

Abstract

The invention relates to the technical field of immunochromatography, in particular to a sample pad processing method for drug abuse detection, a detection method and chromatography test paper. According to the present invention, the sample pad treatment liquid of the immunochromatography test paper for drug abuse detection is innovated, such that the Pluronic F-127-containing sample pad treatment liquid is adopted to treat the sample pad so as to significantly improve the drug abuse item detection sensitivity of the immunochromatography test paper;

Description

Technical Field

[0001] The invention relates to the technical field of immunochromatography, and in particular to a sample pad processing method, a detection method and a chromatography test paper for drug abuse detection. Background Art

[0002] Drug abuse is a social problem faced by the whole world. Common drug abuse tests mainly include methamphetamine (ice), morphine, tetrahydrocannabinol, cocaine and ketamine. With the expansion of the world drug market and the emergence of various new drugs, the cost of drug use has decreased, and the number of drug users has increased year by year. The global monitoring and crackdown on drug abuse is constantly increasing, which also greatly drives the market demand for drug and drug abuse monitoring and detection POCT products.

[0003] At present, there are many methods and instruments for rapid detection of drug abuse, which mainly rely on gas chromatography (GS), high performance liquid chromatography (HPLC), mass spectrometry (MS) and capillary electrophoresis. However, the instruments are expensive, the detection time is too long, and professional technicians are required to operate. Therefore, a fast, accurate and convenient detection method is needed.

[0004] The principle of immunochromatography technology is to use the specific binding reaction of antigen and antibody and the specific color reaction to accurately display the test results on the test paper without the need for complex equipment. For example, the colloidal gold lateral chromatography competition method is used for rapid detection of drug abuse. The small molecule antigen in the sample specifically binds to the recombinant antibody coupled with colloidal gold. As the sample is chromatographed, the antibody-AU conjugate cannot bind to the small molecule antigen-BSA coupling coated on the NC membrane, and a visible red strip cannot be formed on the NC membrane. The color intensity is inversely proportional to the antigen in the sample. After 10 minutes, the chromatography results are interpreted and compared with the color card to determine the quality of protein activity. The method is simple, easy to control, and takes less time.

[0005] The existing immunochromatographic test strips have the problem of insufficient sensitivity and precision in detecting drug abuse substances because the results are affected by substances such as fatty acids and positively charged proteins in the sample. In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a sample pad processing method, a detection method and a chromatography test paper for drug abuse detection.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides a sample pad processing method for drug abuse detection, comprising: contacting a sample pad with a sample pad processing solution; a component of the sample pad processing solution includes Pluronic F-127.

[0009] In a second aspect, an embodiment of the present invention provides a method for detecting drug abuse, which uses an immunochromatographic test strip to detect a sample to be tested, comprising: using the sample pad processing method described in the above embodiment to process the sample pad.

[0010] In a third aspect, an embodiment of the present invention provides a chromatographic test paper for detecting drug abuse, wherein the chromatographic test paper comprises a sample pad, and the sample pad is processed by the sample pad processing method described in the above embodiment.

[0011] In a fourth aspect, an embodiment of the present invention provides the use of Pluronic F-127 in a sample pad treatment solution of a drug abuse detection reagent.

[0012] The present invention has the following beneficial effects:

[0013] The present invention introduces surfactant Pluronic F-127 into the sample pad treatment liquid of the immunochromatographic test paper for drug abuse detection, so that the immunochromatographic test paper can be used for multiple items of drug abuse detection and the sensitivity of these detection items is significantly improved. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0015] The inventors of the present application unexpectedly discovered that by introducing the surfactant Pluronic F-127 into the sample pad treatment solution of the immunochromatographic test strip for drug abuse detection, the immunochromatographic test strip can be used for multiple items of drug abuse detection and the sensitivity of these detection items can be significantly improved.

[0016] In some embodiments, in the sample pad treatment solution, the mass volume percentage of the Pluronic F-127 is 0.1% to 1.5%. The final concentration can be any one of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4% and 1.5%, or any two thereof. The "%" is the mass volume percentage, for example, 1% means 1g per 100ml.

[0017] On the one hand, an embodiment of the present invention provides a sample pad processing method for drug abuse detection, which includes: contacting a sample pad with a sample pad processing liquid; the sample pad processing liquid includes Pluronic F-127. Pluronic F-127 is also known as Poloxamer 407, that is, Polyethylene-polypropylene glycol, which is a polyoxyethylene polyoxypropylene ether triblock copolymer, with a Chinese name of poloxamer, polyether or polyoxyethylene polyoxypropylene, and a molecular weight of about 12.6kD.

