AU2019101573A4 - Kit for detecting hair trace drugs by fluorescence immunity - Google Patents

Abstract

Abstract The invention discloses a kit for detecting hair trace drugs by fluorescence immunity. The kit comprises sample lysate and fluorescent immunochromatographic test strip; Pyrolysis solution is used to dissolve small drug molecules from hair; The fluorescent immunochromatographic test strip comprises a PVC lining plate, a nitrocellulose membrane, a sample pad and an absorption pad, wherein the sample pad, the nitrocellulose membrane and the absorption pad are sequentially and alternately fixed on the PVC lining plate; The sample pad is provided with a drug antibody-quantum dot nanoparticle composite and rabbit IgG- quantum dot nanoparticle composite; Nitrocellulose membrane is provided with a detection line and a quality control line, wherein the detection line is coated with a drug antigen-bovine serum albumin complex, and the quality control line is coated with sheep anti-rabbit antibody. The invention can quickly, accurately and sensitively detect drugs in hair. Figure 1

Description

Technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a kit for detecting hair trace drugs by fluorescence immunity.

Background art [0002] From the most primitive opium poppy, processed into opium, cocaine and heroin, to the present methamphetamine, morphine and marijuana, drugs have always been close to people's lives. Drugs not only affect the health of drug addicts, but also do great harm to their families, society and country. In order to fight against drugs, governments of various countries have invested a large amount of money. China has also invested a large amount of manpower, material resources and financial resources in rescuing and treating drug addicts, carrying out anti-drug education and scientific research, and stepping up anti-drug efforts. However, how to make effective, fast, accurate and stable detection for drug addicts has become an urgent problem to be solved.

[0003] With the progress of detection methods, compared with traditional blood, urine and other detection objects, hair detection samples are easy to obtain and easy to operate on site, thus avoiding drug addicts' uncoordinated evidence collection and adulteration of samples, thus becoming an important drug detection object.

[0004] At present, gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography, capillary electrophoresis, immunoassay and other detection methods have been used for drug detection. For example, Chinese patent CN101750458B discloses a method for detecting drug residues in human body, which adopts HPLC-Chip-MS/MS and HPLC-MS/MS to detect drug residues, and has the advantages of low cost, high accuracy and high stability. Chinese patent CN104422762A discloses an enzyme-linked immunosorbent assay kit and a detection method for detecting drugs in urine, saliva, blood and hair. The enzyme-linked immunosorbent assay kit has the characteristics of high specificity, high sensitivity and the like.

[0005] With the worsening drug control situation, the demand for new, effective, rapid, accurate and stable detection methods and devices is further amplified.

Summary of the Invention [0006] The purpose of the present invention is to provide a kit for fluorescence immunoassay of hair trace drugs. The invention can quickly, accurately and sensitively detect drugs in hair.

[0007] In order to achieve the above purpose, the present invention adopts the following technical scheme:

[0008] A kit for fluorescence immunoassay of hair trace drugs comprises sample

2019101573 12 Dec 2019 lysate and fluorescence immunochromatographic test strip.

[0009] Said lysate is used to dissolve small drug molecules from hair;

[0010] said fluorescence immunochromatographic test strip comprises a PVC lining plate, a nitrocellulose membrane, a sample pad and an absorption pad, wherein the said sample pad, the nitrocellulose membrane and the absorption pad are sequentially and alternately fixed on the PVC lining plate;

[0011] The SAID sample pad is provided with a drug antibody-quantum dot nanoparticle composite and a rabbit IgG- quantum dot nanoparticle composite;

[0012] The SAID nitrocellulose membrane is provided with a detection line and a quality control line, wherein the detection line is coated with a drug antigen-bovine serum albumin compound, and the quality control line is coated with sheep anti-rabbit antibody.

[0013] The detection object of the present invention is blood test, urine test or saliva test for hair compared with the conventional detection method, which can overcome the difficulty of drug test and evidence collection and the non-cooperation of the detected personnel.

[0014] In the above kit, the mass ratio of lysate to hair is 100: 1.

[0015] In the above kit, it is preferred that the said lysate comprises a surfactant, keratinase and a buffer solution. Among them, keratinase can corrode the surface of hair to form a rough surface, thus facilitating the dissolution of small drug molecules from the sample to be tested. Surfactants can wash stains on the hair surface so that keratinase can fully contact with the hair and further improve the dissolution rate of drugs. The buffer solution can neutralize the side reaction produced by keratinase decomposing hair and improve the biological activity of keratinase.

[0016] Further, the said surfactant is preferably a nonionic surfactant, such as polyvinyl pyrrolidone (PVP), Triton X 100, Triton X 114, Triton X 405, Tween 20, Tween 60, Tween 80. Compared with ionic surfactants, nonionic surfactants are not easy to affect the ionic strength, thus avoiding affecting the immune response in subsequent reagent strips and improving the accuracy of immune detection.

[0017] Further, the said buffer solvent is preferably a TRIS (sodium tetrahydrate arihvcfrous nazbsih)-EDTA buffer solution. Compared with other buffer systems, the buffer system of the invention has a pH range close to 8, mild properties and higher solution stability.

[0018] Further, the said cracking liquid further includes sodium bicarbonate. The addition of sodium bicarbonate is mainly to further adjust the pH of cracking solution. [0019] Further, the mass percentage of surfactant in said lysate is 1-2%, and the content of keratinase is 10,000-50,000 lU/mL.

