CN106290875B - With the TiO of triple modifications2Nano-wire array is the construction method of the electrochemical immunosensor of support - Google Patents

With the TiO of triple modifications2Nano-wire array is the construction method of the electrochemical immunosensor of support Download PDF

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CN106290875B
CN106290875B CN201610653015.7A CN201610653015A CN106290875B CN 106290875 B CN106290875 B CN 106290875B CN 201610653015 A CN201610653015 A CN 201610653015A CN 106290875 B CN106290875 B CN 106290875B
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tio
nano
wire array
room temperature
pillar
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CN106290875A (en
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刘小强
霍小鹤
刘培培
朱杰
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Henan University
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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
    • G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
    • 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
    • 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
    • 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
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • 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

Abstract

The present invention relates to a kind of TiO with triple modifications2Nano-wire array is the construction method of the electrochemical immunosensor of support, synthesizes fine and close, neat TiO by hydro-thermal method first2Nano-wire array, then it is modified, Mo elements are successfully doped into TiO in modifying process2Nano-wire array is to promote its electron transfer capacity；MoS2Piece is also by successful deposition in electrode surface；Then chitosan solution is added dropwise in modified TiO2Nano line array electrode surface, use double amino crosslinker BS3Capture antibody A b1 is covalently bonded in electrode surface.Simultaneously cylindrical TiO is prepared with hydro-thermal method2Monodimension nanometer material, and by its amination, then use BS3Make crosslinking agent to modify horseradish peroxidase HRP and signal antibody Ab2 in TiO jointly2Nano-pillar surface is to obtain signal antibody label.The electrochemical immunosensor of gained is prepared using this method can quickly determine CEA, and sensitivity is higher, the range of linearity is larger, test limit is relatively low.

Description

With the TiO of triple modifications2 Nano-wire array is the electrochemical immunosensor of support Construction method
Technical field：
The invention belongs to electrochemical immunosensor constructing technology field, and in particular to a kind of TiO with triple modifications2Receive Nanowire arrays are the construction method of the electrochemical immunosensor of support, and the electrochemical immunosensor can be used for detection cancer embryo to resist It is former.
Background technology：
Accurately, screening, diagnosis and treatment of the tumor markers to cancer are delicately detected and all plays vital effect. Carcinomebryonic antigen（Carcinoembryonic antigen, CEA）It is a kind of glycoprotein caused by Colorectal Carcinoma, is a wide spectrum Property tumor markers, it can reflect the presence of kinds of tumors to people, judge the effect of to colorectal cancer, breast cancer and lung cancer, Disease development monitors and rear health evaluating is extremely important, and 97% health adult's change of serum C EA concentration is in 2.5 ng mI-1Below. Many immunoassays, including fluoroimmunoassay, electrochemical enzymatic immunoassay, chemiluminescence immune assay, enzyme linked immunological are inhaled Attached method etc. is widely used in detecting tumor marker.In numerous immunoassays, Electrical chemiluminescence immunoassay analysis method have it is quick, The features such as effectively and sensitive.
With the rapid development of nanometer science and technology, various nano materials, including carbon nanomaterial, graphite Alkene, silica, quantum dot, noble metal etc. are widely used in immunosensor to improve its analytical performance.Received numerous In rice material, TiO2Nano material is big, mechanical due to its excellent chemical stability, excellent biocompatibility, specific surface area Performance is good, simple synthetic method and has attracted extensive concern the advantages that environment friendly.Especially one-dimensional TiO2Nano junction Structure, such as TiO2Nanotube, it is widely used in optical electro-chemistry and electrochemical immunosensor.Zhi-Da Gao et al. use TiO2Nanometer Pipe array is sensor stand, is detected with the signal antibody-Au nano particles of horseradish peroxidase-labeled as signal probe Immunoglobulin concentrations.Due to TiO2Notable tubulose feature --- big surface area and the inner space of nanotube, this immune biography Compared with other ordinary flat electrodes, performance is effectively strengthened sensor.Its concentration range of linearity to rabbit immunoglobulin is 0.1—105 ng mL-1, detect and be limited to 0.01 ng mL-1。
In spite of substantial amounts of research concern TiO2The photoelectrochemical behaviour of nano-wire array, but may due to its electric conductivity compared with Difference, the shortcomings that specific surface area is relatively small, the load capacity of electronics transfer and biomolecule is limited, thus few people are used In the preparation of electrochemical sensor.It is worth noting that, it is reported that chemical doping metal ion can as a kind of efficient method To promote TiO2Deng the separation of charge and electric conductivity of semiconductor nano material.Such as Wang et al. is prepared for W by hydro-thermal method and mixed Miscellaneous TiO2Core-shell structure copolymer nano-wire array.In etching/regrowth process, W elements are doped into TiO2To improve its photoelectricity in shell Performance.And after electrochemical impedance result also shows W doping, TiO2Nano-wire array electric conductivity significantly increases.
