CN110208072B - Human hair rapid lysis solution for trace drug detection and application thereof - Google Patents
Human hair rapid lysis solution for trace drug detection and application thereof Download PDFInfo
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Abstract
The invention discloses a human hair rapid cracking liquid for trace drug detection and an application thereof, and the human hair rapid cracking liquid for trace drug detection comprises the following raw material components: 50-100nM alkaline protease AH-101, 0.5-2% thioglycolic acid, 25-75mM glycine, 30-60mM Tris-hydrochloride buffer, 40-80mM NaCl-NaOH buffer and 0.5-1.5% polyethylene glycol octylphenyl ether (Triton X-100); wherein nM is nanomole per liter, mM is millimole per liter,% is volume percent. The invention can rapidly extract trace drug micromolecules from human hair samples, and has high drug release rate, small impurity influence, no toxicity and pollution; the method can be used under common experimental conditions, not only can fully crack keratin, but also has rapid cracking speed, and the whole cracking time only needs 90 seconds, so that drug molecules are effectively released.
Description
Technical Field
The invention relates to a human hair rapid lysis solution for trace drug detection and application thereof, belonging to the field of in vivo drug detection.
Background
Drug abuse (drug abuse) refers to the repeated use of some drugs with dependence or potential for dependence in large quantities, regardless of the medical purpose, by the drug user who takes the form of self-administration, resulting in mental and physical dependence, causing confusion and some abnormal behavior. Drug abuse is also known as "drug abuse" or "drug abuse". Drug abuse is a significant problem that seriously jeopardizes social safety and human life health. According to statistics, the number of people taking novel drugs in China increases by about 30% every year, wherein teenagers account for 86%. In order to effectively attack drug crimes and cooperate with the drug prohibition work of law enforcement departments such as public security and the like, researchers have established a series of accurate, rapid and high-sensitivity methods for qualitatively and quantitatively analyzing and detecting novel drugs. At present, the immune colloidal gold technology, the gas chromatography-mass spectrometry and the high performance liquid chromatography-mass spectrometry are mainly applied at home and abroad to detect and identify the urine and saliva of the virus-absorbing personnel. Once the drug is ingested, traces are left in biological detection materials such as urine, blood, saliva and the like, which becomes evidence for identifying drug-intake illegal behaviors. Different types of test materials have different drug absorption tracing periods. For example, the period of tracing the drug in urine generally does not exceed 3 days, and once the urine test is positive, the drug is generally used by the tested person within 3 days; the positive result of blood test can indicate that the tested person has used the drug within several hours (generally no more than 24 hours). Once the time limit is exceeded, no drug component can be detected in blood and urine. Therefore, it is urgently needed to establish a detection method capable of reflecting the use condition of drugs in a long time so as to control drug addicts.
The small drug molecules enter dense capillary blood vessels around hair follicles such as human hair, pubic hair, axillary hair, chest hair or leg hair, and are fixed by keratin in the hair follicles, and the hair containing the small drug molecules will grow out of the surface of the scalp in about 3 days. Due to the slow growth rate of hair, small drug molecules can be stably existed in the hair for a long time, and the content of the drug and metabolites thereof in the hair is low, namely ng/mg. The hair is used as a special carrier of the drug micromolecules, and has the unique advantages of easy acquisition and storage of samples, stable result, long detection time limit, capability of reflecting the use condition of the drug for a long time (more than six months), and the like. The hair of suspected drug addicts is detected, so that evidence can be provided for the abuse behavior of drugs, and the ingestion type and time of the drugs can be determined. Therefore, in the decision on modification (drug addiction identification method) executed from 4/1 in 2017, the detection of drug components in human hair samples is an important basis for public security to identify drug addiction.
Because the drugs in the hair are embedded in the keratin, the hair is firstly digested by organic solvents such as acidolysis, alkaline hydrolysis and methanol, so that the small molecules of the drugs are released and enter the human solution in a free state. In general, the background of acid hydrolysis hair products is low, but the release efficiency of drug molecules of the object to be detected is lower than that of the alkali hydrolysis method; the hair can be completely dissolved by alkaline hydrolysis, so that the drug molecules to be detected are completely released, but the detection is influenced because the protein impurity content in the extracting solution is high. The ultrasonic hydrolysis of organic solvents such as methanol, acetonitrile and the like has the problems of environmental pollution, complex operation, difficult popularization of a basic layer and the like. Compared with urine or blood poison with the molecular content of mu g/ml, the content of the poison and metabolites thereof in hair is very low, namely ng/mg, so that how to safely, quickly and efficiently extract the poison micromolecules enriched in human hair samples becomes the current primary technical problem.
