Sample pad treatment method, detection method and chromatographic test strip for drug abuse detection

Abstract

The present application relates to the field of immunochromatography, in particular to a sample pad treatment method, a detection method and a chromatography test paper for drug abuse detection, and discloses an immunochromatography test paper for drug abuse detection, which is characterized by a sample pad treatment liquid containing Pluronic F-127.

Description

Technical Field

[0001] This invention relates to the field of immunochromatographic technology, and more specifically, to a sample pad processing method, a detection method, and a chromatographic test strip for drug abuse detection. Background Technology

[0002] Currently, there are many methods and instruments for rapid detection of drug abuse, mainly relying on gas chromatography (GS), high performance liquid chromatography (HPLC), mass spectrometry (MS), and capillary electrophoresis. However, these methods are expensive, take too long to detect, and require professional technicians to operate. Therefore, there is a need for a rapid, accurate, and convenient detection method.

[0003] The principle of immunochromatography is to utilize the specific binding reaction between antigens and antibodies, and to accurately display the test results on test strips using a specific colorimetric reaction, without the need for complex equipment. For example, the colloidal gold lateral chromatography competitive method can be used for rapid detection of drug abuse. Small molecule antigens in the sample specifically bind to recombinant antibodies conjugated with colloidal gold. As the sample chromatographically progresses, the antibody-AU conjugate cannot bind to the small molecule antigen-BSA coupler coated on the NC membrane, thus failing to form a visible red band on the NC membrane. The intensity of the color development is inversely proportional to the antigen in the sample. The chromatographic results are interpreted after 10 minutes, and compared with the color chart to determine the quality of protein activity. The method is simple, easy to control, and relatively quick.

[0004] Existing immunochromatographic test strips for detecting substances of drug abuse suffer from insufficient sensitivity and accuracy due to the influence of substances such as fatty acids and positively charged proteins in the sample. Therefore, this invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a sample pad processing method, a detection method, and a chromatography test strip for drug abuse detection.

[0006] This invention is implemented as follows:

[0007] In a first aspect, embodiments of the present invention provide a sample pad treatment method for drug abuse detection, comprising: contacting the sample pad with a sample pad treatment solution; wherein the sample pad treatment solution comprises Pluronic F-127.

[0008] Secondly, embodiments of the present invention provide a method for detecting drug abuse, which uses immunochromatographic test strips to detect the sample to be tested, comprising: processing the sample pad using the sample pad processing method described in the foregoing embodiments.

[0009] In a third aspect, the embodiments of the present application provide a chromatographic test paper for detecting drug abuse, which comprises a sample pad, wherein the sample pad is treated by the sample pad treatment method described in the foregoing embodiments.

[0010] In a fourth aspect, the embodiments of the present application provide an application of Pluronic F-127 to a sample pad treatment solution of a drug abuse detection reagent.

[0011] The present application has the following beneficial effects:

[0012] The present application introduces surfactant Pluronic F-127 into a sample pad treatment solution of an 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

[0013] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If specific conditions are not specified in the embodiments, the conditions are performed according to conventional conditions or manufacturer's recommended conditions. If the used reagents or instruments are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0014] The inventors of the present application accidentally found that by introducing surfactant Pluronic F-127 into a sample pad treatment solution of an immunochromatographic test paper for drug abuse detection, the immunochromatographic test paper can be used for multiple items of drug abuse detection, and the sensitivity of these detection items is significantly improved.

[0015] In some embodiments, in the sample pad treatment solution, the mass-volume percentage of Pluronic F-127 is 0.1% to 1.5%. The final concentration can be specifically 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 a range between any two of them. The "%" is mass-volume percentage, for example, 1% means 1g per 100ml.

[0016] In one aspect, the present application provides a sample pad processing method for drug abuse detection, comprising: contacting a sample pad with a sample pad processing solution; and the sample pad processing solution comprises Pluronic F-127. Pluronic F-127 is also known as Poloxamer 407, i.e. Polyethylene-polypropylene glycol, which is a polyoxyethylene polyoxypropylene ether triblock copolymer, also known as Poloxamer, polyether or polyoxyethylene polyoxypropylene in Chinese, with a molecular weight of about 12.6kD.

[0017] In some embodiments, the sample can be any sample suspected of containing the component to be detected, without particular limitation, such as a throat swab sample, an oropharyngeal swab sample, a nasopharyngeal swab sample, a sputum sample, an alveolar lavage fluid sample, a peripheral blood sample, a plasma sample, or a serum sample.

