CN106841639A - A kind of method that female phenol and bisphenol-A are detected based on double antibody sandwich method - Google Patents
A kind of method that female phenol and bisphenol-A are detected based on double antibody sandwich method Download PDFInfo
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- CN106841639A CN106841639A CN201710010686.6A CN201710010686A CN106841639A CN 106841639 A CN106841639 A CN 106841639A CN 201710010686 A CN201710010686 A CN 201710010686A CN 106841639 A CN106841639 A CN 106841639A
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- bisphenol
- phenol
- hexestrol
- female
- female phenol
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- IISBACLAFKSPIT-UHFFFAOYSA-N bisphenol A Chemical compound C=1C=C(O)C=CC=1C(C)(C)C1=CC=C(O)C=C1 IISBACLAFKSPIT-UHFFFAOYSA-N 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 60
- 229940106691 bisphenol a Drugs 0.000 title claims abstract description 44
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 title claims abstract description 37
- 229950001996 hexestrol Drugs 0.000 claims abstract description 42
- 238000001514 detection method Methods 0.000 claims abstract description 37
- PBBGSZCBWVPOOL-HDICACEKSA-N 4-[(1r,2s)-1-ethyl-2-(4-hydroxyphenyl)butyl]phenol Chemical compound C1([C@H](CC)[C@H](CC)C=2C=CC(O)=CC=2)=CC=C(O)C=C1 PBBGSZCBWVPOOL-HDICACEKSA-N 0.000 claims abstract description 32
- RGLYKWWBQGJZGM-ISLYRVAYSA-N diethylstilbestrol Chemical compound C=1C=C(O)C=CC=1C(/CC)=C(\CC)C1=CC=C(O)C=C1 RGLYKWWBQGJZGM-ISLYRVAYSA-N 0.000 claims abstract description 25
- 229960000452 diethylstilbestrol Drugs 0.000 claims abstract description 24
- NFDFQCUYFHCNBW-SCGPFSFSSA-N dienestrol Chemical compound C=1C=C(O)C=CC=1\C(=C/C)\C(=C\C)\C1=CC=C(O)C=C1 NFDFQCUYFHCNBW-SCGPFSFSSA-N 0.000 claims abstract description 22
- 229960003839 dienestrol Drugs 0.000 claims abstract description 22
- 238000012360 testing method Methods 0.000 claims abstract description 8
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000010931 gold Substances 0.000 claims abstract description 4
- 229910052737 gold Inorganic materials 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 36
- 230000004044 response Effects 0.000 claims description 28
- 238000001903 differential pulse voltammetry Methods 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 17
- 230000004048 modification Effects 0.000 claims description 16
- 238000012986 modification Methods 0.000 claims description 16
- 239000008055 phosphate buffer solution Substances 0.000 claims description 16
- 238000012417 linear regression Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000002245 particle Substances 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 8
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 8
- 235000013305 food Nutrition 0.000 claims description 8
- PCLIMKBDDGJMGD-UHFFFAOYSA-N N-bromosuccinimide Chemical compound BrN1C(=O)CCC1=O PCLIMKBDDGJMGD-UHFFFAOYSA-N 0.000 claims description 6
- 229910021397 glassy carbon Inorganic materials 0.000 claims description 6
- 238000007654 immersion Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000002689 soil Substances 0.000 claims description 5
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 4
- 238000001338 self-assembly Methods 0.000 claims description 4
- SJUCACGNNJFHLB-UHFFFAOYSA-N O=C1N[ClH](=O)NC2=C1NC(=O)N2 Chemical compound O=C1N[ClH](=O)NC2=C1NC(=O)N2 SJUCACGNNJFHLB-UHFFFAOYSA-N 0.000 claims description 3
- 238000001548 drop coating Methods 0.000 claims description 3
- 239000004033 plastic Substances 0.000 claims description 3
- 229920003023 plastic Polymers 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 claims description 2
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- ZYUVGYBAPZYKSA-UHFFFAOYSA-N 5-(3-hydroxybutan-2-yl)-4-methylbenzene-1,3-diol Chemical compound CC(O)C(C)C1=CC(O)=CC(O)=C1C ZYUVGYBAPZYKSA-UHFFFAOYSA-N 0.000 claims 2
- NHADDZMCASKINP-HTRCEHHLSA-N decarboxydihydrocitrinin Natural products C1=C(O)C(C)=C2[C@H](C)[C@@H](C)OCC2=C1O NHADDZMCASKINP-HTRCEHHLSA-N 0.000 claims 2
- BPMFZUMJYQTVII-UHFFFAOYSA-N guanidinoacetic acid Chemical compound NC(=N)NCC(O)=O BPMFZUMJYQTVII-UHFFFAOYSA-N 0.000 claims 2
- 125000003396 thiol group Chemical class [H]S* 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000009795 derivation Methods 0.000 abstract 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 18
