CN106290875A - TiO with triple modifications2nano-wire array is the construction method of the electrochemical immunosensor of support - Google Patents

TiO with triple modifications2nano-wire array is the construction method of the electrochemical immunosensor of support Download PDF

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CN106290875A
CN106290875A CN201610653015.7A CN201610653015A CN106290875A CN 106290875 A CN106290875 A CN 106290875A CN 201610653015 A CN201610653015 A CN 201610653015A CN 106290875 A CN106290875 A CN 106290875A
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tio
nano
wire array
room temperature
pbs
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CN106290875B (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, first passes through water heat transfer and goes out densification, neat TiO2Nano-wire array, then modified to it, in modifying process, Mo element is successfully doped into TiO2Nano-wire array is to promote its electron transfer capacity；MoS2Sheet also by successful deposition in electrode surface；Then chitosan solution is dripped in modified TiO2Nano line array electrode surface, uses double amino crosslinker BS3Capture antibody A b1 is covalently bonded in electrode surface.Prepare cylindrical TiO by hydro-thermal method simultaneously2Monodimension nanometer material, and by its amination, then use BS3Make cross-linking agent jointly to modify horseradish peroxidase HRP and signal antibody Ab2 in TiO2Nano-pillar surface is to obtain signal antibody label.The electrochemical immunosensor using the method to prepare gained can quickly measure CEA, and sensitivity is higher, the range of linearity is relatively big, detection limit is relatively low.

Description

TiO with triple modifications2Nano-wire array is the electrochemical immunosensor of support Construction method
Technical field:
The invention belongs to electrochemical immunosensor constructing technology field, be specifically related to a kind of TiO with triple modifications2Nano wire Array is the construction method of the electrochemical immunosensor of support, and this electrochemical immunosensor can be used for detecting carcinoembryonic antigen.
Background technology:
Accurately, delicately detection tumor markers to the screening of cancer, diagnose and treat and all play vital effect.Cancer embryo Antigen (carcinoembryonic antigen, CEA) is a kind of glycoprotein that Colorectal Carcinoma produces, and is that a broad spectrum activity swells Tumor markers, it can reflect the existence of kinds of tumors, Outcome measure, the state of an illness to colorectal cancer, breast carcinoma and pulmonary carcinoma to people Development monitoring and rear health evaluating are extremely important, and health adult's change of serum C EA concentration of 97% is at 2.5 ng mI-1Below.Many Immunoassay, including fluorescence immunoassay, electrochemical enzymatic immunoassay, chemiluminescence immune assay, enzyme linked immunosorbent assay It is widely used in detecting tumor marker Deng all.In numerous immunoassays, Electrical chemiluminescence immunoassay analysis method has quick, effective With the feature such as sensitive.
Along with developing rapidly of nanometer science and technology, various nano materials, including carbon nanomaterial, graphite Alkene, silicon dioxide, quantum dot, noble metal etc. are widely used in immunosensor improving its analytical performance.Receive numerous In rice material, TiO2Nano material is big, mechanical due to its excellent chemical stability, excellent biocompatibility, specific surface area The advantages such as performance is good, simple synthetic method and environment friendly have attracted to pay close attention to widely.The most one-dimensional TiO2Nano junction Structure, such as TiO2Nanotube, is widely used in Optical Electro-Chemistry and electrochemical immunosensor.Zhi-Da Gao et al. uses TiO2Nanometer Pipe array is sensor stand, detects as signal probe with the signal antibody-Au nano-particle of horseradish peroxidase-labeled Immunoglobulin concentrations.Due to TiO2Surface area that the notable tubulose feature of nanotube is big and inner space, this immunity passes Sensor is compared with other ordinary flat electrodes, and performance is effectively strengthened.The concentration range of linearity of rabbit immunoglobulin is by it 0.1—105 ng mL-1, detection is limited to 0.01 ng mL-1。
TiO is paid close attention to despite substantial amounts of research2The photoelectrochemical behaviour of nano-wire array, but it is likely to be due to its electric conductivity relatively The shortcoming that difference, specific surface area are relatively small, limits the load capacity of electron transfer and biomolecule, thus few people is used Preparation in electrochemical sensor.It should be noted that and it is reported, chemical doping metal ion can as the efficient method of one To promote TiO2Separation of charge and electric conductivity Deng semiconductor nano material.Such as Wang et al. is prepared for W by hydro-thermal method and mixes Miscellaneous TiO2Core-shell structure copolymer nano-wire array.In etching/regrowth process, W element is doped into TiO2To improve its photoelectricity in shell Performance.And after electrochemical impedance result also shows W doping, TiO2Nano-wire array electric conductivity is obviously enhanced.
