CN105486873B - With TiO2Nanometer tube composite materials are the construction method of the electrochemical immunosensor of oriented load support and trace labelling thing - Google Patents

With TiO2Nanometer tube composite materials are the construction method of the electrochemical immunosensor of oriented load support and trace labelling thing Download PDF

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CN105486873B
CN105486873B CN201510893519.1A CN201510893519A CN105486873B CN 105486873 B CN105486873 B CN 105486873B CN 201510893519 A CN201510893519 A CN 201510893519A CN 105486873 B CN105486873 B CN 105486873B
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tnt
pbs
pani
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solution
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CN105486873A (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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles

Abstract

The present invention relates to one kind is with TiO2Nanometer tube composite materials are the construction method of the electrochemical immunosensor of oriented load support and trace labelling thing, which finally synthesizes GNPs PANI TNT composites by hydro-thermal method, chemical oxidative polymerization and trisodium citrate reduction method respectively, is scattered in chitosan solution and Deca is in electrode surface.With BS3Protein G ' is covalently bind in into shitosan surface for double amino crosslinkers, for oriented load capture antibody (Ab1).BS3Also it is used for combining horseradish peroxidase（HRP）And signal antibody（Ab2）To prepare trace labelling thing.The electrochemical immunosensor that gained is prepared using the inventive method can quickly determine α AFPs, and sensitivity is higher, the range of linearity is larger, test limit is relatively low.

Description

With TiO2Nanometer tube composite materials are the electrification of oriented load support and trace labelling thing Learn the construction method of immunosensor
Technical field
The invention belongs to electrochemical immunosensor constructing technology field, and in particular to one kind is with TiO2Nanotube composite Expect the construction method of the electrochemical immunosensor for oriented load support and trace labelling thing, the electrochemical immunosensor can For detecting α-fetoprotein.
Background technology
Accurately, the protein related to disease is delicately detected to many modern study fields, including biochemistry, biology Medical science and clinical diagnosises are all most important.Especially morning of the Clinical detection protein related to tumor and infectious disease to disease Phase is detected and reliability prediction is most important.Alpha-fetoprotein（α-fetoprotein, AFP）It is one kind master of fetal development early stage Serum albumin is wanted, internal in the adult of health, its value is usually less than 25 ng/ml, and the rising of Serum Alpha Fetoprotein is to constitutional Diagnosing cancer of liver has great importance.Many immunoassays, including fluorescence immunoassay, electrochemical enzymatic immunoassay, chemistry Luminescence immunoassay, enzyme linked immunosorbent assay etc. are widely used in detecting tumor marker.In numerous immunoassays, electricity Chemo-immunity analytic process is most quick, effective and sensitive analysis method.
With developing rapidly for nanometer science and technology, various nano materials, including carbon nanomaterial, Graphene Nano material, quantum dot, noble metal etc. are widely used in immunosensor for improving its analytical performance.For example, Juan Immunosensor probe is made at the bionical interface that Tang et al. carbon nano-particles are modified, and uses erose gold nano grain Used as trace labelling thing, for detecting AFP, its range of linearity is 0.02-4.0 ng/mL to the HRP-anti-AFP of labelling, is detected It is limited to 10 pg/mL.Ganesh K. Parshetti et al. gold nano/chitosan-modified glass-carbon electrode, uses dumb-bell shape Au- Fe3O4Heterojunction structure marking signal antibody A b2, for detecting AFP, its range of linearity is 0.01-40 ng/mL, and detection is limited to 2.3 pg/mL.But there is the problems such as range of linearity is narrower, and test limit is higher, stability is relatively low in these immunosensors, because This needs with new technique, method and material to prepare the immunosensor with more preferable performance.
In numerous nano materials, TiO2Nanotube is as its specific surface area is big, the letter of good mechanical stability, synthetic method Just, the inherent advantage such as environment friendly, nontoxic, tubular structure and excellent biocompatibility has attracted extensive concern.So And, due to TiO2The surface inertness of nano material, it is difficult to antibody, antigen are directly carried on its surface.To overcome these shortcomings, Cross-linking method, covalent coupling method and investment etc. are used in TiO2Fixing biological molecules in nano material.Importantly, TiO2Nanotube is never used as trace labelling thing in immunoassay.
BS3It is a kind of pair of amino crosslinker, the features such as with water solublity, non-cracking performance, film impermeability, it contains one Terminal amino group reactive group（Sulfo-NHS ester groups）, can be with any reaction of the molecule containing primary amine group.Therefore, which extensively should For the crosslinking of biomolecule.