Graphene has excited the research interest for preparing similar nano material in the successful application of different field.It is reported that MoS2Comprising a Mo metal level and two S layers, Mo metal levels are clipped between two S layers by Van der Waals force.Because it is unique Geometry, MoS2As one of typical graphene analog.Importantly, MoS2With approximately excellent with graphene Feature is easily modified on performance, such as excellent Electronic Performance, special optical characteristics, excellent mechanical performance and surface.Although MoS2In many fields, such as transistor, fuel cell, energy storage etc. are obtained for extensive use, but it is in terms of electrochemical sensor Application it is also very limited.Vasilescu et al. is prepared for one kind by MoS2The nano material formed with graphene quantum dot is used for Load enzyme molecule.The electric conductivity of carbon-based screen printing electrode is in modification MoS2Greatly enhanced after-graphene quantum dot, while be also Laccase provides a biocompatible scaffold.The laccase biosensor is to caffeic acid in concentration range 0.38-100 uM There is good response, its detection is limited to 0.32 μM, and sensitivity is 17.92 nA uM-1。
BS3It is a kind of double amino crosslinkers, there is water-soluble, non-cracking performance, film impermeability, it contains one Terminal amino group reactive group（Sulfo-NHS ester groups）, can be reacted with any molecule containing primary amine group.Therefore, it extensively should Crosslinking for biomolecule.
The present invention is prepared for the TiO of triple modifications by two step hydro-thermal reactions2Nano-wire array.Specifically, the first step Hydro-thermal reaction prepares TiO2Nano-wire array；Second step realizes " etching, doping and deposition " simultaneously, MoS2Piece is deposited on quarter Erosion/doped TiO2Nano-wire array surface.Then the TiO by chitosan solution dropwise addition after modification2Nano-wire array table Face, with BS3For amino crosslinker its surface is covalently bind in by antibody A b1 is captured.Hydro-thermal method also be used to prepare cylindrical TiO2 Nano-pillar, BS3Also be used to combine HRP and signal antibody to prepare trace labelling thing.Triple modified TiO2Nano-wire array Multinomial performance be significantly improved, and be used as a kind of new immune sensor platform support.Its advantage includes：First, carve Erosion helps lend some impetus to electronics transfer plus Mo doping, strengthens electric conductivity；In addition, dimensional thinlayer MoS2The deposition of piece can not only strengthen Electric conductivity, and electrode specific surface area can be strengthened, help to adsorb more biomolecule.It is thus new prepared by the present invention Immunosensor support has excellent electric conductivity, big specific surface area, is favorably improved detection range and sensitivity, at present also It there are no relevant report.
