CN104865240A - Disposable nanometer electrogenerated chemiluminescence two-component immune sensor and preparation method thereof - Google Patents
Disposable nanometer electrogenerated chemiluminescence two-component immune sensor and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a disposable nanometer electrogenerated chemiluminescence two-component immune sensor and a preparation method thereof. A monothiol compound and a bisthiol compound wrapped QDs electrogenerated chemiluminescence potential difference distinguish and ITO electrode space distinguish are used to achieve object detection. The sensor has a three-electrode structure; Ag / AgCl or saturated calomel electrode is used as a reference electrode, Pt electrode is used as an auxiliary electrode, and two pieces of modified ITO conductive glass capable of specifically identifying the two components are placed back to back as a working electrode for the two-component immune sensor; and an object to be measured is crosslinked to the surface of the working electrode by a sandwich immunoassay reaction, and finally crosslinked with horseradish peroxidase.
Description
Technical field
The present invention relates to a kind of two component immunosensors based on electrogenerated chemiluminescence (ECL) the signal spike of two kinds of different shell quantum dots (quantum dots, QDs) and preparation method thereof.Sensor realizes the detection of object to be measured according to the luminous current potential resolution of two kinds of QDs materials and the spatial discrimination of ITO electro-conductive glass.
Background technology
ECL process refers to by electrochemical method, electronics or cave is injected to the luminophor of electrode surface or its coreagent, by the collision generation electron transmission of oxidized and reduced intermediate, thus produce luminophor excited state, when ground state is returned in transition, produce optical radiation.QDs, also known as nanocrystalline, is made up of a limited number of atom, and three dimension are all in nanometer scale.QDs is generally spherical or class is spherical, usually be made up of IIB ~ VIA or IIIA ~ VA element, particle diameter generally between 1 ~ 10nm, because electronics and hole are by quantum confinement, continuous print band structure becomes the discrete energy levels structure with molecular characterization, can emitting fluorescence after being excited.Based on quantum effect, QDs is at solar cell, and luminescent device, the fields such as optical bio mark are with a wide range of applications.
In recent years, QDs is also widely used in field of bioanalysis as ECL luminophor.Multiple coreagent auxiliary under, constructed a large amount of biology sensors based on QDs ECL technology, for albumen, nucleic acid and cell detection, all obtained good sensitivity and repeatability.Compare traditional containing Ru compound, the nanometer ECL system that QDs builds as luminophor has following advantage: 1) luminous current potential adjusts by the change of surface structure; 2) ECL system can be assisted by multiple coreagent, usually just can obtain stronger ECL radiation under physiological ph conditions; 3) surface structure can provide approach crosslinked with biomolecule further, is more conducive to the structure of bioanalysis new method.In research in the past; the cadmium telluride quantum dot (CdTe QDs) of novel pair of sulfhydryl compound parcel obtains lower luminous current potential in cathodic scan process; for dimercaptosuccinic acid (DMSA) as protective agent; the luminous current potential of DMSA-CdTe QDs negative electrode ECL is ~-0.90V; compare the CdTe QDs of single sulfhydryl compound parcel of widespread use; its ECL spike potential generally ~-1.2V is even more negative; difference can reach at least 300mV, and luminosity curve is not overlapping at spike potential place.This spike potential difference provides good approach for two component detects.
Multicomponent analysis has important clinical meaning.Such as hepatocellular carcinoma is the cancer that fatal rate is number three, and more early diagnosis, the exercisable possibility of the therapeutic modality such as liver transfer operation, operation is larger, greatly can improve the survival rate of patient, improve its life quality.For hypotype (AFP-L3 heteroplasmon) affine with LcA in alpha-fetoprotein (AFP), Serum AFP-L3% can be used as independently predictor, especially AFP expresses negative people at highest risk, answers strong suspicion hepatocellular carcinoma when AFP-L3% >=10%.The AFP-L3% detection method of current widespread use is agglutinin affinity column electrophoresis detection, and program is complicated comparatively speaking, needs to detect respectively AFP and AFP-L3.Meanwhile, the Monitoring lower-cut of low-abundance protein AFP-L3 limits by the detectability of methodology and instrument.Therefore how can detect AFP and AFP-L3 simultaneously, obtain AFP-L3% easily, and have higher sensitivity and lower testing cost, just significant.
