Hair drug kit and detection method

Abstract

The invention provides a hair drug kit and a detection method, and belongs to the technical field of hair drug detection. The kit body is provided with three parallel detection areas; the detection area comprises a sample inlet, a reaction chamber and a waste liquid collection cavity. The method comprises the following steps: sample collection, sample treatment, detection operation and result judgment. The kit has the characteristics of high sensitivity, high specificity, simplicity in operation, multiple functions and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of hair drug detection, and particularly relates to a hair drug detection kit and a detection method. Background Art

[0002] The problem of drug abuse seriously endangers personal health, family happiness and social stability. Hair drug detection, as an effective detection means, can reflect the drug use situation over a long period of time, and has the advantages of convenient sampling, easy sample preservation, long detection window period, etc. At present, there are some deficiencies in the existing hair drug detection kits on the market. The detection sensitivity of some kits is relatively low, and it is difficult to detect trace amounts of drugs and their metabolites in hair; some kits have insufficient specificity and are easily interfered by other substances, resulting in false positive or false negative results; there are also some kits with a relatively complex operation process, which require professional technical personnel and expensive instrument equipment, and are not conducive to on-site rapid detection and large-scale screening. Therefore, it is of great practical significance to develop a hair drug detection kit with high sensitivity, high specificity and simple operation. Summary of the Invention

[0003] The purpose of the present invention is to provide a hair drug detection kit and a detection method.

[0004] To solve the above technical problems, the purpose of the present invention is achieved as follows:

[0005] A hair drug detection kit includes: a kit body; the kit body is provided with three juxtaposed detection areas; the detection area includes a sample inlet, a reaction chamber and a waste liquid collection chamber;

[0006] The sample inlet is formed by the depression of the kit body and is close to the first end of the kit body; a closable cover is provided at the sample inlet;

[0007] The reaction chamber is opened inside the kit body, the first end is communicated with the sample inlet, the second end is close to the second end of the kit body, and the second end of the reaction chamber is lower than the first end; the bottom surface of the reaction chamber is covered with a polydimethylsiloxane material to form a bottom layer; a glass substrate is compounded on the surface of the bottom layer; a metal thin film layer is provided on the surface of the glass substrate; a specific antibody molecular layer for drugs and their metabolites is provided on the surface of the metal thin film layer; a through hole is opened at the second end of the reaction chamber; the through hole extends downward from the top surface of the kit body to communicate with the reaction chamber;

[0008] The waste liquid collection chamber is opened inside the kit body and is located below the reaction chamber; the first end of the waste liquid collection chamber is communicated with the second end of the reaction chamber, and the second end extends to the first end of the kit body, and the second end of the waste liquid collection chamber is lower than its first end;

[0009] A transparent area is provided on the surface of the kit body; the transparent area faces the reaction chamber.

[0010] On the basis of the above solution and as a preferred solution of the above solution, the inner wall of the reaction chamber is coated with polyethylene glycol to reduce non-specific adsorption.

[0011] On the basis of the above solution and as a preferred solution of the above solution, the preparation method of the specific antibody molecular layer includes: forming a COOH-terminated SAM layer by soaking in an 11-mercaptoundecanoic acid solution; and then conjugating the specific antibody to the SAM layer by the EDC / NHS cross-linking method.

[0012] On the basis of the above solution and as a preferred solution of the above solution, the diameter of the sample inlet is 3-5 mm; the width of the reaction chamber is 2.5-4.5 mm, the length is 30-50 mm; and the depth is 2-3 mm.

[0013] On the basis of the above solution and as a preferred solution of the above solution, a slideway is provided in the kit body; the slideway extends from the second end of the kit body to the connection between the reaction chamber and the waste liquid collection chamber; a sealing baffle is slidably arranged in the slideway; the sealing baffle can cut off the connection between the reaction chamber and the waste liquid collection chamber.

[0014] A method for detecting drugs in hair includes the following steps:

[0015] S1. Sample collection: Use hair scissors to collect a hair sample with a length of about 1-3 cm from the occipital part of the head close to the scalp of the tested person, and the collection amount is 50-100 mg;

[0016] S2. Sample treatment: Put the collected hair sample into an extraction tube, add an appropriate amount of extraction reagent to the extraction tube to completely immerse the hair sample, and the addition amount of the extraction reagent is adjusted according to the amount of the hair sample. Add 0.5-1 ml of extraction reagent for every 10 mg of hair;

[0017] After sealing the extraction tube, place it on an oscillator and oscillate for 30-60 minutes to fully dissolve the drugs and their metabolites in the hair into the extraction reagent; the oscillation frequency is 100-200 rpm;