The content of the invention：
Present invention aims at develop a kind of TiO with triple modifications2Nano-wire array passes for the electro-chemistry immunity of support The construction method of sensor, the electrochemical immunosensor can rapidly determine carcinomebryonic antigen, and sensitivity is higher, the range of linearity It is larger, test limit is relatively low.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of TiO with triple modifications2Nano-wire array is the construction method of the electrochemical immunosensor of support, and it is wrapped Include following steps：
1. the TiO of triple modifications2The preparation of nano-wire array：
TiO2The preparation of nano-wire array：The dense HCl of 13 mL are diluted with 15 mL deionized waters, stir 5 at room temperature Min, after adding 300 μ L butyl titanates, continue to stir 15 min to obtain settled solution；By gained settled solution and totally FTO substrates are transferred in reactor together, and 12 h are reacted at 150 DEG C；Room temperature is cooled to, FTO substrates is taken out, uses deionized water Wash and in N2Flow down drying, one layer of white film of uniform fold on FTO substrates, as TiO2Nano-wire array；
TiO2While nano-wire array " etching, doping and deposition "：By 30 mL ultra-pure waters, 0.01 M hydroxylamine hydrochlorides, The water vulcanized sodium of 0.005 M nine, 0.002 M Sodium Molybdate Dihydrates, 50 μ L polyethylene glycol and the above-mentioned TiO being prepared2Nanometer linear array Row are placed in reactor together, and 24 h are reacted at 250 DEG C, are cooled to room temperature, take out FTO substrates, be washed with deionized and N2Flow down drying；It was observed that gained TiO2Covered with one layer of black deposit, as MoS on nano-wire array2Nanometer sheet, the FTO Substrate is designated as modified FTO electrodes；
2. the preparation of signal antibody label：
TiO2The preparation of nano-pillar：1.8 mL titanium tetrachlorides are slowly added into the beaker containing 19 mL ultra-pure waters, frozen water 10 min are stirred at 0-5 DEG C of bath and obtain white suspension；1.8 mL chloroforms are added, continue to stir 10 min, are then transferred to anti- Answer in kettle, 160 DEG C of reaction 12h, be cooled to room temperature, white precipitate is collected by centrifugation and washs to neutrality, then 60 DEG C of vacuum drying 12 h, produce TiO2Nano-pillar, store for future use at room temperature；
TiO2The amination of nano-pillar：By 100 mg TiO2Nano-pillar is placed in containing 20 mL ethanol, the ammoniacal liquor of 1 mL 28% and 4 In the mixed liquor of mL 3- aminopropyl triethoxysilanes, 12h is stirred at room temperature to prevent TiO2Nanometer column sedimentation, is then centrifuged for Separation, abandoning supernatant, the washing of gained white depositions, after drying, store for future use at room temperature, be designated as NH2-TiO2Nano-pillar；
Horseradish peroxidase ︱ aminations TiO2The preparation of nano-pillar ︱ signal antibody Ab2 bioconjugate bodies：By 2 mg BS3 It is dissolved in 1 mL PBSs and obtains solution A, then by 3 mg NH2-TiO2Nano-pillar is scattered in solution A, under stirring Add the mgmL of 300 μ L 2-1The horseradish peroxidase aqueous solution and be incubated 30 min at room temperature；Then 20 μ L are added to contain 0.5 mg·mL-1Signal antibody Ab2 PBS, 8 h are stirred at 4 DEG C, centrifuge, gained sediment PBS Wash and close 30 min at room temperature with the PBS containing 2% bovine serum albumin, after finally being washed with PBS, point Dissipate in PBSs of 1.0 mL containing 0.1% bovine serum albumin, it is standby, it is designated as HRP ︱ NH2- TiO2Nano-pillar ︱ Ab2；
2. the structure of immunosensor：
First, the wt% of 30 μ L 0.3 chitosan-acetic acid solution is added dropwise in modified FTO electrode surfaces, at room temperature from So dry, 30 μ L are then added dropwise and contain 2 mgmL-1 BS3PBS and be incubated 1 h at room temperature, 30 μ L are then added dropwise again 0.38 mg·mL-1Capture antibody A b1, be incubated 1 h at room temperature, then after being incubated 12h under 4 DEG C, 100% moisture-saturated environment, Washed respectively with lavation buffer solution and PBS, then 30 min are incubated with 20 μ L Block buffers, it is finally slow with washing respectively Fliud flushing and PBS wash 3 min, are stored for future use at 4 DEG C.
Specifically, step 3. in, the lavation buffer solution is：Containing 0.05 %（w/v）Tween 20 PBS （0.05 M, pH 7.0）；The Block buffer is：Containing 5 %（w/v）Bovine serum albumin（BSA）PBS（0.05 M, pH 7.0）.
Compared to the prior art, the beneficial effect of the inventive method：
TiO is prepared for by hydro-thermal method2Nano-wire array, then using " etching, doping and deposition " technology simultaneously to it Triple modifications are carried out, in this modifying process, Mo elements are successfully doped into TiO2Nano-wire array, electronics biography can be effectively improved Pass speed；Meanwhile dimensional thinlayer MoS2Piece, in electrode surface, can not only be strengthened electric conductivity, be also greatly enhanced by successful deposition Electrode specific surface area, helps to load further amounts of biomolecule, so as to improve the sensitivity of the sensor.