[0018] In some embodiments, the sample can be any sample suspected of containing the component to be tested, without particular limitation, such as a throat swab sample, an oropharyngeal swab sample, a nasopharyngeal swab sample, a sputum sample, a bronchoalveolar lavage fluid sample, a peripheral blood sample, a plasma sample, or a serum sample.

[0019] In some embodiments, the sample is a sample collected by a throat swab or a sample collected by a nasopharyngeal swab, but is not limited thereto.

[0020] In some embodiments, the drug abuse detection object includes: any one or more of morphine, barbiturates, alcohol, cocaine, marijuana, amphetamines, ketamines and hallucinogens.

[0021] In some embodiments, the detection object includes: any one or more of methylenedioxymethamphetamine (MDMA), barbiturates (BAR), phencyclidine (PCP), ethyl glucuronide (ETG), cocaine (COC), amphetamine (AMP), methadone (MTD), benzodiazepine (BZO) and methamphetamine (MET).

[0022] In some embodiments, the sample pad treatment solution further comprises components of: at least one of a buffer, a protein, a salt, and a polymer substance.

[0023] In some embodiments, the buffer is selected from any one of Tris-HCl, phosphate buffer and borate buffer.

[0024] In some embodiments, the final concentration of the buffer is 10-100 mM, and the pH is 6.0-10.0. The final concentration can be any one of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mM or a range between any two thereof; the pH can be any one of 6.0, 7.0, 8.0, 9.0 and 10.0 or a range between any two thereof.

[0025] In some embodiments, the protein is selected from any one of Casein and BSA.

[0026] In some embodiments, in the sample pad treatment solution, the final concentration (mass volume percentage) of the protein is 0.1% to 1%. The final concentration can be any one of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1%, or any two thereof. The "%" is the mass volume percentage, for example, 1% means 1g per 100ml.

[0027] In some embodiments, the salt is selected from any one of NaCl and magnesium sulfate;

[0028] In some embodiments, in the sample pad treatment solution, the final concentration of magnesium sulfate is 50-150 mM, and the final concentration can be any one of 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, and 150 mM, or a range between any two of the above.

[0029] In some embodiments, in the sample pad treatment solution, the final concentration of NaCl is 0.8% to 1.5%. The final concentration can be any one of 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4% and 1.5%, or any two thereof. The "%" is a mass volume percentage.

[0030] In some embodiments, the polymer substance is selected from any one of PVP, PVA and PEG.

[0031] In some embodiments, in the sample pad treatment solution, the final concentration of the polymer substance is 0.5% to 1.5%. The final concentration can be any one of 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4% and 1.5%, or any two of them. The "%" is the mass volume percentage.

[0032] In some embodiments, the method of contacting the sample pad with the sample pad treatment solution is selected from any one of the following:

[0033] (1) When preparing a reagent strip, adding the sample pad treatment solution to a sample pad of an immunochromatography device;

[0034] (2) mixing the sample with the sample pad treatment solution and adding the mixture to the sample pad of the immunochromatography;

[0035] (3) When using the reagent strip, first add the sample pad treatment solution to the sample pad of the immunochromatography, and then add the sample.

[0036] On the other hand, an embodiment of the present invention further provides a method for detecting drug abuse, using an immunochromatographic test strip to detect a sample to be tested, which includes: using the sample pad processing method described in any of the above embodiments to process the sample pad. In some embodiments, the detection specifically includes: before or during the addition of the sample to the sample pad, using the sample pad processing method described in any of the above embodiments to process the sample and / or the sample pad.

[0037] In some embodiments, the drug abuse detection object includes: any one or more of morphine, barbiturates, alcohol, cocaine, marijuana, amphetamines, ketamines and hallucinogens.

[0038] In some embodiments, the detection object includes: any one or more of methylenedioxymethamphetamine, barbiturates, phencyclidine, ethyl glucuronide, cocaine, amphetamine, methadone, benzodiazepine and methamphetamine.

[0039] On the other hand, the present invention also provides a chromatography test paper for detecting drug abuse, wherein the chromatography test paper includes a sample pad, and the sample pad is processed by the sample pad processing method described in any of the above embodiments. In some embodiments, the chromatography test paper also includes: a bottom plate, a conjugate pad, a chromatography membrane, and a water absorbent pad.

[0040] In some embodiments, the method for preparing the chromatography test paper comprises: sequentially overlapping a sample pad, a conjugate pad, a chromatography membrane, and a water absorbent pad on a bottom plate.