[0018] In some embodiments, the sample is a throat swab sample or a nasopharyngeal swab sample, but is not limited thereto.

[0019] In some embodiments, the detection object of drug abuse includes any one or more of morphine, barbiturates, alcohol, cocaine, Indian hemp, amphetamines, ketamine, and hallucinogens.

[0020] In some embodiments, the detection object includes any one or more of methylenedioxymethamphetamine (MDMA), barbiturates (BAR), phencyclidine (PCP), ethyl glucuronide (ETG), cocaine (COC), amphetamines (AMP), methadone (MTD), benzodiazepines (BZO), and methamphetamine (MET).

[0021] In some embodiments, the sample pad processing solution further comprises at least one of a buffer, a protein, a salt, and a high molecular weight substance.

[0022] In some embodiments, the buffer is selected from any one of Tris-HCl, phosphate buffer, and borate buffer.

[0023] In some embodiments, the buffer has a final concentration of 10-100mM and a pH of 6.0-10.0. The final concentration can be specifically any one of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100mM, or a range between any two of them; and the pH can be specifically any one of 6.0, 7.0, 8.0, 9.0, and 10.0, or a range between any two of them.

[0024] In some embodiments, the protein is selected from any one of Casein and BSA.

[0025] In some embodiments, the final concentration (mass / volume percentage) of the protein in the sample pad treatment solution is 0.1% to 1%. The final concentration can be specifically any one of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1%, or a range between any two of them. The "%" is mass / volume percentage, for example, 1% means 1 g per 100 ml.

[0026] In some embodiments, the salt is selected from any one of NaCl and magnesium sulfate;

[0027] In some embodiments, the final concentration of the magnesium sulfate in the sample pad treatment solution is 50 to 150 mM. The final concentration can be specifically any one of 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, or a range between any two of them.

[0028] In some embodiments, the final concentration of the NaCl in the sample pad treatment solution is 0.8% to 1.5%. The final concentration can be specifically any one of 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, and 1.5%, or a range between any two of them. The "%" is mass / volume percentage.

[0029] In some embodiments, the high molecular substance is selected from any one of PVP, PVA, and PEG.

[0030] In some embodiments, the final concentration of the high molecular substance in the sample pad treatment solution is 0.5% to 1.5%. The final concentration can be specifically 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 a range between any two of them. The "%" is mass / volume percentage.

[0031] In some embodiments, the way of contacting the sample pad with the sample pad treatment solution is selected from any one of the following:

[0032] (1) When preparing the reagent strip, the sample pad treatment solution is added to the sample pad of the immunochromatography;

[0033] (2) After mixing the sample with the sample pad treatment solution, the sample pad treatment solution is added to the sample pad of the immunochromatography;

[0034] (3) When using the reagent strip, the sample pad treatment solution is added to the sample pad of the immunochromatography first, and then the sample is added.

[0035] In another aspect, the embodiments of the present application also provide a method for detecting drug abuse, which adopts an immunochromatographic test paper to detect a sample, and comprises: treating a sample pad by using the sample pad treatment method according to any of the foregoing embodiments. In some embodiments, the detection specifically comprises: treating the sample and / or the sample pad by using the sample pad treatment method according to any of the foregoing embodiments before or during the sample being added to the sample pad.

[0036] In some embodiments, the detection object of the drug abuse comprises any one or more of morphine, barbiturate, alcohol, cocaine, Indian hemp, amphetamine, ketamine and hallucinogen.

[0037] In some embodiments, the detection object comprises any one or more of methylenedioxymethamphetamine, barbiturate, phencyclidine, ethyl glucuronide, cocaine, amphetamine, methadone, benzodiazepine and methamphetamine.

[0038] In another aspect, the embodiments of the present application also provide a chromatographic test paper for detecting drug abuse, which comprises a sample pad treated by using the sample pad treatment method according to any of the foregoing embodiments. In some embodiments, the chromatographic test paper further comprises a base plate, a conjugate pad, a chromatographic membrane and a water absorption pad.

[0039] In some embodiments, the preparation method of the chromatographic test paper comprises: sequentially connecting the sample pad, the conjugate pad, the chromatographic membrane and the water absorption pad on the base plate.

[0040] In addition, the embodiments of the present application also provide an application of Pluronic F-127 as a component of a sample pad treatment solution in the preparation of a lateral chromatographic test paper for detecting drug abuse.

[0041] In another aspect, the embodiments of the present application also provide an application of Pluronic F-127 in the preparation of a sample pad treatment solution of a drug abuse detection reagent.