Graphene has excited preparation to be similar to the research interest of nano material in the successful Application of different field.It is reported, MoS2Comprising a Mo metal level and two S layers, Mo metal level is clipped between two S layers by Van der Waals force.Owing to it is unique Geometry, MoS2Become one of typical Graphene analog.It is essential that MoS2Have excellent with what Graphene approximated Performance, feature is easily modified on Electronic Performance, special optical characteristics, excellent mechanical performance and surface as excellent.Although MoS2In a lot of fields, such as transistor, fuel cell, energy storage etc. are obtained for extensively application, but it is in terms of electrochemical sensor Application the most limited.Vasilescu et al. is prepared for a kind of by MoS2The nano material constituted with graphene quantum dot is used for Load enzyme molecule.The electric conductivity of carbon back screen printing electrode is modifying MoS2It is greatly enhanced after-graphene quantum dot, is also simultaneously Laccase provides a biocompatible scaffold.This laccase biosensor to caffeic acid in concentration range 0.38-100 uM Having well response, its detection is limited to 0.32 μM, and sensitivity is 17.92 nA uM-1。
BS3Being a kind of double amino crosslinker, have the features such as water solublity, non-cracking performance, film impermeability, it contains one Terminal amino group reactive group (Sulfo-NHS ester group), can react with any molecule containing primary amine group.Therefore, it extensively should Crosslinking for biomolecule.
The present invention is prepared for the TiO of triple modification by two step hydro-thermal reactions2Nano-wire array.Specifically, the first step Hydro-thermal reaction prepares TiO2Nano-wire array；Second step achieves simultaneously " etch, adulterate and deposit ", MoS2Sheet is deposited on quarter Erosion/doped TiO2Nano-wire array surface.Then chitosan solution is dripped TiO after modification2Nano-wire array table Face, with BS3For amino crosslinker, capture antibody A b1 is covalently bind in its surface.Hydro-thermal method is also used for preparing cylindrical TiO2 Nano-pillar, BS3Also it is used for combining 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, carves Erosion helps lend some impetus to electron transfer plus Mo doping, strengthens electric conductivity；It addition, dimensional thinlayer MoS2The deposition of sheet can not only strengthen Electric conductivity, and electrode specific surface area can be strengthened, contribute to adsorbing more biomolecule.Thus novel 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 There are no relevant report.
Summary of the invention:
Present invention aim at developing a kind of TiO with triple modifications2Nano-wire array is the electrochemical immunosensor of support Construction method, this electrochemical immunosensor can measure carcinoembryonic antigen rapidly, and sensitivity is higher, the range of linearity relatively Greatly, detection limit is relatively low.
