CN112198315A - 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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- CN112198315A CN112198315A CN202010954796.XA CN202010954796A CN112198315A CN 112198315 A CN112198315 A CN 112198315A CN 202010954796 A CN202010954796 A CN 202010954796A CN 112198315 A CN112198315 A CN 112198315A
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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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- 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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- 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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- 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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Abstract
The invention relates to the technical field of immunodiagnosis, and discloses a method for collecting and detecting drugs of abuse in a nail or toenail sample aiming at the blank of the prior art, 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 or toenail pretreatment: adding the fingernail or the toenail into a composite treatment liquid for reaction, wherein the composite treatment liquid contains a disulfide bond reducing agent, a hydrogen bond blocking agent, a fingernail or toenail swelling agent and a buffer system; (3) nail or toenail stabilization treatment: adding the protective agent into the composite treatment liquid dropwise to obtain a to-be-tested agent; (4) detection of drugs of abuse: and (3) detecting the test agent obtained in the step (2) by adopting an abuse drug detection reagent strip. The test paper and the complete detection method are prepared for the first time, and the test paper has the advantages of simple and rapid operation, visual and accurate result display and strong specificity.
Description
Technical Field
The invention relates to the technical field of immunodiagnosis, in particular to a method for collecting and detecting drugs of abuse in a nail or toenail sample.
Background
In judicial identification practice, many cases and classes abuse drugs. When the forensic toxicology analysis is carried out on the biological material, various biological materials have the characteristics and the application range, wherein the detection research on unconventional biological detection materials, such as hair, tears, sweat, saliva and the like, is also carried out step by step. With the development of the current analysis technology, the application of the non-conventional biological material for testing on the forensic toxicant analysis becomes feasible. The nail or toenail has been used in the past as a forensic evidence in practical cases, and as such, exogenous contamination determines whether toxic substances are administered, nail defects are used to identify whether fighting has occurred, etc., and in vivo drug abuse analysis, the nail or toenail has not received much attention as an auxiliary unconventional biological test material.
Nails are tissues consisting of 80 to 90 layers of dead cells stacked, and the tooth-boring component is keratin (of which about 4/5 is hard keratin and about 1/5 is soft keratin), and also contains moisture in a total amount of 10 to 30% and a trace amount of fat. Walters et al and Murdan et al, in 1982 and 2002, respectively, suggest that nails or toenails are physically and chemically similar to hydrophilic gel films worn in certain proportions. The complete nail or toenail is made up of a nail unit and a nail plate. The nail unit is the part that supports and creates the nail plate, and is divided into three parts by the arch hatching (boundary of semilunar scar edge) and the yellow line "boundary when the nail or toenail protrudes from the nail bed" in order from the nail root, and is the nail matrix domain, the nail bed and the free edge in order from the nail root. The nail plate is the part which is used for forensic toxicant analysis examination materials and is collectively called nail or toenail, and the part is divided into three parts from top to bottom: the upper deck, the middle deck and the lower deck.
The growth rate of nails is affected by many factors, such as age, nutrition, disease, climate, etc. Generally, the growth rate of the nail is 1.9 to 4.4 mm/month. And the difference between the same family members is not great. Typically, when time is inferred from the nail, growth rate can be calculated as 0.1 mm/day or 3 mm/month; the growth rate of the toenail is about 1/2-1/3 of the toenail, and is 0.03-0.04 mm/day. According to the growth rate, it takes about 5 months for the normal nail to finish growing, while 12-18 months for the toenail. In the life of a person, the growth of the fingernails is fastest at the age of 10-14 years, and the growth speed is reduced with the age after the age of 20 years.
The nail plate is generated from a population of cells that grow entirely on the methyl matrix region. The inherent artery and vein of the finger palm on the radial side and the ulnar side of the finger tip form a finger tip blood vessel network at the tail end of the finger, the blood vessel network and a lymphatic vessel form a nutrition supply system of a methyl cytoplasm cell group, then the cell group is continuously metabolized to generate an formazan cell, and the formazan cell is pushed to move forward to form a nail plate. The nail bed is directly adhered to the periosteum of the nail or the toenail, depends on the metabolism of the blood circulation system on the periosteum, does not participate in the generation and growth of the nail or the toenail, and only promotes the forward movement of the nail plate. The route of drug abuse into the nail is retained after the nail or toenail formation initial stage methyl substance domain metabolism permeates the nail root part of the nail plate of the human nail or toenail; the nail bed does not interfere with the content of the nail's or toenail's inclusion property domains. In the absence of external contaminants and the like, the penaeid thinks that the route of abuse in the body into the nail or toenail should be only at the methyl group.
