CN112198315B - Method for collecting and detecting abused drugs in nail or toenail sample - Google Patents
Method for collecting and detecting abused drugs in nail or toenail sample Download PDFInfo
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- CN112198315B CN112198315B CN202010954796.XA CN202010954796A CN112198315B CN 112198315 B CN112198315 B CN 112198315B CN 202010954796 A CN202010954796 A CN 202010954796A CN 112198315 B CN112198315 B CN 112198315B
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- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
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- Cell Biology (AREA)
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- Biotechnology (AREA)
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- Food Science & Technology (AREA)
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- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Nanotechnology (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract
The invention relates to the technical field of immunodiagnosis, and discloses a method for collecting and detecting abused drugs in nail or toenail samples, which specifically comprises the following steps: (1) nail or toenail sample collection: obtaining a nail or toenail sample by a physical mechanical method; (2) nail pretreatment: adding nails into a composite treatment liquid for reaction, wherein the composite treatment liquid contains disulfide bond reducing agent, hydrogen bond blocking agent, nail swelling agent and buffer system; (3) nail stabilization treatment: adding a protective agent into the composite treatment liquid to obtain a reagent to be tested; (4) abuse drug detection: and (3) detecting the reagent to be detected obtained in the step (2) by using a drug abuse detection reagent strip. The test paper and the whole set of test method are prepared for the first time, and the test paper is simple and convenient and quick to operate, visual and accurate in result display and strong in specificity.
Description
Technical Field
The invention relates to the technical field of immunodiagnosis, in particular to a method for collecting and detecting abused drugs in nail or toenail samples.
Background
In judicial identification practice, many cases and various classes of drugs of abuse. When the biological material is subjected to forensic poison analysis, various biological materials have the characteristics and the application range, wherein detection researches on unconventional biological detection materials such as hair, tears, sweat, saliva and the like are also gradually in progress. With the development of analytical techniques now, the use of non-conventional biological assay materials for forensic poison analysis has also become feasible. Nails have been used in practical cases as court practice evidence, such as determining whether to poison, defect of nails to identify the occurrence of fight, etc., and in vivo abuse analysis, nails have not received widespread attention as a class of auxiliary non-routine biological specimens.
Nails are tissues composed of 80-90 layers of layered dead cells, and the tooth decay component is keratin (of which about 4/5 is hard keratin and about 1/5 is soft keratin), and also contains 10% -30% of water and trace fat. Walters et al and Murdan et al, in 1982 and 2002, respectively, suggested that nails or toenails are physically and chemically similar to hydrophilic gel films that were worn in a certain proportion. The complete nail or toenail is composed of a nail unit and a nail plate together. The nail unit is the part that supports and produces the nail plate, which is divided into three parts from the nail root in turn by the arc line (the boundary of the edge of the semilunar mark) and the yellow line "the boundary when the nail or toenail extends out of the nail bed", which are the methyl region, nail bed and free edge in turn from the nail root. The nail plate, the part for forensic poison analysis specimens, collectively referred to as nails or toenails, is divided into three parts from top to bottom: upper deck, middle layer and lower deck.
Nail or toenail growth rate is affected by a number of factors, such as age, nutrition, disease, climate, etc. Generally, the nail growth rate is 1.9 to 4.4 mm/month. And the same family members have little difference. Typically, the growth rate can be calculated as 0.1 mm/day or 3 mm/month when the time is inferred by the nail; the growth rate of the toenail is about 1/2 to 1/3 of the nail and is 0.03 to 0.04 mm/day. From this growth rate estimate, normal nails take about 5 months to complete growth, while toenails take 12-18 months. The fingernails grow fastest in the life of the manned person at 10 to 14 years of age, and the growth rate decreases with age after 20 years of age.
Deck production relies entirely on cell populations in the methyl region of the growth. The metacarpal artery and vein on the radial and ulnar sides of the finger end form the finger end vascular network at the finger end, which together with the lymphatic vessels form the nutrient supply system for the methyl cell population, which then continues to replace Xie Shengcheng nail-forming cells, which push forward to form the nail plate. The nail bed is directly adhered to the periosteum of the nail, and is not involved in the growth of the nail by virtue of the metabolism of the blood circulation system on the periosteum, and only promotes the forward movement of the nail plate. The route of abuse of drugs into the nail is that the nail matrix domain metabolism remains after penetration into the nail root region of a human nail at the early stages of nail formation; the nail bed does not interfere with the content of the inclusion property domain of the nail or toenail. Under the condition of excluding external pollution and other factors, the pencil owner considers that the way of the abused drugs in the body entering the nails or toenails is only at the methyl mass.
At present, today, the diversification and the complicacy of the case of the related toxicity are forced to solve the actual problem, and forensic poison analysis fields of all countries in the world are gradually changed to non-conventional detection materials. Non-conventional test materials such as saliva, hair, sweat, etc. are already available. The nail or toenail has the characteristics of convenient material taking, no damage, difficult substitution and the like, so that the nail or toenail has obvious special advantages when being used as a legal medical expert poison analysis unconventional biological detection material. Many countries have developed studies in this regard. Nail abuse analysis has so far only been possible by GC-MS/MS and LC-MS/MS, and the nail must be pre-treated to collect, wash, mill, hydrolyze (digest), extract clean up to use these instruments. The operation is extremely complex and takes a long time, at least more than 2 hours are needed, the instrument equipment is expensive, and hundreds of thousands to hundreds of thousands of detection equipment are needed; the field environment is high, a fixed laboratory is needed, and the laboratory is not movable; is difficult to popularize and use in the base layer. Thus, there is a great need to develop a simpler and faster method of collecting and detecting drugs of abuse of nails.
Disclosure of Invention
Aiming at the blank of the prior art, the invention provides a method for collecting and detecting abused drugs in nail or toenail samples, fills the technical blank, prepares the detection test paper and the whole set of detection method for the first time, and has the characteristics of simple and rapid operation, visual and accurate result display, strong specificity, simple preparation process, convenient carrying and storage, reduced investment and detection cost, wide application range and the like.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method of collecting and detecting drugs of abuse in nail or toenail samples comprising the steps of:
(1) Nail or toenail sample collection: obtaining a nail or toenail sample by a physical mechanical method;
(2) Nail or toenail pretreatment: adding nails into a composite treatment liquid for reaction, wherein the composite treatment liquid contains disulfide bond reducing agent, hydrogen bond blocking agent, nail swelling agent and buffer system;
(3) Nail stabilization treatment: adding a protective agent into the composite treatment liquid to obtain a reagent to be tested; (4) abuse drug detection: and (3) detecting the reagent to be detected obtained in the step (2) by using a drug abuse detection reagent strip.