- 229910052757 nitrogen Inorganic materials 0.000 description 9
- 238000010586 diagram Methods 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 235000013336 milk Nutrition 0.000 description 6
- 239000008267 milk Substances 0.000 description 6
- 210000004080 milk Anatomy 0.000 description 6
- 235000015277 pork Nutrition 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
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- 239000012153 distilled water Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- GPEHQHXBPDGGDP-UHFFFAOYSA-N acetonitrile;propan-2-one Chemical compound CC#N.CC(C)=O GPEHQHXBPDGGDP-UHFFFAOYSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
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- 238000004519 manufacturing process Methods 0.000 description 2
- 235000008476 powdered milk Nutrition 0.000 description 2
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 2
- LIKMAJRDDDTEIG-UHFFFAOYSA-N 1-hexene Chemical compound CCCCC=C LIKMAJRDDDTEIG-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000032170 Congenital Abnormalities Diseases 0.000 description 1
- RPNUMPOLZDHAAY-UHFFFAOYSA-N Diethylenetriamine Chemical compound NCCNCCN RPNUMPOLZDHAAY-UHFFFAOYSA-N 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004070 electrodeposition Methods 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000001076 estrogenic effect Effects 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical compound CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- 238000004313 potentiometry Methods 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000083 pulse voltammetry Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
Classifications
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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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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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
- G01N33/531—Production of immunochemical test materials
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Hematology (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a kind of method that female phenol and bisphenol-A are detected based on double antibody sandwich method.Described method is:The electrochemical immunosensor of surface finish nano gold TGA hexestrol monoclonal antibody is prepared first, then using double antibody sandwich method to testing sample in female phenol or bisphenol-A detect, by the analysis and derivation of related data, you can obtain the concentration of female phenol or bisphenol-A.The detection of the method detection diethylstilbestrol that the present invention is provided is limited to 0.06ng/mL, and the range of linearity is 0.5~1000ng/mL;Detect that the detection of hexestrol is limited to 0.052ng/mL, the range of linearity is 0.5~1000ng/mL;Detect that the detection of dienestrol is limited to 0.047ng/mL, the range of linearity is 0.5~500ng/mL;Detect that the detection of bisphenol-A is limited to 0.037ng/mL, the range of linearity is 0.5~500ng/mL.Detection method of the present invention is quick, efficient, sensitivity is high, with very low test limit and the range of linearity wider.
Description
Technical field
The invention belongs to food safety detection and technical field of analytical chemistry, it is related to a kind of electricity based on double antibody sandwich method
Chemo-immunity detection method, specifically a kind of method that female phenol and bisphenol-A are detected based on double antibody sandwich method.
Background technology
Diethylstilbestrol (diethylstilbestrol, DES), hexestrol (hexestrol, HEX) and dienestrol
(dienestrol, DIEN) (structure is shown in formula 1) belongs to artificial synthesized estrogenic chemicalses (hereinafter referred to as female phenol), with promotion
Growth of animal, improve food conversion ratio and reduce Fatty synthesis etc. effect, be once widely used in the sixties in 20th century animal husbandry life
Produce.This kind of artificial hormone can disturb the hormonal balance of human normal by food chain, endanger human health, such as cause organism metabolism
The serious problem such as disorder, dysplasia, breast cancer and fetal anomaly, all prohibites in animal in the country such as China and America and Europe
Used in breeding process.