Disclosure of Invention
The invention provides a human hair rapid cracking liquid for trace drug detection and application thereof.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a human hair rapid lysis solution for trace drug detection is characterized in that: the raw material components comprise: 50-100nM Alkaline Protease AH-101(Alkaline Protease AH-101), 0.5-2% Thioglycolic Acid (Thioglycolic Acid, pH 11), 25-75mM glycine, 30-60mM Tris-HCl buffer (Tris-HCl, pH 7.8), 40-80mM NaCl-NaOH buffer (NaCl-NaOH, pH 10.5) and 0.5-1.5% polyethylene glycol octylphenyl ether (Triton X-100); wherein nM is nanomole per liter, mM is millimole per liter,% is volume percent.
The nM and mM equimolar concentrations refer to the molar content of each component in the lysate, wherein the molar concentration of the tris buffer refers to the molar content of tris hydrochloride in the lysate, and the molar concentration of the sodium chloride-sodium hydroxide buffer refers to the molar content of sodium chloride in the lysate; % is volume percent and refers to the volume percent of the corresponding component in the lysate.
The applicant finds that alkaline protease AH-101 can hydrolyze protein peptide bonds under alkaline conditions, decompose keratin and release drug small molecules; the thioglycolic acid is used as a reducing agent and has a promoting effect on improving the activity of the keratinase; glycine has zwitterions of amino and carboxyl, and has strong buffering property; the tris hydrochloride buffer solution can provide an effective buffering environment; a sodium chloride-sodium hydroxide buffer can provide a suitable ionic strength; polyoxyethylene octyl phenyl ether is a non-ionic surfactant that can solubilize lipids to increase the permeability of cell membranes to antibodies.
In the technical scheme of the application, the inventor creatively uses alkaline protease AH-101 to be matched with thioglycolic acid to decompose keratin in hair and release drug small molecules. The lysis solution can be used under common experimental conditions, not only can fully crack keratin, but also has rapid cracking speed, and the whole cracking time only needs 90 seconds.
Further preferably, the raw material components of the human hair rapid lysis solution for trace drug detection comprise: 60-80nM Alkaline Protease AH-101(Alkaline Protease AH-101), 0.75-1.5% Thioglycolic Acid (Thioglycolic Acid, pH 11), 40-60mM glycine, 40-50mM Tris-HCl buffer (Tris-HCl, pH 7.8), 45-65mM NaCl-NaOH buffer (NaCl-NaOH, pH 10.5) and 0.75-1% polyethylene glycol octylphenyl ether (Triton X-100). The lysate can fully and effectively crack keratin in hair and accelerate the release process of small drug molecules.
Further preferably, the raw material components of the human hair rapid lysis solution for trace drug detection further comprise: 50-100mM Dithiothreitol (DTT). Dithiothreitol can act as a reducing agent for disulfide bonds, and can increase alkaline protease activity. The lysate can quickly and effectively extract the small molecule components of the drugs in the hair.
As the most preferable scheme of the application, the raw material components of the human hair rapid lysis solution for trace drug detection comprise: 65nM Alkaline Protease AH-101(Alkaline Protease AH-101), 0.8% Thioglycolic Acid (Thioglycolic Acid, pH 11), 50mM glycine, 45mM Tris-HCl buffer (pH 7.8), 50mM NaCl-sodium hydroxide buffer (NaCl-NaOH, pH 10.5), 0.8% Polyethyleneglycol octylphenyl ether (Triton X-100) and 80mM Dithiothreitol (DTT).
The lysate can be used for effectively extracting small drug molecules such as O6-monoacetylmorphine, morphine, methamphetamine, amphetamine, 3, 4-methylenedioxyamphetamine, 3, 4-methylenedioxymethamphetamine, ketamine, norketamine and the like in human hair samples, no additional equipment is needed for pretreatment of the hair samples, and the extraction time is shortened to be within 90 seconds, so that the rapid detection of law enforcement agencies in places such as entertainment venues, major activity venues and the like becomes possible.
The rapid human hair lysate for trace drug detection can be used for extracting drug components in human hair.
The hair of human body is hair, pubic hair, axillary hair, chest hair or leg hair. Preferably hair.
More preferably, the human hair is a hair having a length of the back of the top of the head of less than 3cm from the root. Hair longer than 3cm is cut 3cm from the root end.