The invention describes nail or toenail sample collection, sample component exposure and protection, and ensures effective exposure and protection of components to be detected by a reasonable and effective method, thereby ensuring the accuracy of results. In the above operation steps, the compound treatment liquid in the step (2) and the protective agent in the step (3) are key technologies of the invention, and the compound treatment liquid can rapidly hydrolyze nails, so that drug molecules embedded in nail gaps are fully exposed to obtain substances to be detected, and the protective agent can protect the exposed substances to be detected from external influences.
Preferably, in the step (1), the physical mechanical method is scraping the nail or toenail cover or cutting the nail or toenail.
Preferably, the pH of the compound treatment solution in the step (2) is 8.0-10.0, wherein the disulfide bond reducing agent comprises tris (2-carboxyethyl) phosphine hydrochloride with the concentration of 28-29.5 mg/mL and beta-sodium thioglycolate with the concentration of 0.2-0.22 mg/mL; the hydrogen bond blocking agent is dodecyl sulfuric acid with the concentration of 0.5-0.7 g/mL; the expanding agent is urea with the concentration of 0.45-0.5 g/mL; the buffer system is TRIS buffer system with the concentration of 0.02 mol-1.0 mol, wherein the concentration is the final concentration of the compound treatment liquid.
Preferably, the reaction time after the nail or toenail is added to the composite treatment liquid in the step (2) is 5-10min.
The composite treating fluid treats nail or toenail samples, adopts a principle of a reduction method, wherein a disulfide bond reducing agent can selectively reduce disulfide bonds in keratin in nail into sulfhydryl groups, and damages secondary and tertiary structures of the keratin, so as to improve the solubility of the protein; the hydrogen bond blocking agent can protect the newly generated sulfhydryl group from being reoxidized into disulfide bond, and can also be used as a surfactant to reduce the surface tension of keratin molecules and further increase the solubility; nail expanders can allow the keratin to continue to swell until partially dissolved.
Preferably, in the step (3), the protective agent is alpha-cyclodextrin with the concentration of 0.12-0.25 mg/mL, and the volume ratio of the compound treatment fluid to the protective agent is 2:0.1-0.3.
The protective agent utilizes the characteristic that the outer edge of the alpha-cyclodextrin is hydrophilic, and the inner cavity is hydrophobic, can provide a hydrophobic binding site like enzyme, is used as a host for encapsulating the substance to be detected, can protect the substance to be detected exposed through hydrolysis of the composite treatment liquid, and can prevent the substance to be detected from being interfered by the outside to cause the deviation of the detection result.
Preferably, the abused drug detection reagent strip in the step (4) comprises a bottom plate, a sample pad, a red nano microsphere abused drug release pad, a polyester cellulose membrane and a water absorption pad are sequentially arranged along the length of the bottom plate, a parallel detection line T and a quality control line C are arranged on the polyester cellulose membrane, and a monoclonal antibody marked by a result is fixed on the red nano microsphere abused drug release pad.
The principle of the invention is red nanometer microsphere immunochromatography and competition method, when the sample contains abused drug to be detected, the antigen can be combined with the mouse anti-abused drug monoclonal antibody-red nanometer microsphere conjugate in the red nanometer microsphere abused drug treatment pad after sample addition to form an antigen-antibody complex, as the mouse anti-abused drug monoclonal antibody-red nanometer microsphere conjugate in the red nanometer microsphere abused drug treatment pad is combined with the abused drug to be detected, along with the progress of the chromatographic process, the excessive mouse anti-abused drug monoclonal antibody-red nanometer microsphere conjugate is not captured by the bovine serum albumin coated on the nitrocellulose membrane to be detected, the quality control line is red, the detection line has no color appearance, the color is rich, clear and discernible, no auxiliary equipment is needed, and the operation is convenient.
By using the test paper, the sample of the nail or the toenail containing the abused drug to be detected can be detected. Detecting the sample by using a test strip, and when the detection line T and the quality control line C both have red strips, indicating that the nail or toenail sample has no abused drug to be detected; if only the quality control line C shows a red band, indicating that the nail or toenail sample contains the abused drug to be detected; if the quality control line C does not have a red color band, the test strip result is invalid.
Preferably, the detection line T and the quality control line C are obtained by: spraying 0.1-2.0mg/ml of abused drug coupled bovine serum albumin conjugate and 1.0-2.0mg/ml of goat anti-rabbit IgG conjugate solution on a nitrocellulose membrane at a speed of 1.0-1.2ul/cm to respectively serve as a detection line T and a quality control line C, and drying at 43-47 ℃ to obtain the detection line T and the quality control line C.
Preferably, the preparation process of the red nano microsphere abused drug release pad comprises the following steps:
A. preparing a red nanometer microsphere particle solution: dissolving chloroauric acid in deionized water to obtain a chloroauric acid solution with the concentration of 0.05-0.5g/ml, heating deionized purified water to 84-86 ℃, adding chloroauric acid into the solution according to the ratio of 1:500-1500, continuously heating to boil, rapidly adding a reducing agent trisodium citrate for reacting for 5-7min, stopping heating, and naturally cooling to room temperature;
B. Marking red nanometer microspheres: measuring red nanometer microsphere particle solution, adding K 2 CO 3 Adjusting the solution to isoelectric point, adding a toxin detection antibody according to a mark proportion of 1:25-1:15, fully reacting for 25-30min, adding 0.1-0.3mol/L BSA for reacting for 30-35min, adding EDC activator with concentration of 0.5-1.5mg/ml, and reacting for 15-20min;
C. and (3) centrifugal treatment: centrifuging the reacted biological raw material-red nanometer microsphere conjugate solution for 30-40min at 10000-10500r/min, removing supernatant, and collecting the centrifuged solution by using the redissolved solution to obtain the finished product.
The red microsphere is 30-80nm in size, gold ions in chloroauric acid are reduced by using trisodium citrate as a reducing agent, EDC is added for activation, BSA is added for blocking redundant sites, a mouse anti-abuse drug monoclonal antibody to be marked is added to be detected, the monoclonal antibody is physically adsorbed on the surface of the gold ions, supernatant is removed through centrifugation, a re-solvent is used for re-dissolution, and the concentration of a conjugate is detected by using an ultraviolet wind-light photometer and is represented by an OD value. The red nanometer microsphere conjugate plays a role in color development in the whole operation stage, and the metal ions and the protein are combined mainly through electrostatic adsorption in the coupling process.
