CN102520163A - Determination method for carbohydrate allergen - Google Patents
Determination method for carbohydrate allergen Download PDFInfo
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- CN102520163A CN102520163A CN2011103648328A CN201110364832A CN102520163A CN 102520163 A CN102520163 A CN 102520163A CN 2011103648328 A CN2011103648328 A CN 2011103648328A CN 201110364832 A CN201110364832 A CN 201110364832A CN 102520163 A CN102520163 A CN 102520163A
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- carbohydrate
- chip
- anaphylactogen
- zinc oxide
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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 the medical biotechnology field, specifically, the invention describes a kind of detection method of carbohydrate anaphylactogen.
Background technology
Food hypersenstivity is that people produce the I allergic reaction type of IgE mediation to food, and clinical manifestation is 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, causes grave danger to health of people.In allergic reaction, be to cause more than 90% by eight group foods such as eggs, peanut, breast classes.At present also very limited to the detection method of anaphylactogen in some food, the immune response that mainly contains based on some anaphylactogens detects.But this method exists cost high, and in addition for some anaphylactogens, corresponding antibody or antigen are difficult to obtain, thereby has 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 anaphylactogen is provided; This method utilizes zinc paste mark carbohydrate anaphylactogen to detect low concentration carbohydrate anaphylactogen, and is low to the detectability of corresponding carbohydrate anaphylactogen, selectivity good, highly sensitive, good stability.
Technical scheme:At the assay method of carbohydrate anaphylactogen of the present invention, utilize Zinc oxide quantum dot mark carbohydrate anaphylactogen; Immobilization one deck agglutinin on the chip of while in electrolytic cell; In the carbohydrate anaphylactogen solution to be measured of a series of concentration known; Make mark carbohydrate anaphylactogen and the agglutinin on the chip of Zinc oxide quantum dot carry out specificity and combine, be connected to the Zinc oxide quantum dot that is marked on the carbohydrate anaphylactogen on the chip; Be dissolved as zinc ion to Zinc oxide quantum dot with acid solution then, measure the concentration of the zinc ion of stripping, set up the relation between zinc ion concentration and the carbohydrate allergen concentration with stripping voltammetry, thus definite 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 anaphylactogen solution of 50～200 microlitres, 1 nanograms/milliliter, at room temperature shakes 10min, centrifugal, using pH value is 6.8 ~ 7.5 PBS cleaning three times.Add 500 μ L, 1 ~ 2% w/v bovine serum albumin again, 15 ~ 35
oC is concussion 10 ~ 30min down, and is centrifugal, and using pH value is 6.8 ~ 7.5 PBS cleaning three times; Finally be scattered in 50 ~ 150 μ L, 0.05 ~ 0.1% v/v triton x-100 phosphate buffer solution, the time spent is not stored in the refrigerator heat-insulation layer;
B. with 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 5 ~ 10min respectively; Take out the back and use the clear water wash clean, dry; The ethanolic solution of putting into the 3-amine propyl group Ethoxysilane of 5%v/v then soaks 2 ~ 5 h, wash clean, dries, and putting into 10 ~ 15mL again, to contain 1 ~ 2%v/v glutaraldehyde pH value be 6.8 ~ 7.5 PBS, in 4
oC leaves standstill 8 ~ 12 h, subsequently with washed with de-ionized water, dry, for use;
C. the chip after will modifying is installed in the electrolytic cell of aperture less than the chip length of side, and it is that 6.8 ~ 7.5 PBS drops on the above-mentioned chip of handling well that 50 μ L are contained 1 ~ 2 mg/mL agglutinin pH value, and in 4
oC leaves standstill 8 ~ 12 h, uses washed with de-ionized water subsequently; Then said chip is soaked 0.5 ~ 1 h with containing 0.5 ~ 1% w/v bovine serum albumin and 0.05 ~ 0.1% v/v Tween-20, use washed with de-ionized water, for use subsequently;
D. the testing sample of handling well is dropped on the chip for preparing, place 15 ~ 50min, with washed with de-ionized water chip surface 3 times;
E. in electrolytic cell, inject pH value and be the Zinc oxide quantum dot on 1 ~ 3 the dissolve with hydrochloric acid solution chip, and it is inferior to use pH value to be that 1 ~ 3 hydrochloric acid solution is given a baby a bath on the third day after its birth, merging lysate and washing lotion add the pH value and are 6.8 ~ 7.5 PBS to 1 ~ 3 milliliter;
F. using platinum electrode is to electrode, and silver/silver chloride electrode is a contrast electrode, and mercury film electrode is a working electrode, and-0.8 ~-1.5 volt of enrichment 90 ~ 150 seconds is surveyed the stripping peak of zinc with square wave voltammetry, and 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 volt-ampere peak of the corresponding zinc of a series of carbohydrate allergen concentration, set up the corresponding relation between peak point current and the carbohydrate allergen concentration.