At present, with the diversification and complication of cases involving in drugs, the analysis view of forensic toxicants is gradually shifted to unconventional inspection of materials by countries in the world in order to solve the requirement of practical problems. The existing unconventional test materials comprise saliva, hair, sweat and the like. The nail or toenail has the characteristics of convenient material acquisition, no damage, difficult replacement and the like, so that the nail or toenail has the unique advantages when being used as an unconventional biological inspection material for forensic toxicant analysis. Many countries have now developed research in this regard. Until now, the analysis of drugs of abuse on nails can only be detected and analyzed by GC-MS/MS and LC-MS/MS, and the nails or toenails must be pre-treated, including collection, washing, grinding, hydrolysis (digestion), extraction and purification, before being detected by using the instruments. The operation is extremely complex and takes long time, at least more than 2 hours are needed, the instrument and equipment are expensive, and the detection equipment is used in case of hundreds of thousands to hundreds of cases; the field environment requirement is high, a fixed laboratory is needed, and the mobile device is not movable; difficult to be popularized and used in the basic layer. Therefore, there is an urgent need to develop a more convenient and faster method for collecting and detecting drugs of abuse on nails or toenails.
Disclosure of Invention
Aiming at the blank of the prior art, the invention provides a method for collecting and detecting abused drugs in a nail or toenail sample, fills the technical blank, prepares the detection test paper and a complete 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 purpose, the invention adopts the following technical scheme:
a method for collecting and detecting drugs of abuse in a nail or toenail sample 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 the fingernail or the toenail into a composite treatment liquid for reaction, wherein the composite treatment liquid contains a disulfide bond reducing agent, a hydrogen bond blocking agent, a fingernail or toenail swelling agent and a buffer system;
(3) nail or toenail stabilization treatment: adding the protective agent into the composite treatment liquid dropwise to obtain a to-be-tested agent; (4) detection of drugs of abuse: and (3) detecting the test agent obtained in the step (2) by adopting an abuse drug detection reagent strip.
The invention expounds the collection of nail or toenail samples, the exposure and the protection of sample components, ensures the effective exposure and protection of the components to be tested by a reasonable and effective method and ensures the accuracy of results. In the above operation steps, the compound treatment solution of step (2) and the protective agent of step (3) are key technologies of the invention, the compound treatment solution of the invention can rapidly hydrolyze nails, so that drug molecules embedded in gaps of nails are fully exposed to obtain the substance to be detected, and the protective agent can protect the exposed substance to be detected from external influences.
Preferably, in step (1), the physical mechanical method is a nail scraping or nail covering or nail cutting.
Preferably, the pH of the composite treatment liquid 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 blocker 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 having a concentration of 0.02mol to 1.0mol, where the concentration is the final concentration of the composite treatment liquid.
Preferably, the reaction time after the compound treatment liquid is added to the nail or toenail in the step (2) is 5 to 10 min.
The composite treating fluid is used for treating nail or toenail samples, and adopts a reduction method principle, wherein a disulfide bond reducing agent can selectively reduce disulfide bonds in keratin in the toenail into sulfydryl, so that secondary and tertiary structures of the keratin are destroyed, and the solubility of protein is improved; the hydrogen bond blocking agent can protect the newly generated sulfydryl to avoid reoxidation to a disulfide bond, and can be used as a surfactant to reduce the surface tension of keratin molecules and further increase the solubility; nail expanders may allow the keratin to continue to swell until partially dissolved.
Preferably, the protective agent in the step (3) is alpha-cyclodextrin with the concentration of 0.12-0.25 mg/mL, and the volume ratio of the composite treatment solution 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 of the alpha-cyclodextrin is hydrophobic, can provide hydrophobic binding sites like enzyme, is used as a host for encapsulating a substance to be detected, can protect the substance to be detected exposed by hydrolysis of the composite treatment solution, and prevents the substance to be detected from being interfered by the outside so as to cause the deviation of a detection result.
Preferably, the abuse drug detection reagent strip in the step (4) comprises a bottom plate, and a sample pad, a red nano-microsphere abuse drug release pad, a polyester cellulose membrane and a water absorption pad are sequentially arranged along the length of the bottom plate, wherein the polyester cellulose membrane is provided with parallel detection lines T and quality control lines C, and a monoclonal antibody with a result mark is fixed on the red nano-microsphere abuse drug release pad.
The principle applied by the invention is red nano-microsphere immunochromatography and competition method, when a sample contains a drug to be abused, after sample application, the antigen can be firstly combined with the monoclonal antibody-red nano-microsphere conjugate of the mouse anti-abused drug to be detected in the red nano-microsphere drug abuse processing pad to form an antigen-antibody complex, because the monoclonal antibody-red nano-microsphere conjugate of the mouse anti-abused drug to be detected in the red nano-microsphere drug abuse processing pad is combined with the drug to be abused, along with the progress of the chromatography process, the monoclonal antibody-red nano-microsphere conjugate of the mouse anti-abused drug to be detected without surplus and the bovine serum albumin to be abused drug coated on the nitrocellulose membrane are captured, the quality control line is red, the detection line is colorless, the color is rich and clear and identifiable, and does not need any auxiliary equipment, convenient operation.
The test paper can be used for detecting the fingernail or the toenail containing a sample to be detected with the abused drugs. Detecting the sample by using a test strip, and when red strips appear on the detection line T and the quality control line C, indicating that the fingernail or toenail sample has no abuse drug to be detected; if only the quality control line C shows a red strip, the fingernail or toenail sample contains the abuse drug to be detected; and if the quality control line C does not have a red color strip, the result of the test strip is invalid.