Preferably, the volume ratio of the red nanoparticle solution, the BSA and the EDC activator is 1:4-5:3-4.
Preferably, the redissolution in the step C is prepared from 5-5.2mg/ml Casein and 0.05-0.1mol Tris-HCL according to a volume ratio of 2: 0.8-1.2.
The re-dissolved solution is used as a solvent after the marked red microsphere particles are coupled with biological raw materials, wherein a Tris-HCL buffer system provides a proper solution system for the coupling of the red microsphere particles and the biological raw materials, and the addition of Casein in the Tris-HCL buffer system can effectively seal redundant binding sites on the biological raw materials and can effectively reduce false positive conditions.
Therefore, the invention has the following beneficial effects:
(1) The method for collecting and detecting the abused drugs in the nail sample is provided, the components in the nail sample are effectively extracted and separated, the prepared test paper is short in detection time, the requirement of on-site detection can be met only by 5-10min, and the current technical blank is filled;
(2) The operation is simple and convenient, and other equipment and instruments are not needed; the detection result is displayed intuitively, can be judged by naked eyes, and is suitable for individuals to use; the detection efficiency is high, the detection result is more direct, and the defect that the existing detection instrument is expensive and inconvenient is avoided;
(3) The preparation process is simple, the detection test paper strip can be stored at normal temperature, no special equipment is needed, the test paper strip is kept dry, the storage period is long, and the storage cost is low.
Drawings
FIG. 1 is a schematic diagram of the test paper structure of the present invention.
FIG. 2 is a schematic diagram of the operational steps of the present invention.
In the figure: 1. a sample pad; 2. the red nano-microsphere abuses the drug release pad; 3. a detection line T; 4. a quality control line C; 5. a polyester cellulose film; 6. water absorption pad, 7, bottom plate.
Detailed Description
The invention is further described below in connection with the following detailed description.
General examples
A method of collecting and detecting drugs of abuse in nail or toenail samples comprising the steps of:
(1) Nail or toenail sample collection: obtaining (scraping or clipping) nail or toenail samples by physical mechanical means;
(2) Nail or toenail pretreatment: adding nails into a composite treatment liquid for reaction for 5-10min, wherein the composite treatment liquid contains disulfide bond reducing agent, hydrogen bond blocking agent, nail swelling agent and buffer system; the pH value of the compound treatment solution is 8.0-10.0, wherein the disulfide bond reducing agent comprises tri (2-carboxyethyl) phosphine hydrochloride with the concentration of 28-29.5 mg/mL and beta-sodium thioglycolate with the concentration of 0.2-0.22 mg/mL; the hydrogen bond blocking agent is dodecyl sulfuric acid with the concentration of 0.5-0.7 g/mL; the expanding agent is urea with the concentration of 0.45-0.5 g/mL; the buffer system is TRIS buffer system with the concentration of 0.02 mol-1.0 mol, wherein the concentration is the final concentration of the compound treatment liquid.
(3) Nail stabilization treatment: adding a protective agent into the composite treatment liquid to obtain a reagent to be tested; the protective agent is alpha-cyclodextrin with the concentration of 0.12-0.25 mg/mL, and the volume ratio of the composite treatment liquid to the protective agent is 2:0.1-0.3.
(4) Drug abuse detection: and (3) detecting the reagent to be detected obtained in the step (2) by using a drug abuse detection reagent strip.
The abused drug detection reagent strip comprises a bottom plate 7, a sample pad 1, a red nanometer microsphere abused drug release pad 2, a polyester cellulose membrane 5 and a water absorption pad 6 are sequentially arranged along the length of the bottom plate 7, a parallel detection line T3 and a quality control line C4 are arranged on the polyester cellulose membrane 5, and a monoclonal antibody marked by a result is fixed on the red nanometer microsphere abused drug release pad 2. The detection line T3 and the quality control line C4 are obtained through the following steps: spraying 0.1-2.0mg/ml of abused drug coupled bovine serum albumin conjugate and 1.0-2.0mg/ml of goat anti-rabbit IgG conjugate solution on a nitrocellulose membrane at a speed of 1.0-1.2ul/cm to respectively serve as a detection line T and a quality control line C, and drying at 43-47 ℃ to obtain the detection line T and the quality control line C.
The preparation process of the red nanometer microsphere abused drug release pad 2 comprises the following steps:
A. Preparing a red nanometer microsphere particle solution: dissolving chloroauric acid in deionized water to obtain a chloroauric acid solution with the concentration of 0.05-0.5g/ml, heating deionized purified water to 84-86 ℃, adding chloroauric acid into the solution according to the ratio of 1:500-1500, continuously heating to boil, rapidly adding a reducing agent trisodium citrate for reacting for 5-7min, stopping heating, and naturally cooling to room temperature;
B. marking red nanometer microspheres: measuring red nanometer microsphere particle solution, adding K 2 CO 3 Adjusting the solution to isoelectric point, adding a toxin detection antibody according to a mark proportion of 1:25-1:15, fully reacting for 25-30min, adding 0.1-0.3mol/L BSA for reacting for 30-35min, adding EDC activator with concentration of 0.5-1.5mg/ml, and reacting for 15-20min; the volume ratio of the red nanoparticle solution to the BSA to the EDC activator is 1:4-5:3-4;
C. and (3) centrifugal treatment: centrifuging the reacted biological raw material-red nanometer microsphere conjugate solution for 30-40min at 10000-10500r/min, removing supernatant, and collecting the centrifuged solution by using a double-dissolving solution (a solution prepared by mixing 5-5.2mg/ml Casein and 0.05-0.1mol Tris-HCL according to a volume ratio of 2:0.8-1.2), thus obtaining the finished product.
Example 1
A method of collecting and detecting drugs of abuse in nail or toenail samples comprising the steps of:
(1) Nail or toenail sample collection: cutting nail or toenail samples;
(2) Nail or toenail pretreatment: adding nails into a composite treatment liquid for reaction for 8min, wherein the composite treatment liquid contains disulfide bond reducing agent, hydrogen bond blocking agent, nail swelling agent and buffer system; the pH value of the compound treatment solution is 9, wherein the disulfide bond reducing agent comprises tris (2-carboxyethyl) phosphine hydrochloride with the concentration of 29mg/mL and beta-sodium thioglycolate with the concentration of 0.21 mg/mL; the hydrogen bond blocking agent is dodecyl sulfuric acid with the concentration of 0.6 g/mL; the swelling agent is urea with the concentration of 0.48 g/mL; the buffer system is TRIS buffer system with concentration of 0.5mol, wherein the concentration is the final concentration of the compound treatment solution.