Brief description of the drawings：
Fig. 1 is TiO2Nano-wire array, TiO2The SEM figures and TEM figures of nano-pillar, wherein A and B are respectively pure TiO2Nanometer The SEM plans and sectional view of linear array；C and D is respectively triple modified TiO of different amplification2Nano-wire array SEM plans；E and F is respectively TiO2The SEM figures and TEM figures of nano-pillar；
Fig. 2 is the XRD spectrum of different materials, and wherein a is FTO substrates;B is pure TiO2Nano-wire array；C is in chloroazotic acid Surface MoS is removed after 24 h of middle immersion2The TiO of etching/doping afterwards2Nano-wire array；D is triple modified TiO2Receive Nanowire arrays；
Fig. 3 is nyquist diagram, and wherein a is naked FTO substrates；B is triple modified TiO2Nano-wire array；C is three The modified TiO of weight2Nano-wire array ︱ chitosan ︱ BS3︱ Ab1；D is triple modified TiO2Nano-wire array ︱ chitosans ︱ BS3︱ Ab1 ︱ BSA；E is triple modified TiO2Nano-wire array ︱ chitosan ︱ BS3The Ab2 of ︱ Ab1 ︱ BSA ︱ CEA ︱ marks；F is Pure TiO2The FTO substrates of nano-wire array modification；
Fig. 4 is influence of the pH value of test solution to immunosensor current-responsive of the present invention；
Fig. 5 is that CEA cultivates influence of the time to immunosensor current-responsive in electrode surface；
Fig. 6 is the optimization of capture antibody A b1 concentration；
Fig. 7 is the interference that disturbance thing determines to CEA；
Fig. 8 is reduction current with the increased change curve of CEA concentration.
Embodiment：
Technical scheme is further discussed in detail with reference to embodiments, but protection scope of the present invention It is not limited thereto.
In following embodiments, used BS3Purchased from Sigma-Aldrich Co., Ltd, capture antibody A b1, signal resist Body Ab2 is purchased from Chengdu double fluid Zheng Long biochemical products research department.
Embodiment 1：
A kind of TiO with triple modifications2Nano-wire array is the construction method of the electrochemical immunosensor of support, and it is wrapped Include following steps：
1. the TiO of triple modifications2The preparation of nano-wire array：
TiO2The preparation of nano-wire array：
Before synthesis, several FTO substrates are first used into acetone, ethanol and milli-Q water successively, in N2Flow down and be dried for standby.Will The dense HCl of 13 mL (37 wt %) are diluted with 15 mL deionized waters, stir 5 min at room temperature.Then 300 μ L metatitanic acid fourths are added Ester, continue to stir 15 min to obtain settled solution.Gained settled solution is shifted together with the FTO substrates of above-mentioned washes clean To the autoclave of 50 mL polytetrafluoroethyllining linings, 12 h are reacted at 150 DEG C.Room temperature is cooled to, takes out FTO substrates, It is washed with deionized and in N2Flow down drying.Uniform fold has one layer of white film on FTO substrates, after proved by a variety of methods As TiO2Nano-wire array.
TiO2While nano-wire array " etching, doping and deposition "：By 30 mL ultra-pure waters, 0.01 M hydroxylamine hydrochlorides, The water vulcanized sodium of 0.005 M nine, 0.002 M Sodium Molybdate Dihydrates, 50 μ L polyethylene glycol and above-mentioned preparation-obtained TiO2Nano wire Array is placed in the autoclave of 50 mL polytetrafluoroethyllining linings together, and 24 h are reacted at 250 DEG C, is cooled to room temperature, is taken out FTO substrates, it is washed with deionized and in N2Flow down drying.It was observed that gained TiO2Covered with one layer of black on nano-wire array Deposit, after by a variety of methods prove be MoS2Nanometer sheet, the FTO substrates are designated as modified FTO electrodes.
2. the preparation of signal antibody Ab2 labels：
TiO2The preparation of nano-pillar：1.8 mL titanium tetrachlorides are slowly added into the beaker containing 19 mL ultra-pure waters, ice The min of strong stirring 10 obtains white suspension at 0-5 DEG C of water-bath；1.8 mL chloroforms are added, continue to stir 10 min, then It is transferred in the autoclave of polytetrafluoroethyllining lining, 160 DEG C of 12 h of reaction.Room temperature is cooled to, white precipitate is collected by centrifugation, point Do not washed with deionized water and absolute ethyl alcohol to neutrality, then 60 DEG C of 12 h of vacuum drying, produce TiO2Nano-pillar, store up at room temperature Deposit standby.