[0041] In addition, the embodiments of the present invention also provide the use of Pluronic F-127 as a component of a sample pad treatment solution in the preparation of a lateral flow test paper for detecting drug abuse.

[0042] On the other hand, the embodiments of the present invention also provide the use of Pluronic F-127 in preparing a sample pad treatment solution for a drug abuse detection reagent.

[0043] In some embodiments, Pluronic F-127 and a polymer substance are used in preparing a sample pad treatment solution for a drug abuse detection reagent.

[0044] On the other hand, an embodiment of the present invention further provides a sample pad treatment solution for a drug abuse detection reagent, which at least includes Pluronic F-127.

[0045] In some embodiments, the sample pad treating solution of the drug abuse detection reagent comprises at least Pluronic F-127 and a polymer substance.

[0046] In some embodiments, the detection employs a competition method.

[0047] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0048] Example 1

[0049] The whole process of preparing immunochromatographic reagent strips:

[0050] (1) Coating:

[0051] Dilute the coating proteins (T-line coating protein and C-line coating protein) to a certain concentration with coating diluent, and use a gold dot film sprayer to evenly draw detection lines on the NC membrane (T line and C line, both at 1.0 μl / cm), taking care not to scratch the membrane, and then put it into a 50-degree air drying oven for drying for more than 4 hours before use.

[0052] (2) Marking:

[0053] 1ml 4 / 10,000AU, add a certain amount of 0.2mol / L K2CO3, shake well for 1min, then add the labeled protein, shake well for 1min, and finally add the blocking agent, shake well for 1min. Collect the precipitate after centrifugation at 10,000 rpm / 7min in a Xiangyi desktop centrifuge, and concentrate the precipitate to 1 / 10 of the labeled volume. The number of centrifugal revolutions can be adjusted appropriately according to the centrifugal effect. The supernatant after centrifugation is required to be light pink or colorless, and there is no irresolvable precipitate.

[0054] (3) Gold spraying: The complex of protein and colloidal gold labeled in (2) is diluted to a specified concentration and then sprayed with gold, and dried for at least 2 hours.

[0055] (4) Sample pad preparation: Spread 2.4 ml of the prepared sample pad treatment solution on a 1.7 cm wide and 30 cm long glass fiber and dry at 50°C for more than 2 h.

[0056] (5) Assembly: Assemble the sample pad, base plate, absorbent paper, conjugate pad, coating film, etc., cut into 3.0 mm pieces, and make the corresponding immunoassay reagent strips.

[0057] Example 2

[0058] 2.1 Base fluid 1

[0059] Formula of basic solution 1: 25mM Tris-HCl pH8.0, 0.3% Casein, 0.9% NaCl, 1% PEG-20000

[0060] 2.2 Sample pad treatment reagent based on base liquid

[0061] Formula of base solution 1-1: add 1% S6 to base solution 1

[0062] Formula of base solution 1-2: add 1% S9 to base solution 1

[0063] Formula of Base Solution 1-3: Add 1% SDS to Base Solution 1

[0064] Formula for Base Solution 1-4: Add 1% Triton X-405 to Base Solution 1

[0065] Formula for Base Fluid 1-5: Add 1% Pluronic F-127 to Base Fluid 1

[0066] Formula for Base Fluid 1-6: Add 1% Sodium N-lauroyl Sarcosinate to Base Fluid 1

[0067] Formula for Base Solution 1-7: Add 1% Triton X-100 to Base Solution 1

[0068] Formula for Base Fluid 1-8: Add 1% Tween-20 to Base Fluid 1

[0069] Formula for Base Fluid 1-9: Add 1% span-80 to Base Fluid 1

[0070] Formula for Base Fluid 1-10: Add 1% CTAB to Base Fluid 1

[0071] In order to explore the surfactant components in the sample pad treatment solution, the performance of commercially available surfactants and Pluronic F-127 were compared, and the obtained sample pad treatment solution was as described above. The above components were used as sample pad treatment solution to treat the sample pad, and after drying, it was assembled into an immunochromatographic device. During the test, the gradient diluted MDMA positive quality control product and negative quality control product were dripped onto the treated sample pad. The colloidal gold card reading value was recorded. The smaller the value, the stronger the color development, indicating a higher sensitivity.

[0072] The results are shown in Table 1. Only when Pluronic F-127 is added to the base solution 1, the sensitivity is significantly improved, and the band elimination can be achieved in the competitive detection of MDMA.

