CN105973876A - Electrochemiluminescence sensor for detecting dopamine, and production method and application thereof - Google Patents
Electrochemiluminescence sensor for detecting dopamine, and production method and application thereof Download PDFInfo
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- CN105973876A CN105973876A CN201610286963.1A CN201610286963A CN105973876A CN 105973876 A CN105973876 A CN 105973876A CN 201610286963 A CN201610286963 A CN 201610286963A CN 105973876 A CN105973876 A CN 105973876A
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- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 title claims abstract description 100
- 229960003638 dopamine Drugs 0.000 title claims abstract description 50
- 238000004519 manufacturing process Methods 0.000 title abstract 3
- 238000001514 detection method Methods 0.000 claims abstract description 26
- 239000000758 substrate Substances 0.000 claims abstract description 23
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 17
- 102000010909 Monoamine Oxidase Human genes 0.000 claims abstract description 15
- 108010062431 Monoamine oxidase Proteins 0.000 claims abstract description 15
- ZOXJIQNURSAHRV-UHFFFAOYSA-N [4-(4-azaniumylphenyl)phenyl]azanium;sulfate Chemical compound [O-]S([O-])(=O)=O.C1=CC([NH3+])=CC=C1C1=CC=C([NH3+])C=C1 ZOXJIQNURSAHRV-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000004020 conductor Substances 0.000 claims abstract 2
- 239000002953 phosphate buffered saline Substances 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 21
- 238000002360 preparation method Methods 0.000 claims description 19
- 239000002131 composite material Substances 0.000 claims description 17
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 15
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 12
- 238000002484 cyclic voltammetry Methods 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 9
- 229910021607 Silver chloride Inorganic materials 0.000 claims description 7
- 239000011521 glass Substances 0.000 claims description 6
- 229910052697 platinum Inorganic materials 0.000 claims description 6
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 5
- 230000004087 circulation Effects 0.000 claims description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 5
- 239000010452 phosphate Substances 0.000 claims description 5
- 239000000337 buffer salt Substances 0.000 claims description 4
- 230000004044 response Effects 0.000 claims description 4
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 claims description 3
- 229910021538 borax Inorganic materials 0.000 claims description 2
- 239000008366 buffered solution Substances 0.000 claims description 2
- 230000005611 electricity Effects 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 239000004328 sodium tetraborate Substances 0.000 claims description 2
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 abstract description 14
- 230000035945 sensitivity Effects 0.000 abstract description 8
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 abstract description 7
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 abstract description 7
- 229960005070 ascorbic acid Drugs 0.000 abstract description 7
- 229940116269 uric acid Drugs 0.000 abstract description 7
- 238000004458 analytical method Methods 0.000 abstract description 6
- 235000010323 ascorbic acid Nutrition 0.000 abstract description 6
- 239000011668 ascorbic acid Substances 0.000 abstract description 6
- 230000008569 process Effects 0.000 abstract description 5
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 108090000790 Enzymes Proteins 0.000 abstract description 4
- 150000001875 compounds Chemical class 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000000975 bioactive effect Effects 0.000 abstract description 2
- 230000036039 immunity Effects 0.000 abstract 1
- 230000000379 polymerizing effect Effects 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 18
- 238000004020 luminiscence type Methods 0.000 description 9
- 239000000463 material Substances 0.000 description 7
- 239000010931 gold Substances 0.000 description 6
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 5
- 229910052737 gold Inorganic materials 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 125000004122 cyclic group Chemical group 0.000 description 4
- 229910000397 disodium phosphate Inorganic materials 0.000 description 4
- 238000006116 polymerization reaction Methods 0.000 description 4
- 229910000162 sodium phosphate Inorganic materials 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 3
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 3
- DZGWFCGJZKJUFP-UHFFFAOYSA-N Tyramine Natural products NCCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-N 0.000 description 3
