CN110470843A - Quantum dot fluorescence immuno-chromatographic test paper strip and preparation method thereof for illicit drugs inspection - Google Patents
Quantum dot fluorescence immuno-chromatographic test paper strip and preparation method thereof for illicit drugs inspection Download PDFInfo
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- CN110470843A CN110470843A CN201910083128.1A CN201910083128A CN110470843A CN 110470843 A CN110470843 A CN 110470843A CN 201910083128 A CN201910083128 A CN 201910083128A CN 110470843 A CN110470843 A CN 110470843A
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- quantum dot
- test paper
- nitrocellulose filter
- pad
- illicit drugs
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- 239000002096 quantum dot Substances 0.000 title claims abstract description 68
- 238000012360 testing method Methods 0.000 title claims abstract description 59
- 238000007689 inspection Methods 0.000 title claims abstract description 34
- 239000002117 illicit drug Substances 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 64
- 229940079593 drug Drugs 0.000 claims abstract description 50
- 239000003814 drug Substances 0.000 claims abstract description 50
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 42
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 40
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 17
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 9
- 239000011248 coating agent Substances 0.000 claims abstract description 6
- 238000000576 coating method Methods 0.000 claims abstract description 6
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical group O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 claims description 36
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical compound O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 claims description 18
- 229960001252 methamphetamine Drugs 0.000 claims description 15
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 239000002245 particle Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 11
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 10
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 229910021538 borax Inorganic materials 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 10
- 239000013078 crystal Substances 0.000 claims description 10
- 239000012467 final product Substances 0.000 claims description 10
- 229960003299 ketamine Drugs 0.000 claims description 10
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 9
- 229960005181 morphine Drugs 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 239000012190 activator Substances 0.000 claims description 5
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 5
- 239000004327 boric acid Substances 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 229940126619 mouse monoclonal antibody Drugs 0.000 claims description 5
- 229920006267 polyester film Polymers 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 5
- 239000013049 sediment Substances 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 239000013589 supplement Substances 0.000 claims description 5
- 238000005507 spraying Methods 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 210000004209 hair Anatomy 0.000 description 41
- 239000000123 paper Substances 0.000 description 38
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 15
- 238000003317 immunochromatography Methods 0.000 description 11
- 238000002965 ELISA Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000012634 fragment Substances 0.000 description 5
- 230000029087 digestion Effects 0.000 description 4
- 231100000640 hair analysis Toxicity 0.000 description 4
- 239000002574 poison Substances 0.000 description 4
- 231100000614 poison Toxicity 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 239000012620 biological material Substances 0.000 description 3
- 239000000084 colloidal system Substances 0.000 description 3
- 238000003255 drug test Methods 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 239000004005 microsphere Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000000643 oven drying Methods 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 206010010774 Constipation Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- GVGLGOZIDCSQPN-PVHGPHFFSA-N Heroin Chemical compound O([C@H]1[C@H](C=C[C@H]23)OC(C)=O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4OC(C)=O GVGLGOZIDCSQPN-PVHGPHFFSA-N 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000007813 chromatographic assay Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001427 coherent effect Effects 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229960002069 diamorphine Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000005424 photoluminescence Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- 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
-
- 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
Abstract
The invention discloses the quantum dot fluorescence immuno-chromatographic test paper strips for illicit drugs inspection, including bottom liner, nitrocellulose filter is pasted on bottom liner, nitrocellulose film outer surface side is viscous to set release pad and sample pad, the nitrocellulose film outer surface other side is viscous to be equipped with blotting paper, nitrocellulose filter is equipped with the detection zone close to release pad and the control zone close to blotting paper, and detection zone is coated with BSA and is coupled drug numerator, and control zone is coated with goat-anti rabbit polyclonal antibody.The invention also discloses the preparation methods of the quantum dot fluorescence immuno-chromatographic test paper strip for illicit drugs inspection, including S1, quantum dot-labeled antibody and the preparation of fluorescence bonding pad；S2, BSA are coupled drug numerator and the how anti-coating nitrocellulose filter of goat-anti rabbit；S3, reagent strip assembling.The advantages of quantum dot fluorescence immuno-chromatographic test paper strip disclosed by the invention for illicit drugs inspection has detection quickly, easy to operate, and high sensitivity has a wide range of application.