[0073] Table 1 Test results of MDMA samples and control samples prepared with reagent strips using basic treatment solution

[0074]

[0075] Note: The above is the colloidal gold card reading. When the result is C, it indicates that the test line or the control line has a color change. The smaller the number after C, the deeper the color change and the more obvious the color development. When the result is B, it indicates that the color of the untested line or the control line has not changed. The competition method requires that the positive quality control can eliminate the band at a certain concentration (that is, when the color is B). The lower the corresponding positive quality control concentration is when the band is eliminated, the higher the sensitivity is.

[0076] Example 3

[0077] In order to explore the best solution for sample pad treatment solution for different project detection, a more in-depth comparative analysis of the components of different sample pad treatment solutions was conducted. The sample pad treatment solution was prepared according to the solution in Table 2 below.

[0078] Table 2 Improved formula of sample pad treatment solution

[0079]

[0080]

[0081] Note: “%” in the table refers to mass volume percentage.

[0082] The above components were used as sample pad treatment liquid to treat the sample pad, and after drying, they were assembled into an immunochromatographic device. During the test, the gradient-diluted MDMA positive quality control product and negative quality control product were dripped onto the treated sample pad. The results were recorded as colloidal gold card readings, and the specific results are shown in Table 3 below.

[0083] Table 3 Test results of MDMA samples and control samples prepared with reagent strips using improved treatment solution

[0084]

[0085] Example 4

[0086] The application of the reagent strips processed with the sample pad treatment solution in detecting other drug abuse items is shown in the following table.

[0087] Table 4. Results of BAR, COC, MTD, and BZO detection by sample pad treatment solution

[0088]

[0089]

[0090] Table 5. Results of ETG, AMP, and MET detection by reagent strips treated with sample pad treatment solution

[0091]

[0092] Table 6. PCP test results of sample pad treatment solution treated reagent strips

[0093]

[0094] In addition, the applicant further discovered that using the sample pad treatment solution containing Pluronic F-127 in the example in non-competitive immunoassay can still significantly improve the detection effect.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A sample pad processing method for drug abuse detection, characterized in that: It includes: contacting the sample pad with a sample pad treatment solution; The sample pad treatment solution comprises Pluronic F-127.

2. The sample pad processing method according to claim 1, characterized in that: The mass volume percentage of the Pluronic F-127 is 0.1% to 1.5%.

3. The sample pad processing method according to claim 1, characterized in that: The drug abuse detection objects include: any one or more of morphine, barbiturates, alcohol, cocaine, marijuana, amphetamines, ketamines and hallucinogens.

4. The sample pad processing method according to claim 3, characterized in that: The detection objects include: any one or more of methylenedioxymethamphetamine, barbiturates, phencyclidine, ethyl glucuronide, cocaine, amphetamine, methadone, benzodiazepine and methamphetamine.

5. The sample pad processing method according to claim 1, characterized in that: The components of the sample pad treatment solution also include polymer substances; Optionally, the polymer substance is selected from any one of PVP, PVA and PEG; Optionally, in the sample pad treatment solution, the mass volume percentage of the polymer substance is: 0.5% to 1.5%; The sample pad treatment solution further comprises at least one of a buffer, a protein and a salt.

6. The sample pad processing method according to any one of claims 1 to 5, characterized in that: The method of contacting the sample pad with the sample pad treatment solution is selected from any one of the following: (1) When preparing a reagent strip, adding the sample pad treatment solution to a sample pad of an immunochromatography device; (2) mixing the sample with the sample pad treatment solution and adding the mixture to the sample pad of the immunochromatography; (3) When using the reagent strip, first add the sample pad treatment solution to the sample pad of the immunochromatography, and then add the sample.

7. A method for detecting drug abuse, using immunochromatographic test paper to detect a sample to be tested, characterized in that: It includes: The sample pad is treated by the sample pad treatment method according to any one of claims 1 to 6.

8. The method according to claim 7, characterized in that The drug abuse detection objects include: any one or more of morphine, barbiturates, alcohol, cocaine, marijuana, amphetamines, ketamines and hallucinogens.

9. The method according to claim 8, characterized in that The detection objects include: any one or more of methylenedioxymethamphetamine, barbiturates, phencyclidine, ethyl glucuronide, cocaine, amphetamine, methadone, benzodiazepine and methamphetamine.

10. A chromatographic test paper for detecting drug abuse, characterized in that: The chromatography test paper comprises a sample pad, and the sample pad is processed by the sample pad processing method according to any one of claims 1 to 6.

11. Application of Pluronic F-127 in the preparation of sample pad treatment solution for drug abuse detection reagents.

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