Summary of the invention
The invention discloses a kind of disposable nanometer electrogenerated chemiluminescence bi-component immunosensor and preparation method thereof, based on the ECL character of nano material QDs, by the different surfaces micromechanism that single sulfydryl and two sulfhydryl protected shell bring, realize ECL spike potential and differentiate; By the planar structure of ITO electro-conductive glass, implementation space is differentiated; AFP and AFP-L3 can be detected simultaneously, obtain AFP-L3% easily, and have higher sensitivity and lower testing cost.
Technical scheme of the present invention is: the two component immunosensor of a kind of disposable nanometer electrogenerated chemiluminescence, for three-electrode structure, Ag/AgCl or saturated calomel electrode are as contrast electrode, Pt electrode is as auxiliary electrode, and using the modification of the two panels of placing back-to-back process, the ITO electro-conductive glass of specific recognition two kinds of components is as the working electrode of two component immunosensor; Described working electrode is back-to-back two panels ITO electro-conductive glass, and the surface of two panels ITO electro-conductive glass is respectively by DMSA-CdTe QDs solution and TiO
2-GSH-CdTe QDs compound suspension applies and dries the uniformly light-emitting body thin film of formation, luminophor film covers chitosan film again, and then successively with Streptavidin, AFP and AFP-L3 antibody modification film, exposed chitosan film is closed with BSA, object AFP to be measured and AFP-L3 antigen react the ito glass surface being linked to respective specific recognition respectively by antigen and antibody specific, the two component immunosensor working electrode of the antibody linked acquisition of AFP and AFP-L3 that is last and horseradish peroxidase-labeled.
The preparation method of described working electrode is:
The first step: preparation DMSA-CdTe QDs and GSH-CdTe QDs: pass through CdCl
2cd source is provided, NaBH as presoma
4the NaHTe be obtained by reacting with Te powder provides Te source as presoma, and in the aqueous systems that protective agent DMSA or GSH exists, one-step synthesis method obtains DMSA-CdTe QDs or GSH-CdTe QDs; Centrifugal concentrating purifying; GSH-CdTe QDs and Detitanium-ore-type TiO
2nano particle ultrasonic acquisition TiO
2-GSH-CdTe QDs compound;
Second step: prepare luminophor film: by DMSA-CdTe QDs solution and TiO
2-GSH-CdTe QDs compound suspension drips respectively and is coated in two panels ITO electrode surface, and room temperature is dried, and obtains uniformly light-emitting body thin film; 3rd step: prepare chitosan film: cover chitosan solution respectively on luminophor film, room temperature is dried; Then use glutaraldehyde solution room temperature activation chitosan film;
4th step: Streptavidin is modified: slowly rinsed completely by glutaraldehyde solution by washing lotion, streptavidin drips and is coated in chitosan film surface, reacts under room temperature, then spends the night at 4 DEG C;
5th step: AFP and AFP-L3 antibody modification: slowly will remain streptavidin solution by washing lotion and rinse completely; Biotin labeling AFP and AFP-L3 antibody are dripped respectively be coated in streptavidin modify film on, 37 DEG C of reactions;
6th step: BSA closes: slowly will remain antibody-solutions by washing lotion and rinse completely, BSA solution drops in the film surface through AFP and AFP-L3 antibody modification, closes exposed chitosan film, 37 DEG C of reactions;
7th step: AFP and AFP-L3 Modified antigen: slowly will remain BSA solution by washing lotion and rinse completely, drips the ITO electrode surface being coated in respective specific recognition, 37 DEG C of reactions respectively by the solution of AFP and AFP-L3 antigen;
8th step: it is antibody linked that horseradish peroxidase is modified: slowly residual antigen solution is rinsed completely by washing lotion, AFP and the AFP-L3 antibody that horseradish peroxidase is modified drips the ITO conductive glass surface being coated onto the 7th step and handling well, 37 DEG C of reactions, slowly rinse completely by washing lotion, placed back-to-back by the two panels ITO electro-conductive glass obtained, workplace outwards obtains working electrode.