(3) Nail stabilization treatment: adding a protective agent into the composite treatment liquid to obtain a reagent to be tested; the protective agent is alpha-cyclodextrin with the concentration of 0.2mg/mL, and the volume ratio of the composite treatment liquid to the protective agent is 2:0.2.
(4) Drug abuse detection: and (3) detecting the reagent to be detected obtained in the step (2) by using a drug abuse detection reagent strip.
The abused drug detection reagent strip comprises a bottom plate 7, a sample pad 1, a red nanometer microsphere abused drug release pad 2, a polyester cellulose membrane 5 and a water absorption pad 6 are sequentially arranged along the length of the bottom plate 7, a parallel detection line T3 and a quality control line C4 are arranged on the polyester cellulose membrane 5, and a monoclonal antibody marked by a result is fixed on the red nanometer microsphere abused drug release pad 2. The detection line T3 and the quality control line C4 are obtained through the following steps: spraying 1.2mg/ml of abused drug coupled bovine serum albumin conjugate and 1.5mg/ml of goat anti-rabbit IgG conjugate solution on a nitrocellulose membrane at a speed of 1.1ul/cm to be respectively used as a detection line T and a quality control line C, and drying at 45 ℃ to obtain the detection line T and the quality control line C.
The preparation process of the red nanometer microsphere abused drug release pad 2 comprises the following steps:
A. preparing a red nanometer microsphere particle solution: dissolving chloroauric acid in deionized water to obtain a chloroauric acid solution with the concentration of 0.25g/ml, heating deionized purified water to 85 ℃, adding chloroauric acid into the solution according to the ratio of 1:1000, continuously heating to boil, rapidly adding a reducing agent trisodium citrate for reaction for 6min, stopping heating, and naturally cooling to room temperature;
B. marking red nanometer microspheres: measuring red nanometer microsphere particle solution, adding K 2 CO 3 Adjusting the solution to isoelectric point, adding a toxin detection antibody according to a mark ratio of 1:20, fully reacting for 28min, adding 0.2mol/L BSA for reacting for 32min, adding EDC activator with concentration of 1mg/ml, and reacting for 18min; the volume ratio of the red nanoparticle solution to the BSA to the EDC activator is 1:4.5:3.5;
C. and (3) centrifugal treatment: centrifuging the reacted rabbit anti-nicotine polyclonal antibody-red nanometer microsphere conjugate solution for 35min at 10250r/min, removing supernatant, and collecting the centrifuged solution by using a redissolved solution (a solution of 5.1mg/ml Casein and 0.5mol Tris-HCL mixed according to a volume ratio of 2:1) to obtain a finished product.
Finger (toe) nicotine detection reagent:
The reagent takes rabbit anti-nicotine polyclonal antibody as biological raw material.
Collecting nails or toenails of a tested person, wherein a smoker 30 persons and a non-smoker 30 persons; the nicotine detection kit is directly used for detection, and the detection result is as follows:
example 2
A method of collecting and detecting drugs of abuse in nail or toenail samples comprising the steps of:
(1) Nail or toenail sample collection: scraping nail or toenail samples;
(2) Nail or toenail pretreatment: adding nails into a composite treatment liquid for reaction for 5min, wherein the composite treatment liquid contains disulfide bond reducing agent, hydrogen bond blocking agent, nail swelling agent and buffer system; the pH of the compound treatment solution is 8.0, wherein the disulfide bond reducing agent comprises tris (2-carboxyethyl) phosphine hydrochloride with the concentration of 29.5mg/mL and beta-sodium thioglycolate with the concentration of 0.2 mg/mL; the hydrogen bond blocking agent is dodecyl sulfuric acid with the concentration of 0.5 g/mL; the swelling agent is urea with the concentration of 0.5 g/mL; the buffer system was TRIS buffer system with a concentration of 0.02mol, where the concentration is the final concentration of the complex treatment solution.
(3) Nail stabilization treatment: adding a protective agent into the composite treatment liquid to obtain a reagent to be tested; the protective agent is alpha-cyclodextrin with the concentration of 0.12mg/mL, and the volume ratio of the composite treatment liquid to the protective agent is 2:0.3.
(4) Drug abuse detection: and (3) detecting the reagent to be detected obtained in the step (2) by using a drug abuse detection reagent strip.
The abused drug detection reagent strip comprises a bottom plate 7, a sample pad 1, a red nanometer microsphere abused drug release pad 2, a polyester cellulose membrane 5 and a water absorption pad 6 are sequentially arranged along the length of the bottom plate 7, a parallel detection line T3 and a quality control line C4 are arranged on the polyester cellulose membrane 5, and a monoclonal antibody marked by a result is fixed on the red nanometer microsphere abused drug release pad 2. The detection line T3 and the quality control line C4 are obtained through the following steps: and respectively spraying 0.1mg/ml of abused drug coupled bovine serum albumin conjugate and 1.0mg/ml of goat anti-rabbit IgG conjugate solution on a nitrocellulose membrane at a speed of 1.2ul/cm to respectively serve as a detection line T and a quality control line C, and drying at 43 ℃ to obtain the detection line T and the quality control line C.
The preparation process of the red nanometer microsphere abused drug release pad 2 comprises the following steps:
A. preparing a red nanometer microsphere particle solution: dissolving chloroauric acid in deionized water to obtain a chloroauric acid solution with the concentration of 0.05g/ml, heating deionized purified water to 84 ℃, adding chloroauric acid into the solution according to the amount of 1:500, continuously heating to boil, rapidly adding a reducing agent trisodium citrate for reaction for 5min, stopping heating, and naturally cooling to room temperature;
B. Marking red nanometer microspheres: measuring red nanometer microsphere particle solution, adding K 2 CO 3 Regulating the solution to isoelectric point, and adding drugs according to the marking proportion of 1:25The detection antibody is fully reacted for 25min, then 0.1mol/L BSA is added for reaction for 30-35min, meanwhile EDC activator with the concentration of 0.5-1.5mg/ml is added for reaction for 15-20min; the volume ratio of the red nanoparticle solution to the BSA to the EDC activator is 1:4:4, a step of;
C. and (3) centrifugal treatment: centrifuging the reacted rabbit anti-nicotine polyclonal antibody-red nanometer microsphere conjugate solution for 30min at 10000r/min, removing supernatant, and collecting the centrifuged solution by using a redissolved solution (5 mg/ml solution of Casein and 0.1mol Tris-HCL mixed according to the volume ratio of 2:0.8), thus obtaining the finished product.