Protein A and Protein G can be used to oriented load antibody.Qiang Zhu et al. modify working electrode with Nafion, By Protein A be adsorbed in electrode surface for load capture antibody A b1, with a large amount of gold nano grains of load and carboxyl functional group Graphene film two anti-@redox probes are loaded as label, for multiplexing detection AFP, CEA and Streptococcus suis 2（SS2）.Wherein the range of linearity of AFP is 0.016-50 ng/mL, detects and is limited to 5.4 pg/mL.Jeremy M. Fowler Et al. be also carried out a series of report researchs, they are support with thiolated protein G loads more in immunosensor surface orientation Capture antibody A b1 of a large amount, tests for immunoassay.However, as Protein G can also be combined with the Fab fragments of IgG, this The oriented load of antibody may be destroyed, so as to cause interference with.The egg that these shortcomings can be produced by gene truncated protein G White G ' and be addressed, Protein G ' still has affinity to IgG, but it has lacked albumin, Fab fragments and film combination position Point.As Protein G ' can only be with reference to the Fc fragments of IgG, therefore it can be more efficiently used for orientation capture antibody A b1, and make it Antigen binding site be exposed to outside and carry out combining target antigen.
The present invention is finally synthesized by hydro-thermal method, chemical oxidative polymerization and trisodium citrate reduction method respectively GNPs-PANI-TNT composites, are scattered in chitosan solution and Deca is in electrode surface.With BS3It is crosslinked for amino Protein G ' is covalently bind in shitosan surface by agent, captures antibody A b1 for oriented load.BS3Also it is used for reference to HRP and letter Number antibody is preparing trace labelling thing.Instant invention overcomes the range of linearity occurred in existing report is narrower, test limit it is higher with And the shortcomings of less stable, yet there are no relevant report.
The content of the invention
Present invention aim to overcome that prior art defect, there is provided one kind is with TiO2Nanometer tube composite materials are oriented load The construction method of the electrochemical immunosensor of support and trace labelling thing, the electrochemical immunosensor rapidly can be determined Alpha-fetoprotein, and sensitivity is higher, the range of linearity is larger, test limit is relatively low.
For achieving the above object, the present invention is adopted the following technical scheme that：
One kind is with TiO2Nanometer tube composite materials are the electrochemical immunosensor of oriented load support and trace labelling thing Construction method, which comprises the steps：
1. the preparation of GNPs-PANI-TNT composites：
TiO2The preparation of nanotube：By 0.6 g TiO2After nano-particle and 120 mL, the NaOH aqueous solution of 10 M, It is transferred in reactor, 48 h of hydro-thermal reaction at 120 ± 5 DEG C, abandoning supernatant, after gained white semifinished product washing, at 60 DEG C Under the conditions of be dried 2 h, it is standby, be designated as TNTs；
Polyaniline-TiO2The synthesis of nanometer tube composite materials：By 0.2660 g TNTs, 8.57 mL, 0.1 M aniline hydrochloric acid The mixed in hydrochloric acid of 0.1 M of liquid and 10 mL, under 0-5 DEG C of ice bath, 30 min of magnetic agitation is obtaining uniform dispersion liquid；So In 2 h, the Ammonium persulfate. hydrochloric acid solution of 8.57 mL, 0.1 M, resulting solution is added to continue to react 3 h under ice bath afterwards, room temperature is quiet Put overnight, solid-liquid separation, it is that grind into powder after 3 h is dried at 7,60 DEG C that gained sediment is washed with deionized to pH, standby With being designated as PANI-TNT；
Gold nano grain-polyaniline-TiO2The preparation of nanometer tube composite materials：Take 18 mg PANI-TNT and 18 mL to surpass Pure water, mixes, adds 1 mL, the HAuCl of 0.01 M under agitation4Solution and 1 mL, the end-capping reagent Fructus Citri Limoniae of 0.01 M Sour three sodium solutions, then add the NaBH of 0.1 M of 1 mL brand-news in 20 min4 Solution, continues 0.5 h of stirring under room temperature After stand overnight, centrifugation, precipitate washing after, be dried overnight at 60 DEG C, it is standby, be designated as GNPs-PANI-TNT；
2. the preparation of marking signal antibody A b2：
Amination TiO2The preparation of nanotube：By 100 mg TNTs and 20 mL ethanol, 1 mL, 28% ammonia and 4 mL 3- Aminopropyl triethoxysilane（APTES）After mixing, mechanical agitation overnight to prevent TNTs from precipitating, centrifugation, supernatant discarded Liquid, after the washing of gained solid residue, drying, stores for future use under room temperature, is designated as NH2-TNT；
