CN105784992A - Method for constructing electrochemical immunosensor employing TNS-MWCNT (TiO2 nanosheet-multi walled carbon nanotube) composite material as antibody and tracing marker carrier - Google Patents

Method for constructing electrochemical immunosensor employing TNS-MWCNT (TiO2 nanosheet-multi walled carbon nanotube) composite material as antibody and tracing marker carrier Download PDF

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CN105784992A
CN105784992A CN201610315599.7A CN201610315599A CN105784992A CN 105784992 A CN105784992 A CN 105784992A CN 201610315599 A CN201610315599 A CN 201610315599A CN 105784992 A CN105784992 A CN 105784992A
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mwcnt
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刘小强
刘培培
霍小鹤
朱杰
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Henan University
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    • G01—MEASURING; TESTING
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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

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Abstract

The invention discloses a method for constructing an electrochemical immunosensor employing a TNS-MWCNT (TiO2 nanosheet-multi walled carbon nanotube) composite material as an antibody and a tracing marker carrier. The method comprises the following steps: preparing the TNS-MWCNT composite material by virtue of a hydrothermal method, performing amination, dispersing the TNS-MWCNT composite material in a Nafion solution and adding dropwise the mixed solution on the surface of an electrode; covalently binding a capture antibody (Abl) to the surface of NH2-TNS-MWCNT for sandwich measurement by adopting an amino crosslinking agent BS3 as a bi-amino crosslinking agent. In addition, BS3 is further used for fixing HRP (horse radish peroxidase) and a signal antibody (Ab2) on the surface of the NH2-TNS-MWCNT to prepare a tracing marker. The electrochemical immunosensor prepared by the method can be used for rapidly measuring alpha-fetoproteins, and is high in sensitivity, wide in linear range and low in detection limit.

Description

Exempt from TNS-MWCNT composite as antibody with the electrochemistry of trace labelling thing carrier Epidemic disease sensor construction method
Technical field
The invention belongs to electrochemical immunosensor constructing technology field, be specifically related to a kind of with TiO2Nanometer sheet (TNS)- Multi-walled carbon nano-tubes (MWCNT) composite is the structure side of the electrochemical immunosensor of antibody and trace labelling thing carrier Method, this electrochemical immunosensor can be used for detecting alpha-fetoprotein (α-fetoprotein, AFP).
Background technology
CLIA, Enzyme-linked Immunosorbent Assay test (ELISA), fluorescence immunoassay and radioimmunoassay method The medical field such as screening for cancer, in early days medical diagnosis on disease, disease treatment curative effect it has been widely used in etc. various immunoassaies. But, these routine immunization assay method processes are more complicated, and the detection time is longer, often further relate to multistep and cultivate and cleaned Journey, and generally these method of immunity must use the fine equipment of laboratory and instrument.On the contrary, in recent years, due to Electrochemical immunosensor show in analyzing test easy to operate, preparation is simple, fast, highly sensitive, performance of analyzing speed The significant advantage such as excellent thus cause relevant scholar and study interest greatly.And, electrochemical immunosensor is extensively regarded For the analysis method sensitiveer than spectrophotography and Optical Analysis Method, this is because spectrophotography or Optical Analysis Method are to light Source requires harsher and needs monochromator and optical detector；Meanwhile, when by spectrophotography or Optical Analysis Method to muddy Or colored sample obtains the information of mistake sometimes when testing.