Preferably, the detection line T and the quality control line C are obtained by the following steps: respectively 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 the 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 abuse drug release pad comprises the following steps:
A. preparing a red nanoparticle 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 according to the proportion of 1: 500-1500, continuously heating to boil, rapidly adding a reducing agent trisodium citrate to react for 5-7min, stopping heating, and naturally cooling to room temperature;
B. marking red nano microspheres: measuring red nanoparticle solution, adding K2CO3Adjusting the solution to isoelectric point, adding toxin detection antibody according to the labeling ratio of 1:25-1:15, reacting for 25-30min, adding 0.1-0.3mol/L BSA, reacting for 30-35min, adding 0.5-1.5mg/ml EDC activator, and reacting for 15-20 min;
C. and (3) centrifugal treatment: centrifuging the reacted biological raw material-red nano microsphere conjugate solution for 30-40min at 10000-10500r/min, removing supernatant, and collecting the centrifuged solution by using a redissolution to obtain a finished product.
The red microsphere is generally 30-80nm in size, chloroauric acid is used as a raw material, gold ions in the chloroauric acid are reduced by a reducing agent trisodium citrate, EDC is added for activation, BSA is used for blocking redundant sites, a rat anti-abuse drug monoclonal antibody to be marked is added, the monoclonal antibody is physically adsorbed on the surface of the gold ions, supernatant is removed by centrifugation, a re-dissolving agent is used for re-dissolving, the concentration of a conjugate is detected by an ultraviolet spectrophotometer, and the OD value is used for expressing. In the whole operation stage, the red nano microsphere conjugate plays a role in color development, and in the coupling process, metal ions and protein are combined mainly through electrostatic adsorption.
Preferably, the volume ratio of the red nanoparticle solution to the BSA and EDC activator is 1: 4-5: 3-4.
Preferably, the reconstituted solution in step C is 5-5.2mg/ml Casein and 0.05-0.1mol Tris-HCL in a volume ratio of 2: 0.8-1.2 mixed solution.
The redissolution is used as a solvent after the marked red microsphere particles are coupled with the biological raw material, wherein a Tris-HCL buffer system provides a proper solution system for coupling the red microsphere particles with the biological raw material, and Casein is added into the Tris-HCL buffer system to effectively seal redundant binding sites on the biological raw material, so that the false positive condition can be effectively reduced.
Therefore, the invention has the following beneficial effects:
(1) the method for collecting and detecting the abuse drugs in the nail or toenail sample is provided, the components in the nail or toenail sample are effectively extracted and separated, the prepared test paper is short in detection time, only needs 5-10min, can meet the requirement of field detection, and fills the technical blank at present;
(2) the operation is simple and convenient, and other equipment and instruments are not needed; the detection result is displayed visually, can be judged by naked eyes and is suitable for personal 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 test strip can be stored at normal temperature, special equipment and instruments are not needed, the test strip only needs to be kept dry, the storage life is long, and the storage cost is low.
Drawings
FIG. 1 is a schematic diagram of the structure of the test strip of the present invention.
FIG. 2 is a schematic diagram of the operation steps of the present invention.
In the figure: 1. a sample pad; 2. red nano-microspheres abuse the drug release pad; 3. detecting a line T; 4. a quality control line C; 5. a polyester cellulose film; 6. absorbent pad, 7, bottom plate.
Detailed Description
The invention is further described with reference to specific embodiments.
General examples
A method for collecting and detecting drugs of abuse in a nail or toenail sample comprising the steps of:
(1) nail or toenail sample collection: obtaining (scraping or clipping) a nail or toenail sample by a physical mechanical method;
(2) nail or toenail pretreatment: adding the fingernails or the toenails into the composite treatment liquid for reaction for 5-10min, wherein the composite treatment liquid contains a disulfide bond reducing agent, a hydrogen bond blocking agent, a fingernail or toenail swelling agent and a buffer system; the pH value of the composite treatment liquid 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 blocker 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 having a concentration of 0.02mol to 1.0mol, where the concentration is the final concentration of the composite treatment liquid.
(3) Nail or toenail stabilization treatment: adding the protective agent into the composite treatment liquid dropwise to obtain a to-be-tested agent; the protective agent is alpha-cyclodextrin with the concentration of 0.12-0.25 mg/mL, and the volume ratio of the composite treatment solution to the protective agent is 2: 0.1-0.3.
(4) Detection of drugs of abuse: and (3) detecting the test agent obtained in the step (2) by using an abuse drug detection reagent strip.