Horseradish peroxidase ︱ aminations TiO2The preparation of nanotube ︱ signal antibody bioconjugate bodies：By 2 mg BS3It is molten Solution obtains solution A in 0.5 mL, the PBS of 0.02 M, then by 3 mg NH2- TNT is scattered in solution A, stirring Lower addition 350 μ L, 2 mgmL-1Horseradish peroxidase（HRP）Aqueous solution is simultaneously incubated 30 min, Ran Houjia at room temperature Enter 20 μ L containing 0.6 mgmL-1The PBS of signal antibody Ab2（0.02 M）, at 4 DEG C, stir 4 h, centrifugation, institute Obtain precipitate to be washed with PBS and use the PBS containing 2% bovine serum albumin to close 30 min at room temperature, finally After being washed with PBS, it is scattered in the 1.0 mL PBSs containing 0.1% bovine serum albumin, it is standby, it is designated as HRP ︱ NH2- TNT ︱ Ab2；
3. the structure of immunosensor：
First, by 5 mg steps, 1. gained GNPs-PANI-TNT is scattered in HAc solution of 1 mL containing 0.2% shitosan In, ultrasonic 20 min takes 5 μ L dispersant liquid drops and is added in the good glassy carbon electrode surface of pretreatment, room temperature to obtain uniform dispersion liquid It is lower to dry naturally；Then 2 mgmL are contained in 10 μ L of glassy carbon electrode surface Deca-1 BS3PBS（0.02 M）And 1 h, 10 μ L of subsequent Deca, 1 mgmL are incubated under room temperature-1 Protein G ', be incubated 60 min under room temperature, immerse PBS Then the glass-carbon electrode is incubated 30 min with 5 μ L Block buffers, then uses lavation buffer solution respectively by 3 min of middle washing （PBST）3 min are washed with PBS, finally by 5 μ L, 0.44 mgmL-1Capture antibody A b1 Deca in glass carbon electricity 1 h is incubated under pole surface, room temperature, then after overnight incubation under 4 DEG C, 100% moisture-saturated environment, respectively with lavation buffer solution and PBS is washed, and obtains final product electrochemical immunosensor.
Specifically, step 3. in, the lavation buffer solution（PBST）For：Containing 0.05 %（w/v）The PBS bufferings of Tween 20 Liquid（0.05 M, pH 7.4）；
Step 3. in, the Block buffer is：Containing 5 %（w/v）Bovine serum albumin（BSA）PBS（0.05 M, pH 7.4）.
Compared to the prior art, the inventive method beneficial effect of the invention：
GNPs-PANI-TNT composites are introduced in electrode surface, the electric conductivity and biology of the immunosensor is increased The compatibility.By Deca Protein G ' energy oriented load Ab1, the joint efficiency of Ab1 is improved.Meanwhile, with Protein A phases Than Protein G ' have the orientation capture antibodies ability of the wider suitability and Geng Gao.Using TiO2Nanotube specific surface area Greatly, the advantage of good biocompatibility, by BS3Enzyme and antibody are fixed on into amination TiO2Nanotube surface is used as labeled immunoglobulin Label, can improve the load capacity of enzyme and antibody, so as to improve sensitivity and the test limit of the sensor.
Description of the drawings
Test collection of illustrative plates of the Fig. 1 for different materials, wherein, A is schemed for the SEM of TNTs, and B is schemed for the TEM of TNTs, and C is PANI- The SEM figures of TNT, D and E are respectively the SEM figures of GNPs-PANI-TNT composites and its partial enlargement, and F is GNPs-PANI- The EDX figures of TNT composites；
XRD spectrums of the Fig. 2 for different materials, wherein, a is TNTs, b is PANI, and c is GNPs-PANI-TNT；
FT-IR collection of illustrative plates of the Fig. 3 for different materials, wherein, a is TNTs, and b is NH2- TNT, c are PANI, and d is GNPs- PANI-TNT；
Fig. 4 is nyquist diagram, wherein, a is naked GCE, and b is GNPs-PANI-TNT ︱ shitosans, and c is GNPs-PANI- TNT ︱ shitosan ︱ Protein Gs ', d are GNPs-PANI-TNT ︱ shitosan ︱ Protein G ' ︱ BSA, and e is GNPs-PANI-TNT ︱ shitosan ︱ Protein G ' ︱ BSA ︱ Ab1, f are the GCE of GNPs-PANI-TNT ︱ shitosan ︱ Protein G ' ︱ BSA ︱ Ab1 ︱ AFP ︱ Ab2 modifications；
Fig. 5 is impact of the pH value of test solution to immunosensor current-responsive of the present invention；
Fig. 6 is impact of the cultivation time to immunosensor current-responsive of the present invention；
Fig. 7 is the change curve that reduction peak current increases with AFP concentration；
Fig. 8 is specificity of the immunosensor of the present invention to the test solution of different compositions, wherein, A：5 ngmL-1AFP； B：5 ngmL-1 AFP + 100 ngmL-1 CEA；C：5ngmL-1 AFP + 100 ngmL-1PSA；D：5 ngmL-1 AFP + 100 ngmL-1CA125；E：5 ngmL-1 AFP + 100 ngmL-1IgG；F：5 ngmL-1 AFP + 100 ngmL-1 BSA。
Specific embodiment
With reference to embodiments technical scheme is further discussed in detail, but protection scope of the present invention It is not limited thereto.