For achieving 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, it include as Lower step:
The TiO of the most triple modifications2The preparation of nano-wire array:
TiO2The preparation of nano-wire array: the 13 dense HCl of mL are diluted with 15 mL deionized waters, stirs 5 min under room temperature, add After entering 300 μ L butyl titanates, continue stirring 15 min to obtain settled solution；By gained settled solution and clean FTO substrate It is transferred to together in reactor, at 150 DEG C, reacts 12 h；It is cooled to room temperature, takes out FTO substrate, be washed with deionized also At N2Flowing down drying, on FTO substrate, one layer of white film of uniform fold, is TiO2Nano-wire array；
TiO2" etch, adulterate and deposit " while nano-wire array: by 30 mL ultra-pure waters, 0.01 M oxammonium hydrochloride., 0.005 M nine water cure sodium, 0.002 M Sodium Molybdate Dihydrate, 50 μ L Polyethylene Glycol and the above-mentioned TiO prepared2Nanometer linear array Row are placed in reactor together, react 24 h at 250 DEG C, are cooled to room temperature, take out FTO substrate, be washed with deionized and N2Flow down drying；Observe gained TiO2It is coated with one layer of black deposit on nano-wire array, is MoS2Nanometer sheet, this FTO Substrate is designated as the FTO electrode of modification；
2. the preparation of signal antibody label:
TiO2The preparation of nano-pillar: 1.8 mL titanium tetrachlorides are slowly added in the beaker containing 19 mL ultra-pure waters, ice-water bath 0- Stir 10 min at 5 DEG C and obtain white suspension；Add 1.8 mL chloroforms, continue stirring 10 min, then proceed to reactor In, 160 DEG C of reaction 12h, it is cooled to room temperature, centrifugal collection white precipitate also washs to neutral, then 60 DEG C of vacuum drying 12 h, Obtain TiO2Nano-pillar, room temperature for storage is standby；
TiO2The amination of nano-pillar: by 100 mg TiO2Nano-pillar is placed in containing 20 mL ethanol, 1 mL 28% ammonia and 4 mL In the mixed liquor of 3-aminopropyl triethoxysilane, stir 12h under room temperature to prevent TiO2Nano-pillar settles, and is then centrifuged for point From, abandoning supernatant, the washing of gained white depositions, dried, room temperature for storage is standby, is designated as NH2-TiO2Nano-pillar；
Horseradish peroxidase amination TiO2The preparation of nano-pillar signal antibody Ab2 bioconjugate body: by 2 mg BS3Molten Solution obtains solution A in 1 mL PBS, then by 3 mg NH2-TiO2Nano-pillar is scattered in solution A, adds under stirring Enter 300 μ L 2 mg mL-1Horseradish peroxidase aqueous solution and at room temperature hatch 30 min；It is subsequently adding 20 μ L containing 0.5 mg·mL-1The PBS of signal antibody Ab2, stirs 8 h, centrifugation at 4 DEG C, gained precipitate PBS washs And at room temperature close 30 min with the PBS containing 2% bovine serum albumin, after finally washing with PBS, it is scattered in In 1.0 mL PBS containing 0.1% bovine serum albumin, standby, it is designated as HRP NH2- TiO2Nano-pillar Ab2；
2. the structure of immunosensor:
First, the chitosan-acetic acid solution of 30 μ L 0.3 wt% is dripped at modified FTO electrode surface, naturally dries in the air under room temperature Dry, then drip 30 μ L containing 2 mg mL-1 BS3PBS and at room temperature hatch 1 h, drip 30 μ L the most again 0.38 mg·mL-1Capture antibody A b1, incubated at room temperature 1 h, then 4 DEG C, hatch 12h under 100% moisture-saturated environment after, Respectively with lavation buffer solution and PBS washing, then hatch 30 min with 20 μ L Block buffer, slow with washing the most respectively Rush liquid and PBS washs 3 min, store for future use at 4 DEG C.
Concrete, step 3. in, described lavation buffer solution is: containing 0.05 %(w/v) PBS of Tween 20 (0.05 M, pH 7.0)；Described Block buffer is: containing 5 %(w/v) PBS of bovine serum albumin (BSA) (0.05 M, PH 7.0).
Compared to the prior art, the beneficial effect of the inventive method:
It is prepared for TiO by hydro-thermal method2Nano-wire array, then uses " etch, adulterate and deposit " technology simultaneously to carry out it Triple modifications, in this modifying process, Mo element is successfully doped into TiO2Nano-wire array, can be effectively improved electron transmission speed Rate；Meanwhile, dimensional thinlayer MoS2Sheet in electrode surface by successful deposition, can not only be strengthened electric conductivity, also drastically increase electricity Ultimate ratio surface area, contributes to loading further amounts of biomolecule, thus improves the sensitivity of this sensor.
