CN112748240A - Kit for detecting drugs in hair and detection method thereof - Google Patents
Kit for detecting drugs in hair and detection method thereof Download PDFInfo
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- CN112748240A CN112748240A CN202011543524.7A CN202011543524A CN112748240A CN 112748240 A CN112748240 A CN 112748240A CN 202011543524 A CN202011543524 A CN 202011543524A CN 112748240 A CN112748240 A CN 112748240A
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- 238000001514 detection method Methods 0.000 title claims abstract description 54
- 229940079593 drug Drugs 0.000 title claims abstract description 26
- 239000003814 drug Substances 0.000 title claims abstract description 26
- 210000004209 hair Anatomy 0.000 title claims abstract description 25
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 19
- 239000006166 lysate Substances 0.000 claims abstract description 17
- 239000000427 antigen Substances 0.000 claims abstract description 13
- 102000036639 antigens Human genes 0.000 claims abstract description 13
- 108091007433 antigens Proteins 0.000 claims abstract description 13
- 229940127121 immunoconjugate Drugs 0.000 claims abstract description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 9
- 108010067770 Endopeptidase K Proteins 0.000 claims abstract description 9
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- 229910021389 graphene Inorganic materials 0.000 claims abstract description 9
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000000758 substrate Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 8
- 231100000640 hair analysis Toxicity 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 8
- 238000004108 freeze drying Methods 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 238000012216 screening Methods 0.000 abstract description 2
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- 238000005516 engineering process Methods 0.000 description 5
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
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- PVXVWWANJIWJOO-UHFFFAOYSA-N 1-(1,3-benzodioxol-5-yl)-N-ethylpropan-2-amine Chemical compound CCNC(C)CC1=CC=C2OCOC2=C1 PVXVWWANJIWJOO-UHFFFAOYSA-N 0.000 description 1
- BEQZHFIKTBVCAU-UHFFFAOYSA-N 2-amino-2-(2-chlorophenyl)-1-cyclohexanone Chemical compound C=1C=CC=C(Cl)C=1C1(N)CCCCC1=O BEQZHFIKTBVCAU-UHFFFAOYSA-N 0.000 description 1
- SHXWCVYOXRDMCX-UHFFFAOYSA-N 3,4-methylenedioxymethamphetamine Chemical compound CNC(C)CC1=CC=C2OCOC2=C1 SHXWCVYOXRDMCX-UHFFFAOYSA-N 0.000 description 1
- VBGLYOIFKLUMQG-UHFFFAOYSA-N Cannabinol Chemical compound C1=C(C)C=C2C3=C(O)C=C(CCCCC)C=C3OC(C)(C)C2=C1 VBGLYOIFKLUMQG-UHFFFAOYSA-N 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
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- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
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- 230000000694 effects Effects 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 239000011487 hemp Substances 0.000 description 1
- OROGSEYTTFOCAN-UHFFFAOYSA-N hydrocodone Natural products C1C(N(CCC234)C)C2C=CC(O)C3OC2=C4C1=CC=C2OC OROGSEYTTFOCAN-UHFFFAOYSA-N 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
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- MYWUZJCMWCOHBA-UHFFFAOYSA-N n-methyl-1-phenylpropan-2-amine Chemical compound CNC(C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-UHFFFAOYSA-N 0.000 description 1
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- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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Abstract
The invention discloses a kit for detecting drugs in hair, which comprises lysate and a detection reagent; wherein the lysis solution comprises 10mM PBS buffer solution with pH of 7-9, 20mM DTT and 100ug/mL proteinase K; the detection reagent comprises a DNA1-Met antigen conjugate, a DNA2-Met antibody conjugate, DNA3 and a graphene oxide combined antioxidant; the detection method comprises the following steps: s1: preparing a reagent; s2: pre-treating; s3: carrying out a first reaction; s4: carrying out a second reaction; s5: and (6) detecting. The kit is simple to operate, can detect the drugs aiming at the hair, can obtain a detection report within about 5 minutes, can detect one or more drugs at one time, and is suitable for screening.
Description
Technical Field
The invention belongs to the technical field of drug detection, and particularly relates to a kit for detecting drugs in hair and a detection method thereof.