Bisphenol-A (bisphenol A, BPA) (structure is shown in formula 1) has just been widely used in since the sixties manufacturing plastics
(milk) bottle, the cup with sucking device of child, food and beverage (milk powder) tank inner side coating, from mineral water bottle, medicine equipment to food bag
Dress inside, there is its figure, can say it is ubiquitous.The structure and property of bisphenol-A seemingly, can cause endocrine with female phenols
Imbalance, in addition caused by cancer and metabolic disturbance obesity be recognized as it is associated.European Union thinks the feeding bottle meeting containing bisphenol-A
Sex premature is induced, from 2 days March in 2011, forbids baby bottles of the production containing bisphenol-A.
Mainly there are high performance liquid chromatography, liquid chromatograph mass spectrography currently used for the method for the detection of female phenol and bisphenol-A
Method, capillary electrophoresis etc..But these analysis methods need large-sized analytic instrument, detection time is long, high cost, therefore have must
Set up more efficient, quick, sensitive analysis test method.
Double antibody sandwich method is normally used for the analysis inspection of macromolecular antigen (such as protein, virus, tumor markers)
Survey, and micromolecular compound is due to small volume, generally cannot be in combination with two antibody, therefore micromolecular compound is typically difficult
Detected with double antibody sandwich method.Female phenol and bisphenol-A are all the difunctional linear molecule with symmetrical structure, from theory
On say can be with two antibody in combination with being basis that the present invention detects female phenol and bisphenol-A using double antibody sandwich method.
The content of the invention
The invention provides a kind of female phenol of detection based on double antibody sandwich method and the method for bisphenol-A, described method is fast
Speed, efficiently, sensitivity it is high, with very low test limit (0.037~0.060ng/mL) and the range of linearity wider (0.5~
1000ng/mL or 0.5~500ng/mL).
The technical solution adopted in the present invention comprises the following steps:
1) preparation of the electrochemical immunosensor of nm of gold-TGA-hexestrol monoclonal antibody modification:First will
Nanogold particle deposits to the glassy carbon electrode surface handled well by electrochemical reducing, then by self-assembly method by sulfydryl second
Hexestrol monoclonal antibody is finally coupled to electrode surface and repaiied by acid modification to nanogold particle surface using activated carboxyl method
On the nanogold particle of decorations, electrochemical immunosensor is obtained;
2) preparation of standard liquid:The phosphate buffer solution containing female phenol or bisphenol-A is prepared, as standard liquid, will be contained
There is the standard liquid of female phenol that concentration is 0 or bisphenol-A as blank standard specimen;
3) foundation of working curve:First by step 1) electrochemical immunosensor immersion step 2) standard is molten
It is incubated in liquid, electrochemical immunosensor is rinsed with phosphate buffer solution after incubation, is then immersed in oneself containing 0.5mg/L
It is incubated in the solution of the female phenol monoclonal antibody of alkane, is rinsed with phosphate buffer solution afterwards, then by it in K3[Fe(CN)6] molten
Differential pulse voltammetry scanning is carried out in liquid, its response current is recorded;The corresponding response current of blank standard specimen is designated as I0, contain female phenol
Or the response current of bisphenol-A standard specimen is designated as Ix, the changing value Δ I of response current is equal to IxWith I0Difference absolute value;By the Δ
I is depicted as Δ I-lgC working curves with the logarithm value lgC of female phenol or bisphenol A concentration in standard liquid, is obtained using linear regression method
To Δ I-lgC equations of linear regression;
4) measure of female phenol and bisphenol-A:Configuration the phosphate buffer solution containing testing sample, according to step 3) identical
Method is incubated and differential pulse voltammetry scanning to the immunosensor, recording responses electric current;According to the change of response current
Change value Δ I and Δ I-lgC equations of linear regression, obtain the content of female phenol and bisphenol-A.