- 230000005518 electrochemistry Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229930182817 methionine Natural products 0.000 description 3
- 229960003732 tyramine Drugs 0.000 description 3
- DZGWFCGJZKJUFP-UHFFFAOYSA-O tyraminium Chemical compound [NH3+]CCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-O 0.000 description 3
- 108091023037 Aptamer Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- MRNHPUHPBOKKQT-UHFFFAOYSA-N indium;tin;hydrate Chemical compound O.[In].[Sn] MRNHPUHPBOKKQT-UHFFFAOYSA-N 0.000 description 2
- 238000002796 luminescence method Methods 0.000 description 2
- 239000002114 nanocomposite Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 238000006862 quantum yield reaction Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- DBZTWYNCZRQPOU-UHFFFAOYSA-N 1,1'-biphenyl;sulfuric acid Chemical group OS(O)(=O)=O.C1=CC=CC=C1C1=CC=CC=C1 DBZTWYNCZRQPOU-UHFFFAOYSA-N 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- 241000122824 Aspergillus ochraceus Species 0.000 description 1
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 102100020743 Dipeptidase 1 Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000002211 L-ascorbic acid Substances 0.000 description 1
- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- VYLQGYLYRQKMFU-UHFFFAOYSA-N Ochratoxin A Natural products CC1Cc2c(Cl)cc(CNC(Cc3ccccc3)C(=O)O)cc2C(=O)O1 VYLQGYLYRQKMFU-UHFFFAOYSA-N 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 101710084578 Short neurotoxin 1 Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 101710182532 Toxin a Proteins 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- DMVOXQPQNTYEKQ-UHFFFAOYSA-N biphenyl-4-amine Chemical compound C1=CC(N)=CC=C1C1=CC=CC=C1 DMVOXQPQNTYEKQ-UHFFFAOYSA-N 0.000 description 1
- 230000003925 brain function Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 150000003943 catecholamines Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- ZZVUWRFHKOJYTH-UHFFFAOYSA-N diphenhydramine Chemical compound C=1C=CC=CC=1C(OCCN(C)C)C1=CC=CC=C1 ZZVUWRFHKOJYTH-UHFFFAOYSA-N 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000001378 electrochemiluminescence detection Methods 0.000 description 1
- 239000007772 electrode material Substances 0.000 description 1
- 238000005401 electroluminescence Methods 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- -1 graphite alkene Chemical class 0.000 description 1
- NTOLGSSKLPLTDW-UHFFFAOYSA-N hydrogen sulfate;phenylazanium Chemical compound OS(O)(=O)=O.NC1=CC=CC=C1 NTOLGSSKLPLTDW-UHFFFAOYSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000002514 liquid chromatography mass spectrum Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002135 nanosheet Substances 0.000 description 1
- 230000007830 nerve conduction Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- RWQKHEORZBHNRI-BMIGLBTASA-N ochratoxin A Chemical compound C([C@H](NC(=O)C1=CC(Cl)=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 RWQKHEORZBHNRI-BMIGLBTASA-N 0.000 description 1
- DAEYIVCTQUFNTM-UHFFFAOYSA-N ochratoxin B Natural products OC1=C2C(=O)OC(C)CC2=CC=C1C(=O)NC(C(O)=O)CC1=CC=CC=C1 DAEYIVCTQUFNTM-UHFFFAOYSA-N 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- JRKICGRDRMAZLK-UHFFFAOYSA-L peroxydisulfate Chemical compound [O-]S(=O)(=O)OOS([O-])(=O)=O JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 description 1
- 230000005622 photoelectricity Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 238000004832 voltammetry Methods 0.000 description 1
- 239000011787 zinc oxide Substances 0.000 description 1
Classifications
-
- 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
Landscapes
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides an electrochemiluminescence sensor for detecting dopamine. The electrochemiluminescence sensor comprises an electrode, the electrode comprises a substrate, a compound layer and a modifying layer, the substrate is made of a conductive material, the compound layer grows on the surface of the conductive substrate and is formed through polymerizing luminol and benzidine sulfate, and the modifying layer is formed by tyramine oxidase modified on the surface of the compound layer. The invention also provides a production method and an application of the electrochemiluminescence sensor. A bioactive enzyme is used in electrochemiluminescence analysis in the invention to effectively solve the problem of poor selectivity of electrochemiluminescence methods. The production method and the detection method of the electrochemiluminescence sensor have the advantages of simplicity, high sensitivity, no influences of uric acid or ascorbic acid on the dopamine detection process, and good interference immunity.