In following embodiments, used BS3, Protein G ' be purchased from Sigma-Aldrich company limited, capture antibody Ab1, signal antibody Ab2 are purchased from Chengdu double fluid Zheng Long biochemical products research department.
Embodiment 1
One kind is with TiO2Nanometer tube composite materials are the electrochemical immunosensor of oriented load support and trace labelling thing Construction method, which comprises the steps：
1. the preparation of GNPs-PANI-TNT composites：
TiO2The preparation of nanotube：By 0.6 g TiO2After nano-particle and 120 mL, the NaOH aqueous solution of 10 M, It is transferred in the autoclave with politef as liner, 48 h of hydro-thermal reaction at 120 DEG C, abandoning supernatant, gained are white 0.1 M HCl of color semifinished product are washed three times, and it is 7 that then deionized water is washed till pH, and products obtained therefrom uses milli-Q water again Three times, centrifugation is dried 2 h under the conditions of 60 DEG C, standby, is designated as TNTs；
Polyaniline-TiO2The synthesis of nanometer tube composite materials：By 0.2660 g TNTs, 0.1 M containing 8.57 mL aniline The mixed in hydrochloric acid of 0.1 M of hydrochloric acid and 10 mL, under 0-5 DEG C of ice bath, 30 min of magnetic agitation is obtaining uniform dispersion liquid； Then the Ammonium persulfate. hydrochloric acid solution of 8.57 mL, 0.1 M is added dropwise in 2 h, resulting solution continues to react 3 h under ice bath, It is stored at room temperature overnight, solid-liquid separation, it is 7 that gained bottle green precipitate is washed with deionized to pH, 60 in vacuum drying oven Grind into powder after 3 h is dried at DEG C, it is standby, it is designated as PANI-TNT；
Gold nano grain-polyaniline-TiO2The preparation of nanometer tube composite materials：Take 18 mg PANI-TNT and 18 mL to surpass Pure water, mixes, adds 1 mL, the HAuCl of 0.01 M under agitation4Solution and 1 mL, the end-capping reagent Fructus Citri Limoniae of 0.01 M Sour three sodium solutions, are then added dropwise over the NaBH of 0.1 M of 1 mL brand-news in 20 min4 Solution（0-4 DEG C）, under room temperature Stand overnight after continuing 0.5 h of stirring, centrifugation, after precipitate ultra-pure water and washing with alcohol, 60 in vacuum drying oven It is dried overnight at DEG C, it is standby, it is designated as GNPs-PANI-TNT；
2. the preparation of marking signal antibody A b2：
Amination TiO2The preparation of nanotube：By 100 mg TNTs and 20 mL ethanol, 1 mL, 28% ammonia and 4 mL 3- Aminopropyl triethoxysilane（APTES）After mixing, mechanical agitation overnight to prevent TNTs from precipitating, centrifugation, supernatant discarded Liquid, gained solid residue milli-Q water three times, be dried under the conditions of 60 DEG C after, store for future use under room temperature, be designated as NH2- TNT；
Horseradish peroxidase ︱ aminations TiO2The preparation of nanotube ︱ signal antibody bioconjugate bodies：By 2 mg BS3It is molten Solution is in 0.5 mL, the PBS of 0.02 M（pH 7.4）Middle acquisition solution A, then by 3 mg NH2- TNT is scattered in solution In A, stirring is lower to add 350 μ L, 2 mgmL-1HRP aqueous solutions and cultivate 30 min at room temperature, be subsequently adding 20 μ L and contain 0.6 mg·mL-1The PBS of signal antibody Ab2（0.02 M）, at 4 DEG C, it is slowly stirred 4 h, centrifugation, gained precipitation Thing is washed with PBS and uses the PBS containing 2% bovine serum albumin to close 30 min at room temperature, finally slow with PBS After rushing liquid washing, it is scattered in the 1.0 mL PBSs containing 0.1% bovine serum albumin, it is standby, it is designated as HRP ︱ NH2- TNT ︱ Ab2；
3. the structure of immunosensor：
First, by 5 mg steps, 1. 0.2 M HAcs of gained GNPs-PANI-TNT and 1 mL containing 0.2% shitosan is molten Liquid mixes, and ultrasonic 20 min takes 5 μ L dispersant liquid drops and is added in the good glass-carbon electrode of pretreatment to obtain uniform dispersion liquid, then （GCE）Surface, dries under room temperature naturally；Then in 10 μ L of glassy carbon electrode surface Deca, 2 mgmL-1 BS3PBS （0.02 M）And 1 h is incubated at room temperature, immediately 10 μ L of Deca, 1 mgmL-1 Protein G ', be incubated 60 under room temperature Min, washs 3 min in immersion PBS, then is incubated 30 min to close with 5 μ L Block buffers by the glass-carbon electrode The nonspecific activity site that may be remained, then 3 min are washed with lavation buffer solution and PBS respectively, finally by 5 μ L 0.44 mg·mL-1Capture antibody A b1 Deca 1 h is incubated under the glassy carbon electrode surface, room temperature, then in 4 DEG C, 100% humidity Under saturated environment after overnight incubation, washed respectively to remove the capture antibody of physical absorption with lavation buffer solution and PBS Ab1, obtains final product electrochemical immunosensor.Store for future use at 4 DEG C.
