CN105891483B - A kind of preparation method of the unmarked electrochemical immunosensor based on graphene parcel polystyrene composite Nano ball - Google Patents
A kind of preparation method of the unmarked electrochemical immunosensor based on graphene parcel polystyrene composite Nano ball Download PDFInfo
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- CN105891483B CN105891483B CN201610211760.6A CN201610211760A CN105891483B CN 105891483 B CN105891483 B CN 105891483B CN 201610211760 A CN201610211760 A CN 201610211760A CN 105891483 B CN105891483 B CN 105891483B
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- 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
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
Abstract
The invention provides a kind of preparation method of the unmarked electrochemical immunosensor based on graphene parcel polystyrene composite Nano ball, it is related to electrochemical immunoanalytical field.Synthesizing graphite alkene parcel polystyrene composite Nano ball first, by its biological functional and modified using Streptavidin in glassy carbon electrode surface, special affinity interaction by streptavidin to biotin, biotinylated antibody is fixed on functionalization interface, is closed to obtain unmarked electrochemical immunosensor with bovine serum albumin.Graphene parcel polystyrene composite Nano ball electrochemical interface has big specific surface area, good biocompatibility, shows excellent chemical property.With the unmarked electrochemical immunosensor obtained by the composite Nano ball, in potassium ferricyanide solution system, the high sensitivity markless detection to tumor markers can be quickly and easily realized.
Description
Technical field
The invention belongs to field of immunology and electrochemical analysis techniques field, is specifically that a kind of graphene that is based on wraps up polyphenyl
The preparation method and the detection technique in tumor markers of the unmarked electrochemical immunosensor of ethene composite Nano ball.
Background technology
Nineteen ninety, Henry etc. propose the concept of immunosensor, and it is to exempt from highly sensitive sensing technology with specificity
Epidemic disease determination techniques combine, for detecting the Novel Biosensor of antigen-antibody reaction.Tumor markers refers to by tumour
Cell produces, and the material related to tumor tissues property, these materials are present in the karyon of tumor tissues and tumour cell, kytoplasm
With on after birth or secretion in body fluid.The presence of tumor markers or changes of contents can prompt presence and its stage of tumour.
Electrochemical analysis techniques and immunoassay techniques are combined by electrochemical immunosensor, and because making, simple, loss is small and flexible
The advantages that portable and to receive much concern be more ripe a kind of immunosensor of current most study, be widely used in tumour mark
The detection of will thing.
Unmarked immunoassay is when being specifically bound using antibody with determined antigen, to cause surface physicses or chemistry
Performance changes, and according to influence relational implementation immune detection of the testing concentration to the change, divides relative to marking type is immune
Analysis, greatly simplifie preparation and operating process, have it is easy to operate, cost is cheap, the advantages such as detection time is short.
Nano material, especially new nano material and nano composite material, due to its special layer of structure, have
Stronger adsorption capacity, good capacity of orientation and bio-compatibility, and be widely used in biology sensor.Graphene is one
Kind only have a carbon atom thickness two-dimensional material, due to the special construction of graphene, graphene show excellent electric conductivity,
High-specific surface area, the fast transportation of electronics, good mechanical property, stable calorifics and special chemical property.Will
Graphene with polystyrene spheres are compound obtains globular graphite alkene composite, the premium properties based on the composite nano materials can
To apply well in field of biosensors.There is very strong specific affinity interaction, and one between biotin and avidin
Individual streptavidin can combine four biotins, and nano structural material is carried out into biological functional using Streptavidin, will
It is applied in biology sensor, can improve sensitivity, and reaches the effect of signal amplification.