Accompanying drawing illustrates:
Fig. 1 is TiO2Nano-wire array, TiO2The SEM figure of nano-pillar and TEM scheme, and wherein A and B is respectively pure TiO2Nanometer linear array The SEM plane graph of row and sectional view；C and D is respectively the triple modified TiO of different amplification2Nano-wire array SEM puts down Face figure；E and F is respectively TiO2The SEM figure of nano-pillar and TEM scheme；
Fig. 2 is the XRD figure spectrum of different materials, and wherein a is FTO substrate;B is pure TiO2Nano-wire array；C is to soak in chloroazotic acid Surface MoS is removed after steeping 24 h2After the TiO of only etching/doping2Nano-wire array；D is triple modified TiO2Nano wire Array；
Fig. 3 is nyquist diagram, and wherein a is naked FTO substrate；B is triple modified TiO2Nano-wire array；C is triple changing TiO after property2Nano-wire array chitosan BS3Ab1；D is triple modified TiO2Nano-wire array chitosan BS3? Ab1 BSA；E is triple modified TiO2Nano-wire array chitosan BS3The Ab2 of Ab1 BSA CEA labelling；F is pure TiO2The FTO substrate that nano-wire array is modified；
Fig. 4 is the impact on immunosensor current-responsive of the present invention of the pH value of test solution；
Fig. 5 is that CEA cultivates the time impact on immunosensor current-responsive at electrode surface；
Fig. 6 is the optimization of capture antibody A b1 concentration；
Fig. 7 is the interference that CEA is measured by disturbance thing；
Fig. 8 is the change curve that reduction current increases with CEA concentration.
Detailed description of the invention:
Below in conjunction with embodiment, technical scheme is discussed in detail further, but protection scope of the present invention is not It is confined to this.
In following embodiment, used BS3Purchased from Sigma-Aldrich company limited, capture antibody A b1, signal resist Body Ab2 is purchased from double fluid Zheng Long biochemical product research department, Chengdu.
Embodiment 1:
A kind of TiO with triple modifications2Nano-wire array is the construction method of the electrochemical immunosensor of support, it include as Lower step:
The TiO of the most triple modifications2The preparation of nano-wire array:
TiO2The preparation of nano-wire array:
Before synthesis, first by several FTO substrates successively with acetone, ethanol and milli-Q water, at N2Flow down dry for standby.By 13 The dense HCl of mL (37 wt %) dilutes with 15 mL deionized waters, stirs 5 min under room temperature.It is subsequently adding 300 μ L butyl titanates, Continue stirring 15 min to obtain settled solution.Gained settled solution is transferred to together with the FTO substrate of above-mentioned washes clean In 50 mL teflon-lined autoclaves, at 150 DEG C, react 12 h.It is cooled to room temperature, takes out FTO substrate, use Deionized water wash at N2Flow down drying.On FTO substrate, uniform fold has one layer of white film, after proved i.e. by multiple method For TiO2Nano-wire array.
TiO2" etch, adulterate and deposit " while nano-wire array: by 30 mL ultra-pure waters, 0.01 M oxammonium hydrochloride., 0.005 M nine water cure sodium, 0.002 M Sodium Molybdate Dihydrate, 50 μ L Polyethylene Glycol and above-mentioned preparation-obtained TiO2Nano wire Array is placed in 50 mL teflon-lined autoclaves together, reacts 24 h, be cooled to room temperature at 250 DEG C, takes out FTO substrate, is washed with deionized and at N2Flow down drying.Observe gained TiO2One layer of black it is coated with on nano-wire array Deposit, after by multiple method prove be MoS2Nanometer sheet, this FTO substrate is designated as the FTO electrode of modification.