Description
Technical field
The invention belongs to field of medical examination, be related to for illicit drugs inspection quantum dot fluorescence immuno-chromatographic test paper strip and its
Preparation method.
Background technique
Drug testing refers to the principle and technology of applied chemistry, physics, biology and modern instrumental analysis, to the kind of drugs
Class, composition and content quickly, accurately identify and measurement.The sample of common drug testing includes the drugs of various forms can
Substance and biological material are doubted, wherein common sample has urine, blood, saliva and hair.
Current widely used biological material drug testing analysis method has: colloid gold immune lateral chromatography method, enzyme
Linked immunosorbent assay, gas chromatography-mass spectrography etc..Wherein: colloidal gold immunochromatographimethod has many advantages, such as quick, simplicity, but sensitivity
It is lower, it is suitable for the higher biological material of drugs concentration such as urine specimen.Enzyme-linked immunization (ELISA) is although method detection sensitivity
Better than colloid gold immune test paper item, but complex for operation step, the conditions such as experimental situation, operator are required it is high, can not be carry-on
It carries and is applied to on-site test.Although gas phase and liquid phase chromatograph-mass spectrometer coupling method high sensitivity, high specificity, instrument and equipment
Volume is big, expensive, requires height to conditions such as experimental situation, operators, and single detection time is long, costly.
In traditional tachysynthesis detection method, both at home and abroad mainly using colloid gold label, fluorescent microsphere label, when
Between fluorescence differentiate the test strips of the methods of microballoon label, these test strips all have the defects that different degrees of, such as colloidal gold system
Standby more difficult, particle diameter is more difficult to control uniformly, and stability is poor, and sensitivity is low；Fluorescent microsphere label is uneven, testing result weight
Existing property is poor；Time-resolved fluorescence microsphere particle is bigger than normal to cause fluorescence reaction insufficient, frequently results in false yang constipation fruit and occurs.
Quantum dot is the aggregate of the atom and molecule on nanoscale, mainly by II-VI group and iii-v element
Composition uniform or Core-shell Structure Nanoparticles, be the nano material of quasi-zero dimension, it has the advantage that exciting light spectrum width and send out
Ejected wave length is narrow, and the exciting light that any wavelength less than its launch wavelength 10nm can be used is excited, to avoid adjacent spy
Survey the crosstalk in channel；Different-grain diameter is different with its launch wavelength of the quantum dot of composition, can be used for multiple labeling detection；Fluorescence efficiency
Height, fluorescence intensity is strong, and stability is good, and bio-compatibility is good, to the active fanout free region of large biological molecule sample, especially by each
Specific connection can be carried out after kind chemical modification.Therefore, it is widely applied in the excellent fluorescence property of quantum dot
In biomedical detection and diagnosis.
Summary of the invention
The first purpose of this invention is to provide a kind of quantum dot fluorescence immuno-chromatographic test paper strip for illicit drugs inspection,
It can play that quantum dot fluorescence immuno-chromatographic assay technology is quick, and easy to operate, high sensitivity a little, avoids existing detection skill
The shortcomings that art and shortcoming.Second object of the present invention is the open quantum dot fluorescence immunochromatography for being used for illicit drugs inspection
The preparation method of test strips.
Technical solution: it for the quantum dot fluorescence immuno-chromatographic test paper strip of illicit drugs inspection, including bottom liner, is pasted on bottom liner
There is nitrocellulose filter, the side of the outer surface of the nitrocellulose filter is viscous to set release pad and sample pad, the cellulose nitrate
The other side of the outer surface of plain film is viscous to be equipped with blotting paper, and nitrocellulose filter is equipped with close to the detection zone of release pad and close to water suction
The control zone of paper, detection zone are coated with BSA and are coupled drug numerator, and control zone is coated with goat-anti rabbit polyclonal antibody.
Further, the illicit drugs inspection mouse monoclonal antibody of release pad spraying quantum dot fluorescent particle markers.