Specifically, step 3. in, the lavation buffer solution（PBST）For：Containing 0.05 %（w/v）The PBS bufferings of Tween 20 Liquid（0.05 M, pH 7.4）；
Step 3. in, the Block buffer is：Containing 5 %（w/v）Bovine serum albumin（BSA）PBS（0.05 M, pH 7.4）.
4. test process：
To carry out immunoreation and electro-chemical test, the electrochemical immunosensor obtained by above-mentioned preparation is first different with 5 μ L The AFP diluents of concentration or blood serum sample are incubated 50 min at room temperature, then use lavation buffer solution respectively（PBST）It is slow with PBS Rush liquid and wash 1.5 min.Again with 5 μ L steps 2. products therefrom HRP ︱ NH2- TNT ︱ Ab2 are incubated 50 min at room temperature, and divide Lavation buffer solution is not used（PBST）3 min are washed with PBS.Subsequently, by the electrochemical immunosensor, reference electrode, Electrode is placed in the electrochemical cell containing 10 mL PBSs, then by hydroquinone（2 mM of ultimate density）And hydrogen peroxide （1 mM of ultimate density）Inject in this battery, in the range of 50 mV of pulse amplitude, 50 ms of pulse width, 0.2～-0.2 V Carry out differential pulse voltammetry（DPV）Scanning, to quantitative determine AFP.
The sign of composite：
To characterize the group of the pattern and trielement composite material of TNTs, PANI-TNT, GNPs-PANI-TNT composite Into Fig. 1 illustrates the TEM figures of their SEM figures and TNTs.Can be seen that from the A of Fig. 1, prepared TNTs is dispersed, With clearly elongated tube-like condition, the about hundreds of nanometer of its length, diameter are less than 15 nm.And, it is obvious without finding P25 granules, this shows TiO2Nano-particle is fully converted to TiO Jing after hydro-thermal reaction2Nanotube.Also illustrate in the B of Fig. 1 Characterizing its form, TNTs has elongated tube-like condition to the TEM figures of TNTs as can be seen from Figure, and its specific surface area is big, very It is suitable for loading biomolecule as the label of electrochemical immunosensor.Can be clearly seen when aniline oxygen in the C of Fig. 1 After change is aggregated on TNTs, bundled per several TNTs at random, this is covered in what is caused on TNTs mainly due to PANI. C in Fig. 1 has been well demonstrated that Jing after chemical oxidising polymerisation PANI-TNT composites successfully synthesize.When GNPs is by chemistry After reducing process is deposited on PANI-TNT composites, the structure and pattern of GNPs-PANI-TNT composites are illustrated in Fig. 1's In D and E, the homodisperse spherical particle in PANI-TNT nano composite materials surface is clearly visible from figure, and without any poly- Phenomenon is closed, about 13 nm of average diameter of particles this demonstrate that GNPs is successfully supported in PANI-TNT nano materials.
To probe into the elementary composition of GNPs-PANI-TNT nano composite materials, the present invention has also carried out EDX analyses（Such as Fig. 1 In F shown in）.From TNTs, C peaks are from PANI at Ti peaks and O peaks in figure.However, the another of PANI is not observed from figure One characteristic peak-N peak, this is as it is together with one of peak overlapping of Ti elements.Additionally, going back at -2.10 keV It was observed that the GNP peaks of a high intensity.The result of these EDX collection of illustrative plates further demonstrates the successful conjunction of PANI-TNT composites Into and GNPs be successfully carried on PANI surfaces, this is consistent with SEM characterization results.