[0018] After the oscillation is completed, put the extraction tube into a centrifuge and centrifuge at a speed of 3000-5000 rpm for 10-15 minutes to precipitate the hair residue;

[0019] Take the supernatant as the sample to be detected;

[0020] S3. Detection operation: Use a pipetting instrument to aspirate an appropriate amount of the sample to be detected and inject it into the reaction chamber through the injection port of the test kit; the injection volume of the sample is 0.15 - 0.7 ml;

[0021] Start the timer and let the sample react with the immobilized antibody in the reaction chamber for 10 - 15 minutes to allow the target drug to fully bind to the antibody;

[0022] Use a pipetting instrument to aspirate an appropriate amount of the signal amplification reagent and inject it into the reaction chamber to further react with the bound complex for 10 - 15 minutes; the injection volume of the signal amplification reagent is 0.15 - 0.7 ml;

[0023] Use a reader to read the change in SPR signal in the detection area on the bottom surface of the reaction chamber and record the detection result;

[0024] Meanwhile, inject solutions containing known concentrations of the target drug and blank solutions without the target drug into two other detection areas respectively as the positive control group and the negative control group;

[0025] S4. Result interpretation: Compare the detection result with the results of the positive control and the negative control; if the detection signal intensity is higher than that of the negative control and reaches or exceeds the preset threshold, it is determined to be positive, indicating that the hair sample contains the target drug; if the detection signal intensity is similar to or lower than the threshold of the negative control, it is determined to be negative, indicating that the target drug is not detected in the hair sample.

[0026] The beneficial effects of the present invention are as follows:

[0027] 1. High sensitivity: The present invention uses a secondary antibody labeled with gold nanoparticles for signal amplification and combines SPR sensing technology, which can significantly improve the detection sensitivity and can accurately detect trace amounts of drugs and their metabolites in hair.

[0028] 2. High specificity: The specific antibody immobilized on the bottom surface of the reaction chamber of the test kit can accurately recognize the target drug and its metabolites, effectively avoiding interference from other substances, improving the detection specificity, and reducing the occurrence of false positive and false negative results.

[0029] 3. Simple operation: The test kit has a simple composition and a clear operation process, and does not require complex instrument equipment and professional technical personnel.

[0030] 4. Multifunctional detection: The test kit can simultaneously detect multiple common drugs and their metabolites, meeting the detection requirements in different scenarios. Description of the Drawings

[0031] Figure 1 It is a schematic structural diagram of the present invention.

[0032] Figure 2 It is a cross-sectional view of the present invention.

[0033] In the figure: 1. Kit body; 2. Reaction chamber; 3. Waste liquid collection chamber; 4. Sealing cover; 5. Through hole; 6. Transparent area; 7. Sealing baffle; 8. Sampling port. Detailed implementation mode

[0034] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0035] As Figure 1 and Figure 2 shown, a hair drug test kit includes: a kit body 1, the kit body 1 is provided with three juxtaposed detection areas, and the detection areas include a sampling port 8, a reaction chamber 2 and a waste liquid collection chamber 3.

[0036] Among them, the sampling port 8 is circular, formed by the depression of the kit body 1, and is close to the first end of the kit body 1. A closable sealing cover 4 is provided at the sampling port 8. Preferably, the diameter of the sampling port 8 is 3-5 mm.

[0037] The reaction chamber 2 is opened inside the kit body 1, the first end is communicated with the sampling port 8, the second end is close to the second end of the kit body 1, and the second end of the reaction chamber 2 is lower than the first end, making it in an inclined state. Preferably, the width of the reaction chamber 2 is 2.5-4.5 mm, the length is 30-50 mm; the depth is 2-3 mm.

[0038] The bottom surface of the reaction chamber 2 is paved with polydimethylsiloxane material to form a bottom layer, a glass substrate is compounded on the surface of the bottom layer, a metal thin film layer is provided on the surface of the glass substrate, and a specific antibody molecule layer for drugs and their metabolites is provided on the surface of the metal thin film layer. In order to realize the detection of specific drugs in hair, molecules with specific recognition ability, such as antibodies, aptamers, etc., are modified on the metal film surface. These molecules can specifically bind to target drug molecules. When the drug molecules in the hair sample come into contact with the detection area after specific treatment, the drug molecules will bind to the specific molecules on the modification layer, thereby causing a change in the refractive index near the metal surface and generating a detectable SPR signal.

[0039] Among them, the preparation method of the specific antibody molecule layer includes: forming a COOH-terminated SAM layer by soaking in 11-mercaptoundecanoic acid solution; then coupling the specific antibody to the SAM layer by the EDC / NHS cross-linking method.