The preparation method of quantum dot fluorescence immuno-chromatographic test paper strip for illicit drugs inspection, comprising the following steps:
S1, quantum dot-labeled antibody and the preparation of fluorescence bonding pad
(1) it is that 10M carboxyl quantum dot is added in reactor by the concentration of 100 μ l, supplements 400 μ l borate buffer solutions,
Mixed liquor is obtained after mixing evenly, subsequently into step (2)；
(2) the drugs antibody that 20 μ l concentration to be marked is 1mg/ml is separately added into the mixed liquor obtained to step (1)
Molecule forms mixed liquor after stirring at low speed, subsequently into step (3)；
(3) 25 μ l activator EDC are added in the mixed liquor obtained to step (2), continue to react at room temperature 2h, reaction terminates
Afterwards, 8000rpm is centrifuged 3min, removes the reunion sediment being likely to occur, stays supernatant, subsequently into step (4)；
(4) supernatant that step (3) obtains 5 times are concentrated and purified with super filter tube to obtain comprising quantum dot-labeled drugs antibody
The final product of molecule, each cocnentration factor be not less than 10, then will comprising quantum dot-labeled drugs antibody molecule final product redissolve in
5ml concentration is to obtain mixed liquor in 0.05mol/L sodium borate buffer liquid；
(5) mixed liquor that step (4) obtains is sprayed onto release pad with the dosage of 0.1~0.3ml/cm, 37 degrees Celsius of bakings
Case is dry for 24 hours, and envelope is spare；
S2, BSA are coupled drug numerator and the how anti-coating nitrocellulose filter of goat-anti rabbit
It is that 0.05mol/L sodium borate buffer liquid is dilute that BSA, which is coupled drug numerator, goat-anti rabbit polyclonal antibody with concentration, respectively
Releasing to concentration is 0.03~0.3mg/mL, detection zone and the control being then sprayed onto respectively with the dosage of 1ml/cm on nitrocellulose filter
Area processed, 45 DEG C of drying and processing 12h, envelope are spare；
S3, reagent strip assembling
Nitrocellulose filter, sample pad, release pad and blotting paper overlap joint mutually successively are pasted on bottom liner, is tried
Cardboard is cut into the test strips that width is 3.78~4mm as requested.
Further, the configuration method of the borate buffer solution in step (1) are as follows: by boric acid 1.2368g, sodium chloride
0.2925g is added to the container, and adds distilled water, and constant volume sets 100ml, after mixing evenly.
Further, the drugs antibody molecule in step (2) be morphine Abs molecule or crystal methamphetamine antibody molecule or
Ketamine antibody molecule.
Further, the release pad in step (5) is polyester film fluorescence release pad.
Testing principle of the invention is by quantum dot fluorescent particles and antibody covalent bond, when this quantum dot fluorescent particles mark
The compound that the antibody of note is formed after in detection sample in conjunction with corresponding drug numerator, with BSA coated on nitrocellulose filter
It is coupled drug numerator and antibody competition combines, quantum dot fluorescent particles marker is assembled in corresponding position, in swashing for ultraviolet light
It gives, its launch wavelength is detected, for rapid quantitative detection.
The utility model has the advantages that the quantum dot fluorescence immuno-chromatographic test paper strip disclosed by the invention for illicit drugs inspection has detection fast
Speed, it is easy to operate, the advantages of high sensitivity, has a wide range of application, can be used in the drugs of whole blood, saliva, urine, hair equal samples
The quick detection that individual event detects or an inspection is multinomial.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the quantum dot fluorescence immuno-chromatographic test paper strip disclosed by the invention for illicit drugs inspection；
Wherein:
1- bottom liner 2- sample pad
3- release pad 4- nitrocellulose filter
5- blotting paper 6- detection zone
The control zone 7-
Specific embodiment:
Detailed description of specific embodiments of the present invention below.
Specific embodiment 1
As shown in Figure 1, the quantum dot fluorescence immuno-chromatographic test paper strip of illicit drugs inspection, including bottom liner 1 are used for, on bottom liner 1
It is pasted with nitrocellulose filter 4, the side of the outer surface of the nitrocellulose filter 4 is viscous to set release pad 3 and sample pad 2, described
The other side of the outer surface of nitrocellulose filter 4 is viscous to be equipped with blotting paper 5, and nitrocellulose filter 4 is equipped with the detection close to release pad 3
The control zone 7 in area 6 and close blotting paper 5, detection zone 6 are coated with BSA and are coupled drug numerator, and control zone 7 is coated with goat-anti rabbit polyclonal
Antibody.
