CN102520162B - Method for detecting glycoprotein - Google Patents
Method for detecting glycoprotein Download PDFInfo
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- CN102520162B CN102520162B CN201110364700.5A CN201110364700A CN102520162B CN 102520162 B CN102520162 B CN 102520162B CN 201110364700 A CN201110364700 A CN 201110364700A CN 102520162 B CN102520162 B CN 102520162B
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- glycoprotein
- chip
- zinc oxide
- oxide quantum
- quantum dot
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Abstract
The invention discloses a method for detecting glycoprotein. The method comprises the following steps of: marking the glycoprotein by using zinc oxide quantum dots; meanwhile, immobilizing an agglutinin layer on a chip in an electrolytic cell; specifically binding the glycoprotein marked by the zinc oxide quantum dots with agglutinin on the chip in a series of glycoprotein solutions to be determined of known concentrations; connecting the zinc oxide quantum dots marked on the glycoprotein to the chip; and determining the agglutinin by using dissolving voltammetry of marking the glycoprotein with the zinc oxide quantum dots. Since the marked glycoprotein has different binding capacities to the glycoprotein to be determined and the agglutinin, the glycoprotein marked on the chip is substituted by the glycoprotein to be determined of different concentrations, and the concentration of zinc ions which are remained on the chip and dissolved out is determined by using the dissolving voltammetry to establish relation between the concentration of the zinc ions and the concentration of the glycoprotein so as to determine the concentration of the glycoprotein to be determined.
Description
Technical field
The invention belongs to the medical biotechnology field, specifically, the invention describes a kind of preparation and detection method of glycoprotein detection chip.
Background technology
Glycoprotein in biomolecule plays an important role in a lot of vital movements and metabolism adjusting. the common generation, the more particularly glycoprotein relevant to cancer with indicating some diseases of the glycosylated variation of some protein surfaces.The importance of glycoprotein in life science has also caused the glycoprotein group fast-developing.This subject main task is exactly to analyze the associated sugars family of some glycoprotein.Traditional analytical approach comprises mass spectrum and chromatogram.Although these methods are very ripe, also have some problems, such as equipment itself is expensive, analyze consuming timely, need the professional to operate.Thereby also hindered universal in glycoprotein detects of these technology.
Summary of the invention
technical matters:the present invention is directed to above-mentioned technical matters, a kind of detection method of glycoprotein be provided, to the detection of corresponding glycoprotein have that detectability is low, selectivity good, highly sensitive and characteristics easily.
technical scheme:the method of detection glycoprotein of the present invention is utilized Zinc oxide quantum dot mark glycoprotein; Immobilization one deck agglutinin on the chip of while in electrolytic cell, in the glycoprotein solution to be measured of a series of concentration known, make mark glycoprotein and the agglutinin on chip of Zinc oxide quantum dot carry out specific binding, the Zinc oxide quantum dot be marked on glycoprotein is connected on chip, adopt the stripping voltammetry agglutinin assay method of Zinc oxide quantum dot mark glycoprotein, concrete steps are described as again:
1.) 0.5 ~ 5 milligram of Zinc oxide quantum dot is distributed in the glycoprotein solution of 50 ~ 200 microlitre 100 ~ 200 nanograms/milliliter, at room temperature shakes 10min, centrifugal, the phosphate buffered solution that is 6.8 ~ 7.5 by the pH value is cleaned three times; Add again 500 μ L 1% w/v bovine serum albumins, 15 ~ 37
oshake 10 ~ 30min under C, centrifugal, the phosphate buffered solution that is 6.8 ~ 7.5 by the pH value is cleaned three times; Finally be scattered in the phosphate buffered solution that 50 ~ 150 μ L 0.05 ~ 0.1% v/v triton x-100 pH values are 6.8 ~ 7.5, the used time is not stored in the refrigerator heat-insulation layer;
2.) 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 2 ~ 5% v/v soaks 2 ~ 5 h, wash clean, dries, then puts into 10 ~ 15mL and contain 1 ~ 2% v/v
glutaraldehydethe phosphate buffered solution that the pH value is 6.8 ~ 7.5, in 4
ostanding 8 ~ 12 h of C, subsequently by washed with de-ionized water, dry, stand-by;
3.) chip after modifying is arranged on to an aperture and is less than in the electrolytic cell of the chip length of side, 50 μ L are contained to the phosphate buffered solution that 1 ~ 2mg/mL agglutinin pH value is 6.8 ~ 7.5 and drop 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;
4.) get 50 ~ 100 uL in 1 step and drop on the chip that 3 steps prepare, place 15 ~ 50min, with washed with de-ionized water chip surface 3 times;
5.) detection method, drop in testing sample on the chip in 4 steps, places 15 ~ 50min, uses washed with de-ionized water chip surface 3 times;
6.), to injecting the Zinc oxide quantum dot stayed on dissolve with hydrochloric acid solution the 5th step chip that pH value is 1 ~ 3 in electrolytic cell, pH1 for well ~ 3 hydrochloric acid solutions are washed three times, merging lysate and washing lotion, the phosphate buffered solution to 1 that to add the pH value be 6.8 ~ 7.5 ~ 3 milliliters;
7. 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;
8.) change the 5th step glycoprotein concentration, repeat the 7th step, measure the peak point current at the Stripping Voltammetry peak of the zinc that a series of glycoprotein relative concentrations answer, set up the corresponding relation between peak point current and glycoprotein concentration.
