CN106932568A - A kind of preparation method and application based on interface transform electrochemical luminescence immunosensor - Google Patents
A kind of preparation method and application based on interface transform electrochemical luminescence immunosensor Download PDFInfo
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- 238000004020 luminiscence type Methods 0.000 title claims abstract description 32
- 229920001690 polydopamine Polymers 0.000 claims abstract description 29
- 229920000767 polyaniline Polymers 0.000 claims abstract description 21
- 239000000090 biomarker Substances 0.000 claims abstract description 14
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims description 76
- 229910052799 carbon Inorganic materials 0.000 claims description 27
- 239000004005 microsphere Substances 0.000 claims description 26
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 25
- 239000000047 product Substances 0.000 claims description 17
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 17
- 239000012498 ultrapure water Substances 0.000 claims description 17
- 239000003643 water by type Substances 0.000 claims description 17
- 238000004140 cleaning Methods 0.000 claims description 11
- -1 4°Hatch 1 h under C Substances 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 239000006227 byproduct Substances 0.000 claims description 8
- 239000000427 antigen Substances 0.000 claims description 6
- 108091007433 antigens Proteins 0.000 claims description 6
- 102000036639 antigens Human genes 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 5
- VYFYYTLLBUKUHU-UHFFFAOYSA-N Dopamine Natural products NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical class CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 5
- 229910002567 K2S2O8 Inorganic materials 0.000 claims description 5
- 239000012901 Milli-Q water Substances 0.000 claims description 5
- 229910021607 Silver chloride Inorganic materials 0.000 claims description 5
- 239000007983 Tris buffer Substances 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 238000011095 buffer preparation Methods 0.000 claims description 5
- 125000004122 cyclic group Chemical group 0.000 claims description 5
- 229960003638 dopamine Drugs 0.000 claims description 5
- 238000001548 drop coating Methods 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 5
- 238000005498 polishing Methods 0.000 claims description 5
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
- 206010028980 Neoplasm Diseases 0.000 claims description 3
- 102000004877 Insulin Human genes 0.000 claims description 2
- 108090001061 Insulin Proteins 0.000 claims description 2
- 239000012491 analyte Substances 0.000 claims description 2
- 238000001354 calcination Methods 0.000 claims description 2
- 210000004907 gland Anatomy 0.000 claims description 2
- 229940125396 insulin Drugs 0.000 claims description 2
- 102100023635 Alpha-fetoprotein Human genes 0.000 claims 1
- 102100021768 Phosphoserine aminotransferase Human genes 0.000 claims 1
- 108010072866 Prostate-Specific Antigen Proteins 0.000 claims 1
- 108010026331 alpha-Fetoproteins Proteins 0.000 claims 1
- 210000000481 breast Anatomy 0.000 claims 1
- 201000011510 cancer Diseases 0.000 claims 1
- 230000012447 hatching Effects 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 claims 1
- 239000002086 nanomaterial Substances 0.000 abstract description 8
- 238000000034 method Methods 0.000 abstract description 7
- 238000003837 high-temperature calcination Methods 0.000 abstract description 4
- 239000000758 substrate Substances 0.000 abstract description 3
- 239000011521 glass Substances 0.000 abstract description 2
- 239000004065 semiconductor Substances 0.000 abstract description 2
- 238000001179 sorption measurement Methods 0.000 abstract 1
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- 230000005518 electrochemistry Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
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- 238000006243 chemical reaction Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- MHUWZNTUIIFHAS-CLFAGFIQSA-N dioleoyl phosphatidic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC MHUWZNTUIIFHAS-CLFAGFIQSA-N 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- Immunology (AREA)
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- Engineering & Computer Science (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
Interface transform electrochemical luminescence immunosensor preparation method and application is based on the invention discloses one kind.The present invention is substrate using polyaniline golden nano-complexes, with reference to the unique adsorption function of carrier poly-dopamine microballoon, by the immobilized interface to electrode of electrochemical luminescence semiconductor nano material, obtains good luminous signal.Poly-dopamine microballoon and the good biocompatibility of polyaniline golden nano-complexes, sessile antibody, then by ito glass high-temperature calcination are utilized simultaneously.This technology is not only easy to operate, obtained sensor has preferable electrochemical luminescence performance, and also achieve the detection signal of different biomarkers is preserved for a long time, solve nano material and the problems such as biomolecule lacks simple and effective electrode fixing means.The method goes for the preparation of various biomarker immunosensors, is with a wide range of applications in scientific research and clinic.