Background
At present, biological detection samples for quick examination of drug addicts mainly comprise urine, saliva and hair. Compared with urine and saliva, the hair inspection material has the advantages of stable property, convenient material taking, easy storage, long inspection time limit, wide application range, less pollution chance and the like. The metabolism of the drug in the hair is slow, the detection window period can reach half a year, the sampling process is convenient to supervise, and the adulteration or exchange of the detected material can be prevented. The rapid detection technology for trace amount of drugs in hair mainly comprises enzyme-linked immunosorbent assay (ELISA), colloidal gold, time-resolved fluorescence immunochromatography and quantum dot immunochromatography. The technology can not detect quickly (less than 10 minutes), has strong specificity, high sensitivity (reaching 0.2ng/mg) and long traceable time.
In order to overcome the defects of the technology, I research and develop a homogeneous luminescence detection technology. Homogeneous immunodetection is realized by utilizing an ortho-position touching principle, and meanwhile, the detection of macromolecular protein is converted into the detection of nucleic acid by using a DNA auxiliary protein detection method, and the detection sensitivity is greatly improved by combining various mature nucleic acid amplification strategies. In the aspect of photoelectric detection, the project adopts a unique measuring module which consists of a photon counting detector and a counting unit. By adopting intelligent control, after the incubation of the sample and the reagent is finished, the light quantum is automatically captured and amplified through the PMT, the signal conversion and the standard curve method analysis are carried out, the concentration data of the object to be detected in the sample is obtained, the first detection result is obtained within 5 minutes at the fastest speed, and the sample can be continuously added in batches, and the result can be obtained in batches.
Compared with the existing drug detection means, the project has great innovativeness in the aspects of accuracy, quick result output (the result is output in the fastest 5 minutes), high flux, cost reduction (the comprehensive cost is greatly reduced by more than 50%), field operation and the like, solves the three pain points of backward qualitative determination, complex manual operation and high detection cost of the drug detection industry, and meets the requirement of drug detection.
Drugs contemplated by the present invention include, but are not limited to, meso06-monoacetylmorphine, morphine, codeine, MAMP, AMP, MDMA, MDA, MDEA, cocaine, benzoylekonin, ketamine, norketamine, 9-tetrahydrocannabinol, cannabidiol, cannabinol, and the like.
Disclosure of Invention
The present invention is directed to a kit for detecting drugs in hair and a detection method thereof, so as to solve the problems mentioned in the background art.
In order to achieve the purpose, the invention provides the following technical scheme: a kit for detecting drugs in hair comprises lysate and detection reagent;
wherein the lysis solution comprises 10mM PBS buffer solution with pH of 7-9, 20mM DTT and 100ug/mL proteinase K;
the detection reagent comprises a DNA1-Met antigen conjugate, a DNA2-Met antibody conjugate, DNA3 and a graphene oxide combined antioxidant (GO-AOD);
the sequence of the DNA1-Met antigen conjugate is as follows:
SEQ ID NO.1：5’-ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C7-3’；
the sequence of the DNA2-Met antibody conjugate is as follows:
SEQ ID NO.2：5’-NH2C6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC-3’；
the sequence of the DNA3 is as follows:
SEQ ID NO.3：5’-AE-NH2C6-CGATCTCAGCAACTCAGCAGCG-3’。
a method for detecting a drug in hair according to claim 1, comprising the following steps in order:
s1: preparing a reagent: mixing 20mM DTT and 100ug/mL proteinase K into 10mM PBS buffer solution with the pH of 7-9, uniformly mixing to obtain lysate for later use, mixing DNA1-Met antigen conjugate, DNA2-Met antibody conjugate, DNA3 and graphene oxide combined antioxidant at the preferable concentrations of 1nM, 10nM, 0.1 muM and 20 mug/mL to obtain a detection reagent, subpackaging the detection reagent into 200 muL tubes, and freeze-drying for later use;
s2: pretreatment: weighing 20mg of hair, and cutting into small sections of 3-5mm to obtain a hair sample to be detected;
s3: reaction I: putting the hair sample to be tested obtained in the step into a test tube, adding 2ml of lysate into the test tube, and standing for 5min at normal temperature for later use;
s4: and (2) reaction II: adding 200 mu L of lysate in the step into a reaction tube loaded with a detection reagent, and incubating for 5min at 37 ℃;
s5: and (3) detection: and adding a chemiluminescent substrate into the reaction tube in the step, uniformly mixing, and adding 200 mu L of chemiluminescent substrate into a chemiluminescence instrument for detection to obtain a detection result.