Description
Technical field
The invention belongs to detection field, be specifically related to a kind of electrochemical luminous sensor and preparation thereof
And application.
Background technology
Dopamine is a kind of naturally occurring catecholamine, is a kind of important in mammal body
Nerve conduction mediator, most important to many basic physiological functions and higher brain function.Dopamine
Have very important regulation effect to body, its concentration level in vivo is combined with parkinson
Close the neurodegenerative diseases such as disease closely related.In view of the physiological function that dopamine is important, accurately
Measure its content in vivo and the prophylactic treatment of disease is become particularly important.
The method of on-line determination dopamine concentration has a lot, including chemiluminescence, electrochemical process,
The methods such as combined with liquid chromatography mass spectrum, surface plasma body resonant vibration and surface-enhanced Raman, such as
Patent CN201310479300.84 Zinc oxide nano sheet ball/foamy graphite alkene composite is made
Detect for dopamine for electrode material.These means there is a problem that it in testing
Cost higher, measure process time-consumingly the longest, and some test need sample is carried out pre-place
Manage and need special test environment.
The analysis method that electrochemiluminescence analysis method combines with electrochemistry as chemiluminescence,
Owing to the area of space of its luminescence-producing reaction concentrates on the near zone of electrode surface, have luminous anti-
The controlling answered is strong and sensitivity advantages of higher, therefore suffers from the extensive concern of analysis worker.
Electrochemiluminescence analysis has equipment and builds simply, and detection signal background noise is low, high sensitivity
And the advantage such as wider detection range, can be used to detect glucose, beta-lactamase and
The macromolecule organics such as DNA, such as patent CN201510482877.3 nano composite material
The preparation method of electrochemical luminous sensor, describe the electrochemistry of a kind of nano composite material
The preparation method of luminescence sensor, is deposited on composite luminol-AuBSA-anti-CEA
The electrode surface of gold, obtains the electrochemiluminescence immune sensing to antigen CEA with targeting
Device.Patent CN201010271247.9 electrochemiluminescence aptamer sensor detection Aspergillus ochraceus
The method of toxin A, modifies naked gold electrode surfaces by nano Au particle, then at working electrode table
Upper single stranded DNA is modified in face, then the aptamers being marked with different luminol (ABEI) is modified electricity
Surface, pole, adds hydrogen peroxide, detects electroluminescence signal, to detect ochratoxin A.Specially
Profit CN201510594214.03 provides electroluminescent chemistry based on methionine gold nano cluster
Luminescence sensor, with methionine protection gold nano cluster material as luminous body, persulfate
Ion is coreagent, and the gold nano cluster protected by methionine is modified on glass-carbon electrode,
Carry out electrochemiluminescence test, and realize the detection to dopamine.This sensor is to dopamine
The range of linearity of detection is 0.1～4 μm ol/L and 4～25 μm ol/L, and detection is limited to 0.032 μ
mol/L。
In the research of Electrochemiluminescince detection dopamine concentration, preparing of detection sensor is outstanding
For important.Owing to luminol and Polyluminol have electro-chemical activity and efficient luminescent properties,
It is the most extensive in the application of electrochemiluminescence detection dopamine concentration.But luminol and poly-Shandong
The quantum yield of minot is the highest, and detection sensitivity is relatively low.Simultaneously at live body environment middle and high concentration
Ascorbic acid and uric acid coexist with dopamine, owing to the oxidizing potential of three is much like, the most right
The detection of dopamine interferes.Therefore research high sensitivity and the selective electrochemiluminescence of height
Sensor will advance the development of dopamine detection field significantly.
Summary of the invention
The present invention proposes a kind of electrochemiluminescence sensing for dopamine detection innovatively
Device, its object is to overcome existing dopamine detection method to involve great expense, time-consumingly long, sensitivity
Low, the shortcoming such as poor selectivity.For problem above, the present invention is by luminol and benzidine sulfate
Polymerization, with the Photophysics of benzidine sulfate modulation luminol, to improve it in dopamine inspection
Quantum yield in survey, strengthens luminous intensity, thus improves its detection sensitivity.
It is another object of the present invention to propose the electrochemical luminous sensor of described detection dopamine
Preparation method.