The content of the invention
It is an object of the invention to provide a kind of unmarked electrochemistry based on graphene parcel polystyrene composite Nano ball
The preparation method of immunosensor.Its basic ideas is：First synthesizing graphite alkene wraps up polystyrene composite Nano ball, utilizes strepto-
Graphene is wrapped up polystyrene composite Nano ball biological functional and modified in the glassy carbon electrode surface of cleaning by Avidin, is passed through
Biotinylated antibody is fixed on functionalization interface to the special affinity interaction of biotin, uses cow's serum by streptavidin
Protein blocking obtains the unmarked electrochemical immunosensor.Obtained sensor is incubated into antigen, antibody and antigentic specificity
The immune complex that reaction is formed hinders electron transmission, so as to cause response current to reduce, reduction and antigen using electric signal
The direct linear relationship of concentration, it is possible to achieve to the high sensitivity markless detection of tumor markers.
Synthesizing graphite alkene wraps up polystyrene composite Nano ball:
The styrene monomer dissolved with initiator azodiisobutyronitrile is added to ethanol water, is stirred at room temperature, N2After bubbling
System temperature is increased to 70 DEG C, constant temperature constant speed pre-reaction 2h, methacryloxypropyl ethylene chlorination ammonium is added, in N2Under atmosphere
Continue to react 22h, polystyrene spheres suspension liquid will be obtained after product centrifuge washing.By graphene oxide suspension and polyphenyl second
Alkene ball suspension liquid mixing ultrasound, adds deionized water, is stirred at room temperature, and system is warming up into 95 DEG C, adds hydrazine hydrate, is returned
Stream reaction, graphene parcel polystyrene composite Nano ball suspension, graphene parcel polyphenyl will be obtained after product centrifuge washing
Ethene composite Nano spherolite footpath is 150-200nm.
Prepare unmarked electrochemical immunosensor and specifically include following steps：
(1) graphene is wrapped up into polystyrene composite Nano ball ultrasonic disperse in secondary deionized water, takes graphene bag
Wrap up in polystyrene composite Nano ball suspension mixed in equal volume with chitosan solution and ultrasonic disperse it is uniform then above-mentioned mixing is molten
Liquid mixes in equal volume with solution of streptavidin；
(2) glass-carbon electrode is first polished with alumina powder, after the alumina powder that residual is rinsed out with secondary deionized water, so
It is cleaned by ultrasonic glass-carbon electrode with ethanol and redistilled water successively afterwards, finally dries up under a nitrogen；
(3) graphene of streptomysin biological functional is wrapped up into the mixed solution drop coating of polystyrene composite Nano ball in upper
The glassy carbon electrode surface of cleaning is stated, is placed in refrigerating and is dried under low temperature environment；
(4) by biotinylated antibody drop coating on the decorative material surface of above-mentioned streptomysin biological functional, at room temperature instead
Ying Hou, rinsed with phosphate buffer solution.
(5) confining liquid bovine serum albumin drop coating after closing at room temperature, is used into phosphate-buffered in above-mentioned modification layer surface
Solution rinses.
Further, in the step of preparation method of the unmarked electrochemical immunosensor (1), the polyphenyl second
Alkene/graphene composite particle dosage is 2.0~4.0mg/mL, and the concentration of chitosan solution is 1.0~2.0wt%, and strepto- is affine
The concentration of element is 400~600 μ g/mL, and in step (2), described alumina powder particle diameter is 0.05mm.
Further, in the step of preparation method of the unmarked electrochemical immunosensor (3), the refrigeration low temperature
For 4 DEG C；In step (4), the biotinylated antibody concentration is 1~10 μ g/mL.
Further, in the step of preparation method of the unmarked electrochemical immunosensor (5), the cow's serum egg
White solution concentration is 1.0~2.0wt%.
The invention also discloses a kind of unmarked electro-chemistry immunity based on graphene parcel polystyrene composite Nano ball
Analysis method, including following analytical procedure：
A. the unmarked electrification of polystyrene composite Nano ball will be wrapped up based on graphene as claimed in claim 1 or 2
Learn immunosensor to educate in the incubation liquid temperature of antigen, then rinsed with phosphate buffer solution；
B. using obtained electrochemica biological sensor as working electrode, saturated calomel electrode is as auxiliary electrode, platinized platinum electricity
Pole is used as to electrode, and in potassium ferricyanide test fluid, electrochemical signals are detected with differential pulse voltametry.