The reagent takes rabbit anti-nicotine polyclonal antibody as biological raw material.
Collecting nails or toenails of a tested person, wherein a smoker 30 persons and a non-smoker 30 persons; the nicotine detection kit is directly used for detection, and the detection result is as follows:
example 3
A method of collecting and detecting drugs of abuse in nail or toenail samples comprising the steps of:
(1) Nail or toenail sample collection: scraping nail or toenail samples;
(2) Nail or toenail pretreatment: adding nails into a composite treatment liquid for reaction for 10min, wherein the composite treatment liquid contains disulfide bond reducing agent, hydrogen bond blocking agent, nail swelling agent and buffer system; the pH of the compound treatment solution is 10.0, wherein the disulfide bond reducing agent comprises tris (2-carboxyethyl) phosphine hydrochloride with the concentration of 29.5mg/mL and beta-sodium thioglycolate with the concentration of 0.22 mg/mL; the hydrogen bond blocking agent is dodecyl sulfuric acid with the concentration of 0.7 g/mL; the swelling agent is urea with the concentration of 0.5 g/mL; the buffer system was TRIS buffer system with a concentration of 0.02mol, where the concentration is the final concentration of the complex treatment solution.
(3) Nail stabilization treatment: adding a protective agent into the composite treatment liquid to obtain a reagent to be tested; the protective agent is alpha-cyclodextrin with the concentration of 0.25mg/mL, and the volume ratio of the composite treatment liquid to the protective agent is 2:0.1.
(4) Drug abuse detection: and (3) detecting the reagent to be detected obtained in the step (2) by using a drug abuse detection reagent strip.
The abused drug detection reagent strip comprises a bottom plate 7, a sample pad 1, a red nanometer microsphere abused drug release pad 2, a polyester cellulose membrane 5 and a water absorption pad 6 are sequentially arranged along the length of the bottom plate 7, a parallel detection line T3 and a quality control line C4 are arranged on the polyester cellulose membrane 5, and a monoclonal antibody marked by a result is fixed on the red nanometer microsphere abused drug release pad 2. The detection line T3 and the quality control line C4 are obtained through the following steps: 02.0mg/ml of abused drug coupled bovine serum albumin conjugate and 1.0mg/ml of goat anti-rabbit IgG conjugate solution are respectively sprayed on a nitrocellulose membrane at a speed of 1.2ul/cm to be respectively used as a detection line T and a quality control line C, and the detection line T and the quality control line C are obtained by drying at 43 ℃.
The preparation process of the red nanometer microsphere abused drug release pad 2 comprises the following steps:
A. Preparing a red nanometer microsphere particle solution: dissolving chloroauric acid in deionized water to obtain a chloroauric acid solution with the concentration of 0.5g/ml, heating deionized purified water to 84 ℃, adding chloroauric acid into the solution according to the amount of 1:1500, continuously heating to boil, rapidly adding a reducing agent trisodium citrate for reaction for 7min, stopping heating, and naturally cooling to room temperature;
B. marking red nanometer microspheres: measuring red nanometer microsphere particle solution, adding K 2 CO 3 Adjusting the solution to isoelectric point, adding a toxin detection antibody according to a mark ratio of 1:15, fully reacting for 30min, adding 0.3mol/L BSA for reacting for 35min, adding EDC activator with concentration of 0.5mg/ml, and reacting for 20min; the volume ratio of the red nanoparticle solution to the BSA to the EDC activator is 1:5:3, a step of;
C. and (3) centrifugal treatment: centrifuging the reacted rabbit anti-nicotine polyclonal antibody-red nanometer microsphere conjugate solution for 30min at 10500r/min, removing supernatant, and collecting the centrifuged solution by using a redissolved solution (a solution of 5.2mg/ml Casein and 0.05mol Tris-HCL according to a volume ratio of 2:1.2), thereby obtaining a finished product.
The reagent takes rabbit anti-nicotine polyclonal antibody as biological raw material.
Collecting nails or toenails of a tested person, wherein a smoker 30 persons and a non-smoker 30 persons; the nicotine detection kit is directly used for detection, and the detection result is as follows:
Example 4
A method of collecting and detecting drugs of abuse in nail or toenail samples comprising the steps of:
(1) Nail or toenail sample collection: cutting nail or toenail samples;
(2) Nail or toenail pretreatment: adding nails into a composite treatment liquid for reaction for 8min, wherein the composite treatment liquid contains disulfide bond reducing agent, hydrogen bond blocking agent, nail swelling agent and buffer system; the pH value of the compound treatment solution is 9, wherein the disulfide bond reducing agent comprises tris (2-carboxyethyl) phosphine hydrochloride with the concentration of 29mg/mL and beta-sodium thioglycolate with the concentration of 0.21 mg/mL; the hydrogen bond blocking agent is dodecyl sulfuric acid with the concentration of 0.6 g/mL; the swelling agent is urea with the concentration of 0.48 g/mL; the buffer system is TRIS buffer system with concentration of 0.5mol, wherein the concentration is the final concentration of the compound treatment solution.
(3) Nail stabilization treatment: adding a protective agent into the composite treatment liquid to obtain a reagent to be tested; the protective agent is alpha-cyclodextrin with the concentration of 0.2mg/mL, and the volume ratio of the composite treatment liquid to the protective agent is 2:0.2.
(4) Drug abuse detection: and (3) detecting the reagent to be detected obtained in the step (2) by using a drug abuse detection reagent strip.
The abused drug detection reagent strip comprises a bottom plate 7, a sample pad 1, a red nanometer microsphere abused drug release pad 2, a polyester cellulose membrane 5 and a water absorption pad 6 are sequentially arranged along the length of the bottom plate 7, a parallel detection line T3 and a quality control line C4 are arranged on the polyester cellulose membrane 5, and a monoclonal antibody marked by a result is fixed on the red nanometer microsphere abused drug release pad 2. The detection line T3 and the quality control line C4 are obtained through the following steps: spraying 1.2mg/ml of abused drug coupled bovine serum albumin conjugate and 1.5mg/ml of goat anti-rabbit IgG conjugate solution on a nitrocellulose membrane at a speed of 1.1ul/cm to be respectively used as a detection line T and a quality control line C, and drying at 45 ℃ to obtain the detection line T and the quality control line C.
