CN104090116A - Preparation method for bovine gamma interferon impedance type immunosensor based on zinc oxide nano-materials - Google Patents
Preparation method for bovine gamma interferon impedance type immunosensor based on zinc oxide nano-materials Download PDFInfo
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- CN104090116A CN104090116A CN201410348998.4A CN201410348998A CN104090116A CN 104090116 A CN104090116 A CN 104090116A CN 201410348998 A CN201410348998 A CN 201410348998A CN 104090116 A CN104090116 A CN 104090116A
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- electrode
- gamma interferon
- zinc oxide
- immunosensor
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6866—Interferon
-
- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
Abstract
The invention discloses a preparation method for a bovine gamma interferon impedance type immunosensor based on zinc oxide nano-materials, and belongs to the technical field of electrochemical immunoassay. A glassy carbon electrode is modified by the zinc oxide nano-materials with different morphologies and excellent performances, and a bovine gamma interferon antibody is fixed on the electrode, so that a novel electrochemical impedance type immunosensor is prepared. The novel electrochemical impedance type immunosensor can be applied to unmarked electrochemical immunoassay of bovine gamma interferon. The immunosensor does not need to be marked, is simple, quick, low in cost, high in sensitivity, good in reproducibility and good in stability, and can be used for early diagnosing of bovine tuberculosis and research of a bovine cellular immunity mechanism.
Description
Technical field
The invention belongs to the technology of preparing of electrochemical immunoanalytical technical field, particularly sensor.
Background technology
Tuberculosis is that milk cow health and galactopoiesis quality are affected to one of the most serious communicate illness.Ox gamma interferon (BoIFN-γ) is under specific inducer spread effect, by a class of T emiocytosis, has the bioactive glycoprotein such as antiviral, antitumor and immunoloregulation function, is to find the earliest, pay close attention to maximum cell factors.
At present, biological analysis method is commonly used in the detection of ox gamma interferon, and these biological analysis method sensitivity are low, effort, consuming time and be difficult to standardization.In addition, they are responsive to the buffering agent and other cell factor that detect in sample, are easily interfered.Though Enzyme Linked Immunoadsorbent Assay is also established to detect ox gamma interferon, but the same complex operation of these methods, effort, consuming time, and sensitivity can not be satisfactory.
Due to ox gamma interferon molecular weight, and the amount in body body fluid is few, and the method distance of sensitivity clinical practice application of measuring at present ox gamma interferon also has larger gap.Therefore, current urgent need is set up a kind of hypersensitivity, fast, method detects ox gamma interferon reliably, for the early diagnosis of perlsucht provides a platform.
The features such as in recent years, electrochemical impedance immunoassay is because it is without mark, highly sensitive, and specificity is good, and sample consumption is little, simple and quick, become a kind of extremely competitive in-situ check and test method.
Nano zinc oxide material is the nano material with unique chemistry, biology, physical and mechanical property, for electrochemical immunoanalytical research provides recent studies on approach.
Summary of the invention
First object of the present invention is the defect that proposes to overcome above-mentioned ox gamma interferon detection method, builds an ox gamma interferon electrochemical impedance immunosensor based on different shape zinc oxide nano material.
Technical scheme of the present invention is: by after nano zinc oxide material modified glassy carbon electrode, then on electrode fixing ox gamma interferon antibody, obtain electrochemical impedance immunosensor.
The present invention adopts the nano zinc oxide material of different-shape, function admirable, such as bar-shaped zinc paste, grain-like zinc-oxide and tubular ZnO nano material etc., it is large that nano zinc oxide material has specific surface area, the advantages such as good biocompatibility, utilize its fixedly ox gamma interferon antibody, are conducive to keep the biologically active of antibody, widen the range of linearity of detection, improve immunity and answer specificity and reaction efficiency, reduce non-specific adsorption, improve detection sensitivity.The present invention utilizes its fixedly ox gamma interferon antibody, realizes the quantitative detection to ox gamma interferon.