The abuse drug detection reagent strip comprises a bottom plate 7, wherein a sample pad 1, a red nano microsphere abuse 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 with a result mark is fixed on the red nano microsphere abuse drug release pad 2. The detection line T3 and the quality control line C4 are obtained by the following steps: respectively 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 the 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 nano-microsphere abuse drug release pad 2 comprises the following steps:
A. preparing a red nanoparticle 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 according to the proportion of 1: 500-1500, continuously heating to boil, rapidly adding a reducing agent trisodium citrate to react for 5-7min, stopping heating, and naturally cooling to room temperature;
B. markingRed nano-microspheres: measuring red nanoparticle solution, adding K2CO3Adjusting the solution to isoelectric point, adding toxin detection antibody according to the labeling ratio of 1:25-1:15, reacting for 25-30min, adding 0.1-0.3mol/L BSA, reacting for 30-35min, adding 0.5-1.5mg/ml EDC activator, and reacting for 15-20 min; the volume ratio of the red nanoparticle solution to the BSA and EDC activator is 1: 4-5: 3-4;
C. and (3) centrifugal treatment: centrifuging the reacted biological raw material-red nano microsphere conjugate solution for 30-40min at 10000- & 10500r/min, removing supernatant, and collecting the centrifuged solution by using a redissolution (a solution in which Casein and 0.05-0.1mol of Tris-HCL are mixed according to a volume ratio of 2: 0.8-1.2), thereby obtaining a finished product.
Example 1
A method for collecting and detecting drugs of abuse in a nail or toenail sample comprising the steps of:
(1) nail or toenail sample collection: clipping a nail or toenail sample;
(2) nail or toenail pretreatment: adding the fingernails or the toenails into a composite treatment liquid for reacting for 8min, wherein the composite treatment liquid contains a disulfide bond reducing agent, a hydrogen bond blocking agent, a fingernail or toenail swelling agent and a buffer system; the pH value of the composite treatment liquid 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 blocker is dodecyl sulfuric acid with the concentration of 0.6 g/mL; the expanding agent is urea with the concentration of 0.48 g/mL; the buffer system was a TRIS buffer system having a concentration of 0.5mol, where the concentration is the final concentration of the composite treatment liquid.
(3) Nail or toenail stabilization treatment: adding the protective agent into the composite treatment liquid dropwise to obtain a to-be-tested agent; the protective agent is alpha-cyclodextrin with the concentration of 0.2mg/mL, and the volume ratio of the composite treatment solution to the protective agent is 2: 0.2.
(4) detection of drugs of abuse: and (3) detecting the test agent obtained in the step (2) by using an abuse drug detection reagent strip.
The abuse drug detection reagent strip comprises a bottom plate 7, wherein a sample pad 1, a red nano microsphere abuse 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 with a result mark is fixed on the red nano microsphere abuse drug release pad 2. The detection line T3 and the quality control line C4 are obtained by the following steps: respectively 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 the speed of 1.1ul/cm to respectively serve 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 nano-microsphere abuse drug release pad 2 comprises the following steps:
A. preparing a red nanoparticle 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 according to the amount of 1:1000, continuously heating to boiling, rapidly adding a reducing agent trisodium citrate, reacting for 6min, stopping heating, and naturally cooling to room temperature;
B. marking red nano microspheres: measuring red nanoparticle solution, adding K2CO3Adjusting the solution to isoelectric point, adding toxin detection antibody according to the labeling ratio of 1:20, reacting for 28min, adding 0.2mol/L BSA, reacting for 32min, adding 1mg/ml EDC activator, and reacting for 18 min; the volume ratio of the red nanoparticle solution to the BSA and EDC activator is 1: 4.5: 3.5;
C. and (3) centrifugal treatment: and (3) centrifuging the reacted rabbit anti-nicotine polyclonal antibody-red nano microsphere conjugate solution for 35min at 10250r/min, removing supernatant, and collecting the centrifuged solution by using a redissolution (a solution prepared by mixing Casein and 0.5mol Tris-HCL in a volume ratio of 2: 1) to obtain a finished product.
Nail nicotine detection reagent:
the reagent uses rabbit anti-nicotine polyclonal antibody as biological raw material.
Collecting fingernails or toenails of tested persons, wherein 30 smokers and 30 non-smokers exist; the detection is directly carried out by using a nicotine detection kit, and the detection result is as follows:
example 2
A method for collecting and detecting drugs of abuse in a nail or toenail sample comprising the steps of:
(1) nail or toenail sample collection: scraping a nail or toenail sample;
(2) nail or toenail pretreatment: adding the fingernails or the toenails into a composite treatment liquid for reaction for 5min, wherein the composite treatment liquid contains a disulfide bond reducing agent, a hydrogen bond blocking agent, a fingernail or toenail swelling agent and a buffer system; the pH value of the composite treatment liquid 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 blocker is dodecyl sulfuric acid with the concentration of 0.5 g/mL; the expanding agent is urea with the concentration of 0.5 g/mL; the buffer system was a TRIS buffer system having a concentration of 0.02mol, where the concentration is the final concentration of the composite treatment liquid.
(3) Nail or toenail stabilization treatment: adding the protective agent into the composite treatment liquid dropwise to obtain a to-be-tested agent; the protective agent is alpha-cyclodextrin with the concentration of 0.12mg/mL, and the volume ratio of the composite treatment solution to the protective agent is 2: 0.3.
(4) detection of drugs of abuse: and (3) detecting the test agent obtained in the step (2) by using an abuse drug detection reagent strip.