Description
Technical field
The present invention relates to fields such as nano science, bio-sensing detection technique, electrochemical luminescences, and in particular to one kind is based on
The preparation method and application of interface transform electrochemical luminescence immunosensor.
Background technology
In recent decades, biology sensor flourishes, and is that early clinical diagnosis bring critical breakthrough, highly sensitive
Degree, the biology sensor of high selectivity, are always the study hotspot of domestic and international analytical chemistry worker, in environmental monitoring, food
The fields such as safety, clinical analysis have broad application prospects.Electrochemical luminescence sensor has that sensitivity is high, is easy to miniaturization
The characteristics of there is big advantage in actual applications, and the electrode sensing interface of stable response is to build this kind of electrochemistry hair
The key of optical sensor.
Nano material has superior electric conductivity, larger specific surface area and good bio-compatibility, extensive
It is applied to the structure of electrochemical luminescence sensor.In building process, both can be as substrate, it is also possible to marked as biology
Remember the amplification signal of thing.But nano material build sensor during exist come off from sensing interface, to substrate selective
The problems such as difference, low biometric identification capabilities.In addition, the electrochemical immunosensor of early stage generally carries out enzyme by antigen or antibody
Mark carrys out detection signal, but enzyme is higher to environmental requirement, and particularly enzyme is easily inactivated after being attached to solid carrier surface.It is based on
What the nano material with enzymatic activity built causes the great research interest of people without enzyme electrochemical immunosensor.Cause
This realizes the preservation steady in a long-term of luminous signal using the technology of interface transform when sensor is built, and can solve a nanometer material
Material problems present in sensor application, for the development and innovation of electrochemical luminescence sensor provide new thinking,
With highly important Research Significance.
The present invention is base material using polyaniline golden nano-complexes, is with the poly-dopamine microballoon with unique texture
Carrier, realizes fixation and High Efficiency Luminescence of the electrochemical luminescence semiconductor nano material in electrode interface.Poly- DOPA is utilized simultaneously
Amine microballoon and the good biocompatibility of polyaniline golden nano-complexes, fixed biomarker realize the structure of sensor.Profit
Electrochemical luminescence immunosensor is built with ito glass high-temperature calcination, the method is not only operated easily, with electrochemistry higher
Luminescent properties, and detection signal to different biomarkers preserved for a long time, solves nano material and biomolecule
The problems such as lacking simply and effectively electrode fixing means.The method goes for various biomarker immunosensors
Prepare, be with a wide range of applications in scientific research and clinic.
The content of the invention
An object of the present invention is to provide a kind of system of interface transform electrochemical luminescence immunosensor simple and easy to apply
Preparation Method, both can also realize quantitative determination to the detection of biomarker efficient and sensible.
The second object of the present invention is that polyaniline golden nano-complexes have good fixation to secondary antibody label, is led to
Cross high-temperature calcination, solve electrode modified material come off, poor reproducibility the problems such as.
The third object of the present invention is to provide permanent preservation, inexpensive, general biological marker object detecting method, is electrification
Learn application of the electrochemiluminescent immunoassay sensor in clinic and technical foundation is provided.
Technical scheme is as follows：
1. it is a kind of to be based on interface transform electrochemical luminescence immunosensor preparation method and application, it is characterised in that including following
Step：
（1）6 μ L polyaniline golden nano-complexes are dropped in the ITO electrode surface for the treatment of, 0.5 cm × 3.5 cm of activation,
In 4°Stored for future use in C refrigerators；
（2）By the μ g mL of 6 μ L 10-1Antibody-solutions drop coating is in step（1）Obtained electrode surface, 4°Hatch 1 h in C refrigerators；
（3）5 μ L are added dropwise, the bovine serum albumin solution that mass fraction is 1 % closes nonspecific activity site, in 4°C ice
Hatch 1 h in case, ultra-pure water cleaning is dried；A series of life of various concentrations that 6 μ L concentration are 0.001 ~ 20 ng/mL is added dropwise
Thing mark solution, 4°Hatch 1 h under C, ultra-pure water is cleaned, dried；
（4）The antibody-solutions of 6 μ L poly-dopamines Nano microspheres load azotized carbon nano piece mark, 4 are added dropwise°Hatch 1 in C refrigerators
H, ultra-pure water cleaning is dried, afterwards 400°30 min are calcined under the conditions of C, as working electrode, electrochemical luminescence survey is carried out
Examination.