[0020] In the above kit, it is preferred that the fluorescence emission wavelength range of said quantum dot nanoparticles is 600-620 nm, and more preferably, the emission wavelength is 610nm. The wavelength range of the excitation light is 360-430nm, and more preferably, the wavelength of the excitation light is 365nm.

[0021] In the above kit, it is preferable that said quantum dot nanoparticles are quantum dots formed by a single compound or composite quantum dots assembled by several compounds. More preferably, the said compound is selected from one or more

2019101573 12 Dec 2019 of ZnS, CdS, HgS, MgS, CdSe, ZnSe, ZnCdSe.

[0022] The fluorescence immunochromatography kit of the present invention adopts quantum dot nanoparticles as fluorescence emitting substances, and quantum dots show two continuous excitation spectra in optical performance, and the emission spectra are narrow and symmetrical. High fluorescence intensity, long fluorescence life and good stability. Can overcome the defects of conventional fluorescent materials such as rare earth microspheres, latex microspheres and the like, signal fading blocks, unobvious signal gradients and the like. The fluorescence signal displayed by the fluorescence immune test strip of the invention cannot be obviously attenuated within 2 hours, and the gradient of the signal is very obvious. Can effectively delaminate, make the fluorescence signal tested more stable and convenient for detection, and improve the sensitivity and accuracy of detection. In addition, the stable fluorescence, strong signal and obvious change of quantum dots also make the conversion effect of fluorescence signal value good, which is conducive to quantitative analysis.

[0023] The detection principle of the fluorescent immunochromatographic test strip of the present invention is competition law, and antigen coated by T line (detection line) and drug antigen in the sample to be detected compete to combine drug antibody-quantum dot nanoparticle composite. When there is no drug antigen in the sample to be tested, the drug antibody-quantum dot nanocomposite is first combined with the T-wire coated drug antigen-bovine serum albumin complex in the swimming process. A fluorescence signal measurable by a hair fluorescence immunoassay instrument is formed at the T line; When drug antigen exists in the sample to be detected, the drug antigen, coated drug antigen and drug antigen-bovine serum albumin complex in the sample compete for binding. At this time, the signal intensity at the T-line will gradually decrease with the increase of drug antigen in the sample. However, the rabbit IgG- quantum dot nanoparticle composite will continue to swim and combine with the goat anti-rabbit antibody coated by the quality control line (line C) to form a stable signal at the line C; If no signal is detected at line c, the test strip is discarded and cannot be used again.

[0024] The invention can adopt a matched fluorescence detection analyzer, and can respectively introduce relevant value curves according to each reagent batch to quickly inject samples to detect readings. The control time is within 10 seconds, and the fluorescence signal can be converted into the concentration value of the corresponding drug or metabolite component and clearly displayed on the display screen. The instrument can store more than 500 sets of test results and can print relevant results in real time. Including the corresponding reagent batch number, specimen number, test time, and tester code and other information.

[0025] Drugs detectable by the present invention include morphine, virus, ketamine, etc.

[0026] The invention has the following advantages:

[0027] 1) The fluorescence immunoassay drug detection kit of the invention adopts hair as a detection sample to quantitatively detect the drug content in the hair, has simple sampling and overcomes the problem of difficult drug detection and sampling;

2019101573 12 Dec 2019 [0028] 2) The present invention uses a lysate composed of surfactant, keratinase and buffer solution to dissolve drugs in hair. The combined action of surfactant, keratinase and buffer solution can effectively improve the dissolution rate and dissolution rate of drugs in hair.

[0029] 3) The invention uses quantum dots for fluorescence labeling, which has stable fluorescence, strong signal and obvious change, and can better convert fluorescence signals into values. The drug content can be quantitatively analyzed, and results can be quickly obtained through a fluorescence analyzer.

[0030] 4) The kit of the invention is used for detecting the drug content in hair, and the detection result has high accuracy, high precision, wide detection range, high stability and strong anti-interference capability (such as drugs, shampoo, hair conditioner, hair gel and the like).

Brief description of drawings [0031] Figure 1 is a schematic structural diagram of the fluorescent immunochromatographic test strip of the present invention, wherein 1 is a PVC lining plate, 2 is a sample pad, 3 is a nitrocellulose membrane, 4 is an absorption pad, 5 is a detection line, and 6 is a quality control line.

Detailed description of the preferred embodiment [0032] The following specific examples further illustrate the methods and technical solutions provided by the present invention but should not be construed as limiting the present invention.

[0033] I. Kit for Fluorescent Immunoassay of Drugs [0034] the kit comprises sample lysate and fluorescent immunochromatographic test strip (see fig. 1 for the structure); The lysate is used to dissolve small drug molecules from the sample to be tested; The fluorescent immunochromatographic test strip comprises a PVC lining plate 1, a nitrocellulose membrane 3, a sample pad 2 and an absorption pad 4, wherein the sample pad, the nitrocellulose membrane and the absorption pad are sequentially and alternately fixed on the PVC lining plate; The sample pad is provided with a drug antibody-quantum dot nanoparticle composite and a rabbit IgG-quantum dot nanoparticle composite; The nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a drug antigen-bovine serum albumin compound, and the quality control line is coated with sheep anti-rabbit antibody.