Further, release pad 3 sprays the illicit drugs inspection mouse monoclonal antibody of quantum dot fluorescent particle markers.
The preparation method of quantum dot fluorescence immuno-chromatographic test paper strip for illicit drugs inspection, comprising the following steps:
S1, quantum dot-labeled antibody and the preparation of fluorescence bonding pad
(1) it is that 10M carboxyl quantum dot is added in reactor by the concentration of 100 μ l, supplements 400 μ l borate buffer solutions,
Mixed liquor is obtained after mixing evenly, subsequently into step (2)；
(2) the drugs antibody that 20 μ l concentration to be marked is 1mg/ml is separately added into the mixed liquor obtained to step (1)
Molecule forms mixed liquor after stirring at low speed, subsequently into step (3)；
(3) 25 μ l activator EDC are added in the mixed liquor obtained to step (2), continue to react at room temperature 2h, reaction terminates
Afterwards, 8000rpm is centrifuged 3min, removes the reunion sediment being likely to occur, stays supernatant, subsequently into step (4)；
(4) supernatant that step (3) obtains 5 times are concentrated and purified with super filter tube to obtain comprising quantum dot-labeled drugs antibody
The final product of molecule, each cocnentration factor be not less than 10, then will comprising quantum dot-labeled drugs antibody molecule final product redissolve in
5ml concentration is to obtain mixed liquor in 0.05mol/L sodium borate buffer liquid；
(5) mixed liquor that step (4) obtains is sprayed onto release pad with the dosage of 0.2ml/cm, 37 degrees Celsius of oven dryings
For 24 hours, envelope, it is spare；
S2, BSA are coupled drug numerator and the how anti-coating nitrocellulose filter of goat-anti rabbit
It is that 0.05mol/L sodium borate buffer liquid is dilute that BSA, which is coupled drug numerator, goat-anti rabbit polyclonal antibody with concentration, respectively
Releasing to concentration is 0.2mg/mL, is then sprayed onto detection zone and control zone on nitrocellulose filter respectively with the dosage of 1ml/cm,
45 DEG C of drying and processing 12h, envelope are spare；
S3, reagent strip assembling
Nitrocellulose filter, sample pad, release pad and blotting paper overlap joint mutually successively are pasted on bottom liner, is tried
Cardboard is cut into the test strips that width is 3.9mm as requested.
Further, the configuration method of the borate buffer solution in step (1) are as follows: by boric acid 1.2368g, sodium chloride
0.2925g is added to the container, and adds distilled water, and constant volume sets 100ml, after mixing evenly.
Further, the drugs antibody molecule in step (2) is morphine Abs molecule.
Further, the release pad in step (5) is polyester film fluorescence release pad.
Specific embodiment 2
As shown in Figure 1, the quantum dot fluorescence immuno-chromatographic test paper strip of illicit drugs inspection, including bottom liner 1 are used for, on bottom liner 1
It is pasted with nitrocellulose filter 4, the side of the outer surface of the nitrocellulose filter 4 is viscous to set release pad 3 and sample pad 2, described
The other side of the outer surface of nitrocellulose filter 4 is viscous to be equipped with blotting paper 5, and nitrocellulose filter 4 is equipped with the detection close to release pad 3
The control zone 7 in area 6 and close blotting paper 5, detection zone 6 are coated with BSA and are coupled drug numerator, and control zone 7 is coated with goat-anti rabbit polyclonal
Antibody.
Further, release pad 3 sprays the illicit drugs inspection mouse monoclonal antibody of quantum dot fluorescent particle markers.