beneficial effect:compared with prior art, the present invention has the following advantages:
1. labeling process and the process in leaching of Zinc oxide quantum dot of the present invention to glycoprotein, simple to operate, mild condition, and the combination of quantum dot and glycoprotein is firmly stable.Be very beneficial for keeping the biologically active of glycoprotein.
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 glycoprotein concentration accurately.Be conducive to reduce the detectability that glycoprotein detects, improve detection sensitivity.Make quick and precisely to detect low concentration glycoprotein and become possibility.
The accompanying drawing explanation
Following accompanying drawing, for the specific embodiment of the invention scheme is described, limits and be not used in the scope of the invention defined by claims.
Figure l is glycoprotein detection chip assembling schematic diagram.
Fig. 2 is the glycoprotein chip detection result of standard model.
Embodiment
In the present invention, utilize quantum dot-labeled glycoprotein.Immobilization one deck agglutinin on chip simultaneously, make mark glycoprotein and the agglutinin on chip of Zinc oxide quantum dot carry out specific binding, the Zinc oxide quantum dot be marked on glycoprotein is connected on chip.Then remove to detect glycoprotein to be measured with chip, because the glycoprotein of mark and glycoprotein to be measured and the difference of leukemia, the glycoprotein to be measured of variable concentrations can replace the glycoprotein of mark on chip, the concentration of the zinc ion stayed on the chip with the Voltammetry stripping, set up the relation between zinc ion concentration and glycoprotein concentration, thereby determine glycoprotein concentration to be measured.Technical solution of the present invention is: adopt lectin chip to detect glycoprotein to be measured to the difference of quantum dot-labeled and unlabelled Glycoprotein binding ability, the concrete grammar process is as accompanying drawing l, and determination step is described as:
(as Fig. 1)
1,0.5 milligram of Zinc oxide quantum dot is distributed in the carcinomebryonic antigen solution of 200 μ L 100 ng/mL, at room temperature shake 10min, centrifugal, the phosphate buffered solution that is 7.0 by the 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 the pH value is cleaned three times.Finally be scattered in 50 μ L 0.1% (v/v) triton x-100 phosphate buffer solutions, the used time is not stored in the refrigerator heat-insulation layer.
2, by glass sheet (1x1x0.2mm) 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 the ethanolic solution of putting into 5% (v/v) 3-amine propyl group Ethoxysilane soaks 5 h, wash clean, dries, then puts into and contain the phosphate buffered solution that 2% (v/v) glutaraldehyde pH value is 7.0, in 4
ostanding 12 h of C, subsequently by washed with de-ionized water, dry, stand-by.
3, the aperture that is arranged on after modifying is less than in the electrolytic cell of the chip length of side, 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, get 50uL in 1 step and drop on the chip that 3 steps prepare, place 15min, with washed with de-ionized water chip surface 3 times.
5, detection method, drop in the 10ng/mL carcinomebryonic antigen on the chip in 4 steps, places 15min, uses washed with de-ionized water chip surface 3 times.
6, 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 5 step chips.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 concentration are the 0.32M sodium hydroxide solution.Then use Electrochemical Detection,
7, 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.
8, change the 5th step glycoprotein concentration, repeat the 7th step, measure the peak point current at the Stripping Voltammetry peak of the zinc that a series of glycoprotein relative concentrations answer, set up the corresponding relation between peak point current and glycoprotein concentration.(Fig. 2).
The Stripping Voltammetry peak point current of the zinc that above process records and the corresponding relation between glycoprotein concentration are the important basic datas of the inventive method, detect for carry out actual clinical by this inventive method the theoretical foundation that provides concentration to judge, to utilizing the inventive method to carry out clinical detection fast and accurately, extremely important meaning is arranged.
Above-mentioned agglutinin and glycoprotein are ConA and carcinomebryonic antigen thereof.
Said chip is titanium dioxide silicon chip or glass sheet.