Said chip is titanium dioxide silicon chip or glass sheet.
The stripping volt-ampere peak point current of the zinc that above process records and the corresponding relation between the carbohydrate allergen concentration are the important basic datas of the inventive method; For carry out the theoretical foundation that actual clinical detects provides concentration to judge with this inventive method, Clinical detection has extremely important meaning to utilizing the inventive method to carry out fast and accurately.
Above-mentioned agglutinin and carbohydrate anaphylactogen are ConA and ovum gallinaceum mucin 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 and the process in leaching of carbohydrate anaphylactogen, and is simple to operate, mild condition, and combining of quantum dot and carbohydrate anaphylactogen is firmly stable.Be very beneficial for keeping the biologically active of carbohydrate anaphylactogen.
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 be prone to survey and peak point current strong, be easy to set up the curved line relation between the peak point current and carbohydrate allergen concentration accurately.Help reducing the detectability that the carbohydrate anaphylactogen detects, improve detection sensitivity.Make and quick and precisely detect low concentration carbohydrate anaphylactogen and become possibility.
Description of drawings
Attached drawings is used to explain specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Figure l is a sensitinogen detecting chip assembling synoptic diagram.
Fig. 2 is the anaphylactogen chip detection result of standard model.
Embodiment
(like Fig. 1)
1,0.5 milligram of Zinc oxide quantum dot is distributed in the ovum gallinaceum mucin solution of 50 microlitres, 1 nanograms/milliliter, at room temperature shake 10min, centrifugal, using pH value is 7.0 PBS cleaning three times.Add 500 μ L 1% (w/v) bovine serum albumins again, shake 10min under the room temperature, centrifugal, using pH value is 7.0 PBS cleaning three times.Finally be scattered in 50 μ L 0.1% (v/v) triton x-100 pH values and be 7.0 PBS, the time spent is not stored in the 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
2(1:1:6 v/v) soaks 10min respectively to O.Take out the back and use the clear water wash clean, dry.The ethanolic solution of putting into 5% (v/v) 3-amine propyl group Ethoxysilane then soaks 5 h, wash clean, dries, and puts into that to contain 2% (v/v) glutaraldehyde pH value be 7.0 PBS again, in 4
oC leaves standstill 12 h, subsequently with washed with de-ionized water, dry, for use.
3, detection chip preparation, with being installed in the electrolytic cell of aperture less than the chip length of side after modifying, it is that 7.0 PBS drops on the above-mentioned chip of handling well that 50 μ L are contained 1 mg/mL ConA pH value, and in 4
oC leaves standstill 12 h, uses washed with de-ionized water subsequently.Then with said chip with containing 1% (w/v) bovine serum albumin and 0.05 % (v/v) Tween-20 soaks 1 h, use washed with de-ionized water, for use subsequently.
4, detection method on the ConA chip for preparing, is placed 15min with the ovum gallinaceum mucin sample drop of handling well, with washed with de-ionized water chip surface 3 times.
5; In electrolytic cell, inject 1800 μ L pH values and be 2 the ultrasonic 10min of hydrochloric acid solution to dissolve the Zinc oxide quantum dot on the chip; Go up subsequently that to transfer to electrolytic cell and add 2000 μ L pH values successively like solution be 7.01 PBS, 4uL 10 ppm mercuric nitrates (II).50 μ L sodium hydroxide solutions (0.32 M).Use Electrochemical Detection then,
6, using platinum electrode is to electrode, and silver/silver chloride electrode is a contrast electrode, and mercury film electrode is a working electrode ,-1.3 V electricity enrichment 2 min, and with the stripping peak of square wave voltammetry survey zinc, current potential is about-1.1 volts.
7, change the 1st step ovum gallinaceum mucin concentration, repeated for the 4th, 5,6 steps, measure the peak point current at the stripping volt-ampere peak of the corresponding zinc of a series of ovum gallinaceum mucin concentration, set up the corresponding relation (Fig. 2) between peak point current and the ovum gallinaceum mucin concentration.