The preparation process of the red nanometer microsphere abused drug release pad 2 comprises the following steps:
A. preparing a red nanometer microsphere particle solution: dissolving chloroauric acid in deionized water to obtain a chloroauric acid solution with the concentration of 0.25g/ml, heating deionized purified water to 85 ℃, adding chloroauric acid into the solution according to the ratio of 1:1000, continuously heating to boil, rapidly adding a reducing agent trisodium citrate for reaction for 6min, stopping heating, and naturally cooling to room temperature;
B. marking red nanometer microspheres: measuring red nanometer microsphere particle solution, adding K 2 CO 3 Adjusting the solution to isoelectric point, adding a toxin detection antibody according to a mark ratio of 1:20, fully reacting for 28min, adding 0.2mol/L BSA for reacting for 32min, adding EDC activator with concentration of 1mg/ml, and reacting for 18min; the volume ratio of the red nanoparticle solution to the BSA to the EDC activator is 1:4.5:3.5;
C. and (3) centrifugal treatment: centrifuging the reacted mouse anti-morphine monoclonal antibody-red nanometer microsphere conjugate solution for 35min at 10250r/min, removing supernatant, and collecting the centrifuged solution by using a redissolved solution (a solution of 5.1mg/ml Casein and 0.5mol Tris-HCL mixed according to a volume ratio of 2:1) to obtain a finished product.
The reagent takes a mouse anti-morphine monoclonal antibody as a biological raw material.
Collecting nails or toenails of a tested person, wherein 15 patients who inhale morphine and 25 patients who do not inhale morphine; the detection is directly carried out by using a finger (toe) morphine detection kit, and the detection results are as follows:
example 5
A method of collecting and detecting drugs of abuse in nail or toenail samples comprising the steps of:
(1) Nail or toenail sample collection: cutting nail or toenail samples;
(2) Nail or toenail pretreatment: adding nails into a composite treatment liquid for reaction for 8min, wherein the composite treatment liquid contains disulfide bond reducing agent, hydrogen bond blocking agent, nail swelling agent and buffer system; the pH value of the compound treatment solution is 9, wherein the disulfide bond reducing agent comprises tris (2-carboxyethyl) phosphine hydrochloride with the concentration of 29mg/mL and beta-sodium thioglycolate with the concentration of 0.21 mg/mL; the hydrogen bond blocking agent is dodecyl sulfuric acid with the concentration of 0.6 g/mL; the swelling agent is urea with the concentration of 0.48 g/mL; the buffer system is TRIS buffer system with concentration of 0.5mol, wherein the concentration is the final concentration of the compound treatment solution.
(3) Nail stabilization treatment: adding a protective agent into the composite treatment liquid to obtain a reagent to be tested; the protective agent is alpha-cyclodextrin with the concentration of 0.2mg/mL, and the volume ratio of the composite treatment liquid to the protective agent is 2:0.2.
(4) Drug abuse detection: and (3) detecting the reagent to be detected obtained in the step (2) by using a drug abuse detection reagent strip.
The abused drug detection reagent strip comprises a bottom plate 7, a sample pad 1, a red nanometer microsphere abused drug release pad 2, a polyester cellulose membrane 5 and a water absorption pad 6 are sequentially arranged along the length of the bottom plate 7, a parallel detection line T3 and a quality control line C4 are arranged on the polyester cellulose membrane 5, and a monoclonal antibody marked by a result is fixed on the red nanometer microsphere abused drug release pad 2. The detection line T3 and the quality control line C4 are obtained through the following steps: spraying 1.2mg/ml of abused drug coupled bovine serum albumin conjugate and 1.5mg/ml of goat anti-rabbit IgG conjugate solution on a nitrocellulose membrane at a speed of 1.1ul/cm to be respectively used as a detection line T and a quality control line C, and drying at 45 ℃ to obtain the detection line T and the quality control line C.
The preparation process of the red nanometer microsphere abused drug release pad 2 comprises the following steps:
A. Preparing a red nanometer microsphere particle solution: dissolving chloroauric acid in deionized water to obtain a chloroauric acid solution with the concentration of 0.25g/ml, heating deionized purified water to 85 ℃, adding chloroauric acid into the solution according to the ratio of 1:1000, continuously heating to boil, rapidly adding a reducing agent trisodium citrate for reaction for 6min, stopping heating, and naturally cooling to room temperature;
B. marking red nanometer microspheres: measuring red nanometer microsphere particle solution, adding K 2 CO 3 Adjusting the solution to isoelectric point, adding a toxin detection antibody according to a mark ratio of 1:20, fully reacting for 28min, adding 0.2mol/L BSA for reacting for 32min, adding EDC activator with concentration of 1mg/ml, and reacting for 18min; the volume ratio of the red nanoparticle solution to the BSA to the EDC activator is 1:4.5:3.5;
C. and (3) centrifugal treatment: centrifuging the reacted mouse anti-chlorthalidone monoclonal antibody-red nanometer microsphere conjugate solution for 35min according to 10250r/min, removing supernatant, and collecting the centrifuged solution by using a redissolved solution (a solution of 5.1mg/ml Casein and 0.5mol Tris-HCL according to a volume ratio of 2:1), thus obtaining a finished product.
The reagent takes a mouse anti-chloraminoketone monoclonal antibody as a biological raw material.
Collecting nails or toenails of a tested person, wherein 10 persons of patients taking chlorthalidone and 30 persons of patients not taking morphine are collected; the detection is directly carried out by using a finger (toe) mechlorethamine detection kit, and the detection result is as follows:
Example 6
A method of collecting and detecting drugs of abuse in nail or toenail samples comprising the steps of:
(1) Nail or toenail sample collection: cutting nail or toenail samples;
(2) Nail or toenail pretreatment: adding nails into a composite treatment liquid for reaction for 8min, wherein the composite treatment liquid contains disulfide bond reducing agent, hydrogen bond blocking agent, nail swelling agent and buffer system; the pH value of the compound treatment solution is 9, wherein the disulfide bond reducing agent comprises tris (2-carboxyethyl) phosphine hydrochloride with the concentration of 29mg/mL and beta-sodium thioglycolate with the concentration of 0.21 mg/mL; the hydrogen bond blocking agent is dodecyl sulfuric acid with the concentration of 0.6 g/mL; the swelling agent is urea with the concentration of 0.48 g/mL; the buffer system is TRIS buffer system with concentration of 0.5mol, wherein the concentration is the final concentration of the compound treatment solution.