Claims (1)
1. a method that detects glycoprotein, is characterized in that utilizing Zinc oxide quantum dot mark glycoprotein; Immobilization one deck agglutinin on the chip of while in electrolytic cell, in the glycoprotein solution to be measured of a series of concentration known, make mark glycoprotein and the agglutinin on chip of Zinc oxide quantum dot carry out specific binding, the Zinc oxide quantum dot be marked on glycoprotein is connected on chip, adopt the stripping voltammetry agglutinin assay method of Zinc oxide quantum dot mark glycoprotein, concrete steps are described as again:
1.) 0.5～5 milligram of Zinc oxide quantum dot is distributed in the glycoprotein solution of 50～200 microlitre 100～200 nanograms/milliliter, at room temperature shakes 10min, centrifugal, the phosphate buffered solution that is 6.8～7.5 by the pH value is cleaned three times; Add 500 μ L1%w/v bovine serum albumins again, shake 10～30min under 15～37 ℃, centrifugal, the phosphate buffered solution that is 6.8～7.5 by the pH value is cleaned three times; Finally be scattered in the phosphate buffered solution that 50～150 μ L0.05～0.1%v/v triton x-100 pH value is 6.8～7.5, the used time is not stored in the refrigerator heat-insulation layer;
2.) 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 2～5%v/v soaks 2～5h, wash clean, dry, put into again 10～15mL and contain the phosphate buffered solution that 1～2%v/v glutaraldehyde pH value is 6.8～7.5, in 4 ℃ of standing 8～12h, subsequently by washed with de-ionized water, dry, stand-by;
3.) chip after modifying being arranged on to an aperture is less than in the electrolytic cell of the chip length of side, 50 μ L are contained to the phosphate buffered solution that 1～2mg/mL agglutinin pH value is 6.8～7.5 to be dropped 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;
4.) get 50～100 μ L in 1 step and drop on the chip that 3 steps prepare, place 15～50min, with washed with de-ionized water chip surface 3 times;
5.) detection method, drop in testing sample on the chip in 4 steps, places 15～50min, uses washed with de-ionized water chip surface 3 times;
6.) in electrolytic cell, inject the Zinc oxide quantum dot stayed on dissolve with hydrochloric acid solution the 5th step chip that the pH value is 1～3, and wash three times with pH1～3 hydrochloric acid solutions, merge lysate and washing lotion, the phosphate buffered solution to 1 that to add the pH value be 6.8～7.5～3 milliliters;
7. 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;
8.) change the 5th step glycoprotein concentration, repeat the 7th step, measure the peak point current at the Stripping Voltammetry peak of the zinc that a series of glycoprotein relative concentrations answer, set up the corresponding relation between peak point current and glycoprotein concentration.
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| CN201110364700.5A CN102520162B (en) | 2011-11-17 | 2011-11-17 | Method for detecting glycoprotein |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103645325A (en) * | 2013-12-12 | 2014-03-19 | 复旦大学 | Method for multiply detecting carbohydrate chain structure of glycoprotein through antibody-assisted lectin liquid-phase suspension chip |
| CN103674918A (en) * | 2013-12-12 | 2014-03-26 | 复旦大学 | Method for detecting glycoprotein carbohydrate chain structure based on lectin liquid suspension chip |
| CN104614417B (en) * | 2014-02-17 | 2016-10-19 | 安阳师范学院 | A kind of electrochemical method for glycoprotein detection |
| CN110420671A (en) * | 2019-06-26 | 2019-11-08 | 南京科瑞芯生物科技有限公司 | A kind of agglutinin micro-array chip and preparation method thereof |
| CN113834871B (en) * | 2021-09-18 | 2024-05-28 | 北京中医药大学 | Method for rapidly analyzing low-molecular sugar based on paper spray mass spectrum and application thereof |
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| CN101271078A (en) * | 2008-04-03 | 2008-09-24 | 东南大学 | Production method of biological chemistry sensor |
| CN101587071A (en) * | 2009-07-01 | 2009-11-25 | 东南大学 | Fluorescence immunoassay method of using zinc oxide quantum dots to mark antibody |
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| CN102141538A (en) * | 2010-12-06 | 2011-08-03 | 山东大学 | Method for determining concentration of phosphite by cyclic voltammetry |
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| US8262998B2 (en) * | 2005-04-15 | 2012-09-11 | Branislav Vlahovic | Detection methods and detection devices based on the quantum confinement effects |
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| CN101271078A (en) * | 2008-04-03 | 2008-09-24 | 东南大学 | Production method of biological chemistry sensor |
| 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 |
| CN102141538A (en) * | 2010-12-06 | 2011-08-03 | 山东大学 | Method for determining concentration of phosphite by cyclic voltammetry |
Non-Patent Citations (2)
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| 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》;20110228;第30卷(第1期);第164-169页 * |
| 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》.2011,第30卷(第1期), |
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