[0035] II. Preparation Methods of Drug Antibody-Quantum Dot Nanoparticle Complex and Rabbit IgG-Quantum Dot Nanoparticle Complex:

[0036] (1) fluorescent microspheres (i.e., quantum dot nanoparticles formed by one or more of ZnS, CdS, HgS, MgS, CdSe, ZnSe, ZnCdSe) are prepared into 5 wt% working concentration;

[0037] (2) Add the antibody to be labeled (drug antibody or rabbit IgG) in an amount of lOgg/ml and stir for 30min;

[0038] (3) sealing with sealing solution, stirring for 30min, wherein the sealing solution is 5 wt% casein solution;

2019101573 12 Dec 2019 [0039] (4) Centrifuge at 4°C with a centrifuge speed of lOOOOr/min for 30min, and then remove the supernatant;

[0040] (5) Dissolve the precipitate with polymer, and add 0.1ml of precipitate to 1ml of polymer solution. The polymer may be polystyrene, polybutene, etc.

[0041] 3. The production steps of the fluorescent immunochromatographic test strip are as follows [0042] 1) The drug antigen-bovine serum albumin complex and sheep anti-rabbit antibody are respectively spotted on the test area (T) and the quality control area (C) of the nitrocellulose membrane by a membrane spotting machine, and are fully dried, so that the nitrocellulose membrane firmly adsorbs the raw materials.

[0043] 2) The drug antibody-quantum dot nanoparticle composite and rabbit IgGquantum dot nanoparticle composite solution are mixed evenly and then processed on a sample pad, and are fully dried.

[0044] 3) The above nitrocellulose film is compounded on a PVC plastic sheet.

[0045] 4) Place the composite plastic sheet on a cutting machine and cut it into a single piece of test paper.

[0046] 5) Load a single test paper into a plastic box for matching use.

[0047] 6) Place the plastic box, desiccant and dropper into the packaging bag, seal and wait for inspection.

[0048] 7) The sensitivity, specificity and stability of the products to be tested are qualified for random inspection before leaving the factory.

[0049] IV. Detection Methods of Fluorescent Immunoassay Kits [0050] Select the hair within 0.5 cm from the scalp, take 5mg of hair and cut it into l-2mm in length (about 10 pieces in length of 10 cm) with scissors, and cut it into l-2mm small pieces on the folded sampling paper.

[0051] Pour the chopped hair into a bottle filled with hair lysate, close the bottle cap, shake and mix for 1 minute. Absorb the supernatant of the hair lysate with a dropper, and drop 2 drops into the sample addition hole of the reagent plate.

[0052] The fluorescence detection analyzer detects the fluorescence signal of the test strip, converts the fluorescence signal into an electrical signal, and then automatically calculates the concentration through the standard curve information on each batch of test strip ID cards, and displays the concentration on the display screen of the instrument.

[0053] V Performance Study of the Products of the Invention [0054] The fluorescent marker is a particle formed by embedding quantum dots ZnCdSe/ZnS in polystyrene microspheres.

[0055] The formula of cracking solution is as follows: [0056]

Name of chemical agent	Dosage g/L
Sodium tetrahnrate arihvcfrous NazBsih	10
Keratinase	5
PVP-10	10
Tralaton-100	15
Sodium Bicarbonate	20

2019101573 12 Dec 2019

EDTA

5

[0057] 1. Linear Range [0058] Dilute morphine enterprise reference products into Ong/mg, 0.05ng/mg, 0.Ing/mg, Ing/mg, 2.5ng/mg, 5ng/mg, lOng/mg, 15ng/mg, 20ng/mg respectively. Then, according to the detection steps in the instruction manual, respectively detect morphine/methamphetamine/ketamine hair rapid detection reagent products in 3 batches, and take the average value of 3 samples for each concentration detection. Linear correlation coefficient R>0.99 and relative deviation of 10% are required. The results are shown in Table 1 below.

[0059] Table 1 Detection Results of Morphine Linear Range [0057] 1. Linear Range [0058] Dilute morphine enterprise references to Ong/mg, 0.05ng/mg, 0. Ing/mg, Ing/mg, 2.5ng/mg, 5ng/mg, 1 Ong/mg, 15ng/mg, 20ng/mg respectively. Then, according to the detection steps in the instruction manual, three batches of morphine/methamphetamine/chloramine hair rapid detection reagent products are respectively detected, and the average value is taken for three samples of each concentration detection. Linear correlation coefficient R>0.99 and relative deviation of 10% are required. The results are shown in Table 1 below.

[0059] Table 1 Detection Results of Morphine Linear Range [0060]

Refere	Lot 1 :MOP201706001	Lot 2:MOP201706002	Lot 3 :MOP201706003
nee	Detection result	Avera	Relati	Detection result	Avera	Relati	Detection result	Avera	Relati
conce			ge	ve			ge	ve			ge	ve
ntratio			value	deviati			value	deviati			value	deviat
n				on%				on%				ion%
Ong/m	1	0.02	0.03	/	1	0.04	0.03	/	1	0.03	0.04	/
g	2	0.04			2	0.02			2	0.04
	3	0.02			3	0.02			3	0.04
0.05ng	1	0.03	0.03	-40	-40	0.03	0.03	-40	1	0.03	0.03	-40
/mg	2	0.03			2	0.03			2	0.03
	3	0.04			3	0.03			3	0.04
0.1 ng/	1	0.1	0.1	0	1	0.1	0.1	0	1	0.1	0.1	0
mg	2	0.1			2	0.1			2	0.1
	3	0.1			3	0.1			3	0.1
lng/m	1	0.97	1.02	2	1	0.96	0.99	-1	1	0.92	0.95	-5
g	2	1.09			2	0.93			2	0.95
	3	0.99			3	1.07			3	0.99
2.5ng/	1	2.54	2.56	2.4	1	2.41	2.47	-1.2	1	2.63	2.46	-1.6
mg	2	2.52			2	2.53			2	2.42
	3	2.61			3	2.47			3	2.32
5ng/m	1	5.38	5.12	2.4	1	4.66	5.17	3.4	1	5.35	4.86	-2.8
g	2	4.79			2	5.43			2	4.67
	3	5.18			3	5.42			3	4.57