Preferably, the chemiluminescent substrates are hydrogen peroxide and sodium hydroxide.
The invention has the technical effects and advantages that:
1. the kit is simple to operate, and compared with a mass spectrometry method, the time and the cost are greatly saved;
2. by adopting a chemiluminescence technology, the sensitivity is far higher than that of common colloidal gold and Elisa;
3. the reagent is in a freeze-dried state, so that the storage and the transportation are facilitated;
4. carrying out drug detection on the hair, and taking a detection report in about 5 minutes;
5. one or more drugs can be tested at one time, and the kit is suitable for screening.
Drawings
FIG. 1 is a schematic view of the detection principle of the present invention;
FIG. 2 is a diagram showing the binding of DNA1, DNA2 and DNA3 according to the present invention.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1:
detection of Methamphetamine (MET) in hair:
a kit for detecting drugs in hair comprises lysate and detection reagent;
wherein the lysis solution comprises 10mM PBS buffer solution with pH of 7-9, 20mM DTT and 100ug/mL proteinase K;
the detection reagent comprises a DNA1-Met antigen conjugate, a DNA2-Met antibody conjugate, DNA3 and a graphene oxide combined antioxidant (GO-AOD);
the sequence of the DNA1-Met antigen conjugate is as follows:
SEQ ID NO.1：5’-ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C7-3’；
the sequence of the DNA2-Met antibody conjugate is as follows:
SEQ ID NO.2：5’-NH2C6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC-3’；
the sequence of DNA3 is as follows:
SEQ ID NO.3：5’-AE-NH2C6-CGATCTCAGCAACTCAGCAGCG-3’。
a method for detecting a drug in hair according to claim 1, comprising the following steps in order:
s1: preparing a reagent: mixing 20mM DTT and 100ug/mL proteinase K into 10mM PBS buffer solution with the pH of 7-9, uniformly mixing to obtain lysate for later use, mixing DNA1-Met antigen conjugate, DNA2-Met antibody conjugate, DNA3 and graphene oxide combined antioxidant at the preferable concentrations of 1nM, 10nM, 0.1 muM and 20 mug/mL to obtain a detection reagent, subpackaging the detection reagent into 200 muL tubes, and freeze-drying for later use;
s2: pretreatment: weighing 20mg of hair, and cutting into small sections of 3-5mm to obtain a hair sample to be detected;
s3: reaction I: putting the hair sample to be tested obtained in the step into a test tube, adding 2ml of lysate into the test tube, and standing for 5min at normal temperature for later use;
s4: and (2) reaction II: adding 200 mu L of lysate in the step into a reaction tube loaded with a detection reagent, and incubating for 5min at 37 ℃;
s5: and (3) detection: adding chemiluminescence substrate into the reaction tube in the above step, adding 200 μ L chemiluminescence substrate by HSCL-10000 chemiluminescence apparatus, immediately detecting chemiluminescence signal of the solution by photomultiplier tube (PMT) for 3s, and determining whether the sample contains methamphetamine according to the recorded chemiluminescence value (RLU).
Specifically, the chemiluminescent substrates are hydrogen peroxide and sodium hydroxide.
Example 2:
detection of Morphine (MOP), cocaine (COC), Ketamine (KET), hemp (THC) in hair:
a kit for detecting drugs in hair comprises lysate and detection reagent;
wherein the lysis solution comprises 10mM PBS buffer solution with pH of 7-9, 20mM DTT and 100ug/mL proteinase K;
the detection reagent comprises a DNA1-Met antigen conjugate, a DNA2-Met antibody conjugate, DNA3 and a graphene oxide combined antioxidant (GO-AOD);
the sequence of the DNA1-Met antigen conjugate is as follows:
SEQ ID NO.1：5’-ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C7-3’；
the sequence of the DNA2-Met antibody conjugate is as follows:
SEQ ID NO.2：5’-NH2C6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC-3’；
the sequence of DNA3 is as follows:
SEQ ID NO.3：5’-AE-NH2C6-CGATCTCAGCAACTCAGCAGCG-3’。
a method for detecting a drug in hair according to claim 1, comprising the following steps in order:
s1: preparing a reagent: mixing 20mM DTT and 100ug/mL proteinase K into 10mM PBS buffer solution with the pH of 7-9, uniformly mixing to obtain lysate for later use, mixing DNA1-Met antigen conjugate, DNA2-Met antibody conjugate, DNA3 and graphene oxide combined antioxidant at the preferable concentrations of 1nM, 10nM, 0.1 muM and 20 mug/mL to obtain a detection reagent, subpackaging the detection reagent into 200 muL tubes, and freeze-drying for later use;
s2: pretreatment: weighing 20mg of hair, and cutting into small sections of 3-5mm to obtain a hair sample to be detected;
s3: reaction I: putting the hair sample to be tested obtained in the step into a test tube, adding 2ml of lysate into the test tube, and standing for 5min at normal temperature for later use;
s4: and (2) reaction II: adding 200 mu L of lysate in the step into a reaction tube loaded with a detection reagent, and incubating for 5min at 37 ℃;
s5: and (3) detection: adding chemiluminescence substrate into the reaction tube, adding 200 μ L chemiluminescence substrate with HSCL-10000 chemiluminescence apparatus, immediately detecting chemiluminescence signal of the solution with photomultiplier tube (PMT) for 3s, and determining whether the sample contains at least one of the above drugs according to the recorded chemiluminescence value (RLU).