Claims (3)
1. the assay method of a carbohydrate anaphylactogen is characterized in that utilizing Zinc oxide quantum dot mark carbohydrate anaphylactogen; Immobilization one deck agglutinin on the chip of while in electrolytic cell; In the carbohydrate anaphylactogen solution to be measured of a series of concentration known; Make mark carbohydrate anaphylactogen and the agglutinin on the chip of Zinc oxide quantum dot carry out specificity and combine, be connected to the Zinc oxide quantum dot that is marked on the carbohydrate anaphylactogen on the chip; Be dissolved as zinc ion to Zinc oxide quantum dot with acid solution then, measure the concentration of the zinc ion of stripping, set up the relation between zinc ion concentration and the carbohydrate allergen concentration with stripping voltammetry, thus definite carbohydrate allergen concentration to be measured.
2. the assay method of carbohydrate anaphylactogen according to claim 1 is characterized in that concrete assay method is:
A. 0.5～5 milligram of Zinc oxide quantum dot is distributed in the carbohydrate anaphylactogen solution of 50～200 microlitres, 1 nanograms/milliliter, at room temperature shakes 10min, centrifugal, using pH value is 6.8～7.5 PBS cleaning three times.Add 500 μ L1～2%w/v bovine serum albumin again, 15～35 ℃ of following concussion 10～30min, centrifugal, using pH value is 6.8～7.5 PBS cleaning three times; Finally be scattered in 50～150 μ L 0.05～0.1% (v/v) triton x-100 phosphate buffer solutions, the time spent is not stored in the refrigerator heat-insulation layer;
B. with titanium dioxide silicon chip or glass sheet ultrasonic 5min respectively in acetone, second alcohol and water respectively, then at 60～80 ℃ NH
4OH/H
2O
2/ H
2O 1: 1: 5～7, v/v and HCl/H
2O
2/ H
2O 1: 1: 4～6, and v/v soaks 5～10min respectively; Take out the back and use the clear water wash clean, dry; The ethanolic solution of putting into the 3-amine propyl group Ethoxysilane of 5%v/v then soaks 2～5h; Wash clean, dry, putting into 10～15mL again, to contain 1～2%v/v glutaraldehyde pH value be 6.8～7.5 PBS, leaves standstill 8～12h in 4 ℃; Subsequently with washed with de-ionized water, dry, for use;
C. the chip after will modifying is installed in the electrolytic cell of aperture less than the chip length of side; It is that 6.8～7.5 PBS drops on the above-mentioned chip of handling well that 50 μ L are contained 1～2mg/mL agglutinin pH value; And leave standstill 8～12h in 4 ℃, use washed with de-ionized water subsequently; Then said chip is soaked 0.5～1h with containing 0.5～1%w/v bovine serum albumin and 0.05～0.1%v/v Tween-20, use washed with de-ionized water, for use subsequently;
D. the testing sample of handling well is dropped on the chip for preparing, place 15～50min, with washed with de-ionized water chip surface 3 times;
E. in electrolytic cell, inject pH value and be the Zinc oxide quantum dot on 1～3 the dissolve with hydrochloric acid solution chip, and it is inferior to use pH value to be that 1～3 hydrochloric acid solution is given a baby a bath on the third day after its birth, merging lysate and washing lotion add the pH value and are 6.8～7.5 PBS to 1～3 milliliter;
F. using platinum electrode is to electrode, and silver/silver chloride electrode is a contrast electrode, and mercury film electrode is a working electrode, and-0.8～-1.5 volt of enrichment 90～150 seconds is surveyed the stripping peak of zinc with square wave voltammetry, and 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 volt-ampere peak of the corresponding zinc of a series of carbohydrate allergen concentration, set up the corresponding relation between peak point current and the carbohydrate allergen concentration.
3. the stripping voltammetric measuring method of Zinc oxide quantum dot mark carbohydrate anaphylactogen according to claim 2 is characterized in that said chip is titanium dioxide silicon chip or glass sheet.
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Citations (6)
| 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 |
| US20090321261A1 (en) * | 2005-04-15 | 2009-12-31 | Dr. Branislav Vlahovic | Detection methods and detection devices based on the quantum confinement effects |
| 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 |
-
2011
- 2011-11-17 CN CN201110364832.8A patent/CN102520163B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090321261A1 (en) * | 2005-04-15 | 2009-12-31 | Dr. Branislav Vlahovic | Detection methods and detection devices based on the quantum confinement effects |
| 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 (2)
| Title |
|---|
| 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)》, vol. 30, no. 1, 28 February 2011 (2011-02-28) * |
| 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》, 31 December 2010 (2010-12-31) * |
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