2019101573 12 Dec 2019

10ng/ mg	1	10.81	10.43	4.3	1	9.38	9.47	-5.3	1	10.18	10.29	2.9
2	9.86	2	9.62	2	9.8
3	10.61	3	9.41	3	10.89
15ng/ mg	1	11.57	12.44	-17.07	1	12.52	11.61	-22.6	1	12.31	12.12	-19.2
2	12.98	2	11.26	2	13.16
3	12.77	3	11.06	3	10.9
20ng/ mg	1	16.9	15.99	-20.05	1	16.1	15.78	-21.1	1	17.79	16.43	-17.85
2	15.39	2	14.25	2	14.91
3	15.67	3	17	3	16.58

[0061] [0062] According to the detection results, the relative deviation is >10% at a concentration of <0.1 ng/mg, and the relative deviation is >10% at a concentration of >10 ng/mg, which is not satisfactory; in the range of 0.1-10 ng/ml, the product is linear. The correlation coefficient R 0.99, the relative deviation is ± 10%, so the linear range of the three products is 0.1-10 ng / mg.

[0063] 2, accuracy [0064] Take three batches of the morphine hair rapid detection reagent product, and according to the detection steps of the specification, respectively, test the standard products of 2.5 ng/mg and 5 ng/mg, and each of the concentration detection 6 samples is averaged and the relative deviation is calculated. . The results are shown in Table 2 below:

[0065] Table 2 morphine accuracy test results [0066]

Refere	Lot 1 :MOP201706001	Lot 2:MOP201706002	Lot 3 :MOP201706003
nee	Detection result	Avera	Relati	Detection result	Avera	Relati	Detection result	Avera	Relati
conce			ge	ve			ge	ve			ge	ve
ntratio			value	deviati			value	deviati			value	deviat
n				on%				on%				ion%
2.5ng/	1	2.45	2.47	-1.2	1	2.46	2.48	-0.8	1	2.53	2.53	1.03
mg	2	2.41			2	2.45			2	2.46
	3	2.48			3	2.43			3	2.62
	4	2.58			4	2.55			4	2.58
	5	2.45			5	2.58			5	2.43
	6	2.43			6	2.40			6	2.55
5ng/m	1	5.11	4.97	-0.6	1	4.68	4.97	-0.6	1	5.21	5.01	0.12
g	2	4.86			2	4.87			2	5.11
	3	5.01			3	5.02			3	4.90
	4	5.21			4	4.95			4	4.91
	5	4.87			5	5.05			5	5.21
	6	4.76			6	5.22			6	4.71
	2	15.39			2	14.25			2	14.91

2019101573 12 Dec 2019

3

15.67

3

17

3

16.58

[0066] [0067] According to the detection results, the accuracy of the morphine hair rapid detection reagent is within±10% for all concentration relative deviations.

[0068] 3. Precision [0069] Take 10 samples of 3 batches of morphine hair rapid detection reagent, and according to the detection steps in the specification, detect the standard substance with concentration of 2.5ng/mg and 5ng/mg and calculate the coefficient of variation CV. The results are shown in Table 3 below:

[0070] Table 3 Morphine Precision Test Results

Refere nee conce ntratio n	Lot 1 :MOP201706001	Lot 2:MOP201706002	Lot 3 :MOP201706003
Detection result	CV%	Detection result	CV%	Detection result	CV%
2.5ng/ mg 5ng/m g	1	2.41	3.49	1	2.57	3.63	1	2.41	3.82
2	2.39	2	2.46	2	2.37
3	2.57	3	2.41	3	2.63
4	2.51	4	2.56	4	2.46
5	2.53	5	2.38	5	2.43
6	2.46	6	2.43	6	2.61
7	2.66	7	2.58	7	2.39
8	2.47	8	2.45	8	2.54
9	2.63	9	2.39	9	2.46
10	2.53	10	2.63	10	2.38
	1	4.81	4.09	1	4.91	3.58	1	5.25	4.23
2	5.11	2	4.69	2	4.90
3	4.65	3	5.23	3	5.30
4	5.20	4	5.02	4	5.05
5	4.98	5	4.79	5	4.79
6	4.74	6	5.05	6	5.00
7	5.06	7	5.22	7	4.80
8	4.91	8	4.85	8	4.69
9	5.15	9	5.10	9	5.13
10	5.25	10	5.05	10	5.21

[0071] [0072] [0073] According to the detection results, the intra-batch precision CV of morphine hair rapid detection reagent is < 10%.