Concrete preparation method comprises the following steps:
1) nano zinc oxide material is scattered in deionized water, forms the intermixture that contains zinc paste;
2) intermixture that contains zinc paste is coated on equably to clean glass-carbon electrode surface, after drying, again in glass-carbon electrode surface uniform apply ox gamma interferon antibody, then be placed in dry under the temperature environment of 4 ℃ after, again in glass-carbon electrode surface uniform modify one deck naphthols film, finally, adopt bovine serum albumin sealing avtive spot.
By above technique, material and antibody firmly can be fixed on electrode, make the electrochemical impedance immunosensor for detection of ox gamma interferon.The immunosensor making is placed in to detection system, adds ox gamma interferon antigen, after incubation, flushing, detect its electrochemical impedance signal.The method that the sensor of preparing with this method is tested without mark, simple, fast, low, highly sensitive, the reappearance of cost and good stability, can be for the early diagnosis of perlsucht and the research of ox cellular immunity mechanism.
In addition, in order to improve the accuracy of detection, before the intermixture that the present invention also contains zinc paste in coating, by glass-carbon electrode, it with particle diameter, is first the alumina powder polishing of 0.05 mm, with deionized water rinsing, fall after residual alumina powder again, put into diluted nitric acid aqueous solution ultrasonic cleaning, finally with ethanol and redistilled water, clean glass-carbon electrode successively, with this, obtain the clean glass-carbon electrode in surface.
In addition, the present invention also proposes another goal of the invention: the application of the electrochemical impedance immunosensor that above inventive method makes in ox gamma interferon detects.
Its technical scheme is: to contain 5.0 mM [Fe (CN)
6]
3-/4-with 0.1 M of 0.1 M KCl, the PBS solution that pH value is 7.0 be in test fluid, the electrochemical impedance immunosensor making of take is working electrode, saturated calomel electrode is as auxiliary electrode, platinum plate electrode is as to electrode, add ox gamma interferon antigen, after 37.5 ℃ of incubation 40 min, take out working electrode and rinse, then the electrochemical impedance signal of testing electrode.
Because the present invention is optimized the pH of buffer solution, heated culture temperature and incubative time, and obtain best ox gamma interferon testing conditions, improved analytical effect.The method without mark, simple, fast, low, highly sensitive, the reappearance of cost and good stability, can be for the early diagnosis of perlsucht and the research of ox cellular immunity mechanism.
Therefore, the present invention utilizes fixedly ox gamma interferon antibody of nano zinc oxide material, build a unmarked electrochemical impedance immunosensor and realize the quantitative detection to ox gamma interferon, significant to the research of the early diagnosis of perlsucht and ox cellular immunity mechanism.
Accompanying drawing explanation
Fig. 1 is ox gamma interferon immunosensor preparation principle figure and unmarked impedance immunoassay schematic diagram.
Fig. 2 is the Linear Fit Chart of the BoIFN-γ immunosensor prepared of the inventive method.
Embodiment
Embodiment 1:
(1) prepare grain-like zinc-oxide nano material.
Taking 0.5 g sub-micron Zinc oxide powder (ZnO) is in the stainless steel cauldron of teflon lined of 50 mL in volume, adds 40 mL 20 % H
2o
2aqueous solution, stirs 5 min.Then be placed in baking oven and at 120 ℃, react 6 h, question response system naturally cools to room temperature, suction filtration, and with deionized water washing for several times, in last baking oven, 80 ℃ of dry 10 h, obtain ZnO
2white powder.
Take the above-mentioned 120 ℃ of made ZnO of hydro-thermal reaction 6 h of 0.5 g
2, be placed in the corundum crucible of 25 ml.Then put it in muffle furnace and at 300 ℃, heat 6 h, question response system is taken out after naturally cooling to room temperature, obtains ZnO white powder.
(2), under ultrasonic, grain-like zinc-oxide nano material is dispersed in deionized water to the zinc paste intermixture that to form zinc oxide content be 1mg/mL.
(3) the alumina powder polishing with 0.05 mm by glass-carbon electrode, with deionized water, rinse out residual alumina powder, put into dilute nitric acid solution ultrasonic cleaning, finally with ethanol and redistilled water, clean glass-carbon electrode surface successively, make pretreated glass-carbon electrode.