The abuse drug detection reagent strip comprises a bottom plate 7, wherein a sample pad 1, a red nano microsphere abuse 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 with a result mark is fixed on the red nano microsphere abuse drug release pad 2. The detection line T3 and the quality control line C4 are obtained by the following steps: 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 the 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 nano-microsphere abuse drug release pad 2 comprises the following steps:
A. preparing a red nanoparticle 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 according to the amount of 1:500, continuously heating to boiling, rapidly adding a reducing agent trisodium citrate, reacting for 5min, stopping heating, and naturally cooling to room temperature;
B. marking red nano microspheres: measuring red nanoparticle solution, adding K2CO3Adjusting the solution to isoelectric point, adding toxin detection antibody according to the labeling ratio of 1:25, reacting for 25min, adding 0.1mol/L BSA, reacting for 30-35min, adding 0.5-1.5mg/ml EDC activator, and reacting for 15-20 min; the volume ratio of the red nanoparticle solution to the BSA and EDC activator is 1: 4: 4;
C. and (3) centrifugal treatment: centrifuging the reacted rabbit anti-nicotine polyclonal antibody-red nano microsphere conjugate solution for 30min at 10000r/min, removing supernatant, and collecting the centrifuged solution by using a redissolution (a solution of 5mg/ml Casein and 0.1mol Tris-HCL mixed according to the volume ratio of 2: 0.8) to obtain a finished product.
The reagent uses rabbit anti-nicotine polyclonal antibody as biological raw material.
Collecting fingernails or toenails of tested persons, wherein 30 smokers and 30 non-smokers exist; the detection is directly carried out by using a nicotine detection kit, and the detection result is as follows:
example 3
A method for collecting and detecting drugs of abuse in a nail or toenail sample comprising the steps of:
(1) nail or toenail sample collection: scraping a nail or toenail sample;
(2) nail or toenail pretreatment: adding the fingernails or the toenails into a composite treatment liquid for reaction for 10min, wherein the composite treatment liquid contains a disulfide bond reducing agent, a hydrogen bond blocking agent, a fingernail or toenail swelling agent and a buffer system; the pH value of the composite treatment liquid 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 blocker is dodecyl sulfuric acid with the concentration of 0.7 g/mL; the expanding agent is urea with the concentration of 0.5 g/mL; the buffer system was a TRIS buffer system having a concentration of 0.02mol, where the concentration is the final concentration of the composite treatment liquid.
(3) Nail or toenail stabilization treatment: adding the protective agent into the composite treatment liquid dropwise to obtain a to-be-tested agent; the protective agent is alpha-cyclodextrin with the concentration of 0.25mg/mL, and the volume ratio of the composite treatment solution to the protective agent is 2: 0.1.
(4) detection of drugs of abuse: and (3) detecting the test agent obtained in the step (2) by using an abuse drug detection reagent strip.
The abuse drug detection reagent strip comprises a bottom plate 7, wherein a sample pad 1, a red nano microsphere abuse 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 with a result mark is fixed on the red nano microsphere abuse drug release pad 2. The detection line T3 and the quality control line C4 are obtained by the following steps: respectively spraying 02.0mg/ml of abused drug-conjugated bovine serum albumin conjugate and 1.0mg/ml of goat anti-rabbit IgG conjugate solution onto 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 nano-microsphere abuse drug release pad 2 comprises the following steps:
A. preparing a red nanoparticle 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 according to the amount of 1:1500 in proportion, continuing heating to boil, quickly adding a reducing agent trisodium citrate to react for 7min, stopping heating, and naturally cooling to room temperature;
B. marking red nano microspheres: measuring red nanoparticle solution, adding K2CO3Adjusting the solution to isoelectric point, adding toxin detection antibody according to the labeling ratio of 1:15, reacting for 30min, adding 0.3mol/L BSA, reacting for 35min, adding 0.5mg/ml EDC activator, and reacting for 20 min; the volume ratio of the red nanoparticle solution to the BSA and EDC activator is 1: 5: 3;
C. and (3) centrifugal treatment: and (3) centrifuging the reacted rabbit anti-nicotine polyclonal antibody-red nano microsphere conjugate solution for 30min at the speed of 10500r/min, removing supernatant, and collecting the centrifuged solution by using a redissolution (a solution prepared by mixing 5.2mg/ml Casein and 0.05mol Tris-HCL in a volume ratio of 2: 1.2) to obtain a finished product.
The reagent uses rabbit anti-nicotine polyclonal antibody as biological raw material.
Collecting fingernails or toenails of tested persons, wherein 30 smokers and 30 non-smokers exist; the detection is directly carried out by using a nicotine detection kit, and the detection result is as follows:
example 4
A method for collecting and detecting drugs of abuse in a nail or toenail sample comprising the steps of:
(1) nail or toenail sample collection: clipping a nail or toenail sample;
(2) nail or toenail pretreatment: adding the fingernails or the toenails into a composite treatment liquid for reacting for 8min, wherein the composite treatment liquid contains a disulfide bond reducing agent, a hydrogen bond blocking agent, a fingernail or toenail swelling agent and a buffer system; the pH value of the composite treatment liquid 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 blocker is dodecyl sulfuric acid with the concentration of 0.6 g/mL; the expanding agent is urea with the concentration of 0.48 g/mL; the buffer system was a TRIS buffer system having a concentration of 0.5mol, where the concentration is the final concentration of the composite treatment liquid.