So far, although substantial amounts of electrochemical immunosensor is developed, but still need to them analytical Can carry out being greatly improved meeting the demand that the mankind are growing.In order to realize this target, different nano materials such as magnetic Particle, noble metal nanometer material, quantum dot, carbon nanomaterial and oxide-based nanomaterial are used for electrochemical immunosensor In development.The properties such as significant electricity, chemistry, geometry and electron transmission are shown, they usual quilts due to these materials Capture antibody is fixed as carrier platform.Such as: Malhotra et al. prepares a kind of electrochemical immunosensor and achieves The mensuration of interleukin-6 in squamous cancer cell in enemy, cervical region cell.This immunosensor is with high connductivity, high-ratio surface Long-pending and dense setting SWCN (SWCNT) bundle is matrix load capture antibody, analyte antigen and capture antibody phase In conjunction with, then constitute sandwich type sensor with the signal antibody effect of horseradish peroxidase (HRP) labelling.Immunity passes The high sensitivity of sensor is to amplify signal by the enzyme catalysis on SWCNT bundle carrier platform to realize, and can make human leucocyte The interleukin-6 detection limit in Sanguis Bovis seu Bubali is pure as little as 30 pg mL-1.Nano material is in addition to as base load biomolecule, also Can be as carrier loaded signal antibody and enzyme, to reach the purpose that signal amplifies.Such as: Xu et al. constructs one with Jenner Rice rod is as the luminol cathodic electrochromic chemiluminescence immunoassay sensor of antibody and Glucose Oxidase.Owing to it is good Biocompatibility, efficient electro catalytic activity and big specific surface area, gold nanorods can load substantial amounts of electrochemiluminescence mark Note thing and biomolecule, amplify electroluminescence signal, thus improve the performance of biosensor.Electroluminescence signal and prostate Specific antigen is linear, and the concentration range of detection is 10 pg mL-1 - 8 ng mL-1, detection limit is low reaches 8 pg mL-1。
Titanium dioxide nano material is due to its high surface area and the feature of even pore distribution and at bioelectronics sensor Research and development field causes increasing concern.Titanium dioxide nano material can increase the biomolecule on sensor surface and bear Carry, and provide biocompatible carrier microenvironment for biomolecule.Such as: gold nano grain (GNP) modifies titanate radical nanopipe (TNT) composite is used to prepare a kind of glucoseoxidase (GOD) biosensor, and this composite nano materials directly facilitates GOD electron transfer rate is (at 180 mV s-1Sweeping electron transfer rate constant under speed is 7.1 s-1) and to glucose Electro catalytic activity.Result shows, this sensor shows low detection limit (5.1 mA mM to glucose -1) and the wide range of linearity (0.01-1.2 mM).But, titanium dioxide nano material is poor due to its electric conductivity and application in biological electronics always It is suppressed, so that improve the performance of material.It is reported, CNT is with its quick electron transfer capacity, highly sensitive Degree and biological electro catalysis ability are widely used in biological electronics.Therefore, TiO2-CNT composite passes at electrochemica biological The application of sensor and immunosensor has great potential value.
The present invention is prepared for amidized titanium dioxide nanoplate (TNS)-multi-walled carbon nano-tubes (MWCNT) by hydro-thermal method Complex.This composite can be used as carrier platform load capture antibody simultaneously and resist as carrier loaded HRP and signal Body.Amidized TNS-MWCNT complex and biomolecule are by amino crosslinker (double (thiosuccimide bases) pungent two Sodium salt) (BS3) combine.After being reacted by sandwich immunoassay, it is possible to use Differential Pulse Voltammetry (DPV) technology is come Detection tumor markers alpha-fetoprotein.
Summary of the invention
Present invention aim to overcome that prior art defect, it is provided that a kind of with TiO2Nanometer sheet (TNS)-multi-walled carbon nano-tubes (MWCNT) composite is the construction method of electrochemical immunosensor of antibody and trace labelling thing carrier, and this electrochemistry is exempted from Epidemic disease sensor can measure alpha-fetoprotein rapidly, and sensitivity is higher, the range of linearity is relatively big, detection limit is relatively low.
For achieving the above object, the present invention adopts the following technical scheme that
It is a kind of with TNS-MWCNT composite as antibody with the electrochemical immunosensor construction method of trace labelling thing carrier, It comprises the steps:
①NH2The preparation of-TNS-MWCNT composite:
The preparation of TNS-MWCNT (prepares TiO by hydrolysis isopropyl titanate on MWCNT surface under diethylenetriamines catalytic action2 Nanometer sheet): the MWCNTs weighing 50 mg is dispersed in the isopropanol of 40 mL, ultrasonic 20 min, is subsequently added the sub-second of 50 μ L bis- Base triamine (DETA), after stirring, adds 3.6 mL isopropyl titanates (TIP), is transferred to by mixed liquor after being again stirring for uniformly In politef reactor, react 18-28 h in 180-220 DEG C；After reaction terminates, taking out, be cooled to room temperature, black sinks Shallow lake thing through centrifugation, washing with alcohol, 70 DEG C be dried, i.e. obtain composite nano materials TNS-MWCNT；
The preparation of amination TNS-MWCNT: by 120 mg TNS-MWCNT and 20 mL ethanol, 1 mL 28% ammonia and 5 mL 3- Aminopropyl triethoxysilane mixing after, mechanical agitation overnight (12h) to prevent TNS-MWCNT from precipitating, centrifugation, discard Clear liquid, the washing of gained solid residue, dried, room temperature for storage is standby, is designated as NH2-TNS-MWCNT；
2. the preparation of trace labelling thing:
HRP NH2The preparation of-TNS-MWCNT Ab2 trace labelling thing: by 2 mg BS3(double (thiosuccimide bases) are pungent Two sodium salt) it is dissolved in the PBS of 0.5 mL 0.02 M acquisition solution A, then by 3 mg NH2-TNS- MWCNT is scattered in solution A, and stirring is lower adds 400 μ L 2.5 mg mL-1Horseradish peroxidase aqueous solution and in room temperature Under hatch 30 min, be subsequently adding 20 μ L containing 1.0 mg mL-1The PBS of signal antibody Ab2, stirs 4 h at 4 DEG C, Centrifugation, the washing of gained precipitate lavation buffer solution also at room temperature closes 30 min, finally with washing with Block buffer After washing buffer solution, it is scattered in the 1.0 mL PBS containing 0.1% bovine serum albumin, standby, it is designated as HRP NH2- TNS-MWCNT Ab2；
3. the structure of immunosensor:
By 6 mg step 1. gained NH2-TNS-MWCNT is scattered in 1 mL 1% Nafion solution, and ultrasonic 20 min are to obtain Uniform dispersion liquid, takes 5 μ L dispersant liquid drops and is added in the glassy carbon electrode surface that pretreatment is good, naturally dry under room temperature；Then at glass Carbon electrodes drips 10 μ L containing 2 mg mL-1 BS3PBS and at room temperature hatch 1 h, drip 5 μ L subsequently 0.5 mg·mL-1 Capture antibody A b1, incubated at room temperature 60 min, immerse in PBS and wash 3 min, then should Glass-carbon electrode hatches 30 min with 5 μ L Block buffer, then washs 3 min, i.e. with lavation buffer solution and PBS respectively Obtain electrochemical immunosensor.Gained electrochemical immunosensor is standby under 4 DEG C and 100% moisture-saturated environment.
Step 2. and 3. in, described Block buffer is: containing 2 %(w/v) PBS of bovine serum albumin (BSA) (0.05 M, pH 7.4)；Described lavation buffer solution is: containing 0.05 %(w/v) PBS (0.05 M, the pH of Tween 20 7.4).
Compared to the prior art, the inventive method beneficial effect of the invention:
NH is introduced at electrode surface2-TNS-MWCNT composite, adds electric conductivity and the bio-compatible of this immunosensor Property, considerably increase the load capacity of capture antibody A b1, improve the joint efficiency of antibody and antigen.Utilize NH simultaneously2-TNS- MWCNT composite specific surface area is big, the advantage of good biocompatibility, by BS3Enzyme HRP and signal antibody Ab2 is fixed on NH2-TNS-MWCNT surface is used as labeled immunoglobulin label, can improve the load capacity of enzyme and antibody, thus improve this sensor Sensitivity and detection limit.
Accompanying drawing explanation
Fig. 1 is the high power Electronic Speculum collection of illustrative plates of different materials: wherein, and A is the transmission electron microscope picture (TEM) of MWCNT, and B is TNS- MWCNT composite transmission electron microscope picture, C is the transmission electron microscope picture of the partial enlargement of TNS-MWCNT composite, and D is TNS- MWCNT composite scanning electron microscope (SEM) photograph (SEM)；
Fig. 2 is X-ray diffraction (XRD) collection of illustrative plates of different materials, wherein, a be MWCNTs, b be TiO2Nanoparticle, c is TNS- MWCNT composite.
Fig. 3 is Fourier infrared (FT-IR) collection of illustrative plates of different materials, and wherein, a is that MWCNTs, b are to infrared spectrogram TiO2Nanoparticle, c is TNS-MWCNT composite；
Fig. 4 is that the x-ray photoelectron power spectrum (XPS) of TNS-MWCNT composite is analyzed: A is that XPS composes scanning entirely；B, C and D is respectively the high-resolution XPS spectrum of C1s, O1s and Ti2p；
Fig. 5 is nyquist diagram, and wherein, a is naked GCE, and b is TNS-MWCNT GCE, and c is TiO2GCE, d are Ab1 TNS- MWCNT GCE, e be BSA Ab1 TNS-MWCNT GCE, f be Ab2 AFP BSA Ab1 TNS-MWCNT GCE；
In Fig. 6, A is the impact on immunosensor current-responsive of the present invention of the Ab1 concentration value；B be HRP on trace labelling thing with The impact of the concentration comparison immunosensor of the present invention current-responsive of Ab2；C is that immunosensor current-responsive of the present invention is with AFP The change curve that concentration increases；D is the linear relationship chart of immunosensor current-responsive of the present invention and the logarithm of AFP 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 It is not limited thereto.