2. the preparation of polyaniline golden nano-complexes solution, it is characterised in that comprise the following steps：
By the HAuCl of the mM of 5 mL 0.8 ~ 2.04Solution is placed in 45°10 min are preheated in C water-baths, is slowly added to afterwards
Aniline-the cyclohexane solution of the mM of 0.5 mL 20, react 12 h, by the rpm of product 11000 be centrifuged 10 min, wash three times, most
The polyaniline golden nano-complexes that will be obtained afterwards are distributed in 3 mL ultra-pure waters, and 4°C is preserved.
3. poly-dopamine Nano microsphere loads the preparation of azotized carbon nano piece solution, it is characterised in that comprise the following steps：
（1）The preparation of poly-dopamine Nano microsphere solution
80 ~ 120 mg Dopamine hydrochlorides are added to the Tris cushioning liquid and 30 ~ 70 mL isopropanols of 80 ~ 120 mL pH=8.8
Mixed liquor in, lucifuge reacts 24 h at room temperature, by product centrifugation and with milli-Q water three times, afterwards divides product
It is scattered in 5 mL ultra-pure waters；
（2）The preparation of azotized carbon nano piece solution
5 g dicyandiamides are placed in Muffle furnace, and 550°C burns 4 h, heating rate 5°C/min, is cooled to room temperature afterwards, weighs
100 mg carbonitrides are placed in 100 mL ultra-pure waters, first with wall-breaking machine 10 ~ 20 min of polishing, then the h of ultrasonic disperse 4 ~ 8,
The rpm of product 5000 is centrifuged 10 min, upper solution is standby；
（3）Poly-dopamine Nano microsphere loads the preparation of azotized carbon nano piece solution
1 mL poly-dopamine Nano microspheres solution adds 2 mL azotized carbon nano piece solution, ultrasonic 15 min to be placed in 4°Shaken under C
6 h are swung, be distributed to product in 1 mL ultra-pure waters afterwards by centrifugation.
4. a kind of to be based on interface transform electrochemical luminescence immunosensor preparation method and application, the sensor of preparation is used for
Biological marker analyte detection, it is characterised in that step is as follows：Using the ITO electrode after calcining as working electrode, Ag/AgCl electrodes
As reference electrode, Pt electrodes as to electrode, with 50 mmolL of the PBS buffer preparations that pH is 7.4-1K2S2O8
Solution determines with cyclic voltammetric pattern the luminous letter under -1.1 ~ 0 V potentials as bottom liquid on electrochemical luminescence work station
Number, realize the detection to biomarker.
Useful achievement of the invention
The preparation method of 1 sensor is not only operated easily, with low cost, and with efficient electrochemical luminescence performance, solution
Nano material of having determined and biomolecule lack simple and effectively electrode fixing means problem.
2 poly-dopamine microsphere surfaces contain abundant functional group, are easy to fix biomarker and antibody, in sensing interface
As important carrier during conversion, it is to avoid complicated antibody fixation procedure and the deactivation prob for thus causing.
3 polyaniline golden nano-complexes have good fixation to secondary antibody label, by high-temperature calcination so that passing
Sensor stable luminescent property, solve electrode modified material come off, poor reproducibility the problems such as.
4 the method go for the preparation of various biomarker immunosensors, have in scientific research and clinic wide
General application prospect.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.
A kind of preparation method based on interface transform electrochemical luminescence immunosensor of embodiment 1
（1）6 μ L polyaniline golden nano-complexes are dropped in the ITO electrode surface for the treatment of, 0.5 cm × 3.5 cm of activation,
In 4°Stored for future use in C refrigerators；
（2）By the μ g mL of 6 μ L 10-1Insulin antibody solution drop coating is in step（1）Obtained electrode surface, 4°Incubated in C refrigerators
Change 1 h；
（3）5 μ L are added dropwise, the bovine serum albumin solution that mass fraction is 1 % closes nonspecific activity site, in 4°C ice
Hatch 1 h in case, ultra-pure water cleaning is dried；A series of pancreas of various concentrations that 6 μ L concentration are 0.001 ~ 20 ng/mL is added dropwise
Island element solution, 4°Hatch 1 h under C, ultra-pure water is cleaned, dried；
（4）The antibody-solutions of 6 μ L poly-dopamines Nano microspheres load azotized carbon nano piece mark, 4 are added dropwise°Hatch 1 in C refrigerators
H, ultra-pure water cleaning is dried, afterwards 400°30 min are calcined under the conditions of C, using ITO electrode as working electrode, Ag/AgCl electricity
Pole as reference electrode, Pt electrodes as to electrode, with 50 mmolmL of the PBS buffer preparations that pH is 7.4-1's
K2S2O8Solution determines with cyclic voltammetric pattern the hair under -1.1 ~ 0 V potentials as bottom liquid on electrochemical luminescence work station
Optical signal, realizes the detection to insulin.
