CN102520163B - Determination method for carbohydrate allergen - Google Patents
Determination method for carbohydrate allergen Download PDFInfo
- Publication number
- CN102520163B CN102520163B CN201110364832.8A CN201110364832A CN102520163B CN 102520163 B CN102520163 B CN 102520163B CN 201110364832 A CN201110364832 A CN 201110364832A CN 102520163 B CN102520163 B CN 102520163B
- Authority
- CN
- China
- Prior art keywords
- carbohydrate
- allergen
- chip
- concentration
- zinc oxide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a determination method for a carbohydrate allergen. The method comprises the following steps of: marking the carbohydrate allergen by using zinc oxide quantum dots; immobilizing an agglutinin layer on a chip in an electrolytic cell; specifically binding the carbohydrate allergen marked by the zinc oxide quantum dots with agglutinin on the chip in a series of carbohydrate allergen solutions to be determined of known concentrations; connecting the zinc oxide quantum dots marked on the carbohydrate allergen to the chip; dissolving the zinc oxide quantum dots into zinc ions by using acidic solution; determining concentration of the dissolved zinc ions by using dissolving voltammetry; and establishing relation between the concentration of the zinc ions and the concentration of the carbohydrate allergen so as to determine the concentration of the carbohydrate allergen to be determined.
Description
Technical field
The invention belongs to medical biotechnology field, specifically, the invention describes a kind of detection method of carbohydrate allergen.
Background technology
Food hypersenstivity is that people produce the I allergic reaction type of IgE mediation to food, and clinical manifestation is the multiple symptoms such as nettle rash, asthma, stomachache and diarrhoea, serious caused shock.In general, food allergen is albumen or the glycoprotein of molecular weight between 10 ~ 70 kD.Along with social development, the variation of people's dietary structure, the irritated incidence of disease increases day by day, to health of people, causes grave danger.In allergic reaction, by eight group foods such as eggs, peanut, newborn classes, caused.At present also very limited to the detection method of anaphylactogen in some food, the immune response mainly containing based on some anaphylactogens detects.But this method exists cost high, in addition for some anaphylactogens, corresponding antibody or antigen are difficult to obtain, thereby have also hindered the application of immunoassay technology in anaphylactogen detects.
Summary of the invention
technical matters:the present invention is directed to above-mentioned technical matters, a kind of stripping voltammetry detection chip of Zinc oxide quantum dot mark carbohydrate allergen is provided, the method utilizes zinc paste mark carbohydrate allergen to detect low concentration carbohydrate allergen, low to the detectability of corresponding carbohydrate allergen, selectivity good, highly sensitive, good stability.
technical scheme:at the assay method of carbohydrate allergen of the present invention, utilize Zinc oxide quantum dot mark carbohydrate allergen; Immobilization one deck agglutinin on the chip of while in electrolytic cell, in the carbohydrate allergen solution to be measured of a series of concentration known, make mark carbohydrate allergen and the agglutinin on chip of Zinc oxide quantum dot carry out specific binding, the Zinc oxide quantum dot being marked on carbohydrate allergen is connected on chip; Then with acid solution, Zinc oxide quantum dot is dissolved as to zinc ion, uses the concentration of the zinc ion of Voltammetry stripping, set up the relation between zinc ion concentration and carbohydrate allergen concentration, thereby determine carbohydrate allergen concentration to be measured.