The mass percent concentration of described chitosan solution is 0.025%, and the mass percent concentration of glutaraldehyde solution is 0.1%, and the concentration of Streptavidin is 1mg mL
-1, the mass percent concentration of BSA solution is 5%.
The preparation method of the two component immunosensor of described disposable nanometer electrogenerated chemiluminescence, prepare ITO working electrode by Sandwich immunoassay, concrete grammar is:
The first step: preparation DMSA-CdTe QDs and GSH-CdTe QDs: pass through CdCl
2cd source is provided, NaBH as presoma
4the NaHTe be obtained by reacting with Te powder provides Te source as presoma; Centrifugal concentrating purifying; GSH-CdTe QDs and Detitanium-ore-type TiO
2the ultrasonic acquisition TiO of nano particle (NPs)
2-GSH-CdTe QDs compound;
Second step: prepare luminophor film: by DMSA-CdTe QDs solution and TiO
2-GSH-CdTe QDs compound suspension drips respectively and is coated in two panels ITO electrode surface, and room temperature is dried, and obtains uniformly light-emitting body thin film;
3rd step: prepare chitosan film: cover chitosan solution respectively on luminophor film, room temperature is dried; Then use glutaraldehyde solution room temperature activation chitosan film;
4th step: Streptavidin is modified: slowly rinsed completely by glutaraldehyde solution by washing lotion, streptavidin drips and is coated in chitosan film surface, reacts under room temperature, then spends the night at 4 DEG C;
5th step: AFP and AFP-L3 antibody modification: slowly will remain streptavidin solution by washing lotion and rinse completely; Biotin labeling AFP and AFP-L3 antibody are dripped respectively be coated in streptavidin modify film on, 37 DEG C of reactions;
6th step: BSA closes: slowly will remain antibody-solutions by washing lotion and rinse completely, BSA solution drops in the film surface through AFP and AFP-L3 antibody modification, closes exposed chitosan film, 37 DEG C of reactions;
7th step: AFP and AFP-L3 Modified antigen: slowly will remain BSA solution by washing lotion and rinse completely, drips the ITO electrode surface being coated in respective specific recognition, 37 DEG C of reactions respectively by the solution of AFP and AFP-L3 antigen;
8th step: it is antibody linked that horseradish peroxidase is modified: slowly residual antigen solution is rinsed completely by washing lotion, AFP and the AFP-L3 antibody that horseradish peroxidase is modified drips the ITO conductive glass surface being coated onto the 7th step and handling well, 37 DEG C of reactions, slowly rinse completely by washing lotion, placed back-to-back by the two panels ITO electro-conductive glass obtained, workplace outwards obtains working electrode.
Apply the detection method of disposable nanometer electrogenerated chemiluminescence bi-component immunosensor, select to be suitable for supporting electrolyte, application three-electrode system, detect the electrochemiluminescence signal of two component immunosensor, record the electrogenerated chemiluminescence peak intensity that one group of surface adopts the working electrode be obtained by reacting containing variable concentrations antigenic solution, application origin 6.1 software matching peak intensity-Log (antigen concentration) value working curve and concentration computing formula, formulae discovery analytic target concentration is calculated by recorded electrogenerated chemiluminescence curve peak intensity being brought into concentration in actual detection.
Described electrolyte is KCl or KNO
3.
Principle: the different surfaces micromechanism that the present invention utilizes two sulfydryls and single sulfydryl shell protective agent to cause, obtains the quanta point material of different luminous current potential.Electrogenerated chemiluminescence (ECL) potential difference (PD) of bi-material can reach ~ 360mV.Utilize the feature of indium tin oxide (ITO) electro-conductive glass, using two panels ITO electro-conductive glass as working electrode, be positioned over above photomultiplier optical window back-to-back, the two resolution in current potential-region can be realized, build disposable pair of component immunosensor.Further with the coupling of enzyme cycle signal amplifying technique, greatly reduce the detectability of bioanalysis, thus realize hypersensitivity detection.Two component ECL immunosensor has great significance for the application such as joint-detection of disease marker hypotype content detection or two kinds of disease markers.