The electrochemical immunosensor of the nm of gold-TGA-hexestrol monoclonal antibody modification is using following
It is prepared by method:To clean first during clean glass-carbon electrode immersion mass percentage concentration is 0.5% chlorauric acid solution, using perseverance
Gold chloride is reduced to nanogold particle so as to be deposited on glassy carbon electrode surface by potentiometry;Then glass-carbon electrode is placed in 1mmol/
In the TGA aqueous solution of L, by the method for self assembly in nanogold particle surface modification TGA;Again by glass-carbon electrode
1- (3- the dimethylamino-propyls) -3- ethyls carbon two for being placed in the N-bromosuccinimide containing 0.1mol/L and 0.1mol/L is sub-
In 2- (N- morpholines) ethanesulfonic acid buffer of amine hydrochlorate, activated carboxyl;Glassy carbon electrode surface after above-mentioned activated carboxyl
Drop coating mass concentration is the hexestrol monoclonal antibody of 0.5mg/L, is reacted under the conditions of 4 DEG C, and described nm of gold-sulfydryl is obtained
The electrochemical immunosensor of acetic acid-hexestrol monoclonal antibody modification.
The female phenol is respectively diethylstilbestrol, hexestrol and dienestrol.
Using 3 times of the measured value standard deviation of the blank standard specimen as sample detection limit, repeat step 3) can draw it is female
The test limit of phenol or bisphenol-A sample.
The method of the present invention can be used for the detection of female phenol and bisphenol-A in various actual samples, the actual sample include but
It is not limited to food, soil, water quality, plastics etc..
The method that the present invention is provided is used for the detection of female phenol and bisphenol-A, and its test limit is respectively diethylstilbestrol 0.060ng/
ML, hexestrol 0.052ng/mL, dienestrol 0.047ng/mL, bisphenol-A 0.037ng/mL;The range of linearity is that hexene is female
0.5~1000ng/mL of phenol and hexestrol, 0.5~500ng/mL of dienestrol and bisphenol-A.
The present invention has following beneficial effect:As a result of double antibody sandwich method, detection sensitivity is improve, detected
Limit very low (0.037~0.060ng/mL), while having the range of linearity (0.5~1000ng/mL or 0.5~500ng/ wider
mL)。
Protection scope of the present invention is not limited with specific embodiment, but is defined in the claims.
Brief description of the drawings
Fig. 1 is the DPV curve maps that electrochemical immunosensor is detected to diethylstilbestrol.
Fig. 2 is the working curve diagram of the changing value Δ I and diethylstilbestrol log concentration value lgC of response current.
Fig. 3 is the DPV curve maps that electrochemical immunosensor is detected to hexestrol.
Fig. 4 is the working curve diagram of the changing value Δ I and hexestrol log concentration value lgC of response current.
Fig. 5 is the DPV curve maps that electrochemical immunosensor is detected to dienestrol.
Fig. 6 is the working curve diagram of the changing value Δ I and dienestrol log concentration value lgC of response current.
Fig. 7 is the DPV curve maps that electrochemical immunosensor is detected to bisphenol-A.
Fig. 8 is the working curve diagram of the changing value Δ I and bisphenol A concentration logarithm value lgC of response current.
Specific embodiment
Technical solutions according to the invention are further described in detail below by specific embodiment combination accompanying drawing,
But it is necessary to note that following examples are served only for the description to the content of the invention, do not constitute limiting the scope of the invention.