Further, in step a, incubative time is 30~40min.
Further, in step b, potassium ferricyanide test fluid is by 5mM K3[Fe(CN)6]/K4[Fe(CN)6] and 0.1M KCl
Mixed liquor forms, and the pH of solution is 6.5, volume 10mL.
The present invention compared with prior art, has advantages below：
(1) material that the present invention uses wraps up polystyrene composite Nano ball for graphene, and one side graphene has excellent
Different electric conductivity, high-specific surface area, electronics fast transportation the advantages that, it is affine more strepto-s can be fixed on interface
Element, fixed more antibody, can also promote the transmission of electronics, so as to build more efficient sensing interface, on the other hand by stone
Black alkene is wrapped on polystyrene spheres, and polystyrene spheres particle diameter is 150-200nm, compared with micron order polystyrene spheres, this hair
The graphene parcel polystyrene composite Nano ball electrochemical interface of bright synthesis has big specific surface area, good bio-compatible
Property, show excellent chemical property.
(2) in addition, biotin-Streptavidin system with high specific and a molecule Streptavidin due to that can tie
Four molecular biosciences element is closed, can be fixed on the graphene parcel polystyrene composite Nano ball interface of Streptavidin functionalization
Substantial amounts of biotinylated antibody, so as to build more efficient sensing platform, the immune complex formed can significantly suppress
Electron Transfer on interface.
Brief description of the drawings
Fig. 1 is making and the immunoassay schematic diagram of electrochemical immunosensor of the present invention.
Fig. 2 is the linearity curve of AFP standard samples of the present invention detection.
Specific implementation method
In order to preferably illustrate the technical process of the present invention and purpose, below in conjunction with the accompanying drawings and embodiment is to this hair
It is bright to describe in detail.
Embodiment 1
The invention provides a kind of unmarked electro-chemistry immunities of AFP based on graphene parcel polystyrene composite Nano ball
The preparation method of sensor, the preparation method of the unmarked immunosensors of the AFP comprise the following steps：
(1) graphene parcel polystyrene composite Nano ball is prepared
The ethanol water that 136g volume fractions are 30% is taken to be subsequently added into 250mL three-necked flask dissolved with 0.5g
The styrene monomer 27mL of initiator azodiisobutyronitrile, is stirred at room temperature (400r/min), N2By system temperature after bubbling 30min
70 DEG C are increased to, constant temperature constant speed pre-reaction 2h.0.75g methacryloxypropyl ethylene chlorination ammoniums are added, in N2Continue under atmosphere
22h is reacted, polystyrene spheres suspension liquid will be obtained after product centrifuge washing.10mL 0.1wt% graphene oxide is suspended
The dispersion liquid of liquid and 5g 10wt% polystyrene particle mixes, ultrasonic 30min, then adds 120mL deionizations
Water, 6h is stirred at room temperature, system is warming up to 95 DEG C, adds 200 μ L 80wt% hydrazine hydrate, back flow reaction 4h, by product
Graphene parcel polystyrene composite Nano ball suspension is obtained after centrifuge washing.
(2) graphene that concentration is 2.0mg/mL is wrapped up into polystyrene composite Nano ball suspension with 1.0wt% shells to gather
Ultrasonic disperse after sugar juice mixes in equal volume.
(3) the above-mentioned mixed liquors of 30 μ L and the μ g/mL solution of streptavidin of 30 μ L 600 is taken to mix in equal volume.
(4) the uniform drop coating of mixed solution of the above-mentioned Streptavidin functionalization of 5 μ L is taken in the glassy carbon electrode surface of cleaning, so
After be placed at 4 DEG C dry.
(5) the biotinylated uniform drop coatings of AFP antibody of the μ g/mL of 10 μ L 1 are taken in the surface of above-mentioned decorative material, at room temperature
1h is incubated, is rinsed with phosphate buffer solution.