In following embodiment, used BS3, 5% Nafion solution purchased from Sigma-Aldrich company limited, MWCNTs is purchased from Chengdu Organical Chemical Co., Ltd., Chinese Academy of Sciences, and capture antibody A b1, signal antibody Ab2 are purchased from Chengdu pair Liu Zhenglong biochemical product research department.
Embodiment 1
It is a kind of with TNS-MWCNT composite as antibody with the electrochemical immunosensor construction method of trace labelling thing carrier, It comprises the steps:
①NH2The preparation of-TNS-MWCNT composite:
The preparation of TNS-MWCNT: the MWCNTs weighing 50 mg is dispersed in the isopropanol of 40 mL, ultrasonic 20 min add subsequently Enter 50 μ L diethylenetriamines (DETA), after stirring, add 3.6 mL isopropyl titanates (TIP, purity 97%), again stir After mixing uniformly, mixed liquor is transferred in politef reactor, reacts 24h in 200 DEG C；After reaction terminates, take out, cooling To room temperature, black precipitate through centrifugation, washing with alcohol, 70 DEG C be dried, i.e. obtain composite nano materials TNS-MWCNT；
The preparation of amination TNS-MWCNT: by 120 mg TNS-MWCNT and 20 mL ethanol, 1 mL 28% ammonia and 5 mL 3- Aminopropyl triethoxysilane mixing after, mechanical agitation overnight (12h) to prevent TNS-MWCNT from precipitating, centrifugation, discard Clear liquid, the washing of gained solid residue, dried, room temperature for storage is standby, is designated as NH2-TNS-MWCNT；
2. the preparation of trace labelling thing:
HRP NH2The preparation of-TNS-MWCNT Ab2 trace labelling thing: by 2 mg BS3It is dissolved in the PBS of 0.5 mL 0.02 M Buffer obtains solution A, then by 3 mg NH2-TNS-MWCNT is scattered in solution A, and stirring is lower adds 400 μ L 2.5 mg·mL-1Horseradish peroxidase aqueous solution and at room temperature hatch 30 min, be subsequently adding 20 μ L containing 1.0 mg mL-1The PBS of signal antibody Ab2, stirs 4 h, centrifugation at 4 DEG C, gained precipitate lavation buffer solution washs and uses Block buffer at room temperature closes 30 min, after finally washing with lavation buffer solution, is scattered in containing 0.1% bovine serum albumin In 1.0 mL PBS, standby, it is designated as HRP NH2-TNS-MWCNT Ab2；
3. the structure of immunosensor:
By 6 mg step 1. gained NH2-TNS-MWCNT is scattered in 1 mL 1% Nafion solution, and ultrasonic 20 min are to obtain Uniform dispersion liquid, takes 5 μ L dispersant liquid drops and is added in the glassy carbon electrode surface that pretreatment is good, naturally dry under room temperature；Then at glass Carbon electrodes drips 10 μ L containing 2 mg mL-1 BS3PBS and at room temperature hatch 1 h, drip 5 μ L subsequently 0.5 mg·mL-1 Capture antibody A b1, incubated at room temperature 60 min, immerse in PBS and wash 3 min, then should Glass-carbon electrode hatches 30 min with 5 μ L Block buffer, then washs 3 min, i.e. with lavation buffer solution and PBS respectively Obtain electrochemical immunosensor.Gained electrochemical immunosensor is standby under 4 DEG C and 100% moisture-saturated environment.
Step 2. and 3. in, described Block buffer is: containing 2 %(w/v) PBS of bovine serum albumin (BSA) (0.05 M, pH 7.4)；Described lavation buffer solution is: containing 0.05 %(w/v) PBS (0.05 M, the pH of Tween 20 7.4).
4. test process:
For carrying out immunoreation and electro-chemical test, the above-mentioned electrochemical immunosensor of gained of preparing is first with 5 μ L variable concentrations AFP 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 5 μ L step 2. products therefrom HRP NH2-TNS-MWCNT Ab2 at room temperature hatches 50 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 10 mL PBS, then by hydroquinone (ultimate density 2 mM) and hydrogen peroxide (ultimate density 1 mM) inject in this battery, carry out difference arteries and veins in pulse amplitude 50 mV, pulse width 50 ms, 0.2～-0.2 in the range of V Rush volt-ampere (DPV) scanning, to quantitative determine AFP.