To the hypotype that LcA is affine in alpha-fetoprotein (AFP) composition, namely AFP-L3 heteroplasmon can be used as blood serum designated object, and the prompting early stage to hepatocellular carcinoma, the especially people at highest risk of AFP negative, have very high sensitivity and specificity.Be detected as example with AFP and AFP-L3 simultaneously, through layer assembly (assembling process of sensor characterizes confirmation by atomic force microscope), the surperficial successively modification through capture antibody, determined antigen, enzyme labelled antibody of working electrode ITO electrode of sensor, realized by the specific recognition between Ag-Ab, final formation horseradish peroxidase (HRP) active interface, can realize enzyme cycle signal and amplify strategy.Under the effect of substrate p-dihydroxy-benzene (HQ), in cathodic scan process, ECL coreagent is consumed by HRP catalytic substrate oxidation reaction, and ECL intensity reduces, and ECL intensity becomes negative correlation with the amount being linked to sensor surface HRP; And the amount being linked to the surperficial HRP labelled antibody of sensor becomes positive correlation with the amount of detected object (AFP, AFP-L3), therefore, ECL peak intensity becomes negative correlation with detected object concentration, detects drawing curve by the antigen of a series of concentration known.Under enzyme cycle signal amplifies assisting of strategy, Monitoring lower-cut reaches pg mL
-1level, demonstrates the detection sensitivity of superelevation.Detect through AFP and AFP-L3 simultaneously, the ratio of AFP-L3 in total AFP can be obtained easily, i.e. AFP-L3%.During unknown concentration sample detection, read-0.90V and-1.30V on ECL curve and locate ECL luminous intensity, bring the concentration that namely concentration computing formula obtains AFP and AFP-L3 into, calculate AFP-L3%.Through actual sample check and evaluation, sensor has good detection sensitivity and repeatability.
Beneficial effect:
1, it is simple that two amounts that the present invention uses puts material preparation method, commercialization anatase crystal TiO
2it is convenient that nano particle and ITO electro-conductive glass are bought, and cheap.
2, the two component immunosensors preparation that builds of the present invention and operating process simple, experiment, all in room temperature, conventional 4 DEG C of refrigerators and carry out in 37 DEG C of incubation casees, is avoided chemistry and the bio-modification process of complexity, is easy to preservation, signal stabilization.
3, it is high that the immunosensor prepared by the present invention detects degree of accuracy, application electrogenerated chemiluminescence technology, the structure of the enzyme cycle signal amplification strategy of institute's coupling is incorporated in the preparation of sensor and completes, do not increase operation steps, obtain high detection sensitivity, overcome the defect that classic method is subject to the restriction of instrument Monitoring lower-cut.
4, one-time detection process obtains the concentration of AFP and AFP-L3 in sample to be tested simultaneously, calculates AFP-L3%, can overcome the defect of traditional AFP-L3% detection method complex steps.
5, this sensor is single use, cheap, and to temperature-insensitive, is beneficial to the exploitation of the other new detecting method of bed especially.
Accompanying drawing explanation
Fig. 1 .DMSA-CdTe QDs and TiO
2-GSH-CdTe QDs compound modifies ITO electrode ECL curve (A) and cyclic voltammetry curve (B).A: simultaneously scan; B: scanning DMSA-CdTe QDs modifies ITO electrode separately; C: scan TiO separately
2-GSH-CdTe QDs compound modifies ITO electrode.Illustration: continuous 10 weeks scanning ECL curves.ITO electrode modified by two amounts point material does not have overlapping at respective spike potential place ECL curve, does not therefore produce interference mutually.The two ECL intensity is substantially suitable.In cyclic voltammetry scan, the ITO electrode that two amounts point material is modified, at-1.3V place, sweep current intensity equals sweep current intensity sum respectively substantially simultaneously.(ECL Intensity-electrogenerated chemiluminescence intensity, Current-strength of current, Potential-current potential, Time-time)
Fig. 2. (A) TiO
2nanocrystal transmission electron microscope image; (B) TiO
2-GSH-CdTe QDs compound transmission electron microscope image.