(3) Nail stabilization treatment: adding a protective agent into the composite treatment liquid to obtain a reagent to be tested; the protective agent is alpha-cyclodextrin with the concentration of 0.2mg/mL, and the volume ratio of the composite treatment liquid to the protective agent is 2:0.2.
(4) Drug abuse detection: and (3) detecting the reagent to be detected obtained in the step (2) by using a drug abuse detection reagent strip.
The abused drug detection reagent strip comprises a bottom plate 7, a sample pad 1, a red nanometer microsphere abused drug release pad 2, a polyester cellulose membrane 5 and a water absorption pad 6 are sequentially arranged along the length of the bottom plate 7, a parallel detection line T3 and a quality control line C4 are arranged on the polyester cellulose membrane 5, and a monoclonal antibody marked by a result is fixed on the red nanometer microsphere abused drug release pad 2. The detection line T3 and the quality control line C4 are obtained through the following steps: spraying 1.2mg/ml of abused drug coupled bovine serum albumin conjugate and 1.5mg/ml of goat anti-rabbit IgG conjugate solution on a nitrocellulose membrane at a speed of 1.1ul/cm to be respectively used as a detection line T and a quality control line C, and drying at 45 ℃ to obtain the detection line T and the quality control line C.
The preparation process of the red nanometer microsphere abused drug release pad 2 comprises the following steps:
A. preparing a red nanometer microsphere particle solution: dissolving chloroauric acid in deionized water to obtain a chloroauric acid solution with the concentration of 0.25g/ml, heating deionized purified water to 85 ℃, adding chloroauric acid into the solution according to the ratio of 1:1000, continuously heating to boil, rapidly adding a reducing agent trisodium citrate for reaction for 6min, stopping heating, and naturally cooling to room temperature;
B. marking red nanometer microspheres: measuring a red nanometer microsphere particle solution, adding a K2CO3 solution to adjust to an isoelectric point, adding a toxin detection antibody according to a labeling proportion of 1:20, fully reacting for 28min, adding 0.2mol/L BSA to react for 32min, adding EDC activator with the concentration of 1mg/ml, and reacting for 18min; the volume ratio of the red nanoparticle solution to the BSA to the EDC activator is 1:4.5:3.5;
C. And (3) centrifugal treatment: centrifuging the reacted mouse anti-methamphetamine monoclonal antibody-red nanometer microsphere conjugate solution for 35min at 10250r/min, removing supernatant, and collecting the centrifuged solution by using a redissolution (a solution of 5.1mg/ml Casein and 0.5mol Tris-HCL mixed according to a volume ratio of 2:1) to obtain a finished product. Finger (toe) methamphetamine (toxalbumin) detection reagent:
the reagent takes a mouse anti-methamphetamine monoclonal antibody as a biological raw material.
Collecting nails or toenails of a tested person, wherein 15 people of patients with ice toxin are inhaled and 25 people of patients without ice toxin are not inhaled; directly detecting with a finger (toe) nail ice toxin detection kit, and the detection result is as follows:
example 7
A method of collecting and detecting drugs of abuse in nail or toenail samples comprising the steps of:
(1) Nail or toenail sample collection: cutting nail or toenail samples;
(2) Nail or toenail pretreatment: adding nails into a composite treatment liquid for reaction for 8min, wherein the composite treatment liquid contains disulfide bond reducing agent, hydrogen bond blocking agent, nail swelling agent and buffer system; the pH value of the compound treatment solution is 9, wherein the disulfide bond reducing agent comprises tris (2-carboxyethyl) phosphine hydrochloride with the concentration of 29mg/mL and beta-sodium thioglycolate with the concentration of 0.21 mg/mL; the hydrogen bond blocking agent is dodecyl sulfuric acid with the concentration of 0.6 g/mL; the swelling agent is urea with the concentration of 0.48 g/mL; the buffer system is TRIS buffer system with concentration of 0.5mol, wherein the concentration is the final concentration of the compound treatment solution.
(3) Nail stabilization treatment: adding a protective agent into the composite treatment liquid to obtain a reagent to be tested; the protective agent is alpha-cyclodextrin with the concentration of 0.2mg/mL, and the volume ratio of the composite treatment liquid to the protective agent is 2:0.2.
(4) Drug abuse detection: and (3) detecting the reagent to be detected obtained in the step (2) by using a drug abuse detection reagent strip.
The abused drug detection reagent strip comprises a bottom plate 7, a sample pad 1, a red nanometer microsphere abused drug release pad 2, a polyester cellulose membrane 5 and a water absorption pad 6 are sequentially arranged along the length of the bottom plate 7, a parallel detection line T3 and a quality control line C4 are arranged on the polyester cellulose membrane 5, and a monoclonal antibody marked by a result is fixed on the red nanometer microsphere abused drug release pad 2. The detection line T3 and the quality control line C4 are obtained through the following steps: spraying 1.2mg/ml of abused drug coupled bovine serum albumin conjugate and 1.5mg/ml of goat anti-rabbit IgG conjugate solution on a nitrocellulose membrane at a speed of 1.1ul/cm to be respectively used as a detection line T and a quality control line C, and drying at 45 ℃ to obtain the detection line T and the quality control line C.
The preparation process of the red nanometer microsphere abused drug release pad 2 comprises the following steps:
A. Preparing a red nanometer microsphere particle solution: dissolving chloroauric acid in deionized water to obtain a chloroauric acid solution with the concentration of 0.25g/ml, heating deionized purified water to 85 ℃, adding chloroauric acid into the solution according to the ratio of 1:1000, continuously heating to boil, rapidly adding a reducing agent trisodium citrate for reaction for 6min, stopping heating, and naturally cooling to room temperature;
B. marking red nanometer microspheres: measuring red nanometer microsphere particle solution, adding K 2 CO 3 Adjusting the solution to isoelectric point, adding a toxin detection antibody according to a mark ratio of 1:20, fully reacting for 28min, adding 0.2mol/L BSA for reacting for 32min, adding EDC activator with concentration of 1mg/ml, and reacting for 18min; the volume ratio of the red nanoparticle solution to the BSA to the EDC activator is 1:4.5:3.5;
C. and (3) centrifugal treatment: centrifuging the reacted mouse anti-cocaine monoclonal antibody-red nanometer microsphere conjugate solution for 35min at 10250r/min, removing supernatant, and collecting the centrifuged solution by using a redissolved solution (a solution of 5.1mg/ml Casein and 0.5mol Tris-HCL mixed according to a volume ratio of 2:1) to obtain a finished product.