Detection of the embodiment 1 to diethylstilbestrol standard sample
1) preparation of electrochemical immunosensor:
The glass-carbon electrode of a diameter of 3mm is polished with the alumina powder of 0.3 μm and 0.05 μm on polishing flannelet successively,
It is cleaned by ultrasonic 30s with absolute ethyl alcohol-distilled water (V/V=1/1), distilled water successively, then it is clean with distilled water flushing.By above-mentioned electricity
Carry out constant potential electrochemical deposition (voltage be -0.2V, sedimentation time 60s) in the chlorauric acid solution of pole insertion 0.5%, spend from
Sub- water immerses in the mercaptoacetic acid solution of 1mmol/L after rinsing well, is reacted 8 hours at being placed in 37 DEG C.Above-mentioned electrode is steamed
Distilled water is placed in 1- (the 3- dimethylaminos of N-bromosuccinimide (NHS) and 0.1mol/L containing 0.1mol/L after rinsing well
Propyl group) -3- ethyl-carbodiimide hydrochlorides (EDC) 2- (N- morpholines) ethyl sulfonic acid (MES) cushioning liquid in, activated carboxyl 40
Minute.μ L hexestrols monoclonal antibody (0.5mg/L) of electrode surface drop coating 50 after above-mentioned activated carboxyl, is placed in refrigerator
4 DEG C overnight after, rinsed well and be immersed in 5% bovine serum albumin(BSA) (BSA) solution with deionized water, in 37 DEG C of baking ovens
It is incubated 30 minutes, to close remaining avtive spot, you can obtain nm of gold-TGA-hexestrol monoclonal antibody and repair
The electrochemical immunosensor of decorations.
2) detection of diethylstilbestrol standard sample
By step 1) described in electrochemical immunosensor immersion contain a series of various concentrations (including concentration is zero)
Diethylstilbestrol standard liquid in, 37 DEG C be incubated 30 minutes, rinsed well with phosphate buffer solution, be then immersed in hexestrol
It is incubated 30 minutes at 37 DEG C in monoclonal antibody (0.5mg/L) solution, 2mmol/L is placed in after being rinsed well with phosphate buffer solution
K3[Fe(CN)6] differential pulse voltammetry (DPV) scanning is carried out in solution, record corresponding response current.DPV curve maps are such as
Shown in Fig. 1, concentration is followed successively by 0ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 20ng/ to curve from top to bottom in figure
ML, 100ng/mL, 200ng/mL and 1000ng/mL.
Corresponding response current is designated as I when by diethylstilbestrol concentration being zero0, and corresponding to various concentrations diethylstilbestrol
Response current is designated as IX, calculate response current changing value Δ I=| IX-I0|, with Δ I to diethylstilbestrol log concentration value lg C
(ng/mL) mapping can obtain Δ I-lgC working curves.Working curve diagram using linear regression method as shown in Fig. 2 obtain its Δ I-
LgC equations of linear regression are Δ I (μ A)=0.90073+3.91741lgC (ng/mL), the concentration of diethylstilbestrol 0.5~
LgC is directly proportional to Δ I in the range of 1000ng/mL, and linearly dependent coefficient is 0.99467.With blank standard specimen measured value standard deviation
3 times (3 σ) as sample detection limit, be repeated 10 times experiment and draw, method described above detection diethylstilbestrol sample detection be limited to
0.060ng/mL。
Detection of the embodiment 2 to hexestrol standard sample
With with the step 1 of embodiment 1) identical method prepares nm of gold-TGA-hexestrol monoclonal antibody
The electrochemical immunosensor of modification, is dipped in the hexestrol standard containing a series of various concentrations (including concentration is zero)
In solution, then with the step 2 of embodiment 1) identical method carries out DPV and scans and data record and processed.DPV curve maps
As shown in figure 3, in figure curve from top to bottom concentration be followed successively by 0ng/mL, 0.5ng/mL, 1ng/mL, 10ng/mL, 20ng/mL,
50ng/mL, 100ng/mL, 200ng/mL, 500ng/mL and 1000ng/mL.Meet with a response current variation value Δ I and hexestrol
Log concentration value lgC is linear when hexestrol concentration is between 0.5~1000ng/mL, working curve diagram such as Fig. 4 institutes
Show, its equation of linear regression is Δ I (μ A)=2.94885+4.53102lgC (ng/mL), and linearly dependent coefficient is 0.99420.
Limited as sample detection using 3 times (3 σ) of blank standard specimen measured value standard deviation, be repeated 10 times experiment and draw, method described above inspection
The sample detection for surveying hexestrol is limited to 0.052ng/mL.