Fig. 3. two component immunosensor ITO electrode surface-assembled process atomic force microscope (AFM) image (for AFP-L3).A:TiO
2-GSH-CdTe QDs compound/shitosan/streptavidin modifies ITO electrode interface (interface undulation ~ 43nm); B:A+ biotin labeling AFP-L3 antibody modification ITO electrode interface (interface undulation ~ 600nm); C:B+BSA closes exposed shitosan electrode interface (interface undulation becomes level and smooth); D:C+AFP-L3 electrode interface (interface undulation ~ 600nm); E:D+HRP marks AFP-L3 antibody modification ITO electrode interface (~ 750nm).The fluctuating at interface confirms the realization of layer assembly process.
Fig. 4. (A) AFP-L3 detects circulation ECL curve: (a) 0.00324, (b) 0.324, (c) 32.4ng mL
-1; (B) AFP detects circulation ECL curve: (a) 0.001, (b) 0.01, (c) 20ng mL
-1; (C) AFP-L3 and AFP detects cyclic voltammetry curve simultaneously: (a) AFP-L30.324ng mL
-1/ AFP 0.1ng mL
-1, (b) AFP-L33.24ngmL
-1/ AFP 1ng mL
-1.AFP-L3 (D) and AFP (E) testing curve.
Embodiment
The present invention is based on two component nanometer ECL immunosensors of two amounts sub-point (QDs) material construction, utilize the QDs ECL potential difference resolution of single sulfhydryl compound and two sulfhydryl compound parcel and the spatial discrimination of indium tin oxide (ITO) electrode to realize two kinds of objects to detect simultaneously, for three-electrode structure, Ag/AgCl electrode is as contrast electrode, Pt electrode is as auxiliary electrode, and using the modification of the two panels of placing back-to-back process, the ITO electro-conductive glass of specific recognition two kinds of components is as the working electrode of two component immunosensor.By sandwich method immune response, the horseradish peroxidase (HRP) being linked to ITO electrode surface after layer assembly implements enzyme cycle signal amplification strategy.Concrete steps are:
The first step: pass through CdCl
2cd source is provided, NaBH as presoma
4the NaHTe be obtained by reacting with Te powder provides Te source as presoma, and in the aqueous systems that protective agent dimercaptosuccinic acid (DMSA) or glutathione (GSH) exist, one-step synthesis method obtains DMSA-CdTe QDs or GSH-CdTe QDs.[list of references Anal.Chem.2007,79,8055-8060.] is by centrifugal concentrating purifying and be adjusted to desired concn.GSH-CdTe QDs and Detitanium-ore-type TiO
2the ultrasonic acquisition TiO of nano particle (NPs)
2-GSH-CdTe QDs compound.
Second step: by DMSA-CdTe QDs solution and TiO
2-GSH-CdTe QDs compound suspension drips respectively and is coated in two panels ITO electrode surface, and room temperature is dried, and obtains uniformly light-emitting body thin film.
3rd step: cover 10 μ L 0.025% shitosan (chitosan) solution on luminophor film respectively, room temperature is dried.Then use 20 μ L 0.1% glutaraldehyde solution room temperature activation chitosan film 2 hours.
4th step: slowly glutaraldehyde solution is rinsed completely by washing lotion, 15 μ L 1mg mL
-1streptavidin drips and is coated in two panels ITO electrode chitosan film surface, reacts 1 hour under room temperature, then spends the night at 4 DEG C.
5th step: slowly ITO surface liquid is rinsed completely by washing lotion.20 μ L biotin labeling AFP and AFP-L3 antibody drip respectively be coated in two panels ITO electrode streptavidin modify film on, 37 DEG C reaction 1 hour.