[0042] In some embodiments, an application of Pluronic F-127 and a high molecular substance in the preparation of a sample pad treatment solution of a drug abuse detection reagent.

[0043] In another aspect, the embodiments of the present application also provide a sample pad treatment solution of a drug abuse detection reagent, which at least comprises Pluronic F-127.

[0044] In some embodiments, a sample pad treatment solution of a drug abuse detection reagent, which at least comprises Pluronic F-127 and a high molecular substance.

[0045] In some embodiments, the detection employs a competition method.

[0046] The features and nature of the present application will become more apparent from the detailed description set forth below, taken in conjunction with the accompanying drawings.

[0047] Example 1

[0048] Full process of preparing immunochromatography reagent strip:

[0049] (1) Coating:

[0050] Coat the coating proteins (T-line coating protein and C-line coating protein) with coating diluent to a certain concentration, draw the detection lines (T-line and C-line, both 1.0 μl / cm) on the NC membrane with a gold spraying point membrane instrument, and make sure not to scratch the membrane, then put it into a 50-degree air-drying oven for drying for more than 4h before use.

[0051] (2) Labeling:

[0052] 1ml 4 / 10,000AU, add a certain amount of 0.2mol / L K2CO3, shake for 1min, then add the labeled protein, shake for 1min, finally add the blocking agent, shake for 1min. After centrifugation at 10,000rpm for 7min, collect the precipitate, and concentrate the precipitate to 1 / 10 of the labeling volume. The centrifugation speed can be adjusted according to the centrifugation effect, and the supernatant should be light pink or colorless after centrifugation, and there should be no resoluble precipitate.

[0053] (3) Gold spraying: spray the complex of the labeled protein and colloidal gold in (2) after dilution to the specified concentration, dry, and dry for at least 2h.

[0054] (4) Sample pad preparation: spread the prepared sample pad treatment solution on the glass fiber with a width of 1.7cm and a length of 30cm, and dry at 50℃ for more than 2h.

[0055] (5) Assembly: assemble the sample pad, bottom plate, absorbent paper, combination pad, coated membrane, etc., cut them into 3.0mm, and make corresponding immunodetection reagent strips.

[0056] Example 2

[0057] 2.1 Base solution 1

[0058] The formula of base solution 1: 25mM Tris-HCl pH8.0, 0.3% Casein, 0.9% NaCl, 1% PEG-20000

[0059] 2.2 Sample pad treatment reagent based on base solution

[0060] The formula of base solution 1-1: add 1% S6 to base solution 1

[0061] Formulation of base solution 1-2: 1% S9 was added to base solution 1

[0062] Formulation of base solution 1-3: 1% SDS was added to base solution 1

[0063] Formulation of base solution 1-4: 1% Triton X-405 was added to base solution 1

[0064] Formulation of base solution 1-5: 1% Pluronic F-127 was added to base solution 1

[0065] Formulation of base solution 1-6: 1% N-lauroylsarcosine sodium was added to base solution 1

[0066] Formulation of base solution 1-7: 1% Triton X-100 was added to base solution 1

[0067] Formulation of base solution 1-8: 1% Tween-20 was added to base solution 1

[0068] Formulation of base solution 1-9: 1% span-80 was added to base solution 1

[0069] Formulation of base solution 1-10: 1% CTAB was added to base solution 1

[0070] 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 sample pad treatment solution obtained is as described above. The above components were used as sample pad treatment solution to treat sample pads, which were dried and assembled into immunochromatographic devices. When detecting, the gradient-diluted MDMA positive and negative quality control samples were added to the treated sample pads. The colloidal gold color card reading value was recorded, and the smaller the value, the stronger the color development, indicating higher sensitivity.

[0071] The results in Table 1 show that only when Pluronic F-127 is added to base solution 1, the sensitivity is significantly improved, and at the same time, the band can be eliminated in the competitive detection of MDMA.

[0072] Table 1: Detection results of reagent strips prepared from base treatment solutions on MDMA samples and control samples

[0073]

[0074] Note: The above is the colloidal gold color card reading value, when the result is C, it means that the detection line or control line has color change, the smaller the numerical value after C, the darker the color change, the more obvious the color development, and when the result is B, it means that the detection line or control line has no color change; the competition method requires that a certain concentration of positive control can eliminate the band (i.e. color development is B), and when the band is eliminated, the lower the concentration of the positive control, the higher the sensitivity.

[0075] Example 3

[0076] In order to explore the best sample pad treatment liquid scheme for different project detection, the components of different sample pad treatment liquids were further compared and analyzed. The sample pad treatment liquid was prepared according to the scheme in Table 2 below.

