CN109507257A - The electrochemical immunosensor of G-Chi-CuNPs nano-complex preparation and its application - Google Patents

The electrochemical immunosensor of G-Chi-CuNPs nano-complex preparation and its application Download PDF

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CN109507257A
CN109507257A CN201811368645.5A CN201811368645A CN109507257A CN 109507257 A CN109507257 A CN 109507257A CN 201811368645 A CN201811368645 A CN 201811368645A CN 109507257 A CN109507257 A CN 109507257A
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cunps
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谢芝勋
黄娇玲
谢丽基
谢志勤
邓显文
范晴
罗思思
曾婷婷
张艳芳
王盛
张民秀
刘加波
庞耀珊
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Guangxi Veterinary Research Institute
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Abstract

The present invention is using chitosan (Chi) grapheme modified (G) and loads copper nano-particle (CuNPs), graphene-chitosan-copper nano-particle (G-Chi-CuNPs) nano-complex is obtained for modified electrode, then sessile antibody constructs electrochemical immunosensor.Electrochemical immunosensor combines electrochemical analysis method and immunoassay, has the advantages that quick, easy to operate distinctive detection of electrochemical analysis, high sensitivity, Miniaturized instrument, it may have the characteristics of distinctive Ag-Ab is specifically bound in immune response.Pass through the advantages of electrochemical immunosensor constructed by the anti-NDV polyclonal antibody of fixation is good to the detection specificity of NDV and high sensitivity, detection sensitivity 102EID50 provides a kind of novel quick detection method for NDV.

Description

The electrochemical immunosensor of G-Chi-CuNPs nano-complex preparation and its application
Technical field
This application involves technical field of biological, in particular to a kind of G-Chi-CuNPs nano-complex system Standby electrochemical immunosensor and its application.
Background technique
Electrochemical immunosensor had both had distinctive sensitive, quick, the simple and potential instrument of electrochemical analysis can The advantages that micromation, while also having the characteristics of high specificity in immunoassay.In recent years, electrochemical immunosensor is in disease Pathogenic microorganism quickly detect in attention of the application by scholars, be successfully applied to environmental analysis, food work at present Industry and clinical medicine etc..Graphene is with the tightly packed carbon material into two-dimentional special construction of carbon atom, it has biggish Specific surface area and good electric conductivity are the high-quality potentiality materials applied to the Research of Immunosensor.
But the disadvantages of sensor of existing report is there are still structure is complicated, and preparation is difficult or expensive using nano material, because How efficiently, cleanly material is fixed to the direction on sensor as research worker's effort by this, they convert not Material together, the electrochemical sensor that a series of new has been prepared using different method of modifying.Metal nano material is not only Suitable microenvironment, and its good compatibility to microbial molecules recognition component are created for the fixation of biomolecule, compares table Area is big, may be used as carrier or plays the role of probe；In addition, metal nano material electric conductivity is strong, microorganism can be enhanced Electronics transfer between activated centre and electrode surface improves electrochemical reaction speed, and then improves the sensitivity of detection, can also The activity of intensified response extends the service life of biosensor, therefore metal nano material both includes your gold such as gold, silver, platinum Belong to material, is widely used in electrochemical immunosensor.Nano silver (AgNP) has good electric conductivity, optical Energy, catalytic performance etc., electrochemical reaction can be accelerated by being fixed on sensor surface, therefore this research team has applied for G- Chi-AgNP sensor, but silver belongs to precious metal material after all, scarcity of resources is expensive.
Method currently used for detection virus has Virus Isolation, Enzyme-linked Immunosorbent Assay (ELISA), reverse transcription polymerase Chain reaction (RT-PCR), amplification of nucleic acid sequences and real-time fluorescence quantitative PCR, but these methods are deposited in practical applications In certain defect, but respectively there is shortcoming, wherein Virus Isolation is the most accurate, but trivial operations, takes a long time. ELISA takes a long time, and needs to contact the harmful substances such as EB, amplification of nucleic acid sequences technology behaviour when RT-PCR technology is complicated and electrophoresis Make cumbersome, instrument and expensive reagents needed for real-time fluorescence quantitative PCR, these are insufficient or shortcoming affects their reality and answers With.Therefore, it is necessary to establish a kind of easy, quick, sensitive electrochemical immunosensor technology detection virus.