A kind of preparation method based on interface transform electrochemical luminescence immunosensor of embodiment 2
（1）6 μ L polyaniline golden nano-complexes are dropped in the ITO electrode surface for the treatment of, 0.5 cm × 3.5 cm of activation,
In 4°Stored for future use in C refrigerators；
（2）By the μ g mL of 6 μ L 10-1PSA antibody-solutions drop coating is in step（1）Obtained electrode surface, 4°Hatch 1 h in C refrigerators；
（3）5 μ L are added dropwise, the bovine serum albumin solution that mass fraction is 1 % closes nonspecific activity site, in 4°C ice
Hatch 1 h in case, ultra-pure water cleaning is dried；Be added dropwise 6 μ L concentration for 0.001 ~ 20 ng/mL it is a series of without concentration before
Row gland specific antigen solution, 4°Hatch 1 h under C, ultra-pure water is cleaned, dried；
（4）The antibody-solutions of 6 μ L poly-dopamines Nano microspheres load azotized carbon nano piece mark, 4 are added dropwise°Hatch 1 in C refrigerators
H, ultra-pure water cleaning is dried, afterwards 400°30 min are calcined under the conditions of C, using ITO electrode as working electrode, Ag/AgCl electricity
Pole as reference electrode, Pt electrodes as to electrode, with 50 mmolmL of the PBS buffer preparations that pH is 7.4-1's
K2S2O8Solution determines with cyclic voltammetric pattern the hair under -1.1 ~ 0 V potentials as bottom liquid on electrochemical luminescence work station
Optical signal, realizes the detection to PSA.
A kind of preparation method based on interface transform electrochemical luminescence immunosensor of embodiment 3
（1）6 μ L polyaniline golden nano-complexes are dropped in the ITO electrode surface for the treatment of, 0.5 cm × 3.5 cm of activation,
In 4°Stored for future use in C refrigerators；
（2）By the μ g mL of 6 μ L 10-1Carcinomebryonic antigen antibody-solutions drop coating is in step（1）Obtained electrode surface, 4°In C refrigerators
Hatch 1 h；
（3）5 μ L are added dropwise, the bovine serum albumin solution that mass fraction is 1 % closes nonspecific activity site, in 4°C ice
Hatch 1 h in case, ultra-pure water cleaning is dried；A series of cancers without concentration that 6 μ L concentration are 0.001 ~ 20 ng/mL are added dropwise
Embryonal antigen solution, 4°Hatch 1 h under C, ultra-pure water is cleaned, dried；
（4）The antibody-solutions of 6 μ L poly-dopamines Nano microspheres load azotized carbon nano piece mark, 4 are added dropwise°Hatch 1 in C refrigerators
H, ultra-pure water cleaning is dried, afterwards 400°30 min are calcined under the conditions of C, using ITO electrode as working electrode, Ag/AgCl electricity
Pole as reference electrode, Pt electrodes as to electrode, with 50 mmolmL of the PBS buffer preparations that pH is 7.4-1's
K2S2O8Solution determines with cyclic voltammetric pattern the hair under -1.1 ~ 0 V potentials as bottom liquid on electrochemical luminescence work station
Optical signal, realizes the detection to carcinomebryonic antigen.
The preparation of the polyaniline golden nano-complexes solution of embodiment 4
By the HAuCl of the mM of 5 mL 0.84Solution is placed in 45°10 min are preheated in C water-baths, 0.5 mL is slowly added to afterwards
Aniline-the cyclohexane solution of 20 mM, reacts 12 h, and the rpm of product 11000 is centrifuged into 10 min, washes three times, will finally obtain
Polyaniline golden nano-complexes be distributed in 3 mL ultra-pure waters, 4°C is preserved.
The preparation of the polyaniline golden nano-complexes solution of embodiment 5
By the HAuCl of the mM of 5 mL 1.04Solution is placed in 45°10 min are preheated in C water-baths, 0.5 mL is slowly added to afterwards
Aniline-the cyclohexane solution of 20 mM, reacts 12 h, and the rpm of product 11000 is centrifuged into 10 min, washes three times, will finally obtain
Polyaniline golden nano-complexes be distributed in 3 mL ultra-pure waters, 4°C is preserved.