2. the preparation of signal antibody Ab2 label:
TiO2The preparation of nano-pillar: 1.8 mL titanium tetrachlorides are slowly added in the beaker containing 19 mL ultra-pure waters, ice-water bath At 0-5 DEG C, strong stirring 10 min obtains white suspension；Add 1.8 mL chloroforms, continue stirring 10 min, then proceed to In teflon-lined autoclave, 160 DEG C of reaction 12 h.It is cooled to room temperature, centrifugal white precipitate of collecting, difference The most neutral with deionized water and absolute ethanol washing, then 60 DEG C of vacuum drying 12 h, obtain TiO2Nano-pillar, room temperature for storage Standby.
TiO2The amination of nano-pillar: by 100 mg TiO2Nano-pillar is placed in containing 20 mL ethanol, 1 mL 28% ammonia and 4 In the mixed liquor of mL 3-aminopropyl triethoxysilane, it is stirred overnight (12h) under room temperature to prevent TiO2Nano-pillar settles.So Rear centrifugation, abandoning supernatant, the washing of gained white depositions, dried, room temperature for storage is standby, is designated as NH2-TiO2Receive Meter Zhu.
Horseradish peroxidase amination TiO2The preparation of nano-pillar signal antibody Ab2 bioconjugate body: by 2 mg BS3 It is dissolved in phosphate (PBS, the 0.02 M) buffer of 1 mL acquisition solution A, then by 3 mg NH2-TiO2Nano-pillar is disperseed In solution A, stirring is lower adds 300 μ L 2 mg mL-1Horseradish peroxidase aqueous solution and at room temperature hatch 30 min.It is subsequently adding 20 μ L containing 0.5 mg mL-1The PBS of signal antibody Ab2, stirs 8 h, centrifugation, institute at 4 DEG C Obtain the washing of precipitate PBS and at room temperature close 30 min with the PBS containing 2% bovine serum albumin.Finally use After PBS washing, it is scattered in 1.0 mL PBS containing 0.1% bovine serum albumin, standby, it is designated as HRP NH2- TiO2Nano-pillar Ab2.
3. the structure of immunosensor:
First, the chitosan-acetic acid solution of 30 μ L 0.3 wt% is dripped at modified FTO electrode surface, naturally dries in the air under room temperature Dry, then drip 30 μ L containing 2 mg mL-1 BS3PBS and at room temperature hatch 1 h, drip 30 μ L the most again 0.38 mg·mL-1Capture antibody A b1, incubated at room temperature 1 h, then 4 DEG C, overnight incubation under 100% moisture-saturated environment (12h), after, respectively with lavation buffer solution and PBS washing, then hatch 30 min with 20 μ L Block buffer, finally distinguish Wash 3 min with lavation buffer solution and PBS, store for future use at 4 DEG C.
Step 3. in, described lavation buffer solution is: containing 0.05 %(w/v) PBS (0.05 M, the pH of Tween 20 7.0)；Described Block buffer is: containing 5 %(w/v) PBS (0.05 M, pH 7.0) of bovine serum albumin (BSA).