In order to ensure the detection precision, the dosage of the human hair rapid lysis solution for detecting trace drugs is 100 +/-10 ul per mg of hair.
The method for extracting the narcotic in the hair sample by using the rapid human hair lysate for trace narcotic detection comprises the following steps:
step one, hair sample collection: collecting hair according to the standards for inspecting hair samples of drug-related personnel, placing the collected hair samples in a clean paper bag or an aluminum foil bag, and recording individual information, medication history, length characteristics and other special treatment conditions;
step two, physically crushing the hair sample: cutting the collected hair sample into 0.5-2mm thin sections by using scissors to prepare hair sample powder;
step three, preparing hair mixed liquid: absorbing the lysate according to the proportion of 500ul per 5mg of hair sample, placing the lysate in a 1.5ml centrifuge tube to be mixed with the hair sample powder, and ultrasonically oscillating for 90-180 seconds to prepare hair mixed solution;
step four, taking 5 drops of the hair mixed liquid prepared in the step three, dripping the 5 drops of the hair mixed liquid into a round hole of a test strip, standing for 3 +/-0.2 minutes, and then detecting by using a SuperTest 1000 series whole biological sample drug analyzer, wherein the detection result shows that the hair sample contains more than or equal to 0.2ng/mg of drugs such as O6-monoacetylmorphine, morphine, methamphetamine, amphetamine, 3, 4-methylenedioxyamphetamine, 3, 4-methylenedioxymethamphetamine, ketamine, norketamine and the like, and less than 0.2ng/mg of the drugs is negative.
The test strip of the application can be directly used by the existing test strip, and the operation method of the test strip is the same as that of the existing method.
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.
The prior art is referred to in the art for techniques not mentioned in the present invention.
The rapid human hair lysate for trace drug detection can rapidly extract trace drug micromolecules from a human hair sample, and has the advantages of high drug release rate, small impurity influence, no toxicity and pollution; the method can be used under common experimental conditions, not only can fully crack keratin, but also has rapid cracking speed, and the whole cracking time only needs 90 seconds, so that drug molecules are effectively released; the hair fast cracking solution can fully and effectively crack keratin in hair and accelerate the release process of drug micromolecules.
Drawings
FIG. 1 is a graph showing the results of detection in example 1;
FIG. 2 is a graph showing the results of detection in example 2;
FIG. 3 is a graph showing the results of detection in example 3.
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
The hair drug detection instrument used in the following examples is a Supertest 1000 series full biological sample drug analyzer and a matched detection reagent produced by Jiangsu Subo detection technology Co.
The hair samples are all from suspected drug addicts, and the hair samples with positive legal test are obtained.
The lysis solution used in each example was prepared as follows: 65nM Alkaline Protease AH-101(Alkaline Protease AH-101), 0.8% Thioglycolic Acid (Thioglycolic Acid, pH 11), 50mM glycine, 45mM Tris-HCl buffer (pH 7.8), 50mM NaCl-sodium hydroxide buffer (NaCl-NaOH, pH 10.5), 0.8% polyethylene glycol octylphenyl ether (Triton X-100) and 80mM dithiothreitol.
Example 1: detection of amphetamine drugs in hairs of drug-involved persons
Treating and detecting the hair of the virus-associated person according to the formula of the lysate, wherein the operation steps are as follows:
step one, hair sample collection: collecting hair roots of monitored people according to the standard for testing hair samples of drug-related personnel, marking hair roots and hair tails, and adopting hair 3cm away from the roots, 30 hair roots and about 5mg hair roots;
step two, physically crushing the hair sample: cutting the collected hair sample into thin sections of 0.5mm-1mm by using scissors to prepare hair sample powder;
step three, preparing a mixed solution for releasing the poison in the hair: absorbing the lysate according to the proportion of 500ul per 5mg of hair sample, placing the lysate in a 1.5ml centrifuge tube to be mixed with the hair sample powder, and oscillating for 90 seconds by ultrasonic waves (the frequency is 25KHZ and the power is 300W) to prepare a drug release mixed solution in the hair;
step four, taking 5 drops (about 100uL) of the drug release mixed solution in the hair prepared in the step three, vertically dropping the drug release mixed solution into a sample adding hole of an ice toxin detection reagent plate, standing for 3 minutes, inserting the drug release mixed solution into a clamping groove of a Supertest 1000 series full biological sample drug analyzer, clicking a detection option in a main interface of the analyzer, entering a detection interface, selecting a detection option and a sample type, finally clicking a quick detection option at the lower left corner of a screen for detection, giving a result after 6 seconds, simultaneously giving a concentration value and a negative and positive result (displaying that the content is more than or equal to 0.2ng/mg as positive and less than 0.2ng/mg as negative), clicking a printing button, and printing the result; note that: if the result is abnormal (no result is displayed), the instrument reading is recommended to be carried out again.