Concrete assay method is:
A. 0.5～5 milligram of Zinc oxide quantum dot is distributed in the carbohydrate allergen solution of 50～200 microlitre 1 nanograms/milliliter, at room temperature shakes 10min, centrifugal, the phosphate buffered solution that is 6.8 ~ 7.5 by pH value is cleaned three times.Add again 500 μ L 1 ~ 2% w/v bovine serum albumins, 15 ~ 35
ounder C, shake 10 ~ 30min, centrifugal, the phosphate buffered solution that is 6.8 ~ 7.5 by pH value is cleaned three times; Finally be scattered in 50 ~ 150 μ L 0.05 ~ 0.1% v/v triton x-100 phosphate buffer solutions, the used time is not stored in refrigerator heat-insulation layer;
B. by titanium dioxide silicon chip or glass sheet ultrasonic 5min respectively in acetone, second alcohol and water respectively, then at the NH of 60 ~ 80 ° of C
4oH/H
2o
2/ H
2o 1:1:5 ~ 7, v/v and HCl/H
2o
2/ H
2o 1:1:4 ~ 6, v/v soaks respectively 5 ~ 10min; Take out the rear clear water wash clean of using, dry; Then the ethanolic solution of putting into the 3-amine propyl group Ethoxysilane of 5%v/v soaks 2 ~ 5 h, wash clean, dries, then puts into the phosphate buffered solution that 10 ~ 15mL is 6.8 ~ 7.5 containing 1 ~ 2%v/v glutaraldehyde pH value, in 4
ostanding 8 ~ 12 h of C, subsequently by washed with de-ionized water, dry, stand-by;
C. the chip after modifying is arranged on to an aperture and is less than in the electrolytic cell of the chip length of side, the phosphate buffered solution that is 6.8 ~ 7.5 containing 1 ~ 2 mg/mL agglutinin pH value by 50 μ L drops on the above-mentioned chip of handling well, and in 4
ostanding 8 ~ 12 h of C, use washed with de-ionized water subsequently; Then said chip is soaked to 0.5 ~ 1 h with containing 0.5 ~ 1% w/v bovine serum albumin and 0.05 ~ 0.1% v/v Tween-20, use subsequently washed with de-ionized water, stand-by;
D. the testing sample of handling well is dropped on the chip preparing, place 15 ~ 50min, use washed with de-ionized water chip surface 3 times;
E. to injecting pH value in electrolytic cell, be the Zinc oxide quantum dot on 1 ~ 3 dissolve with hydrochloric acid solution chip, and be that 1 ~ 3 hydrochloric acid solution is washed three times by pH value, merge lysate and washing lotion, add pH value and be 6.8 ~ 7.5 phosphate buffered solution to 1 ~ 3 milliliter;
F. with platinum electrode, be to electrode, silver/silver chloride electrode is contrast electrode, and mercury film electrode is working electrode ,-0.8 ~-1.5 volt of enrichment 90 ~ 150 seconds, and with the stripping peak of square wave voltammetry survey zinc, current potential is about-1.1 volts;
G. change the 1st step carbohydrate allergen concentration, repeat e, f, g step, measure the peak point current at the Stripping Voltammetry peak of the zinc that a series of carbohydrate allergen relative concentrations answer, set up the corresponding relation between peak point current and carbohydrate allergen concentration.
Described chip is titanium dioxide silicon chip or glass sheet.
The Stripping Voltammetry peak point current of the zinc that above process records and the corresponding relation between carbohydrate allergen concentration are the important basic datas of the inventive method, for carry out actual clinical by this inventive method, detect the theoretical foundation that provides concentration to judge, to utilizing the inventive method to carry out clinical detection fast and accurately, have extremely important meaning.
Above-mentioned agglutinin and carbohydrate allergen are ConA and ovomucoid thereof.
Said chip is titanium dioxide silicon chip or glass sheet.
beneficial effect:compared with prior art, the present invention has the following advantages:
1, Zinc oxide quantum dot of the present invention is to the labeling process of carbohydrate allergen and process in leaching, simple to operate, mild condition, and the combination of quantum dot and carbohydrate allergen is firmly stable.Be very beneficial for keeping the biologically active of carbohydrate allergen.
2, the present invention uses stripping voltammetry to survey zinc ion concentration, is easy to the enrichment of zinc on mercury electrode, the stripping peak of zinc easily survey and peak point current strong, be easy to set up the curved line relation between peak point current and carbohydrate allergen concentration accurately.Be conducive to reduce the detectability that carbohydrate allergen detects, improve detection sensitivity.Make quick and precisely to detect low concentration carbohydrate allergen and become possibility.
Accompanying drawing explanation
Following accompanying drawing is used for illustrating specific embodiment of the invention scheme, and be not used in, limits the scope of the invention being defined by claims.
Figure l is sensitinogen detecting chip assembling schematic diagram.
Fig. 2 is the anaphylactogen chip detection result of standard model.