TiO2The amination of nano-pillar：By 100 mg TiO2Nano-pillar is placed in containing 20 mL ethanol, the ammoniacal liquor of 1 mL 28% and 4 In the mixed liquor of mL 3- aminopropyl triethoxysilanes, it is stirred overnight at room temperature（12h）To prevent TiO2Nanometer column sedimentation.So After centrifuge, abandoning supernatant, gained white depositions washing, dry after, store for future use at room temperature, be designated as NH2-TiO2Receive Meter Zhu.
Horseradish peroxidase ︱ aminations TiO2The preparation of nano-pillar ︱ signal antibody Ab2 bioconjugate bodies：By 2 mg BS3 It is dissolved in 1 mL phosphate（PBS, 0.02 M）Solution A is obtained in buffer solution, then by 3 mg NH2-TiO2Nano-pillar is disperseed In solution A, lower addition 300 μ L 2 mgmL are stirred-1The horseradish peroxidase aqueous solution and be incubated 30 at room temperature min.Then 20 μ L are added and contain 0.5 mgmL-1Signal antibody Ab2 PBS, 8 h are stirred at 4 DEG C, centrifuge, institute Sediment is obtained to be washed with PBS and close 30 min at room temperature with the PBS containing 2% bovine serum albumin.Finally use After PBS washing, it is scattered in PBSs of 1.0 mL containing 0.1% bovine serum albumin, it is standby, it is designated as HRP ︱ NH2- TiO2Nano-pillar ︱ Ab2.
3. the structure of immunosensor：
First, the wt% of 30 μ L 0.3 chitosan-acetic acid solution is added dropwise in modified FTO electrode surfaces, at room temperature from So dry, 30 μ L are then added dropwise and contain 2 mgmL-1 BS3PBS and be incubated 1 h at room temperature, 30 μ L are then added dropwise again 0.38 mg·mL-1Capture antibody A b1, be incubated 1 h at room temperature, then be incubated overnight under 4 DEG C, 100% moisture-saturated environment （12h）Afterwards, washed respectively with lavation buffer solution and PBS, then 30 min are incubated with 20 μ L Block buffers, finally distinguished 3 min are washed with lavation buffer solution and PBS, are stored for future use at 4 DEG C.
Step 3. in, the lavation buffer solution is：Containing 0.05 %（w/v）Tween 20 PBS（0.05 M, pH 7.0）；The Block buffer is：Containing 5 %（w/v）Bovine serum albumin（BSA）PBS（0.05 M, pH 7.0）.
4. test process：
To carry out immune response and electro-chemical test, the electrochemical immunosensor obtained by above-mentioned preparation is first different with 30 μ L The CEA dilutions or blood serum sample of concentration are incubated 50 min at room temperature, are then washed respectively with lavation buffer solution and PBS Wash 1.5 min.Again with 30 μ L steps 2. products therefrom HRP ︱ NH2-TiO2Nano-pillar ︱ Ab2 are incubated 60 min at room temperature, and divide 3 min are not washed with lavation buffer solution and PBS.Then, by the electrochemical immunosensor, reference electrode, to electrode It is placed in the electrochemical cell containing 5 mL PBSs, then by quinhydrones（The mM of final concentration 2）And hydrogen peroxide（Final concentration 1 mM）Inject in this battery, carry out time current curve scanning under -0.2 V respectively before and after hydrogen peroxide is added, with quantitative Determine CEA.
SEM and TEM is used to characterize TiO2Nano-wire array and TiO2The pattern of nano-pillar.As shown in figure 1, pure TiO2Receive The SEM plans of nanowire arrays（A）With SEM sectional views（B）After showing hydro-thermal reaction, the white film on FTO surfaces be it is fine and close, The nano-wire array of vertical arrangement, they possess rectangular cross section, its average diameter and length respectively may be about 150-180 nm and 1.5-2μm." etching, doping, deposition " modified, TiO simultaneously2Nano-wire array surface covers one layer of black deposit, such as SEM schemes（C）,（D）Shown, they are in honeycomb type layer structure, and its surface area is very big, contribute to the substantial amounts of biology of further load Molecule.SEM and TEM figures are additionally operable to characterize prepared TiO2Nano-pillar, as SEM schemes（E）, TEM figures（F）It is shown, all TiO2Receive Meter Zhu Wei cylindrical structurals, its average diameter are about 20 nm, and average length is about 180 nm, and this contributes to the negative of biomolecule Carry.