[0077] Table 2 Sample pad treatment liquid improvement formula table

[0078]

[0079] Note: The "%" in the table is the mass volume percentage.

[0080] The above components were used as sample pad treatment liquid to treat sample pad, and after drying, the sample pad was assembled into an immunochromatographic device. The gradient diluted MDMA positive control and negative control were added to the treated sample pad during detection. The result was recorded as colloidal gold color card reading value, and the specific results are shown in Table 3 below.

[0081] Table 3 Detection results of improved treatment liquid preparation reagent strip on MDMA sample and control sample

[0082]

[0083] Example 4

[0084] The reagent strip treated by sample pad treatment liquid was applied to detect other drug abuse projects, and the results are shown in the following table.

[0085] Table 4. Results of sample pad treatment liquid treated reagent strip in detecting BAR, COC, MTD and BZO

[0086]

[0087] Table 5. Results of sample pad treatment liquid treated reagent strip in detecting ETG, AMP and MET

[0088]

[0089] Table 6. Results of sample pad treatment liquid treated reagent strip in detecting PCP

[0090]

[0091] In addition, the applicant further found that the sample pad treatment liquid containing Pluronic F-127 in the examples can still significantly improve the detection effect when used in non-competitive immunoassay.

[0092] The preferred embodiments of the present application have been described above with the preferred embodiments, but the present application is not limited to the above examples, and various modifications and changes can be made by those skilled in the art. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A sample pad treatment method for drug abuse detection, characterized by, It comprises: contacting the sample pad with a sample pad treatment solution; the components of the sample pad treatment solution consist of Pluronic F-127, a buffer, a protein, a salt and a high molecular substance; the mass-volume percentage of Pluronic F-127 is 0.1%-1.5%; the salt is NaCl, and the mass-volume percentage of NaCl is 0.8%-1.5%; the protein is Casein, and the mass-volume percentage of Casein is 0.1%-1%; the high molecular substance is PVP-40 or PVA, and the mass-volume percentage of the high molecular substance is 0.5%-1.5%; the buffer is selected from any one of Tris-HCl, phosphate buffer, borate buffer and glycine-NaOH.

2. The sample pad processing method according to claim 1, characterized by, The detection object of drug abuse includes any one or more of morphine, barbiturates, alcohol, cocaine, Indian hemp, amphetamines, ketamine and hallucinogens.

3. The sample pad treatment method according to claim 2, characterized by, The detection object includes any one or more of methylenedioxymethamphetamine, barbiturates, phencyclidine, ethyl glucuronide, cocaine, amphetamines, methadone, benzodiazepines and methamphetamine.

4. The method of claim 1, 2 or 3, wherein The way 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, the sample pad treatment solution is added to the sample pad of immunochromatography; (2) After mixing the sample with the sample pad treatment solution, the sample pad of immunochromatography is added; (3) When using the reagent strip, the sample pad treatment solution is added to the sample pad of immunochromatography first, and then the sample is added.

5. A method for detecting drug abuse, wherein an immuno-chromatographic test strip is used to detect a sample to be tested, characterized in that, It comprises: The sample pad is treated by the sample pad treatment method according to any one of claims 1-4.

6. The method of claim 5, wherein, The detection object of drug abuse includes any one or more of morphine, barbiturates, alcohol, cocaine, Indian hemp, amphetamines, ketamine and hallucinogens.

7. The method of claim 6, wherein, The detection object includes any one or more of methylenedioxymethamphetamine, barbiturates, phencyclidine, ethyl glucuronide, cocaine, amphetamines, methadone, benzodiazepines and methamphetamine.

8. A chromatographic test strip for detecting drug abuse, characterized in that The chromatography test paper comprises a sample pad, which is treated by the sample pad treatment method according to any one of claims 1-4.

9. Use of Pluronic F-127, a buffer, a protein, a salt, and a high molecular weight substance in the manufacture of a sample pad treatment solution for a drug of abuse test, the components of the sample pad treatment solution consisting of: Pluronic F-127, a buffer, a protein, a salt and a high molecular substance; the mass-volume percentage of Pluronic F-127 is 0.1%-1.5%, the salt is NaCl, and the mass-volume percentage of NaCl is 0.8%-1.5%; the protein is Casein, and the mass-volume percentage of Casein is 0.1%-1%; the high molecular substance is PVP-40, and the mass-volume percentage of the high molecular substance is 0.5%-1.5%; and the buffer is selected from any one of Tris-HCl, phosphate buffer, borate buffer and glycine-NaOH.

Images