The preparation of the polyaniline golden nano-complexes solution of embodiment 6
By the HAuCl of the mM of 5 mL 2.04Solution is placed in 45°10 min are preheated in C water-baths, 0.5 mL is slowly added to afterwards
Aniline-the cyclohexane solution of 20 mM, reacts 12 h, and the rpm of product 11000 is centrifuged into 10 min, washes three times, will finally obtain
Polyaniline golden nano-complexes be distributed in 3 mL ultra-pure waters, 4°C is preserved.
The poly-dopamine Nano microsphere of embodiment 7 loads the preparation of azotized carbon nano piece solution
（1）The preparation of poly-dopamine Nano microsphere solution
80 mg Dopamine hydrochlorides are added in the mixed liquor of the Tris cushioning liquid of 80 mL pH=8.8 and 30 mL isopropanols,
Lucifuge reacts 24 h at room temperature, by product centrifugation and with milli-Q water three times, product is distributed into 5 mL afterwards and is surpassed
In pure water；
（2）The preparation of azotized carbon nano piece solution
5 g dicyandiamides are placed in Muffle furnace, and 550°C burns 4 h, heating rate 5°C/min, is cooled to room temperature afterwards, weighs
100 mg carbonitrides are placed in 100 mL ultra-pure waters, first with wall-breaking machine 10 min of polishing, then the h of ultrasonic disperse 4, by product 5000
Rpm is centrifuged 10 min, and upper solution is standby；
（3）Poly-dopamine Nano microsphere loads the preparation of azotized carbon nano piece solution
1 mL poly-dopamine Nano microspheres solution adds 2 mL azotized carbon nano piece solution, ultrasonic 15 min to be placed in 4°Shaken under C
6 h are swung, be distributed to product in 1 mL ultra-pure waters afterwards by centrifugation.
The poly-dopamine Nano microsphere of embodiment 8 loads the preparation of azotized carbon nano piece solution
（1）The preparation of poly-dopamine Nano microsphere solution
100 mg Dopamine hydrochlorides are added to the Tris cushioning liquid of 100 mL pH=8.8 and the mixed liquor of 50 mL isopropanols
In, lucifuge reacts 24 h at room temperature, by product centrifugation and with milli-Q water three times, product is distributed into 5 mL afterwards
In ultra-pure water；
（2）The preparation of azotized carbon nano piece solution
5 g dicyandiamides are placed in Muffle furnace, and 550°C burns 4 h, heating rate 5°C/min, is cooled to room temperature afterwards, weighs
100 mg carbonitrides are placed in 100 mL ultra-pure waters, first with wall-breaking machine 15 min of polishing, then the h of ultrasonic disperse 6, by product 3000
Rpm is centrifuged 10 min, and upper solution is standby；
（3）Poly-dopamine Nano microsphere loads the preparation of azotized carbon nano piece solution
1 mL poly-dopamine Nano microspheres solution adds 2 mL azotized carbon nano piece solution, ultrasonic 15 min to be placed in 4°Shaken under C
6 h are swung, be distributed to product in 1 mL ultra-pure waters afterwards by centrifugation.
The poly-dopamine Nano microsphere of embodiment 9 loads the preparation of azotized carbon nano piece solution
（1）The preparation of poly-dopamine Nano microsphere solution
120 mg Dopamine hydrochlorides are added to the Tris cushioning liquid of 120 mL pH=8.8 and the mixed liquor of 70 mL isopropanols
In, lucifuge reacts 24 h at room temperature, by product centrifugation and with milli-Q water three times, product is distributed into 5 mL afterwards
In ultra-pure water；
（2）The preparation of azotized carbon nano piece solution
5 g dicyandiamides are placed in Muffle furnace, and 550°C burns 4 h, heating rate 5°C/min, is cooled to room temperature afterwards, weighs
100 mg carbonitrides are placed in 100 mL ultra-pure waters, first with wall-breaking machine 20 min of polishing, then the h of ultrasonic disperse 8, by product 3000
Rpm is centrifuged 10 min, and upper solution is standby；
（3）Poly-dopamine Nano microsphere loads the preparation of azotized carbon nano piece solution
1 mL poly-dopamine Nano microspheres solution adds 2 mL azotized carbon nano piece solution, ultrasonic 15 min to be placed in 4°Shaken under C
6 h are swung, be distributed to product in 1 mL ultra-pure waters afterwards by centrifugation.