(3) Nail or toenail stabilization treatment: adding the protective agent into the composite treatment liquid dropwise to obtain a to-be-tested agent; the protective agent is alpha-cyclodextrin with the concentration of 0.2mg/mL, and the volume ratio of the composite treatment solution to the protective agent is 2: 0.2.
(4) detection of drugs of abuse: and (3) detecting the test agent obtained in the step (2) by using an abuse drug detection reagent strip.
The abuse drug detection reagent strip comprises a bottom plate 7, wherein a sample pad 1, a red nano microsphere abuse 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 with a result mark is fixed on the red nano microsphere abuse drug release pad 2. The detection line T3 and the quality control line C4 are obtained by the following steps: respectively 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 the speed of 1.1ul/cm to respectively serve 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 nano-microsphere abuse drug release pad 2 comprises the following steps:
A. preparing a red nanoparticle 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 according to the amount of 1:1000, continuously heating to boiling, rapidly adding a reducing agent trisodium citrate, reacting for 6min, stopping heating, and naturally cooling to room temperature;
B. marking red nano microspheres: measuring red nanoparticle solution, adding K2CO3Adjusting the solution to isoelectric point, adding toxin detection antibody according to the labeling ratio of 1:20, reacting for 28min, adding 0.2mol/L BSA, reacting for 32min, adding 1mg/ml EDC activator, and reacting for 18 min; the volume ratio of the red nanoparticle solution to the BSA and EDC activator is 1: 4.5: 3.5;
C. and (3) centrifugal treatment: and (3) centrifuging the reacted mouse anti-morphine monoclonal antibody-red nano microsphere conjugate solution for 35min at 10250r/min, removing supernatant, and collecting the centrifuged solution by using a redissolution (a solution prepared by mixing 5.1mg/ml Casein and 0.5mol Tris-HCL in 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 fingernails or toenails of tested persons, wherein 15 persons who absorb morphine and 25 persons who do not absorb morphine are collected; the detection is directly carried out by using a finger (toe) morphine detection kit, and the detection result is as follows:
example 5
A method for collecting and detecting drugs of abuse in a nail or toenail sample comprising the steps of:
(1) nail or toenail sample collection: clipping a nail or toenail sample;
(2) nail or toenail pretreatment: adding the fingernails or the toenails into a composite treatment liquid for reacting for 8min, wherein the composite treatment liquid contains a disulfide bond reducing agent, a hydrogen bond blocking agent, a fingernail or toenail swelling agent and a buffer system; the pH value of the composite treatment liquid 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 blocker is dodecyl sulfuric acid with the concentration of 0.6 g/mL; the expanding agent is urea with the concentration of 0.48 g/mL; the buffer system was a TRIS buffer system having a concentration of 0.5mol, where the concentration is the final concentration of the composite treatment liquid.
(3) Nail or toenail stabilization treatment: adding the protective agent into the composite treatment liquid dropwise to obtain a to-be-tested agent; the protective agent is alpha-cyclodextrin with the concentration of 0.2mg/mL, and the volume ratio of the composite treatment solution to the protective agent is 2: 0.2.
(4) detection of drugs of abuse: and (3) detecting the test agent obtained in the step (2) by using an abuse drug detection reagent strip.
The abuse drug detection reagent strip comprises a bottom plate 7, wherein a sample pad 1, a red nano microsphere abuse 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 with a result mark is fixed on the red nano microsphere abuse drug release pad 2. The detection line T3 and the quality control line C4 are obtained by the following steps: respectively 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 the speed of 1.1ul/cm to respectively serve 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 nano-microsphere abuse drug release pad 2 comprises the following steps:
A. preparing a red nanoparticle 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 according to the amount of 1:1000, continuously heating to boiling, rapidly adding a reducing agent trisodium citrate, reacting for 6min, stopping heating, and naturally cooling to room temperature;
B. marking red nano microspheres: measuring red nanoparticle solution, adding K2CO3Adjusting the solution to isoelectric point, adding toxin detection antibody according to the labeling ratio of 1:20, reacting for 28min, adding 0.2mol/L BSA, reacting for 32min, adding 1mg/ml EDC activator, and reacting for 18 min; the volume ratio of the red nanoparticle solution to the BSA and EDC activator is 1: 4.5: 3.5;
C. and (3) centrifugal treatment: and (3) centrifuging the reacted mouse anti-chlorammoniate monoclonal antibody-red nano microsphere conjugate solution for 35min at 10250r/min, removing supernatant, and collecting the centrifuged solution by using a redissolution (a solution prepared by mixing Casein (5.1 mg/ml) and 0.5mol of Tris-HCL (Tris-HCl) in a volume ratio of 2: 1) to obtain a finished product.
The reagent takes a mouse anti-chlorthalidone monoclonal antibody as a biological raw material.