The experimental result is shown in figure 1, and the result shows that according to the lysate proportioning scheme of the example, the positive amphetamine drugs in the hair of the drug-related personnel can be effectively and accurately detected, the influence of impurities is avoided, and the detection result is consistent with the detection result of dissolving by using an organic solvent.
Example 2: detection of morphine drugs in hair of drug-involved person
Step one, hair sample collection: collecting hair roots of monitored people according to the standard for testing hair samples of drug-related personnel, marking hair roots and hair tails, and adopting hair 3cm away from the roots, 30 hair roots and about 5mg hair roots;
step two, physically crushing the hair sample: cutting the collected hair sample into thin sections of 0.5mm-1mm by using scissors to prepare hair sample powder;
step three, preparing a mixed solution for releasing the poison in the hair: absorbing lysate according to the proportion of 500ul per 5mg of hair sample, placing the lysate in a 1.5ml centrifuge tube to be mixed with the hair sample powder, and ultrasonically oscillating for 90 seconds to prepare a drug release mixed solution in the hair;
step four, taking 5 drops (about 100uL) of the drug release mixed solution in the hair prepared in the step three, vertically dropping the mixed solution into a sample adding hole of a morphine detection reagent plate, standing for 3 minutes, inserting the mixed solution into a clamping groove of a Supertest 1000 series full biological sample drug analyzer, clicking a detection option in a main interface of the analyzer, entering a detection interface, selecting a detection option and a sample type, finally clicking a quick detection option at the lower left corner of a screen for detection, giving a result after 6 seconds, simultaneously giving a concentration value and a negative and positive result (displaying that the content is more than or equal to 0.2ng/mg as positive and less than 0.2ng/mg as negative), clicking a printing button, and printing the result; note that: if the result is abnormal (no result is displayed), the instrument reading is recommended to be carried out again.
The experimental results are shown in fig. 2, and the results show that the scheme of the lysate proportioning of the example can effectively and accurately detect that morphine narcotics in hairs of drug-involved persons are positive and are consistent with the detection result of dissolving by using an organic solvent.
Example 3: detection of ketamine narcotic in hairs of narcotic-involved personnel
Step one, hair sample collection: collecting hair roots of monitored people according to the standard for testing hair samples of drug-related personnel, marking hair roots and hair tails, and adopting hair 3cm away from the roots, 30 hair roots and about 5mg hair roots;
step two, physically crushing the hair sample: cutting the collected hair sample into thin sections of 0.5mm-1mm by using scissors to prepare hair sample powder;
step three, preparing a mixed solution for releasing the poison in the hair: absorbing lysate according to the proportion of 500ul per 5mg of hair sample, placing the lysate in a 1.5ml centrifuge tube to be mixed with the hair sample powder, and ultrasonically oscillating for 90 seconds to prepare a drug release mixed solution in the hair;
step four, taking 5 drops (about 100uL) of the drug release mixed solution in the hair prepared in the step three, vertically dropping the drug release mixed solution into a sample adding hole of an ice toxin detection reagent plate, standing for 3 minutes, inserting the drug release mixed solution into a clamping groove of a Supertest 1000 series full biological sample drug analyzer, clicking a detection option in a main interface of the analyzer, entering a detection interface, selecting a detection option and a sample type, finally clicking a quick detection option at the lower left corner of a screen for detection, giving a result after 6 seconds, simultaneously giving a concentration value and a negative and positive result (displaying that the content is more than or equal to 0.2ng/mg as positive and less than 0.2ng/mg as negative), clicking a printing button, and printing the result; note that: if the result is abnormal (no result is displayed), the instrument reading is recommended to be carried out again.
The experimental result is shown in fig. 3, and the result shows that the ketamine drug in the hair of the virus-involved person can be effectively and accurately detected to be positive according to the lysate proportioning scheme of the example, and the result is consistent with the detection result of dissolving by using an organic solvent.