To characterize TiO2Nano-wire array before modified after crystalline structure and composition, Fig. 2 illustrates their XRD.Fig. 2 A illustrates the diffraction maximum of FTO substrates and marked with asterisk to be compared with facilitating.Fig. 2 b are pure TiO2The XRD of nano-wire array, Two characteristic diffraction peaks wherein at 36.1 ° and 62.7 ° correspond to cubic rutile TiO respectively2(101) and (002) crystal face.It is triple modified, as shown in Fig. 2 d, TiO2The MoS of nano-wire array surface deposition2At 14.5 °, 33.8 ° and Occur three obvious characteristic diffraction peaks at 59.5 °, correspond to MoS respectively2(002), (100) and (110) crystal face. Immersion removes surface MoS in chloroazotic acid2Afterwards, the TiO of the etching/doping2The XRD spectrum of nano-wire array（Fig. 2 c）With pure TiO2Receive Nanowire arrays（Fig. 2 b）There is similar diffraction maximum.With pure TiO2Nano-wire array is compared, and all diffraction maximums are without obvious skew.
Electrochemical impedance（EIS）Method monitors the assembling process of immunosensor：
As the effective tool for assessing dynamic process and modified electrode interface performance, electrochemical impedance is used to monitoring originally Inventive embodiments 1 are prepared detecting the assembling process of CEA electrochemical immunosensor.
Impedance experiment is in the 5 mM K containing 0.1 M KCl electrolyte3[Fe ︱ CN)6] ︱ K4[Fe(CN)6] enter in solution OK, it is 0.230 V to apply DC voltage, the mV of laminated thereto 5 alternating voltage.Impedance experiment is all in 100 KHz~100 Record nyquist diagram in mHz frequency ranges.All nyquist diagrams in Fig. 3 include a semicircle in high frequency region, and it is right Electronics is answered to shift resistance（Ret）；A straight line portion is showed in low frequency range, its corresponding diffusion controlled process.Half circular diameter is with repairing The potassium ferricyanide electron transmission coefficient of decorations electrode surface is inversely proportional, and this provides a vision for our judgement material electric conductivity and sentenced Disconnected standard.As shown in figure 3, naked FTO electrodes（Curve a）The semicircle of a very little is showed, represents sheet resistance very little, belongs to expansion Dissipate control process.In contrast, pure TiO2Nano-wire array（Curve f）Radius is maximum, shows TiO2Electric conductivity is poor.But together When " etching, doping, deposition " modified, TiO2Nano-wire array（Curve b）It is obviously reduced, shows while Mo- is adulterated and MoS2It is heavy Product greatly strengthen TiO2The electric conductivity of nano-wire array.In curve c-e, triple modified TiO2Nano line array electrode is successively Ab1, BSA and marking signal antibody A b2 are modified, it can be seen that along with progressively modification, half circular diameter Gradually increase, this hinders electrochemical probe to electrode surface mainly due to the low electric conductivity and steric effect of protein Electron transmission, while also show various materials and be assembled in electrode surface layer by layer.
The optimization of electrochemical immunosensor testing conditions:
The pH value of test solution is to influence a key factor of enzyme sensor performance, because strong acid and strong alkali environment can Destroy the activity of biomolecule.Influence of the pH value of solution to immunosensor current-responsive is as shown in Figure 4.Current-responsive value is in pH Quickly increase with the increase of pH value in the range of 5.0-7.0, after pH is more than 7.0, start to reduce.Therefore, containing 0.10 M KCl 0.05 M PBS（pH 7.0）Buffer solution is selected as detecting liquid for detecting AFP.
Cultivation times of the CEA on immunosensor surface be influence Immunosensor Analysis performance another it is important because Element.At room temperature, the current-responsive value of CEA immunosensors is cultivated the increase of time with AFP and dramatically increased, more than 50 min After basically reach a steady state value（As shown in Figure 5）, this shows that the capture antibody A b1 of AFP and electrode surface combination reaches full With.Therefore, 50 min are elected to be to the cultivation time of this interlayer type immunoassay.
The capture antibody A b1 concentration of electrode surface is also optimized, as shown in fig. 6, just starting CEA immunosensors Current-responsive value dramatically increases with the increase of Ab1 concentration, when Ab1 concentration is more than 0.38 mg mL-1Afterwards, current-responsive value is basic It is constant, or even also slightly reduce, illustrate 0.38 mg mL-1For Ab1 concentration saturation values, electrode surface can not be carried on after exceeding, Therefore 0.38 mg mL-1It is chosen to be capture antibody A b1 optimum concentration value.