Fig. 2 illustrates TNTs（Curve a）、PANI（Curve b）And GNPs-PANI-TNT（Curve c）Typical XRD figure Spectrum.In curve aFive characteristic diffraction peaks occurred at 9.0 °, 24.4 °, 28.1 °, 48.5 ° and 62.6 ° are corresponded to respectively Rhombic form TNTs's（200）,（110）,（600）,（020）With（002）Crystal face.PANI existsTo there is a crystallization at 25.3 ° Peak,To have two amorphous characteristic peaks at 14.7 ° and 20.5 °（Curve b）.At 25.3 °, the intensity of peak crystallization substantially compares two Individual amorphous peak intensity, this shows aniline atacamitum（ES-PANI）With local-crystalized structure.It is local-crystalized to stem from ES-PANI It is interiorAcross chain accumulation, thus ES-PANI possesses good electric charge transfer and electric conductivity.GNPs-PANI-TNT（Curve c） Except existing containing TNTs characteristic peaks and ES-PANIOutside for two small peaks at 16.3 ° and 20.1 °, also existFor The characteristic peak of several gold is illustrated at 38.5 °, 44.5 ° and 64.7 °, they correspond to (110) of gold nano grain respectively, And (220) crystal face diffraction (200).Curve c shows GNPs by successful deposition in PANI-TNT surfaces and trielement composite material It is successfully formed.It should be noted that stemming from TNT's and ES-PANIPeak position is equipped with small skew, and this is likely due to Strong interaction between PANI and TNT and the crystal formation that causes changes.
Fig. 3 is TNT（Curve a）、NH2-TNT（Curve b）、PANI（Curve c）And GNPs-PANI-TNT（Curve d）FT- IR collection of illustrative plates, it provides structural information of nano material, it was demonstrated that the formation of composite.TNTs is in 3000-3700 cm-1's One width absworption peak and 1631 cm-1A spike, be attributed to surface hydroxy vibration.In addition, 400-700 cm-1 Another characteristic absorption peak belong to the Ti-O stretching vibrations and Ti-O-Ti bridge vibrations of nano material（Curve a）.Such as curve Shown in b, primary amine groups（-NH2）In 3400-3500 cm-1The symmetrical and asymmetric stretching vibration peak at place is stretched with the O-H on TNTs Absorption band overlaps.1625 cm-1With 1508 cm-1The peak at place belongs to 1625 cm in N-H flexural vibrations peaks, and curve b-1 The peak at place is substantially than 1631 cm in curve a-1The absorption peak intensity of place's adsorbed water molecule, these show the successful amination of TNTs. In curve c, 1299 cm-1, 1125 cm-1With 797 cm-1The absworption peak at place is respectively from secondary aromatic amine, N=Q=N（Q represents quinone Ring）Flexible and aromatic C-H out-of-plane bending vibrations.In 1563 cm-1With 1474 cm-1The peak at place be belonging respectively to quinone ring and The C=C stretching vibrations of phenyl ring.These infrared signature absorption peaks show that aniline successful conversion is polyaniline.Importantly, 1242 cm-1The peak at place belongs to C-N+•Stretching vibration peak, this again demonstrates the presence of ES-PANI.GNPs-PANI-TNT is illustrated The characteristic absorption peak of PANI（700-1600 cm-1）And the absorption of vibrations band of Ti-O-Ti and Ti-O（400-700 cm-1）（It is bent Line d）.As expected, GNPs does not produce obvious FT-IR absworption peaks.However, the formation of trielement composite material causes The skew of PANI characteristic absorption peak position, wherein 1563,1474,1299 and 1125 cm-1 (the absworption peak of curve b) It is respectively moved to 1580,1497,1309 and 1147 cm-1（Curve c）.These displacements are by strong between PANI and TNTs Interaction cause.
Electrochemical impedance（EIS）Method monitors the assembling process of immunosensor：
Electrochemical impedance collection of illustrative plates（EIS）It is the effective tool for assessing dynamic process and modified electrode interface performance. Therefore, electrochemical impedance is used to detect the preparation-obtained electrochemistry immuno-sensing to detect AFP of the embodiment of the present invention 1 The assembling process of device.
Impedance experiment is in the 5 mM K containing 0.1 M electrolyte KCl3[Fe(CN)6] ︱ K4[Fe(CN)6] in carry out, and The alternating voltage of 5 mV is superimposed on the DC voltage of 0.209 V.Nai Kuisi is recorded in 100 KHz～100 mHz frequency ranges Special figure, as shown in Figure 4.All nyquist diagrams in Fig. 4 are presented a semicircle in high frequency region, its correspondence electron transfer resistance （Ret）；A straight line portion is included in low frequency range, its correspondence diffusion controlled process.Half circular diameter and the ferrum cyanogen on modified electrode surface Change potassium electron transmission coefficient to be inversely proportional to.The attachment of nano material and biomolecule defines an electronics and mass transfer barrier layer, it Diffusion of the potassium ferricyanide to electrode surface is hindered, thus reduces electron transmission coefficient.As expecting, naked GCE electrodes（Curve a）There is the semicircle of a very little, it represents sheet resistance very little, correspondence diffusion controlled process.In curve b-f, naked GCE electrodes GNPs-PANI-TNT ︱ shitosan dispersions, Protein G ', BSA, capture antibody A b1 and signal antibody Ab2 are modified successively, It can be seen that along with progressively modification, half circular diameter gradually increases, and shows that various materials are assembled in electricity layer by layer Pole surface.