Collecting fingernails or toenails of tested persons, wherein 10 persons suffer from chlorammonia ketone absorption and 30 persons suffer from morphine non-absorption; the kit is directly used for detecting the dactylicarbazone (dactylicarbazone), and the detection result is as follows:
example 6
A method for collecting and detecting drugs of abuse in a nail or toenail sample comprising the steps of:
(1) nail or toenail sample collection: clipping a nail or toenail sample;
(2) nail or toenail pretreatment: adding the fingernails or the toenails into a composite treatment liquid for reacting for 8min, wherein the composite treatment liquid contains a disulfide bond reducing agent, a hydrogen bond blocking agent, a fingernail or toenail swelling agent and a buffer system; the pH value of the composite treatment liquid 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 blocker is dodecyl sulfuric acid with the concentration of 0.6 g/mL; the expanding agent is urea with the concentration of 0.48 g/mL; the buffer system was a TRIS buffer system having a concentration of 0.5mol, where the concentration is the final concentration of the composite treatment liquid.
(3) Nail or toenail stabilization treatment: adding the protective agent into the composite treatment liquid dropwise to obtain a to-be-tested agent; the protective agent is alpha-cyclodextrin with the concentration of 0.2mg/mL, and the volume ratio of the composite treatment solution to the protective agent is 2: 0.2.
(4) detection of drugs of abuse: and (3) detecting the test agent obtained in the step (2) by using an abuse drug detection reagent strip.
The abuse drug detection reagent strip comprises a bottom plate 7, wherein a sample pad 1, a red nano microsphere abuse 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 with a result mark is fixed on the red nano microsphere abuse drug release pad 2. The detection line T3 and the quality control line C4 are obtained by the following steps: respectively 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 the speed of 1.1ul/cm to respectively serve 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 nano-microsphere abuse drug release pad 2 comprises the following steps:
A. preparing a red nanoparticle 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 according to the amount of 1:1000, continuously heating to boiling, rapidly adding a reducing agent trisodium citrate, reacting for 6min, stopping heating, and naturally cooling to room temperature;
B. marking red nano microspheres: weighing red nanoparticle solution, adding K2CO3 solution to adjust to isoelectric point, adding toxin detection antibody according to a labeling ratio of 1:20, reacting for 28min, adding 0.2mol/L BSA for reacting for 32min, adding EDC activator with concentration of 1mg/ml, and reacting for 18 min; the volume ratio of the red nanoparticle solution to the BSA and EDC activator is 1: 4.5: 3.5;
C. and (3) centrifugal treatment: and (3) centrifuging the reacted murine anti-methamphetamine monoclonal antibody-red nano microsphere conjugate solution for 35min at 10250r/min, removing supernatant, and collecting the centrifuged solution by using a redissolution (a solution prepared by mixing 5.1mg/ml Casein and 0.5mol Tris-HCL in a volume ratio of 2: 1), thereby obtaining a finished product. Detection reagent for methamphetamine (methamphetamine):
the reagent takes a murine anti-methamphetamine monoclonal antibody as a biological raw material.
Collecting fingernails or toenails of a tested person, wherein 15 persons of patients who take the ice toxin and 25 persons of patients who do not take the ice toxin; the kit is directly used for detecting the nail (toe) methamphetamine, and the detection result is as follows:
example 7
A method for collecting and detecting drugs of abuse in a nail or toenail sample comprising the steps of:
(1) nail or toenail sample collection: clipping a nail or toenail sample;
(2) nail or toenail pretreatment: adding the fingernails or the toenails into a composite treatment liquid for reacting for 8min, wherein the composite treatment liquid contains a disulfide bond reducing agent, a hydrogen bond blocking agent, a fingernail or toenail swelling agent and a buffer system; the pH value of the composite treatment liquid 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 blocker is dodecyl sulfuric acid with the concentration of 0.6 g/mL; the expanding agent is urea with the concentration of 0.48 g/mL; the buffer system was a TRIS buffer system having a concentration of 0.5mol, where the concentration is the final concentration of the composite treatment liquid.
(3) Nail or toenail stabilization treatment: adding the protective agent into the composite treatment liquid dropwise to obtain a to-be-tested agent; the protective agent is alpha-cyclodextrin with the concentration of 0.2mg/mL, and the volume ratio of the composite treatment solution to the protective agent is 2: 0.2.
(4) detection of drugs of abuse: and (3) detecting the test agent obtained in the step (2) by using an abuse drug detection reagent strip.
The abuse drug detection reagent strip comprises a bottom plate 7, wherein a sample pad 1, a red nano microsphere abuse 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 with a result mark is fixed on the red nano microsphere abuse drug release pad 2. The detection line T3 and the quality control line C4 are obtained by the following steps: respectively 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 the speed of 1.1ul/cm to respectively serve 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 nano-microsphere abuse drug release pad 2 comprises the following steps:
A. preparing a red nanoparticle 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 according to the amount of 1:1000, continuously heating to boiling, rapidly adding a reducing agent trisodium citrate, reacting for 6min, stopping heating, and naturally cooling to room temperature;
B. marking red nano microspheres: measuring red nanoparticle solution, adding K2CO3Adjusting the solution to isoelectric point, adding toxin detection antibody according to the labeling ratio of 1:20, reacting for 28min, adding 0.2mol/L BSA, reacting for 32min, adding 1mg/ml EDC activator, and reacting for 18 min; the volume ratio of the red nanoparticle solution to the BSA and EDC activator is 1: 4.5: 3.5;
C. and (3) centrifugal treatment: and (3) centrifuging the reacted mouse anti-cocaine monoclonal antibody-red nano microsphere conjugate solution for 35min at 10250r/min, removing supernatant, and collecting the centrifuged solution by using a redissolution (a solution prepared by mixing 5.1mg/ml Casein and 0.5mol Tris-HCL in a volume ratio of 2: 1) to obtain a finished product.