[0074] 4. Minimum detection degree [0075] Take 10 morphine hair rapid detection reagents of 3 batches each, and detect

2019101573 12 Dec 2019 the standard substance matrix according to the detection steps in the specification, and calculate the average X and standard deviation SD of the determination results. Results are shown in Table 4:

[0076] Table 4 Detection Results of Minimum Detection Limit of Morphine

Re	Lot 1 :MOP201706001	Lot 2 :MOP201706002	Lot 3 :MOP201706003
fer	Detection	Ave	Sta	Minima	Detection	Ave	Sta	Minima	Detection	Ave	Rel
en	result		rag	nd	m	result		rag	nd	m	result		rag	ativ
ce			e	ard	detection			e	ard	detection			e	e
ma			val	de	limit			val	de	limit			val	dev
ter			ue	via				ue	via				ue	iati
ial				tio					tio					on
				n					n					%
2.5	1	0.0	0.0	0.0	0.07	1	0.0	0.0	0.0	0.07	1	0.0	0.0	0.4	00
ng/		4	1	4			3	1	4			5	1		7
mg	2	0.0				2	0.0				2	0.0
5 ng		2					4					2
/mg	3	0.0				3	0.0				3	0.0
		6					5					4
	4	0.0				4	0.0				4	0.0
		5					4					5
	5	0.0				5	0.0				5	0.0
		4					5					6
	6	0.0				6	0.0				6	0.0
		6					2					6
	7	0.0				7	0.0				7	0.0
		3					3					2
	8	0.0				8	0.0				8	0.0
		4					6					5
	9	0.0				9	0.0				9	0.0
		4					4					5
	10	0.0				10	0.0				10	0.0
		5					2					3

[0077] [0078] [0079] According to the detection results, the minimum detection limit (X+3SD) of morphine hair rapid detection reagent is less than or equal to (X+3SD)< 0.1 ng/mg.

[0080] 5. Interference Experiment

2019101573 12 Dec 2019 [0081] 5.1, drug substance interference [0082] Take 8 parts of morphine hair rapid detection reagents of 3 batches, respectively, and carry out detection on Methamphetamine, Methadone, Caffeine, Diazepam, Pheno Barbita, Ketamine, Tramadol and Ranitidine with a concentration of 10pg/mL respectively according to the detection steps in the specification, and each cross substance shall be detected once. The results are required to be negative. The results are shown in Table 5 below:

[0083] Table 5 Detection Results of Drug Substance Interference

Interfering substance	Detection result
Lotl:MGP201706001	Lot2:MOP201706002	Lot3:MOP201706003
Methamphetamine	0.05	0.04	0.06
Methadone	0.04	0.03	0.05
Caffeine	0.06	0.03	0.05
Diazepam	0.04	0.03	0.04
Phenobarbital	0.06	0.04	0.04
Ketamine	0.03	0.04	0.05
Tramadol	0.05	0.05	0.03
Ranitidine	0.05	0.04	0.03

[0084] [0085] According to the detection results, the morphine hair rapid detection reagent has no interference when the concentration of the interfering substance is 10 ug/ml. [0086] 5.2, shampoo conditioner interference [0087] Randomly take a batch of 72 parts of hair rapid detection reagent, then take 3 parts of negative hair and 3 parts of positive hair respectively, wash the hair samples with shampoo and conditioner respectively, rinse with tap water for 3 times (simulate the normal shampoo process). After that, the cleaned hair was tested, and the unwashed hair samples were taken as the control, one for each. The results are shown in Table 6-7 below:

[0088] Table 6 Effect of Shampoo on Rapid Detection Reagents for Hair

Shampoo brand	Hair samples	Is it cleaned?	Morphine test results	Methamphet amine test results	Ketamine test results
Shampoo 1	Negative sample 1	No	0.03	0.5	0.05
Yes	0.06	0.05	0.04
Negative sample 2	No	0.03	0.04	0.04
Yes	0.03	0.05	0.04
Negative sample 3	No	0.06	0.06	0.06
Yes	0.06	0.06	0.03
Positive sample 1	No	2.65	4.86	2.78
Yes	2.97	4.57	2.65
Positive sample 2	No	0.2	2.28	3.93
Yes	0.22	2.1	3.88

2019101573 12 Dec 2019

	Positive sample 3	No	1.37	1.12	0.81
Yes	1.4	0.92	0.92
Shampoo 2	Negative sample 1	No	0.06	0.03	0.05
Yes	0.04	0.06	0.05
Negative sample 2	No	0.03	0.05	0.06
Yes	0.06	0.03	0.03
Negative sample 3	No	0.05	0.03	0.03
Yes	0.05	0.06	0.04
Positive sample 1	No	2.95	4.42	2.99
Yes	2.68	4.41	3.11
Positive sample 2	No	0.2	2.26	3.91
Yes	0.2	2.14	3.73
Positive sample 3	No	1.44	0.94	0.92
Yes	1.39	1.02	0.83
Shampoo 3	Negative sample 1	No	0.06	0.05	0.04
Yes	0.03	0.06	0.03
Negative sample 2	No	0.03	0.03	0.04
Yes	0.05	0.05	0.05
Negative sample 3	No	0.05	0.03	0.05
Yes	0.04	0.04	0.04
Positive sample 1	No	2.88	4.05	2.97
Yes	2.98	4.38	2.66
Positive sample 2	No	0.22	2.28	3.64
Yes	0.22	2.01	3.48
Positive sample 3	No	1.53	0.95	0.9
Yes	1.4	1.1	0.94

[0089] [0091] the results showed that shampoo had no effect on the rapid detection of morphine, methamphetamine and ketamine.