[0040] At the same time, the inner wall of the reaction chamber 2 is coated with polyethylene glycol to reduce non-specific adsorption.

[0041] A through hole 5 is opened at the second end of the reaction chamber 2, and the through hole 5 extends downward from the top surface of the kit body 1 to communicate with the reaction chamber 2 for pressure relief.

[0042] The waste liquid collection chamber 3 is opened inside the kit body 1 and is located below the reaction chamber 2. The first end of the waste liquid collection chamber 3 is communicated with the second end of the reaction chamber 2, and the second end extends to the first end portion of the kit body 1. Moreover, the second end of the waste liquid collection chamber 3 is lower than its first end, presenting an inclined structure to facilitate the waste liquid to flow into the bottom of the waste liquid collection chamber 3.

[0043] A transparent area 6 is provided on the surface of the kit body 1, and the transparent area 6 faces the reaction chamber 2 directly. The transparent area 6 is made of glass.

[0044] A slideway is opened inside the kit body 1; the slideway extends from the second end portion of the kit body 1 to the connection part where the reaction chamber 2 is communicated with the waste liquid collection chamber 3; a sealing baffle 7 is slidably arranged inside the slideway; the sealing baffle 7 can cut off the communication between the reaction chamber 2 and the waste liquid collection chamber 3 so that the sample stays in the reaction chamber 2.

[0045] A method for detecting drugs in hair includes the following steps:

[0046] S1. Sample collection: Use a hair clipper to collect a hair sample with a length of about 1 - 3 cm from the occipital part close to the scalp of the tested person, and the collection amount is 50 - 100 mg;

[0047] S2. Sample treatment: Put the collected hair sample into an extraction tube, add an appropriate amount of extraction reagent to the extraction tube to completely immerse the hair sample. The addition amount of the extraction reagent is adjusted according to the amount of the hair sample. For every 10 mg of hair, 0.5 - 1 ml of extraction reagent is added. The main components of the extractant are methanol, ethyl acetate and buffer solution. Methanol and ethyl acetate can effectively dissolve the drugs and their metabolites in the hair, and the buffer solution is used to maintain the pH value of the solution stable to ensure the extraction effect. The preferred formula is: methanol accounts for 40% - 60% of the total volume, ethyl acetate accounts for 30% - 50% of the total volume, and the buffer solution accounts for 10% - 20% of the total volume.

[0048] After sealing the extraction tube, place it on an oscillator and oscillate for 30 - 60 minutes to fully dissolve the drugs and their metabolites in the hair into the extraction reagent; the oscillation frequency is 100 - 200 rpm;

[0049] After the oscillation ends, put the extraction tube into a centrifuge and centrifuge at a speed of 3000 - 5000 rpm for 10 - 15 minutes to precipitate the hair residues;

[0050] Take the supernatant as the sample to be detected.

[0051] S3. Detection operation: Use a pipetting device to suck an appropriate amount of the sample to be detected and inject it into the reaction chamber through the injection port of the kit. The sample injection amount is 0.15 - 0.7 ml;

[0052] Start the timer and let the sample react with the immobilized antibody in the reaction chamber for 10 - 15 minutes to allow the target drug to fully bind to the antibody.

[0053] Use a pipetting instrument to aspirate an appropriate amount of signal amplification reagent and inject it into the reaction chamber to further react with the bound complex for 10 - 15 minutes; the injection volume of the signal amplification reagent is 0.15 - 0.7 ml. The signal amplifier is a solution of a secondary antibody labeled with nanogold. Nanogold particles have good optical properties and biocompatibility, and a secondary antibody against the target drug antibody is linked to its surface by chemical coupling. When the target drug binds to the antibody immobilized in the reaction chamber, the nanogold-labeled secondary antibody will further bind to this complex to form a "sandwich" structure, thereby amplifying the detection signal.

[0054] Use a reader to read the change in SPR signal in the detection area on the bottom surface of the reaction chamber and record the detection result;

[0055] Meanwhile, inject solutions containing the target drug with known concentrations and blank solutions without the target drug into two other detection areas respectively as the positive control group and the negative control group.

[0056] S4. Result interpretation: Compare the detection result with the results of the positive control and the negative control; if the detection signal intensity is higher than that of the negative control and reaches or exceeds the preset threshold, it is determined to be positive, indicating that the hair sample contains the target drug; if the detection signal intensity is similar to or lower than the threshold of the negative control, it is determined to be negative, indicating that the target drug is not detected in the hair sample.

[0057] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative labor. Therefore, all technical solutions that can be obtained by those skilled in the art in this technical field based on the concept of the present invention through logical analysis, reasoning, or limited experiments on the basis of the prior art should fall within the protection scope determined by the claims.