The preparation method of quantum dot fluorescence immuno-chromatographic test paper strip for illicit drugs inspection, comprising the following steps:
S1, quantum dot-labeled antibody and the preparation of fluorescence bonding pad
(1) it is that 10M carboxyl quantum dot is added in reactor by the concentration of 100 μ l, supplements 400 μ l borate buffer solutions,
Mixed liquor is obtained after mixing evenly, subsequently into step (2)；
(2) the drugs antibody that 20 μ l concentration to be marked is 1mg/ml is separately added into the mixed liquor obtained to step (1)
Molecule forms mixed liquor after stirring at low speed, subsequently into step (3)；
(3) 25 μ l activator EDC are added in the mixed liquor obtained to step (2), continue to react at room temperature 2h, reaction terminates
Afterwards, 8000rpm is centrifuged 3min, removes the reunion sediment being likely to occur, stays supernatant, subsequently into step (4)；
(4) supernatant that step (3) obtains 5 times are concentrated and purified with super filter tube to obtain comprising quantum dot-labeled drugs antibody
The final product of molecule, each cocnentration factor be not less than 10, then will comprising quantum dot-labeled drugs antibody molecule final product redissolve in
5ml concentration is to obtain mixed liquor in 0.05mol/L sodium borate buffer liquid；
(5) mixed liquor that step (4) obtains is sprayed onto release pad with the dosage of 0.1ml/cm, 37 degrees Celsius of oven dryings
For 24 hours, envelope, it is spare；
S2, BSA are coupled drug numerator and the how anti-coating nitrocellulose filter of goat-anti rabbit
It is that 0.05mol/L sodium borate buffer liquid is dilute that BSA, which is coupled drug numerator, goat-anti rabbit polyclonal antibody with concentration, respectively
Releasing to concentration is 0.03mg/mL, is then sprayed onto detection zone and control zone on nitrocellulose filter respectively with the dosage of 1ml/cm,
45 DEG C of drying and processing 12h, envelope are spare；
S3, reagent strip assembling
Nitrocellulose filter, sample pad, release pad and blotting paper overlap joint mutually successively are pasted on bottom liner, is tried
Cardboard is cut into the test strips that width is 3.78mm as requested.
Further, the configuration method of the borate buffer solution in step (1) are as follows: by boric acid 1.2368g, sodium chloride
0.2925g is added to the container, and adds distilled water, and constant volume sets 100ml, after mixing evenly.
Further, the drugs antibody molecule in step (2) is crystal methamphetamine antibody molecule.
Further, the release pad in step (5) is polyester film fluorescence release pad.
Specific embodiment 3
As shown in Figure 1, the quantum dot fluorescence immuno-chromatographic test paper strip of illicit drugs inspection, including bottom liner 1 are used for, on bottom liner 1
It is pasted with nitrocellulose filter 4, the side of the outer surface of the nitrocellulose filter 4 is viscous to set release pad 3 and sample pad 2, described
The other side of the outer surface of nitrocellulose filter 4 is viscous to be equipped with blotting paper 5, and nitrocellulose filter 4 is equipped with the detection close to release pad 3
The control zone 7 in area 6 and close blotting paper 5, detection zone 6 are coated with BSA and are coupled drug numerator, and control zone 7 is coated with goat-anti rabbit polyclonal
Antibody.
Further, release pad 3 sprays the illicit drugs inspection mouse monoclonal antibody of quantum dot fluorescent particle markers.