[0092] Table 7 Effect of conditioner on hair rapid detection reagent

Brand of conditioner	Hair samples	Is it cleaned?	Morphine test results	Methamphet amine test results	Ketamine test results
Hair conditioner 1	Negative sample 4	No	0.06	0.03	0.03
Yes	0.03	0.05	0.04
Negative	No	0.06	0.06	0.04

2019101573 12 Dec 2019

	sample 5	Yes	0.06	0.03	0.03
Negative sample 6	No	0.06	0.05	0.03
Yes	0.06	0.04	0.06
Positive sample 4	No	3.98	2.55	3.62
Yes	4.2	2.43	3.16
Positive sample 5	No	1.3	0.83	0.97
Yes	1.39	0.99	0.99
Positive sample 6	No	2.78	1.81	1.45
Yes	2.7	1.99	1.39
Hair conditioner 2	Negative sample 4	No	0.03	0.03	0.05
Yes	0.05	0.06	0.04
Negative sample 5	No	0.05	0.04	0.04
Yes	0.06	0.03	0.06
Negative sample 6	No	0.04	0.04	0.06
Yes	0.06	0.03	0.05
Positive sample 4	No	3.74	2.2	3.64
Yes	3.93	2.25	3.47
Positive sample 5	No	1.46	0.82	0.95
Yes	1.35	0.82	0.93
Positive sample 6	No	2.66	2.06	1.3
Yes	2.39	2.05	1.28
Hair conditioner 3	Negative sample 4	No	0.03	0.03	0.06
Yes	0.06	0.06	0.05
Negative sample 5	No	0.06	0.05	0.05
Yes	0.04	.04	0.06
Negative sample 6	No	0.05	0.06	0.03
Yes	0.06	0.03	0.05
Positive sample 4	No	3.81	2.19	3.17
Yes	3.71	2.32	3.46
Positive sample 5	No	1.49	0.9	0.87
Yes	1.28	0.86	0.94
Positive sample 6	No	2.31	1.99	1.23
Yes	2.48	1.88	1.46

[0093] [0094] [0095] The results showed that conditioner had no effect on the rapid detection of morphine, methamphetamine and ketamine.

[0096] 5.3 interference of hair gel products [0097] Randomly take 36 parts of a batch of hair rapid detection reagent, then take 3 parts of positive hair and 3 parts of negative hair respectively, simulate the normal use process of hair gel, spray the hair gel on the hair or smear it on the hair, and take the uncoated hair sample as a control, and do one time each. The results are shown in

2019101573 12 Dec 2019

Table 8 below:

[0098] Table 8 Effect of Hair Gel on Rapid Detection Reagents for Hair [0099]

Hair products spleen	Hair samples	Whether to use hair spray	Morphine test results	Methamphet amine test results	Ketamine test results
Hair gel 1	Negative sample 7	No	0.04	0.05	0.04
Yes	0.05	0.04	0.04
Negative sample 8	No	0.06	0.05	0.03
Yes	0.04	0.05	0.04
Negative sample 9	No	0.06	0.04	0.05
Yes	0.06	0.05	0.05
Positive sample 7	No	4.08	0.43	4.1
Yes	3.68	0.47	4.4
Positive sample 8	No	5.66	0.9	5.56
Yes	6.7	0.84	5.76
Positive sample 9	No	1.79	6.71	2.19
Yes	2.02	7.28	2.14
Hair gel 2	Negative sample 7	No	0.05	0.06	0.05
Yes	0.05	0.03	0.04
Negative sample 8	No	0.05	0.05	0.03
Yes	0.04	0.04	0.05
Negative sample 9	No	0.06	0.04	0.04
Yes	0.04	0.05	0.04
Positive sample 7	No	4.13	0.5	4.83
Yes	3.51	0.45	4.56
Positive sample 8	No	6.34	0.93	5.82
Yes	6.39	0.99	5.62
Positive sample 9	No	1.99	7.82	2.15
Yes	1.95	7.83	2.27
Hair gel 3	Negative sample 7	No	0.06	0.06	0.05
Yes	0.05	0.04	0.06
Negative sample 8	No	0.06	0.05	0.06
Yes	0.04	0.05	0.03
Negative sample 9	No	0.06	0.06	0.03
Yes	0.05	0.05	0.03
Positive sample 7	No	3.7	0.51	4.18
Yes	3.51	0.46	4.2
Positive sample 8	No	5.93	1.01	6.23
Yes	5.71	0.95	5.89
Positive sample 9	No	1.77	7.59	2.21
Yes	1.95	7.11	2.37

[0100]

2019101573 12 Dec 2019 [0101] The results showed that hair gel had no effect on morphine hair rapid detection reagent, methamphetamine hair rapid detection reagent and ketamine hair rapid detection reagent.

[0102] 6. Stability [0103] 400 parts of finished products for each batch of each product are taken out and put into a normal temperature environment, and the detection buffer solution is stored at 2-8°C. during each detection, all the detection buffer solution is taken out from the refrigerator at 2-8 deg c, opened, restored to room temperature for detection, and the detection buffer solution is put back into the environment condition of 2-8 °C after detection. The testing time was 0, 3, 6, 9, 12, 18, 24, 30, 31 months. (If the product is found to be unstable during the test, it needs to be verified with the currently prepared detection buffer, indicating whether it is the stability reason of the finished product or the reason of the detection buffer). The testing items are as follows:

[0104] 1) Linearity: Select the internal standards with concentrations of O.lng/mg, Ing/mg, 2.5ng/mg, 5ng/mg and lOng/mg for measurement.