Specifically, the chemiluminescent substrates are hydrogen peroxide and sodium hydroxide.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (3)
1. A kit for detecting drugs in hair, characterized in that: comprises lysate and detection reagent;
wherein the lysis solution comprises 10mM PBS buffer solution with pH of 7-9, 20mM DTT and 100ug/mL proteinase K;
the detection reagent comprises a DNA1-Met antigen conjugate, a DNA2-Met antibody conjugate, DNA3 and a graphene oxide combined antioxidant;
the sequence of the DNA1-Met antigen conjugate is as follows:
SEQ ID NO.1：5’-ACGCTGAGTTATCAACGACTTTTTTTATCACATCAGGCTCTAGCGTATGCTATTG-NH2C7-3’；
the sequence of the DNA2-Met antibody conjugate is as follows:
SEQ ID NO.2：5’-NH2C6-TACGTCCAGAACTTTACCAAACCACACCCTTTTTTTGTCGTTGGCTGAGATTC-3’；
the sequence of the DNA3 is as follows:
SEQ ID NO.3：5’-AE-NH2C6-CGATCTCAGCAACTCAGCAGCG-3’。
2. a method for detecting drugs in hair according to claim 1, characterized in that: the method comprises the following steps in sequence:
s1: preparing a reagent: mixing 20mM DTT and 100ug/mL proteinase K into 10mM PBS buffer solution with the pH of 7-9, uniformly mixing to obtain lysate for later use, mixing DNA1-Met antigen conjugate, DNA2-Met antibody conjugate, DNA3 and graphene oxide combined antioxidant to obtain a detection reagent, subpackaging the detection reagent into 200 mu L tubes, and freeze-drying for later use;
s2: pretreatment: weighing 20mg of hair, and cutting into small sections of 3-5mm to obtain a hair sample to be detected;
s3: reaction I: putting the hair sample to be tested obtained in the step into a test tube, adding 2ml of lysate into the test tube, and standing for 5min at normal temperature for later use;
s4: and (2) reaction II: adding 200 mu L of lysate in the step into a reaction tube loaded with a detection reagent, and incubating for 5min at 37 ℃;
s5: and (3) detection: and adding a chemiluminescent substrate into the reaction tube in the step, uniformly mixing, and adding 200 mu L of chemiluminescent substrate into a chemiluminescence instrument for detection to obtain a detection result.
3. The method according to claim 2, wherein the method comprises the steps of: the chemiluminescent substrates are hydrogen peroxide and sodium hydroxide.
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| CN202011543524.7A CN112748240A (en) | 2020-12-24 | 2020-12-24 | Kit for detecting drugs in hair and detection method thereof |
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| CN202011543524.7A CN112748240A (en) | 2020-12-24 | 2020-12-24 | Kit for detecting drugs in hair and detection method thereof |
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| CN114414796A (en) * | 2021-12-09 | 2022-04-29 | 杭州莱和生物技术有限公司 | Fluorescence immunoassay method for trace amount of drugs in hair |
| US20240006076A1 (en) * | 2022-05-02 | 2024-01-04 | Kpn Innovations, Llc. | Apparatus and method for determining toxic load quantifiers |
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| US20240006076A1 (en) * | 2022-05-02 | 2024-01-04 | Kpn Innovations, Llc. | Apparatus and method for determining toxic load quantifiers |
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