Claims (5)
1. a kind of preparation method and application based on interface transform electrochemical luminescence immunosensor, it is characterised in that including with
Lower step：
（1）6 μ L polyaniline golden nano-complexes are dropped in the ITO electrode surface for the treatment of, 0.5 cm × 3.5 cm of activation,
In 4°Stored for future use in C refrigerators；
（2）By the μ g mL of 6 μ L 10-1Antibody-solutions drop coating is in step（1）Obtained electrode surface, 4°Hatch 1 h in C refrigerators；
（3）5 μ L are added dropwise, the bovine serum albumin solution that mass fraction is 1 % closes nonspecific activity site, in 4°C refrigerators
1 h of middle hatching, ultra-pure water cleaning is dried；A series of biology of various concentrations that 6 μ L concentration are 0.001 ~ 20 ng/mL is added dropwise
Mark solution, 4°Hatch 1 h under C, ultra-pure water cleaning is dried；
（4）The antibody-solutions of 6 μ L poly-dopamines Nano microspheres load azotized carbon nano piece mark, 4 are added dropwise°Hatch 1 in C refrigerators
H, ultra-pure water cleaning is dried, afterwards 400°30 min are calcined under the conditions of C, as working electrode, electrochemical luminescence survey is carried out
Examination.
2. a kind of preparation method and application based on interface transform electrochemical luminescence immunosensor as claimed in claim 1,
The preparation of the polyaniline golden nano-complexes solution, it is characterised in that comprise the following steps：
By the HAuCl of the mM of 5 mL 0.8 ~ 2.04Solution is placed in 45°10 min are preheated in C water-baths, 0.5 is slowly added to afterwards
Aniline-the cyclohexane solution of the mM of mL 20, reacts 12 h, and the rpm of product 11000 is centrifuged into 10 min, washes three times, finally will
The polyaniline golden nano-complexes for obtaining are distributed in 3 mL ultra-pure waters, and 4°C is preserved.
3. a kind of preparation method and application based on interface transform electrochemical luminescence immunosensor as claimed in claim 1,
The poly-dopamine Nano microsphere loads the preparation of azotized carbon nano piece solution, it is characterised in that comprise the following steps：
（1）The preparation of poly-dopamine Nano microsphere solution
80 ~ 120 mg Dopamine hydrochlorides are added to the Tris cushioning liquid and 30 ~ 70 mL isopropanols of 80 ~ 120 mL pH=8.8
Mixed liquor in, lucifuge reacts 24 h at room temperature, by product centrifugation and with milli-Q water three times, afterwards divides product
It is scattered in 5 mL ultra-pure waters；
（2）The preparation of azotized carbon nano piece solution
5 g dicyandiamides are placed in Muffle furnace, and 550°C burns 4 h, heating rate 5 °C/min, is cooled to room temperature afterwards, weighs
100 mg carbonitrides are placed in 100 mL ultra-pure waters, first with wall-breaking machine 10 ~ 20 min of polishing, then the h of ultrasonic disperse 4 ~ 8, will
The rpm of product 5000 is centrifuged 10 min, and upper solution is standby；
（3）Poly-dopamine Nano microsphere loads the preparation of azotized carbon nano piece solution
1 mL poly-dopamine Nano microspheres solution adds 2 mL azotized carbon nano piece solution, ultrasonic 15 min to be placed in 4°Shaken under C
6 h are swung, be distributed to product in 1 mL ultra-pure waters afterwards by centrifugation.
4. a kind of preparation method based on interface transform electrochemical luminescence immunosensor according to claim 1 and should
With, it is characterised in that described biomarker is carcinomebryonic antigen, PSA, insulin, alpha-fetoprotein, breast
One kind in gland cancer tumor susceptibility gene.
5. a kind of preparation method based on interface transform electrochemical luminescence immunosensor according to claim 1 and should
With the sensor of preparation is used for biological marker analyte detection, it is characterised in that step is as follows：Using the ITO electrode after calcining as work
Make electrode, Ag/AgCl electrodes as reference electrode, Pt electrodes as to electrode, with the PBS buffer preparations that pH is 7.4
50 mmol·L-1K2S2O8Solution determines -1.1 ~ 0 on electrochemical luminescence work station as bottom liquid with cyclic voltammetric pattern
Luminous signal under V potentials, realizes the detection to biomarker.
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| CN110133266A (en) * | 2019-05-22 | 2019-08-16 | 太原理工大学 | The method of the preparation of the conjugate of cancer embryo antibody and polyaniline@gold and its building photo-thermal immunosensor detection carcinomebryonic antigen |
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