Third object of the present invention is to propose the application of described electrochemical luminous sensor.
The technical scheme realizing above-mentioned purpose of the present invention is:
A kind of electrochemical luminous sensor detecting dopamine, described electrochemical luminous sensor bag
Including electrode, described electrode includes substrate, composite layer and decorative layer, and described substrate is conduction material
Material, described composite layer is grown on described conductive substrates surface, by luminol and sulphuric acid biphenyl
Amine is polymerized, and described decorative layer is made up of the tyramine oxidase modified in composite layer surface.
Owing to tyramine oxidase can be with selective catalytic oxidation dopamine, generated in-situ H2O2
Can the electrochemiluminescence of notable enhanced sensitivity luminol-benzidine sulfate, tyramine is aoxidized by the present invention
Enzyme is fixed on the compound film electrode surface recognition element as dopamine, by the choosing of enzymic catalytic reaction
The susceptiveness of selecting property and poly-(luminol-benzidine sulfate) electrochemical luminescence method is combined in one
Rise, constitute quick, sensitive, the sensor of selectivity much higher bar amine concentration mensuration.
The preparation method of electrochemical luminous sensor of the present invention, including step:
1) in phosphate buffered saline(PBS), configuration luminol and benzidine sulfate solution, use
Cyclic voltammetry, prepares poly-(luminol-benzidine sulfate) composite layer in conductive substrates,
2) above-mentioned electrode is taken out, is dried, configure tyramine oxidase solution with buffer solution,
By cyclic voltammetry in described step 1) poly-(luminol-benzidine sulfate) complex of obtaining
Tyramine oxidase decorative layer is fixed on layer surface.
Preferably, described step 1) in, the pH value of phosphate buffered saline(PBS) is 5.5～8.5,
In cyclic voltammetry preparation, with conductive substrates as working electrode, platinized platinum is to electrode, saturated sweet
Mercury electrode is reference electrode, and described conductive substrates is the FTO (SnO of doped with fluorine2Electrically conducting transparent
Glass) or ITO (tin indium oxide) electro-conductive glass.
Poly-(luminol-benzidine sulfate) film properties is affected by the pH value of phosphate buffered saline(PBS)
Less, prepare in the range of pH=5.5 set forth in the present invention～8.5 and all can obtain poly-(Rumi
Promise-benzidine sulfate) film.
Described step 1) in, in buffer salt solution, the molar concentration of luminol is 1～10mmol/L,
The molar concentration of benzidine sulfate is 1.5～15mmol/L.
It is highly preferred that the molar ratio of luminol and benzidine sulfate is 3:2～2:3.
Wherein, described step 1) in, conductive substrates is prepared poly-(luminol-sulphuric acid biphenyl
Amine) composite layer time, the potential range of cyclic voltammetry scan is-0.2V～1.0V, scanning circle
Several 30～100 circles, sweep speed 80～200mV/s.
Wherein, described step 2) in, described buffer solution be pH be the phosphate of 6.5～7.5
Buffer solution, containing the tyramine oxidation that concentration is 0.05～2mg/mL in described buffer solution
Enzyme.
Further, described step 2) in, the potential range of cyclic voltammetry scan is-0.2V～0.6
V, the scanning number of turns 10～30 circle, sweep speed 80～150mV/s.
The present invention also proposes the application of described electrochemical luminous sensor.
The method of the electrochemical luminous sensor detection dopamine that the application present invention proposes, by be measured
The pH value of solution is adjusted to 6.8～7.2, under the conditions of lucifuge, is sensed by described electrochemiluminescence
The electrode of device is placed in solution to be measured, applies square wave current potential, and potential range is-0.5～-0.9V (vs.
Ag/AgCl), the response signal of electrode is measured.
Preferably, square wave current potential can be selected with 0V ,-0.5～-0.9V (vs.Ag/AgCl) two
The voltage of ladder is a circulation, circulates 2～5 times.
Wherein, the pH value of solution to be measured is regulated by phosphate-buffered salt or borax buffer solution.
Wherein, in solution to be measured the content of dopamine at more than 0.5nmol/L.