4. test process:
For carrying out immunoreation and electro-chemical test, the above-mentioned electrochemical immunosensor of gained of preparing is first with 30 μ L variable concentrations CEA diluent or blood serum sample at room temperature hatch 50 min, the most respectively with lavation buffer solution and PBS washing 1.5 min.Again with 30 μ L step 2. products therefrom HRP NH2-TiO2Nano-pillar Ab2 at room temperature hatches 60 min, and difference 3 min are washed with lavation buffer solution and PBS.Subsequently, by this electrochemical immunosensor, reference electrode, electrode is put In the electrochemical cell containing 5 mL PBS, then by hydroquinone (final concentration 2 mM) and hydrogen peroxide (final concentration 1 mM) Inject in this battery, before and after adding hydrogen peroxide, under-0.2 V, carry out time current curve scanning respectively, with quantitative determination CEA。
SEM and TEM is used to characterize TiO2Nano-wire array and TiO2The pattern of nano-pillar.As it is shown in figure 1, pure TiO2Receive After the SEM plane graph (A) of nanowire arrays and SEM sectional view (B) all show hydro-thermal reaction, the white film on FTO surface be fine and close, Nano-wire array arranged vertically, they have rectangular cross section, its average diameter and length respectively may be about 150-180 nm and 1.5-2μm." etch, adulterate, deposit " is modified simultaneously, TiO2Nano-wire array surface covers one layer of black deposit, as SEM schemes (C), and shown in (D), they are honeycomb type layer structure, and its surface area is very big, contributes to loading substantial amounts of biology further Molecule.SEM and TEM figure is additionally operable to characterize prepared TiO2Nano-pillar, as SEM schemes (E), shown in TEM figure (F), all TiO2Receive Meter Zhu Wei cylindrical structural, its average diameter is about 20 nm, and average length is about 180 nm, and this contributes to the negative of biomolecule Carry.
For characterizing TiO2Nano-wire array before modified after crystalline structure and composition, Fig. 2 illustrates their XRD figure.Fig. 2 A illustrates the diffraction maximum of FTO substrate and compares to facilitate with asterisk labelling.Fig. 2 b is pure TiO2The XRD figure of nano-wire array, Wherein two characteristic diffraction peaks the most corresponding four directions rutile TiO at 36.1 ° with 62.7 °2(101) and (002) crystal face.Triple modified, as shown in Fig. 2 d, TiO2The MoS of nano-wire array surface deposition2At 14.5 °, 33.8 ° Three obvious characteristic diffraction peaks are occurred in that, the most corresponding MoS at 59.5 °2(002), (100) and (110) brilliant Face.Chloroazotic acid soaks and removes surface MoS2After, the TiO of this etching/doping2XRD figure spectrum (Fig. 2 c) of nano-wire array is with pure TiO2Nano-wire array (Fig. 2 b) has similar diffraction maximum.With pure TiO2Nano-wire array is compared, and all diffraction maximums are without the most partially Move.
The assembling process of electrochemical impedance (EIS) method monitoring immunosensor:
As assessment dynamic process and the effective tool of modified electrode interface performance, electrochemical impedance is used to monitor the present invention Embodiment 1 is prepared in order to detect the assembling process of the electrochemical immunosensor of CEA.
Impedance experiment is at the 5 mM K containing 0.1 M KCl electrolyte3[Fe CN)6] K4[Fe(CN)6] solution enters OK, applied DC voltage is 0.230 V, the alternating voltage of laminated thereto 5 mV.Impedance experiment is all at 100 KHz～100 Record nyquist diagram in mHz frequency range.All nyquist diagrams in Fig. 3 all comprise a semicircle at high frequency region, and it is right Answer electron transfer resistance (Ret)；A straight line portion is presented, its corresponding diffusion controlled process in low frequency range.Half circular diameter with repair The potassium ferricyanide electron transmission coefficient of decorations electrode surface is inversely proportional to, for us, this judges that material conductivity provides a vision and sentences Disconnected standard.As it is shown on figure 3, naked FTO electrode (curve a) presents a semicircle the least, represents sheet resistance the least, belongs to expansion Dissipate control process.In contrast, pure TiO2(curve f) radius is maximum, shows TiO for nano-wire array2Electric conductivity is poor.But, with Time " etch, adulterate, deposit " modified, TiO2(curve b) is obviously reduced nano-wire array, shows Mo-doping simultaneously and MoS2Heavy Amass and greatly strengthen TiO2The electric conductivity of nano-wire array.In curve c-e, triple modified TiO2Nano line array electrode is successively It is modified Ab1, BSA and marking signal antibody A b2, it can be seen that along with progressively modification, half circular diameter Being gradually increased, this, mainly due to the low electric conductivity of protein and steric effect, hinders electrochemical probe to electrode surface Electron transmission, also show various material simultaneously and is assembled in electrode surface layer by layer.