Embodiment
(as Fig. 1)
1,0.5 milligram of Zinc oxide quantum dot is distributed in the ovomucoid solution of 50 microlitre 1 nanograms/milliliter, at room temperature shake 10min, centrifugal, the phosphate buffered solution that is 7.0 by pH value is cleaned three times.Add 500 μ L 1% (w/v) bovine serum albumins again, under room temperature, shake 10min, centrifugal, the phosphate buffered solution that is 7.0 by pH value is cleaned three times.Finally be scattered in the phosphate buffered solution that 50 μ L 0.1% (v/v) triton x-100 pH values are 7.0, the used time is not stored in refrigerator heat-insulation layer.
2, chip is modified, and silicon chip (1x1x0.2mm) is ultrasonic 5min respectively in acetone, second alcohol and water respectively, then at the NH of 80 ° of C
4oH/H
2o
2/ H
2o (1:1:7, v/v) and HCl/H
2o
2/ H
2o (1:1:6, v/v) soaks respectively 10min.Take out the rear clear water wash clean of using, dry.Then put into 5%(v/v) ethanolic solution of 3-amine propyl group Ethoxysilane soaks 5 h, wash clean, dry, then put into contain 2%(v/v) the glutaraldehyde pH value phosphate buffered solution that is 7.0, in 4
ostanding 12 h of C, subsequently by washed with de-ionized water, dry, stand-by.
3, detection chip preparation, is less than the aperture that is arranged on after modifying in the electrolytic cell of the chip length of side, and 50 μ L are contained to the phosphate buffered solution that 1 mg/mL ConA pH value is 7.0 and drop on the above-mentioned chip of handling well, and in 4
ostanding 12 h of C, use washed with de-ionized water subsequently.Then said chip is soaked to 1 h with containing 1% (w/v) bovine serum albumin and 0.05 % (v/v) Tween-20, use subsequently washed with de-ionized water, stand-by.
4, detection method, on the ConA chip preparing, places 15min by the ovomucoid sample drop of handling well, uses washed with de-ionized water chip surface 3 times.
5, to injecting the ultrasonic 10min of hydrochloric acid solution that 1800 μ L pH values are 2 in electrolytic cell to dissolve the Zinc oxide quantum dot on chip, go up subsequently and transfer to electrolytic cell and add successively the phosphate buffered solution that 2000 μ L pH values are 7.01,4uL 10 ppm mercuric nitrates (II) as solution.50 μ L sodium hydroxide solutions (0.32 M).Then use Electrochemical Detection,
6, with platinum electrode, be to electrode, silver/silver chloride electrode is contrast electrode, mercury film electrode is working electrode ,-1.3 V electricity enrichment 2 min, with the stripping peak of square wave voltammetry survey zinc, current potential is about-1.1 volts.
7, change the 1st step ovomucoid concentration, repeat the 4th, 5,6 steps, measure the peak point current at the Stripping Voltammetry peak of the zinc that a series of ovomucoid relative concentrations answer, set up the corresponding relation (Fig. 2) between peak point current and ovomucoid concentration.
Claims (1)
1. an assay method for carbohydrate allergen, is characterized in that utilizing Zinc oxide quantum dot mark carbohydrate allergen; Immobilization one deck agglutinin on the chip of while in electrolytic cell, in the carbohydrate allergen solution to be measured of a series of concentration known, make mark carbohydrate allergen and the agglutinin on chip of Zinc oxide quantum dot carry out specific binding, the Zinc oxide quantum dot being marked on carbohydrate allergen is connected on chip; Then with acid solution, Zinc oxide quantum dot is dissolved as to zinc ion, uses the concentration of the zinc ion of Voltammetry stripping, set up the relation between zinc ion concentration and carbohydrate allergen concentration, thereby determine carbohydrate allergen concentration to be measured; The assay method of described carbohydrate allergen specifically comprises the following steps:
A. 0.5～5 milligram of Zinc oxide quantum dot is distributed in the carbohydrate allergen solution of 50～200 microlitre 1 nanograms/milliliter, at room temperature shake 10min, centrifugal, the phosphate buffered solution that is 6.8～7.5 by pH value is cleaned three times, add again 500 μ L1～2%w/v bovine serum albumins, at 15～35 ℃, shake 10～30min, centrifugal, the phosphate buffered solution that is 6.8～7.5 by pH value is cleaned three times; Finally be scattered in 50～150 μ L0.05～0.1%v/v triton x-100 phosphate buffer solutions, the used time is not stored in refrigerator heat-insulation layer;