[0105] 2) Minimum detection limit: Take 10 parts of the product and carry out detection on the standard substance matrix in the preparation enterprise according to the detection steps.

[0106] Table 9 Linear Test Results of Morphine Finished Products (Test Time: 0 Months) [0107]

Refere	Lot 1 :MOP201706001	Lot 2:MOP201706002	Lot 3 :MOP201706003
nee	Detection result	Avera	Relati	Detection result	Avera	Relati	Detection result	Avera	Relati
conce			ge	ve			ge	ve			ge	ve
ntratio			value	deviati			value	deviati			value	deviat
n				on%				on%				ion%
0.1 ng/	1	0.1	0.1	0	1	0.1	0.1	0	1	0.1	0.1	0
mg	2	0.1			2	0.1			2	0.1
	3	0.1			3	0.1			3	0.1
Ing/m	1	1.09	1.06	6	1	1.05	1.01	1	1	0.98	0.99	-1
g	2	1.05			2	1.03			2	1.03
	3	1.05			3	0.96			3	0.97
2.5ng/	1	2.65	2.6	4	1	2.32	2.39	-4.4	1	2.37	2.44	-2.4
mg	2	2.52			2	2.44			2	2.28
	3	2.64			3	2.4			3	2.66
5ng/m	1	4.64	4.78	-4.4	1	5.32	5.08	1.6	1	5.26	4.96	-0.8
g	2	4.68			2	4.65			2	5.05
	3	5.01			3	5.26			3	4.56
10ng/	1	9.23	9.72	-2.8	1	10.56	10.44	4.4	1	10.51	9.92	-0.8
mg	2	9.81			2	10.5			2	9.43
	3	10.13			3	10.22			3	9.83

2019101573 12 Dec 2019 [0108] Table 10 Linear Test Results of Morphine Finished Products (Test Time: 3 Months) [0109]

Refere	Lot 1 :MOP201706001	Lot 2:MOP201706002	Lot 3 :MOP201706003
nee	Detection result	Avera	Relati	Detection result	Avera	Relati	Detection result	Avera	Relati
conce			ge	ve			ge	ve			ge	ve
ntratio			value	deviati			value	deviati			value	deviat
n				on%				on%				ion%
0.1 ng/	1	0.1	0.1	0	1	0.1	0.1	0	1	0.1	0.1	0
mg	2	0.1			2	0.1			2	0.1
	3	0.1			3	0.1			3	0.1
Ing/m	1	1.02	1.05	5	1	0.91	0.98	-2	1	0.95	0.99	-1
g	2	1.09			2	0.98			2	1.07
	3	1.05			3	1.06			3	0.96
2.5ng/	1	2.3	2.49	-0.4	1	2.71	2.53	1.2	1	2.65	2.57	2.8
mg	2	2.66			2	2.53			2	2.39
	3	2.52			3	2.36			3	2.67
5ng/m	1	4.57	4.69	-6.2	1	4.96	5.27	5.4	1	4.91	5.06	1.2
g	2	4.9			2	5.42			2	5.42
	3	4.59			3	5.43			3	4.86
10ng/	1	9.93	9.9	-1	1	10.34	9.92	-0.8	1	9.51	9.9	-1
mg	2	10.53			2	9.61			2	10.89
	3	9.23			3	9.8			3	9.3

[0110] [0111] Table 11 Linear Test Results of Morphine Finished Products (Test Time: 6 Months) [0112]

Refere	Lot 1 :MOP201706001	Lot 2:MOP201706002	Lot 3 :MOP201706003
nee	Detection result	Avera	Relati	Detection result	Avera	Relati	Detection result	Avera	Relati
conce			ge	ve			ge	ve			ge	ve
ntratio			value	deviati			value	deviati			value	deviat
n				on%				on%				ion%
0.1 ng/	1	0.1	0.1	0	1	0.1	0.1	0	1	0.1	0.1	0
mg	2	0.1			2	0.1			2	0.1
	3	0.1			3	0.1			3	0.1

2019101573 12 Dec 2019

lng/m g	1	0.96	0.94	-6	1	1.07	1.04	4	1	0.98	0.99	-1
2	0.92	2	0.98	2	1
3	0.94	3	1.07	3	0.99
2.5ng/ mg	1	2.65	2.61	4.4	1	2.48	2.59	3.6	1	2.65	2.52	0.8
2	2.54	2	2.58	2	2.59
3	2.65	3	2.72	3	2.33
5ng/m g	1	5.34	5.15	3	1	4.82	5.03	0.6	1	5.08	4.76	-4.8
2	4.79	2	4.99	2	4.57
3	5.31	3	5.27	3	4.64
10ng/ mg	1	10.47	10.19	1.9	1	9.92	9.74	-2.6	1	9.61	9.52	-4.8
2	10.69	2	9.31	2	9.5
3	9.52	3	10.00	3	9.45

[0114] Table 12 Linear Test Results of Morphine Finished Products (Test Time: 9 Months) [0115]