The reagent takes a mouse anti-cocaine monoclonal antibody as a biological raw material.
Collecting nails or toenails of a tested person, wherein 5 persons of a cocaine patient are sucked and 25 persons of a cocaine patient are not sucked; the detection is directly carried out by using a finger (toe) nail cocaine detection kit, and the detection result is as follows:
From the data of examples 1-7, it can be seen that the above requirements can be met in all respects only with solutions within the scope of the claims of the present invention, leading to an optimized detection scheme. And the corresponding negative effects are brought to the change of the proportion, the replacement/addition of the raw materials or the change of the feeding sequence.
The raw materials and equipment used in the invention are common raw materials and equipment in the field unless specified otherwise; the methods used in the present invention are conventional in the art unless otherwise specified.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.
Claims (9)
1. A method for collecting and detecting drugs of abuse in nail samples, comprising the steps of:
(1) Nail or toenail sample collection: obtaining a nail or toenail sample by a physical mechanical method;
(2) Nail or toenail pretreatment: adding nails into a composite treatment liquid for reaction, wherein the composite treatment liquid contains disulfide bond reducing agent, hydrogen bond blocking agent, nail swelling agent and buffer system; the pH value of the composite treatment liquid is 8.0-10.0, wherein the disulfide bond reducing agent comprises tri (2-carboxyethyl) phosphine hydrochloride with the concentration of 28-29.5 mg/mL and beta-sodium thioglycolate with the concentration of 0.2-0.22 mg/mL; the hydrogen bond blocking agent is dodecyl sulfuric acid with the concentration of 0.5-0.7 g/mL; the expanding agent is urea with the concentration of 0.45-0.5 g/mL; the buffer system is a TRIS buffer system with the concentration of 0.02 mol-1.0 mol, and the concentration is the final concentration of the composite treatment liquid;
(3) Nail stabilization treatment: adding a protective agent into the composite treatment liquid to obtain a reagent to be tested; the protective agent is alpha-cyclodextrin with the concentration of 0.12-0.25 mg/mL;
(4) Drug abuse detection: and (3) detecting the reagent to be detected obtained in the step (2) by using a drug abuse detection reagent strip.
2. A method of collecting and detecting drugs of abuse in nail samples according to claim 1, wherein in step (1) the physical mechanical means is scraping or clipping the nail cover.
3. The method of claim 1, wherein the reaction time of the nail or toenail in step (2) after the addition of the complex treatment fluid is from 5 to 10 minutes.
4. The method for collecting and detecting drugs of abuse in nail samples according to claim 1, wherein the volume ratio of the complex treatment fluid to the protectant of step (3) is 2: 0.1-0.3.
5. The method for collecting and detecting abused drugs in nail samples according to claim 1, wherein the abused drug detection reagent strip in the step (4) comprises a bottom plate (7), a sample pad (1), a red nano microsphere abused drug release pad (2), a polyester cellulose membrane (5) and a water absorption pad (6) are sequentially arranged along the length of the bottom plate (7), a parallel detection line T (3) and a quality control line C (4) are arranged on the polyester cellulose membrane (5), and monoclonal antibodies marked by results are fixed on the red nano microsphere abused drug release pad (2).
6. The method for collecting and detecting drugs of abuse in nail samples according to claim 5, wherein the detection line T (3) and the quality control line C (4) are obtained by: spraying 0.1-2.0mg/ml of abused drug coupled bovine serum albumin conjugate and 1.0-2.0mg/ml of goat anti-rabbit IgG conjugate solution on a nitrocellulose membrane at a speed of 1.0-1.2ul/cm to respectively serve as a detection line T and a quality control line C, and drying at 43-47 ℃ to obtain the detection line T and the quality control line C.
7. The method for collecting and detecting abused drugs in nail samples according to claim 5, wherein the preparation process of the red nano microsphere abused drug release pad (2) is as follows:
A. preparing a red nanometer microsphere particle solution: dissolving chloroauric acid in deionized water to obtain a chloroauric acid solution with the concentration of 0.05-0.5g/ml, heating deionized purified water to 84-86 ℃, adding chloroauric acid into the solution according to the ratio of 1:500-1500, continuously heating to boil, rapidly adding a reducing agent trisodium citrate for reacting for 5-7min, stopping heating, and naturally cooling to room temperature;
B. marking red nanometer microspheres: measuring red nanometer microsphere particle solution, adding K 2 CO 3 Adjusting the solution to isoelectric point, adding a toxin detection antibody according to a mark proportion of 1:25-1:15, fully reacting for 25-30min, adding 0.1-0.3mol/L BSA for reacting for 30-35min, adding EDC activator with concentration of 0.5-1.5mg/ml, and reacting for 15-20min;
C. and (3) centrifugal treatment: centrifuging the reacted biological raw material-red nanometer microsphere conjugate solution for 30-40min at 10000-10500r/min, removing supernatant, and collecting the centrifuged solution by using the redissolved solution to obtain the finished product.
8. The method of claim 7, wherein the volume ratio of red nanoparticle solution, BSA and EDC activator is 1:4-5:3-4.
9. The method for collecting and detecting drugs of abuse in nail samples according to claim 7, wherein the reconstituted solution in step C is 5-5.2mg/ml Casein to 0.05-0.1mol Tris-HCL in a volume ratio of 2: 0.8-1.2.
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| US6949344B1 (en) * | 1987-12-28 | 2005-09-27 | Psychemical Corporation | Hair analysis method |
| CN102333546A (en) * | 2009-02-26 | 2012-01-25 | 巴斯夫欧洲公司 | Compositions, use and method for the use of surface active proteins in topical drug delivery across keratin |
| CN103175716A (en) * | 2011-12-20 | 2013-06-26 | 司法部司法鉴定科学技术研究所 | Preprocessing method for nail/toenail poison analysis |
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| US6949344B1 (en) * | 1987-12-28 | 2005-09-27 | Psychemical Corporation | Hair analysis method |
| CN102333546A (en) * | 2009-02-26 | 2012-01-25 | 巴斯夫欧洲公司 | Compositions, use and method for the use of surface active proteins in topical drug delivery across keratin |
| CN103175716A (en) * | 2011-12-20 | 2013-06-26 | 司法部司法鉴定科学技术研究所 | Preprocessing method for nail/toenail poison analysis |
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