The optimization of electrochemical immunosensor testing conditions:
The pH value of test solution is a key factor for affecting enzyme sensor performance, because strong acid and strong alkali environment can The activity of destruction biomolecule.Impact of the pH value of solution to immunosensor current-responsive is as shown in Figure 5.Current-responsive value is in pH Quickly increase with the increase of pH value in the range of 4.0-7.0, after pH is more than 7.0, start to reduce.Therefore, containing 0.10 M's 0.05 M pH, 7.0 PBSs are selected as detecting that liquid is used to detect AFP.
Cultivation times of the AFP on GNPs-PANI-TNT ︱ shitosan ︱ Protein G ' ︱ BSA ︱ Ab1 is to affect immunosensor Another key factor of analytical performance.At room temperature, the current-responsive value of AFP immunosensors cultivates the increasing of time with AFP Plus and dramatically increase, more than a steady state value is basically reached after 50 min（As shown in Figure 6）, this shows AFP with electrode surface The combination of capture antibody A b1 reaches saturation.Therefore, 50 min are elected to be the cultivation time of this sandwich type immunoassay.
Analytical performance is assessed:
HRP becomes widely studied in electrochemical immunoanalytical due to its stability, high catalytic property and hypersensitivity Spike enzyme.Here, the present invention passes through bisamination reagent BS3HRP and capture antibody are carried on into TiO2Nanotube surface is formed The trace labelling thing of immunoassay.Before experiment, liquid 15 min of high pure nitrogen deoxygenation is detected, nitrogen is kept in detection process Environment.In H2O2With the help of, HRP catalysis hydroquinone is converted into benzoquinone, and subsequent benzoquinone is reduced electrochemically as hydroquinone to provide detection Signal, shown in following two reaction equation：
Benzoquinone+2H+ + 2e−→ hydroquinone.
In optimal conditions, differential pulse voltammetry（DPV）It is used to assess the analytical performance of immunosensor.Such as Fig. 7 institutes Show, reduction peak current increases with the increase of AFP concentration, calibration curve（Illustration in Fig. 7）Show that peak current is dense with analyte There is good linear relationship between the logarithm of degree, the range of linearity is the ng/mL of 0.01 ng/mL to 350, correlation coefficient is 0.994 (n=4). under conditions of signal to noise ratio is 3, test limit as little as 1.5 pg/mL of AFP, this is not only below what is reported before Panimmunity analysis method, is also less than those and has the immunosensor for amplifying strategy.
The repeatability of immunosensor, specificity, stability and the application in blood serum sample
Invention further contemplates embodiment 1 prepares the specificity of gained interlayer type electrochemical immunosensor, reappears Property and stability.To assess the specificity of the immunosensor, invention introduces several possible chaff interferences, including cancer embryo is anti- It is former（CEA）, carcinoma of prostate proteantigen（PSA）, tumor antigen 125（CA125）, immunoglobulin G（IgG）With Ox blood serum egg In vain（BSA）.This immunosensor is respectively with containing above-mentioned one of which chaff interference（100 ng·mL-1）5 ngmL-1 AFP Incubation.As shown in figure 8, compared with thing is not interfered with, being less than 5% by the curent change that chaff interference causes, this shows this immune sensing Utensil has good selectivity.
In order to probe into the accuracy and repeatability of this immunosensor, present invention same sample prepares five electrodes respectively Research test, gained relative standard deviation are carried out under the same conditions（RSD）For 5.2%.Show this immunosensor accuracy It is good with repeatability.
Additionally, the stability of this immunosensor is rung also by the electric current measured before and after they are stored under the conditions of 4 DEG C 3 weeks Should be assessed.As a result show, after 3 weeks, 92.1% current-responsive is retained, it is good that this shows that this immunosensor has Stability.
The immunosensor is assessed by standard addition method to the suitability that actual blood serum sample is analyzed.Experiment knot Fruit is displayed in table 1.The response rate/verification and measurement ratio is 96.4% -106.0%, and this shows that the electrochemical immunosensor of the present invention can It is used for the detection of AFP in human serum during routine clinical is diagnosed.