6th step: slowly ITO surface liquid is rinsed completely by washing lotion.20 μ L 5% bovine serum albumin (BSA) solution drop in the film surface of two panels ITO electrode through AFP and AFP-L3 antibody modification, close exposed chitosan film.Surplus solution, after 1 hour, slowly rinses completely by washing lotion by 37 DEG C of reactions.
7th step: the solution that 20 μ L contain variable concentrations AFP and AFP-L3 antigen drips the ITO conductive glass surface being coated in respective specific recognition respectively, 37 DEG C of reactions obtain the ITO electro-conductive glass of one group of corresponding variable concentrations antigen for 1 hour.
8th step: slowly ITO surface liquid is rinsed completely by washing lotion.AFP and the AFP-L3 antibody that 30 μ L horseradish peroxidases (HRP) mark drips painting respectively, and the ITO electrode of specific recognition is surperficial separately on a sensor, and 37 DEG C are reacted 1 hour.Washing lotion is slowly rinsed completely.
9th step: select to be suitable for supporting electrolyte, application three-electrode system, the ITO electrode that the two panels of AFP and the AFP-L3 antigen of the one group of corresponding variable concentrations the 8th step obtained is placed back-to-back detects the electrochemiluminescence signal of two component immunosensor as working electrode, by cathode circulation voltammetric scan record not synantigen (detected object) concentration time electrogenerated chemiluminescence peak intensity.Wherein AFP specificity ECL glow peak provides for DMSA-CdTe QDs, and spike potential is-0.90V; AFP-L3 specificity ECL glow peak is TiO
2-GSH-CdTe QDs compound provides, and spike potential is-1.30V.Application origin 6.1 software matching peak intensity-Log (antigen concentration) value working curve and concentration computing formula.Formulae discovery analytic target concentration is calculated by the electrogenerated chemiluminescence curve peak intensity recorded in cathode circulation voltammetric scan being brought into concentration in actual detection.In the Tris-HCl buffer solution of 0.1M pH 7.5, with the KNO of 0.1M
3for supporting electrolyte, in 0 ~-1.3V scope, carry out cyclic voltammetry scan.The concentration selected in 7th step is respectively AFP-L3:0.00324,0.0324,0.324,3.24,32.4,324ng mL
-1; AFP:0.001,0.01,0.1,1,20ng mL
-1.After origin 6.1 matching, AFP-L3 testing curve is y=6484-1848x (R=0.994); AFP testing curve is y=1202.5-599x (R=0.993).Wherein y is ECL luminous intensity, and x is Log (antigen concentration).Amplify [list of references Anal.Chem.2010,82,7351-7356.] through enzyme cycle signal, the Monitoring lower-cut of AFP-L3 and AFP can reach 3.24pg mL
-1with 1.0pg mL
-1.In reality detects, measured-1.30V and-0.90V is located ECL intensity and brings working curve into, calculate the concentration of AFP-L3 and AFP, the two ratio is AFP-L3% content.As shown in Figure 4.
Claims (6)
1. the two component immunosensor of disposable nanometer electrogenerated chemiluminescence, it is characterized in that, for three-electrode structure, Ag/AgCl or saturated calomel electrode are as contrast electrode, Pt electrode is as auxiliary electrode, and using the modification of the two panels of placing back-to-back process, the ITO electro-conductive glass of specific recognition two kinds of components is as the working electrode of two component immunosensor; Described working electrode is back-to-back two panels ITO electro-conductive glass, and the surface of two panels ITO electro-conductive glass is respectively by DMSA-CdTe QDs solution and TiO
2-GSH-CdTe QDs compound suspension applies and dries the uniformly light-emitting body thin film of formation, luminophor film covers chitosan film again, and then successively with Streptavidin, AFP and AFP-L3 antibody modification film, exposed chitosan film is closed with BSA, object AFP to be measured and AFP-L3 antigen react the ito glass surface being linked to respective specific recognition respectively by antigen and antibody specific, the two component immunosensor working electrode of the antibody linked acquisition of AFP and AFP-L3 that is last and horseradish peroxidase-labeled.