(4) intermixture that contains grain-like zinc-oxide is evenly dripped and be applied to pretreated glass-carbon electrode surface, after being placed on and drying in 4 ℃ of refrigerators, get again the anti-BoIFN-γ of 5.0 μ L 200 μ g/mL, it is coated on electrode, as for dry 12 h in 4 ℃ of refrigerators, at electrode surface, drip the naphthols that is coated with 5.0 μ L 1% afterwards.After it is dry, electrode is put into and is contained i.e. 10 mg/mL of 1 wt%() PBS of bovine serum albumin, in 37.5 ℃ of water-baths, incubation 30 min are with the avtive spot on enclosed-electrode, with deionized water, wash away bovine serum albumin free on electrode afterwards, make BoIFN-γ immunosensor, be placed in 4 ℃ of refrigerators, store stand-by.
(5) the BoIFN-γ immunosensor making is placed in to detection system, adds ox gamma interferon antigen incubation BoIFN-γ antigenic solution 40 min under 37.5 ℃ of conditions, with detecting its electrochemical impedance signal after deionized water rinsing.
(6) Fig. 2 is the Linear Fit Chart of the BoIFN-γ immunosensor of embodiment 1 preparation, in figure: horizontal ordinate is the value after ox gamma interferon antigen concentration is taken the logarithm, ordinate is the relative changing value of the impedance that variable concentrations antigen is corresponding, and relative changing value's computing formula is:
, Y=74.31+16.34X is linear work equation, is linearly dependent coefficient.
Visible, and the present invention adopts, be Electrode with Electrochemical Impedance Spectroscopy, [the Fe (CN) in test fluid
6]
3-/4-be exactly active probe molecule, thus without mark because without mark, thus with regard to fairly simple, quick, cost is low, highly sensitive, reappearance and good stability.
Embodiment 2:
(1) prepare bar-shaped nano zinc oxide material.
Get the zinc nitrate (Zn (NO of 3.0 mmol
3)
26H
2o) be placed in the stainless steel cauldron that volume is the teflon lined of 100 mL, add 80mL ethanol solution, make the compactedness of poly-tetrem alkene reaction still reach 80%, at room temperature stir 10min, the NaOH (NaOH) that takes 30.0 mmol adds in above-mentioned solution, stirs 1h to dissolving completely under room temperature.Then by it as for reacting 12h in electric heating constant-temperature blowing drying box under 120 ° of C, question response system is taken out after naturally cooling to room temperature, suction filtration, with deionized water and absolute ethyl alcohol, clean for several times, suction filtration is complete is placed in 80 ° of C baking oven inner drying 12h by sample, can obtain the bar-shaped nano zinc oxide material of white powder after oven dry.
(2) bar-shaped nano zinc oxide material is dispersed in deionized water under ultrasonic to the zinc paste intermixture that formation zinc oxide content is 1mg/mL.
(3) the alumina powder polishing with 0.05 mm by glass-carbon electrode, rinses out residual alumina powder with deionized water, puts into dilute nitric acid solution ultrasonic cleaning, finally uses successively ethanol and redistilled water cleaning electrode surface, makes pretreated electrode.
(4) intermixture that contains bar-shaped zinc paste is evenly dripped and be applied to the glass-carbon electrode surface that pre-service is good, after being placed on and drying in 4 ℃ of refrigerators, get again the anti-BoIFN-γ of 5.0 μ L 200 μ g/mL, it is coated on electrode, as for dry 12 h in 4 ℃ of refrigerators, at electrode surface, drip the naphthols that is coated with 5.0 μ L 1% afterwards.After it is dry, electrode is put into and is contained i.e. 10 mg/mL of 1 wt%() PBS of bovine serum albumin, in 37.0 ℃ of water-baths, incubation 30 min are with the avtive spot on enclosed-electrode, with deionized water, wash away bovine serum albumin free on electrode afterwards, make BoIFN-γ immunosensor, be placed in 4 ℃ of refrigerators, store stand-by.
(5) by BoIFN-γ antigenic solution 40 min of the immunosensor preparing incubation variable concentrations at 37.5 ℃, with detecting its electrochemical impedance signal after deionized water rinsing.