Claims

1. A hair drug kit, characterized in that: include: A reagent kit body (1); the reagent kit body (1) is provided with three parallel detection areas; the detection areas include a sample inlet (8), a reaction chamber (2) and a waste liquid collection chamber (3); The sample inlet (8) is formed by a depression of the reagent box body (1) and is close to the first end of the reagent box body (1); an openable and closable cover (4) is provided at the sample inlet (8); The reaction chamber (2) is arranged inside the reagent box body (1), the first end of which is connected to the injection port (8), the second end of which is close to the second end of the reagent box body (1), and the second end of the reaction chamber (2) is lower than the first end; the bottom surface of the reaction chamber (2) is paved with a polydimethylsiloxane material to form a bottom layer; the surface of the bottom layer is composited with a glass substrate; the surface of the glass substrate is provided with a metal film layer; the surface of the metal film layer is provided with a specific antibody molecule layer for drugs and their metabolites; the second end of the reaction chamber (2) is provided with a through hole (5); the through hole (5) extends downward from the top surface of the reagent box body (1) to communicate with the reaction chamber (2); The waste liquid collection chamber (3) is opened inside the reagent box body (1) and is located below the reaction chamber (2); the first end of the waste liquid collection chamber (3) is connected to the second end of the reaction chamber (2), the second end extends to the first end of the reagent box body (1), and the second end of the waste liquid collection chamber (3) is lower than the first end; A transparent area (6) is provided on the surface of the reagent kit body (1); the transparent area (6) faces the reaction chamber (2).

2. A hair drug kit according to claim 1, characterized in that: The inner wall of the reaction chamber (2) is coated with polyethylene glycol to reduce non-specific adsorption.

3. A hair drug kit according to claim 1, characterized in that: The preparation method of the specific antibody molecular layer comprises: forming a COOH-terminated SAM layer by soaking in 11-mercaptoundecanoic acid solution; and coupling the specific antibody to the SAM layer by using an EDC / NHS cross-linking method.

4. A hair drug kit according to claim 1, characterized in that: The diameter of the injection port (8) is 3-5 mm; the width of the reaction chamber (2) is 2.5-4.5 mm, the length is 30-50 mm, and the depth is 2-3 mm.

5. A hair drug kit according to claim 1, characterized in that: A slide is provided in the reagent box body (1); the slide extends from the second end of the reagent box body (1) to the connection point between the reaction chamber (2) and the waste liquid collection chamber (3); a sealing plate (7) is slidably arranged in the slide; the sealing plate (7) can cut off the connection between the reaction chamber (2) and the waste liquid collection chamber (3).

6. A hair drug detection method, characterized in that: The following steps are involved: S1. Sample collection: Use a hair scissor to collect a hair sample of about 1-3 cm in length from the occipital part of the subject's head close to the scalp. The collection volume is 50-100 mg. S2. Sample processing: Put the collected hair sample into an extraction tube, add an appropriate amount of extraction reagent into the extraction tube to completely immerse the hair sample. The amount of extraction reagent added is adjusted according to the amount of the hair sample. 0.5-1 ml of extraction reagent is added for every 10 mg of hair. After sealing the extraction tube, place it on an oscillator and oscillate for 30-60 minutes to allow the drugs and their metabolites in the hair to fully dissolve into the extraction reagent; the oscillation frequency is 100-200 rpm; After shaking, place the extraction tube in a centrifuge and centrifuge at 3000-5000 rpm for 10-15 minutes to precipitate the hair residue; The supernatant was taken as the sample to be tested; S3. Detection operation: Use a pipette to draw an appropriate amount of the sample to be tested and inject it into the reaction chamber through the injection port of the reagent kit; the sample injection volume is 0.15-0.7 ml; Start the timer and allow the sample to react with the fixed antibody in the reaction chamber for 10-15 minutes to allow the target drug to fully bind to the antibody; Use a pipette to draw up an appropriate amount of signal amplification reagent, inject it into the reaction chamber, and further react with the bound complex for 10-15 minutes; the injection volume of the signal amplification reagent is 0.15-0.7 ml; Use a reader to read the SPR signal changes in the detection area on the bottom surface of the reaction chamber and record the detection results; At the same time, a solution containing a known concentration of the target drug and a blank solution without the target drug were injected into the other two test areas as a positive control group and a negative control group respectively; S4. Result interpretation: Compare the test results with those of the positive control and negative control; if the test signal intensity is higher than that of the negative control and reaches or exceeds the preset threshold, it is judged as positive, indicating that the target drug is contained in the hair sample; if the test signal intensity is close to that of the negative control or lower than the threshold, it is judged as negative, indicating that the target drug is not detected in the hair sample.

Images