2. the two component immunosensor of disposable nanometer electrogenerated chemiluminescence as claimed in claim 1, it is characterized in that, the preparation method of described working electrode is:
The first step: preparation DMSA-CdTe QDs and GSH-CdTe QDs: pass through CdCl
2cd source is provided, NaBH as presoma
4the NaHTe be obtained by reacting with Te powder provides Te source as presoma, and in the aqueous systems that protective agent DMSA or GSH exists, one-step synthesis method obtains DMSA-CdTe QDs or GSH-CdTe QDs; Centrifugal concentrating purifying; GSH-CdTe QDs and Detitanium-ore-type TiO
2nano particle ultrasonic acquisition TiO
2-GSH-CdTe QDs compound;
Second step: prepare luminophor film: by DMSA-CdTe QDs solution and TiO
2-GSH-CdTe QDs compound suspension drips respectively and is coated in two panels ITO electrode surface, and room temperature is dried, and obtains uniformly light-emitting body thin film;
3rd step: prepare chitosan film: cover chitosan solution respectively on luminophor film, room temperature is dried; Then use glutaraldehyde solution room temperature activation chitosan film;
4th step: Streptavidin is modified: slowly rinsed completely by glutaraldehyde solution by washing lotion, streptavidin drips and is coated in chitosan film surface, reacts under room temperature, then 4
ospend the night under C;
5th step: AFP and AFP-L3 antibody modification: slowly will remain streptavidin solution by washing lotion and rinse completely; Biotin labeling AFP and AFP-L3 antibody are dripped respectively be coated in streptavidin modify film on, 37
oc reacts;
6th step: BSA closes: slowly will remain antibody-solutions by washing lotion and rinse completely, BSA solution drops in the film surface through AFP and AFP-L3 antibody modification, closes exposed chitosan film, 37
oc reacts;
7th step: AFP and AFP-L3 Modified antigen: slowly will remain BSA solution by washing lotion and rinse completely, drips the ITO electrode surface being coated in respective specific recognition, 37 respectively by the solution of AFP and AFP-L3 antigen
oc reacts;
8th step: it is antibody linked that horseradish peroxidase is modified: slowly rinse completely by residual antigen solution by washing lotion, AFP and the AFP-L3 antibody that horseradish peroxidase is modified drips the ITO conductive glass surface being coated onto the 7th step and handling well, 37
oc reacts, and slowly rinse completely by washing lotion, placed back-to-back by the two panels ITO electro-conductive glass obtained, workplace outwards obtains working electrode.
3. the two component immunosensor of disposable nanometer electrogenerated chemiluminescence as claimed in claim 2, it is characterized in that, the mass percent concentration of described chitosan solution is 0.025%, and the mass percent concentration of glutaraldehyde solution is 0.1%, and the concentration of Streptavidin is 1 mg mL
-1, the mass percent concentration of BSA solution is 5%.