Newcastle disease is one of the epidemic disease for endangering aviculture most serious, diagnostic method have clinical diagnosis, Virus Isolation, Blood clotting-hemagglutination-inhibition test (HA-HI test) and reverse transcription-polymerase chain reaction (RT-PCR) etc..Wherein, clinical Diagnosis can only tentative diagnosis.Virus Isolation and blood clotting-hemagglutination-inhibition test are both needed to carry out virus purification, take a long time, no Adapt to the requirement of clinical quick diagnosis.Need to contact the harmful substances such as EB when RT-PCR technology is complicated and electrophoresis, these are insufficient or Shortcoming affects their practical application.Therefore, it is necessary to establish a kind of easy, quick, sensitive electrochemical immunosensor Technology detects newcastle disease virus (NDV).
Summary of the invention
To solve the above problems, the present invention provides a kind of preparation method of G-Chi-CuNPs nano-complex, comprising: G-Chi suspension is obtained using chitosan (Chi) package graphene (G), CuSO is added in the G-Chi suspension4It is molten Liquid, 95 DEG C of reactions are completed.
Further, the preparation method of G-Chi-CuNPs nano-complex includes:
(1) every 100mL volume fraction is that the chitosan of 0.1g is added in 1.0% acetum, and magnetic agitation 1h is obtained 0.1wt% crust amine aqueous solution；
(2) graphene of 2.5mg is mixed into crust amine aqueous solution described in every 25mL, ultrasonic 1h is stirred for 24 hours under room temperature, Obtain stable G-Chi suspension；
(3) every 20mL10mM CuSO4Solution is mixed with G-Chi suspension described in 25ml, stirs 3h, then water-bath adds Heat obtains the G-Chi-CuNPs nano-complex to 95 DEG C of the reaction was continued 0.5h.
The present invention also provides a kind of G-Chi-CuNPs nano-complexes, are prepared using aforementioned preparation process.
The present invention also provides a kind of electrochemical immunosensors, including working electrode, and working electrode is through G-Chi- Simultaneously sessile antibody, antibody can be specifically bound the modification of CuNPs nano-complex with antigen corresponding in sample to be tested.
Further, electrochemical immunosensor preparation method includes:
G-Chi-CuNPs nano-complex is coated onto working electrode surface, antibody is coated after drying, is used after reaction The closing of BSA solution, is cleaned.
Further, electrochemical immunosensor preparation method specifically includes:
G-Chi-CuNPs nano-complex is taken to be coated onto working electrode surface, naturally dry is at 4 DEG C to get G-Chi- CuNPs modifies working electrode；
Antibody is applied in G-Chi-CuNPs modification working electrode surface, is placed at 4 DEG C and reacts 8 hours, then to impregnate BSA molten 1h is closed at 37 DEG C of liquid, taking-up is cleaned with secondary deionized water.
Specifically, the working electrode includes glass-carbon electrode and/or gold electrode.
Again specifically, working electrode is that glass-carbon electrode uses Al before coating G-Chi-CuNPs nano-complex2O3It throws Glass-carbon electrode is polishing to after mirror surface and is cleaned with deionized water by light powder, then successively with secondary deionized water, dehydrated alcohol, secondary go Ionized water ultrasound 5min；Then glass-carbon electrode is placed in 0.5molL- 1H2SO4Cyclic voltammetry scan, scanning speed are carried out in solution Degree is 50mV/s, and voltage range is -0.3 ~+1.5V, until stablizing to cyclic voltammogram.
Sensor is platinum electrode to electrode, and reference electrode is saturated calomel electrode.
The present invention also provides the applications of G-Chi-CuNPs nano-complex and preparation method thereof, are applied particularly to electrification Learn the preparation of immunosensor.
The present invention also provides the applications of any electrochemical immunosensor of the present invention, are applied particularly to viral diagnosis Field.
The present invention also provides a kind of electrochemical immunosensor for detecting newcastle disease virus, the sensor selection present invention appoints Electrochemical immunosensor described in one, wherein antibody is anti-NDV polyclonal antibody.Specifically, anti-NDV polyclonal antibody is The Anti-Newcastle Disease virus antibody (ab34402) of abcam.