The sign of composite:
Fig. 1 is the high power Electronic Speculum collection of illustrative plates of different materials: wherein, and A is the transmission electron microscope picture of MWCNT, and B is TNS-MWCNT composite wood Material transmission electron microscope picture, C is the transmission electron microscope picture of the partial enlargement of TNS-MWCNT composite, and D is TNS-MWCNT composite Scanning electron microscope (SEM) photograph.Can be seen that from the A of Fig. 1 MWCNT is hollow and the tubular structure showing slightly bending, its overall diameter is about 20-30nm, long about 0.5-1 μm.The B of Fig. 1 is the TEM figure of TNS-MWCNT binary complex, it can be seen that in The MWCNT of hollow tube-like structure is completely by dense translucent TiO2Nanometer sheet is covered, this translucent TiO2Nanometer sheet be It is synthesized by hydrolysis isopropyl titanate under the effect of diethylenetriamines catalyst；DETA simultaneously also can be effectively by TiO2Receive Rice sheet is assembled in carboxyl modified MWCNTs surface to form one-dimensional porous composite construction.In Fig. 1, the TEM figure of C confirms especially In TNS-MWCNT composite, MWCNTs is by dense TiO2Nanometer sheet surrounds, but only is difficult to determine TiO from TEM figure2's Flake nano structure, then, TiO2SEM figure be used to accurately determine TiO2Nanostructured.From the D of Fig. 1 it can be seen that TiO2In showing slightly the flake of bending, completely the same with its TEM figure.From TEM and SEM figure all it can be seen that TNSs and The contact on MWCNTs surface is very close, and this explanation TNSs successful deposition is on MWCNTs.
Fig. 2 is MWCNTs, TiO2Nanoparticle and the XRD spectra of TNS-MWCNT composite, respectively with curve a, b and C represents.Find out that MWCNTs has two obvious characteristic diffraction peaks in 25.9 and 43.6 from curve a, the most corresponding MWCNT's And (100) crystal face (002)；TiO2The XRD spectra of nanoparticle (curve b) 25.3,37.9,48.0,54.1, 54.9, all there is diffraction maximum at 62.7,68.8,70.2,75.0 and 82.8, the most corresponding anatase TiO2's (1 0 1)、 (0 0 4)、 (2 0 0)、(1 0 5)、 (2 1 1)、 (2 0 4)、 (1 1 6)、 (2 2 0)、 (2 1 5) and (2 2 4) crystal face；TiO is then contained in the curve c of Fig. 22Nanoparticle and all of diffraction maximum of MWCNTs, Illustrate that TNS-MWCNT complex is successfully synthesized.But, the peak strength of composite drops compared with independent component significantly Low, it means that the interaction between two kinds of components has damaged their crystal structure slightly.
Fig. 3 gives MWCNTs(curve a), TiO2Nanoparticle (curve b) and TNS-MWCNT composite (curve C) infrared spectrogram, can draw the structural information of relevant nanometer material from infrared spectrogram.The curve a of Fig. 3 is 3440 cm-1With 1630 cm-1Near two obvious absworption peaks occur, correspond respectively on MWCNTs surface the stretching vibration of-OH and Bending vibration.And the two absworption peak all can be observed at curve b and c, illustrate at TiO2Nanoparticle and TNS-MWCNT are multiple Compound all contains-OH functional group.In the curve b of Fig. 3, TiO2Nanoparticle is at 400 and 800 cm-1Strong absworption peak respectively Belonging to stretching vibration and the bridge vibration of Ti-O-Ti key of Ti-O key, the two absworption peak is at TNS-MWCNT composite Infrared spectrogram (can also be found in Fig. 3 curve c).Therefore, Fig. 3 may certify that TNS-MWCNT composite nano materials by Success synthesizes.
It is appreciated that by x-ray photoelectron power spectrum (XPS) surface-element of sample forms and chemical bonding state.Fig. 4 Middle A gives the full spectrogram of XPS of TNS-MWCNT composite, from spectrogram it can be seen that sample mainly contains C1s, O1s, Ti2p Three kinds of peaks, illustrate that sample is mainly elementary composition by C, O and Ti.B in the XPS spectrum figure such as Fig. 4 of C1s in TNS-MWCNT composite Shown in, it is positioned at the peak at 284.1,285.2,286.4 and 288.8 eV and corresponds respectively to the C atom of four kinds of different chemical states. Such as, the peak correspondence graphite ene-type C=C/C-C structure near 284.1eV；Peak near 285.2eV then belongs to carbon nanometer Defective C atom in pipe；Additionally, correspond respectively to C-O and C=O chemical bond at the peak of 286.4eV and 288.8eV.By Fig. 4 In C it can be seen that O1s XPS spectrum figure 532.4 and 530.7eV occur two obvious peaks, the two peak corresponds respectively to Lattice Oxygen [Ti-O in the C=O structure of O atom and TNS6] structure.It addition, the XPS spectrum figure of Ti2p because of its spin-spin splitting in Reveal two energy level peaks (see D in Fig. 4), be positioned at the most corresponding TNS-MWCNT complex in energy level peak of 459.1eV and 464.6 eV Middle Ti2p3/2And Ti2p1/2Combination energy.Above XPS spectrum figure clearly illustrates that TNS has been completely deposited on MWCNT surface Form binary complex.