B. by titanium dioxide silicon chip or glass sheet ultrasonic 5min respectively in acetone, second alcohol and water respectively, then at the NH of 60～80 ℃
4oH/H
2o
2/ H
2o1:1:5～7, v/v and HCl/H
2o
2/ H
2o1:1:4～6, v/v soaks respectively 5～10min; Take out the rear clear water wash clean of using, dry; Then the ethanolic solution of putting into the 3-amine propyl group Ethoxysilane of 5%v/v soaks 2～5h, wash clean, dry, then put into the phosphate buffered solution that 10～15mL is 6.8～7.5 containing 1～2%v/v glutaraldehyde pH value, in 4 ℃ of standing 8～12h, subsequently by washed with de-ionized water, dry, stand-by;
C. the chip after modifying being arranged on to an aperture is less than in the electrolytic cell of the chip length of side, the phosphate buffered solution that is 6.8～7.5 containing 1～2mg/mL agglutinin pH value by 50 μ L drops on the above-mentioned chip of handling well, and in 4 ℃ of standing 8～12h, use subsequently washed with de-ionized water; Then said chip is soaked to 0.5～1h with containing 0.5～1%w/v bovine serum albumin and 0.05～0.1%v/v Tween-20, use subsequently washed with de-ionized water, stand-by;
D. the testing sample of handling well is dropped on the chip preparing, place 15～50min, use washed with de-ionized water chip surface 3 times;
E. to injecting pH value in electrolytic cell, be the Zinc oxide quantum dot on 1～3 dissolve with hydrochloric acid solution chip, and be that 1～3 hydrochloric acid solution is washed three times by pH value, merge lysate and washing lotion, add pH value and be 6.8～7.5 phosphate buffered solution to 1～3 milliliter;
F. with platinum electrode, be to electrode, silver/silver chloride electrode is contrast electrode, and mercury film electrode is working electrode ,-0.8～-1.5 volt of enrichment 90～150 seconds, and with the stripping peak of square wave voltammetry survey zinc, current potential is about-1.1 volts;
G. change the 1st step carbohydrate allergen concentration, repeat e, f, g step, measure the peak point current at the Stripping Voltammetry peak of the zinc that a series of carbohydrate allergen relative concentrations answer, set up the corresponding relation between peak point current and carbohydrate allergen concentration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110364832.8A CN102520163B (en) | 2011-11-17 | 2011-11-17 | Determination method for carbohydrate allergen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110364832.8A CN102520163B (en) | 2011-11-17 | 2011-11-17 | Determination method for carbohydrate allergen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102520163A CN102520163A (en) | 2012-06-27 |
| CN102520163B true CN102520163B (en) | 2014-03-12 |
Family
ID=46291151
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110364832.8A Active CN102520163B (en) | 2011-11-17 | 2011-11-17 | Determination method for carbohydrate allergen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102520163B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101109749A (en) * | 2007-08-07 | 2008-01-23 | 南京大学 | Multifunctional immune chip and preparing method thereof and its application in immunity detection |
| CN101526523A (en) * | 2009-03-27 | 2009-09-09 | 东南大学 | Preparation for cadmium antimonide quantum dot immune marker and detection method for electrochemical sandwich immune |
| CN101526531A (en) * | 2009-03-20 | 2009-09-09 | 东南大学 | Method for immunoassay by utilizing zinc oxide quantum dots |
| CN101587071A (en) * | 2009-07-01 | 2009-11-25 | 东南大学 | Fluorescence immunoassay method of using zinc oxide quantum dots to mark antibody |
| CN101672845A (en) * | 2009-09-23 | 2010-03-17 | 东南大学 | Immunoassay method for antibodies labeled with zinc-oxide quantum dots by stripping voltammetry |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8262998B2 (en) * | 2005-04-15 | 2012-09-11 | Branislav Vlahovic | Detection methods and detection devices based on the quantum confinement effects |
-
2011