The preparation method of quantum dot fluorescence immuno-chromatographic test paper strip for illicit drugs inspection, comprising the following steps:
S1, quantum dot-labeled antibody and the preparation of fluorescence bonding pad
(1) it is that 10M carboxyl quantum dot is added in reactor by the concentration of 100 μ l, supplements 400 μ l borate buffer solutions,
Mixed liquor is obtained after mixing evenly, subsequently into step (2)；
(2) the drugs antibody that 20 μ l concentration to be marked is 1mg/ml is separately added into the mixed liquor obtained to step (1)
Molecule forms mixed liquor after stirring at low speed, subsequently into step (3)；
(3) 25 μ l activator EDC are added in the mixed liquor obtained to step (2), continue to react at room temperature 2h, reaction terminates
Afterwards, 8000rpm is centrifuged 3min, removes the reunion sediment being likely to occur, stays supernatant, subsequently into step (4)；
(4) supernatant that step (3) obtains 5 times are concentrated and purified with super filter tube to obtain comprising quantum dot-labeled drugs antibody
The final product of molecule, each cocnentration factor be not less than 10, then will comprising quantum dot-labeled drugs antibody molecule final product redissolve in
5ml concentration is to obtain mixed liquor in 0.05mol/L sodium borate buffer liquid；
(5) mixed liquor that step (4) obtains is sprayed onto release pad with the dosage of 0.3ml/cm, 37 degrees Celsius of oven dryings
For 24 hours, envelope, it is spare；
S2, BSA are coupled drug numerator and the how anti-coating nitrocellulose filter of goat-anti rabbit
It is that 0.05mol/L sodium borate buffer liquid is dilute that BSA, which is coupled drug numerator, goat-anti rabbit polyclonal antibody with concentration, respectively
Releasing to concentration is 0.3mg/mL, is then sprayed onto detection zone and control zone on nitrocellulose filter respectively with the dosage of 1ml/cm,
45 DEG C of drying and processing 12h, envelope are spare；
S3, reagent strip assembling
Nitrocellulose filter, sample pad, release pad and blotting paper overlap joint mutually successively are pasted on bottom liner, is tried
Cardboard is cut into the test strips that width is 4mm as requested.
Further, the configuration method of the borate buffer solution in step (1) are as follows: by boric acid 1.2368g, sodium chloride
0.2925g is added to the container, and adds distilled water, and constant volume sets 100ml, after mixing evenly.
Further, the drugs antibody molecule in step (2) is ketamine antibody molecule.
Further, the release pad in step (5) is polyester film fluorescence release pad.
The quantum dot fluorescence immuno-chromatographic test paper strip for illicit drugs inspection of specific embodiment 1-3 preparation is established in competitiveness
In the principle of immunochromatography, detect in hair or other samples whether contain Poison using quantum dot fluorescent particle markers principle.
When being free of drugs and its catabolite in hair solution, the antibody conjugates of quantum dot fluorescent particles label are in capillary siphonage
Under, it is moved forward together with solution along film.When reaching detection zone (T) of fixed Poison conjugate.Quantum dot fluorescent particles mark
Remember that antibody and pre-coated drugs association reaction form compound, wavelength is used to be excited for the light of 365nm, acquisition wavelength is
The signal light of 615nm is converted to fluorescence intensity, therefore, shows that the band of photoluminescence line illustrates in sample in yin at detection zone (T)
Property.When containing Poison in sample, by with a certain amount of antibody of drug numerator competitive binding for being coated on detection zone, prevent
The combination of quantum dot fluorescent particles labelled antibody and the pre-coated drug numerator on detection zone, to weaken fluorescence intensity.Finally
According to T/C value, the corresponding drugs content on standard curve is read.No matter there are No Poison and its drop in sample at control zone (C)
Product is solved, develops the color, illustrates that detection plate performance is normal；If (C) does not develop the color for control zone, illustrate this test board performance failure.
For the quantitative standard of the quantum dot fluorescence immuno-chromatographic test paper strip for illicit drugs inspection of specific embodiment 1-3 preparation
Exactness detection
Concentration is respectively configured are as follows: 0ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/
The MOP/MET/KET standard solution of mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, using corresponding detection reagent
Box is detected, and testing result is as follows:
For the stability of the quantum dot fluorescence immuno-chromatographic test paper strip for illicit drugs inspection of specific embodiment 1-3 preparation
Detection
Crystal methamphetamine hair detection kit, morphine hair detection kit, ketamine hair detection kit is each
100 boxes are placed 6 months under the conditions of room temperature, drying, being protected from light；It simultaneously will be by crystal methamphetamine hair detection kit, morphine hair