The beneficial effects of the present invention is:
Method based on electrochemiluminescence test dopamine concentration, is different from traditional use Rumi
Promise or Polyluminol are as electrochemical luminous sensor, and the present invention selects to join luminol and sulphuric acid
Aniline carries out electrochemical polymerization, utilizes benzidine sulfate modulation to strengthen the optical physics of luminol
Can, and with tyramine oxidase as dopamine identify material modify the poly-(Shandong in above-mentioned preparation
Minot-benzidine sulfate) on composite membrane, make and send out for the electrochemistry detecting dopamine concentration
Optical sensor.
The present invention will have bioactive enzyme and be used for electrochemiluminescence analysis, can effectively solve
The problem of electrochemical luminescence method poor selectivity.The preparation of electrochemical sensor of the present invention and detection
Method is simple, highly sensitive, not by the shadow of uric acid and ascorbic acid etc. during dopamine detection
Ringing, anti-interference is good.
Accompanying drawing explanation
Fig. 1 a is embodiment of the present invention 1A) middle poly-(luminol-benzidine sulfate) film prepared
AFM picture；Fig. 1 b is embodiment of the present invention 1B) the middle tyramine oxidase-poly-(Shandong prepared
Minot-benzidine sulfate), i.e. the AFM picture of the electrochemical luminous sensor in the present invention；
Fig. 2 a is embodiment of the present invention 1A) in use cyclic voltammetry prepare poly-(luminol-
Benzidine sulfate) the course of reaction curve of film；Fig. 2 b is embodiment of the present invention 1B) in use follow
Ring voltammetry is in inventive embodiments 1A) in poly-(luminol-benzidine sulfate) film surface fix
The course of reaction curve of tyramine oxidase；
Fig. 3 a is the different dopamine concentration of electrochemical luminous sensor detection in the embodiment of the present invention 2
Time electrochemical luminescence signals；Fig. 3 b is the electrochemiluminescence of sensor in the embodiment of the present invention 2
Signal intensity is with the change of dopamine concentration and linear fit situation；
Fig. 4 is the anti-interference test result in the embodiment of the present invention 3, and specially sensor is in difference
Electrochemical luminescence signals intensity in solution, wherein DA is dopamine, and AA is ascorbic acid,
UA is uric acid.
Fig. 5 is that the present invention proposes electrochemical sensor structural representation.In figure, 1 is conductive substrates,
2 is composite layer, and 3 is decorative layer.
Detailed description of the invention
Now with following most preferred embodiment, the present invention is described, but is not limited to the model of the present invention
Enclose.
If no special instructions, the means used in embodiment are technology hands commonly used in the art
Section.
Embodiment 1:
In the present embodiment, the preparation method of electrochemical luminous sensor comprises the steps
A) preparation of poly-(luminol-benzidine sulfate) film
Configuration phosphate buffered saline(PBS), by changing 0.1mol/L NaH2PO4And 0.1mol/L
Na2HPO4The pH value of the proportion adjustment phosphate buffered saline(PBS) of aqueous solution is 5.7.Test knot
Fruit shows, solution ph is less on the impact of poly-(luminol-benzidine sulfate) film properties,
Prepare in the range of pH=5.5～8.5 and all can obtain poly-(luminol-benzidine sulfate) film；By Shandong
Minot and the benzidine sulfate above-mentioned phosphate buffered solution of addition are configured to reaction solution, specifically
Concentration is 8mmol/L luminol and 12mmol/L benzidine sulfate, selects FTO conduction glass
Glass makees substrate, and with FTO as working electrode, platinized platinum is to electrode, and saturated calomel electrode is ginseng
Ratio electrode, uses cyclic voltammetric electrochemical polymerization method above-mentioned luminol and benzidine sulfate to be polymerized
In FTO substrate, cyclic voltammetric parameter is set to scanning step 100mV/s, sweep limits
-0.2V～1.0V, cycle-index 40, the curve of polymerization process is as shown in Figure 2 a.By above-mentioned
Composite membrane cleans, and is dried, obtains poly-(luminol-benzidine sulfate) film, its afm image
As shown in Figure 1a (coordinate unit of Fig. 1 is μm).