The assembling process of electrochemical impedance (EIS) method monitoring immunosensor:
Electrochemical impedance collection of illustrative plates is used to assess dynamic process and the effective tool of modified electrode interface performance.Therefore, electrification Learn impedance and be used to the assembling process of research electrochemical immunosensor prepared by the embodiment of the present invention 1.
Impedance is tested at the 5 mM K containing 0.1 M KCl electrolyte3[Fe(CN)6] K4[Fe(CN)6Carry out in], and On the DC voltage of 0.209 V, the alternating voltage of superposition 5 mV is as excitation signal.In 100 KHz～100 mHz frequency ranges Interior record nyquist diagram, as shown in Figure 5.All nyquist diagrams in Fig. 5 present a semicircle at high frequency region, its corresponding electricity Sub-transfer resistance (Ret)；A straight line portion is comprised, its corresponding diffusion controlled process in low frequency range.Half circular diameter and modified electrode The electron transmission resistance of the potassium ferricyanide on surface is directly proportional.As expected, (curve a) is at the semicircle of high frequency region for naked GCE electrode Diameter is the least, represents sheet resistance the least, it was demonstrated that completely by diffusion process control.In curve b, d, e and f, naked GCE electrode depends on Secondary it is modified TNS-MWCNT Nafion dispersion, capture antibody A b1, BSA and marking signal antibody A b2, can from figure To find out, along with progressively modification, half circular diameter is gradually increased, and shows that various material is assembled in electrode surface layer by layer. The attachment at electrode surface of nano material and biomolecule defines an electronics and mass transfer barrier layer, and it hinders the potassium ferricyanide To the diffusion of electrode surface, thus reduce electron transmission coefficient.
The optimization of electrochemical immunosensor testing conditions:
Due in the capture concentration of antibody and trace labelling thing traget antibody and the concentration of HRP than all affecting the strong of detection signal Degree, therefore before Performance Evaluation, reply reaction condition is optimized to reach optimum determining effect.As shown in A in Fig. 6, The concentration of Ab1 reaches 0.5 mg mL-1Before, the DPV electric current of this patent immunosensor, in dramatically increasing trend, omits subsequently Reduce.Along with the increase of Ab1 concentration, increasing AFP is captured antibodies, thus increasing trace labelling thing It is fixed on electrode surface, thus produces stronger detection signal.But, too much Ab1 concentrates on electrode surface and will produce Space steric effect, hinders the binding of label, can cause the reduction of DPV electric current.Therefore the concentration of Ab1 is set as 0.5 mg mL-1。
Additionally, due to add HRP amount on detection signal have appreciable impact, therefore the HRP on trace labelling thing with The concentration ratio of Ab2 is also required to optimize.From the B of Fig. 6 it can be seen that when starting, electric current increases along with the increase of HRP/Ab2 ratio Adding, when ratio is 50:1, electric current reaches maximum, and and then electric current has declined, and this is probably due to the HRP/ of higher proportion Ab2 hinders the combination of antigen and Ab2.It is to say, the HRP of excess can hinder the combination of Ab2 and antigen.Therefore HRP/Ab2 Ratio is set as 50:1.