Detection of the embodiment 3 to dienestrol standard sample
With with the step 1 of embodiment 1) identical method prepares nm of gold-TGA-hexestrol monoclonal antibody
The electrochemical immunosensor of modification, is dipped in the dienestrol mark containing a series of various concentrations (including concentration is zero)
In quasi- solution, then with the step 2 of embodiment 1) identical method carries out DPV and scans and data record and processed.DPV curves
Figure as shown in figure 5, in figure curve from top to bottom concentration be followed successively by 0ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL,
20ng/mL, 50ng/mL, 100ng/mL, 200ng/mL and 500ng/mL.Meet with a response current variation value Δ I and dienestrol
Log concentration value lgC is linear when dienestrol concentration is between 0.5~500ng/mL, working curve diagram such as Fig. 6
Shown, its equation of linear regression is Δ I (μ A)=3.58971+4.99674lgC (ng/mL), and linearly dependent coefficient is
0.99713.Limited as sample detection using 3 times (3 σ) of blank standard specimen measured value standard deviation, be repeated 10 times experiment and draw, the above
The sample detection for stating method detection dienestrol is limited to 0.047ng/mL.
Detection of the embodiment 4 to bisphenol-A standard sample
With with the step 1 of embodiment 1) identical method prepares nm of gold-TGA-hexestrol monoclonal antibody
The electrochemical immunosensor of modification, is dipped in the bisphenol-A standard containing a series of various concentrations (including concentration is zero) molten
In liquid, then with the step 2 of embodiment 1) identical method carries out DPV and scans and data record and processed.DPV curve maps are such as
Shown in Fig. 7, concentration is followed successively by 0ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 20ng/ to curve from top to bottom in figure
ML, 50ng/mL, 100ng/mL, 200ng/mL and 500ng/mL.Meet with a response current variation value Δ I and bisphenol A concentration logarithm value
LgC is linear when bisphenol A concentration is between 0.5~500ng/mL, and working curve diagram is as shown in figure 8, its linear regression
Equation is Δ I (μ A)=6.10178+6.4287lgC (ng/mL), and linearly dependent coefficient is 0.99314.Determined with blank standard specimen
3 times (3 σ) for being worth standard deviation are limited as sample detection, are repeated 10 times experiment and are drawn, method described above detects the sample of bisphenol-A
Detection is limited to 0.037ng/mL.
The measure of mark-on diethylstilbestrol in the milk powder of embodiment 5
1) treatment of powdered milk sample:1 ± 0.0050g milk powder is weighed in the sample cell to 10mL, diethylstilbestrol mark is added
Supernatant, in being centrifuged 10 minutes under 2000r/m, is transferred to nitrogen by quasi- solution and 6mL n-hexanes, mixture ultrasonic disperse 30 minutes
In blowpipe, residue repeats to extract 1 time with the identical extract solution of 3mL, and supernatant merges in nitrogen blowpipe.Extract blows condition in nitrogen
Under at a temperature of 50 DEG C concentration and evaporation, concentrate add the pH of 1mL for after the dissolving of 7.4 phosphate buffer solutions for electrochemical credit
Analysis.
2) in powdered milk sample mark-on diethylstilbestrol measure:The milk powder extract solution sample of different volumes is taken respectively, is added to
A series of Incubating Solutions that 200 μ L are containing various concentrations diethylstilbestrol and cumulative volume are configured in phosphate buffer solution.To receive
The electrochemical immunosensor of meter Jin/TGA/hexestrol monoclonal antibody modification is immersed in above-mentioned Incubating Solution, at 37 DEG C
It is incubated 30 minutes, is rinsed well with phosphate buffer solution, is then immersed in hexestrol monoclonal antibody (0.5mg/L) solution
37 DEG C are incubated 30 minutes, and the K of 2mmol/L is placed in after being rinsed well with phosphate buffer solution3[Fe(CN)6] difference is carried out in solution
Pulse Voltammetry (DPV) is scanned, and records corresponding response current.By using and step 2 in embodiment 1) identical method obtains
The Δ I-lgC working curves of the response current changing value Δ I for arriving and diethylstilbestrol log concentration value lgC, calculate diethylstilbestrol
Concentration, detect rate of recovery result such as table 1.