B) tyramine oxidase is fixing
Configuration phosphate buffered saline(PBS), by changing 0.1mol/L NaH2PO4And 0.1mol/L
Na2HPO4The pH value of the proportion adjustment phosphate buffered saline(PBS) of aqueous solution is 7, uses described phosphorus
Tyramine oxidase is made into reactant liquor with the ratio of 0.1mg/mL by acid buffering saline solution.With step
A) poly-(luminol-benzidine sulfate) film being grown in FTO surface prepared in is that substrate is
Working electrode, platinized platinum is to electrode, and saturated calomel electrode is reference electrode, uses cyclic voltammetric
Above-mentioned tyramine oxidase is fixed in substrate by method, and concrete cyclic voltammetric parameter is set to scanning step
Long 100mV/s, sweep limits-0.2V～0.6V, cycle-index 20, response curve such as figure
Shown in 2b.By sample clean, it is dried, obtains complexes membrane, i.e. electrochemical luminous sensor,
Its afm image is as shown in Figure 1 b.
The electrode structure such as Fig. 5 prepared, conductive substrates 1 is FTO electro-conductive glass, composite layer
2 is to be polymerized by luminol and benzidine sulfate, and decorative layer 3 is by modifying in composite layer table
The tyramine oxidase in face is constituted.
Embodiment 2
The electrochemical luminous sensor that embodiment 1 prepares is for the method detecting dopamine concentration
Comprise the steps:
Test is carried out at ambient temperature, before test, configures phosphate buffered saline(PBS), by changing
Become 0.1mol/L NaH2PO4With 0.1mol/L Na2HPO4The proportion adjustment phosphoric acid of aqueous solution
The pH value of buffer salt solution is 7, adds dopamine and make it in described phosphate buffered saline(PBS)
Concentration is 0.5nmol/L, 1nmol/L, 10nmol/L and 20nmol/L respectively, to implement
In example 1, the electrochemical luminous sensor of preparation is working electrode, and platinum filament is to electrode, Ag/AgCl
(saturated KCl) is reference electrode, above-mentioned system is placed in the camera bellows of Weak-luminescence instrument, to
The upper system following square wave current potential of applying: (13s, 0V) and (4s ,-0.85V) (vs.Ag/AgCl),
Two square waves are a circulation, and work four circulations.First to 0.1mol/L in test process,
The phosphate buffered saline(PBS) of pH=7 is tested, to obtain blank signal, then by above-mentioned phosphorus
Acid buffering saline solution changes the dopamine solution of above-mentioned four kinds of concentration into, and above system is carried out photoelectricity
Chemiluminescent assay, obtains sample signal, and the electrochemiluminescence intensity finally giving sensor is
Sample signal deducts blank signal.Fig. 3 a show described electrochemical luminous sensor to difference
The dopamine response signal of concentration, along with the increase of dopamine concentration, luminous signal increases the most therewith
By force, the detection of dopamine is limited and has reached 0.5nmol/L by this sensor.By dopamine concentration with
Electrochemical luminescence signals is mapped, line linearity matching of going forward side by side (Fig. 3 b, coefficient R=0.9991),
The vertical coordinate of Fig. 3 is electrochemiluminescence intensity in test process, at 1nmol/L～20nmol/L
In the range of luminous intensity and the dopamine concentration of sensor present good linear relationship.
Embodiment 3
The electrochemical luminous sensor that the present invention proposes is used for detecting dopamine at high concentration uric acid
As follows with the selectivity test under ascorbic acid existence condition:
Test is carried out at ambient temperature, before test, configures phosphate buffered saline(PBS), by changing
Become 0.1mol/L NaH2PO4With 0.1mol/L Na2HPO4The proportion adjustment phosphoric acid of aqueous solution
The pH value of buffer salt solution is 7, adds 1nmol/L dopamine, 100nmol/L in the solution
Uric acid and 100nmol/L ascorbic acid are made into interference test solution, to make in embodiment 1
Standby electrochemical luminous sensor is working electrode, and platinum filament is to electrode, and Ag/AgCl is (saturated
KCl) it is reference electrode, above-mentioned system is placed in the camera bellows of Weak-luminescence instrument, it is applied such as
Lower square wave current potential: (13s, 0V) and (4s ,-0.85V) (vs.Ag/AgCl), tests four circulations.