Analytical performance is assessed:
HRP, due to its stability, high catalytic performance and hypersensitivity, becomes wide variety of in electrochemical immunoanalytical and shows Track marker enzyme.The present invention passes through bisamination reagent BS3HRP and Ab2 is carried on NH2-TNS-MWCNT surface defines immunity The trace labelling thing analyzed.This trace labelling thing forms three-layer sandwich structure after being combined with antigen and is used for analyzing detection.Before test, Detection liquid high pure nitrogen deoxygenation 15 min, keeps nitrogen environment during detection.At H2O2With the help of, HRP is catalyzed hydroquinone Being converted into benzoquinone, benzoquinone is reduced electrochemically as hydroquinone to provide detection signal subsequently, shown in following two reaction equations:
。
In optimal conditions, differential pulse voltammetry (DPV) is used to assess the analytical performance of immunosensor.Such as C in Fig. 6 Shown in, reduction peak current increases with the increase of AFP concentration, calibration curve (D in Fig. 6) display peak current and analyte concentration Having good linear relationship between logarithm, the range of linearity is 0.005 ng/mL to 320 ng/mL, and correlation coefficient is 0.994 (n=4).Under conditions of signal to noise ratio is 3, detection limit as little as 2.0 pg/mL of AFP, before the performance of inventive sensor is better than The panimmunity of report analyzes method and immunosensor.
The repeatability of immunosensor, specificity, stability and the application in blood serum sample
Invention further contemplates embodiment 1 and prepare the specificity of gained electrochemical immunosensor, repeatability and stability. For assessing the specificity of this immunosensor, invention introduces several possible chaff interference, including carcinoembryonic antigen (CEA), front Row adenocarcinoma proteantigen (PSA), tumor antigen 125(CA125), immunoglobulin G (IgG) and bovine serum albumin (BSA).This exempts from Epidemic disease sensor is respectively with containing above-mentioned one of which chaff interference (100 ng mL-1) 5 ng mL-1 AFP is hatched.With the most dry Disturb thing to compare, chaff interference the curent change caused is less than 5.4%, shows that this immunosensor has good selectivity.
In order to probe into accuracy and the repeatability of this immunosensor, the present invention uses same procedure and step to prepare respectively Six electrodes, carries out research test under same experimental conditions, and gained relative standard deviation (RSD) is 4.9%.Show this immunity Sensor accuracy and repeatability are good.
Additionally, the stability of this immunosensor is also by the electric current measured before and after they store two weeks under the conditions of 4 DEG C Response is assessed.Result shows, after two weeks, the current-responsive of 92.7% is retained, and this shows that this immunosensor has Good stability.
The suitability of actual blood serum sample analysis is assessed by this immunosensor by standard addition method.Experiment knot Fruit display is in Table 1.The response rate/verification and measurement ratio is 96.2%-104.8%, and this shows that the electrochemical immunosensor of the present invention can It is used for the detection of AFP in human serum in routine clinical diagnosis.
Table 1 immunosensor of the present invention detection adds the AFP in human serum
。

Claims (2)

1. one kind with TNS-MWCNT composite as antibody with the electrochemical immunosensor structure side of trace labelling thing carrier Method, it is characterised in that comprise the steps:
①NH2The preparation of-TNS-MWCNT composite:
The preparation of TNS-MWCNT: the MWCNTs weighing 50 mg is dispersed in ultrasonic 20 min in the isopropanol of 40 mL, is subsequently added 50 μ L diethylenetriamines, after stirring, add 3.6 mL isopropyl titanates, are transferred to by mixed liquor after being again stirring for uniformly In reactor, react 18-28 h in 180-220 DEG C；After reaction terminates, take out, be cooled to room temperature, black precipitate through from The heart separates, washs, is dried, and i.e. obtains composite nano materials TNS-MWCNT；
The preparation of amination TNS-MWCNT: by 120 mg TNS-MWCNT and 20 mL ethanol, 1 mL 28% ammonia and 5 mL 3- After aminopropyl triethoxysilane mixing, mechanical agitation 12h, centrifugation, abandoning supernatant, the washing of gained solid residue, After drying, room temperature for storage is standby, is designated as NH2-TNS-MWCNT；
2. the preparation of trace labelling thing:
HRP NH2The preparation of-TNS-MWCNT Ab2 trace labelling thing: by 2 mg BS3It is dissolved in the PBS of 0.5 mL 0.02 M Buffer obtains solution A, then by 3 mg NH2-TNS-MWCNT is scattered in solution A, and stirring is lower adds 400 μ L 2.5 mg·mL-1Horseradish peroxidase aqueous solution and at room temperature hatch 30 min, be subsequently adding 20 μ L containing 1.0 mg mL-1The PBS of signal antibody Ab2, stirs 4 h, centrifugation at 4 DEG C, gained precipitate lavation buffer solution washs and uses Block buffer at room temperature closes 30 min, after finally washing with lavation buffer solution, is scattered in containing 0.1% bovine serum albumin In 1.0 mL PBS, standby, it is designated as HRP NH2-TNS-MWCNT Ab2；
3. the structure of immunosensor:
By 6 mg step 1. gained NH2-TNS-MWCNT is scattered in 1 mL and contains in 1% Nafion solution, and ultrasonic 20 min are to obtain To uniform dispersion liquid, take 5 μ L dispersant liquid drops and be added in the glassy carbon electrode surface that pretreatment is good, naturally dry under room temperature；Then exist Glassy carbon electrode surface drips 10 μ L containing 2 mg mL-1 BS3PBS and at room temperature hatch 1 h, drip 5 μ subsequently L 0.5 mg·mL-1 Capture antibody A b1, incubated at room temperature 60 min, immerse in PBS and wash 3 min, then will This glass-carbon electrode hatches 30 min with 5 μ L Block buffer, then washs 3 min with lavation buffer solution and PBS respectively, Obtain electrochemical immunosensor.