The optimization of electrochemical immunosensor testing conditions:
The pH value of test solution is the key factor affecting enzyme sensor performance, because strong acid and strong alkali environment all can destroy The activity of biomolecule.PH value of solution is on the impact of immunosensor current-responsive as shown in Figure 4.Current-responsive value is at pH 5.0- Quickly increase along with the increase of pH value in the range of 7.0, after pH is more than 7.0, start to reduce.Therefore, containing 0.10 M KCl's 0.05 M PBS(pH 7.0) buffer be selected as detect liquid for detecting AFP.
CEA the cultivation time on immunosensor surface be affect another of Immunosensor Analysis performance important because of Element.At room temperature, the current-responsive value of CEA immunosensor is cultivated the increase of time with AFP and is dramatically increased, more than 50 min After basically reach a steady state value (as shown in Figure 5), this shows that AFP reaches full with the combination of capture antibody A b1 of electrode surface With.Therefore, 50 min are elected to be the cultivation time of this sandwich type immunoassay.
The capture antibody A b1 concentration of electrode surface has been also carried out optimizing, and as shown in Figure 6, has just started CEA immunosensor Current-responsive value dramatically increases, when Ab1 concentration is more than 0.38 mg mL with the increase of Ab1 concentration-1After, current-responsive value is basic Constant, the most slightly reduce, 0.38 mg mL is described-1For Ab1 concentration saturation value, electrode surface after exceeding, cannot be carried on, Therefore 0.38 mg mL-1It is chosen to be the optimum concentration value of capture antibody A b1.
Detection by quantitative CEA:
Based on specific sandwich type immunoreation and H2O2For the HRP catalytic reaction of medium, shown in following two reaction equations:
Benzoquinone+2H+ + 2e−→ hydroquinone.
This CEA immunosensor is used for detection by quantitative CEA with optimal conditions, as it is shown in fig. 7, add H2O2After, this biography Sensor electric current is rapidly reached stable, and reduction peak current increases with the increase of CEA concentration.Calibration curve (illustration in Fig. 7) shows Showing there is good linear relationship between peak current and analyte concentration, the range of linearity is 0.001 ng/mL to 150 ng/mL, Correlation coefficient is 0.996 (n=4). under conditions of signal to noise ratio is 3, detection limit as little as 0.5 pg/mL of CEA, this is than a lot of reports The immunosensor in road is the lowest.
Repeatability, selectivity, stability test:
In order to probe into accuracy and the repeatability of this immunosensor, the present invention prepares 4 electrodes the most respectively, Being analyzed test under the same terms, acquired results relative standard deviation (RSD) is 5.6 %, shows that this immunosensor has Good accuracy and repeatability.
For assessing the specificity of this immunosensor, invention introduces several possible chaff interference, including α-first tire egg In vain (AFP), carcinoma of prostate proteantigen (PSA), tumor antigen 125(CA125), immunoglobulin G (IgG) and Ox blood serum egg In vain (BSA).This immunosensor is respectively with containing above-mentioned one of which chaff interference (100 ng mL-1) 1 ng mL-1 CEA Hatch.As shown in Figure 8, wherein A is that pure CEA is respectively CEA and AFP, PSA, CA125, IgG without chaff interference, B, C, D, E, F, The mixture of BSA.Do not interfere with thing to compare, chaff interference the curent change caused is less than 5.2 %, and this shows this immunosensor There is good selectivity.
Additionally, the stability of this immunosensor is rung also by the electric current measured before and after they store 2 weeks under the conditions of 4 DEG C Should be assessed.Result shows, after 2 weeks, the current-responsive of 90.7 % is retained, and it is good that this shows that this immunosensor has Good stability.