Quantitatively detect CEA:
Based on the immune response of specific sandwich type and H2O2For the HRP catalytic reactions of medium, shown in following two reaction equation：
Benzoquinones+2H+ + 2e−→ quinhydrones.
The CEA immunosensors are used for quantitative detection CEA with optimal conditions, as shown in fig. 7, adding H2O2Afterwards, the biography Sensor electric current is rapidly reached stabilization, and reduction peak current increases with the increase of CEA concentration.Calibration curve（Illustration in Fig. 7）It is aobvious Show there is good linear relationship between peak current and analyte concentration, the range of linearity is 0.001 ng/mL to 150 ng/mL, Coefficient correlation be 0.996 (n=4) under conditions of signal to noise ratio is 3, CEA test limit as little as 0.5 pg/mL, this is than many reports The immunosensor in road is low.
Reappearance, selectivity, stability test：
In order to probe into the degree of accuracy of this immunosensor and reappearance, the present invention prepares 4 electricity respectively under the same conditions Pole, analysis test, acquired results relative standard deviation are carried out under the same conditions（RSD）For 5.6 %, show this immune sensing Utensil has good accuracy and reappearance.
To assess the specificity of the immunosensor, invention introduces several possible chaff interferences, including α-first tire egg In vain（AFP）, prostate cancer proteantigen（PSA）, tumour antigen 125（CA125）, immunoglobulin G（IgG）With cow's serum egg In vain（BSA）.This immunosensor is used respectively contains above-mentioned one of which chaff interference（100 ng·mL-1）1 ngmL-1 CEA It is incubated.As shown in figure 8, wherein A, which is pure CEA, is free of chaff interference, B, C, D, E, F is respectively CEA and AFP, PSA, CA125, IgG, BSA mixture.Do not interfere with thing to compare, the curent change as caused by chaff interference is less than 5.2 %, and this shows this immunosensor With good selectivity.
In addition, the stability of this immunosensor is rung also by the electric current measured before and after they are stored 2 weeks under the conditions of 4 DEG C It should be assessed.As a result show, 90.7 % current-responsive is retained after 2 weeks, and it is good that this shows that this immunosensor has Good stability.

Claims (2)

  1. A kind of 1. TiO with triple modifications2Nano-wire array for support electrochemical immunosensor construction method, its feature It is, comprises the following steps：
    1. the TiO of triple modifications2The preparation of nano-wire array：
    TiO2The preparation of nano-wire array：The dense HCl of 13 mL are diluted with 15 mL deionized waters, 5 min is stirred at room temperature, adds After entering 300 μ L butyl titanates, continue to stir 15 min to obtain settled solution；By gained settled solution and clean FTO substrates It is transferred to together in reactor, 12 h is reacted at 150 DEG C；Room temperature is cooled to, FTO substrates is taken out, is washed with deionized simultaneously In N2Flow down drying, one layer of white films of uniform fold on FTO substrates, as TiO2Nano-wire array；
    TiO2While nano-wire array " etching, doping and deposition "：By 30 mL ultra-pure waters, 0.01 M hydroxylamine hydrochlorides, 0.005 The water vulcanized sodium of M nine, 0.002 M Sodium Molybdate Dihydrates, 50 μ L polyethylene glycol and the above-mentioned TiO being prepared2Nano-wire array is together It is placed in reactor, 24 h is reacted at 250 DEG C, be cooled to room temperature, take out FTO substrates, the FTO substrates is designated as modified FTO electricity Pole；It is washed with deionized and in N2Flow down drying；It was observed that gained TiO2Covered with one layer of black sediment on nano-wire array Thing, as MoS2Nanometer sheet；
    2. the preparation of signal antibody label：
    TiO2The preparation of nano-pillar：1.8 mL titanium tetrachlorides are added in the beaker containing 19 mL ultra-pure waters, at 0-5 DEG C of ice-water bath Stir 10 min and obtain white suspension；1.8 mL chloroforms are added, continues to stir 10 min, is then transferred in reactor, 160 DEG C reaction 12h, is cooled to room temperature, white precipitate is collected by centrifugation and washs to neutrality, then 60 DEG C of 12 h of vacuum drying, are produced TiO2Nano-pillar, store for future use at room temperature；