Refere	Lot 1 :MOP201706001	Lot 2:MOP201706002	Lot 3 :MOP201706003
nee	Detection result	Avera	Relati	Detection result	Avera	Relati	Detection result	Avera	Relati
conce			ge	ve			ge	ve			ge	ve
ntratio			value	deviati			value	deviati			value	deviat
n				on%				on%				ion%
0.1 ng/	1	0.1	0.1	0	1	0.1	0.1	0	1	0.1	0.1	0
mg	2	0.1			2	0.1			2	0.1
	3	0.1			3	0.1			3	0.1
lng/m	1	1.05	0.99	-1	1	0.93	1.01	1	1	1.07	0.98	-2
g	2	0.97			2	1.07			2	0.93
	3	0.96			3	1.03			3	0.95
2.5ng/	1	2.69	2.54	1.6	1	2.44	2.38	-4.8	1	2.42	2.51	0.4
mg	2	2.48			2	2.42			2	2.55
	3	2.45			3	2.29			3	2.57
5ng/m	1	5.16	4.99	-0.2	1	5.3	5.19	3.8	1	5.37	5.4	8
g	2	4.88			2	5.38			2	5.44
	3	4.92			3	4.89			3	5.38
10ng/	1	9.83	9.72	-2.7	1	9.46	9.78	-2.2	1	9.47	9.59	-4.1
mg	2	9.25			2	10.44			2	10.14
	3	10.12			3	9.44			3	9.15

[0116] Table 13 Linear Test Results of Morphine Finished Products (Test Time: 12 Months) [0117]

2019101573 12 Dec 2019

Refere	Lot 1 :MOP201706001	Lot 2:MOP201706002	Lot 3 :MOP201706003
nee	Detection result	Avera	Relati	Detection result	Avera	Relati	Detection result	Avera	Relati
conce			ge	ve			ge	ve			ge	ve
ntratio			value	deviati			value	deviati			value	deviat
n				on%				on%				ion%
0.1 ng/	1	0.1	0.1	0	1	0.1	0.1	0	1	0.1	0.1	0
mg	2	0.1			2	0.1			2	0.1
	3	0.1			3	0.1			3	0.1
Ing/m	1	1.04	1.04	4	1	1.05	1.06	6	1	1.08	1.02	2
g	2	1.07			2	1.08			2	1.06
	3	1.02			3	1.05			3	0.92
2.5ng/	1	2.53	2.56	2.4	1	2.5	2.52	0.8	1	2.63	2.48	-0.8
mg	2	2.49			2	2.68			2	2.35
	3	2.65			3	2.38			3	2.46
5ng/m	1	5.38	5.01	0.2	1	5.36	5.12	2.4	1	4.81	4.95	-1
g	2	4.57			2	5.02			2	5.19
	3	5.07			3	4.97			3	4.86
10ng/	1	9.41	9.47	-5.3	1	10.7	10.05	0.5	1	10.84	10.43	4.3
mg	2	9.53			2	9.31			2	10.84
	3	9.48			3	10.4			3	9.61

[0118] [0119] The above description of the embodiments is only for the purpose of helping to understand the method of the present invention and its core ideas. It should be pointed out that for a person of ordinary skill in the technical field, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of the claims of the present invention.

Claims (10)

1. A kit for fluorescence immunoassay of hair trace drugs, characterized by comprising sample lysate and fluorescence immunochromatographic test strip;

Said lysate is used to dissolve small drug molecules from hair;

The said fluorescence immunochromatographic test strip comprises a PVC lining plate, a nitrocellulose membrane, a sample pad and an absorption pad, wherein the said sample pad, the nitrocellulose membrane and the absorption pad are sequentially and alternately fixed on the PVC lining plate;

The said sample pad is provided with a drug antibody-quantum dot nanoparticle composite and a rabbit IgG- quantum dot nanoparticle composite;

The said nitrocellulose membrane is provided with a detection line and a quality control line, wherein the detection line is coated with a drug antigen-bovine serum albumin compound, and the quality control line is coated with sheep anti-rabbit antibody.

2. A kit for fluorescence immunoassay of hair trace drugs according to claim Isaid, characterized in that the mass ratio of lysate to hair is 100: 1.

3. A kit for fluorescence immunoassay of hair trace drugs according to claim 1, wherein Isaid lysate comprises surfactant, keratinase and buffer solution.

4. said according to claim 1, wherein said surfactant is preferably a nonionic surfactant.

5. The kit for fluorescence immunoassay of hair trace drugs according to claim 1, wherein the Isaid buffer solvent is preferably TRIS (sodium tetrahnrate arihvcfrous NazBsih)-EDTA buffer solution.

6. A kit for fluorescence immunoassay of hair trace drugs according to claim 1, wherein Isaid lysate further comprises sodium bicarbonate.

7. The kit for fluorescence immunoassay of hair trace drugs according to claim 1, wherein the mass percentage of surfactant in Isaid lysate is 1-2%, and the content of keratinase is 10000-50000IU/ML.

8. said according to claim 1, wherein the fluorescence emission wavelength range of said quantum dot nanoparticles is 600-620nm, and more preferably, the emission wavelength is 610nm.

9. said according to claim 1, wherein said quantum dot nanoparticles are quantum dots formed by a single compound or composite quantum dots assembled by several compounds.

10. The kit for fluorescence immunoassay of hair trace drugs according to claim 1, wherein the Isaid compound is selected from one or more of ZnS, CdS, HgS, MgS, CdSe, ZnSe, ZnCdSe.