Claims (2)

1. one kind with the TiO of triple modifications2Nano-wire array is the construction method of the electrochemical immunosensor of support, its feature It is, comprises the steps:
The TiO of the most triple modifications2The preparation of nano-wire array:
TiO2The preparation of nano-wire array: the 13 dense HCl of mL are diluted with 15 mL deionized waters, stirs 5 min under room temperature, add After entering 300 μ L butyl titanates, continue stirring 15 min to obtain settled solution；By gained settled solution and clean FTO substrate It is transferred to together in reactor, at 150 DEG C, reacts 12 h；It is cooled to room temperature, takes out FTO substrate, be washed with deionized also At N2Flowing down drying, on FTO substrate, one layer of white films of uniform fold, is TiO2Nano-wire array；
TiO2" etch, adulterate and deposit " while nano-wire array: by 30 mL ultra-pure waters, 0.01 M oxammonium hydrochloride., 0.005 M nine water cure sodium, 0.002 M Sodium Molybdate Dihydrate, 50 μ L Polyethylene Glycol and the above-mentioned TiO prepared2Nano-wire array is together It is placed in reactor, at 250 DEG C, reacts 24 h, be cooled to room temperature, take out FTO substrate, be washed with deionized and at N2Flow down Dry；Observe gained TiO2It is coated with one layer of black deposit on nano-wire array, is MoS2Nanometer sheet, this FTO substrate is remembered For modified FTO electrode；
2. the preparation of signal antibody label:
TiO2The preparation of nano-pillar: 1.8 mL titanium tetrachlorides are joined in the beaker containing 19 mL ultra-pure waters, at ice-water bath 0-5 DEG C Stir 10 min and obtain white suspension；Add 1.8 mL chloroforms, continue stirring 10 min, then proceed in reactor, 160 DEG C reaction 12h, is cooled to room temperature, centrifugal collect white precipitate and washs to neutral, and then 60 DEG C are vacuum dried 12 h, to obtain final product TiO2Nano-pillar, room temperature for storage is standby；
TiO2The amination of nano-pillar: by 100 mg TiO2Nano-pillar is placed in containing 20 mL ethanol, 1 mL 28% ammonia and 4 mL In the mixed liquor of 3-aminopropyl triethoxysilane, stir 12h under room temperature to prevent TiO2Nano-pillar settles, and is then centrifuged for point From, abandoning supernatant, the washing of gained white depositions, dried, room temperature for storage is standby, is designated as NH2-TiO2Nano-pillar；
Horseradish peroxidase amination TiO2The preparation of nano-pillar signal antibody Ab2 bioconjugate body: by 2 mg BS3Dissolve Solution A is obtained, then by 3 mg NH in 1 mL PBS2-TiO2Nano-pillar is scattered in solution A, and stirring is lower to add 300μL 2 mg·mL-1Horseradish peroxidase aqueous solution and at room temperature hatch 30 min；It is subsequently adding 20 μ L containing 0.5 mg·mL-1The PBS of signal antibody Ab2, stirs 8 h, centrifugation at 4 DEG C, gained precipitate PBS washs And at room temperature close 30 min with the PBS containing 2% bovine serum albumin, after finally washing with PBS, it is scattered in In 1.0 mL PBS containing 0.1% bovine serum albumin, standby, it is designated as HRP NH2- TiO2Nano-pillar Ab2；
3. the structure of immunosensor:
First, the chitosan-acetic acid solution of 30 μ L 0.3 wt% is dripped at modified FTO electrode surface, naturally dries in the air under room temperature Dry, then drip 30 μ L containing 2 mg mL-1 BS3PBS and at room temperature hatch 1 h, drip 30 μ L the most again 0.38 mg·mL-1Capture antibody A b1, incubated at room temperature 1 h, then 4 DEG C, hatch 12h under 100% moisture-saturated environment after, Respectively with lavation buffer solution and PBS washing, then hatch 30 min with 20 μ L Block buffer, slow with washing the most respectively Rush liquid and PBS washs 3 min, store for future use at 4 DEG C.
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, described lavation buffer solution is: containing the PBS of 0.05 % Tween 20；Described envelope Closing buffer is: containing the PBS of 5 % bovine serum albumins.
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