    TiO2The amination of nano-pillar：By 100 mg TiO2Nano-pillar is placed in containing 20 mL ethanol, the ammoniacal liquor of 1 mL 28% and 4 mL In the mixed liquor of 3- aminopropyl triethoxysilanes, 12h is stirred at room temperature to prevent TiO2Nanometer column sedimentation, is then centrifuged for point From abandoning supernatant, gained white depositions are washed, after drying, are stored for future use at room temperature, are designated as NH2-TiO2Nano-pillar；
    Horseradish peroxidase ︱ aminations TiO2The preparation of nano-pillar ︱ signal antibody Ab2 bioconjugate bodies：By 2 mg BS3Dissolving Solution A is obtained in 1 mL PBSs, then by 3 mg NH2-TiO2Nano-pillar is scattered in solution A, stirs lower add 300μL 2 mg·mL-1The horseradish peroxidase aqueous solution and be incubated 30 min at room temperature；Then 20 μ L are added containing 0.5 mg·mL-1Signal antibody Ab2 PBS, 8 h being stirred at 4 DEG C, being centrifuged, gained sediment is washed with PBS And 30 min are closed at room temperature with the PBS containing 2% bovine serum albumin, after finally being washed with PBS, it is scattered in It is standby in PBSs of 1.0 mL containing 0.1% bovine serum albumin, it is designated as HRP ︱ NH2- TiO2Nano-pillar ︱ Ab2；
    3. the structure of immunosensor：
    First, the wt% of 30 μ L 0.3 chitosan-acetic acid solution is added dropwise in modified FTO electrode surfaces, dried in the air naturally at room temperature It is dry, 30 μ L are then added dropwise and contain 2 mgmL-1 BS3PBS and be incubated 1 h at room temperature, 30 μ L are then added dropwise again 0.38 mg·mL-1Capture antibody A b1, be incubated 1 h at room temperature, then after being incubated 12h under 4 DEG C, 100% moisture-saturated environment, Washed respectively with lavation buffer solution and PBS, then 30 min are incubated with 20 μ L Block buffers, it is finally slow with washing respectively Fliud flushing and PBS wash 3 min, are stored for future use at 4 DEG C.
  2. 2. as claimed in claim 1 with the TiO of triple modifications2Nano-wire array is the structure of the electrochemical immunosensor of support Method, it is characterised in that step 3. in, the lavation buffer solution is：PBS containing 0.05 % Tween 20；The envelope Closing buffer solution is：PBS containing 5 % bovine serum albumins.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102011193A (en) * 2010-09-21 2011-04-13 南京航空航天大学 Protein modified GaN nanowire array as well as preparation method and application thereof
CN102175859A (en) * 2010-11-29 2011-09-07 浙江理工大学 TiO2 (titanium dioxide) nanowire biosensor chip and system for fast detecting Enteropathogenetic Escherichia coli (EPEC)
CN105758912A (en) * 2016-04-20 2016-07-13 济南大学 Preparation and application of nano TiO2-MoS2 photoelectric Saos-2 cell sensor

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101050790B1 (en) * 2010-01-05 2011-07-20 한국과학기술연구원 Live Cell Activity Assay
WO2012094634A2 (en) * 2011-01-07 2012-07-12 Dune Sciences, Inc. Functionalized carbon membranes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102011193A (en) * 2010-09-21 2011-04-13 南京航空航天大学 Protein modified GaN nanowire array as well as preparation method and application thereof
CN102175859A (en) * 2010-11-29 2011-09-07 浙江理工大学 TiO2 (titanium dioxide) nanowire biosensor chip and system for fast detecting Enteropathogenetic Escherichia coli (EPEC)
CN105758912A (en) * 2016-04-20 2016-07-13 济南大学 Preparation and application of nano TiO2-MoS2 photoelectric Saos-2 cell sensor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Signal-amplified platform for electrochemical immunosensor based on TiO2 nanotube arrays using a HRP tagged antibody-Au nanoparticles as probe;Zhi-Da Gao et al;《Biosensors and Bioelectronics》;20121008;第41卷;第771–775页 *
水热合成TiO2纳米线阵列及其生长机理的研究;唐立丹 等;《可再生能源》;20151231;第33卷(第12期);第1773-1777页 *
钛酸钠纳米线制备TiO2纳米线的反应条件;赵斯琴 等;《材料工程》;20151231;第43卷(第12期);第58-62页 *

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