(6) the conventional biological analysis method of the at present detection of ox gamma interferon, these biological analysis method sensitivity are low, effort, consuming time and be difficult to standardization.In addition, they are responsive to the buffering agent and other cell factor that detect in sample, are easily interfered.Though Enzyme Linked Immunoadsorbent Assay is also established to detect ox gamma interferon, but the same complex operation of these methods, effort, consuming time, and sensitivity can not be satisfactory.And this patent adopts, be Electrode with Electrochemical Impedance Spectroscopy, [the Fe (CN) in test fluid
6]
3-/4-be exactly active probe molecule, thus without mark because without mark, thus with regard to fairly simple, quick, cost is low, highly sensitive, reappearance and good stability.
Claims (4)
1. the preparation method of the ox gamma interferon impedance type immunosensor based on nano zinc oxide material, it is characterized in that after nano zinc oxide material modified glassy carbon electrode, fixing ox gamma interferon antibody on electrode, obtains electrochemical impedance immunosensor again.
2. according to the preparation method described in claim 1, it is characterized in that comprising the following steps:
1) nano zinc oxide material is scattered in deionized water, forms the intermixture that contains zinc paste;
2) intermixture that contains zinc paste is coated on equably to clean glass-carbon electrode surface, after drying, again in glass-carbon electrode surface uniform apply ox gamma interferon antibody, then be placed in dry under the temperature environment of 4 ℃ after, again in glass-carbon electrode surface uniform modify one deck naphthols film, finally, adopt bovine serum albumin sealing avtive spot.
3. preparation method according to claim 2, before the intermixture that it is characterized in that containing zinc paste in coating, by glass-carbon electrode, it with particle diameter, is first the alumina powder polishing of 0.05 mm, with deionized water rinsing, fall after residual alumina powder again, put into diluted nitric acid aqueous solution ultrasonic cleaning, finally with ethanol and redistilled water, clean glass-carbon electrode successively.
4. the electrochemical impedance immunosensor that method makes the as claimed in claim 1 application in ox gamma interferon detects, in test fluid, the electrochemical impedance immunosensor making of take is working electrode, saturated calomel electrode is as auxiliary electrode, platinum plate electrode, as to electrode, adds ox gamma interferon antigen, after incubation, taking-up working electrode rinses, then the electrochemical impedance signal of testing electrode; It is characterized in that testing conditions is:
1) described test fluid is the [Fe (CN) that contains 5.0 mM
6]
3-/4-pBS solution with 0.1 M of 0.1 M KCl;
2) pH of described PBS solution is 7.0;
3) temperature for the mixed system of incubation is 37.5 ℃; The time of incubation is 40 min.
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Cited By (3)
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|---|---|---|---|---|
| CN105784819A (en) * | 2016-03-11 | 2016-07-20 | 青岛大学 | 3D G-N/MoS2-based interferon-gamma aptamer electrode, as well as preparation method and application thereof |
| CN105866105A (en) * | 2016-04-06 | 2016-08-17 | 扬州大学 | Preparation and analysis method for chemiluminiscence imaging immunosensor for detecting multiple chicken cytokines |
| US10883984B1 (en) | 2019-02-20 | 2021-01-05 | Qingdao University | Method for preparing nanocomposite and label-free aptamer electrochemical sensor of gamma-interferon based on the nanocomposite |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105784819A (en) * | 2016-03-11 | 2016-07-20 | 青岛大学 | 3D G-N/MoS2-based interferon-gamma aptamer electrode, as well as preparation method and application thereof |
| CN105784819B (en) * | 2016-03-11 | 2018-02-13 | 青岛大学 | A kind of fit electrode of interferon based on three-dimensional nitrogen-doped graphene/molybdenum disulfide and preparation method and application |
| CN105866105A (en) * | 2016-04-06 | 2016-08-17 | 扬州大学 | Preparation and analysis method for chemiluminiscence imaging immunosensor for detecting multiple chicken cytokines |
| US10883984B1 (en) | 2019-02-20 | 2021-01-05 | Qingdao University | Method for preparing nanocomposite and label-free aptamer electrochemical sensor of gamma-interferon based on the nanocomposite |
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| CN104090116B (en) | 2017-01-25 |
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