4. the preparation method of the two component immunosensor of the arbitrary described disposable nanometer electrogenerated chemiluminescence of claim 1 ~ 3, it is characterized in that, prepare ITO working electrode by Sandwich immunoassay, concrete grammar is:
The first step: preparation DMSA-CdTe QDs and GSH-CdTe QDs: pass through CdCl
2cd source is provided, NaBH as presoma
4the NaHTe be obtained by reacting with Te powder provides Te source as presoma; Centrifugal concentrating purifying; GSH-CdTe QDs and Detitanium-ore-type TiO
2the ultrasonic acquisition TiO of nano particle (NPs)
2-GSH-CdTe QDs compound;
Second step: prepare luminophor film: by DMSA-CdTe QDs solution and TiO
2-GSH-CdTe QDs compound suspension drips respectively and is coated in two panels ITO electrode surface, and room temperature is dried, and obtains uniformly light-emitting body thin film;
3rd step: prepare chitosan film: cover chitosan solution respectively on luminophor film, room temperature is dried; Then use glutaraldehyde solution room temperature activation chitosan film;
4th step: Streptavidin is modified: slowly rinsed completely by glutaraldehyde solution by washing lotion, streptavidin drips and is coated in chitosan film surface, reacts under room temperature, then 4
ospend the night under C;
5th step: AFP and AFP-L3 antibody modification: slowly will remain streptavidin solution by washing lotion and rinse completely; Biotin labeling AFP and AFP-L3 antibody are dripped respectively be coated in streptavidin modify film on, 37
oc reacts;
6th step: BSA closes: slowly will remain antibody-solutions by washing lotion and rinse completely, BSA solution drops in the film surface through AFP and AFP-L3 antibody modification, closes exposed chitosan film, 37
oc reacts;
7th step: AFP and AFP-L3 Modified antigen: slowly will remain BSA solution by washing lotion and rinse completely, drips the ITO electrode surface being coated in respective specific recognition, 37 respectively by the solution of AFP and AFP-L3 antigen
oc reacts;
8th step: it is antibody linked that horseradish peroxidase is modified: slowly rinse completely by residual antigen solution by washing lotion, AFP and the AFP-L3 antibody that horseradish peroxidase is modified drips the ITO conductive glass surface being coated onto the 7th step and handling well, 37
oc reacts, and slowly rinse completely by washing lotion, placed back-to-back by the two panels ITO electro-conductive glass obtained, workplace outwards obtains working electrode.
5. application rights requires the detection method of 1 ~ 3 arbitrary disposable nanometer electrogenerated chemiluminescence bi-component immunosensor, it is characterized in that, select to be suitable for supporting electrolyte, application three-electrode system, detect the electrochemiluminescence signal of two component immunosensor, record the electrogenerated chemiluminescence peak intensity that one group of surface adopts the working electrode be obtained by reacting containing variable concentrations antigenic solution, application origin 6.1 software matching peak intensity-Log(antigen concentration) value working curve and concentration computing formula, formulae discovery analytic target concentration is calculated by recorded electrogenerated chemiluminescence curve peak intensity being brought into concentration in actual detection.
6. detection method as claimed in claim 5, it is characterized in that, described electrolyte is KCl or KNO
3.
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| CN105785043A (en) * | 2016-04-06 | 2016-07-20 | 上海良润生物医药科技有限公司 | Kit for quantitatively detecting AFP-L3% |
| CN108007989A (en) * | 2016-10-28 | 2018-05-08 | 中国科学院烟台海岸带研究所 | Based on detection method of the type polypeptide for identification molecule of splitting |
| CN109470690A (en) * | 2018-10-19 | 2019-03-15 | 浙江大学 | The antigen detection method of electrochemical luminescence is differentiated based on current potential |
| CN110220964A (en) * | 2019-06-05 | 2019-09-10 | 常州大学 | The measurement method of chloride ion in the copper electrolyte of electrolytic copper foil |
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Cited By (7)
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| CN105785043A (en) * | 2016-04-06 | 2016-07-20 | 上海良润生物医药科技有限公司 | Kit for quantitatively detecting AFP-L3% |
| CN105785043B (en) * | 2016-04-06 | 2018-02-02 | 上海良润生物医药科技有限公司 | For quantitatively detecting AFP L3% kit |
| CN108007989A (en) * | 2016-10-28 | 2018-05-08 | 中国科学院烟台海岸带研究所 | Based on detection method of the type polypeptide for identification molecule of splitting |
| CN108007989B (en) * | 2016-10-28 | 2020-04-17 | 中国科学院烟台海岸带研究所 | Detection method based on split polypeptide as recognition molecule |
| CN109470690A (en) * | 2018-10-19 | 2019-03-15 | 浙江大学 | The antigen detection method of electrochemical luminescence is differentiated based on current potential |
| CN110220964A (en) * | 2019-06-05 | 2019-09-10 | 常州大学 | The measurement method of chloride ion in the copper electrolyte of electrolytic copper foil |
| CN110220964B (en) * | 2019-06-05 | 2020-04-28 | 常州大学 | Method for measuring chloride ions in copper electrolyte of electrolytic copper foil |
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| CN104865240B (en) | 2019-11-19 |
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