Specifically, the preparation method of the electrochemical immunosensor of detection newcastle disease virus includes:
(1) every 100mL volume fraction is that the chitosan of 0.1g is added in 1.0% acetum, and magnetic agitation 1h is obtained 0.1wt% crust amine aqueous solution；The graphene of 2.5mg is mixed into crust amine aqueous solution described in every 25mL, ultrasonic 1h is stirred under room temperature It mixes and obtains stable G-Chi suspension for 24 hours；Every 20mL 10mM CuSO4Solution is mixed with G-Chi suspension described in 25ml It closes, stirs 3h, then heating water bath obtains the G-Chi-CuNPs nano-complex to 95 DEG C of the reaction was continued 0.5h.
(2) with 0.05 μm of Al2O3Polishing powder by glass-carbon electrode (GCE,) be polishing to mirror surface after use deionization Water cleaning, then successively use secondary deionized water, dehydrated alcohol, 5 min of secondary deionized water ultrasound.Then glass-carbon electrode is placed in 0.5molL-1H2SO4Carry out cyclic voltammetry scan in solution, scanning speed be 50mV/s (voltage range be -0.3 ~+ 1.5V), until pipetting 10 μ L G-Chi-CuNPs nano-complexes after stablizing to cyclic voltammogram and being coated onto glass-carbon electrode table Face, naturally dry is to get G-Chi-CuNPs modified glassy carbon electrode at 4 DEG C.Sensor is platinum electrode, reference to electrode Electrode is saturated calomel electrode.
(3) to the anti-NDV polyclonal antibody of 10 μ L10 μ g/mL of the G-Chi-CuNPs modified glassy carbon electrode drop coating of preparation (PAb/NDV) it is placed at 4 DEG C and reacts 8 hours, then impregnate and closed at 37 DEG C of solution of 200 μ L1wt% bovine serum albumin(BSA) (BSA) 1h, taking-up are cleaned with secondary deionized water.
The present invention also provides a kind of methods for detecting newcastle disease virus, and sample to be tested is coated in any inspection of the present invention The electrochemical immunosensor surface of newcastle disease virus is surveyed, the detection of peak point current is carried out after 37 DEG C of incubation 30min.
Specifically, the detection of the peak point current includes: containing 0.1molL- 10.01 mol of KCl, pH=7.0 L- 1Differential pulse voltammetry test is carried out in PBS solution, the DVP location parameter of electrochemical workstation includes: electric potential scanning range- 0.3~0.4V, sweep speed 0.05V/s；It is platinum electrode to electrode, reference electrode is saturated calomel electrode.
Unless otherwise specified, the solvent in the solution is water.
Specifically, the method for above-mentioned detection newcastle disease virus does not include the detection side for the purpose of the diagnosing and treating of disease Method.
Beneficial effects of the present invention:
The present invention replaces nano silver using ordinary metallic material Nanometer Copper, has developed novel nano-sensor.Silver belongs to In precious metal material, scarcity of resources is expensive, the exploitation of inventive sensor, achievees the purpose that reduce sensor cost.
The electrochemical immunosensor for the detection newcastle disease virus that the present invention establishes has specific good and high sensitivity Advantage, the detection sensitivity to NDV are 102EID50, specific test the result shows that, this method infective bronchitis (IBV), Infectiousness larynx tracheae (ILTV), H5 subtype avian influenza virus (AIV H5), H7 subtype avian influenza virus (AIV H7), fowl exhales intestines Lonely virus (ARV), infectious bursa of Fabricius virus (IBDV), I group I fowl adenovirus (AAV) no cross reaction are quickly detected in BVDV Field has a good application prospect.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present application, constitutes part of this application, this Shen Illustrative embodiments and their description please are not constituted an undue limitation on the present application for explaining the application.In the accompanying drawings:
Fig. 1 is the cyclic voltammogram of electrode modification process.
Fig. 2 is the differential pulse voltammetry curve graph of electrode modification process.
Fig. 3 is the experimental result picture of influence of the NDV incubation time to the response current of sensor.
Fig. 4 is the electrochemical immunosensor specific detection result figure for detecting newcastle disease virus, wherein 1 is newcastle disease (NDV)；2 be infective bronchitis (IBV)；3 be infectiousness larynx tracheae (ILTV)；4 be H5 subtype avian influenza virus (AIV H5)；5 be H7 subtype avian influenza virus (AIV H7)；6 be Avianreovirus (ARV)；7 be infectious bursa of Fabricius virus (IBD)；8 be I group I fowl adenovirus (AAV).
Fig. 5 is the linear volt-ampere curve figure of various concentration NDV virus, and wherein a represents concentration as 0；B represent concentration as 102EID50；C represents concentration as 103EID50；D represents concentration as 104EID50；E represents concentration as 105EID50。
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material as used in the following examples etc., is commercially available unless otherwise specified.Wherein, embodiment In CuSO used4And bovine serum albumin(BSA) (BSA) is purchased from Sigma company；KMnO4、K4Fe(CN)6、 K3Fe(CN)6、H2SO4、 CH3CH2OH, chitosan, ten two citric acid monohydrate trisodiums etc. are that domestic analysis is pure, and experimental water is secondary deionized water.
Anti- NDV polyclonal antibody (PAb/NDV) as used in the following examples is purchased from abcam；Name of product: Anti- Newcastle Disease virus antibody(ab34402)；Catalog number: ab34402；It is the polyclonal antibody of chicken IgY。
F48E9 plants of NDV strain are purchased from China Veterinary Drugs Supervisory Inst.；Infective bronchitis (IBV), infectiousness larynx tracheae (ILTV), H5 subtype avian influenza virus (AIV H5), H7 subtype avian influenza virus (AIV H7), Avianreovirus (ARV) pass Metachromia bursal disease virus (IBDV), I group I fowl adenovirus (AAV) are saved by Guangxi veterinary institute Biotechnology Experiment room.
Instrument CHI660D electrochemical workstation is purchased from the general day science and technology responsibility Co., Ltd of Beijing China Tech, centrifuge purchase From U.S. Bake Mann, KQ-250DB type numerical control ultrasonic cleaning machine has children outside the state plan Instrument Ltd., vortex mixing purchased from city of Kunshan Device is purchased from Beijing Jin Bei moral Trade Co., Ltd., and DF-101S heat collecting type constant-temperature heating magnetic stirring apparatus gives Hua Yi purchased from Gongyi City Device Co., Ltd, vacuum oven are purchased from Zhengzhou Greatwall Scientific Industrial & Trading Co., Ltd..
In following embodiments by taking the electrochemical immunosensor for detecting newcastle disease virus as an example, embodiment and its illustrate For explaining only the invention, it does not constitute improper limitations of the present invention.
The preparation of 1 G-Chi-CuNPs modified glassy carbon electrode of embodiment
(1) preparation of graphene
Graphene oxide is prepared after improving according to Hummer method, the specific steps are as follows:
Under the conditions of ice-water bath, 1g graphite powder, 2.5g KNO3、100mL H2SO4It is slowly added to 5g permanganic acid after mixing evenly Potassium, is subsequently placed at 35 DEG C of water-bath 2h, and 100mL deionized water is added, and in 95 DEG C of the reaction was continued 1h, observes mixture by palm fibre Brown becomes glassy yellow, and 300mL distilled water is added after being cooled to room temperature, and the H that mass fraction is 30% is added2O2It neutralizes not anti- Then the potassium permanganate answered is washed with the HCl of 0.5mol/L, then be centrifuged repeatedly washing 5 times with secondary deionized water, and vacuum is dry It is dry to get graphite oxide.It weighs 10mg graphite oxide to be placed in a beaker, 100mL is added and goes secondary deionized water, ultrasonic 1h is obtained To graphene oxide (GO).
Again with NaBH4Make reducing agent, is restored to obtain graphene (G) under conditions of 95 DEG C.
(2) preparation of G-Chi-CuNPs nano-complex
The chitosan (Chi) of 0.1g is added in 100mL1.0% (V/V) acetum, at room temperature under the conditions of magnetic force stir 1h is mixed, 0.1wt% crust amine aqueous solution is obtained.Take 25mL crust amine aqueous solution that the graphene (G) of 2.5mg, ultrasonic 1h, room temperature item is added It is stirred under part and obtains stable G-Chi suspension for 24 hours.Take 20mL 10mM CuSO4Solution is added to the above-mentioned G- prepared In Chi suspension, 3h is stirred at room temperature, and then heating water bath obtains G-Chi- to 95 DEG C of the reaction was continued 0.5h CuNPs nano-complex.
(3) preparation of G-Chi-CuNPs modified glassy carbon electrode
With 0.05 μm of Al2O3Polishing powder by glass-carbon electrode (GCE,) be polishing to it is clear with deionized water after mirror surface It washes, then successively uses secondary deionized water, dehydrated alcohol, secondary deionized water ultrasound 5min.Then glass-carbon electrode is placed in 0.5mol·L- 1 H2SO4Carry out cyclic voltammetry scan in solution, scanning speed be 50mV/s (voltage range be -0.3 ~+ 1.5V), until pipetting 10 μ L G-Chi-CuNPs nano-complexes after stablizing to cyclic voltammogram with liquid-transfering gun and being coated onto glass Carbon electrodes, naturally dry is to get G-Chi-CuNPs modified glassy carbon electrode at 4 DEG C.
The preparation of 1 Chi-CuNPs modified glassy carbon electrode of comparative example
(1) preparation of Chi-CuNPs nano-complex
The chitosan (Chi) of 0.1g is added in 100mL1.0% (V/V) acetum, under room temperature magnetic agitation 1h obtains 0.1wt% crust amine aqueous solution.
Take 20mL 10mmoL/L CuSO4Solution is added in 25mL crust amine aqueous solution, stirs 5h at room temperature, so Heating water bath is cooled to room temperature to 95 DEG C of the reaction was continued 0.5h and continues to stir 1h, obtain Chi-CuNPs nano-complex afterwards.
(2) preparation of Chi-CuNPs modified glassy carbon electrode
Specific preparation method is identical as embodiment 1 step (3), and difference is only that will be G-Chi-CuNPs nanometers therein Compound replaces with Chi-CuNPs nano-complex.
The electrochemical Characterization of electrode modification process
Fig. 1 is the cyclic voltammogram of electrode modification process.In Fig. 1, a line is bare glassy carbon electrode in 0.01 molL- 1PBS (contain 0.1molL- 1KCl, pH=7.0) cyclic voltammetry scan curve obtained is carried out in solution.B is using comparative example 1Chi- CuNPs modified glassy carbon electrode scans curve obtained；C is using 1 G-Chi-CuNPs modified glassy carbon electrode of embodiment scanning institute Obtain curve.
From figure 1 it appears that occurring a pair of after glass-carbon electrode covers one layer of G-Chi-CuNPs nano-complex Electrochemical redox absorption peak, when glass-carbon electrode covers one layer of G-Chi-CuNPs nano-complex, electrochemical oxidation is also Former absorption peak obviously increases, this is because G has big specific surface area and stronger conductive capability, accelerates the electricity of electrode surface Son transfer.
Embodiment 2 detects the preparation and detection of the electrochemical immunosensor of newcastle disease virus
(1) preparation of the electrochemical immunosensor of newcastle disease virus is detected
The anti-NDV of the 10 μ L10 μ g/mL of G-Chi-CuNPs modified glassy carbon electrode drop coating prepared to embodiment 1 is polyclonal Antibody (PAb/NDV), which is placed at 4 DEG C, to react 8 hours, then impregnates and seal at 37 DEG C of solution of 200 μ L1wt% bovine serum albumin(BSA) (BSA) 1h is closed, taking-up is cleaned with secondary deionized water.
Sensor is platinum electrode to electrode, and reference electrode is saturated calomel electrode.
(2) it detects
30min is reacted at 15 37 DEG C of μ L sample to be tested of drop coating, washing 3 times is centrifuged repeatedly with secondary deionized water, is placed in 0.01mol·L- 1(PBS contains 0.1molL to phosphate buffer- 1KCl, pH=7.0) in carry out differential pulse voltammetry test (DVP), the DVP location parameter of electrochemical workstation: electric potential scanning range -0.3 ~ 0.4V, sweep speed 0.05V/s.
Fig. 2 is the differential pulse voltammetry figure of electrode modification process.In Fig. 2, a line is glass-carbon electrode in 0.01 molL? 1PBS (contains 0.1molL- 1KCl, pH=7.0) differential pulse voltammetry (DVP) is carried out in solution tests curve obtained.B is to use Comparative example 1Chi-CuNPs modified glassy carbon electrode tests curve obtained；C is to modify glass carbon using embodiment 1G-Chi-CuNPs Electrode scans curve obtained；D is to test curve obtained using after the closing of the anti-NDV polyclonal antibody of embodiment 2；E is using implementation Curve obtained is tested after the closing of 2 bovine serum albumin(BSA) of example；F is to test curve obtained after drop coating sample to be tested NDV in embodiment 2.
From figure 2 it can be seen that there is electrochemistry suction after glass-carbon electrode covers one layer of Chi-CuNPs nano-complex Receive peak.This is because copper nano-particle has electro-chemical activity.When glass-carbon electrode covers one layer G-Chi-CuNPs nanometers again After closing object, electrochemistry absorption peak obviously increases than curve b, this is because G has big specific surface area and stronger conductive energy Power accelerates the electronics transfer of electrode surface, and therefore, the present invention is using G-Chi-CuNPs as glass-carbon electrode decorative material.With Pb/NDV, BSA and NDV (10 is successively fixed afterwards2.5EID50) after, the peak point current of obtained DVP curve is decreased obviously (figure Curve d in 2, e and f), this is because Pb/NDV, BSA and NDV are that protein molecular substance is non-conductive, hinder electrode surface Electronics transfer.
The detection of 3 newcastle disease virus of embodiment
(1) optimization of incubation time
In the preparation process of sensor, the immune response time of NDV has important influence to the response signal of sensor. When the electrode of embodiment 2 is spotted with NDV (105EID50) after 5,10,15,20,30,40,50min are successively reacted in 37 DEG C, so Containing 0.01molL afterwards- 1PBS (contains 0.1molL- 1KCl, pH=7.0) DVP scanning is carried out in solution.Obtained peak electricity Flow valuve and time curve are as shown in figure 3, as the peak point current of the extension working electrode of time constantly drops before 30min Low, current value does not change substantially later, illustrates that immune response is over.Therefore, the present invention selects 30min for NDV's Incubation time.
(2) specificity experiments
The electrochemical immunosensor of the detection newcastle disease virus (NDV) prepared with embodiment 2 is to other pathogen body packets It includes: infective bronchitis (IBV), infectiousness larynx tracheae (ILTV), H5 subtype avian influenza virus (AIV H5), H7 hypotype fowl stream Influenza Virus (AIV H7), Avianreovirus (ARV), infectious bursa of Fabricius virus (IBD), I group I fowl adenovirus (AAV) carry out special Specific assay, as a result as shown in figure 4, the sensor of embodiment 2 only peak point current drop in the presence of newcastle disease virus (NDV) It is low, and change is hardly happened to the peak point current of the detection of other pathogen its CV, i.e., yin is detected as to other pathogen Property, show that sensor specificity is good.
(3) sensitivity experiments
It is 10 to viral liquid hold-up6EID50NDV carry out 10 times of doubling dilutions, the biography for respectively taking 15 μ L to be prepared with embodiment 2 Sensor detection, as a result as shown in figure 5,10- 4Its response current value is still decreased obviously when dilution, therefore the immunosensor Limit of identification be 102EID50。

Claims (10)

  1. The preparation method of 1.G-Chi-CuNPs nano-complex, comprising: obtained using chitosan (Chi) package graphene (G) G-Chi suspension, it is characterised in that the preparation method further includes that CuSO is added in the G-Chi suspension4Solution, 95 DEG C reaction complete.
  2. 2. the preparation method of G-Chi-CuNPs nano-complex according to claim 1, it is characterised in that the preparation Method includes:
    (1) every 100mL volume fraction is that the chitosan of 0.1g is added in 1.0% acetum, and magnetic agitation 1h is obtained 0.1wt% crust amine aqueous solution；
    (2) graphene of 2.5mg is mixed into crust amine aqueous solution described in every 25mL, ultrasonic 1h is stirred for 24 hours under room temperature, obtained Stable G-Chi suspension；
    (3) every 20mL10mMCuSO4Solution is mixed with G-Chi suspension described in 25ml, stirs 3h, then heating water bath to 95 DEG C the reaction was continued 0.5h, obtains the G-Chi-CuNPs nano-complex.
  3. 3. a kind of G-Chi-CuNPs nano-complex, it is characterised in that prepared using preparation method as claimed in claim 1 or 2 It forms.
  4. 4. a kind of electrochemical immunosensor, including working electrode, it is characterised in that the working electrode is through described in claim 3 The modification of G-Chi-CuNPs nano-complex and sessile antibody, the antibody can send out with antigen corresponding in sample to be tested Raw specific binding.
  5. 5. electrochemical immunosensor according to claim 4, which is characterized in that the transducer production method includes:
    The G-Chi-CuNPs nano-complex is coated onto the working electrode surface, the antibody is coated after drying, is reacted After with BSA solution close, clean.
  6. 6. electrochemical immunosensor according to claim 4 or 5, which is characterized in that the transducer production method tool Body includes:
    The G-Chi-CuNPs nano-complex is taken to be coated onto working electrode surface, naturally dry is at 4 DEG C to get G-Chi- CuNPs modifies working electrode；
    Antibody is applied in G-Chi-CuNPs modification working electrode surface, is placed at 4 DEG C and reacts 8 hours, then to impregnate BSA molten 1h is closed at 37 DEG C of liquid, taking-up is cleaned with secondary deionized water.
  7. 7. the described in any item preparation methods of claim 1-2, G-Chi-CuNPs nano-complex as claimed in claim 3 Application, which is characterized in that the preparation applied to electrochemical immunosensor.
  8. 8. the application of the described in any item electrochemical immunosensors of claim 4-6, which is characterized in that be applied to virus inspection Survey field.
  9. 9. a kind of electrochemical immunosensor for detecting newcastle disease virus, it is characterised in that including any one of claim 4-6 institute The electrochemical immunosensor stated, wherein the antibody is anti-NDV polyclonal antibody.
  10. 10. a kind of method for detecting newcastle disease virus, it is characterised in that sample to be tested is coated in sensor as claimed in claim 9 Surface, 37 DEG C be incubated for 30min after carry out peak point current detection.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103235123A (en) * 2013-04-19 2013-08-07 广西壮族自治区兽医研究所 Electrochemical immunosensor for detecting avian reoviruses and production method thereof
CN104569097A (en) * 2014-12-17 2015-04-29 浙江理工大学 Preparation method of copper nanowire graphene composite-modified electrode, as well as applications thereof
CN104588110A (en) * 2014-12-23 2015-05-06 郑州轻工业学院 Grapheme/chitosan/cuprous oxide composite material and preparation method and applications thereof
US20160054252A1 (en) * 2014-08-22 2016-02-25 Samsung Electronics Co., Ltd. Electrochemical Biosensor
CN105784822A (en) * 2016-04-19 2016-07-20 青岛科技大学 Preparation and application methods for electrochemical DNA sensor based on chitosan-graphene/ gold nanoparticle composite membrane

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103235123A (en) * 2013-04-19 2013-08-07 广西壮族自治区兽医研究所 Electrochemical immunosensor for detecting avian reoviruses and production method thereof
US20160054252A1 (en) * 2014-08-22 2016-02-25 Samsung Electronics Co., Ltd. Electrochemical Biosensor
CN104569097A (en) * 2014-12-17 2015-04-29 浙江理工大学 Preparation method of copper nanowire graphene composite-modified electrode, as well as applications thereof
CN104588110A (en) * 2014-12-23 2015-05-06 郑州轻工业学院 Grapheme/chitosan/cuprous oxide composite material and preparation method and applications thereof
CN105784822A (en) * 2016-04-19 2016-07-20 青岛科技大学 Preparation and application methods for electrochemical DNA sensor based on chitosan-graphene/ gold nanoparticle composite membrane

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
QIAN WANG等: "Preparation of reduced graphene oxide/Cu nanoparticle composites through electrophoretic deposition: application for nonenzymatic glucose sensing", 《RSC ADV.》 *
潘芊秀 等: "纳米铜/石墨烯-壳聚糖复合膜修饰电极及其对葡萄糖的直接测定", 《分析测试学报》 *

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