2. pass with TNS-MWCNT composite as antibody with the electro-chemistry immunity of trace labelling thing carrier as claimed in claim 1 Sensor construction method, it is characterised in that step 2. and 3. in, described lavation buffer solution is: containing 0.05 % Tween 20 PBS；Described Block buffer is: containing the PBS of 2 % bovine serum albumins.
CN201610315599.7A 2016-05-13 2016-05-13 Method for constructing electrochemical immunosensor employing TNS-MWCNT (TiO2 nanosheet-multi walled carbon nanotube) composite material as antibody and tracing marker carrier Pending CN105784992A (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107221636A (en) * 2017-05-12 2017-09-29 哈尔滨工业大学 A kind of high performance three-dimensional classification hybrid structure lithium ion battery negative material and preparation method thereof
CN110441528A (en) * 2019-08-01 2019-11-12 济南大学 One kind being based on core-shell structure Mo2The building of the cTnI immunosensor of C@C nano ball
CN112305041A (en) * 2020-09-15 2021-02-02 东莞东阳光医疗智能器件研发有限公司 Multiple quantitative electrochemical immunosensor and construction method thereof
CN113376233A (en) * 2021-07-13 2021-09-10 沈阳师范大学 Novel liposome-based electrode constructed by horseradish peroxidase, preparation method and direct electrochemical application thereof
CN113428893A (en) * 2021-07-14 2021-09-24 陕西科技大学 Carboxylation modified nano titanium dioxide ultraviolet screening agent and preparation method thereof
CN113960139A (en) * 2021-07-13 2022-01-21 沈阳师范大学 Novel modified electrode constructed by liposome-based glucose oxidase, preparation method and direct electrochemical application thereof
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105241939A (en) * 2015-09-16 2016-01-13 山东理工大学 Preparation method for immunosensor based on gold/silver core-shell magnetic graphene adsorption cadmium ion and application
CN105486873A (en) * 2015-12-08 2016-04-13 河南大学 Construction method of electrochemical immunosensor using TiO2 nanotube composite material as a directed load support and tracer marker

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105241939A (en) * 2015-09-16 2016-01-13 山东理工大学 Preparation method for immunosensor based on gold/silver core-shell magnetic graphene adsorption cadmium ion and application
CN105486873A (en) * 2015-12-08 2016-04-13 河南大学 Construction method of electrochemical immunosensor using TiO2 nanotube composite material as a directed load support and tracer marker

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XIAOQIANG LIU等: "DEVELOPMENT OF A PHOTOELECTROCHEMICAL LACTIC DEHYDROGENASE BIOSENSOR USING MULTI-WALL CARBON NANOTUBE -TIO2 NANOPARTICLE COMPOSITE AS COENZYME REGENERATION TOOL", 《ELECTROCHIMICA ACTA》 *

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CN110441528A (en) * 2019-08-01 2019-11-12 济南大学 One kind being based on core-shell structure Mo2The building of the cTnI immunosensor of C@C nano ball
CN110441528B (en) * 2019-08-01 2022-06-07 济南大学 Mo based on core-shell structure2Construction of C @ C nanosphere cardiac troponin I immunosensor
CN112305041A (en) * 2020-09-15 2021-02-02 东莞东阳光医疗智能器件研发有限公司 Multiple quantitative electrochemical immunosensor and construction method thereof
CN112305041B (en) * 2020-09-15 2022-05-27 东莞东阳光医疗智能器件研发有限公司 Multiple quantitative electrochemical immunosensor and construction method thereof
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CN113428893A (en) * 2021-07-14 2021-09-24 陕西科技大学 Carboxylation modified nano titanium dioxide ultraviolet screening agent and preparation method thereof
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