The lysate in each example can effectively crack the small drug molecules such as O6-monoacetylmorphine, morphine, methamphetamine, amphetamine, 3, 4-methylenedioxyamphetamine, 3, 4-methylenedioxymethamphetamine, ketamine, norketamine and the like in hair samples such as human hair, leg hair, pubic hair, axillary hair and the like, and the high-efficiency release of the small drug molecules to be detected in the hair samples can be completed within 90 seconds without additional auxiliary equipment. The invention provides a rapid, safe, efficient, simple and convenient method for extracting small molecules of drugs for detecting trace drugs in human hair.
Claims (9)
1. A human hair rapid lysis solution for trace drug detection is characterized in that: the raw material components comprise: 50-100nM alkaline protease AH-101, 0.5-2% thioglycolic acid, 25-75mM glycine, 30-60mM Tris-hydrochloride buffer, 40-80mM sodium chloride-sodium hydroxide buffer, 0.5-1.5% polyethylene glycol octyl phenyl ether and 50-100mM dithiothreitol; wherein nM is nanomole per liter, mM is millimole per liter,% is volume percent.
2. The rapid human hair lysate for trace drug detection according to claim 1, wherein: the raw material components comprise: 60-80nM alkaline protease AH-101, 0.75-1.5% thioglycolic acid, 40-60mM glycine, 40-50mM Tris-hydrochloride buffer, 45-65mM NaCl-NaOH buffer and 0.75-1% polyethylene glycol octylphenyl ether.
3. An application of a human hair rapid lysate for trace drug detection is characterized in that: the rapid human hair lysate for trace drug detection according to claim 1 or 2, which is used for extracting drug components from human hair.
4. Use according to claim 3, characterized in that: the hair of human body is hair, pubic hair, axillary hair, chest hair or leg hair.
5. The use of claim 4, wherein: the human hair is hair.
6. The use of claim 5, wherein: the human hair is the hair with the length of less than 3 centimeters from the hair root at the back of the top of the head.
7. Use according to any one of claims 3 to 6, wherein: the amount of lysate is 100 + -10 ul per mg of hair.
8. Use according to any one of claims 3 to 6, wherein: the method comprises the following steps:
step one, hair sample collection: collecting hairs according to the Standard for testing the hair samples of the people involved in the poison;
step two, physically crushing the hair sample: crushing the collected hair sample into the length of 0.5-2mm to prepare hair sample powder;
step three, preparing hair mixed liquid: absorbing lysate according to the proportion of 500 +/-10 ul per 5mg of hair sample, uniformly mixing the lysate with hair sample powder, and ultrasonically oscillating for 90-180 seconds;
step four, dropping 4-6 drops of the mixed liquid prepared in the step three into a dropping liquid area of the test strip, standing for 3 +/-0.2 minutes, and then detecting by using a Supertest 1000 series full biological sample drug analyzer, wherein the drug content is positive when being more than or equal to 0.2ng/mg, and the drug content is negative when being less than 0.2 ng/mg.
9. Use according to any one of claims 3 to 6, wherein: the drug component comprises O6-monoacetylmorphine, morphine, methamphetamine, amphetamine, 3, 4-methylenedioxyamphetamine, 3, 4-methylenedioxymethamphetamine, ketamine or norketamine.
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| CN110987548A (en) * | 2019-10-11 | 2020-04-10 | 广州万孚生物技术股份有限公司 | Method for detecting hair poison, pretreatment reagent and kit |
| CN111751537A (en) * | 2020-05-21 | 2020-10-09 | 佛山市顺德区公安局 | Test strip, device and method for detecting trace drugs |
| CN112345658B (en) * | 2020-08-07 | 2022-06-24 | 司法鉴定科学研究院 | Quality control product for analyzing organic toxicants in human hair and preparation method thereof |
| CN112213491B (en) * | 2020-10-10 | 2024-04-12 | 自然资源部第三海洋研究所 | Kit for rapidly detecting drugs in hair and application of kit |
| CN112495458A (en) * | 2020-12-11 | 2021-03-16 | 杭州臻稀生物科技有限公司 | Warm bath device for hair poison detection |
| CN113848102A (en) * | 2021-09-27 | 2021-12-28 | 深圳市心月生物科技有限公司 | Methylamphetamine extracting solution and preparation method thereof |
| CN114295420A (en) * | 2021-12-31 | 2022-04-08 | 广州大陌检测技术有限公司 | Extracting solution for auxiliary detection of trace drugs and preparation and use methods thereof |
| CN115060562B (en) * | 2022-06-27 | 2023-05-05 | 上海凯创生物技术有限公司 | Hair drug trace detection extracting solution and preparation and use methods thereof |
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