Table 1 is the rate of recovery of the diethylstilbestrol concentration in immunosensor detection mark-on milk powder
The measure of mark-on hexestrol in the pork of embodiment 6
1) treatment of pork sample:1 ± 0.0050g porks are weighed in the sample cell to 10mL, hexestrol mark is added
Quasi- solution and 3mL acetonitrile-acetone extract solutions (V:V=4:1), mixture ultrasonic disperse 30 minutes, in 10 points of centrifugation under 2000r/m
Clock, supernatant is transferred in nitrogen blowpipe, and residue repeats to extract 1 time with the identical extract solution of 3mL, and supernatant merges in nitrogen blowpipe
In.Extract under the conditions of nitrogen blows at a temperature of 50 DEG C concentration and evaporation, concentrate add 1mL pH be 7.4 phosphate buffer solutions
It is used for electrochemical analysis after dissolving.
2) in pork sample mark-on hexestrol measure:The pork extract solution sample of different volumes is taken respectively, is added to
A series of Incubating Solutions that 200 μ L are containing various concentrations hexestrol and cumulative volume are configured in phosphate buffer solution.To receive
The electrochemical immunosensor of meter Jin/TGA/hexestrol monoclonal antibody modification is immersed in above-mentioned Incubating Solution, with reality
Apply step 2 in example 1) the Δ I- of the response current changing value Δ I that obtains of identical method and hexestrol log concentration value lgC
LgC working curves, calculate the concentration of hexestrol, detect rate of recovery result such as table 2.
Table 2 is the rate of recovery of the hexestrol concentration in immunosensor detection mark-on pork
According to method substantially the same manner as Example 6, various animal derived foods can be also carried out, including pig, ox, sheep etc. often
With the detection of the diethylstilbestrol, dienestrol contained in the meat products of animal, internal organ etc..
The measure of mark-on dienestrol in the pedotheque of embodiment 7
1) treatment of pedotheque:1 ± 0.0050g soil is weighed, is added in the sample cell of 10mL after grinding, plus
Enter dienestrol standard liquid and 3mL acetonitrile-acetone extract solutions (V:V=4:1), mixture ultrasonic disperse 30 minutes, in
It is centrifuged 10 minutes under 2000r/m, supernatant is transferred in nitrogen blowpipe, residue repeats to extract 1 time with the identical extract solution of 3mL,
Supernatant merges in nitrogen blowpipe.Extract under the conditions of nitrogen blows at a temperature of 50 DEG C concentration and evaporation, concentrate add 1mL pH
To be used for electrochemical analysis after the dissolving of 7.4 phosphate buffer solutions.
2) in pedotheque mark-on dienestrol measure:The soil extract sample of different volumes is taken respectively, is added
A series of Incubating Solutions that 200 μ L are containing various concentrations dienestrol and cumulative volume are configured to in phosphate buffer solution.
The electrochemical immunosensor of nm of gold/TGA/hexestrol monoclonal antibody modification is immersed in above-mentioned Incubating Solution, with
With step 2 in embodiment 1) the response current changing value Δ I that obtains of identical method and dienestrol log concentration value lgC
Δ I-lgC working curves, calculate the concentration of dienestrol, detect rate of recovery result such as table 3.
Table 3 is the rate of recovery of the dienestrol concentration in immunosensor detection mark-on soil
The measure of mark-on bisphenol-A in the water of embodiment 8
1.0mL water samples are measured, the bisphenol-A standard liquid of various concentrations is added, then one is configured to phosphate buffer solution and be
Row are the Incubating Solution of 2.0mL containing various concentrations bisphenol-A and cumulative volume.By nm of gold/TGA/hexestrol monoclonal
The electrochemical immunosensor of antibody modification is immersed in above-mentioned Incubating Solution (200 μ L), with step 2 in embodiment 1) identical side
The Δ I-lgC working curves of the response current changing value Δ I that method is obtained and bisphenol A concentration logarithm value lgC, calculate bisphenol-A
Concentration, detects rate of recovery result such as table 4.