Hair detection kit, each 100 box of ketamine hair detection kit in 42 DEG C, it is dry, be protected from light under the conditions of place two weeks.It takes all kinds of
Sample of hair, is cut the fragment for being 5mm to length, takes about 5mg sample of hair, add by drug addict and negative each 25 of sample of hair
Enter 0.5mL hair digestion solution, after shaking 30s, stands 1min, detected using above-mentioned detection kit.Testing result is as follows:
For specific embodiment 1-3 preparation for illicit drugs inspection the sensitivity of quantum dot fluorescence immuno-chromatographic test paper strip and
Range of linearity detection
Concentration is respectively configured are as follows: 0ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/
The MOP/MET/KET standard solution of mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, using corresponding detection reagent
Box is detected, and testing result is as follows:
The comparison to methamphetamine content in sample of hair is detected with ELISA method and colloidal gold strip
20 parts of methamphetamine personnel sample of hair is sucked in collection, and sample collector acquires sample from number of people top rear portion is cut using scissors
About 10mg is dispensed into 2 dedicated sampling bags of hair sample: one bag be used as test sample, another bag keep sample it is spare, respectively with note
Number notes picture recording close detection information.Sample of hair is cut to the fragment for being 5mm to length, takes about 5mg sample of hair, 0.5mL is added
Hair digestion solution after shaking 30s, stands 1min, using crystal methamphetamine hair detection kit (quantum dot fluorescence immunochromatography
Method) and colloidal gold immuno-chromatography test paper strip detected.Testing result is as follows:
| Detection kit | Drugs classification | Sample number | Detect number | Recall rate (%) |
| Crystal methamphetamine hair detection kit | Methamphetamine | 20 | 20 | 100% |
| Crystal methamphetamine colloidal gold immuno-chromatography test paper strip | Methamphetamine | 20 | 2 | 10% |
| Enzyme linked immunosorbent assay (ELISA) | Methamphetamine | 20 | 9 | 45% |
With the comparison of morphine content in ELISA method and colloidal gold strip detection sample of hair
20 parts of heroin personnel sample of hair is sucked in collection, and sample collector acquires sample from number of people top rear portion is cut using scissors
Product about 10mg is dispensed into 2 dedicated sampling bags of hair sample: one bag is used as test sample, and another bag keeps sample spare, uses respectively
Marking pen records coherent detection information.Sample of hair is cut to the fragment for being 5mm to length, takes about 5mg sample of hair, is added
0.5mL hair digestion solution after shaking 30s, stands 1min, using morphine hair detection kit (quantum dot fluorescence immunochromatography
Method) and colloidal gold immuno-chromatography test paper strip detected.Testing result is as follows:
With the comparison of Ketamine content in ELISA method and colloidal gold strip detection sample of hair
20 parts of Ketamine personnel sample of hair is sucked in collection, and sample collector acquires sample from number of people top rear portion is cut using scissors
About 10mg is dispensed into 2 dedicated sampling bags of hair sample: one bag be used as test sample, another bag keep sample it is spare, respectively with note
Number notes picture recording close detection information.Sample of hair is cut to the fragment for being 5mm to length, takes about 5mg sample of hair, 0.5mL is added
Hair digestion solution after shaking 30s, stands 1min, using ketamine hair detection kit (quantum dot fluorescence immunochromatographic method)
And colloidal gold immuno-chromatography test paper strip is detected.Testing result is as follows:
Different detection methods detect the comparison of negative sample of hair result
Negative 20 parts of sample of hair is collected, sample collector acquires sample about 10mg from number of people top rear portion is cut using scissors,
Be dispensed into 2 dedicated sampling bags of hair sample: one bag is used as test sample, and another bag keeps sample spare, is taken down notes respectively with mark
Picture recording closes detection information.Sample of hair is cut to the fragment for being 5mm to length, takes about 5mg sample of hair, 0.5mL hair is added and disappears
Liquid is solved, after shaking 30s, 1min is stood, is detected using a variety of detection reagents.Testing result is as follows:
| Detection method | Negative sample number | Feminine gender detection number | Recall rate (%) |
| Ketamine hair detection kit | 20 | 0 | 0% |
| Morphine hair detection kit | 20 | 0 | 0% |
| Crystal methamphetamine hair detection kit | 20 | 0 | 0% |
| Ketamine colloidal gold immuno-chromatography test paper strip | 20 | 0 | 0% |
| Morphine colloidal gold immuno-chromatography test paper strip | 20 | 0 | 0% |
| Crystal methamphetamine colloidal gold immuno-chromatography test paper strip | 20 | 0 | 0% |
Embodiments of the present invention are elaborated above.But present invention is not limited to the embodiments described above, In
Technical field those of ordinary skill within the scope of knowledge, can also do without departing from the purpose of the present invention
Various change out.