Table 1 detects the AFP (n=3) added in human serum with immunosensor of the present invention

Claims (2)

1. one kind is with TiO2Nanometer tube composite materials are the structure of the electrochemical immunosensor of oriented load support and trace labelling thing Construction method, it is characterised in that comprise the steps：
1. the preparation of GNPs-PANI-TNT composites：
TiO2The preparation of nanotube：By 0.6 g TiO2After nano-particle and 120 mL, the NaOH aqueous solution of 10 M, turn Move in reactor, 48 h of hydro-thermal reaction at 120 ± 5 DEG C, abandoning supernatant, after gained white semifinished product washing, in 60 DEG C of bars 2 h are dried under part, it is standby, it is designated as TNTs；
Polyaniline-TiO2The synthesis of nanometer tube composite materials：By 0.2660 g TNTs, 8.57 mL, 0.1 M aniline hydrochloric acid solutions, with And the mixed in hydrochloric acid of 10 mL, 0.1 M, under 0-5 DEG C of ice bath, 30 min of magnetic agitation is obtaining uniform dispersion liquid；Then 2 8.57 mL, the Ammonium persulfate. hydrochloric acid solution of 0.1 M, resulting solution is added to continue to react 3 h under ice bath, be stored at room temperature in h Night, solid-liquid separation, it is that grind into powder after 3 h is dried at 7,60 DEG C that gained sediment is washed with deionized to pH, standby, It is designated as PANI-TNT；
Gold nano grain-polyaniline-TiO2The preparation of nanometer tube composite materials：18 mg PANI-TNT and 18 mL ultra-pure waters are taken, Mix, add 1 mL, the HAuCl of 0.01 M under agitation4Solution and 1 mL, the end-capping reagent citric acid three of 0.01 M Sodium solution, then adds the NaBH of 0.1 M of 1 mL brand-news in 20 min4 Solution, it is quiet after 0.5 h of continuation stirring under room temperature Put overnight, centrifugation, after precipitate washing, be dried overnight at 60 DEG C, it is standby, it is designated as GNPs-PANI-TNT；
2. the preparation of marking signal antibody A b2：
Amination TiO2The preparation of nanotube：By 100 mg TNTs and 20 mL ethanol, 1 mL, 28% ammonia and 4 mL 3- ammonia third Ethyl triethoxy silicane alkane mixing after, mechanical agitation overnight to prevent TNTs from precipitating, centrifugation, abandoning supernatant, gained solid After residue washing, drying, store for future use under room temperature, be designated as NH2-TNT；
Horseradish peroxidase ︱ aminations TiO2The preparation of nanotube ︱ signal antibody bioconjugate bodies：By 2 mg BS3It is dissolved in Solution A is obtained in 0.5 mL, the PBS of 0.02 M, then by 3 mg NH2- TNT is scattered in solution A, and stirring is lower to be added Enter 350 μ L, 2 mgmL-1Horseradish peroxidase aqueous solution and be incubated 30 min at room temperature, be subsequently adding 20 μ L and contain 0.6 mg·mL-1The PBS of signal antibody Ab2, stirs 4 h, centrifugation, gained sediment PBS at 4 DEG C Wash and use the PBS containing 2% bovine serum albumin to close 30 min at room temperature, after finally being washed with PBS, point Dissipate in the 1.0 mL PBSs containing 0.1% bovine serum albumin, it is standby, it is designated as HRP ︱ NH2- TNT ︱ Ab2；
3. the structure of immunosensor：
First, by 5 mg steps, 1. gained GNPs-PANI-TNT is scattered in HAc solution of 1 mL containing 0.2% shitosan, is surpassed 20 min of sound takes 5 μ L dispersant liquid drops and is added in the good glassy carbon electrode surface of pretreatment to obtain uniform dispersion liquid, under room temperature certainly So dry；Then 2 mgmL are contained in 10 μ L of glassy carbon electrode surface Deca-1 BS3PBS and be incubated 1 at room temperature 1 mgmL of h, 10 μ L of subsequent Deca-1 Protein G ', be incubated 60 min under room temperature, in immersion PBS, wash 3 min, Then the glass-carbon electrode is incubated into 30 min with 5 μ L Block buffers, then 3 are washed with lavation buffer solution and PBS respectively Min, finally by 5 μ L, 0.44 mgmL-1Capture antibody A b1 Deca 1 h is incubated under the glassy carbon electrode surface, room temperature, Again after overnight incubation under 4 DEG C, 100% moisture-saturated environment, washed with lavation buffer solution and PBS respectively, obtain final product electrification Learn immunosensor.
2. as claimed in claim 1 with TiO2Nanometer tube composite materials are exempted from for the electrochemistry of oriented load support and trace labelling thing The construction method of epidemic disease sensor, it is characterised in that step 3. in, the lavation buffer solution is：Containing 0.05 % Tween's 20 PBS；
Step 3. in, the Block buffer is：PBS containing 5 % bovine serum albumins.
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