First the phosphate buffered saline(PBS) of 0.1mol/L, pH=7 is tested by we, to obtain
Blank signal, then changes the interference test configured before into molten by above-mentioned phosphate buffered saline(PBS)
Liquid, tests above-mentioned system, obtains sample signal, the electrification of the sensor finally given
Learning luminous intensity is that sample signal deducts blank signal.
Fig. 4 reflects interference test result, it is found that at the bar of 100 times of dopamine concentrations
Under part, the electrochemical luminescence signals of uric acid and ascorbic acid well below dopamine, two kinds of interference
Property material can ignore, it follows that prepared electrochemical luminous sensor to dopamine detect
There is good selectivity.
Above embodiment is only to be described the preferred embodiment of the present invention, the most right
The scope of the present invention is defined, on the premise of designing spirit without departing from the present invention, and this area
Various modification that technical scheme is made by ordinary skill technical staff and improvement, all should
Fall in the protection domain that claims of the present invention determines.
Claims (10)
1. detecting an electrochemical luminous sensor for dopamine, described electrochemiluminescence senses
Device includes that electrode, described electrode include substrate, composite layer and decorative layer, it is characterised in that
Described substrate is conductive material, and described composite layer is grown on described conductive substrates surface, by
Luminol and benzidine sulfate are polymerized, and described decorative layer is by modifying in composite layer surface
Tyramine oxidase is constituted.
2. the preparation method of the electrochemical luminous sensor described in claim 1, its feature exists
In, including step:
1) in phosphate buffered saline(PBS), configuration luminol and benzidine sulfate solution, use
Cyclic voltammetry, prepares poly-(luminol-benzidine sulfate) composite layer in conductive substrates,
2) above-mentioned electrode is taken out, is dried, configure tyramine oxidase solution with buffer solution,
By cyclic voltammetry in described step 1) poly-(luminol-benzidine sulfate) complex of obtaining
Tyramine oxidase decorative layer is fixed on layer surface.
Preparation method the most according to claim 2, it is characterised in that described step 1)
In, the pH value of phosphate buffered saline(PBS) is 5.5～8.5, in cyclic voltammetry preparation, with conduction
Substrate is working electrode, and platinized platinum is to electrode, and saturated calomel electrode is reference electrode, described in lead
Electricity substrate is FTO or ITO electro-conductive glass.
Preparation method the most according to claim 2, it is characterised in that described step 1)
In, in buffer salt solution, the molar concentration of luminol is 1～10mmol/L, benzidine sulfate
Molar concentration is 1.5～15mmol/L.
Preparation method the most according to claim 2, it is characterised in that described step 1)
In, when conductive substrates is prepared poly-(luminol-benzidine sulfate) composite layer, circulation
The potential range of voltammetric scan is-0.2V～1.0V, the scanning number of turns 30～100 circle, sweep speed
80～200mV/s.
6. according to the arbitrary described preparation method of claim 2～5, it is characterised in that described
Step 2) in, described buffer solution be pH be the phosphate buffered solution of 6.5～7.5, in institute
State in buffer solution containing the tyramine oxidase that concentration is 0.05～2mg/mL.
7. according to the arbitrary described preparation method of claim 2～5, it is characterised in that described
Step 2) in, the potential range of cyclic voltammetry scan is-0.2V～0.6V, scans the number of turns 10～30
Circle, sweep speed 80～150mV/s.
8. the method for electrochemical luminous sensor detection dopamine described in application claim 1,
It is characterized in that, the pH value of solution to be measured is adjusted to 6.8～7.2, under the conditions of lucifuge, will
The electrode of described electrochemical luminous sensor is placed in solution to be measured, applies square wave current potential, current potential
Scope is-0.5～-0.9V (vs.Ag/AgCl), measures the response signal of electrode.
The method of electrochemical luminous sensor detection dopamine the most according to claim 8,
It is characterized in that, regulate the pH value of solution to be measured by phosphate-buffered salt or borax buffer solution.
The side of electrochemical luminous sensor detection dopamine the most according to claim 8 or claim 9
Method, it is characterised in that in solution to be measured, the content of dopamine is at more than 0.5nmol/L.
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