- 2011-11-17 CN CN201110364832.8A patent/CN102520163B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101109749A (en) * | 2007-08-07 | 2008-01-23 | 南京大学 | Multifunctional immune chip and preparing method thereof and its application in immunity detection |
| CN101526531A (en) * | 2009-03-20 | 2009-09-09 | 东南大学 | Method for immunoassay by utilizing zinc oxide quantum dots |
| CN101526523A (en) * | 2009-03-27 | 2009-09-09 | 东南大学 | Preparation for cadmium antimonide quantum dot immune marker and detection method for electrochemical sandwich immune |
| CN101587071A (en) * | 2009-07-01 | 2009-11-25 | 东南大学 | Fluorescence immunoassay method of using zinc oxide quantum dots to mark antibody |
| CN101672845A (en) * | 2009-09-23 | 2010-03-17 | 东南大学 | Immunoassay method for antibodies labeled with zinc-oxide quantum dots by stripping voltammetry |
Non-Patent Citations (4)
| Title |
|---|
| 基于凝集素识别和ZnO标记的电化学CEA检测;杨池等;《第五届上海国际分析化学研讨会论文摘要》;20101231;第159页 * |
| 基于纳米结构氧化锌的生物传感研究;徐春祥等;《东南大学学报(医学版)》;20110228;第30卷(第1期);第166-167页第4-5节第1-3段，图5 * |
| 徐春祥等.基于纳米结构氧化锌的生物传感研究.《东南大学学报(医学版)》.2011,第30卷(第1期), |
| 杨池等.基于凝集素识别和ZnO标记的电化学CEA检测.《第五届上海国际分析化学研讨会论文摘要》.2010, |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102520163A (en) | 2012-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102520162B (en) | Method for detecting glycoprotein | |
| Kindermann et al. | Urinary corticosterone metabolites and chytridiomycosis disease prevalence in a free-living population of male Stony Creek frogs (Litoria wilcoxii) | |
| CN103940872B (en) | Detect preparation method and the application of two kinds of acute leukemia mark electrochemical sensors simultaneously | |
| CN103033611A (en) | Chemiluminiscence diagnostic kit for sensitization allergens and preparation method thereof | |
| CN102735835B (en) | Disposable whole-blood uric acid detection test strip and manufacturing method thereof | |
| CN108627559B (en) | Immunoassay method based on the Prussian blue self energizing electrochemical sensor building of aluminium- | |
| CN103675279A (en) | Preparation method and application of prostate tumor marker immunosensor | |
| CN104634847A (en) | Electrochemical immunosensing method for detecting Listeria monocytogenes | |
| CN110441295A (en) | One kind is based on ferritin encapsulation Ir (ppy)3Biosensor preparation method | |
| CN102236021A (en) | Human immunodeficiency virus (HIV) antibody time resolved fluoroimmunoassay method and kit | |
| CN103439502A (en) | Rapid specific antibody IgE detection kit and preparation method thereof | |
| CN102520163B (en) | Determination method for carbohydrate allergen | |
| CN101858918B (en) | Method for detecting ractopamine in animal-derived food by microgap array electrode-based electrochemical immunosensor | |
| CN101387641B (en) | Liver fluke rapid immune detection kit for fresh water fishes and detection method | |
| CN104880497B (en) | A kind of electrochemical determination method for the chemically modified electrode and cefalexin for detecting cefalexin | |
| CN101458222B (en) | Preparation of peanut or shrimp allergen rapid detecting sensor and applications | |
| CN101532980A (en) | Enzyme immunosensor for detecting Shigella species and its preparation method and application | |
| CN103163193A (en) | Preparation method of electrochemical sensor for detecting three clenbuterol hydrochloride and application | |
| CN102830103B (en) | Nanometer silver probe modified by fluorescent molecule, kit thereof, and application thereof in detecting human IgE | |
| CN101672845A (en) | Immunoassay method for antibodies labeled with zinc-oxide quantum dots by stripping voltammetry | |
| CN101149384B (en) | Device for detecting avian influenza virus antigen and detection method | |
| CN102507678B (en) | Stripping voltammetry measuring method using quantum dots to mark biological drug and biological marker | |
| CN204359795U (en) | Mite specific IgG4 subclass antibodies detection kit | |
| CN103197059A (en) | Schistosomiasis electrochemistry sensing quick determination kit, detection method and preparation method of kit | |
| CN105628754A (en) | Method for preparing biosensor established based on mesoporous platinum titanium load neutral red and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |