CN105929153B - A kind of preparation method of aflatoxin B1 gold nano well array immunization electrode - Google Patents

A kind of preparation method of aflatoxin B1 gold nano well array immunization electrode Download PDF

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CN105929153B
CN105929153B CN201610263224.0A CN201610263224A CN105929153B CN 105929153 B CN105929153 B CN 105929153B CN 201610263224 A CN201610263224 A CN 201610263224A CN 105929153 B CN105929153 B CN 105929153B
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gold
electrode
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afb1
gold nano
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CN105929153A (en
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曹立新
李小龙
梁晨希
王凯
刘海萍
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Harbin Institute of Technology Weihai
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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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • 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
    • 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
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus

Abstract

The present invention relates to a kind of preparation method of aflatoxin B1 gold nano well array immunization electrode, it uses chemical deposition golden to be deposited on 400 800nm polycarbonate leaching film in die throat diameter, obtain gold nanotubes array body, it is golden to be deposited on 80 200nm polycarbonate leaching film in die throat diameter, gold nanorod array egative film is obtained, gold nano well array electrode is made in assembling；Protein A solution formation albumin A/gold nano well array electrode is added dropwise on gold nanotubes array electrode surface；Then it is put into unmarked AFB1 antibody-solutions, AFB1 antibody/albumin A/gold nano well array electrode is made；And then closing obtains AFB1 immune response electrodes.The present invention makes simple, with three-dimensional structure, and surface area is big, is prevented effectively from the interference of electrochemical response signal caused by unlike material；Antibody fixation is effectively, stable and reliable for performance, and AFB1 sensitive quick measure can be achieved.

Description

A kind of preparation method of aflatoxin B1 gold nano well array immunization electrode
Technical field
The present invention relates to a kind of preparation method of electrode, specifically a kind of aflatoxin B1 gold nano well array is exempted from The preparation method of epidemic disease electrode.
Background technology
Aflatoxin B1（AFB1）It is the toxicity found so far a most strong class biotoxin, with induced mutation, suppression System is immunized and carcinogenesis.Quickly, sensitive, accurate analysis is the effective means for avoiding and reducing AFB1 harm.Current AFB1 points Detection method such as high performance liquid chromatography, enzymoimmunoassay, radioimmunoassay, Gold standard are analysed, immune affinity column is net Change-fluorescence Fast Detection Technique etc., although achieve greater advance, but still in the presence of operation requires high, step is excessively cumbersome, spirit The different defects such as sensitivity is poor, false drop rate is high, expensive equipment.
Identification and combination of the electrochemica biological immunoelectrode based on antigen and antibody, it is simple with quick, sensitive, instrument and equipment The peculiar advantage such as single.The features such as stable high and large biological molecule compatibility of the conductive good, chemical property of gold electrode is good, because This is one of most-often used electrode material in bioelectrochemistry.Gold nano-material specific surface area is big, can load more multispecific antibody and Label, can effectively be amplified to signal, and with good biocompatibility, thus electro-chemistry immunity biography can be effectively improved The sensitivity of sensor.[Liu Y, Qin Z, Wu X, the Jiang H. Biochem. Eng. J., 2006,32 such as Liu: 211-217] one layer of nanogold micelle of self assembly in the two-dimensional gold interdigital electrode modified with 2- aminoothyl mercaptans, by AFB1 antibody It is directly anchored on gold size grain, constructs conductivity type immunosensor, Monitoring lower-cut reaches 0.1 ng/mL.[the Zhou L such as Zhou T, Li R Y, Li Z J, Xia Q F, Fang Y J, Liu J K. Sensor. Actuat. B-Chem., 2012, 174:359-365] nanogold particle is embedded into conducting polymer, improve the mechanical performance of modified electrode.Sharma etc. [Sharma A, Matharu Z, Sumana G, Solanki P R, Kim C G, Malhotra B D. Thin Solid Films, 2010, 519:1213-1218] utilize n-hydroxysuccinimide and ethyldimethyl amine propyl carbodiimide Diimine（EDC）Activate AFB1 antibody（anti-AFB1）On carboxyl and with cysteamine modify gold nano grain be covalently attached, Gold nano grain is covalently then fixed to mercaptobenzoic acid（MBA）Self-assembled film（SAM）On the gold electrode of modification, electricity is constructed Flow pattern immunosensor, Monitoring lower-cut is up to 0.179 ng/mL.[Hu H F, Cao L X, Li Q C, the et al. such as cao RSC Advances. 2015, 5:55209-55217.] people is in the poly- adjacent benzene of golden three-dimensional column nano-array electrode surface modification Diamines, AFB1 antibody is fixed by glutaraldehyde cross-linking, constructs impedance type immunosensor, detects that offline is 0.019 ng/mL.
However, existing AFB1 gold nanos immunosensor also has certain defect at present, it is limited actually detected In application.Such as preparation process complexity, cost height；Distribution and size of the nano-scale gold particle on electrode are difficult to control to；Antibody is fixed Effect is poor, and the space resistance of antibody and antigen binding is high；Limit for height is detected, the quantitative determination range of linearity is narrow, stability difference etc..Improve Maximally effective two approach of AFB1 immunosensor performances are：（1）Improve the performance of base electrode；（2）Ensure antigen or antibody Effectively fixed on electrode, build the molecular recognition film of superior performance.
Nano-array electrode is the aggregate of multiple nano-electrodes.It not only has the high mass transfer rate of single electrode, low double Electric layer charging current, small time constant, small IR drops and high s/n ratio etc. are excellent, and due to thousands of single nano-electrodes Concentrate on a matrix, the shortcomings of overcoming that single nano-electrode response signal is too small, be easily disturbed and be difficult to operation can pole The earth improves sensitivity and the reliability of measurement, reduction operation difficulty and measurement cost.Current people prepare diversified forms Gold-nano array electrode.As one-dimensional granular gold-nano array electrode, two-dimentional banding plate-like gold-nano array electrode, three-dimensional column are received Rice array electrode and gold nanotubes array electrode.Wherein nanometer pipe array electrode has bigger work compared with other array electrodes Property area, more excellent electrocatalysis characteristic in recent years, causes the particular concern of researcher.
Current gold nanotubes array electrode（Or modified electrode）The report of preparation method mainly has following two class：One class be Chemical deposition gold pipe or the overleaf alumina formwork of metal spraying in polycarbonate leaching film nib（Polycarbonate leaching film template）Hole Middle deposited Au pipe, then be fixed in adhesive on base electrode, template is then dissolved, nano-tube array is made（Modification） Electrode.Another kind of is that CNT is modified using nanogold, then it is fixed on base electrode with adhesive.So And, there is deficiency in varying degrees in the immunoelectrode that above method is prepared.（1）Electrode prepared by electrodeposition process, was prepared Journey is complicated, it is difficult to ensure nanotube length, pipe thickness and uniformity；（2）Due to being nano-scale, the nanotube come is exposed Easily snap off；（3）No matter gold nanotubes array electrode prepared by electrodeposition process or chemical deposition, the bottom of its nanotube is It is open, it is base electrode surface with base electrode surface connecting portion, or adhesive, its material is with gold nano tube wall Different（Even if base electrode surfacing is gold, because preparation technology is different from golden tube wall, the surface state of the two is also not With）.Thus electrode is put into solution, and solution can not only be contacted with gold nano tube wall can also be with base electrode surface or gluing Agent is contacted, and electrode function and immune modification surface state are inconsistent, and electrochemical response signal can be caused necessarily to be disturbed, because And have influence on the accuracy of AFB1 immune detections.
The content of the invention
The technical problems to be solved by the invention be overcome above-mentioned the deficiencies in the prior art there is provided one kind make it is simple and direct, into This is low, with three-dimensional structure, and surface area is big, and antibody fixation is effectively, stable and reliable for performance, can be prevented effectively from unlike material and lead The preparation method of the AFB1 gold nano well array immunization electrodes of the electrochemical response signal interference of cause.
The present invention solve above-mentioned technical problem use technical scheme be：A kind of AFB1 gold nanos well array immunization electrode Preparation method, it is characterised in that：It comprises the following steps：
（1）The preparation of gold nanotubes array body：Use chemical deposition in die throat diameter for the poly- of 400-800nm first Deposition in gold, nib is deposited in carbonic ester filter membrane template gold nanotubes, by controlling sedimentation time, makes to deposit Jenner in nib The wall thickness of mitron is 50-200nm；The Gold plated Layer of the polycarbonate leaching film upper surface is then removed, gold nanotubes array master is obtained Body；
（2）The preparation of gold nanorod array egative film：Chemical deposition is used in poly- carbonic acid of the die throat diameter for 80-200nm Gold is deposited in ester filter membrane template, by controlling sedimentation time, makes to be full of solid gold nanorod in nib, obtains gold nanorod battle array Row egative film；
（3）The assembling of gold nano well array electrode：Obtained gold nanorod array egative film lower surface is then passed through into conduction Glue is bonded and fixed on collector, and the tiling of gold nanotubes array body is covered on gold nanorod array egative film, periphery insulation Rubber belt sealing is fixed on the current collector, and gold nano well array electrode is made；
（4）Orient the preparation of the self-assembled modified gold nano well array electrode of albumin A：In gold nanotubes array electrode surface drop Plus 50 μ L 0.2mg/mL Protein A solution, 25-30 DEG C of constant temperature, the reaction time is 50-60min, obtains albumin A/gold nano well Array electrode；
（5）The fixation of antibody：By step（4）Obtained albumin A/gold nano well array electrode is put into 1.2 mg/mL nothing Mark in AFB1 antibody-solutions, 25-30 DEG C of constant temperature, the reaction time is 50-60min, and AFB1 antibody/albumin A/gold nano is made Well array electrode；
（6）Closing：By step（5）Obtained electrode is immersed in 3% calf serum solution, closes nonactive site, permanent Warm 25-30 DEG C, the time is 50-60min, obtains AFB1 immunoelectrodes.
Basic electrode prepared by the present invention -- gold nano well array electrode has 3-D nano, structure, and surface area is big.Due to Tube wall is embedded in polycarbonate leaching film, Stability Analysis of Structures, and intensity is good；The borehole wall is identical with shaft bottom material, can be prevented effectively from different materials The interference of electrochemical response signal caused by matter.Directly make to resist by self assembly fixed protein A in its borehole wall and shaft bottom surface The antigen binding site Fab section of body is sufficiently exposed, and effectively reduction antibody and the space resistance of antigen binding, preferably Its space conformation is kept, the specificity and utilization rate of insolubilized antibody is improved, improves the sensitivity of sensor.What is prepared is unmarked Antigen or antibody need not be marked for immune response electrode, simplify preparation and operating process, are effectively improved analysis Speed, reduces analysis cost.When measuring, immunoelectrode is put to a series of quantitative pass for drawing antigen of various concentrations It is in solution, to be carried out at 25 DEG C after the min of immune response 60, in 2 mmol/L K3Fe(CN)6/K4Fe(CN)6+PBS（pH= 7.00）Middle progress electrochemical impedance spectrometry, using impedance spectrum fitting software ZsimpWin to sensor and various concentrations antigen Impedance spectrum after immune response is fitted calculating, obtains the working curve under finite concentration.The test limit of this method is reachable To 1.0 × 10-10g/mL.Far below the detectability 2ng/g of European Union.Present invention making is simple, cost is low, with three-dimensional knot Structure, surface area is big, and antibody fixation is effectively, stable and reliable for performance, and the specificity and utilization rate of antibody are greatly improved, Neng Goushi The miniaturization of existing detection architecture and integrated, is prevented effectively from the interference of electrochemical response signal caused by unlike material, can be achieved AFB1 rapid sensitive detection.
Embodiment
It is specifically described with reference to the present invention.
A kind of preparation method of aflatoxin B1 gold nano well array immunization electrode, it comprises the following steps：
（1）The preparation of gold nanotubes array body：Use chemical deposition in die throat diameter for the poly- of 400-800nm first Deposition in gold, nib is deposited in carbonic ester filter membrane template gold nanotubes, by controlling sedimentation time, makes to deposit Jenner in nib The wall thickness of mitron is 50-200nm；The Gold plated Layer of the polycarbonate leaching film upper surface is then removed, gold nanotubes array master is obtained Body；
（2）The preparation of gold nanorod array egative film：Chemical deposition is used in poly- carbonic acid of the die throat diameter for 80-200nm Gold is deposited in ester filter membrane template, by controlling sedimentation time, makes to be full of solid gold nanorod in nib, obtains gold nanorod battle array Row egative film；
（3）The assembling of gold nano well array electrode：Obtained gold nanorod array egative film lower surface is then passed through into conduction Glue is bonded and fixed on collector, and the tiling of gold nanotubes array body is covered on gold nanorod array egative film, periphery insulation Rubber belt sealing is fixed on the current collector, and gold nano well array electrode is made；
（4）Orient the preparation of the self-assembled modified gold nano well array electrode of albumin A：In gold nanotubes array electrode surface drop Plus 50 μ L 0.2mg/mL Protein A solution, 25-30 DEG C of constant temperature, the reaction time is 50-60min, obtains albumin A/gold nano well Array electrode；
（5）The fixation of antibody：By step（4）Obtained albumin A/gold nano well array electrode is put into 1.2 mg/mL nothing Mark in AFB1 antibody-solutions, 25-30 DEG C of constant temperature, the reaction time is 50-60min, and AFB1 antibody/albumin A/gold nano is made Well array electrode；
（6）Closing：By step（5）Obtained electrode is immersed in 3% calf serum solution, closes nonactive site, permanent Warm 25-30 DEG C, the time is 50-60min, obtains AFB1 immune response electrodes.
The preparation method of the above-mentioned aflatoxin B1 gold nano well array immunization electrode of the present invention, is comprised the concrete steps that：
(1) preparation of nano-tube array main body：
A. die throat diameter is fixed on non-metal plate or nonmetallic clamping plate for 400-800nm polycarbonate leaching film first On, soaking and washing 5 minutes in methanol are put into, are aided with ultrasonication；
B. the polycarbonate leaching film after methanol soaking and washing is put into containing SnCl2、CF3COOH methanol and water mixed liquid Middle progress sensitization pre-treatment, the sensitization mixed liquor is SnCl2 0.026M, CF3COOH 0.05M, volume ratio 1:1 (v/v's) Methanol and water mixed liquid, temperature are normal temperature, and the time is 40-60 min；
C. the polycarbonate leaching film after sensitization and cleaning is put into the content of silver pre- to be carried out in 0.04 M silver ammino solution It is silver-plated；
D. the polycarbonate leaching film after pre- silver-plated and cleaning is put into gold plating solution and carries out chemical plating, the gold plating solution Main composition and craft of gilding be：Na3Au(SO3)2 0.006M ；Na2SO30.08M；Formaldehyde 0.04M；PH value 9.50~10.50；Temperature：2-5℃；Time：16-26 h；
E. by the polycarbonate leaching film after chemical plating in 20-25% HNO3Soak 12 hours, then cleaned with water in the aqueous solution；
F. the gold-plated film of the polycarbonate leaching film one side after above-mentioned processing is removed, exposes gold nanotubes port, at 150 DEG C Heated 20 minutes in baking oven, eliminate the space between gold nanotubes and polycarbonate leaching film, the wall of gold nanotubes in filter opening nib Thickness is 50-200nm；Obtain gold nanotubes array.
(2) preparation of gold nanorod array egative film：
A. die throat diameter is fixed on non-metal plate or nonmetallic clamping plate for 80-200nm polycarbonate leaching film, be put into Soaking and washing 5 minutes, are aided with ultrasonication in methanol；
B. the polycarbonate leaching film after methanol soaking and washing is put into containing SnCl2、CF3COOH methanol and water mixed liquid Middle progress sensitization pre-treatment, the sensitization mixed liquor is SnCl2 0.026M, CF3COOH 0.05M, volume ratio 1:1 (v/v) Methanol and water mixed liquid, temperature is normal temperature, and the time is 40-60 min；
C. the polycarbonate leaching film after sensitization and cleaning is put into the content of silver pre- to be carried out in 0.04 M silver ammino solution It is silver-plated；
D. the polycarbonate leaching film after pre- silver-plated and cleaning is put into gold plating solution and carries out chemical plating, the gold plating solution Main composition and craft of gilding be：Na3Au(SO3)2 0.005-0.05M；Na2SO30.01-0.1M；Formaldehyde 0.3- 0.8M；PH value 9.50 ~ 10.50；Temperature：2-8 ℃；Time：12-24 h；
E. by the polycarbonate leaching film after chemical plating in 20-25% HNO3Soak 12 hours, then cleaned with water in the aqueous solution；
F. heated 20 minutes in 150 DEG C of baking oven, eliminate the sky between nanowires of gold and polycarbonate leaching film in filter opening Gap, obtains gold nanorod array.
(3) assembling of electrode：
The above-mentioned gold nanorod array egative film lower surface prepared is bonded on conductive aluminum two-sided tape, conductive aluminum is two-sided The another side of adhesive tape is bonded on the metal copper sheet for making collector, and the tiling of gold nanotubes array body is covered in gold nanorod array bottom On piece, it can be reserved according to measurement needs, the upper surface of gold nanotubes array body after certain area, remainder insulating cement Band is sealed and fixed on the current collector, and gold nano well array electrode is made.When so can ensure to put the electrodes into solution, only There is unencapsulated part gold nanotubes array to produce electrochemical response signal.
（4）Orient the preparation of the self-assembled modified gold nano well array electrode of albumin A：In gold nanotubes array electrode surface drop Plus 50 μ L 0.2mg/mL Protein A solution, 25-30 DEG C of constant temperature, the reaction time is 50-60min, obtains albumin A/gold nano well Array electrode.
（5）The fixation of antibody：By step（4）Obtained albumin A/gold nano well array electrode is put into 1.2 mg/mL nothing Mark in AFB1 antibody-solutions, 25-30 DEG C of constant temperature, the reaction time is 50-60min, and AFB1 antibody/albumin A/gold nano is made Well array electrode.
（6）Closing：By step（5）Obtained electrode is immersed in 3% calf serum solution, closes nonactive site, permanent Warm 25-30 DEG C, the time is 50-60min, obtains AFB1 immune response electrodes.
Gold nanotubes array body of the present invention is connected on the current collector by gold nanorod array egative film, in gold The shaft bottom of nano-tube array bottom part body formation same material.The advantage and effect of basic electrode gold nano well array electrode of the present invention It is：(1) there is three-dimensional structure, surface area is bigger, wherein gold nanotubes inner surface not only the skin effect with nano material, The speciality of the nano materials such as bulk effect, quantum size effect, can also carry out work(by method of modifying such as self assembly and chemistry Energyization is designed, and is built nanometer assembling electrode system, is realized the miniaturization of detection architecture and integrated.The electrode is in electrochemica biological Sensor field has special advantage.(2) make simple, stability is high, cost is low.The pipe thickness of gold nanotubes array With the big I of internal diameter by chemical deposition time control, it is additionally, since tube wall and is embedded in polycarbonate leaching film, Stability Analysis of Structures, Intensity is good, is effectively guaranteed the stability of product.(3) shaft bottom of gold nanorod array egative film formation and gold nanotubes array The borehole wall of formation is same material, and gold nanotubes array body and gold nanorod array egative film can be connect therebetween with seamless matching Contacting surface is all gold, and contact is good.The interference for the electrochemical response signal that can be prevented effectively from caused by unlike material.
The present invention, by self assembly fixed protein A, makes the antigen binding site of antibody directly in its borehole wall and shaft bottom surface Fab section is sufficiently exposed, and effectively reduction antibody and the space resistance of antigen binding, preferably keeps its space conformation, The specificity and utilization rate of insolubilized antibody are improved, the sensitivity of sensor is improved.The unmarked immune response electrode prepared is not Need that antigen or antibody is marked, the change of redox chemistry reaction when can directly determine antigen antibody complex formation Change, simplify and prepare and operating process, effectively improve analyze speed, reduce analysis cost.When measuring, immunoelectrode is put To a series of drawing in the quantitative relationship solution of antigen for various concentrations, carried out at 25 DEG C after the min of immune response 60,2 mmol/L K3Fe(CN)6/K4Fe(CN)6+PBS（pH=7.00）Middle progress electrochemical impedance spectrometry, is fitted soft using impedance spectrum Part ZsimpWin is fitted calculating to sensor and the reacted impedance spectrum of various concentrations antigen immune, obtains in finite concentration Under working curve.The test limit of this method can reach 1.0 × 10-10g/mL.Far below the detectability 2ng/g of European Union.
Present invention making is simple, cost is low, with three-dimensional structure, and surface area is big, and antibody fixation is effective, the spy of antibody The opposite sex and utilization rate are greatly improved, stable and reliable for performance, are prevented effectively from the interference of electrochemical response signal caused by unlike material, AFB1 rapid sensitive detection can be achieved.

Claims (1)

1. a kind of preparation method of aflatoxin B1 gold nano well array immunization electrode, it is characterised in that：It includes following step Suddenly：
（1）The preparation of gold nanotubes array body：Chemical deposition is used first in poly- carbonic acid of the die throat diameter for 400-800nm Deposition in gold, nib is deposited in ester filter membrane template gold nanotubes, by controlling sedimentation time, makes to deposit gold nanotubes in nib Wall thickness be 50-200nm；The Gold plated Layer of the polycarbonate leaching film upper surface is then removed, gold nanotubes array body is obtained；
（2）The preparation of gold nanorod array egative film：Chemical deposition is used to be filtered in die throat diameter for 80-200nm makrolon Gold is deposited in film template, by controlling sedimentation time, makes to be full of solid gold nanorod in nib, obtains gold nanorod array bottom Piece；
（3）The assembling of gold nano well array electrode：It is then that obtained gold nanorod array egative film lower surface is gluing by conduction Knot is fixed on the current collector, and the tiling of gold nanotubes array body is covered on gold nanorod array egative film, periphery insulating tape Sealing is fixed on the current collector, and gold nano well array electrode is made；
（4）Orient the preparation of the self-assembled modified gold nano well array electrode of albumin A：50 are added dropwise on gold nanotubes array electrode surface μ L 0.2mg/mL Protein A solution, 25-30 DEG C of constant temperature, the reaction time is 50-60min, obtains albumin A/gold nano well array Electrode；
（5）The fixation of antibody：By step（4）Obtained albumin A/gold nano well array electrode is put into the unmarked of 1.2 mg/mL In AFB1 antibody-solutions, 25-30 DEG C of constant temperature, the reaction time is 50-60min, and AFB1 antibody/albumin A/gold nano well battle array is made Row electrode；
（6）Closing：By step（5）Obtained electrode is immersed in 3% calf serum solution, closes nonactive site, constant temperature 25- 30 DEG C, the time is 50-60min, obtains AFB1 immune response electrodes.
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