Claims (6)
1. being used for the quantum dot fluorescence immuno-chromatographic test paper strip of illicit drugs inspection, which is characterized in that including bottom liner, pasted on bottom liner
There is nitrocellulose filter, the side of the outer surface of the nitrocellulose filter is viscous to set release pad and sample pad, the cellulose nitrate
The other side of the outer surface of plain film is viscous to be equipped with blotting paper, and nitrocellulose filter is equipped with close to the detection zone of release pad and close to water suction
The control zone of paper, detection zone are coated with BSA and are coupled drug numerator, and control zone is coated with goat-anti rabbit polyclonal antibody.
2. the quantum dot fluorescence immuno-chromatographic test paper strip according to claim 1 for illicit drugs inspection, which is characterized in that release
Put the illicit drugs inspection mouse monoclonal antibody of pad spraying quantum dot fluorescent particle markers.
3. the preparation method of the quantum dot fluorescence immuno-chromatographic test paper strip for illicit drugs inspection, which is characterized in that including following step
It is rapid:
S1, quantum dot-labeled antibody and the preparation of fluorescence bonding pad
(1) it is that 10M carboxyl quantum dot is added in reactor by the concentration of 100 μ l, supplements 400 μ l borate buffer solutions, stirring
Mixed liquor is obtained after uniformly, subsequently into step (2)；
(2) the drugs antibody molecule that 20 μ l concentration to be marked is 1mg/ml is separately added into the mixed liquor obtained to step (1),
Mixed liquor is formed after stirring at low speed, subsequently into step (3)；
(3) 25 μ l activator EDC are added in the mixed liquor obtained to step (2), continue to react at room temperature 2h, after reaction,
8000rpm is centrifuged 3min, removes the reunion sediment being likely to occur, stays supernatant, subsequently into step (4)；
(4) supernatant that step (3) obtains 5 times are concentrated and purified with super filter tube to obtain comprising quantum dot-labeled drugs antibody molecule
Final product, each cocnentration factor be not less than 10, then will comprising quantum dot-labeled drugs antibody molecule final product redissolve it is dense in 5ml
Degree is to obtain mixed liquor in 0.05mol/L sodium borate buffer liquid；
(5) mixed liquor that step (4) obtains is sprayed onto release pad with the dosage of 0.1~0.3ml/cm, 37 degrees Celsius of baking ovens are dry
It is dry for 24 hours, envelope is spare；
S2, BSA are coupled drug numerator and the how anti-coating nitrocellulose filter of goat-anti rabbit
It is that 0.05mol/L sodium borate buffer liquid is diluted to that BSA, which is coupled drug numerator, goat-anti rabbit polyclonal antibody with concentration, respectively
Concentration is 0.03~0.3mg/mL, detection zone and the control being then sprayed onto respectively with the dosage of 1ml/cm on nitrocellulose filter
Area, 45 DEG C of drying and processing 12h, envelope are spare；
S3, reagent strip assembling
Nitrocellulose filter, sample pad, release pad and blotting paper overlap joint mutually successively are pasted on bottom liner, obtains test paper
Plate is cut into the test strips that width is 3.78~4mm as requested.
4. the preparation method of the quantum dot fluorescence immuno-chromatographic test paper strip according to claim 3 for illicit drugs inspection,
It is characterized in that, the configuration method of the borate buffer solution in step (1) are as follows: boric acid 1.2368g, sodium chloride 0.2925g are added
In container, distilled water is added, constant volume sets 100ml, after mixing evenly.
5. the preparation method of the quantum dot fluorescence immuno-chromatographic test paper strip according to claim 3 for illicit drugs inspection,
It is characterized in that, the drugs antibody molecule in step (2) is that morphine Abs molecule or crystal methamphetamine antibody molecule or ketamine are anti-
Body molecule.
6. the preparation method of the quantum dot fluorescence immuno-chromatographic test paper strip according to claim 3 for illicit drugs inspection,
It is characterized in that, the release pad in step (5) is polyester film fluorescence release pad.
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