The reagent uses a mouse anti-cocaine monoclonal antibody as a biological raw material.
Collecting fingernails or toenails of the tested persons, wherein 5 persons suffer from cocaine inhalation and 25 persons suffer from cocaine non-inhalation; the detection is directly carried out by using a cocaine detection kit for the nail (toe), and the detection result is as follows:
from the data of examples 1-7, it can be seen that only the solutions within the scope of the claims of the present invention can satisfy the above requirements in all aspects and result in an optimized detection solution. The change of the mixture ratio, the replacement/addition/subtraction of raw materials or the change of the feeding sequence can bring corresponding negative effects.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (10)
1. A method for collecting and detecting drugs of abuse in a nail or toenail sample 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 the fingernail or the toenail into a composite treatment liquid for reaction, wherein the composite treatment liquid contains a disulfide bond reducing agent, a hydrogen bond blocking agent, a fingernail or toenail swelling agent and a buffer system;
(3) nail or toenail stabilization treatment: adding the protective agent into the composite treatment liquid dropwise to obtain a to-be-tested agent;
(4) detection of drugs of abuse: and (3) detecting the test agent obtained in the step (2) by adopting an abuse drug detection reagent strip.
2. The method of claim 1, wherein in step (1), the physical mechanical method is scraping the nail cover or cutting the nail.
3. The method for collecting and detecting drugs of abuse in a nail or toenail sample according to claim 1, wherein the pH of the complex treatment solution of the step (2) is 8.0 to 10.0, wherein the disulfide bond reducing agent comprises tris (2-carboxyethyl) phosphine hydrochloride at a concentration of 28 to 29.5mg/mL and β -sodium thioglycolate at a concentration of 0.2 to 0.22 mg/mL; the hydrogen bond blocker 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 having a concentration of 0.02mol to 1.0mol, where the concentration is the final concentration of the composite treatment liquid.
4. The method for collecting and detecting drugs of abuse in a nail or toenail sample according to claim 1, wherein the reaction time after the nail or toenail is added to the complex treatment solution in the step (2) is 5 to 10 min.
5. The method for collecting and detecting drugs of abuse in a nail or toenail sample according to claim 1, wherein the protective agent in step (3) is alpha-cyclodextrin with a concentration of 0.12-0.25 mg/mL, and the volume ratio of the complex treatment solution to the protective agent is 2: 0.1-0.3.
6. The method for collecting and detecting abused drugs in fingernail or toenail samples according to claim 1, wherein the abused drug detection reagent strip of step (4) comprises a bottom plate (7), and a sample pad (1), a red nanoparticle abuse 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), wherein the polyester cellulose membrane (5) is provided with a parallel detection line T (3) and a quality control line C (4), and a monoclonal antibody with a result mark is fixed on the red nanoparticle abuse drug release pad (2).
7. The method of claim 6, wherein the detection line T (3) and the quality control line C (4) are obtained by: respectively 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 the 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.
8. The method for collecting and detecting drugs of abuse in fingernails or toenails as claimed in claim 6, wherein the red nanosphere drug of abuse release pad (2) is prepared by:
A. preparing a red nanoparticle 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 according to the proportion of 1: 500-1500, continuously heating to boil, rapidly adding a reducing agent trisodium citrate to react for 5-7min, stopping heating, and naturally cooling to room temperature;
B. marking red nano microspheres: measuring red nanoparticle solution, adding K2CO3Adjusting the solution to isoelectric point, adding toxin detection antibody according to the labeling ratio of 1:25-1:15, reacting for 25-30min, adding 0.1-0.3mol/L BSA, reacting for 30-35min, adding 0.5-1.5mg/ml EDC activator, and reacting for 15-20 min;
C. and (3) centrifugal treatment: centrifuging the reacted biological raw material-red nano microsphere conjugate solution for 30-40min at 10000-10500r/min, removing supernatant, and collecting the centrifuged solution by using a redissolution to obtain a finished product.
9. The method of claim 8, wherein the volume ratio of the red nanoparticle solution, BSA and EDC activator is 1: 4-5: 3-4.
10. The method of claim 8, wherein the reconstitution solution of step C is 5-5.2mg/ml Casein and 0.05-0.1mol Tris-HCL at a volume ratio of 2: 0.8-1.2 mixed solution.
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| US6949344B1 (en) * | 1987-12-28 | 2005-09-27 | Psychemical Corporation | Hair analysis method |
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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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