(6) the 10 μ uniform drop coatings of L 1.0wt% bovine serum albumins are taken to close 1h at room temperature in above-mentioned modification layer surface, use
Phosphate buffer solution is rinsed, and the unmarked electrochemical immunosensors of the AFP are made.
Embodiment 2
To the unmarked chemiluminescence immunoassays of AFP based on graphene parcel polystyrene composite Nano ball in embodiment 1
Sensor, which carries out immunoassay method, to be included：
(1) the unmarked immunosensors of AFP obtained above are incubated in the incubation liquid containing different AFP antigens, incubated
Time is 30~40min, is then rinsed with phosphate buffer solution；
(2) using the unmarked immunosensors of obtained AFP as working electrode, saturated calomel electrode is as auxiliary electrode, platinum
Plate electrode is used as to electrode, is containing 5mM K3[Fe(CN)6]/K4[Fe(CN)6] and 0.1M KCl test fluids in, with differential arteries and veins
Rush voltammetry and detect its electrochemical signals.
Such as the linearity curve that Fig. 2 is AFP standard samples.To investigate the reliability of the practical application of the electrochemical sensor,
The detection of human serum actual sample is carried out, and (sample 1-5 reference value is by Jiangsu Prov. Tumour Hospital in table 1 with standard method
There is provided, measured by the Electrogenerated chemiluminescent immunoassay instrument of commercialization) compare, both testing results have well consistent
Property, relative error is less than 10%.As shown in table 1：
The measure of table 1.AFP actual samples
Embodiment 3
The invention provides a kind of unmarked electro-chemistry immunities of CEA based on graphene parcel polystyrene composite Nano ball
Transducer production method, the preparation method of the unmarked immunosensors of the CEA comprise the following steps：
(1) graphene that concentration is 2.0mg/mL is wrapped up into polystyrene composite Nano ball suspension with 1.0wt% shells to gather
Ultrasonic disperse after sugar juice mixes in equal volume.
(2) the above-mentioned mixed liquors of 30 μ L and the μ g/mL solution of streptavidin of 30 μ L 600 is taken to mix in equal volume.
(3) the uniform drop coating of mixed solution of the above-mentioned Streptavidin functionalization of 5 μ L is taken in the glassy carbon electrode surface of cleaning, so
After be placed at 4 DEG C dry.
(4) the biotinylated uniform drop coatings of CEA antibody of the μ g/mL of 10 μ L 1 are taken in the surface of above-mentioned decorative material, at room temperature
1h is incubated, is rinsed with phosphate buffer solution.
(5) the 10 μ uniform drop coatings of L 1.0wt% bovine serum albumins are taken to close 1h at room temperature in above-mentioned modification layer surface, use
Phosphate buffer solution is rinsed, and the unmarked immunosensors of the CEA are made.
The unmarked immunosensors of the CEA being made are incubated in the incubation liquid containing different CEA antigens, this is unmarked to exempt from
Epidemic disease sensor carries out the method such as embodiment 2 of immunoassay.