Table 4 is the rate of recovery of the bisphenol A concentration in immunosensor detection mark-on water sample
Claims (5)
1. a kind of method that female phenol and bisphenol-A are detected based on double antibody sandwich method, is comprised the following steps:
1) preparation of the electrochemical immunosensor of nm of gold-TGA-hexestrol monoclonal antibody modification:First by nanometer
Gold grain deposits to the glassy carbon electrode surface handled well by electrochemical reducing, is then repaiied TGA by self-assembly method
Nanogold particle surface is adornd, hexestrol monoclonal antibody is finally coupled to by electrode face finish using activated carboxyl method
On nanogold particle, electrochemical immunosensor is obtained;
2) preparation of standard liquid:The phosphate buffer solution containing female phenol or bisphenol-A is prepared, as standard liquid, will be containing dense
Spend for 0 female phenol or bisphenol-A standard liquid as blank standard specimen;
3) foundation of working curve:First by step 1) electrochemical immunosensor immersion step 2) in the standard liquid
It is incubated, electrochemical immunosensor is rinsed with phosphate buffer solution after incubation, is then immersed in the hexane containing 0.5mg/L female
It is incubated in the solution of phenol monoclonal antibody, is rinsed with phosphate buffer solution afterwards, then by it in K3[Fe(CN)6] in solution
Differential pulse voltammetry scanning is carried out, its response current is recorded;The corresponding response current of blank standard specimen is designated as I0, contain female phenol or double
The response current of phenol A standard specimens is designated as Ix, the changing value Δ I of response current is equal to IxWith I0Difference absolute value;By the Δ I with
The logarithm value lgC of female phenol or bisphenol A concentration is depicted as Δ I-lgC working curves in standard liquid, is obtained using linear regression method
Δ I-lgC equations of linear regression;
4) measure of female phenol and bisphenol-A:Configuration the phosphate buffer solution containing testing sample, according to step 3) identical method
The immunosensor is incubated and differential pulse voltammetry scanning, recording responses electric current;According to the changing value of response current
Δ I and Δ I-lgC equations of linear regression, obtain the content of female phenol and bisphenol-A.
2. a kind of method that female phenol and bisphenol-A are detected based on double antibody sandwich method according to claim 1, its feature is existed
In the electrochemical immunosensor of the nm of gold-TGA-hexestrol monoclonal antibody modification uses following methods system
It is standby:To clean first during clean glass-carbon electrode immersion mass percentage concentration is 0.5% chlorauric acid solution, using potentiostatic method
Gold chloride is reduced to nanogold particle so as to be deposited on glassy carbon electrode surface;Then glass-carbon electrode is placed in the mercapto of 1mmol/L
In the guanidine-acetic acid aqueous solution, by the method for self assembly in nanogold particle surface modification TGA;Glass-carbon electrode is placed in again
1- (3- dimethylamino-propyls) -3- ethyl carbodiimide salt of N-bromosuccinimide and 0.1mol/L containing 0.1mol/L
In 2- (N- morpholines) ethanesulfonic acid buffer of hydrochlorate, activated carboxyl;Glassy carbon electrode surface drop coating after above-mentioned activated carboxyl
Mass concentration is the hexestrol monoclonal antibody of 0.5mg/L, is reacted under the conditions of 4 DEG C, and described nm of gold-sulfydryl second is obtained
The electrochemical immunosensor of acid-hexestrol monoclonal antibody modification.
3. a kind of method that female phenol and bisphenol-A are detected based on double antibody sandwich method according to claim 1, its feature is existed
In the female phenol is respectively diethylstilbestrol, hexestrol and dienestrol.
4. a kind of method that female phenol and bisphenol-A are detected based on double antibody sandwich method according to claim 1, its feature is existed
In, using 3 times of the measured value standard deviation of the blank standard specimen as sample detection limit, repeat step 3) female phenol or double can be drawn
The test limit of phenol A samples.
5. application of a kind of method based on the female phenol of double antibody sandwich method detection and bisphenol-A in food, soil, water quality, plastics.
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