Claims (6)
1. a kind of preparation method of the unmarked electrochemical immunosensor based on graphene parcel polystyrene composite Nano ball,
Fixed characterized in that, wrapping up polystyrene composite Nano ball modified electrode interface using the graphene that particle diameter is 150-200nm
Antibody molecule, unmarked electrochemical immunosensor is prepared, comprises the following steps：
(1) graphene is wrapped up into polystyrene composite Nano ball ultrasonic disperse in secondary deionized water, takes graphene parcel poly-
Styrene composite Nano ball suspension mixes in equal volume with chitosan solution and ultrasonic disperse is uniform；Again by above-mentioned mixed solution with
Solution of streptavidin mixes in equal volume；
(2) glass-carbon electrode is first polished with alumina powder, after the alumina powder that residual is rinsed out with secondary deionized water, Ran Houyi
It is secondary to be cleaned by ultrasonic glass-carbon electrode with ethanol and redistilled water, finally dry up under a nitrogen；
(3) graphene of obtained streptomysin biological functional in step (1) is taken to wrap up the mixing of polystyrene composite Nano ball
Solution drop coating clean glassy carbon electrode surface of gained in step (2), it is placed in drying in the environment of refrigeration low temperature；
(4) the biotinylated antibody of glassy carbon electrode surface drop coating after step (3) is modified, after reacting at room temperature, phosphoric acid is used
Salt buffer solution rinses；
(5) by bovine serum albumen solution drop coating glassy carbon electrode surface resulting in step (4), after closing at room temperature, use
Phosphate buffer solution is rinsed, and obtains the unmarked electrochemical immunosensor；
In step (1), the polystyrene/graphene composite particle concentration is 2.0~4.0mg/mL, chitosan solution it is dense
Spend for 1.0~2.0wt%, the concentration of Streptavidin is 400~600 μ g/mL；In step (2), described alumina powder particle diameter
For 0.05mm；
In step (1), the styrene monomer dissolved with initiator azodiisobutyronitrile is added to ethanol water, is stirred at room temperature, N2Drum
System temperature is increased to 70 DEG C after bubble, constant temperature constant speed pre-reaction 2h, methacryloxypropyl ethylene chlorination ammonium is added, in N2Atmosphere
Continue to react 22h under enclosing, polystyrene spheres suspension liquid will be obtained after product centrifuge washing.By graphene oxide suspension and gather
Styrene ball suspension liquid mixing ultrasound, adds deionized water, is stirred at room temperature, and system is warming up into 95 DEG C, adds hydration
Hydrazine, back flow reaction, graphene parcel polystyrene composite Nano ball suspension, graphene parcel will be obtained after product centrifuge washing
Polystyrene composite Nano spherolite footpath is 150-200nm.
A kind of 2. unmarked electro-chemistry immunity based on graphene parcel polystyrene composite Nano ball as claimed in claim 1
Transducer production method, it is characterised in that in step (3), the refrigeration low temperature is 4 DEG C；In step (4), the biotinylation
Antibody concentration be 1~10 μ g/mL.
A kind of 3. unmarked electro-chemistry immunity based on graphene parcel polystyrene composite Nano ball as claimed in claim 1
Transducer production method, it is characterised in that in step (5), the bovine serum albumen solution concentration is 1.0~2.0wt%.
4. a kind of unmarked electrochemical immunoanalytical method based on graphene parcel polystyrene composite Nano ball, its feature exist
In, including following analytical procedure：
A. the unmarked electro-chemistry immunity for being wrapped up polystyrene composite Nano ball based on graphene as claimed in claim 1 is passed
Sensor incubates in antigen incubation liquid, is then rinsed with phosphate buffer solution；
B. using the obtained unmarked electrochemical immunosensor as working electrode, saturated calomel electrode is as auxiliary electrode, platinum
Plate electrode is used as to electrode, and in potassium ferricyanide test fluid, electrochemical signals are detected with differential pulse voltametry.
A kind of 5. unmarked electro-chemistry immunity based on graphene parcel polystyrene composite Nano ball as claimed in claim 4
Analysis method, it is characterised in that in step a, incubative time is 30~40min.
A kind of 6. unmarked electro-chemistry immunity based on graphene parcel polystyrene composite Nano ball as claimed in claim 4
Analysis method, it is characterised in that in step b, potassium ferricyanide test fluid is by 5mM K3[Fe(CN)6]/K4[Fe(CN)6] and 0.1M
KCl mixed liquors form, and the pH of solution is 6.5, volume 10mL.
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| CN108169473B (en) * | 2017-12-28 | 2020-07-31 | 重庆医科大学 | Electrochemical immunosensor for C L B detection and preparation method thereof |
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| CN102344700A (en) * | 2011-07-07 | 2012-02-08 | 南京大学 | Preparation method of polystyrene/graphene composite particle |
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| CN105158469B (en) * | 2015-09-18 | 2016-08-31 | 山东理工大学 | A kind of preparation method and application based on biotinylation amination Fe3O4 Yu the immunosensor of Streptavidin |
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